Its planar structure was elucidated by extensive NMR investigations, whereas its absolute configurations, featuring the presence of 36 carbon stereocenters and 30 hydroxy groups, were successfully established by comparison of NMR data of the ozonolyzed products with those of gibbosol A, combined with J-based configuration analysis, Kishi’s universal NMR database, and the modified Mosher’s MTPA ester method. Multi-segment modification was revealed as the smart biosynthetic strategy for the dinoflagellate to create remarkable super-carbon-chain compounds with structural diversity.A super-carbon-chain compound, named gibbosol C, featuring a polyoxygenated C Marine dinoflagellates produce huge organic molecules, particularly super-carbon-chain compounds (SCCCs), which are complex natural products with numerous carbon stereocenters on a long-carbon-chain backbone ,2,3,4. AAmphidinium and Karlodinium, exhibit antifungal, antitumor, antiosteoclastic, and analgesic effects  ions at m/z 784.4814  and 1567.9579 . According to the 1H and 13C NMR data of 1 , 41 methine groups (including 5 olefinic and 33 oxymethine groups), and three non-protonated olefinic carbons.The molecular formula of ata of 1 , four deI , II , and III , were determined by analysis of key 1H–1H COSY, H2BC, and HMBC correlations of 1 –H-10, H2BC correlations from H-7/C-6 and H-7/C-8, and HMBC correlations from H-7/C-9, H3-72/C-8, H3-72/C-9, and H3-72/C-10. The linear connection of C-4 to C-6 and the branched connection of C-5 and C-71 were corroborated by the H2BC correlation from H-6/C-5 and HMBC cross-peaks from H3-71/C-4, H3-71/C-5, H3-71/C-6, and H-7/C-5, whereas the linear connection of C-10 to C-13 and the branched connections from C-11/C-73 and C-13/C-74 were confirmed by HMBC cross-peaks from H-10/C-12, H3-73/C-10, H3-73/C-11, H3-73/C-12, H3-74/C-12, and H3-74/C-13. Taken together, the planar structure of I was elucidated , C-48 (δ 73.2), C-52(δ 76.5), and C-56 (δ 75.76), did not exhibit a deuterium-induced isotope shift. The presence of ether bridges between C-44/C-48 and C-52/C-56, respectively, was further supported by HMBC cross-peaks from H-44/C-48 and H-52/C-56. Taken together, the backbone of III was assembled  and NOE correlations from H3-72/H3-73, H-10/H-12, H3-75/H3-76, H-32/H2-34, and H3-70/H-68 concluded that the geometries of the four carbon–carbon double bonds in 1, viz., C10═C11, C12═C13, C32═C33, and C68═C69, were all E-configured. Based on the above results, the planar structure of 1, containing 30 hydroxy groups and six pendant methyl moieties, was successfully established  groups in 1b  , by(δ 66.4) ,33.JBCA 2+  and [M + H]+ .Gibbosol C (1)  was dissolved in a mixture of CH2Cl2–MeOH . Ozone was bubbled into the above solution at –78 °C for 2 min. An excess amount of NaBH4 was then added and stirred at –78 °C for 3 h. The reaction mixture was purified by a C18 reversed-phase silica gel column , eluted with 10 mL of water followed by 15 mL of MeOH, to afford three fractions. The second fraction was purified by HPLC  to afford three products, viz., fragments 1a , 1b , and 1c  (Gibbosol C (2.7 min) .1a: For 1H and 13C NMR spectroscopic data, see m/z 257.6 [M + Na]+ and 491.6 [2M + Na]+.1b: For 1H and 13C NMR spectroscopic data, see m/z 487.6 [M + H]+ and 509.5 [M + Na]+.1c: For 1H and 13C NMR spectroscopic data, see m/z 869.4 [M + H]+ and 891.3 [M + Na]+.1a (0.3 mg) was treated with (R)-MTPACl (8.0 μL) in dried pyridine (0.8 mL) at room temperature for 10 h. The reaction mixture was concentrated and purified by HPLC  to afford the (S)-MTPA ester 1as (1.1 mg). The (R)-MTPA ester 1ar (1.0 mg) was prepared in the same way. With the aid of key 1H–1H COSY correlations, 1H NMR spectroscopic data of 1as and 1ar were assigned (The fragment assigned .1as: For 1H NMR spectroscopic data, see m/z 1116.4 [M + NH4]+ and 1121.2 [M + Na]+.1ar: For 1H NMR spectroscopic data, see m/z 1116.4 [M + NH4]+ and 1121.3 [M + Na]+.A. gibbosum. Its planar structure and absolute configurations, featuring the presence of 36 carbon stereocenters and 30 hydroxy groups, were successfully established by extensive NMR investigations, ozonolysis of the carbon–carbon double bonds, J-based configuration analysis, Kishi’s universal NMR database, the modified Mosher’s MTPA ester method, and comparison of the NMR data of the ozonolyzed products with those of gibbosol A. Multi-segment modification seems to be the smart biosynthetic strategy for the dinoflagellate to create remarkable SCCCs with diverse structures. Marine dinoflagellates of the genus Amphidinium harbor novel and complex SCCC biosynthetic routes. New integrated chemical, spectroscopic, and computational approaches or intelligent databases should be developed to cope with the stereochemical complexity of SCCCs. Evidently, specific carbon–carbon bond cleavages are an important means for the determination of the relative and absolute configurations of polyol-polyol SCCCs in the future.In summary, a new polyol-polyol SCCC, named gibbosol C, was isolated from the South China Sea dinoflagellate


Penicillium chrysogenum strain S003, a fungus isolated from Red Sea deep sediment, led to the isolation of a cerebroside molecular species LAMA (1) along with three other known compounds, ergosterol (2), epidioxyergosterol (3), and kojic acid (4). The structures of the isolated compounds were elucidated by interpretation of spectral data, including detailed 1D and 2D NMR  and mass spectrometry. The cytotoxic activities of isolated compounds 1–4 against five human carcinoma cells were evaluated using sulforhodamine B (SRB) assay. Compounds 2 and 3 displayed promising cytotoxic profiles against lung cancer (A-549), prostate (DU-145), breast adenocarcinoma (MCF-7), and hepatocellular (HepG2) cell lines, with IC50 values of 21.26, 19.3; 1.50, 6.10; 16.95, 13.6; and 2.89, 3.07 µM, respectively, while they were inactive against HeLa cells. Compounds 1 and 4 showed weak cytotoxic profiles against all cell lines under investigation.Chemical investigation of the ethyl acetate extract of  Penicillium species, which are considered a major source for drug discovery , an additional trisubstituted double bond  in long-chain base residues, and an extra disubstituted double bond  (JH4–H5 = 15.5 Hz) clearly indicated an E geometry for the double bond [δC 33.0 (C-6)]   in fattyble bond , which w0 (C-6)] . By the 15.3 Hz) . The geo δC 22.7 . The 2-ae groups . Furtherde chain  because al type) .− showed a succession of molecular ion peaks , since the aforementioned 13C- NMR signals  and 1H-NMR , in addition to optical rotation value S, 3R, 2′R) conformations −, which were considered as FAME-1, FAME-2, FAME-3, and FAME-4, indicating C-16, C-17, C-18, and C-22 FAMEs, respectively  +, 726 [M + H]+, 724 [M − H]−, and the characteristic fragments at m/z 562 corresponding to the aglycone part in addition to m/z 472 corresponding to glucosylated long chain base  [3), [4) [In addition to LAMA-1, the structures of other known compounds 2–4  were elu2–4 (2) ,29,30, e(2) [3), , and koj[3), [4) ,33,34,351–4 were evaluated for their cytotoxic activity against five selected human cancer cell lines (2 and 3 displayed a promising cytotoxic profile against lung-cancer (A-549), prostate cancer (DU-145), breast adenocarcinoma (MCF-7), and hepatocellular carcinoma (HepG2) cell lines with IC50 values of 21.26, 19.3; 1.50, 6.10; 16.95, 13.6; and 2.89, 3.07 µM, respectively, while they were inactive against HeLa cells. Compounds 1 and 4 showed weak cytotoxic profiles against all cell lines under investigation . After cultivation for 30 days under static conditions, 15 L of the whole broth was filtrated using cheesecloth. Extraction of the broth with ethyl acetate was performed three times, and was further dried under vacuum to afford an ethyl acetate extract (1.4 g), while the mycelia were extracted three times with MeOH. The MeOH solution was partitioned with n-hexane, followed by evaporation to give a MeOH extract and an n-hexane extract (5 g). The MeOH extract was dissolved in water and partitioned with CHCl3, followed by evaporation under reduced pressure to produce an CHCl3 extract (2.8 g). The resulting extracts were subjected to further fractionation and separation to obtain the pure compounds.Fungus 3 extract  from the previous section was chromatographed using CHCl3: MeOH (9.5:0.5~4:6) on SiO2 gel to yield 11 subfractions (C-M-C-1 to C-M-C-11). Fraction C-M-C-7 (325 mg) was subjected to a Sephadex LH-20 column and eluted with MeOH to afford 5 fractions (C-M-C-7-1 to C-M-C-7-5). Fraction C-M-C-7-1 (194 mg) was passed through a SiO2 gel column using CHCl3/MeOH (97:3 to 90:10) to afford two subfractions (C-M-C-7-1-1 to C-M-C-7-1-2). Fraction C-M-C-7-1-2 (80 mg) was chromatographed over a SiO2 gel column using CHCl3/MeOH (95:5) to yield phytoceramide molecular species compound (1) (30 mg). The name LAMA was assigned to compound 1. Compound 1 was fractionated over reversed-phase column chromatography using H2O/MeOH (2:8 to 0.5:9.5) to give LAMA-1.The resulting CHCl3 (1:1) to yield 10 subfractions (C-E-1 to C-E-10). Fraction C-E-4 (0.5 g) was passed through a SiO2 column with increasing EtOAc in n-hexane as eluent to obtain six fractions (C-E-4-1 to C-E-4-6). Fraction C-E-4-3 (195 mg) was subject to further chromatography using n-hexane: EtOAc (9.5:0.5 to 1:9) over a SiO2 gel column to give eight fractions (C-E-4-3-1 to C-E-4-3-8). Fraction C-E-4-3-2 (24 mg) was purified over a silica gel column using n-hexane/EtOAc (9.5:0.5 to 1:1) to get ergosterol (2) (3.1 mg) and epidioxyergosterol (3) (1.4 mg).The EtOAc extract  was eluted on Sephadex LH-20 column with MeOH/CHCl2 gel column using n-hexane/EtOAc (2:8 to 0:10) gradient elution produced seven subfractions (C-E-6-1 to C-E-6-7). Fraction C-E-6-4 (57 mg) was fractionated using MeOH/H2O (6:4) isocratic elution on reversed phase column, followed by gradient elution using n-hexane/EtOAc (2:8~0:10) on SiO2 gel column to obtain kojic acid (4) (20 mg).Chromatography for fraction C-E-6 (190 mg) over the SiOThe cytotoxic activity  of the cPenicillium chrysogenum S003 yielded a cerebroside molecular species (1), and three known compounds: ergosterol (2), epidioxyergosterol (3), and kojic acid (4). The chemical structures of purified compounds 1–4 were characterized using spectroscopic studies and by comparison with available data in the literature. The cytotoxic activities of isolated compounds 1–4 against five human carcinoma cells were evaluated using an SRB assay. Compounds 2 and 3 displayed a promising cytotoxic profiles against lung cancer (A-549), prostate cancer (DU-145), breast adenocarcinoma (MCF-7), and hepatocellular carcinoma (HepG2) cell lines with IC50 values of 21.26, 19.3; 1.50, 6.10; 16.95, 13.6; and 2.89, 3.07 µM, respectively, while they were inactive against HeLa cells. Compounds 1 and 4 showed weak cytotoxic profiles against all cell lines under investigation.Chemical investigation of deep-sediment-derived


