The current work aims to design and synthesis a new series of isatin derivatives and greatly enhances their cytotoxic activity. The derivatives 3-((bromophenyl) imino)-1-(morpholino (pyridine) methyl) indolin-2-one, 2-((oxoindoline) amino) benzoic acid, 3-(thiazolo-imino) indolinone, ethyl-2-((oxoindolin-3-ylidene)amino)-benzothiophene-3-carboxylate, 1-(oxoindoline)-benzo thieno pyrimidin-4(1H)-one, ethyl-2-(2-oxoindoline) hydrazine-1-carboxylate, N-(mercapto-oxo-pyrimidine)-2-(oxoindoline) hydrazine-1-carboxamide, N--2-(oxoindolin-ylidene) hydrazine-carboxamide, 3-((amino-phenyl) amino)-3-hydroxy- indolinone, 3-((amino-phenyl) imino)-indolinone, 2-(2-((oxoindoline) amino) phenyl) isoindolinone, 2-(oxoindoline) hydrazine-carbothioamide, 5′-thioxospirotriazolidin]-one, 5′-amino-spirothiadiazol]-2-one and 3- imino) indolinone were synthesized from the starting material 1-(morpholino (pyridine) methyl) indoline-2,3-dione and evaluated for their in vitro cytotoxic activity against carcinogenic cells. The new chemical structures were evidenced using spectroscopy  and elemental analysis. The results show that compounds imidazoquinoxaline-indolinone, thiazolopyrimidine-oxoindoline, pyrimidine-oxoindoline-hydrazine-carboxamide, spiro thiadiazol]-one and spirotriazolidin]-one have excellent anti-proliferative activities against different human cancer cell lines such as gastric carcinoma cells (MGC-803), breast adenocarcinoma cells (MCF-7), nasopharyngeal carcinoma cells (CNE2) and oral carcinoma cells (KB). Recently, cancer has been spreading among people at a high rate. Thus, cancer is one of the main causes of death. In 2017, the death of nearly 9.6 million people due to different types of cancer made it the second leading cause of death, second only to cardiovascular diseases thieno pyrimidinone; hydrazine-carboxylate; hydrazine-carboxamide; thiazolopyr- imidine; amino-iminoindolinone; thioxospirotriazolidin]-one; spiro thiadiazol]-2-one; and 3-imino) indolinone using recent procedures. The synthesized compounds were studied in vitro to evaluate their anticancer activity against different human cancer cell lines.(2) in excellent yield through stirring and refluxing a mixture of isatin (1) with freshly distilled secondary aliphatic amine as morpholine and isonicotinaldehyde in sodium ethoxide solution at room temperature under control (TLC). The IR spectrum compound (2) showed absorption bands at ν 1735 and 1680 cm−1 conforming to two carbonyl groups. 1H-NMR spectrums of (2) revealed multiplet signals at δ 2.60–3.05 ppm corresponding to eight protons of morpholine and one singlet at δ 6.72 ppm corresponding to one proton of methine proton.In order to perform one-pot synthesis using Mannich bases reaction ,52,53, w13C-NMR spectrum of (2) displayed absorption signals at δ 51.2 and 58.8 ppm conforming to four carbon atoms of the (4CH2) morpholine and 95.4 ppm corresponding to one carbon atom of the methine proton (CH) group.The (3–5) started from 1- (morpholino (pyridin-4-yl) methyl) indoline-2,3-dione (2) through activation of the C3-substituted isatin, which is a carbonyl group, as a good leaving group with the exit water molecule as a requirement for the subsequent SNAr (nucleophilic aromatic substitution) reaction to occur with weak N-nucleophiles such as NH2-heterocycles. Moreover, compound (2) reacted with 4-bromo aniline in methanolic potassium hydroxide solution to yield 3-((4-bromophenyl) imino)-1- (morpholino (3yridine-4-yl) methyl) indolin-2-one (3).In continuation of our research plan in exploring the reactivity of isatin derivatives, we were interested in preparing 3-substituted-indolin-2-one, as expected as anticancer scaffolds. Therefore, syntheses of (2) with 2-aminobenzoic acid and anhydrous potassium carbonate in dry dimethylformamide afforded 2-((1-(morpholino (pyridin3-4-yl) methyl)-2-oxoindolin-3-ylidene) amino) benzoic acid (4).Furthermore, condensation of compound (2) with 2-aminothiazole and anhydrous potassium carbonate in dry dimethylformamide yielded 1-(morpholino (3yridine-4-yl) methyl)-3-(thiazol-2-ylimino) indolin-2-one (5). The IR spectrum of (3) showed absorption bands at ν 1688 cm−1 for the one carbonyl group, and the 13C-NMR spectrum of 3 revealed absorption signals at δ 51.5 and 58.9 ppm corresponding to four carbon atoms of the (4CH2) morpholine, at δ 94.7 ppm corresponding to one carbon atom of the methine proton (CH) group and at δ 164.1 ppm conforming to one carbonyl group.In addition, condensation of compound (4) indicated absorption bands at ν 3750 cm−1 for the one hydroxyl group and at ν 1740 and 1688 cm−1 for two carbonyl groups, and the 1H-NMR spectrum of (4) evidenced one singlet signal at δ 12.60 ppm for the one proton of (OH) group (D2O exchangeable). The 13C-NMR spectrum of (4) exposed absorption signals at δ 52.2 and 59.1 ppm matching four carbon atoms of the (4CH2) morpholine, at δ 95.3 ppm corresponding for one carbon atom of the methine proton (CH) group and at δ 164.7 and 167.1 ppm corresponding to two carbonyl groups.Likewise, the IR spectrum of 1H-NMR spectrums of (5) discovered multiplet signals at δ 2.72–3.18 ppm conforming to eight protons of morpholine, one singlet at δ 6.67 ppm corresponding to one proton of methine proton and two doublet signals at δ 7.78 and 7.85 ppm corresponding for two protons of (J = 6.92 Hz) thiazole ring. The 13C-NMR spectrum of (5) demonstrated absorption signals at δ 52.5 and 59.7 ppm corresponding to four carbon atoms of the (4CH2) morpholine, at 94.4 ppm corresponding to one carbon atom of the methine proton (CH) group and at δ 168.2 ppm conforming to one carbonyl group.Similarly,  evidenced molecular ion peaks at m/z = 323 , 477 , 442  and 405 , respectively. IR, NMR, MS and elemental analysis are elucidated in the experimental section for new compounds and shown in  reacted with ethyl 2-amino-4,5,6,7-tetrahydro benzo[b]thiophene-3-carboxylate in refluxing dimethylformamide with the presence of potassium carbonate afforded ethyl-2-((1-(morpholino (pyridin-4-yl) methyl)-2-oxoind- olin-3-ylidene) amino)-4,5,6,7-tetrahydrobenzo[b] thiophene-3-carboxylate (6). The IR spectrum of (6) indicated the presence of absorption bands at ν 1775 and 1682 cm−1 conforming for two carbonyl groups. The 1H NMR spectrum of (6) displayed multiplet signals at δ = 1.08–1.15 ppm for eight protons of , multiplet signals at δ = 2.75–3.22 ppm for eight protons of , and one singlet signal at δ = 6.62 ppm for one proton of methine proton (CH) group.Furthermore, the compound (6) reacted with formamide in dimethylformamide with the presence of potassium carbonate for a long time under control (TLC) to yield 1-(1-(morpholino(pyridin-4-yl)methyl)-2-oxoindolin-3-yl)-5,6,7,8-tetrahydrobenzo thienopyrimidin-4(1H)-one (7). The 1H-NMR spectrum of (7) exhibited two singlets at δ = 5.10 and 8.15 ppm, corresponding to the one proton of pyrrole and one proton of pyrimidine ring, respectively.Furthermore, the latter compound (2) with ethyl hydrazinecarboxylate in boiling dimethylformamide and potassium carbonate produced Ethyl-2-(1-(morpholino (pyridin-4-yl) methyl)-2-oxoindolin-3-ylidene) hydrazine-1-carboxylate (8). The IR spectrum of 8 showed the presence of broad band absorption at ν 3325 cm−1 for one (NH) group and two peaks in absorption at ν 1691 and 1684 cm−1 for two carbonyl groups, and the 1H-NMR spectrum of (8) displayed a broad singlet at δ 10.50 ppm corresponding to the one proton of (NH) group exchangeable with D2O.Moreover, condensation of compound (8) with 6-amino-2-mercapto- pyrimidin- 4(3H)-one in refluxing and stirring dimethylformamide and an amount of TEA (0.5 mL) as catalysis produced N--2- (1-(morpholi- no (pyridin-4-yl) methyl)-2-oxoindolin-3-ylidene) hydrazine-1-carboxami- de (9). The IR spectrum of (9) displayed three peaks in broad band absorption at ν 3330–3270 cm−1 corresponding to three (3NH) groups and three peaks in absorption at ν 1695, 1688 and 1682 corresponding to three carbonyl groups, and 1H-NMR spectrum of (9) exhibited three broad singlets at δ 9.90, 10.80 and 11.70 ppm corresponding to the three protons of three (3NH) exchangeable with D2O.Furthermore, the treatment of the same compound (9) with chloroacetic acid in a mixture of glacial acetic acid and glacial acetic anhydride in the presence of anhydrous sodium acetate under stirring and refluxing led to N--2-(1-(morpholino(pyridin-4-yl)methyl)-2-oxoindolin-3-ylidene)hydr- azine-1-carboxamide (10). IR spectrum of (10) showed absorption broadband at ν 3325–3280 cm−1 conforming to two (2NH) groups and four peaks at ν 1691, 1686, 1681 and 1675 cm−1 for four carbonyl groups, and 1H-NMR spectrum of (10) exposed two broad singlets at δ 10.10 and 11.60 ppm corresponding to the two protons of two (2NH) groups exchangeable with D2O.Otherwise, condensation of the later compound  revealed molecular ion peaks at m/z = 530 , 513 , 409 , 409  and 546 , respectively. Correct new chemical structures were approved of through spectroscopic, IR, 1H, 13C, -NMR, mass spectra and elemental analysis, shown in (Exp. Part and MS spectrum of We report here the synthesis of new 3-((2-aminophenyl) amino)-indolin-2-one, 3-((2-amino-phenyl) imino)-indolin-2-one and 2-((amino) phenyl) isoindoline-1, 3-dione and describe their anticancer properties.