The synthesis was initiated from ethyl 2-acetate (1) which was converted to corresponding hydrazide, 2, by hydrazine hydrate in methanol. The refluxing of hydrazide, 2, with carbon disulfide in basic medium, resulted in 5-[methyl]-1,3,4-oxadiazol-2-thiol (3). A series of electrophiles, 6a-l, was synthesized by stirring un/substituted anilines (4a-l) with 3-bromopropanoyl chloride (5) in a basic aqueous medium. Finally, the targeted compounds, 7a-l, were acquired by stirring 3 with newly synthesized electrophiles, 6a-l, in DMF using LiH as a base and an activator. The structures of these bi-heterocyclic propanamides were confirmed through spectroscopic techniques, such as IR, 1H-NMR, 13C-NMR, and EI-MS. These molecules were tested for their urease inhibitory potential, whereby, the whole series exhibited very promising activity against this enzyme. Their cytotoxic behavior was ascertained through hemolysis and it was observed that all these were less cytotoxic agents. The in-silico molecular docking analysis of these molecules was also in full agreement with their in-vitro enzyme inhibition data.The synthesis of a novel series of bi-heterocyclic propanamides,  Heterocyclic compounds have been under investigation for a long time because of their important pharmacological properties . ThiazolN-acyl hydrazones, and reaction of N-acylbenzotriazoles with acyl hydrazides . The spots were detected by UV254. Gallonkamp apparatus was used to detect melting points (uncorrected) in capillary tubes. IR spectra  were recorded by KBr pellet method in the Jasco-320-A spectrophotometer. EI-MS spectra were measured on a JEOL JMS-600H instrument with data processing system. 1H-NMR spectra  were recorded at 600 MHz  in DMSO-d6 using the Bruker Advance III 600 As- cend spectrometer using BBO probe. The coupling constant (J) is given in Hz and chemical shift  in ppm. The abbreviations used in interpretation of 1H NMR spectra are as follows: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; br.t, broad triplet; q, quartet; quint, quintet; sex, sextet; sep, septet; m, multiplet; dist., distorted. All the chemicals, along with analytical grade solvents, were purchased from Sigma-Aldrich, Alfa Aesar (Germany), or Merck through local suppliers. Pre-coated silica gel Al-plates were used for TLC with ethyl acetate and Procedure for the synthesis of 5-acetohydrazide (2) 1; 10 g, 0.054 mol) and methanol (200 mL) were taken in a 500 mL RB flask. Hydrazine hydrate  was added drop wise and the mixture was refluxed for 2 h. The reaction progress was observed by TLC using n-hexane and ethyl acetate solvent system (40:60). After completion, the reaction mixture was allowed to cool at room temperature to attain white colored precipitates, which were filtered and washed with methanol to obtain purified hydrazide, 2. Ethyl 2-acetate methyl]-1,3,4-oxadiazol-2-thiol (3)2; 4 g, 0.023 mol) was dissolved in C2H5OH (70 mL) in a 250 mL RB flask at 28 °C and then solid KOH  was dissolved on reflux. Carbon disulphide  was poured drop wise at 28 °C and then the reaction mixture was refluxed again for 5 h. Reaction progress was noted with TLC using n-hexane and ethyl acetate solvent system (7:3). After completion of reaction, excess of ethanol was evaporated and sufficient ice cold distilled water was added followed by addition of dilute HCl to adjust pH of 4-5. Light peach colored precipitates of 5-[methyl]-1,3,4-oxadiazol-2-thiol (3) were filtered and washed with distilled water. 5-acetohydrazide (Synthesis of 3-bromo-N-(un/substituted-phenyl)propanamides (6a-l)4a-l; 0.038 mol) were suspended in 30 mL distilled water in an iodine flask (100 mL) and aqueous Na2CO3 solution  was added. 3-Bromopropanoyl chloride  was added gradually with vigorous manual shaking. Then this mixture was set to stir on magnetic stirrer for 2-3 h. Reaction completion was monitored by TLC. On completion, the excess ice-cold distilled water (60 mL) was added and the resulting precipitates were collected through filtration, washed with distilled water, and dried to get purified electrophiles, 6a-l.The un/substituted anilines methyl]-1,3,4-oxadiazol-2-yl}sulfanyl)-N-(un/substituted-phenyl)propanamides (7a-l) 3; 0.1 g, 0.467 mmol) was dissolved in N,N-dimethyl formamide  in a 100 mL RB flask. Solid LiH (0.005 g) was added and the mixture was stirred for half an hour. Then, different aforementioned electrophiles, 3-bromo-N-(un/substituted-phenyl)propanamides , were added and the mixture was set to stirring for 3-5 h. The progress of reaction was monitored through TLC using n-hexane and ethyl acetate solvent system (80:20). On completion, excess ice-cold distilled water was added and the precipitates obtained were filtered, washed with distilled water, and dried to acquire purified products, 7a-l.5-[methyl]-1,3,4-oxadiazol-2-thiol methyl]-1,3,4-oxadiazol-2-yl}sulfanyl)-N-phenylpropanamide (7a)15H15N5O2S2; Mol. Mass: 361 gmol-1; IR : 3355 (N-H stretching), 3050 (C-H of aromatic ring), 2923 (-CH2- stretching), 1665 (C=O stretching), 1645 (C=N stretching), 1577 (C=C stretching of aromatic ring),; 1H-NMR : 10.06 , 7.59 , 7.30 , 7.04 , 7.01 , 6.42 , 4.05 , 3.48 , 2.89 ; 13C-NMR : 168.73 (C-1’’), 168.68 (C-5’), 165.66 (C-2’), 163.40 (C-2), 143.94 (C-4), 138.38 (C-1’’’), 128.68 (C-3’’’ & 5’’’), 123.22 (C-4’’’), 119.05 (C-2’’’ & C-6’’’), 103.17 (C-5), 35.66 (C-6), 27.68 (C-2’’), 27.52 (C-3’’); EI-MS: m/z 361 [M]+, 269 (C9H9N2O2S2)+, 241 (C8H9N4OS2)+, 214 (C6H5N4OS2)+, 113 (C4H5N2S)+, 93 (C6H7N)+, 77 (C6H5)+, 55 (C3H3O)+.Light brown solid; Yield: 89%; m.p.:190-191 °C; Mol. Formula: C3-methyl)-1,3,4-oxadiazol-2-yl}sulfanyl)-N-(2-methylphenyl)propanamide (7b)18H17N5O2S2; Mol. Mass: 375 gmol-1; IR : 3360 (N-H stretching), 3045 (C-H of aromatic ring), 2935 (-CH2- stretching), 1661 (C=O stretching), 1647 (C=N stretching), 1576 (C=C stretching of aromatic ring); 1H-NMR : 9.42 , 7.37 , 7.20 , 7.15 , 7.08 , 7.02 , 6.42 , 4.05 , 3.47 , 2.90 , 2.17 ; 13C-NMR : 168.65 (C-1’’), 168.58 (C-5’), 165.55 (C-2’), 163.34 (C-2), 143.71 (C-4), 135.97 (C-1’’’), 131.62 (C-2’’’), 130.16 (C-6’’’), 125.79 (C-5’’’), 125.13 (C-3’’’), 124.94 (C-4’’’), 103.11 (C-5), 35.66 (C-6), 27.86 (C-2’’), 27.40 (C-3’’), 17.75 (2’’’-CH3); EI-MS: m/z 375 [M]+, 269 (C9H9N4O2S2)+, 241 (C8H9N4OS2)+, 214 (C6H5N4OS2)+, 195 (C10H13NOS)+, 113 (C4H5N2S)+, 106 (C7H8N) +, 55 (C3H3O)+.Light brown solid; Yield: 85%; m.p.: 201-202 °C; Mol. Formula: C3-methyl]-1,3,4-oxadiazol-2-yl}sulfanyl)-N-(3-methylphenyl)propanamide (7c)16H17N5O2S2; Mol. Mass: 375 gmol-1; IR : 3350 (N-H stretching), 3052 (C-H of aromatic ring), 2923 (-CH2- stretching), 1670 (C=O stretching), 1641 (C=N stretching), 1576 (C=C stretching of aromatic ring); 1H-NMR : 9.48 , 7.32 , 7.29 , 7.25-7.23 , 6.99 , 6.43 , 4.06 , 3.46 , 2.91 , 2.25 ; 13C-NMR : 168.73 (C-1’’), 168.69 (C-5’), 165.66 (C-2’), 163.41 (C-2), 143.90 (C-4), 138.01 (C-1’’’), 137.62 (C-3’’’), 128.68 (5’’’), 123.94 (C-4’’’), 119.64 (C-6’’’), 116.28 (C-2’’’), 103.17 (C-5), 35.66 (C-6), 27.87 (C-2’’), 27.42 (C-3’’), 21.12 (3’’’-CH3); EI-MS: m/z 375 [M]+, 241 (C8H9N4OS2)+, 214 (C6H5N4OS2)+, 195 (C10H13NOS)+, 113 (C4H5N2S)+, 107 (C7H9N)+, 91 (C7H7)+, 55 (C3H3O)+.Light brown solid; Yield: 82%; m.p.: 171-172 °C; Mol. Formula: C3-methyl]-1,3,4-oxadiazol-2-yl}sulfanyl)-N-(4-methylphenyl)propanamide (7d)16H17N5O2S2; Mol. Mass: 375 gmol-1; IR : 3360 (N-H stretching), 3050 (C-H of aromatic ring), 2923 (-CH2- stretching), 1666 (C=O stretching), 1643 (C=N stretching), 1580 (C=C stretching of aromatic ring); 1H-NMR : 9.95 , 7.45 , 7.11 , 7.00 , 6.42 , 4.05 , 3.46 , 2.86 , 2.25 ; 13C-NMR : 168.71 (C-1’’), 168.40 (C-5’), 165.66 (C-2’), 163.40 (C-2), 143.94 (C-4), 136.39 (C-1’’’), 132.10 (C-4’’’), 129.05 (C-3’’’ and C-5’’’), 119.06 (C-2’’’ and C- 6’’’), 103.15 (C-5), 35.59 (C-6), 27.71 (C-2’’), 27.51 (C-3’’), 20.39 (4’’’-CH3); EI-MS: m/z 375 [M]+, 269 (C9H9N4O2S2)+, 241 (C8H9N4OS2)+, 214 (C6H5N4OS2)+, 195 (C10H13NOS) +, 113 (C4H5N2S)+, 107 (C7H9N) +, 55 (C3H3O)+.Light brown solid; Yield: 80 %; m.p.: 177-178 °C; Mol. Formula: C3-methyl]-1,3,4-oxadiazol-2-yl}sulfanyl)-N-propanamide (7e)17H19N5O2S2; Mol. Mass: 389 gmol-1; IR : 3360 (N-H stretching), 3045 (C-H of aromatic ring), 2923 (-CH2- stretching), 1668 (C=O stretching), 1647 (C=N stretching), 1546 (C=C stretching of aromatic ring); 1H-NMR : 9.95 , 7.23 , 7.04 , 7.01 , 6.94 , 6.42 , 4.05 , 3.48 , 2.89 , 2.23 , 2.12 ; 13C-NMR : 168.72 (C-1’’), 168.68 (C-5’), 165.66 (C-2’), 163.40 (C-2), 143.90 (C-4), 134.26 (C-1’’’), 133.47 (C-2’’’), 131.65 (C-4’’’), 130.74 (C-5’’’), 126.35 (C-3’’’), 125.07 (C-6’’’), 103.17 (C-5), 35.66 (C-6), 27.68 (C-2’’), 27.52 (C-3’’), 20.42 (4’’’-CH3), 17.76 (2’’’-CH3); EI-MS: m/z 375 [M]+, 269 (C9H9N4O2S2)+, 241 (C8H9N4OS2)+, 214 (C6H5N4OS2)+, 195 (C10H13NOS)+, 121 (C8H11N)+, 106 (C8H10), 55 (C3H3O)+.Light brown solid; Yield: 80%; m.p.: 171-172 °C; Mol. Formula: C 3-methyl]-1,3,4-oxadiazol-2-yl}sulfanyl)-N-propanamide (7f)17H19N5O2S2; Mol. Mass: 389 gmol-1; IR : 3345 (N-H stretching), 3050 (C-H of aromatic ring), 2923 (-CH2- stretching), 1666 (C=O stretching), 1641 (C=N stretching), 1546 (C=C stretching of aromatic ring); 1H-NMR : 9.40 , 7.59 , 7.30 , 7.06 , 7.01 , 6.42 , 4.05 , 3.48 , 2.89 , 2.18 , 2.16 ; 13C-NMR : 168.73 (C-1’’), 168.68 (C-5’), 165.66 (C-2’), 163.40 (C-2), 143.90 (C-4), 137.99 (C-1’’’), 131.62 (5’’’), 130.61 (C-2’’’), 126.65 (C-3’’’), 123.23 (C-4’’’), 119.05 (C-6’’’), 103.17 (C-5), 35.66 (C-6), 27.68 (C-2’’), 27.52 (C-3’’), 19.60 (5’’’-CH3), 18.80 (2’’’-CH3); EI-MS: m/z 389 [M]+, 269 (C9H9N4O2S2)+, 241 (C8H9N4OS2)+, 214 (C6H5N4OS2)+, 175 (C11H13NO)+, 120 (C8H10N)+, 106 (C8H10), 55 (C3H3O)+.Dull white solid; Yield: 89 %; m.p.: 134-135 °C; Mol. Formula: C3-methyl]-1,3,4-oxadiazol-2-yl}sulfanyl)-N-propanamide (7g)17H19N5O2S2; Mol. Mass: 389 gmol-1; IR : 3360 (N-H stretching), 3020 (C-H of aromatic ring), 2923 (-CH2- stretching), 1667 (C=O stretching), 1651 (C=N stretching), 1580 (C=C stretching of aromatic ring); 1H-NMR : 9.37 , 7.95 , 7.05 , 6.98 , 6.42 , 4.05 , 3.48 , 2.89 , 2.12 ; 13C-NMR : 168.72 (C-1’’), 168.29 (C-5’), 165.65 (C-2’), 163.41 (C-2), 143.94 (C-4), 135.06 (C-2’’’ and C-6’’’), 134.91 (C-1’’’), 127.62 (C-3’’’ & C-5’’’), 126.40 C-4’’’), 103.17 (C-5), 34.62 (C-6), 28.11 (C-2’’), 27.52 (C-3’’), 18.09 (2’’’-CH3 & 6’’’-CH3); EI-MS: m/z 389 [M]+, 269 (C9H9N4O2S2)+, 241 (C8H9N4OS2)+, 214 (C6H5N4OS2)+, 175 (C11H13NO)+, 121 (C8H11N)+, 106 (C8H10), 55 (C3H3O)+.Light brown solid; Yield: 87%; m.p.: 149-150 °C; Mol. Formula: C3-methyl]-1,3,4-oxadiazol-2-yl}sulfanyl)-N-propanamide (7h)17H19N5O2S2; Mol. Mass: 389 gmol-1; IR : 3350 (N-H stretching), 3052 (C-H of aromatic ring), 2923 (-CH2- stretching), 1669 (C=O stretching), 1648 (C=N stretching), 1570 (C=C stretching of aromatic ring); 1H-NMR : 9.96 , 7.27 , 7.22 , 7.04 , 7.00 , 6.41 , 4.05 , 3.46 , 2.85 , 2.20 , 2.16 ; 13C-NMR : 168.71 (C-1’’), 168.63 (C-5’), 165.65 (C-2’), 163.39 (C-2), 143.94 (C-4), 136.70 (C-1’’’), 136.62 (C-3’’’), 131.98 (C-4’’’), 126.35 (C-5’’’), 120.49 (C-2’’’), 120.31 (C- 6’’’), 103.14 (C-5), 35.57 (C-6), 27.73 (C-2’’), 27.51 (C-3’’), 19.59 (3’’’-CH3), 18.75 (4’’’-CH3); EI-MS: m/z 389 [M]+, 269 (C9H9N4O2S2)+, 241 (C8H9N4OS2)+, 214 (C6H5N4OS2)+, 175 (C11H13NO)+, 120 (C8H10N)+, 106 (C8H10), 55 (C3H3O)+.Dark brown solid; Yield: 81%; m.p.: 177-178 °C; Mol. Formula: C3-methyl]-1,3,4-oxadiazol-2-yl}sulfanyl)-N-propanamide (7i)17H19N5O2S2; Mol. Mass: 389 gmol-1; IR : 3350 (N-H stretching), 3050 (C-H of aromatic ring), 2920 (-CH2- stretching), 1667 (C=O stretching), 1651 (C=N stretching), 1580 (C=C stretching of aromatic ring); 1H-NMR : 9.89 , 7.21 , 7.19 , 6.98 , 6.42 , 4.05 , 3.48 , 2.88 , 2.12 ; 13C-NMR : 168.72 (C-1’’), 168.29 (C-5’), 165.65 (C-2’), 163.41 (C-2), 143.94 (C-4), 137.65 (C-1’’’), 135.06 (C-3’’’ and C-5’’’), 127.62 (C-4’’’), 124.34 (C-2’’’ and C-6’’’), 103.17 (C-5), 35.62 (C-6), 28.11 (C-2’’), 27.52 (C-3’’), 21.06 (3’’’-CH3 and 5’’’-CH3); EI-MS: m/z 389 [M]+, 269 (C9H9N4O2S2)+, 241 (C8H9N4OS2)+, 214 (C6H5N4OS2)+, 175 (C11H13NO)+, 121 (C8H11N)+, 106 (C8H10), 55 (C3H3O)+.Light brown solid; Yield: 82%; m.p.: 180-181 °C; Mol. Formula: C3-methyl]-1,3,4-oxadiazol-2-yl}sulfanyl)-N-(2-ethylphenyl)propanamide (7j)17H19N5O2S2; Mol. Mass: 389 gmol-1; IR : 3350 (N-H stretching), 3075 (C-H of aromatic ring), 2930 (-CH2- stretching), 1667 (C=O stretching), 1650 (C=N stretching), 1576 (C=C stretching of aromatic ring); 1H-NMR : δ 9.42 , 7.32 , 7.22 , 7.17-7.12 , 6.99 , 6.41 , 4.05 , 3.47 , 2.90 , 2.56 , 1.09 ; 13C-NMR : 168.99 (C-5’), 168.71 (C-1’’), 165.65 (C-2’), 163.42 (C-2), 143.94 (C-4), 137.91 (C-1’’’), 135.30 (C-2’’’), 128.48 (C-3’’’), 126.03 (C-5’’’), 125.84 (C-4’’’), 125.71 (C-6’’’), 103.15 (C-5), 35.01 (C-6), 27.97 (C-2’’), 27.52 (C-3’’), 23.72 (2’’’-CH2-CH3), 14.21 (2’’’-CH2-CH3); EI-MS: m/z 389 [M]+, 269 (C9H9N4O2S2)+, 241 (C8H9N4OS2)+, 214 (C6H5N4OS2)+, 175 (C11H13NO)+, 121 (C8H11N)+, 106 (C8H10), 55 (C3H3O)+.Light brown solid; Yield: 91%; m.p.: 198-199 °C; Mol. Formula: C3-methyl]-1,3,4-oxadiazol-2-yl}sulfanyl)-N-(4-ethylphenyl)propanamide (7k)17H19N5O2S2; Mol. Mass: 389 gmol-1; IR : 3345 (N-H stretching), 3062 (C-H of aromatic ring), 2923 (-CH2- stretching), 1663 (C=O stretching), 1649 (C=N stretching), 1576 (C=C stretching of aromatic ring); 1H-NMR : 9.96 , 7.47 , 7.13 , 6.99 , 6.41 , 4.04 , 3.45 , 2.86 , 2.54 , 1.14 ; 13C-NMR : 168.71 (C-1’’), 168.41 (C-5’), 165.66 (C-2’), 163.40 (C-2), 143.94 (C-4), 138.59 (C-1’’’), 136.58 (C-4’’’), 127.86 (C-3’’’ and C-5’’’), 119.14 (C-2’’’ and C-6’’’), 103.15 (C-5), 35.59 (C-6), 27.72 (C-2’’), 27.54 (4’’’-CH2-CH3), 27.51 (C-3’’), 15.64 (4’’’-CH2-CH3); EI-MS: m/z 389 [M]+, 269 (C9H9N4O2S2)+, 241 (C8H9N4OS2)+, 214 (C6H5N4OS2)+, 175 (C11H13NO)+, 121 (C8H11N)+, 106 (C8H10), 55 (C3H3O)+.Light brown solid; Yield: 84 %; m.p.: 193-194 °C; Mol. Formula: C3-methyl]-1,3,4-oxadiazol-2-yl}sulfanyl)-N-(4-ethoxyphenyl)propanamide (7l)16H17N5O2S2; Mol. Mass: 375 gmol-1; IR : 3360 (N-H stretching), 3050 (C-H of aromatic ring), 2923 (-CH2- stretching), 1663 (C=O stretching), 1651 (C=N stretching), 1580 (C=C stretching of aromatic ring); 1H-NMR : 9.88 , 7.46 , 6.99 , 6.85 , 6.41 , 4.04 , 3.97 , 3.45 , 2.84 , 1.29 ; 13C-NMR : 168.64 (C-1’’), 168.00 (C-5’), 165.58 (C-2’), 163.33 (C-2), 154.34 (C-4’’’), 143.87 (C-4), 131.89 (C-1’’’), 120.50 (C-2’’’ and C-6’’’), 114.27 (C-3’’’ and C-5’’’), 103.07 (C-5), 62.96 (4’’’-OCH2-CH3) 35.42 (C-6), 27.71 (C-2’’), 27.44 (C-3’’), 14.57 (4’’’-OCH2-CH3); EI-MS: m/z 375 [M]+, 269 (C9H9N4O2S2)+, 241 (C8H9N4OS2)+, 214 (C6H5N4OS2)+, 136 (C8H10NO)+, 121 (C8H9O), 55 (C3H3O)+.Light brown solid; Yield: 80%; m.p.: 177-178 °C; Mol. Formula: CUrease inhibition assay50 values were calculated using the following formula:This enzyme assay is the customized form of the commonly known Berthelot assay , 24. The50 values were calculated using the EZ–Fit Enzyme kinetics software .Where, Control is the total enzyme activity without inhibitor and Test is the activity in the presence of test compound. ICStatistical analysisStatistical analysis was performed by Microsoft Excel 2010 for all the thrice measured values and the results are presented as mean ± SEM.Hemolytic activityBovine blood samples was collected in EDTA, that was diluted with saline (0.9% NaCl), and centrifuge at 1000 ×g for 10 min. The erythrocytes separated diluted in phosphate buffer saline of pH 7.4 and a suspension was made. Add 20 µL of synthetic compounds solution (10 mg/mL) in 180 µL of RBCs suspension and incubate for 30 min at room temperature. PBS was used as negative control and Triton 100-X was taken as positive control , 26. TheMolecular docking7a-l) were drawn in ACD/ChemSketch tool and retrieved in mol format. Furthermore, UCSF Chimera 1.10.1 tool was employed for energy minimization having default parameters such as steepest descent steps 100 with step size 0.02 (Å), conjugate gradient steps 100 with step size 0.02 (Å) and update interval was fixed at 10. Finally, Gasteiger charges were assigned in ligands using Dock Prep to obtain the good structure conformation. Molecular docking experiment was employed on 7a-l, against Jack bean urease by using virtual screening tool PyRx with VINA Wizard approach  was refluxed with hydrazine hydrate in methanol to acquire corresponding hydrazide, 2. This nucleophillic substitution reaction was completed in two hours. The hydrazide, 2, was made to react with carbon disulfide in the presence of KOH, an activator for cyclization, to yield a thiol containing nucleophile, 5-[methyl]-1,3,4-oxadiazol-2-thiol (3). Different electrophiles, 6a-l, were synthesized by stirring un/substituted anilines (4a-l) with 3-bromopropanoyl chloride (5) in a weak basic aqueous medium. Finally, the designed bi-heterocyclic propanamides (7a-l), were acquired by stirring 3 with newly synthesized different electrophiles, 6a-l, in DMF using LiH as a base and an activator. The molecular structures of these derivatives were corroborated by IR, EI-MS, 1H-NMR and 13C-NMR spectral data. The synthesis of these targeted molecules was accomplished in several steps. Ethyl 2-acetate , 3050 (C-H of aromatic ring), 2923 (-CH2- stretching), 1665 (C=O stretching), 1645 (C=N stretching), and 1577 (C=C stretching of aromatic ring) cm-1. The phenyl group attached with nitrogen of amide functionality was clearly depicted by three signals in aromatic region at δ 7.59 , 7.30 , and 7.04  , and 6.42  while the propanamide unit in the molecule was specified by a downfield amidic singlet at δ 10.06  along with two broad-triplets in aliphatic region at δ 3.48 , and 2.89 , symbolic for two interconnected methylenes , was assignable to a methylene uniting the two heterocycles  in the molecule. The structural characterization of one the compounds is discussed hereby in detail for the benefit of the reader. Compound spectrum  and numb H-4’’’) . 2-Aminothylenes . The sig13-C-NMR spectrum  and three methine signals are δ 128.68 (C-3’’’ and 5’’’), 123.22 (C-4’’’), and 119.05 (C-2’’’ and C-6’’’). 2-Amino-1,3-thiazol-4-yl heterocycle was recognized by two quaternary signals at δ 163.40 (C-2), and 143.94 (C-4) along with a methine signal at δ 103.17 (C-5) while the other heterocycle i.e. 1,3,4-oxadiazole was endorsed with two quaternary resonances at δ 168.68 (C-5’), and 165.66 (C-2’) (δ 168.73 (C-1’’) along with two upfield methylene signals at δ 27.68 (C-2’’), and 27.52 (C-3’’). Another signal at δ 35.66 (C-6) was assignable to a methylene connecting the 2-amino-1,3-thiazol-4-yl heterocycle with 1,3,4-oxadiazole heterocyle via the forth position of former to the fifth position of latter  . The prof latter . The presence of these moieties was also apparent from various fragment ion peaks in its EI-MS spectrum , as give7a was confirmed and it was named as 3-methyl]-1,3,4-oxadiazol-2-yl}sulfanyl)-N-phenylpropanamide. A similar approach was implemented for the structural confirmation of other derivatives.Based upon these collective evidences, the structure of Urease inhibition and structure-activity relationship 50 (mM) values as compared to the standard thiourea, as tabulated in 50 value of 21.11 ± 0.12 µM. So, it was pertinent to say that these molecules possessed many-folds better inhibitory potentials as compared to the standard.The synthesized bi-heterocyclic propan-amides were screened against urease and found to have potent inhibitory potential against this enzyme, evident from their much lower IC1 & -R2) attached to phenyl ring (aryl part) on the inhibitory potential. Although the displayed activity is an attribute of a whole molecule, but a limited structure-activity relationship (SAR) was recognized by examining the effect of different groups (-R7a (IC50 = 2.45 ± 0.04 µM) with unsubstituted phenyl ring (aryl part) exhibited very resembling inhibitory potential with mono-substituted molecule 7b (IC50 = 2.64 ± 0.03 µM) in which a small sized methyl group was present at 2-position. Compound 7c (IC50 = 1.32 ± 0.02 µM) with methyl group at 3-position, and 7d (IC50 = 1.75 ± 0.01 µM) with a methyl group at 4-position; however exhibited somewhat greater inhibitory potential relative to 7a. Indeed, the 7c was identified as a second most active compound in the synthetic series. So it was cogent from the present observations that when a small sized group was present at meta or para-position, the molecule was attributed with superb inhibitory potential  and was the most active in synthesized series. A decrease in inhibitory potential (IC50 = 5.18 ± 0.06 µM) was observed in 7f in which the methyl groups were present at 2- and 5-position as compared to the isomer 7g (IC50 = 2.63 ± 0.03 µM) in which these groups were present at 2- and 6-position  was compared with di-ortho substituted molecule 7g (IC50 = 2.63 ± 0.03 µM), it was surprising to know that the additional small sized ortho-substituent in latter molecule was not contributing to vary its inhibitory potential as compared to former molecule in a considerable manner  as compared to that of 7i (IC50 = 3.62 ± 0.01 µM), in which these methyl groups were present at 3- and 5-position  as compared to both para-substituted molecules, 7k (IC50 = 2.85 ± 0.05 µM) and 7l (IC50 = 2.17 ± 0.02 µM). Instead of small sized groups, when medium sized groups were present in the following three compounds , althougmeta-position or the di-substituted molecules, bearing at least one small sized group at para-position, were generally potent inhibitors of the urease enzyme. However, when a medium sized group was present on aryl part of these molecules, no significant variation in their inhibitory potential was observed.  