(2) with benzene-1, 2-diamine in methanol and diethyl amine with stirring and refluxing afforded 3-((2-aminophenyl) amino)-3-hydroxy-1-(morpholino (pyridin-4-yl) methyl) indolin-2- one (11). Its IR of (11) shows three broad peaks at ν 3415, 3370 and 3330 cm−1 corresponding to (NH2), (OH) and (NH) groups, respectively, and a sharp intensity in one peak at ν 1682 cm−1 corresponding to one carbonyl group. The 1H-NMR spectrum of (11) contains three broad signals at δ 6.25, 11.10 and 12.60 ppm for two protons (NH2), one proton (NH) and one proton (OH) groups exchangeable with D2O, respectively.Furthermore, the reaction of (11) in glacial acetic acid with concentrated HCl under (TLC) control afforded 3-((2-aminophenyl)imino)-1- (morpholi- no (pyridin-4-yl) methyl) indolin-2-one (12) in good yield. The IR spectrum compound (12) exhibited absorption bands at ν 3410 cm−1 corresponding to NH2 group and1684 cm−1 corresponding to one carbonyl group. 1H-NMR spectrum of (12) revealed one singlet at δ 6.35 ppm conforming to two protons of NH2 group (D2O exchangeable).Furthermore, stirring and refluxing of the same compound (12) with phthalic anhydrides in absolute methanol containing a few drops of glacial acetic acid afforded 2-(2-((1-(morpho- lino (pyridin-4-yl) methyl)-2-oxoindolin-3-ylidene) amino) phenyl) isoindoline-1, 3-dione (13) in good yield. IR spectrum of (13) showed absorption bands at ν 1688, 1682 and 1677 cm−1 for three carbonyl groups. The 13C-NMR spectrum of (13) exhibited absorption signals at δ 52.3 and 59.1 ppm, corresponding to four carbon atoms of , at δ 93.8 ppm for methine proton (CH) group and at δ 166.1, 168.5 ppm for three carbon atoms of the three carbonyl groups.Moreover, treatment of the latter compound  evidenced molecular ion peaks at m/z = 431 , 413  and 543 , respectively. IR, NMR, MS and elemental analysis were explicated in the experimental section for all compounds and shown in  imino) through reaction of thiosemicarbazide with isatin moieties.(2) in glacial acetic acid was heated and refluxed to produce the target of 2- (1- (morpholino (pyridin-4-yl) methyl)-2-oxoindolin-3-ylidene) hydrazine-1- carbothioamide (14) as key starting material to synthesize many heterocyclic compounds. The IR spectrum of compound (14) exhibited absorption bands at ν 3430 and 3320 cm−1 corresponding to NH2 and NH groups, ν 1678 cm−1 for one carbonyl group and 1475 cm−1 for one (C=S) group. 1H-NMR spectrum of (14) demonstrated one singlet at δ 6.61 ppm corresponding to one proton of methine proton and two singlets at δ 6.90 and 11.35 ppm corresponding to the two protons of (NH2) and one proton of (NH) groups (D2O exchangeable), respectively.Furthermore, the thiosemicarbazide with 1-(morpholino (pyridine) methyl) isatins (2) and thiosemicarbazide in glacial acetic acid under reflux for a long time to form 1-(morpholino (pyridin-4-yl) methyl)-5′-thioxospiro  triazolidin]-2-one (15).Additionally, a new one-pot multi-component reaction for the perfect selective synthesis of the various spiro-indolines-based frameworks has been described. Thus, the multicomponent reaction involves the compound isatin (15) can be prepared in another way, which is to use cycloaddition reactions of the latter compound (14) in dry chloroform or glacial acetic acid with a few drops of triethylamine as catalysis for a long time under reflux and (TLC) control. The IR spectrum compound 15 showed absorption bands at ν 3350–3230 cm−1 compatible with three (3NH) groups and ν 1688 cm−1 conforming to one carbonyl group and ν 1450 cm−1 for one (C=S) group.Furthermore, the same compound 1H-NMR spectrum of 15 revealed three broad singlets at δ 10.10, 10.40 and 11.55 ppm corresponding to three protons of (3NH) groups (D2O exchangeable). The 13C-NMR spectrum of 15 showed absorption signals at δ 97.1 ppm conforming to one spiro-carbon atom of  and δ 168.2 ppm for one carbon atom of the one carbonyl group.H-spirothiadiazol]-2-one (16) was prepared separately using compound (14) and iodine with potassium carbonate in boiling dioxane or DMF for long periods of time.In addition, 5′-amino-1-(morpholino (pyridin-4-yl) methyl)-3′(16) is refluxing of compound (14) in ethanol containing a few drops of triethylamine as catalysis under (TLC) control. The IR spectrum of (16) displayed absorption bands at ν 3420 and 3335 cm−1 matching NH2 and NH groups and ν 1682 cm−1 for one carbonyl group. 1H-NMR spectrum of (16) proved one singlet at δ 6.85 ppm corresponding to two protons of (NH2) and 10.25 ppm corresponding to one proton of (NH) groups exchangeable with D2O. The 13C-NMR spectrum of (16) indicated absorption signals at δ 78.5 ppm corresponding to one spiro-carbon atom of  and δ 167.5 ppm for one carbon atom of the one carbonyl group.Another method for synthesizing a compound H-imid- azo  quinoxalin-1-yl) imino) indolin-2-one (17) was synthesized by nucleophilic aromatic substitution reactions of compound (14) with 2,3-dichloro-quinoxaline by refluxing in absolute ethanol in the presence of triethylamine as catalysis. The IR spectrum of (17) exposed absorption bands at ν 3340 cm−1 corresponding to NH group, 1686 cm−1 compatible with one carbonyl group and ν 1480 cm−1 conforming to one (C=S) group. 1H-NMR spectrum of (17) revealed one singlet at δ 10.60 ppm conforming to one proton of (NH) group (D2O exchangeable).Moreover, 1-(morpholino (pyridin-4-yl) methyl)-3- displayed molecular ion peaks at m/z = 396 , 396 , 396  and 522 , respectively. Correct new chemical structures were evidenced via spectroscopic, infrared, nuclear magnetic resonance, mass spectra and elemental analysis, shown in  ,32,33,5450 values are summarized in ; the MCF-7 ; the CNE2 ; and the KB . Furthermore, some of the compounds that moderate resulted in activities against cancer cells such as 7, 13, 6 and 5. Further, the rest of the compounds in this manuscript have a weak effect against cancer cells. All results concerning the compounds’ anticancer activities in vitro are shown in  these compounds 17, 10, 9, 16 and 15  showed good anticancer activity in vitro. This agrees with previous scientific studies, because it includes many heterocyclic compounds possessing pharmacological activities such as imidazoquinoxaline- iminoindolinone, N-thiazolopyrimidine-oxoindoline, N-(pyrimidine)-oxoindoline- hydrazine-1-carboxamide, spiro thiadiazol]-2-one, thioxospiro triazolidin]-one, oxoindoline-benzothienopyrimidinone, oxoindoline-amino-isoindolinone, oxoindoline-amino-benzo[b]thiophene-3-carboxylate and 1- (morpholino (pyridine) methyl)-3-(thiazolo-imino) indolinone. They have proved potent anticancer activities thiophene-3-carboxylate  and anhydrous potassium carbonate  were added in dry dimethylformamide (40 mL), and the reaction mixture was refluxed for 10–12 h with control by (TLC). After cooling at room temperature, the reaction solution was poured onto ice water. The product precipitate was filtered off, dried and recrystallized from toluene in (81%) yield, forming yellow crystals, with m. p. > 350 °C (dec.). IR  KBr: 3070 , 2960 , 1775 , 1682  and 1631 (C=N), 1590 (C=C); 1HNMR  δ 1.08–1.15 , 1.28–1.35 , 2.75–3.22 , 4.42–4.49 , 6.62 , 7.25–7.33 , 7.55–7.82 , 7.90–7.95 ; 13C NMR (DMSO-6d) δ 18.8 , 21.5, 22.4, 23.1, 24.7 , 52.8, 58.9 , 61.2 , 94.7 , 116.3, 117.9, 123.7, 124.5, 127.1, 128.8, 129.5, 130.9, 138.6, 146.1, 147.7, 149.5, 151.3, 160.8 , 164.2, 166.9 ; MS : m/z = 530 ; Anal. Calc. (Found) for C29H30N4O4S (530.64): C, 65.64 (65.71); H, 5.70 (5.62); N, 10.56 (10.50).To a solution of compound 6  and formamide  was stirred under reflux in dimethylformamide (35 mL) in the presence of anhydrous potassium carbonate (0.015 mol) for 16–18 h under control (TLC). The reaction solution was cooled at room temperature, poured into cold water and neutralized. The solid product formed was filtered off, dried and crystallized from dioxane in (80%) yield, forming yellowish crystals, with m. p. > 350 °C (dec.). IR  KBr: 3075 , 2970 , 1690, 1685  and 1634 (C=N), 1588 (C=C); 1HNMR  δ 110–1.18 , 2.80–3.27 , 5.10 , 6.58 , 7.35–7.44 , 7.59–7.88 , 7.92–7.97 , 8.15 ; 13C NMR (DMSO-6d) δ 21.7, 22.8, 23.4, 24.9 , 52.9, 59.1 , 77.4 , 93.9 , 117.7, 124.1, 124.6, 125.8, 127.5, 127.9, 129.7, 135.8, 138.8, 139.6, 144.5, 146.3, 148.2, 149.7, , 163.8, 167.1 ; MS : m/z = 513 ; Anal. Calc. (Found) for C28H27N5O3S (513.62): C, 65.48 (65.40); H, 5.30 (5.37); N, 13.64 (13.57).A mix of compound 2 , ethyl hydrazinecarboxylate  and anhydrous potassium carbonate  was formed in dry dimethylformamide (45 mL). The reaction mixture was heated under reflux for 11–13 h under control (TLC) and then allowed to cool and poured into cold water. The precipitate was filtered off, dried and recrystallized from n-hexane in (84%) yield, yellowish crystals, m.p = 308–310 °C (dec.). IR  KBr: 3325 , 3055 , 2960 , 1691, 1684 , 1628 (C=N) and 1582 (C=C); 1HNMR  δ 1.29–1.36 , 2.72–3.19 , 4.35–4.42 , 6.55 , 7.32–7.41 , 7.52–7.81 , 7.88–7.95 , 10.50 ; 13C NMR (DMSO-d6) δ 19.1 , 52.6, 58.5 , 61.7 , 93.5 , 116.1, 117.5, 124.1, 124.6, 129.2, 131.8, 132.7, 146.3, 147.5, 149.6 , 161.1, 164.3 ; MS : m/z = 409 ; Anal. Calc. (Found) for C21H23N5O4 (409.45): C, 61.60 (61.67); H, 5.66 (5.60); N, 17.10 (17.17).A mix of compound H)-one  in dry dimethylformamide (40 mL) containing a catalytic amount of TEA (0.5 mL) was stirred and heated under reflux for 13–15 h with (TLC) control. The reaction solution was cooled; the solid precipitate was filtered off, dried and recrystallized from methanol/ DMF in (78%) yield, forming brownish crystals, with m. p. > 350 °C (dec.). IR  KBr: 3330–3270 , 3060 , 2970 , 1695, 1688, 1682 , 1631 (C=N) and 1585 (C=C); 1HNMR  δ 2.66 , 2.75–3.25 , 6.50 , 6.85 , 7.36–7.42 , 7.50–7.80 , 7.85–7.92 , 9.90 , 10.80 , 11.70 ; 13C NMR (DMSO-d6) δ 51.9, 58.7 , 81.5 , 94.2 , 116.3, 117.6, 124.4, 124.8, 129.6, 131.5, 132.1, 146.7, 147.8, 149.7, 153.1, 160.2 , 161.5, 164.1, 166.2 ; MS : m/z = 409 ; Anal. Calc. (Found) for C23H22N8O4S (506.54): C, 54.54 (54.62); H, 4.38 (4.31); N, 22.12 (22.07).A suspension of compound 4  and 6-amino-2-mercaptopyrimidin- 4, chloroacetic acid , and 0.02 mol of anhydrous sodium acetate was stirred under reflux in (40 mL) of glacial acetic acid and (25 mL) of acetic anhydride in a water bath (85–95 °C) for 17–19 h under control (TLC). The reaction mixture was cooled at room temperature and poured into cold water (100 mL). The solid precipitate was filtered off, dried and crystallized from a suitable solvent to yield (10). Method B: A solution of 9  and chloroacetic acid  in dimethylformamide (35 mL) was heated and refluxed for 14–16 h under control (TLC). The final precipitate was filtered off, dried and crystallized from DMF/methanol in good yields (75%), forming yellowish crystals, with m. p. > 350 °C (dec.). IR  KBr: 3325–3280 , 3055 , 2965, 1691, 1686, 1681, 1675 , 1634 (C=N) and 1582 (C=C); 1HNMR  δ 2.78–3.28 , 4.22 , 6.58 , 6.88 , 7.39–7.45 , 7.58–7.87 , 7.91–7.98 ,10.10 , 11.60 ; 13C NMR (DMSO-d6) δ 32.1 , 52.3, 59.1 , 82.3 , 93.5 , 116.1, 117.9, 124.5, 124.7, 129.5, 131.4, 132.5, 146.2, 147.6, 149.5, 153.3, 159.1 , 161.8, 163.5, 165.7, 169.1 ; MS : m/z = 546 ; Anal. Calc. (Found) for C25H22N8O5S (546.56): C, 54.94 (54.86); H, 4.06 (4.01); N, 20.50 (20.59).2 , benzene-1,2-diamine  in absolute methanol (30 mL) and diethylamine  was added. The reaction solution was stirred and refluxed at room temperature for ~8–10 h and monitored by using TLC. The solid formed was filtered off, washed with water, dried and recrystallized from DMF in good yields (83%), forming yellow crystals, with m. p.= 297–300 °C (dec.). IR  KBr: 3415 , 3370–3330 , 3060 , 2950 , 1682 , 1630 (C=N) and 1580 (C=C); 1H NMR  δ 2.70–3.10 , 6.25 , 6.60 , 7.22–7.30 , 7.35–7.80 , 7.85–7.97, 11.10 , 12.60 ;13C NMR (DMSO-d6) δ 51.6, 58.5 , 93.1 , 111.4, 112.1, 114.8, 116.9, 118.2, 120.3, 124.4, 128.1, 128.9, 133.3, 135.2, 135.7, 137.5, 145.2, 147.4, 149.7 , 165.7 ; MS : m/z = 431 ; Anal. Calc. (Found) for C24H25N5O3 (431.50): C, 66.81 (66.73); H, 5.84 (5.75); N, 16.23 (16.32).To a mixture of 11  in glacial acetic acid (30 mL) and concentrated HCl (0.5 mL) was heated and refluxed at 80–90 °C for ~1–2 h. After cooling, the reaction mixture was cooled and poured into ice water. The solid precipitate that formed was filtered off, washed with water, air-dried and crystallized from dioxane in good yields (80%), forming yellowish crystals, with m. p. = 320–322 °C (dec.). IR  KBr: 3410 , 3057 , 2953 , 1684 , 1637 (C=N) and 1583 (C=C); 1H NMR  δ 2.77–3.16 , 6.35 , 6.65 , 7.28–7.37 , 7.40–7.85 , 7.87–7.99 ; 13C NMR (DMSO-d6) δ 51.8, 58.7 , 93.5 , 115.9, 117.4, 117.8, 123.7, 124.1, 124.6, 127.2, 128.4, 129.6, 132.3, 140.8, 142.5, 146.3, 147.1, 149.5, 161.7 , 165.9 ; MS : m/z = 413 ; Anal. Calc. (Found) for C24H23N5O2 (413.48): C, 69.72 (69.80); H, 5.61 (5.55); N, 16.94 (16.88).A solution of compound 12  and phthalic anhydride  in absolute methanol (45 mL) containing a few drops of glacial acetic acid was stirred and heated under reflux for ~10–12 h with (TLC control). The solid precipitate obtained was filtered, dried and recrystallized from DMF in good yields (75%), forming white crystals, with m. p. > 350 °C (dec.). IR  KBr: 3062 , 2958, 1688, 1682, 1677 , 1633(C=N) and 1585 (C=C); 1H NMR  δ 2.80–3.20 , 6.60 , 7.13–7.22 , 7.31–7.72 , 7.75–7.88 , 7.91–7.99 ; 13C NMR (DMSO-d6) δ 52.3, 59.1 , 93.8 , 116.2, 118.5, 122.6, 123.4, 124.3, 124.8, 126.4, 127.7, 129.4, 129.8, 131.4, 131.9, 132.4, 132.7, 146.2, 146.8, 147.6, 149.7, 161.9 , 166.1, 168.5 ; MS : m/z = 543 ; Anal. Calc. (Found) for C32H25N5O4 (543.58): C, 70.71 (70.78); H, 4.64 (4.57); N, 12.88 (12.80).A solution of compound 2  was added. The reaction mixture was heated under reflux for ~5–7 h. The formed solid precipitate was filtered off, washed with ethanol / water and dried, then recrystallized from DMF to obtain yellow crystals in good yield (84%), with m. p. > 350 °C (dec.). IR  KBr: 3430, 3320 (NH2 and NH), 3052 , 2943 , 1678 (C=O), 1629 (C=N), 1582 (C=C), and 1475 (C=S); 1H NMR  δ 2.72–3.13 , 6.61 , 6.90 , 7.22–7.50 , 7.61–7.70 , 7.77–7.90 , 11.35 ; 13C NMR (DMSO-d6) δ 51.4, 59.9 , 94.5 , 116.9, 118.3, 124.1, 124.6, 129.2, 131.5, 132.1, 145.9, 147.1, 149.6 , 165.1 , 178.8 ; MS : m/z = 396 ; Anal. Calc. (Found) for C19H20N6O2S (396.47): C, 57.56 (57.65); H, 5.08 (5.02); N, 21.20 (21.14).To a warm mixture of thiosemicarbazide  in glacial acetic acid (30 mL), 1-(morpholino (pyridine) methyl) indoline-2,3-dione Method A: Compound 14  was stirred under reflux in dry chloroform (35 mL) or glacial acetic acid (30 mL), and triethylamine (1 mL) was added, as a catalyst for a long time (~15–17 h) under TLC control. The mixture was evaporated under reduced pressure. The precipitate produced was washed many times with methanol and crystallized form dioxane to produce (15) in high yields. Method B: This was a one-pot synthesis method. A mixture of thiosemicarbazide  in glacial acetic acid (40 mL) and compound 2  was formed. The reaction solution was heated and under reflux for a long time (~19–21 h) under control (TLC). The final precipitate was filtered off, washed with ethanol/water and dried, then recrystallized from dioxane to obtain yellowish crystals in good yield (78%), with m. p. > 350 °C (dec.). IR  KBr: 3350–3230 (3NH), 3060 , 2950 , 1688 (C=O), 1633 (C=N), 1585 (C=C), and 1450 (C=S); 1H NMR  δ 2.70–3.11 , 6.64 , 7.15–7.44 , 7.55–7.65 , 7.80–7.95 , 10.10 , 10.40 , 11.55 ; 13C NMR (DMSO-d6) δ 51.8, 60.2 , 93.7 , 97.1 , 117.8, 123.1, 124.5, 128.3, 130.6, 136.8, 145.4, 147.5, 149.5 , 168.2 , 180.4 ; MS : m/z = 396 ; Anal. Calc. (Found) for C19H20N6O2S (396.47): C, 57.56 (57.48); H, 5.08 (5.14); N, 21.20 (21.27).14 , iodine  and anhydrous potassium carbonate  was stirred under reflux in dry 1,4-dioxane (40 mL) or dimethylformamide (40 mL), for ~13–15 h under TLC control. The solution was evaporated under reduced pressure. After drying, the precipitate produced was washed with 5% Na2S2O3 (30 mL) and extracted from mixture of CH2Cl2 / MeOH . The final precipitate was dried over anhydrous sodium sulphate and was purified using petroleum ether/ethyl-acetate mixture as eluent by silica gel column chromatography, yielding the synthesis of conforming analogs (16). Method B: A solution of 14  and triethylamine (3 drops) in ethanol (30 mL) was heated under reflux for 6–8 h with TLC control. After cooling, the solid precipitate was collected and crystallized from dioxane to produce yellow crystals in good yield (76%), with m. p. > 350 °C (melted). IR  KBr: 3420, 3335 (NH2 and NH), 3066 , 2957 , 1682 (C=O), 1631 (C=N), 1583 (C=C); 1H NMR  δ 2.68–3.10 , 6.60 , 6.85 , 7.22–7.51 , 7.61–7.75 , 7.78–7.93 , 10.25 ; 13C NMR (DMSO-d6) δ 52.4, 61.7 , 78.5 , 91.6 , 118.2, 124.3, 124.7, 127.9, 128.5, 130.4, 145.1, 146.7, 149.3, 149.9 , 167.5 ; MS : m/z = 396 ; Anal. Calc. (Found) for C19H20N6O2S (396.47): C, 57.56 (57.50); H, 5.08 (5.16); N, 21.20 (21.30).A mixture of compound 14  and 2, 3-dichloroquinoxaline  in absolute ethanol containing (0.2 mL) of triethylamine as a catalyst was refluxed for 22–25 h under control (TLC). The reaction solution was cooled and the deposited solid was filtered off, dried and recrystallized from dimethylformamide to produce yellowish crystals in good yield (74%), with m. p. > 350 °C (melted). IR  KBr: 3340 (NH), 3075 , 2970 , 1686 (C=O), 1634 (C=N), 1587 (C=C), 1480 (C=S); 1H NMR  δ 2.75–3.18 , 6.55 , 7.05–7.67 , 7.70–7.80 , 7.85–7.98 , 10.60 ; 13C NMR (DMSO-d6) δ 52.7, 61.9 , 92.8 , 116.2, 118.5, 124.1, 124.4, 124.6, 124.8, 129.6, 130.4, 131.5, 134.2, 146.8, 147.1, 149.6, 153.4, , 166.2 , 175.1 ; MS : m/z = 522 ; Anal. Calc. (Found) for C27H22N8O2S (522.59): C, 62.06 (62.12); H, 4.24 (4.18); N, 21.44 (21.52).A mixture of compound No humans or animals were used in this study; nevertheless, all the procedures were carried out under the Medical Research Ethics Committee of Mansoura University, Faculty of Pharmacy, Department of Pharmacognosy, 35516, Egypt.No humans or animals were used in the study. The research was conducted according to ethical standards in vitro.Types of human carcinoma cancer cell line  are derived from the National Cancer Institute, Cairo University, Cairo, Egypt, and 5-Fluorouracil and DMSO were purchased from Sigma-Aldrich .H-tetrazoliumbromide (MTT) into a blue-purple formazan product, mainly by mitochondrial reductase activity inside living cells. The cells used in the cytotoxicity assay were cultured in the suitable cell culture medium (RPMI 1640) supplemented with 10% fetal calf serum. Cells suspended in the medium (2Y’ 104/mL) were plated in 96-well culture plates and incubated at 37 °C in a 5% CO2 incubator. After 12 h, the test sample (2 mL) was added to the cells (2Y’ 104) in 96-well plates and cultured at 37 °C for 3 days. The cultured cells were mixed with 20 mL of MTT solution and incubated for 4 h at 37 °C. The supernatant was carefully removed from each well, and 100 mL of DMSO was added to each well to dissolve the formazan crystals, which were formed by the cellular reduction of MTT. After mixing with a mechanical plate mixer, the absorbance of each well was measured by a microplate reader using a test wavelength of 570 nm. The results were expressed as the IC50, which is the concentration of the drugs inducing a 50% inhibition of cell growth of treated cells, when compared to the growth of control cells. Each experiment was performed at least three times. There was good reproducibility between replicate wells with standard errors below 10%. Each experiment was repeated on three different days and conducted in triplicate. The relative cell anti-proliferative was measured according to the following equation: % cytotoxicity = (1 − As/Ab) × 100, where As = absorbance of each sample and Ab = absorbance of the blank. The probity analysis using the SPSS software program  was used to determine each IC50.The in vitro cytotoxicity of the synthesized compounds against different cancer cell lines was measured with the MTT assay, in agreement with the method found in ,32,33,54N-(mercapto-pyrimidine)-2-(oxoindoline) hydrazine-1-carboxamide, N--2-(oxoindoline) hydrazine-1-carboxamide, 3-((amino-phenyl) amino)- 3-hydroxy-indolinone, 3-((amino-phenyl) imino)-indolinone, 2-(2-((oxoindoline) amino) phenyl) isoindolinone, 2-(oxoindoline) hydrazine-1-carbothioamide, 5′-thioxospirotriazolidin]-one, 5′-amino-spiro  thiadiazol]-one and 3-imino) indolinone in good yields. Isatin with imidazo  quinoxaline, thiazolo  pyrimidine, hydrazine-1-carboxamide, spirothiadiazol]-2-one, 5′-thioxospiro triazolidin]-2-one moiety and substituted heteroatoms as nitrogen, oxygen and sulfur showed potent cytotoxic activity against the different types of human carcinoma cells lines . The simplicity of the synthetic methods and the potent activity of the synthesized compounds render these derivatives interesting targets for future development as cytotoxic agents. So, the imidazo  quinoxaline-imino- indolinone is a promising anticancer molecule with diverse effects, including angiopreventive and anti-proliferative effects, in addition to apoptosis-inducing effects.In this article, isatin derivatives (1-(morpholino (pyridin-4-yl) methyl) indoline-2,3-dione) were converted into polyfunctionalized heterocycles with promising cytotoxic activity. Different derivatives were prepared, including 3-((bromophenyl) imino)-1- (morpholino (pyridine) methyl) indolinone, 2-((oxoindoline) amino) benzoic acid, 3-(thi- azoloimino) indolinone, ethyl-2-((oxoindoline) amino)-benzothiophene-3-carboxylate, 1- (oxoindoline)-benzothienopyrimidinone, ethyl-2-(oxoindoline) hydrazine-1-carb- oxylate,


In this study, four new azo-azomethines, R1 = Ph, R2 = phenol, and R3 = pyrazol-Ph-R’ (R = H or NO2), have been synthesized, structurally characterized using X-ray, IR, NMR and UV–Vis techniques, and their antifungal activity evaluated against certified strains of Candida albicans and Cryptococcus neoformans. The antifungal tests revealed a high to moderate inhibitory activity towards both strains, which is regulated as a function of both the presence and the location of the nitro group in the aromatic ring of the series. These biological assays were further complemented with molecular docking studies against three different molecular targets from each fungus strain. Molecular dynamics simulations and binding free energy calculations were performed on the two best molecular docking results for each fungus strain. Better affinity for active sites for nitro compounds at the “meta” and “para” positions was found, making them promising building blocks for the development of new Schiff bases with high antifungal activity.The azo-azomethine imines, R Azomethine compounds (Schiff bases), with the general formula RHC=NR’, are highly relevant ligands in pharmaceutical and medical chemistry. These structures are recognized for their high anticancer, antifungal, antiparasitic, antitubercular, cytotoxic, and herbicidal characteristics ,2,3,4. TE)-(2-Phenylpyrazol-3-yl)iminomethyl]phenol (structure (c) in 3H2N2-Ph azomethine analogs (R = (HOPh)(Me)2- or CH2CHCH2-(PhOH)-, and R″ = H or Me, structures (d) and (e) in R′ substituted azomethine derivatives with functionalized pyrazole rings have revealed both potent and varied biological activities. Compounds such as 2- and 4-[-methyl]-phenol (structures (a) and (b) in C. albicans strains, showing significant antifungal activity compared to the standard drug fluconazole, especially for derivatives prepared from aromatic amines substituted with nitro groups in the para and meta positions (structure (a) in C. albicans strains when the reference drug cycloheximide failed to do so with a concentration of 1 mg mL−1  (100), 424 [M+ +1] (20.33), 425 [M+ +2] (3.72), 408 [M+ -15] (9.66), 366 [M+ −57] (2.15)). FT-IR ATR (cm−1) ν(O-H) 3440, ν(C-H) 3127 (pyrazole), ν(C-H) 3063 (aromatic), ν(C-H) 3005 (imine), νas(C-H) 2960 (t-butyl), νs (C-H) 2862 (t-butyl), ν(C=N) 1598 (imine), ν(C=C) 1576 (aromatic), ν(N=N) 1503 (azo), δas(C-H) 1454 (t-butyl), δs(C-H) 1358 (t-butyl), ν(C-O) 1281. 1H-NMR  1.39 , 6.75 , 7.09 , 7.45 , 7.54 , 7.56 , 7.59 , 7.69 , 7.91 , 8.06 ; 8.25 , 9.26 , 12.58 . 