So, it was postulated, from the structure-activity relationship among these bi-heterocyclic propanamides, that the compound bearing a small sized group at 7d > 7c > 7d > 7j > 7l > 7a > 7h > 7g > 7b > 7k > 7i > 7f .Synthesized compounds can be arranged in the following row according to their inhibitory activity: Hemolytic activityThe cytotoxicity of the synthesized compounds was evaluated through hemolytic assay. Results of percentage hemolysis are shown in Molecular docking analysisDocking energy evaluation of ligands 7a-l, depict good energy values   and bonding interaction pattern such as hydrogen and hydrophobic, respectively. Docking results justified that all compounds, cal/mol) . The sta∆Gbinding = ∆Ggauss + ∆Grepulsion + ∆Ghbond + ∆Ghydrophobic + ∆Gtors (Equation 1)∆G gauss Attractive term for dispersion, two gaussian functions, ∆Grepulsion Square of the distance if closer than a threshold value, ∆G hbond Ramp function - also used for interactions with metal ions, ∆G hydrophobic Ramp function, ∆G tors Proportional to the number of rotatable bondsBinding pocket evaluationin-vitro analysis 7e showed good enzyme inhibition potential therefore, ligand 7e was selected to check the binding interaction pattern. The 7e-docking complex showed that ligand structure showed its penetration inside the binding pocket. The 2-aminothiazole part of 7e showed its infiltration toward nickel metal and adjusted within binding pocket of urease. However, the substituted aryl moiety showed good conformation position in the opening part of binding pocket of target protein (The binding pocket is present in domain 4 where nickel metals are present . Based o protein .Hydrogen bonds analysis between 7e and urease7e against urease. In detail binding analysis it was observed that 7e forms four hydrogen bonds at different residues of target protein. The free amino group of thiazole ring and nitrogen within the cyclic ring were both prone to make hydrogen bonds. Our results showed that 2-aminothiazole and cyclic nitrogen of 7e forms two hydrogen bonds against Ala440 and Asp633 having 2.17 and 1.90 Å, respectively. The oxygen of amide group also forms couple of hydrogen bonds with Gln635 and Ala636 with bonds length 2.95 and 3.00 Å, respectively. The docking complex binding pocket residues also showed good correlation with published data which strengthened our docking results ( results , 31.in-vitro inhibitory results were also coherent with in-silico molecular docking outcomes. These molecules were also very mild cytotoxic towards membrane of the red blood cells. Hence, these molecules might be used as safe and promising drug candidates for urease-related ailments. A series of bi-heterocyclic propanamides was synthesized in appreciable yields. All these molecules demonstrated an excellent inhibitory potential against urease and their


Compounds 2 and 4 showed interestingly efficient and selective antitumor (hCA IX and hCA XII) inhibitor activities . Compounds 4 and 5 showed selective hCA IX inhibitory activity over hCA I , hCA IX/hCA II  and selective hCA XII inhibitory activity over hCA I , hCA XII/hCA II,  respectively compared to AAZ. Compounds 12–17, and 19–20 showed selective inhibitory activity towards hCA IX over hCA I and hCA II, with selectivity ranges of 27–195 and 3.2–19, respectively, while compounds 12, 14–17, and 19 exhibited selective inhibition towards hCA XII over hCA I and hCA II, with selectivity ratios of 48–158 and 5.4–31 respectively, compared to AAZ. Molecular docking analysis was carried out to investigate the selective interactions among the most active derivatives, 17 and 20 and hCAs isoenzymes. Compounds 17 and 20, which are highly selective CA IX and XII inhibitors, exhibited excellent interaction within the putative binding site of both enzymes, comparable to the co-crystallized inhibitors.Quinazoline-linked ethylbenzenesulfonamides inhibiting CA were synthesised.The new molecules potently inhibited the hCA isoforms I, II, IV, and IX.4 and 5 were found to be selective hCA IX/hCA I and hCA IX/hCA II inhibitors.Compounds 4 and 5 were found to be selective hCA XII/hCA I and hCA XII/hCA II inhibitors.Compounds 12–17, 19, and 20 were found to be selective hCA IX/hCA I and hCA IX/hCA II inhibitors.Compounds 12, 14–17, 19 were found to be selective hCA XII/hCA I and hCA XII/hCA II inhibitors.Compounds 4 and 5 are selective hCA IX and XII inhibitors over hCA I  and hCA II . Compounds 12–17, and 19–20 are selective hCA IX inhibitors over hCA I (selectivity ratios of 27-195) and hCA II (selectivity ratios of 3.2-19). Compounds 12, 14–17 and 19 are also selective hCA XII inhibitors over hCA I (selectivity ratios of 48-158) and hCA II (selectivity ratios of 5.4-31).Compounds We evaluated the hCA  inhibitory activity of novel 4-(2-(2-substituted-thio-4-oxoquinazolin-3(4H)-yl)ethyl)benzenesulfonamides (compounds  CAs are classified into eight genetically distinct families, named α-, β-, γ-, δ-, ζ-, η-, ɵ- and ι-CAs,,,,,A, ,N-(4-sulfamoylphenethyl)anildes  constitute the superfamily of metalloenzymes that catalyse the COildes B,  also shoes B, C,  showed sC, 2–20, Here, we studied 2-mercaptoquinazolinone, C,  a slight2.2.1.1H and 13C NMR) spectra were recorded with Bruker 700 MHz spectrometers . Micro-analytical data  were obtained using a Perkin-Elmer 240 analyser  and agreed with the proposed structures within ±0.4% of the theoretical values. Mass spectra were recorded on a Varian TQ 320 GC/MS/MS mass spectrometer . Thione 1 and compounds 8–20 were prepared as described earlier,Melting points were recorded on a Barnstead 9100 electrothermal melting point apparatus (UK). IR spectra (KBr) were recorded on a FT-IR Perkin-Elmer spectrometer . NMR  and potassium carbonate  in 6 ml acetone were stirred at room temperature for one hour. Appropriate phenacyl bromide (1 mmol) was added and the reaction mixture was stirred at room temperature for 9–12 h, filtered, and the crude solid was washed with water, dried and recrystallized from ethanol .A mixture of thione 4-(2-(4-Oxo-2-((2-oxo-2-phenylethyl)thio)quinazolin-3(4H)-yl)ethyl)benzenesulfonamide (2): m.p 246–247°; 94% yield; IR  ν: 3284, 3237 (NH), 1665 (C=O), 1342, 1151 (O=S=O); 1H NMR : δ 8.14 , 8.04 , 7.82 , 7.74 , 7.66 , 7.62 , 7.52 , 7.41 , 7.37 , 6.98 , 4.92 , 4.33 , 3.14 ; 13C NMR : δ 194.04, 160.76, 156.10, 146.92, 143.11, 142.29, 136.90, 135.19, 134.01, 129.67, 129.29, 128.79, 126.92, 126.45, 125.87, 119.08, 45.69, 39.38, 33.67; Ms; m/z (479).4-(2-(2-((2-(4-Bromophenyl)-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)ethyl)benzenesulfonamide (3): m.p 248–248°; 95% yield; IR  ν: 3280, 3236 (NH), 1686 (C=O), 1340, 1153 (O=S=O); 1H NMR : δ 8.07 , 8.04 , 8.85 , 7.81 , 7.68 , 7.52 , 7.41 , 7.37 , 7.00 , 4.89 , 4.32 , 3.13 ; 13 C NMR : δ 193.41, 160.74, 156.02, 146.88, 143.12, 142.27, 135.9266, 135.26, 132.38, 130.81, 129.67, 128.10, 126.93, 126.55, 126.52, 126.45, 125.87, 119.08, 45.72, 39.27, 33.67; Ms; 558.0; Ms; .4-(2-(2-((2-(4-Chlorophenyl)-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)ethyl)benzenesulfonamide (4): m.p 250–251°; 93% yield; IR  ν: 3281, 3239 (NH), 1684 (C=O), 1345, 1159 (O=S=O); 1H NMR : δ 8.15 , 8.04 , 7.82 , 7.70 , 7.67 , 7.52 , 7.41 , 7.37 , 6.99 , 4.89 , 4.32 , 3.13 ; 13 C NMR : δ 193.20, 160.74, 156.03, 146.89, 143.12, 142.28, 138.89, 135.60, 135.25, 130.73, 129.67, 129.43, 126.93, 126.53, 125.86, 119.08, 45.72, 39.28, 33.67; Ms; 514; Ms; .4-(2-(2-((2-(4-Fluorophenyl)-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)ethyl)benzenesulfonamide (5): m.p 249–250°; 92% yield; IR  ν: 3278, 3238(NH), 1666 (C=O), 1342, 1152 (O=S=O); 1H NMR : δ 8.23 , 8.04 , 7 82 , 7.68 , 7.52 , 7.46 , 7.41 , 7.37 , 6.99 , 4.90 , 4.33 , 3.14 ; 13 C NMR : δ 192.73, 166.36, 164.93, 160.7571, 156.07, 146.89, 143.12, 142.28, 135.24, 133.65, 133.64, 131.88, 131.83, 129.67, 126.92, 126.52, 125.87, 119.08, 116.41, 116.29, 45.70, 39.26, 33.67; Ms; m/z (497).4-(2-(4-Oxo-2-((2-oxo-2-(p-tolyl)ethyl)thio)quinazolin-3(4H)-yl)ethyl)benzenesulfonamide (6): m.p 257–258°; 92% yield; IR  ν: 3281, 3237 (NH), 1665 (C=O), 1339, 1150 (O=S=O); 1H NMR : δ 8.04 , 7.82 , 7.68 , 7.52 , 7.42 , 7.37 , 7.04 , 4.90 , 4.33 , 3.14 , 2.44 ; 13 C NMR : δ 193.40, 160.78, 156.12, 146.94, 144.46, 143.12, 142.30, 135.21, 134.33, 129.83, 129.67, 128.93, 126.91, 126.52, 125.95, 119.09, 45.65, 39.41, 33.67, 21.73; Ms; m/z (493).4-(2-(4-Oxo-2-((1-oxo-1-phenylpropan-2-yl)thio)quinazolin-3(4H)-yl)ethyl)benzenesulfonamide (7): m.p 245–246°; 90% yield; IR  ν: 3279, 3237 (NH), 1668 (C=O), 1347, 1154 (O=S=O); 1H NMR : δ 8.16 , 8.29 , 7.80 , 7.73 , 7.65–7.61 , 7.84 , 7.39 , 7.37 , 6.78 , 5.75 , 4.29–4.19 , 3.07 , 1.57 ; 13 C NMR : δ 198.20, 160.67, 155.81, 146.87, 143.12, 142.23, 135.98, 135.15, 134.01, 129.69, 129.34, 128.95, 126.92, 126.57, 126.48, 125.46, 119.13, 46.23, 45.76, 33.60, 16.44; Ms; 493.00; Ms; m/z (493).2.2.,The hCA I, II, IX, and XII isoenzyme inhibition assays were performed according to the reported method using the SX.18 MV-R stopped-flow instrument 2.3.,,,,,The molecular docking protocol was conducted according to the reported methods3.3.1.H)-yl)ethyl)benzenesulfonamide (1) was obtained via the reaction of 4-(2-isothiocyanatoethyl)benzenesulfonamide, triethylamine and 2-aminobenzoic acid in boiling ethanol,1 with potassium carbonate in acetone and different phenacyl bromides produced the corresponding 4-(2-(2-((2-(4-substituted-phenyl)-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)ethyl)benzenesulfonamides 2–7 with 90–95% yield. Various spectroscopic studies were conducted to validate the structures of the newly synthesised compounds, 2–7. The target compounds, 2–6, were validated by the diminishing of the thioamidic proton (NH–C=S) at 13.03 ppm and that of the thione moiety (NH–C=S) at 175.29 ppm, as well as by the presence of the phenacyl carbonyl group (SCH2COAr) at 194.04–192.73 ppm, with singlet peaks at 4.92–4.89 ppm and 39.41–39.26 ppm due to the phenacyl (S2CHCOAr) moiety, in the 1H and 13C NMR spectra, respectively. Additionally, 4-(2-(4-oxo-2-((1-oxo-1-phenylpropan-2-yl)thio)quinazolin-3(4H)-yl)ethyl)benzenesulfonamide (7) was confirmed by presence of the carbonyl group of (S(CH)CH3COAr) at 198.20 ppm in the 13C NMR spectrum, as well as by the quartette (S(CH)CH3COAr) and doublet (S(CH)3CHCOAr) peaks at 5.75 and 1.57 ppm respectively in the 1H NMR spectrum, together with the characteristic peaks (S(CH)CH3COAr) at 46.23 and (S(CH)3CHCOAr) at 16.44 ppm in the 13C NMR spectrum. The ethylbenzenesulfonamide amino group (NH2) (in compounds 2–7) was long-established by the presence of a typical singlet peak at 7.37 ppm in the 1H NMR spectrum. The tails of aliphatic ethylbenzenesulfonamide moiety were fixed by triplet peaks at 4.33–4.32 and 3.15–3.12 ppm in the 1H NMR spectrum and distinctive peaks at 45.72–45.65 and 33.67 ppm in 13C NMR spectrum, respectively. 2-Substituted mercapto-4(3H)-quinazolinones (8–20) were prepared in 90–96% yield by mixing compound 1 and 2-chloro-N-substitutedamide in acetone at room temperature in the presence of potassium carbonate4- towards hCA I, II, IV, and IX isoforms was measured and compared to acetazolamide (AAZ), a typical sulphonamide inhibitor. hCA I was effectively inhibited by compounds 2 and 4–13 with the inhibition-constant (KI) values ranging from 114.5–938.3 nM (AAZ: KI = 250.0 nM). Compounds 3 and 16 showed moderate activity with KI values of 1447.0 and 1697.0 nM, respectively, while compounds 14–15 and 17–20 showed weak activity with KI values ranging from 2048–5467 nM. Compounds 5, 8, 9, 11, 12, and 20 were verified to be effective hCA II inhibitors, with KI values of 25.4–95.4 nM (AAZ: KI = 12.0 nM). Compounds 2, 3, 4, 6, 7, 10, 14, and 16 showed modest hCA II inhibitory activity with KI values ranging between 116.2 and 266.1 nM, whereas compounds 13 and 15 showed a weak inhibitory activity with KI values of 304.6 and 1099.0 nM, respectively. Compounds 2–17 and 20 displayed potent hCA IX inhibitory activity with KI values ranging from 8.0 to 100.4 nM, which were greater than or nearly identical to that of AAZ (KI = 25.0 nM), whereas compounds 18 and 19 showed modest hCA IX inhibitory activity with KI values ranging between 256.4 and 145.1 nM, respectively. 