13C-NMR  29.78 (tBu-C), 34.68 (C-12), 91.12 (C-9), 117.76 (C-45), 119.17 (C-39), 122.44 (C-48), 124.95 (C-26), 127.49 (C-43), 127.62 (C-30), 128.42 (C-40), 129.02 (C-50), 129.21 (C28), 130.54 (C-52), 139.33 (C-25), 145.69 (C-42), 147.35 (C-11), 152.87 (C-47), 162.09 (C-8), 163.12 (C-38), 164.03 (C-36). The atoms were numbered according to 1 200 (4.36), λ2 234 (4.46), λ3 335, λ4 362 (4.44).2-((E)-((3-(tert-butyl)-1-(2-nitrophenyl)-1H-pyrazol-5-yl)imine)methyl)-4-((E)-phenyldiazenyl)phenol (Azo2). Brown. Yield: 75%. Anal Calc for C26H24N6O3: %C 66.65, %H 5.16, %N 17.90, %O 10.24, found: %C 66.74, %H 5.07, %N 18.08, %O 10.13, m.p. 180-182 °C. MS (70 eV) m/z (%) 468 [M+] (82.71), 469 [M+ +1] (27.72), 470 [M+ +2] (4.52), 453 [M+ -15] (3.55), 391 [M+ −77] (0.77), 77 (100), 57 (42.98). FT-IR ATR (cm−1) ν(C-H) 3122 (pyrazole), ν(C-H) 3064 (aromatic), ν(C-H) 3011 (aromatic), νas(C-H) 2964 (t-butyl), νs(C-H) 2861 (t-butyl), ν(C=N) 1604 (imine), ν(C=C) 1586, νas(NO2) 1528, ν(N=N) 1478 (azo), δas(C-H) 1459 (t-butyl), δs(C-H) 1362(t-butyl), νs(NO2) 1352, ν(C-O) 1285. 1H-NMR  1.25 , 6.70 , 7.05 , 7.52 , 7.55 , 7.68 , 7.77 , 7.80 , 7.86 , 7.93 , 8.08 , 8.19 , 9.16 . 13C-NMR  30.26 (tBu-C), 32.40 (C-33), 91.58 (C-27), 118.09 (C-23), 120.59 (C-13), 122.68 (C-38), 125.39 (C-29), 125.68 (C-34), 125.92 (C-19), 128.00 (C-25), 129.48 (C-36), 129.89 (C-44), 134.06 (C-31), 131.53 (C-42), 145.12 (C-14), 145.38 (C-11), 149.27 (C-12), 151.92 (C-15), 153.51 (C-18), 161.18 (C-21), 162.29 (C-17), 163.17 (C-16). The atoms were numbered according to 1 296 (4.52), λ2 233 (4.62), λ3 335 (4.79), λ4 362 (4.64).2-((E)-((3-(tert-butyl)-1-(3-nitrophenyl)-1H-pyrazol-5-yl)imino)methyl)-4-((E)-phenyldiazenyl)phenol (Azo3). Yellow. Yield: 80%. Anal. Calc. for C26H24N6O3: %C 66.65, %H 5.16, %N 17.90, %O 10.24, found: %C 66.71, %H 5.14, %N 17.87, %O 10.25, m.p. 184-186 °C. MS (70 eV) m/z (%) 468 [M+] (100), 469 [M+ +1] (29.24), 470 [M+ +2] (5.19), 453 [M+ −15] (3.21), 391 [M+ −77] (2.13), 57 (32.04). FT-IR ATR (cm−1) ν(C-H) 3123 (pyrazole), ν(C-H) 3061 (aromatic), ν(C-H) 3011 (aromatic), νas(C-H) 2965 (t-butyl), νs(C-H) 2861 (t-butyl), ν(C=N) 1604 (imine), ν(C=C) 1586, νas(NO2) 1529, ν(N=N) 1478 (azo), δas(C-H) 1460 (t-butyl), δs(H-C) 1363 (t-butyl), νs(NO2) 1352, ν(C-O) 1286. 1H-NMR  1.32 , 6.75 , 7.15 , 7.55 , 7.57 , 7.78 , 7.80 , 7.96 , 8.18 , 8.21 , 8.37 , 8.63 , 9.21 . 13C-NMR  30.32 (tBu-C), 32.87 (C-25), 92.77 (C-9), 118.23 (C-42), 118.25 (C-16), 120.96 (C-40), 121.44 (C-19), 125.21 (C-47), 128.24 (C-44), 129.84 (C-54), 129.94 (C-50), 130.89 (C-52), 130.95 (C-23), 131.41 (C-21), 139.76 (C-15), 145.08 (C-46), 147.97 (C-18), 149.66 (C-12), 151.93 (C-49), 160.14 (C-41), 162.20 (C-38), 162.79 (C-8). The atoms were numbered according to 1 196 (4.38), λ2 234 (4.71), λ3 275 (4.49), λ4 335 (4.83), λ5 362 (4.67).2-((E)-((3-(tert-butyl)-1-(4-nitrophenyl)-1H-pyrazol-5-yl)imino)methyl)-4-((E)-phenyldiazenyl)phenol (Azo4). Yellow. Yield 89%. Anal. Calc. for C26H24N6O3: %C 66.65, %H 5.16, %N 17.90, %O 10.24, found: %C 66.62, %H 5.11, %N 17.88, %O 10.17, m.p: 182–184 °C. MS (70 eV) m/z (%) 468 [M+] (100), 469 [M+ +1] (30.26), 470 [M+ +2] (5.27), 453 [(M+ −15] (4.49), 411 [M+ −57] (0.82), 391 [M+ −77] (1.87). FT-IR ATR (cm−1) ν(C-H) 3120 (pyrazole), ν(C-H) 3063 (aromatic), ν(C-H) 3011 (aromatic), νas(C-H) 2959 (t-butyl), νs(C-H) 2863 (t-butyl), ν(C=N) 1605 (imine), ν(C=C) 1592, νas(N-O) 1514 (-NO2), ν(N=N) 1500 (azo), δas(C-H) 1461 (t-butyl), δs(C-H) 1367 (t-butyl), νs(N-O) 1333 (-NO2), ν(C-O) 1279. 1H-NMR  1.35 , 6.77 , 7.15 , 7.85 , 7.53 , 7.56 , 7.99 , 8.03 , 8.36 , 8.37 , 9.23 , 11.87 . 13C-NMR  30.43 (tBu-C), 32.87 (C-36), 93.54 (C-23), 118.21 (C-16), 121.41 (C-10), 122.82 (C-50), 124.10 (C-27), 125.07 (C-29), 125.62 (C-11), 128.35 (C-14), 129.90 (C-52), 131.55 (C-54), 144.44 (C-26), 145.49 (C-31), 145.64 (C-13), 150.35 (C-25), 152.35 (C-49), 161.25 (C-18), 162.56 (C-9), 163.67 (C-22). The atoms were numbered according to 1 196 (5.27), λ2 234 (5.05), λ3 275 (4.90), λ4 335 (5.36), λ5 362 (5.19).2- were purchased from Sigma-Aldrich and Across without further purification. The melting points were determined on a Büchi melting point apparatus. The excitation and emission spectra were obtained in a JASCO 8600 fluorescence spectrophotometer with a FMP-825 microplate reader. Infrared spectra were measured in a Perkin Elmer FT 2000 series spectrophotometer using KBr disks. The NMR spectra were recorded on a Bruker Avance 400 spectrophotometer operating at 400 MHz for Single-crystals suitable for X-ray diffraction of compounds Azo1, Azo2, Azo3, and Azo4 were obtained by slowly evaporating the solutions in toluene at room temperature. Data for the compounds were collected at room temperature (298 K) on a Bruker Apex-II CCD diffractometer using monochromatic graphite MoKα (0.71073Å) radiation. The determination of the cell and the final cell parameters were obtained on all the reflections using the Bruker SAINT software included in the APEX 2 software package. The integration and scaling of the data were carried out using the Bruker SAINT software.The crystalline structures were solved by direct methods by using the Olex2 program  and the Molecular optimizations and harmonic vibration frequencies were calculated in gas phase in the Gaussian09 suite  using th1H and 13C nuclear magnetic shielding tensors (in ppm) relative to the TMS standard were calculated using the gauge-independent atomic orbital (GIAO) method and the implicit continuum model (PCM) and utilizing the same theoretical optimization levels. Vertical electronic transitions were calculated by the time-dependent density functional theory (TD-DFT) at the same level of theory. For the percentage contribution of the boundary orbitals for each vertical transition the GaussView software was used [was used .I), electron affinity (A), hardness (η), smoothness (σ), electronegativity (χ), potential chemistry (µ) and the global electrophilicity index (ω). The mathematical expression for these descriptors together with the corresponding values calculated for the Azo1–Azo4 series are presented in Using Koopman’s theorem ,46,47, tNCPn − BCPn + RCPn − CCPn = 1).The study of the topology of the electron density (ρ) in the isolated molecules extracted from the crystals of the compounds Azo1 to Azo4 was carried out in the AIM2000 program  using waC. albicans and C. neoformans, molecular docking studies were carried out on six different target receptors extracted from these fungal species. For C. albicans we used the sterol 14-alpha demethylase (CaCYP51) protein with PDB code 5FSA [CaTMPK) with PDB code 5UIV [CaNmt) with PDB code 1IYL [CnFTase) with PDB code 3SFX [CnHsp90) with PDB code 7K9S [CnAdSS) with PDB code 5I34 [For the in silico antifungal evaluation of azoimine-pyrazole derivatives (Azo1–Azo4) against strains of ode 5FSA , the thyode 5UIV , and theode 1IYL . For C. ase CaCYP protein ode 7K9S , and theode 5I34 .Ligand and receptor preparations were made using Autodock Tools v1.5.6 software . For recMolecular docking calculations were performed in triplicate and carried out using the Autodock Vina  and Smin+ and Cl− ions were added to neutralize the charges of the system and an excess of ions was added to reach a NaCl concentration of 0.15M. Energy minimization and a series of equilibration stages under NVT and NPT conditions  using a temperature of 310 K was carried out by 250 ps applying position restrictions of 1000 kJ mol nm2. Then, 4 equilibration stages under NPT conditions  using a pressure of 1 bar by 250 ps each one, applying gradually decreased position restrictions  were carried out. A total time of 1.25 ns was implemented in our equilibration stage. Finally, a production stage of 200 ns without restrictions and using a time step of 2 fs was carried out. The temperature was controlled using the V-rescale thermostat [To study the behavior of protein-ligand complexes, we performed molecular dynamics (MD) simulations using the Gromacs 2019 package . For thiermostat  and a tiermostat  and a tibind) using the molecular mechanics/Poisson–Boltzmann surface area (MM/PBSA) method, available in the gmx_MMPBSA tool [bind between a protein and ligand to form a complex can be calculated as seen in the following equations (Equations (1)–(3)) [MM represents the contribution of molecular mechanics energy given by the system in the gas phase, and it can be obtained from the force field implemented in the MD simulations. ΔGsolvation represents the solvation energy given by the sum of the polar contribution (ΔGPB/GB) and nonpolar contribution (ΔGSA). ΔGPB/GB can be obtained using the Poisson–Boltzmann or generalized-Born models, while ΔGSA can be calculated by solvent accessible surface area (SASA). Thus, the sum of ΔEMM and ΔGsolvation represents the enthalpic contribution (ΔH) to ΔGbind [bind calculations, frames from the 100 ns to the end were taken each 100 ps, for a total of 1000 frames per trajectory.To perform the analysis, the root mean square deviation (RMSD) of the backbone