4-(2-(2-(Substituted-thio)-4(3H)-quinazolinon-3-yl)ethyl)benzenesulfonamide derivatives 2, 4, 5, 8, 9, 11, 12, 13, 14, 16 and 17 showed potent hCA XII inhibitory activity with KI values of 2.4–49.1 nM compared to AAZ (KI = 5.7 nM), whereas compounds 3, 6, 7, 10, 15, 18, 19, and 20 exerted moderate hCA XII inhibitory activities with KI values of 59.7–113.4 nM (2 and 4 showed characteristic effective and selective antitumor (hCA IX and hCA XII) carbonic anhydrase inhibitory activity with KI values  and KI values  compared with AAZ . 4-(2-(2-((2-(4-Substituted-phenyl)-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)ethyl)benzenesulfonamides (compounds 2–7) showed high selectivity in the inhibition of hCA IX over hCA I and hCA II , as well as selectivity in the inhibition of hCA XII over hCA I and hCA II  (The CA inhibitory activity of 4-(2-(2-(substituted-thio)-4(3H)-quinazolinon-3-yl)ethyl)benzenesulfonamides (compounds 113.4 nM . On the ctively) .4 and 5 showed high selectivity in the inhibition of hCA IX over hCA I and hCA II, with selectivity ratios of 95.0 and 23.0, respectively for compound 4, and those of 24.0 and 5.8, respectively, for compound 5, compared with AAZ selectivity ratios of 10.0 and 0.5, respectively. Additionally, compounds 4 and 5 showed selective inhibition of hCA XII over hCA I and hCA II with selectivity ratios of 70.0 and 17.0, respectively, for compound 4, and 44.0 and 10.0, respectively, for compound 5, compared with AAZ selectivity ratios of 44.0 and 2.1, respectively. N-(substituted)-2-((4-oxo-3-(4-sulfamoylphenethyl)-3,4-dihydroquinazolin-2-yl)thio)amides (compounds 8–20) showed high selectivity in the inhibition of hCA IX over hCA I and hCA II, with selectivity ratios in the range 3.3–195.0 and 0.7–8.3 respectively, compared with AAZ selectivity ratios of 10.0 and 0.5 respectively, and that of hCA XII over hCA I and hCA II, respectively with selectivity ratios in the range 11.0–158.0 and 1.8–31.0, compared with AAZ selectivity ratios of 44.0 and 2.1 respectively. Compounds 12–17, and 19–20 showed selective inhibition of hCA IX over hCA I and hCA II, with selectivity ratios of 23.0–195.0 and 3.2–19.0, respectively, compared with AAZ selectivity ratios of 10.0 and 0.5. Additionally, Compounds 12, 14–17, and 19 showed selective inhibition of hCA XII over hCA I and hCA II, with selectivity ratios of 48.0–158.0 and 5.4–31.0, respectively, compared with AAZ selectivity ratios of 44.0 and 2.1 respectively.Compounds 3.3.2–20) were potent inhibitors of the hCA isoforms.Several synthesised quinazolinone derivatives (compounds 3.3.1.2), with a KI value of 592.7 nM, was more potent than 4-(2-(2-((1-(4-substituted-phenyl)-1-oxopropan-2-yl)thio)-4-oxoquinazolin-3(4H)-yl)ethyl)benzenesulfonamides 3–4 and 4-(2-(4-oxo-2-((1-oxo-1-phenylpropan-2-yl)thio)quinazolin-3(4H)-yl)ethyl)benzenesulfonamide 7, with KI values of 758.7–1447 nM, but less potent than 4-(2-(2-((1-(4-flouro/4-methyl-phenyl)-1-oxopropan-2-yl)thio)-4-oxoquinazolin-3(4H)-yl)ethyl)benzenesulfonamide 5 and 6 with KI values of 399.5–471.0 nM. (2) Unsubstituted-N-acetamide 8  was more active than the corresponding N-phenylacetamide 9  Substitution of the phenyl ring of N-phenylacetamide 9  resulted in substituted-N-phenylacetamides 10–17 and N-phenylpropanamide 20 with significantly decreased CA inhibitory activity . (4) The hCA I inhibitory activity of N-(4-fluorophenyl)-2-((4-oxo-3-(4-sulfamoylphenethyl)-3,4-dihydroquinazolin-2-yl)thio)acetamide (12), with a KI value of 548.6 nM, was more stronger than the corresponding N-(4-fluorobenzyl)-2-((4-oxo-3-(4-sulfamoylphenethyl)-3,4-dihydroquinazolin-2-yl)thio)acetamide (18) KI of 2048 nM. (5) hCA I inhibition of N-acetamide 11, with a KI value of 726.4 nM, was more powerful than the corresponding N-propanamide 20 with a KI value of 3628 nM.SAR analysis of hCA I inhibition indicated revealed several key features. (1) 4–(2-(4-Oxo-2-((2-oxo-2-phenylethyl)thio)quinazolin-3(4H)-yl)ethyl)benzenesulfonamide (3.3.2.H)-yl)ethyl)benzenesulfonamides 5 and 6 with KI values of 95.4–116.2 nM were more effective than unsubstituted phenyl and other substituted phenyl derivatives, such as compounds 2–4 and 7 with KI values of 140.8–202.6 nM. (2) hCA II inhibition of 2-((2-oxo-2-phenylethyl)thio)quinazolinone 2, with a KI value of 140.8 µM, was stronger than the corresponding 2-((1-oxo-1-phenylpropan-2-yl)thio)quinazolinone 7 with a KI value of 202.6 nM. (3) N-Phenylacetamide 9 with a KI value of 69.7 nM was less potent than unsubstituted-N-acetamide 8 . (4) Substitution of the phenyl ring of N-phenylacetamide 9  resulted in substituted-N-phenylacetamides 10–17 and N-phenylpropanamide 20 with considerably diminished CA II inhibitory activity ; (5) The hCA II inhibitory effect of N-acetamide 11  was less potent than the corresponding N-propanamide 20 . (6) The hCA II inhibitory activity of N-(4-fluorophenyl)acetamide 12, with a KI value of 87.6 nM, was stronger than the corresponding N-(4-fluorobenzyl)acetamide 18 (KI of 2048 nM).The SAR analysis for hCA II inhibition revealed several key features. (1) 4-(2-(2-((1-(4-Fluorophenyl/4-methylphenyl)-1-oxopropan-2-yl)thio)-4-oxoquinazolin-3thio)quinazolinone 7 with KI value of 63.2 nM. (2) The induction of the activating group, such as the 4-methyl group on the phenyl ring of compound 2  led to compound 6, with an increased hCA IX inhibitory activity . (3) The introduction of the deactivating group on phenyl ring of compound 2, such as the 4-bromo group, resulted in compound 3 with diminished hCA IX inhibition activity ; in contrast, the introduction of 4-fluoro/4-chloro groups produced compounds 4–5 with boosted the inhibitory potency of the hCA IX . (4) N-propanamide 20, with a KI value of 18.6 nM, was powerful than the corresponding N-acetamide 11 with a KI value of 66.8 nM. (5) The introduction of activating/deactivating groups on the phenyl ring of compound 2  yielded compounds 10–17 with reduced inhibitory activity , except for compounds 12 and 16, which had improved hCA IX inhibitory potency . (6) Substitution of the phenyl group of compound 12  with a benzyl moiety resulted in compound 18, which had significantly reduced hCA IX inhibitory activity .SAR analysis of hCA IX inhibition revealed several key factors. (1) The 2-((2-oxo-2-phenylethyl)thio)quinazolinone 3.3.4.2, with a KI value of 13.0 nM, was more potent than 2-((1-oxo-1-phenylpropan-2-yl)thio)quinazolinone 7 with a KI value of 76.8 nM. (2) The introduction of a chloro/fluoro group at the phenyl ring, such as in compounds 4 and 5 , improved the hCA XII inhibition activity and was similar to that of compound 2 . (3) The unsubstituted N-acetamide, compound 8,  resulted in more powerful hCA XII inhibition than N-substituted amides, compounds 9–20, . (4) hCA XII inhibition of N-acetamide 11, with a KI value of 31.6 nM, was more powerful than that of the corresponding N-propanamide 20 with a KI value of 66.7 nM. (5) The substitution of the phenyl group of N-(4-fluorophenyl)acetamide 12  with a benzyl moiety resulted in the N-(4-fluorobenzyl)acetamide 18, with sharply reduced CA inhibitory activity .SAR analysis for hCA XII inhibition revealed several key factors. (1) 2-((2-Oxo-2-phenylethyl)thio)quinazolinone 3.4.3.4.1.17 and 20 with the hCAs targets, we performed docking simulations into the binding pockets of the hCA isoforms, IX and XII, using the MOE SuiteTo further investigate the interactions between the selected active compounds 17 and 20 were shown to directly interact with the zinc ion of CA IX and CA XII isoenzymes, via the sulphonamide anion of the active sites of both enzymes. However, the contributions of the quinazoline scaffold and the terminal bulky thioether fragments interaction are different, based on the CA isoform. In CA IX, the quinazoline ring of compound 20 interacts with the Gln71 residue through a stable hydrogen bond, and gets accommodated in the hydrophobic pocket lined by the Val121, Val130, Leu134, and Leu91 residues, thereby stabilising the binding  towards the hCA I, II, IV, and IX isoforms was assessed and compared with acetazolamide (AAZ), a typical sulphonamide inhibitor. Of the different hCA isoforms, hCA I was effectively inhibited by the compounds 2 and 4–13, with inhibition constant (KI) values in the range of 114.5–938.3 nM , while compounds 3 and 14–20 showed moderate to weak CA inhibitory activity with KI values of 1447.0–5467 nM. Compounds 5, 8, 9, 11, 12, and 20 were revealed to be effective hCA II inhibitors, with KI values of 25.4–95.4 nM . Compounds 2, 3, 4, 6, 7, 10, 13, 14, 15, and 16 showed modest to weak hCA II inhibitory activity with KI values ranging between 116.2 and 1099.0 nM. Compounds 2–17 and 20 displayed potent hCA IX inhibitory activity with KI values ranging from 8.0 to 100.4 nM compared to AAZ , whereas compounds 18 and 19 showed modest hCA IX inhibitory activity with KI values ranging between 256.4 and 145.1 nM, respectively. Ethylbenzenesulfonamide derivatives, 2, 4, 5, 8, 9, 11, 12, 13, 14, 16, and 17 showed potent hCA XII inhibitory activities with KI values of 2.4–49.1 nM compared to AAZ , whereas compounds 3, 6, 7, 10, 15, 18, 19, and 20 showed moderate hCA XII inhibitory activities with KI values of 59.7–113.4 nM. Compounds 2 and 4 showed characteristic effective and selective antitumor (hCA IX and hCA XII) carbonic anhydrase inhibitory activity with KI values  and KI values . Compounds 2–7 showed high selectivity ratios for the inhibition of hCA IX over hCA I (15.0–95.0) and hCA IX over hCA II (2.3–23.0), while selectivity ratios of hCA XII over hCA I (5.5–70.0) and hCA XII over hCA II (1.4–17.0). Compounds 4 and 5 displayed selective inhibitory activity towards hCA IX over hCA I with selectivity ratios of 95.0 and 24.0 respectively, and hCA IX over hCA II with selectivity ratios of 23.0 and 5.8 respectively, as well as, selective inhibitory activity for hCA XII over hCA I and hCA XII over hCA II . Compounds 12–17, and 19–20 exhibited selective inhibitory activities towards hCA IX over hCA I and hCA IX over hCA II . In addition, compounds 8, 12, 14–17, and 19 showed selective inhibitory activity towards hCA XII over hCA I and hCA XII over hCA II . Docking study of the selective derivatives, compounds 17 and 20, with the hCAs revealed consistent interactions, particularly selectivity-oriented hydrophobic and aromatic interactions through the S-alkyl substituent.The CA inhibitory activity of 4-(2-(2-(substituted-thio)-4(3H)-quinazolinon-3-yl)ethyl)benzenesulfonamides (compounds Click here for additional data file.