of each protein was calculated using the gmx_rms tool. The root mean square fluctuation (RMSF) of the Cα atoms of each protein was also calculated using the gmx_rmsf tool. Additionally, we calculated the main contacts/interactions between the proteins and each ligand. To do this, we measured the distances between the ligand and all protein residues as a function of time using the gmx_mindist tool. A contact/interaction was considered if the distance was at least 2.5 Å. Then each contact was tracked over time and its frequency was calculated. If the frequency was 100%, it meant that the contact was fully conserved over time. For this analysis, the last 100 ns was used. Finally, to obtain a thermodynamic insight of the protein–ligand interactions, we calculated the binding free-energy (ΔGBSA tool  which isBSA tool . The ΔGb(1)–(3)) :ΔGbind =o ΔGbind ,67. TΔS Candida albicans ATCC 10231 and Cryptococcus neoformans ATCC 32264 from the American Type Culture Collection, . The inocula of cells were adjusted to 1–5 × 103 cells with colony forming units (CFU)/mL according to CLSI [The antifungal activity was determined using the microdilution methods in broth M27-A3 for yeasts and M38-A2 for filamentous fungi following the guidelines of CLSI . The ant to CLSI .For minimum inhibitory concentration (MIC) determination, Broth microdilution techniques were performed in 96-well microplates according to the guidelines of the Clinical and Laboratory Standards Institute for yeasts (M27-A3) . For theMIC50 was defined as the lowest concentration of a compound that showed 50% reduction of the fungal growth control.1-N=N-R2-CH=N-R3  were obtained with a high yield (75–89%) by a straightforward and environmentally friendly synthesis method. They were further characterized by means of GC-MS, DRX, UV–Vis, FTIR, and NMR  physicochemical techniques, as well as by both gas phase and periodical quantum-mechanical electronic DFT methods. These four derivatives, labeled as Azo1 (R3 = -((C3HN2)(tert-butyl)(Ph)), Azo2 (R3 = -((C3HN2)(tert-butyl)(Ph-NO2(ortho))), Azo3 (R3 = -((C3HN2)(tert-butyl)(Ph-NO2(meta))), and Azo4 (R3 = -((C3HN2)(tert-butyl)(Ph-NO2(para))), were biologically evaluated as antifungal agents against certified strains of Candida albicans (ATCC 10231) and Cryptococcus neoformans (ATCC 32264) and demonstrated a moderate-to-high fungicidal activity that was observed to be dependent on the molecular electrophilic character related to the presence and position of the nitro group. In both bioassays, the nitro compound Azo2 (ortho-NO2 derivative), showed an antifungal activity of less than 50% inhibition, while the compounds Azo3 (meta-NO2 derivative) and Azo4 (para-NO2 derivative), in the case of C. albicans, and Azo1 (derivative without NO2) and Azo4, in the case of C. neoformans, showed inhibitions in ranges of 60–90% and 50–100%, respectively. Antifungal in vitro results were computationally complemented by means of molecular docking and molecular dynamics simulations. In silico studies revealed a high affinity of these azo ligands with two proteins extracted from C. albicans (CaTMPK and CaNmt) and two proteins extracted from C. neoformans (CnFTase and CnAdSS). For both CaTMPK and CaNmt proteins, the best affinity for the azo-azomethine compounds follows the trend of the position of the nitro group as para > meta > ortho > not-substituted, which is close to the results observed in the in vitro assays using the C. albicans strain. The higher affinity for compounds Azo4 and Azo3 was associated to the fact that the nitro group was more exposed to interact through hydrogen bonds with protein residues located inside the active site. In consensus, modifications of the nitro group on the ortho and meta positions in azo-pyrazole-azomethines derivatives appear to be a promising strategy in the search for Schiff base compounds with potential antifungal activity.The first four members of a new family of azo-azomethine imines (Schiff bases) with the general formula R


The Cu-catalyzed alkyne-azide 1,3-dipolar cycloaddition variant provides a highly efficient entry to conjugated triazolyl-substituted (oligo)phenothiazine organosilicon derivatives with luminescence and reversible redox characteristics. Furthermore, by in-situ co-condensation synthesis several representative mesoporous MCM-41 type silica hybrid materials with embedded (oligo)phenothiazines are prepared and characterized with respect to their structural and electronic properties. The hybrid materials also can be oxidized to covalently bound embedded radical cations, which are identified by their UV/Vis absorption signature and EPR signals. The mechanistic details of 1,3-dipolar cycloaddition were elucidated by Huisgen and his group almost six decades ago . HoweverBesides application in drug discovery  and lead1 +m/z: 793.3; found: 793.3 (M+).According to GP1 the triazole 3d  was isolated as a brown oil. 1H-NMR : δ 0.62 , 1.20 , 2.03 , 3.42 , 3.80 , 4.37 , 6.90 , 7.45 , 7.60 , 7.74 , 7.87 . 13C-NMR : δ 7.1 (CH2), 17.8 (CH3), 23.8 (CH2), 35.0 (CH3), 52.2 (CH2), 58.1 (CH2), 114.0 (CH), 118.8 (CH), 123.0 (Cquat), 123.7 (CH), 124.4 (CH), 125.3 (Cquat), 128.2 (CH), 131.7 (CH), 144.8 (Cquat), 146.1 (Cquat). UV/Vis (CH2Cl2) λmax (ε): 276 (37800), 334 nm (7200). IR (film): −1. MS (MALDI) calcd. for [C35H53N7O6SSi2]+ m/z: 756.335, found: 756.297 (M+).According to GP1 the bistriazole 3e  was isolated as a brown oil. 1H-NMR : δ 0.62 , 0.88 , 1.20 , 1.33 , 1.46 , 1.83 , 2.03 , 3.80 , 3.89 , 4.37 , 6.93 , 7.57 , 7.63 , 7.73 . 13C-NMR : δ 7.0 (CH2), 13.4 (CH3), 17.8 (CH3), 22.3 (CH2), 23.9 (CH2), 26.2 (CH2), 26.4 (CH2), 31.1 (CH2), 41.7 (CH2), 52.2 (CH2), 58.1 (CH2), 115.2 (CH), 123.8 (CH), 124.2 (Cquat), 124.3 (CH), 125.1 (CH), 144.2 (Cquat), 148.1 (Cquat). UV/Vis (CH2Cl2) λmax (ε): 276 (54500), 335 nm (10100). IR (film): −1. MS (MALDI) calcd. for [C40H63N7O6SSi2]+ m/z: 825.4; found: 825.4 (M+).According to GP1 the bistriazole 3f  was isolated as a brown oil. 1H-NMR : δ 0.64 , 0.88 , 1.33 , 1.46 , 1.82 , 2.02 , 3.55 , 3.88 , 4.36 , 6.93 , 7.32 , 7.35 , 7.56 , 7.62 , 7.72 . 13C-NMR : δ 5.8 (CH2), 13.4 (CH3), 22.3 (CH2), 23.7 (CH2), 26.2 (CH2), 26.4 (CH2), 31.1 (CH3), 47.7 (CH2), 52.1 (CH2), 115.1 (CH), 115.2 (CH), 118.7 (CH), 122.6 (Cquat), 122.8 (Cquat), 123.2 (Cquat), 123.4 (Cquat), 123.9 (CH), 124.2 (CH), 124.3 (CH), 124.8 (CH), 131.6 (Cquat), 142.8 (Cquat), 144.1 (Cquat). UV/Vis (CH2Cl2) λmax (ε): 278 (50700), 336 nm (16100). IR (film): −1. MS (MALDI) calcd. For [C52H70N8O6S2Si2]+ m/z: 1022.44; found: 1022.5 (M+).According to GP1 the bistriazole H-phenothiazine , 1,4-diiodobenzene , bis(dibenzylideneacetone)palladium(0) , tri(tert-butyl)phosphonium tetrafluoroborate , sodium tert-butanolate , and dry 1,4-dioxane (80.0 mL). The dark solution was deaerated by a constant stream of nitrogen via syringe for 10 min. Then, the reaction mixture was heated to reflux for 16 h. After cooling to room temp the solvents were removed in vacuo and the residue was absorbed on celite® and purified by column chromatography on silica gel (n-hexane/ethyl acetate (9:1 → 1:1) to give 1,4-di(10H-phenothiazin-10-yl)benzene : 3061 (w), 3032 (vw), 1884 (vw), 1591 (m), 1556 (m), 1501 (s), 1483 (w), 1460 (vs), 1443 (s), 1406 (vw), 1369 (vw), 1304 (s), 1279 (w), 1254 (s), 1236 (s), 1184 (w), 1165 (vw), 1155 (vw), 1126 (m), 1084 (m), 1042 (s), 1017 (m), 961 (w), 930 (s), 840 (m), 739 (vs), 723 (s), 712 (s), 700 (vw), 664 (m), 627 (vs). Anal. calcd. for C30H20N2S2 [472.6]: C 76.24, H 4.27, N 5.93, S 13.57; Found: C 76.41, H 4.19, N 5.76, S 13.50.In an oven-dried Schlenk tube with a magnetic stir bar were placed 10)benzene ,46 (4.44H-phenothiazin-10-yl)benzene  and glacial acetic acid (823 mL). A solution of elementary bromine  in acetic acid (114 mL) was added dropwise over an hour to the vigorously stirred solution at room temp. The dark brown solution was then stirred at room temp for 16 h. Then a saturated aqueous solution of sodium sulfite  was added and the solution was stirred at room temp for 3 h. The precipitate was collected by suction and was washed three times with methanol. Then the product was dried under vacuo to give compound 4a  as a light yellow powder, Mp 356–358 °C. fR (n-hexane/dichloromethane 9:1): 0.15. 1H-NMR : δ 6.33 , 7.21 , 7.39 , 7.62 . 