Compounds 7a, 7b and 8 exhibited excellent antimicrobial activity against all Gram-positive and Gram-negative bacteria, and the fungal Aspergillus flavus with minimum inhibitory concentration (MIC) values ranged from 0.95 ± 0.22 to 3.25 ± 1.00 μg/mL. Furthermore, cytotoxicity studies against MCF-7 cells revealed that compounds 7a and 7b were the most potent with IC50 values of 10.17 ± 0.65 and 11.59 ± 0.59 μM, respectively. On the other hand, the tested compounds were less toxic against normal kidney epithelial cell lines (Vero cells). The in vitro enzyme inhibition assay of 8 displayed excellent inhibitory activity against Escherichia coli DNA B gyrase and moderate one against E. coli Topoisomerase IV  in comparison with novobiocin . Finally, the molecular docking was done to position compound 8 into the E. coli DNA B and Topoisomerase IV active pockets to explore the probable binding conformation. In summary, compound 8 may serve as a potential dual E. coli DNA B and Topoisomerase IV inhibitor.To develop new antimicrobial agents, a series of novel thiourea derivatives incorporated with different moieties  It is widely known that there is a great demand for discovery of new antibacterial compounds due to the rising and global problem of antibiotic resistance . SearcheMycobacterium tuberculosis pyrimidine (III) increased the activity against both Gram positive and Gram negative bacteria through interfering with protein synthesis [N-arylthiourea IV explored the highest potency against standard and methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis strains through its inhibitory effect on topoisomerase IV [Recently in the search for novel antimicrobial agents, it was found that the reported thiosemicarbazide I significantly inhibits the activity of 14.59 μM . Howeverynthesis . N-arylterase IV . The thierase IV  -2-thioxoimidazolidin-4-one (3), which was opening by refluxing in ethanol/hydrochloric acid to obtain thiourea derivative 2  beside two singlet signals at δ 3.46 and δ 3.79 for 2CH2 protons and a multiplet signals for phenyl protons. On the other hand, IR spectrum of 3 shows high absorption band of cyclic carbonyl group at 1741 cm−1 and its 1HNMR spectrum displays a broad singlet signal for the NH proton that is exchangeable with D2O.Reaction of benzylisothiocyanate vative 2 . The 1HN1 with N-amino imidazole derivative 4 [5a,b [6), and thiosemicarbazide derivatives 7a,b and 8, respectively -1,3,4-thiadiazol-2-yl)-2-phenylacetamide 10, respectively. The latter compound 10 was reacted with salicaldehyde in refluxing ethanol, in the presence of ammonium acetate to give the thiadiazolochromen derivative 11 .Finally, cyclization of ectively . The str2–13 was screened against a panel of two Gram-positive bacteria (Staphylococcus aureus ATCC 29213 and Bacillus subtilis ATCC 6633), two Gram-negative bacteria  and two fungi  using the agar well diffusion method [7a, 7b and 8 revealed excellent improved antimicrobial activity against all Gram-positive and Gram-negative bacteria, and the fungal A. flavus while moderate activity against C. albicans. Moreover, compound 6 displayed higher activities against Gram positive bacteria (S. aureus and B. subtilis) and the Gram-negative P. aeruginosa and weak activity against the remaining strains. Additionally, the promising activity against S. aureus was observed from compounds 9, 12 and 13, however against B. subtilis from compounds 12 and 13. The remaining derivatives demonstrated potencies from moderate to weak in comparison with the reference drugs. The antifungal potency against C. albicans for all tested derivatives ranged from weak to no activity at all. Additionally, it was noticed that compounds 2 and 3 showed no activity against all the screened strains. The most active targets 6, 7a, 7b, 8, 9, 12 and 13 were further investigated for the assignment of the minimum inhibitory concentration (MIC) .The antimicrobial activity of all synthesized compounds n method . Ciproflon (MIC) . Compoun2 or to thioxoimidazolidine moiety in 3 abolished the antimicrobial activity against almost tested strains. Replacement of ethyl acetate in 2 with cyanoacetamide in compound 8 explored the highest antimicrobial activity against all strains except C. albicans. By fixation of thiourea bearing phenyl acetyl group, insertion of 4-benzylidene-5-oxo-2-phenylimidazole on the other side in 6 displayed excellent activity against S. aureus, B. subtilis and P. aeruginosa with MIC values of 5.12 ± 0.05, 2.29 ± 0.05 and 2.48 ± 0.11 μg/mL, respectively. While, insertion of 2-benzamido-3-(phenyl or thienyl)-acryloyl fraction in 7a and 7b, exhibited superior antimicrobial activity against all screened strains (MIC ranged from 1.39 ± 0.50 to 5.70 ± 0.01 μg/mL). Cyclization of the thiosemicarbazide 8 to pyrazolotriazinone 9, 5-cyanomethyl-1,3,4-thiadiazole 10 or thiadiazole chromen 11 dropped the antimicrobial activity. On the other hand, cyclization of 7a and 7b to 2-phenylethylidene-6-oxo-3-phenyl-1,2,4-triazine 12 and 1,3,4-thiadiazol-2-yl-2-thien-2-yl-vinyl-benzamide 13, respectively retained the potency against the Gram positive strains  with remarked drop in the activity against the remaining strains.Regarding to the structure–activity relationship (SAR) study, it was noted that the attachment of phenyl acetyl fragment to thiourea and ethyl acetate groups in compound 6, 7a, 7b, 8, 9, 12 and 13 were evaluated against human breast cancer (MCF-7) as well as normal kidney epithelial cell line (Vero) cells using the MTT method [50 values (µM) and are depicted in 7a > 7b > 8 > 6 > 9 > 12 > 13. Compounds 7a and 7b were the most potent showing the least obtained IC50 values of 10.17 ± 0.65 and 11.59 ± 0.59 μM, respectively. These values were more or less comparable to the tested positive control (cisplatin: IC50 8.89 ± 0.37 μM). On the other hand, the tested compounds were less toxic against normal kidney epithelial cell lines (Vero cells).The cytotoxic activities of the highly active compounds as antimicrobials T method  and cisp7a, 7b and 8 displayed the highest potency as antimicrobial with low toxicity.The data obtained from antimicrobial and cytotoxic screening revealed that compounds 8 as antimicrobial, it was assessed for in vitro inhibition against three kinases, E. coli DNA gyrase B, E. coli Topoisomerase IV and dihydrofolate reductase (DHFR) using the reported procedures [E. coli DNA gyrase B and E. coli Topoisomerase IV, while methotrexate as the standard for DHFR. The results were recorded as IC50 values in μM and listed in In order to validate the mode of action of the highly potent compound ocedures ,24,29. T8 exhibited excellent inhibitory activity against E. coli DNA B gyrase in comparison with novobiocin . Moreover, it showed moderate inhibitory potency against E. coli Topoisomerase IV, about half the potency of novobiocin . On the other hand, compound 8 cannot be considered as the DHFR inhibitor by comparing its IC50 value with that of methotrexate . As expressed in 8 was selected for molecular docking against E. coli DNA gyrase B and Topoisomerase IV using MOE (Molecular Operating Environment) software 10.2008 [E. coli DNA gyrase B and Topoisomerase IV with energy score (S) = −10.77 and −7.88 kcal/mol and root mean standard deviation (RMSD) = 0.86 and 0.79 Å, respectively.Prompted by the kinase inhibitory results, compound  10.2008 . The pro 10.2008 ,31 was dAsp46 and Asp73 and arene-cation interaction with Arg76. While within the ATP binding pocket of Topoisomerase IV, it linked to the four key amino acids Asn1042, Asp1069, Asp1077 and Arg1132 via hydrogen bonding . Moreover, the similar arene–cation interaction with Arg76 was noticed with the centroid of phenyl ring . Furthermore, the phenyl ring also took part in the hydrophobic interaction with Arg1072 through an arene–cation interaction  with the essential amino acids Asn46, Asp73 and Arg76 in the binding pocket of E. coli DNA gyrase B as novobiocin that was responsible for its superior antimicrobial activity through inhibition of E. coli DNA gyrase B (IC50 = 0.33 ± 1.25 µM). On the other hand, the key Asn1042 played a crucial role in stability of compound 8 like novobiocin within the binding pocket of Topoisomerase IV through two hydrogen bond acceptors to exert good inhibitory activity . Furthermore, concerning the previous results, our derivatives could possibly target other receptors than DNA gyrase and topoisomerase IV. This point will be discussed and screened more in the future work.Based on the biological evaluations and molecular docking study we could deduce the following features in the most active compound 1HNMR spectra run at 300 MHz on a GEMINI 300 BB NMR spectrometer using tetramethylsilane (TMS) as internal standard in deuterated dimethylsulphoxide (DMSO-d6) at the main defense chemical laboratory. The mass spectra operating at 70 eV on Shimadzu GCMS-QP-1000EX mass spectrometer at the Regional center for Mycology and Biotechnology of Al-Azhar University. The elemental analyses performed on a Perkin-Elemer 2400 CHN elemental analyzer at the Micro analytical center of Cairo University. Antimicrobial activity was studied at Pharmacology Department, Faculty of Pharmacy, Mansoura University. N-Amino-4-benzylidene-2-phenyl-1H-imidazol-5(4H)-one 4 and 3-aryl-2-(3-phenylureido) acrylohydrazide 5a,b were synthesized according to the method outlined in the literature [Melting points are uncorrected and measured on a Gallenkamp electric melting point apparatus. Infrared spectra carried out using potassium bromide disks on a FTIR Thermo Electron Nicolet 7600 (USA) infrared spectrometer at the central laboratory of faculty of science Ain shams University. terature ,28.1 (0.01 mol) in dry acetonitrile (20 mL) and drops of pyridine was refluxed with ethyl glycinate hydrochloride (0.01 mol) for 4 h. The solvent was vacuum distilled and the residue was treated with cold ice. A solid product was obtained, filtered off and recrystallized from ethanol to give 3. Yield 75%; colorless needles; m.p. 70–72 °C; IR (KBr) : 3245 (NH), 3071 (CHarom), 2994, 2940, 2908 , 1741, 1642 (C=O), 1196 (C=S); 1HNMR (DMSO-d6) δ: 3.45 , 3.74 , 7.17–7.29 , 8.33 ; 13C-NMR (DMSO-d6) δ: 41.2 (ph-CH2), 42.9 (NH-CH2), 126.8, 128.6, 129.5, 136.7 (Ar-C), 171 (C=O), 171.8 (C=S); MS (70 eV) m/z (%): 234 , 192 (9), 177 (11), 161 (14), 119 (18), 91 (16), 77 (25), 69 (92), 43 (100); Anal. calcd for C11H10N2O2S (234.27): C, 56.39; H, 4.30; N, 11.95. Found: C, 56.18; H, 3.93; N, 11.59.A solution of phenyl acetyl isothiocyanate 3 (1 g) in ethanol (25 mL), hydrochloric acid  was added. The reaction mixture was refluxed for 1 h, after cooling the colorless solid product was obtained, filtered off, and recrystallized from ethanol to give compound 2. Yield 92%; m.p. 120–122 °C; IR (KBr) : 3384 (NH), 3062, 3032 (CHarom), 2933, 22858, 2743 , 1721, 1613 (C=O); 1HNMR (DMSO-d6) δ: 1.15 , 3.46 , 3.79 , 4.08 , 7.22 , 7.19–7.29 , 8.45 ; 13C-NMR (DMSO-d6) δ: 14.5 (CH3), 41.3 (ph-CH2), 42.6 (NH-CH2), 60.9 (CH2), 126.8, 128.7, 129.5, 136.6 (Ar-C), 170.4 (C=O), 171.2 (C=S); MS (70 eV) m/z (%): 280 , 263 (100), 207 (17), 193 (33), 178 (22), 134 (50), 103 (32), 91 (90); Anal. calcd for C13H16N2O3S (280.34): C, 55.67; H, 5.75; N, 9.99. Found: C, 55.28; H, 5.39; N, 9.71.To a solution of compound 1 (0.01 mol) and N-aminoimidazole 4 (0.01 mol), carbohydrazide 5a,b (0.01 mol) or cyanoacetohydrazide (0.01 mol) in dry acetonitrile (20 mL) was stirred at room temperature for 2–4 h. The precipitated solid was collected by filtration, dried and crystallized from ethanol to give the corresponding compounds 6, 7a, 7b and 8, respectively.A mixture of compound −1): 3191 (NH), 3059, 3027 (CHarom), 2928 , 1701, 1640 (C=O), 1598 (C=N), 1163 (C=S); 1HNMR (DMSO-d6) δ: 3.68 , 7.25–8.32 , 11.96 , 12.31 ; 13C-NMR (DMSO-d6) δ: 42.9 (CH2), 127.5 (CH=C), 128.7–129.9 (Ar-C), 131.3, 134.3, 134.6, 136.6 (Ar-C), 133 (C=C), 160.6 (C=N), 167.3 (N-C=O), 172.2 (NH-C=O), 182.4 (C=S); MS (70 eV) m/z (%): 440 , 321 (2), 119 (5), 105 (27), 91 (100), 77 (28), 59 (4); Anal. calcd for C25H20N4O2S (440.51): C, 68.16; H, 4.57; N, 12.71. Found: C, 67.76; H, 4.23; N, 12.46.Yellow crystals; yield (90%); m.p. 238–240 °C (EtOH); IR (KBr) : 3247, 3182 (NH), 3061, 3026 (CHarom), 1694, 1642 (C=O), 1599 (C=N), 1153 (C=S); 1HNMR (DMSO-d6) δ: 3.80 , 7.29–8.01 , 10.15 , 10.84 , 11.88 , 12.36 ; 13C-NMR (DMSO-d6) δ: 42.7 (CH2), 127.5, 128.3 (C=C), 128.3-134.7 (Ar-C), 163, 166.6, 173.4 (C=O),178.1 (C=S); MS (70 eV) m/z (%):458 , 353 (2), 265 (6), 237 (4), 208 (2), 134 (2), 119 (12), 105 (62), 91 (49), 77 (100), 64 (10); Anal. calcd for C25H22N4O3S (458.53): C, 65.48; H, 4.83; N, 12.21. Found: C, 65.12; H, 4.44; N, 11.89.White crystals; yield (92%); m.p. 190–192 °C; IR (KBr) : 3268, 3172 (NH), 3078, 3027 (CHarom), 1694, 1664, 1643 (C=O), 1617 (C=N), 1156 (C=S); 1HNMR (DMSO-d6) δ: 3.76 , 7.11–8.05 , 9.95 , 10.65 , 11.84 , 12.44 ; 13C-NMR (DMSO-d6) δ: 42.6 (CH2), 124.5 (CH=C), 127.5–133.9 (Ar-C), 134 (NH-C=C), 134.7 (CH2-C=C), 131.8, 136.6 (S-C=C), 162.1, 166.8, 173.4 (C=O), 177 (C=S); MS (70 eV) m/z (%): 464 , 357 (49), 345 (22), 256 (10), 236 (4), 134 (13), 120 (15),104 (29), 91 (4), 83 (7), 77 (48), 73 (72); Anal. calcd for C23H20N4O3S2 (464.55): C, 59.46; H, 4.33; N, 12.06. Found: C, 59.11; H, 3.95; N, 11.82.White crystals; yield (85%); m.p. 212–214 °C; IR (KBr) : 3282, 3194 (NH), 3012 (CHarom), 2933, 2911 , 1694 (C=O), 1590 (C=N), 1150 (C=S); 1HNMR (DMSO-d6) δ: 3.72 , 3.79 , 7.25–7.33 , 11.94 , 11.77 , 12.13; 13C-NMR (DMSO-d6) δ: 31.7 (2CH-CN) 42.5 (Ph-CH2), 115.8 (CN), 127.4–136.3 (Ar-C), 166.1, 166.7 (C=O), 172.9 (C=S); MS (70 eV) m/z (%): 276 , 117 (66), 96 (2), 77 (19), 63 (100); Anal. calcd for C12H12N4O2S (276.31): C, 52.16; H, 4.37; N, 20.27. Found: C, 51.76; H, 4.09; N, 19.91.White crystals; yield (96%); m.p. 158–160 °C; IR (KBr)  and catalytic amount of sodium hydroxide or hydrochloric acid  in ethanol (20 mL) was refluxed for 2 h. After cooling to room temperature, the reaction mixture was poured into ice, and then acidified with hydrochloric acid (1N) “in case NaOH”. The obtained precipitate was filtered off and recrystallized from ethanol to give the corresponding compounds 9 and 10, respectively. A solution of −1): 3161, 3106 (NH), 3013 (CHarom), 2854 , 1620 (C=O), 1600 (C=N), 1173 (C=S); 1HNMR (DMSO-d6) δ: 3.93 , 5.86 , 7.20–7.34 , 11.69 , 13.65 ; 13C-NMR (DMSO-d6) δ: 40.6 (2CH) 86.1 (C=C-CO), 127, 128.7, 129.6, 136.2 (Ar-C), 145.9 (C=N), 154.1 (N-C=C), 167.2 (C=O), 168.1 (C=S); MS (70 eV) m/z (%): 258 , 167 (2), 201 (5), 199 (8), 91 (100), 82 (2), 77 (10), 57 (2); Anal. calcd for C12H10N4OS (258.29): C, 55.80; H, 3.90; N, 21.69. Found: C, 55.43; H, 3.65; N, 21.30.White powder; yield (81%); m.p. < 300 °C; IR (KBr) : 3148 (NH), 3032 (CHarom), 2945, 2886, 2794 , 2252 (CN), 1681 (C=O), 1558 (C=N); 1HNMR (DMSO-d6) δ: 3.81 , 4.53 , 7.22–7.33 , 12.87 ; 13C-NMR (DMSO-d6) δ: 18.8 (2CH-CN), 41.9 (CH2), 117 (CN), 127.4, 128.9, 129.8, 134.9 (Ar-C), 154.4, 160.2 (S-C=N), 170.2 (C=O); MS (70 eV) m/z (%): 258 , 118 (17), 91 (100), 65 (10); Anal. calcd for C12H10N4OS (258.29): C, 55.80; H, 3.90; N, 21.69. Found: C, 55.46; H, 3.54; N, 21.41.Colorless crystals; yield (58%); m.p. 198–200 °C; IR (KBr) , and salicylaldehyde (0.01 mol) in ethyl alcohol (30 mL), ammonium acetate (0.01 mol) was added, and then heated under reflux for 6 h. The formed solid product was filtered off, dried and recrystallized from dimethylformamide to give compound 11. Pale yellow needles; yield (94%); m.p. < 250 °C; IR (KBr) : 3288 (NH), 3024 (CHarom), 2925, 2856, 2725 , 1692 (C=O), 1659, 16047 (C=N); 1HNMR (DMSO-d6) δ: 3.84 , 7.20–7.74 , 8.57 , 9.01 , 12.71 ; 13C-NMR (DMSO-d6) δ: 42.1 (CH2), 115.5–135.2 (Ar-C), 153.8 (O-C=C), 153.4, 155.8 (S-C=N), 162.4 (C=NH), 169.9 (C=O); MS (70 eV) m/z (%): 363 , 347 (1), 245 (2), 101 (6), 91 (100), 77 (5), 44 (2); Anal. calcd for C19H13N3O3S (363.38): C, 62.80; H, 3.60; N, 11.56. Found: C, 62.45; H, 3.31; N, 11.16.To a mixture of compound 7a (0.01 mol) in alcoholic sodium hydroxide  was refluxed for 3h. The reaction mixture was cooled to room temperature, and then poured into ice/HCl. The formed solid product was filtered off and recrystallized from benzene to give compound 12. Pale brown crystals; yield (65%); m.p. 138–140 °C; IR (KBr) : 3240 (NH), 3034 (CHarom), 2924, 2855 , 1648 (C=O) 1602 (C=N); 1HNMR (DMSO-d6) δ: 3.85 , 7.05–7.97 , 10.12 , 13.52 , 13.64 ; 13C-NMR (DMSO-d6) δ: 41.5, 42.2 (CH2), 114.6, 134.1 (C=C), 127.9–137.4 (Ar-C), 158.3 (C=N), 163, 165.8, 172.7 (C=O), 171.3 (C-OH), 183.8, 188.2 (C=S); MS (70 eV) m/z (%): 440 , 175 (23), 144 (12), 105 (93), 71 (100); Anal. calcd for C25H20N4O2S (440.51): C, 68.16; H, 4.57; N, 12.71. Found: C, 67.79; H, 4.29; N, 12.31.A solution of compound 7b (0.15 gm) in POCl3 (10 mL) was heated under reflux for 2 h. The reaction mixture was cooled at room temperature and poured into ice water. The obtained solid was filtered off and recrystallized from ethanol/water to give compound 13. Green crystals; yield (98%); m.p. 110–112 °C; IR (KBr) : 3120 (NH), 3036 (CHarom), 2925, 2853 , 1694, 1641 (C=O); 1HNMR (DMSO-d6) δ: 3.80 , 7.10–8.05 , 9.73 , 10.31 ; 13C-NMR (DMSO-d6) δ: 41 (CH2), 123.6 (CH=C), 127.1-136.9 (Ar-C), 137.3 (NH-C=C), 158.6, 163.1 (S-C=N), 166.8, 170 (C=O); MS (70 eV) m/z (%): 446 , 327 (23), 230 (10), 105 (93), 91 (20), 65 (100), 44 (18); Anal. calcd for C23H18N4O2S2 (446.54): C, 61.86; H, 4.06; N, 12.54. Found: C, 61.46; H, 3.77; N, 12.18.A solution of compound 2–13 against Staphylococcus aureus ATCC 29213, Bacillus subtilis ATCC 6633 as Gram-positive bacteria, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 as Gram-negative bacteria and Candida albicans ATCC 10231, Aspergillus flavus ATCC 46283 as fungi. At the first, 100 μL of the test bacteria/fungi were grown in 10 mL of fresh media until they reached a count of approximately 108 cells/ml for bacteria or 105 cells/mL for fungi. One milliliter of each sample (at 0.5 mg/mL) was added to each well (10 mm diameter holes cut in the agar gel). The plates were incubated for 24 h at 37 °C (for bacteria and yeast) and for 72 h at 27 °C (for filamentous fungi), each test was repeated three times. After incubation, the microorganism’s growth was observed. Ciprofloxacin and clotrimazole were used as standard antibacterial and antifungal drugs, respectively. The resulting inhibition zone diameters were measured in millimeters using the diffusion technique [6, 7a, 7b, 8, 9, 12 and 13 were further investigated to determine their antimicrobial activity expressed in terms of minimum inhibitory concentration (MIC) using the modified agar well diffusion method that mentioned above. The different concentrations (triplicate) of each compound were tested and compared with standard drugs. The antimicrobial assay was performed in vitro for the target compounds 6, 7a, 7b, 8, 9, 12 and 13 were assessed against both human breast cancer (MCF-7) and normal kidney epithelial cell lines (Vero cells) that were obtained from Sigma-Aldrich Chemical Company, St. Louis, MO, USA. The experiment performed using MTT assay and cisplatin as a standard following the previously mentioned techniques [The cytotoxic activities of the highly potent derivatives as antimicrobials chniques .8 was carried out in the confirmatory diagnostic unit, Vacsera, Egypt. The screening performed against E. coli DNA gyrase B, E. coli Topoisomerase IV and dihydrofolate reductase enzymes. E. coli DNA gyrase microplate assay kit , E. coli topoisomerase IV decatenation kit  and dihydrofolate reductase inhibitor screening Kit (Colorimetric) Bio Vision have been used for the anticipated assay according to the optimized protocol by the manufacturer. The used reference drugs were novobiocin for E. coli DNA gyrase B and E. coli Topoisomerase IV and methotrexate for DHFR according to the previously mentioned methods [50 values in The in vitro enzyme inhibition assessment for the most active derivative  methods ,24,29. T8 was drawn by chem. Draw. Then, the protonated 3D was built using standard bond angles and lengths, with the MOE 10.2008 software [E. coli DNA gyrase B and Topoisomerase IV with their ligand novobiocin were downloaded (PDB code: 1AJ6 and 1S14), respectively [−1Å−1 with the MMFF94x force field and the partial charges were automatically calculated. The structures of the two enzymes were prepared for molecular docking using the Protonate 3D protocol in MOE with the default options. Triangle Matcher placement method and London dG scoring function were applied in the docking protocol. Firstly, the validation processes were confirmed by docking of the original ligand, followed by docking of the compound 8 into the active sites after removing the co-crystallized ligand according to the reported procedure [The 2D structure of the newly synthesized compound software , followiectively ,31. All rocedure . 2–13 bearing different heterocyclic systems was synthesized and screened for their biological activities. Compound 8 showed significant antimicrobial activity against almost tested strains with inhibition zone diameters (in mm) ranging from 42 ± 0.30 to 18 ± 0.41 and MIC values ranged from 0.95 ± 0.22 to 3.25 ± 1.00 μg/mL comparing with the reference drugs. Furthermore, the cytotoxic screening against MCF-7 cancer cells compared to normal kidney epithelial cell lines (Vero cells) revealed the potential cytotoxic effects of the synthesized derivatives. Based on the promising in vitro inhibition results of compound 8 against E. coli DNA B gyrase and Topoisomerase IV, the thiosemicarbazide derivative 8 bearing cyano group via acetamide moiety illustrated good fitting and favorable binding interactions in the docking study in comparison with the native ligand, novobiocin.A series of novel thiourea derivatives


Closo-o-carboranyl compounds bearing the ortho-type perfectly distorted or planar terphenyl rings  and their nido-derivatives  were synthesized and fully characterized using multinuclear NMR spectroscopy and elemental analysis. Although the emission spectra of both closo-compounds exhibited intriguing emission patterns in solution at 298 and 77 K, in the film state, closo-DT mainly exhibited a π-π* local excitation (LE)-based emission in the high-energy region, whereas closo-PT produced an intense emission in the low-energy region corresponding to an intramolecular charge transfer (ICT) transition. In particular, the positive solvatochromic effect of closo-PT and theoretical calculation results at the first excited (S1) optimized structure of both closo-compounds strongly suggest that these dual-emissive bands at the high- and low-energy can be assigned to each π-π* LE and ICT transition. Interestingly, both the nido-compounds, nido-DT and nido-PT, exhibited the only LE-based emission in solution at 298 K due to the anionic character of the nido-o-carborane cages, which cannot cause the ICT transitions. The specific emissive features of nido-compounds indicate that the emissive color of closo-PT in solution at 298 K is completely different from that of nido-PT. As a result, the deboronation of closo-PT upon exposure to increasing concentrations of fluoride anion exhibits a dramatic ratiometric color change from orange to deep blue via turn-off of the ICT-based emission. Consequently, the color change response of the luminescence by the alternation of the intrinsic electronic transitions via deboronation as well as the structural feature of terphenyl rings indicates the potential of the developed closo-o-carboranyl compounds that exhibit the intense ICT-based emission, as naked-eye-detectable chemodosimeters for fluoride ion sensing. Closo-ortho-carboranes  are well-known boron-cluster components of three-dimensional (3D) icosahedral analogs. Recently, closo-ortho-carboranes have attracted significant attention as new molecular scaffolds of steric and electronic substituents for luminescent organic and organometallic compounds due to their unique photophysical properties and reasonable thermal and electrochemical stabilities originating from the o-carborane unit fluorene-based closo-o-carboranyl compound (PT), with planar o-terphenyl rings, which possessed an intense emission from the ICT transition. Subsequently, the designed closo-o-compounds, as well as the nido-o-carboranyl compounds, were prepared and fully characterized. The comparison of the photophysical properties of these closo- and nido-compounds indicated that the deboronation of the o-carborane moiety and the structural feature of the appended terphenyl rings may deactivate the ICT transition and also quenching of the emission, thereby providing a novel method for fluoride sensing.On the basis of inducing significant changes in the electronic properties through the conversion of  results   and nido-o- (nido-DT and nido-PT) carboranyl compounds, where the o-carborane cages are substituted at both the ends by terphenyl moieties, are outlined in DT1 and PT1 produced the ethynyltrimethylsilane-substituted terphenyl compounds DT2 and PT2, respectively, in high yields (62% for DT2 and 83% for PT2). The mild base (K2CO3)-mediated deprotection of the trimethylsilyl protecting groups of DT2 and PT2 furnished DT3 and PT3, respectively, which were then subjected to decaborane (B10H14)-promoted cage-forming reactions in the presence of Et2S  in THF at 60 °C led to the conversion of the closo-carboranes to the nido-species; nido-DT is the (NBu4)2-salt of the nido-form of closo-DT, and nido-PT is the (NBu4)2-salt of the nido-form of closo-PT  NMR spectroscopy  in the 1H{11B} NMR spectra of both nido-DT and nido-PT are characteristic of the B–H–B bridge protons of nido-o-carborane moieties. The 11B{1H} NMR signals of nido-DT and nido-PT at δ ca. −8 to −37 ppm, which are shifted significantly upfield due to the anionic character of the nido-o-carboranes, clearly confirmed the presence of the nido-o-carboranyl boron atoms.All of the prepared troscopy  and elemcloso- and nido-o-carboranyl were investigated using UV/Vis absorption and photoluminescence (PL) spectroscopies  results vide infra). Indeed, these ICT-based low-energy absorption bands were not present in the spectra of nido-DT and nido-PT, due to which those absorption spectra were slightly blue-shifted , this high-energy emission appears to originate from the π–π* LE transitions of the central terphenyl moieties. In contrast, the low-energy emission is closely associated with ICT transitions between the o-carborane cages and the terphenyl rings. Furthermore, the emission spectrum of closo-DT in THF at 298 K exhibited an intense emission in the high-energy region at λem = 350 nm due to π−π* LE transitions based on the central phenyl rings. The fact that the high-energy emission band of closo-DT was consistently maintained in a variety of solvents of different polarities , and the PL spectra in the rigid state  (PMMA)) showed an enhanced low energy emission (λem = 482 nm in THF at 77 K and λem = 492 nm in the film) that tailed to 550 nm. The emission band for closo-PT around 500 nm was also significantly increased in the rigid state (THF at 77 K and in film), indicating that the electronic transition for both closo-compounds are governed by non-radiative process in solution state at 298 K. This behavior originates from the increased efficiency of the radiative decay associated with the ICT transition in the rigid molecular state, which restricts structural fluctuations such as C–C bond variations in the o-carborane cage fluorene (PT1), were prepared as reported in the literature [2Cl2 and THF-d8, purchased from Cambridge Isotope Laboratories, were dried over activated molecular sieves (5 Å). All nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance 400 spectrometer  at ambient temperature. Chemical shifts are given in ppm and are referenced against external Me4Si (1H and 13C) or BF3·Et2O (11B). Elemental analysis was performed on an EA3000 instrument (Eurovector) at the Central Laboratory of Kangwon National University. UV–Vis absorption and photoluminescence (PL) spectra were recorded on Jasco V-530  and Horiba FluoroMax-4P spectrophotometers , respectively. Fluorescence decay lifetimes (τobs) were measured using a time-correlated single-photon counting spectrometer  equipped with an EPL 375 ps pulsed semiconductor diode laser as the excitation source and a microchannel plate photomultiplier tube (200–850 nm) as the detector, at 298 K. The absolute PL quantum yields (Фem) were obtained with an absolute PL quantum yield spectrophotometer  at 298 K.All operations were performed under an inert nitrogen atmosphere using standard Schlenk and glove-box techniques. Anhydrous solvents , copper iodide (15 mg), and Pd(PPh3)2Cl2 (62 mg) at 25 °C. After stirring for 15 min, ethynyltrimethylsilane  was added, and the reaction mixture was heated at 90 °C with stirring for 24 h. After cooling to 25 °C, the volatiles were removed by rotary evaporation to afford a dark brown residue. The crude product was purified by column chromatography on silica gel  to yield DT2 as a yellow solid, 0.28 g (yield = 62%). 1H-NMR (CD2Cl2): δ 7.51 , 7.32 , 7.13 , 2.25 , 0.27 3). 13C-NMR (CD2Cl2): δ 142.35, 140.71, 133.04, 132.05, 131.97, 129.60, 121.97, 105.26 (acetylene-C), 94.83 (acetylene-C), 20.01 (–CH3), 0.03 (–Si(CH3)3). Anal. Calcd. for C30H34Si2: C, 79.94; H, 7.60. Found: C, 79.87; H, 7.49.Triethylamine (16 mL) was added via cannulation to a mixture of PT2 was prepared according to a procedure analogous to that used for DT2, with PT1 , copper iodide (15 mg), Pd(PPh3)2Cl2 (62 mg), and ethynyltrimethylsilane , and was isolated as a yellow solid . 1H-NMR (CD2Cl2): δ 7.78 , 7.71 , 7.55 , 7.45 , 1.53 , 0.27 3). 13C-NMR (CD2Cl2): δ 154.55, 154.16, 140.07, 138.92, 131.40, 126.63, 121.79, 120.08, 114.99, 106.24 (acetylene-C), 94.53 (acetylene-C), 46.96 (–C(CH3)2), 27.37 (–CH3), 0.07 (–Si(CH3)3). Anal. Calcd. for C34H38Si2: C, 81.21; H, 7.62. Found: C, 80.99; H, 7.55.2CO3  was dissolved in methanol (10 mL) and added to a solution of DT2  in DCM (5 mL). After stirring for 2 h at 25 °C, the resulting mixture was treated with DCM (50 mL) and the organic layer was separated. The aqueous layer was further extracted with DCM (20 × 2 mL). The combined organic extracts were dried over MgSO4, filtered, and evaporated to dryness to afford a white residue. The crude product was purified by washing with n-hexane (10 mL) to yield DT3 as a white solid, 0.13 g (yield = 84%). 1H-NMR (CD2Cl2): δ 7.56 , 7.34 , 7.13 , 3.18 , 2.26 . 13C-NMR (CD2Cl2): δ 142.68, 140.68, 133.06, 132.24, 132.07, 129.66, 120.90, 83.81 (acetylene-C), 77.69 (acetylene-C), 20.01 (–CH3). Anal. Calcd. for C24H18: C, 94.08; H, 5.92. Found: C, 93.77; H, 5.62.KPT3 was prepared according to a procedure analogous to that used for DT3 with PT2  and K2CO3 , and was isolated as a white solid . 1H-NMR (CD2Cl2): δ 7.80 , 7.74 , 7.59 , 7.50 , 3.20 , 1.54 . 13C-NMR (CD2Cl2): δ 154.59, 154.15, 140.34, 138.91, 131.64, 126.88, 120.72, 120.15, 115.06, 84.70 (acetylene-C), 77.44 (acetylene-C), 46.98 (–C(CH3)2), 27.36 (–CH3). Anal. Calcd. for C28H22: C, 93.81; H, 6.19. Found: C, 93.77; H, 6.04.2S  was added at 25 °C to a solution of decaborane  and DT3  in toluene (20 mL). After heating to reflux, the reaction mixture was further stirred for 72 h. The solvent and volatiles were removed under vacuum and methanol (10 mL) was added. The resulting solid was filtered and redissolved in toluene. The crude product upon washing with n-hexane (15 mL), afforded closo-DT as a white solid (47 mg. Yield = 43%). 1H{11B} NMR (THF-d8): δ 7.66 , 7.38 , 7.12 , 5.13 , 2.54 , 2.39 , 2.30 , 2.23 . 13C-NMR (THF-d8): δ 144.05, 140.59, 133.27, 133.24, 132.34, 130.14, 128.04, 77.58 (CB-C), 61.49 (CB-C), 19.74 (–CH3). 11B{1H} NMR (THF-d8): δ −4.44 (3B), −6.57 (1B), −10.84 (5B), −12.77 (7B), −14.67 (4B). Anal. Calcd. for C28H38B20: C, 56.92; H, 6.48. Found: C, 56.79; H, 6.33.Excess EtCloso-PT was prepared according to a procedure analogous to that used for closo-DT, with decaborane , PT3 , and Et2S (2.5 equiv.). The crude product upon washing with n-hexane (15 mL), afforded closo-PT as a white solid . 1H{11B} NMR (THF-d8): δ 7.94 , 7.81 , 7.67 , 7.58 , 5.14 , 2.56 , 2.50 , 2.39 , 2.30 , 1.54 . 13C-NMR (THF-d8): δ 155.41, 154.80, 141.67, 138.98, 133.36, 127.39, 122.52, 120.66, 115.65, 78.45 (CB-C), 61.55 (CB-C), 47.50 (–C(CH3)2), 27.08 (–CH3). 11B{1H} NMR (THF-d8): δ −2.73 (3B), −4.64 (1B), −9.09 (5B), −10.77 (7B), −12.86 (4B). Anal. Calcd. for C32H42B20: C, 59.79; H, 6.59. Found: C, 59.87; H, 6.45.Closo-DT  was dissolved in 0.3 mL of a 0.2 M solution of n-tetrabutylammonium fluoride (TBAF) in THF at 25 °C. The reaction mixture was heated to reflux (60 °C) and stirred for 2 h. After cooling to 25 °C, the resulting mixture was treated with 50mL of distilled water and 50 mL of DCM, and the organic portion was separated. The aqueous layer was further extracted with DCM (20 mL). The combined organic portions were dried over MgSO4, filtered, and concentrated to dryness, affording a pale yellow residue. The crude product upon washing with methanol (15 mL), afforded nido-DT as a white solid . 1H{11B} NMR: δ 7.28 , 7.13 , 7.07 , 3.11 , 2.36 , 2.25 , 2.12 , 1.88 , 1.82 , 1.62 , 1.43 , 1.26 , 1.02 , −2.36 . 13C NMR (CD2Cl2): δ 144.65, 140.78, 138.40, 132.83, 132.21, 128.64, 126.71, 59.43 (n-butyl-CH2), 24.29 (n-butyl-CH2), 20.18 (–CH3), 20.10 (n-butyl-CH2), 13.76 (n-butyl-CH3). 11B{1H} NMR (CD2Cl2): δ −8.97 (3B), −10.43 (2B), −13.79 (1B), −18.28 (3B), −19.50 (1B), −23.00 (1B), −32.95 (3B), −36.10 (4B). Anal. Calcd. for C60H110B18N2: C, 68.37; H, 10.52; N, 2.66. Found: C, 68.11; H, 10.42; N, 2.54.nido-DT was employed using closo-PT  and 0.23 mL of a 0.2 M solution of TBAF in THF. The crude product, upon washing with methanol (15 mL), afforded nido-PT as a white solid . 1H{11B} NMR (CD2Cl2): δ 7.65 , 7.49 , 7.32 , 7.23 , 3.08 , 2.39 , 2.12 , 2.00 , 1.89 , 1.60 , 1.48 , 1.41 , 1.31 , 1.26 , 1.00 , −2.34 . 13C-NMR (CD2Cl2): δ 153.80, 153.54, 145.25, 138.62, 136.55, 126.10, 121.40, 118.72, 114.00, 59.40 (n-butyl-CH2), 46.71 (–C(CH3)2), 27.74 (–CH3), 24.27 (n-butyl-CH2), 20.09 (n-butyl-CH2), 13.75 (n-butyl-CH3). 11B{1H} NMR (CD2Cl2): δ −8.91 (3B), −10.45 (2B), −13.68 (1B), −18.46 (3B), −19.47 (1B), −23.09 (1B), −32.95 (3B), −36.05 (4B). Anal. Calcd. for C64H114B18N2: C, 69.49; H, 10.39; N, 2.53. Found: C, 69.30; H, 10.16; N, 2.39.A procedure analogous to that for closo- and nido-o-carbornyl compounds were performed in degassed organic solvents with a 1 cm quartz cuvette (3.0 × 10−5 M) at 298 K. PL measurements for the closo-compounds were also performed in THF at 77 K and in the film state (5 wt% doped in PMMA) on 1.5 × 1.5 cm quartz plates (thickness = 1 mm) at 298 K.The solution-phase UV–Vis absorption and PL measurements of the 0) and first excited (S1) states of both closo-o-carboranyl compounds (closo-DT and closo-PT) in THF were obtained using the B3LYP/6-31G [0 geometries as well as the optimized geometries of the S1 states were calculated using time-dependent density functional theory (TD-DFT) [The optimized geometries for the ground   level of(TD-DFT)  at the s(TD-DFT) ,84. All (TD-DFT) . The per(TD-DFT) . Visuali(TD-DFT) .closo- (closo-DT and closo-PT) and nido- (nido-DT and nido-PT) o-carboranyl compounds. Although closo-DT exhibited strong π–π* LE-based emission in THF at 298 K in the high-energy region, closo-PT demonstrated intense emission in the low-energy region that was attributable to the ICT transitions involving the o-carborane cage. Interestingly, both nido-compounds exhibited LE-based emission alone in the same condition due to the anionic character of the nido-o-carborane cages, which cannot cause the ICT transitions. Consequently, the successful deboronation of closo-PT to nido-PT upon exposure to increasing concentration of fluoride anion leads to ratiometric emission color change from orange to deep-blue in solution. Such results strongly imply that the fine-tuning of electronic and structural features, which can control the ICT-based emission, shows the potential of closo-o-carboranyl compounds as candidates for naked-eye-detectable chemodosimeters for fluoride ion-sensing.We herein reported the preparation and characterization of distorted and planar terphenyl-based