13C-NMR : δ 114.8 (Cquat), 119.0 (CH), 122.6 (Cquat), 128.8 (CH), 130.3 (CH), 131.6 (CH), 139.6 (Cquat), 142.2 (Cquat). MS (EI) m/z: 792 , 790 , 788 , 786 , 784 , 711 , 709 , 707 , 705 , 630 , 628 , 626 , 358 , 356 , 354 , 272 , 196 . IR −1]: 3917 (vw), 2959 (vw), 2857 (vw), 2332 (w), 2305 (vw), 1582 (m), 1504 (s), 1481 (m), 1452 (vs), 1406 (w), 1379 (m), 1346 (vw), 1308 (s), 1279 (w), 1265 (w), 1254 (m), 1242 (m), 1200 (vw), 1186 (vw), 1152 (vw), 1123 (vw), 1096 (m), 1078 (w), 1067 (vw), 1018 (w), 951 (vw), 920 (w), 866 (m), 856 (m), 839 (m), 820 (vw), 797 (vs), 754 (m), 735 (w), 716 (vw), 685 (w), 669 (w). Anal. calcd. for C30H16N2S2Br4 [788.2]: C 45.72, H 2.05, N 3.55, S 8.13; Found: C 45.83, H 1.99, N 3.46, S 7.95.In an oven-dried 250 mL two-necked round bottom flask with a magnetic stir bar were placed 1,4-di, powdered potassium hydroxide , and dry N,N-dimethylformamide (30.0 mL) under nitrogen. The red solution was stirred at 60 °C (oil bath) for 2 d. Then a solution of α,α′-dibromo-para-xylene  in N,N-dimethylformamide (20.0 mL) was added dropwise to the solution. The reaction mixture was stirred at 80 °C for 16 h. After cooling to room temp the solvent was removed in vacuo. The crude product was triturated with water (2 × 200 mL) and ethanol (100 mL). After drying under vacuo compound 4b +, 814 , 360 , 358 , 356 , 278 , 276 , 196 , 104 . IR −1]: 2384 (vw), 2349 (vw), 2253 (vw), 1586 (w), 1485 (w), 1456 (vs), 1435 (w), 1387 (w), 1348 (m), 1296 (vw), 1258 (s), 1223 (m), 1157 (vw), 1121 (w), 1107 (m), 1082 (vw), 1045 (vw), 1007 (m), 982 (vw), 966 (vw), 930 (w), 825 (s), 795 (vs), 741 (s), 652 (m). Anal. calcd. for C32H20Br4N2S2 [816.3]: C 47.09, H 2.47, N 3.43, S 7.86; Found: C 46.96, H 2.60, N 3.47, S 7.62.In an oven-dried Schlenk tube with a magnetic stir bar were placed 3,7-dibromo-10pound 4b  , CuI (20.0 mol%), PdCl2(PPh3)2 (20.0 mol%), dry tetrahydrofuran (26.4 mL/mmol) and piperidine (13.5 mL/mmol). The reaction mixture was deaerated by a constant stream of nitrogen by a syringe for 10 min. Then, (trimethylsilyl)acetylene (5) (7.40 equivs) was added to the reaction mixture, which was subsequently heated at 67 °C (oil bath) under nitrogen for 21 h. After cooling to room temp, the solvents were removed under reduced pressure and the residue 6 was triturated with diethylether. The precipitated salts were separated by filtration and the solvents of the filtrate were evaporated in vacuo. The residue was purified by chromatography on silica gel. The product was dissolved in tetrahydrofuran (10.5 mL/mmol) and a 1.0 m solution of TBAF (6.00 equivs) was added. After stirring at room temp for 1 h, the solvents were removed in vacuo and the product 7 was purified by suspension in water, filtration and drying of the solid under vacuo.In an oven-dried screw-capped Schlenk vessel with a magnetic stir bar under nitrogen atmosphere were placed the tetrahalogenated dyad 4a , PdCl2(PPh3)2 , CuI , THF (140 mL), piperidine (72 mL), and (trimethylsilyl)-acetylene  were reacted to give compound 7a  as an orange powder after chromatography on silica gel (hexane/dichloromethane 9:1), Mp > 400 °C (dec.). fR (hexane/dichloromethane 4:1): 0.45. 1H-NMR : δ 3.50 , 6.33 , 7.04 , 7.16 , 7.71 . 13C-NMR : δ 81.0 (CH), 82.5 (Cquat), 116.4 (CH), 119.4 (Cquat), 125.3 (CH), 129.5 (CH), 131.3 (Cquat), 133.2 (CH), 139.6 (Cquat), 143.0 (Cquat). MS (MALDI-TOF) calcd. for [C38H20N2S2]+ m/z: 568.110; Found: 568.120. IR −1]: 3287 (m), 2104 (m), 1599 (m), 1582 (m), 1503 (w), 1495 (w), 1468 (vw), 1387 (s), 1312 (vw), 1256 (vw), 1190 (w), 1152 (w), 1096 (w), 1077 (w), 1065 (vw), 1045 (vw), 1020 (m), 937 (m), 880 (s), 866 (w), 806 (vw), 779 (vw), 745 (w), 729 (w), 710 (w), 667 (s), 648 (vw). Anal. calcd. for C38H20N2S2 (568.7): C 80.25, H 3.54, N 4.93, S 11.27; Found: C 80.01, H 3.60, N 4.71, S 10.99.According to GP2 compound 4b , PdCl2(PPh3)2 , CuI , THF (92 mL), piperidine (47 mL), and (trimethylsilyl)acetylene  were reacted to give compound 7b  as an orange powder after chromatography on silica gel (hexane/dichloromethane 9:1), Mp >400 °C (dec.). fR (hexane/dichloromethane 4:1): 0.29. 1H-NMR : δ 3.47 , 5.11 , 6.66 , 7.08 , 7.16 , 7.27 . 13C-NMR : δ 79.0 (CH), 83.5 (CH), 116.6 (CH), 118.1 (Cquat), 124.0 (Cquat), 128.1 (CH), 130.9 (CH), 132.2 (CH), 136.5 (Cquat), 145.5 (Cquat). MS (MALDI-TOF) calcd. for [C40H24N2S2]+ m/z: 596.770; Found: 597.160. IR −1]: 3298 (w), 3055 (vw), 2108 (vw), 1599 (w), 1464 (vs), 1456 (s), 1400 (m), 1354 (m), 1339 (w), 1300 (w), 1256 (m), 1223 (s), 1163 (w), 1123 (w), 1007 (w), 1065 (w), 1011 (w), 893 (m), 881 (m), 816 (s), 745 (w), 704 (w), 633 (s), 604 (m). Anal. calcd. for C40H24N2S2 [596.8] C 80.51, H 4.05, N 4.69, S 10.74; Found: C 79.65, H 4.25, N 4.37, S 10.53.According to GP2 compound 7 (1.00 equiv), CuI  and dry DMF (22.4 mL/mmol). After addition of (3-azidopropyl)tri-ethoxysilane (2a) , the turbid reaction mixture was stirred at room temp for 16 h, while the reaction mixture turned red. The solvent was removed under reduced pressure and the residue was purified by chromatography on silica gel.In an oven-dried screw-capped Schlenk vessel with a magnetic stir bar under nitrogen atmosphere were placed the tetrakis dyad 7a , CuI , CuI , (3-azidopropyl)triethoxysilane (2a) , and DMF (27 mL) were reacted to give compound 8a  as a yellow crystalline solid after chromatography on silica gel (hexane/acetone 6:4), Mp > 350 °C (dec.). fR (n-hexane/acetone 6:4): 0.15. 1H-NMR : δ 0.62 , 1.19 , 2.02 , 3.80 , 4.36 , 6.57 , 7.54 , 7.63 , 7.67 , 8.09 . 13C-NMR : δ 14.1 (CH2), 18.7 (CH3), 20.8 (CH2), 53.1 (CH2), 59.1 (CH2), 118.7 (CH), 120.2 (CH), 123.4 (Cquat), 124.6 (CH), 125.0 (CH), 128.1 (Cquat), 132.1 (CH), 141.9 (Cquat), 143.8 (Cquat), 146.8 (Cquat). MS (MALDI-TOF) calcd. for [C74H104N14O12S2Si4]+ m/z: 1558.190; Found: 1556.650. IR −1]: 2953 (m), 2922 (m), 2853 (m), 1653 (vw), 1601 (vw), 1458 (w), 1437 (w), 1364 (w), 1310 (w), 1271 (m), 1248 (vs), 1233 (s), 1167 (m), 1119 (w), 1086 (m), 1034 (s), 945 (w), 930 (w), 912 (vw), 874 (w), 858 (vw), 800 (m), 768 (m), 743 (vw), 723 (vw), 700 (vw), 644 (s), 637 (s), 621 (m).According to GP 3 compound 7b , CuI , CuI , (3-azidopropyl)triethoxysilane (2a) , and DMF (15 mL) were reacted to give compound 8b  as yellow crystals after chromatography on silica gel (hexane/acetone 6:4), Mp >320 °C (dec.). fR (hexane/acetone 6:4): 0.17. 1H-NMR : δ 0.65 , 1.18 , 2.73 , 3.82 , 4.44 , 4.46 , 6.61 , 7.56 , 7.67 , 7.77 , 8.29 . 13C-NMR : δ 8.2 (CH2), 18.7 (CH3), 25.1 (CH2),52.3 (CH2), 53.1 (CH2), 59.0 (CH2), 118.6 (CH), 120.9 (CH), 122.6 (Cquat), 124.6 (CH), 125.3 (CH), 127.9 (Cquat), 132.8 (CH), 141.7 (Cquat), 143.9 (Cquat), 146.7 (Cquat). MS (MALDI-TOF) calcd. for [C76H108N14O12S2Si4]+ m/z: 1586.250; Found: 1586.000. IR −1]: 2818 (vw), 2365 (w), 1597 (vw), 1456 (vs), 1414 (w), 1331 (m), 1258 (m), 1242 (m), 1198 (w), 1155 (w), 1140 (m), 1107 (w), 1038 (w), 1009 (vw), 961 (vw), 910 (vw), 872 (w), 862 (w), 806 (s), 745 (s), 725 (m), 706 (m), 650 (w).According to GP 3 compound 3b in methanol and tetraethylorthosilicate (TEOS) was added dropwise. The composition of the mixture of TEOS:3b:CTAB:EtNH2:methanol:H2O in molar ratio was 1.0:0.085:0.14:2.4:2.0:100. The reaction mixture was stirred at room temp for 24 h to obtain a homogenous mixture of the organosilane with TEOS. Then the mixture was stirred at 100 °C for 24 h. The product was collected by filtration, washed thoroughly with deionized water to pH-Neutrality. After drying at 50 °C under vacuo a powdery material was obtained. For extraction of the surfactant (CTAB) 1.0 g of the powder was suspended twice in ethanol (80 mL) and aqueous HCl  and heated to reflux for 8 h. After cooling to room temp, the solid was filtered, washed with ethanol and dichloromethane and finally dried at 50 °C under vacuo. The material 3b@MCM-41 was obtained as a yellow powder. Anal. calcd for 3b@MCM-41: C 20.63, H 3.34, N, 3.94; which corresponds to a loading of 0.70 mmol/g.An aqueous solution of cetyltrimethylammonium bromide (CTAB) was mixed with ethylamine under stirring. Then, a solution of precursor 3c in methanol/THF and tetraethylorthosilicate (TEOS) was added dropwise. The composition of the mixture of TEOS:3c:CTAB:EtNH2:methanol:THF:H2O in molar ratio was 1.0:0.03:0.14:2.4:2.0:2.0:100. The reaction mixture was stirred at room temp for 24 h to obtain a homogenous mixture of the organosilane with TEOS. Then the mixture was stirred at 100 °C for 24 h. The product was collected by filtration, washed thoroughly with deionized water to pH-Neutrality. After drying at 50 °C under vacuo a powdery material was obtained. For extraction of the surfactant (CTAB), 1.0 g of the powder was suspended twice in ethanol (80 mL) and aqueous HCl  and heated to reflux temp for 8 h. After cooling to room temp, the solid was filtered, washed with ethanol and dichloromethane and finally dried at 50 °C under vacuo. The material 3c-1@MCM-41 was obtained as a yellow powder. Anal. calcd for 3c-1@MCM-41: C 19.92, H 2.92, N, 1.93; which corresponds to a loading of 0.28 mmol/g.An aqueous solution of cetyltrimethylammonium bromide (CTAB) was mixed with ethylamine under stirring. Then a solution of precursor 3c in methanol/THF and tetraethylorthosilicate (TEOS) was added dropwise. The composition of mixture TEOS:3c:CTAB:EtNH2:methanol:THF:H2O in molar ratio was 1.0:0.055:0.14:2.4:2.0:2.0:100. The reaction mixture was stirred at room temp for 24 h to obtain a homogenous mixture of the organosilane with TEOS. Then the mixture was stirred at 100 °C for 24 h. The product was collected by filtration, washed thoroughly with deionized water to pH-neutrality. After drying at 50 °C under vacuo a powderous material was obtained. For extraction of the surfactant (CTAB) 1.0 g of the powder was twice suspended in ethanol (80 mL) and aqueous 36% HCl (1.0 mL) and heated to reflux temp for 8 h. After cooling to room temp the solid was filtered, washed with ethanol and dichloromethane and finally dried at 50 °C under vacuo. The material 3c-2@MCM-41 was obtained as a yellow powder. Anal. calcd for 3c-2@MCM-41: C 27.79, H 2.97, N, 3.04; which corresponds to a loading of 0.43 mmol/g.An aqueous solution of cetyltrimethylammonium bromide (CTAB) was mixed with ethylamine under stirring. Then, a solution of precursor 3d in methanol/THF and tetraethylorthosilicate (TEOS) was added dropwise. The composition of the mixture of TEOS:3d:CTAB:EtNH2:methanol:THF:H2O in molar ratio was 1.0:0.04:0.14:2.4:2.0:2.0:100. The reaction mixture was stirred at room temp for 24 h to obtain a homogenous mixture of the organosilane with TEOS. Then the mixture was stirred at 100 °C for 24 h. The pro-duct was collected by filtration, washed thoroughly with deionized water to pH-Neutrality. After drying at 50 °C under vacuo a powdery material was obtained. For extraction of the surfactant (CTAB), 1.0 g of the powder was suspended twice in ethanol (80 mL) and aqueous HCl  and heated to reflux temp for 8 h. After cooling to room temp, the solid was filtered, washed with ethanol and dichloromethane and finally dried at 50 °C under vacuo. The material 3d@MCM-41 was obtained as a yellow powder. Anal. calcd for 3d@MCM-41: C 12.67, H 2.82, N, 2.78; which corresponds to a loading of 0.28 mmol/g.An aqueous solution of cetyltrimethylammonium bromide (CTAB) was mixed with ethylamine under stirring. Then, a solution of precursor In summary, we have successfully employed the CuAAC, a copper catalyzed variation of (3+2)-cycloadditions, for synthesizing triazole conjugated phenothiazines bearing propyl trialkoxylsilyl moieties as precursors for the synthesis of mesoporous silica hybrid materials by the in-situ co-condensation strategy. The precursor materials are all intensively luminescent by triazole substitution and reversible redox systems according to cyclic voltammetry. These favorable electronic properties can be directly included in the mesoporous hybrid materials as well. Exemplarily, four different materials were synthesized and extensively characterized for XRD, nitrogen absorption-desorption isotherms, TEM, solid state CP-MAS NMR and IR spectroscopy to gain an insight into mesoporous structuring and molecular ligation in the silica framework. The mesoporous periodicity of the obtained materials largely depends on the molecular dimensions of the employed precursors in the templated in-situ condensation synthesis. The electronic properties of the hybrid materials are affected by the molecular properties of the constituents, however, more dense packing and embedding leads to further cooperativity, e.g., intense fluorescence. Also, the stability of chemically oxidized materials displaying the formation of radical cations sets the stage for devising chemically and electrochemically reversible electrophores embedded in stabilizing silica frameworks. Further studies will be directed to develop periodically mesoporous materials with switchable charge transport properties.


Elsholtzia bodinieri led to the isolation of seven new triterpenoid saponins, Bodiniosides S–Y . Their strictures were elucidated on the basis of spectroscopic techniques, including HSQC, HSBC, and HSQC–TOCSY experiments, together with acid hydrolysis and GC analysis. The anti-influenza activities of compounds 1–7 were evaluated against A/WSN/33/2009 (H1N1) virus in MDCK cells. The results showed that compounds 2 and 5 exhibited moderate anti-influenza activities against A/WSN/33/2009 (H1N1), with inhibition rates of 35.33% and 24.08%, respectively.Investigation of the n-BuOH extract of the aerial parts of  Elsholtzia bodinieri Vaniot (Chinese name “Dongzisu”), belonging to the taxonomically diverse group of the family Labiatae, is a medicinal plant that grows in Yunnan and Guizhou Provinces in China. It is commonly known as “yashuacao” and is used as a traditional Chinese medicine for the treatment of cough, headache, pharyngitis, fever and hepatitis , 11 methylenes containing an oxygenated one , 5 methines including an oxygenated one and 1 unsaturated one , as well as 8 quaternary carbons  and a unsaturated one ) were observed , 6.09 , 5.16 , 4.97  and 4.86 , which correlated with anomeric carbon signals at δC 101.0, 94.5, 107.2, 107.1 and 105.3 in the HSQC spectrum, respectively, suggesting the presence of five sugar moieties. Acid hydrolysis of 1 with 1 M HCl produced L-rhamnose (Rha), D-glucose (Glc), and D-xylose (Xyl) as sugar residues by GC chromatography with the corresponding trimethylsilylated L-cysteine derivatives. Since NMR signals of five sugar units have undesirable overlapped effects, the HMQC-TOCSY experiment was successfully used to distinguish and assign the 1H and 13C NMR signals of each sugar moiety. The correlations from the anomeric proton signal at δH 4.97 to three carbon signals at δC 75.3, 78.4, and 67.1, as well as from three proton signals at δH 4.22, 4.06, and 3.75 to the anomeric carbon, suggested the presence of D-xylopyranose. In a similar way, the 1H and 13C NMR signals for D-glucopyranosyl and L-rhamnopyranosyl were assigned. In addition, the JH1, H2 coupling constants of four anomeric proton signals at δH 6.09 , 5.16 , 4.97  and 4.86  suggested the β anomeric configuration for the xylopyranosyl and glucopyranosyl units. The inspection of the anomeric proton  deduced the α anomeric configuration for the L-rhamnopyranosyl unit -β-D-glucopyranoside. Thus, the structure of compound 1 was elucidated to be 3-O-β-D-xylopyranosyl-23-acetyloxy-olean-12-en-28-oic acid 28-O-β-D-glucopyranosyl(1→6)-[β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl(1→2)]-β-D-glucopyranoside, a new oleanane triterpenoid saponin, named Bodinioside S.The sequence of the glycoside chains connected to C-3 and C-28 was established by analysis of the HMBC correlations . The abs2 was obtained as white amorphous powder. Its positive HR-ESI-MS spectrum indicated the molecular formula to be C60H96O27 by the observation quasi-molecular ion peak [M − H]− at m/z 1247.6070 and with the help of the NMR spectroscopic date, indicating 13 degrees of unsaturation. Detailed comparison of the 1H and 13C NMR spectral data , 6.14 , 5.62 (br. s), 5.12  and 4.86  correlated with anomeric carbon signals at δC 101.2, 94.4, 109.7, 107.0, and 107.2 in the HSQC spectrum, respectively, suggesting the presence of five sugar residues: one rhamnopyranosyl (Rha), one arabinopyranosyl (Ara), one xylopyranosyl (Xyl) and two glucopyranosyl (Glc and Glc’) units. The Xyl unit was still linked to C-3 (δC 82.8) of the aglycone based on the HMBC correlation between HXyl-1 (δH 4.86) of Xyl and C-3 (δc 82.8). A series of HMBC correlations from HGlc-1 (δH 6.14) to C-28, from HRha-1 (δH 6.47) to CGlc-2 (δc 76.3), from HAra-1 (δH 5.62) to CGlc-6 (δc 68.3), and from HGlc′-1 (δH 5.12) to CRha-4 (δc 85.7) enabled the sugar chain of C-28 to be assigned as α-L -arabinopyranosyl(1→6)-[β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl(1→2)]-β-D-glucopyranoside. From the foregoing evidence, the structure of 1 was unequivocally determined to be 3-O-β-D-xylopyranosyl-23-acetyloxy-olean-12-en-28-oic acid 28-O-α-L-arabinopyranosyl(1→6)-[β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside, and named Bodinioside T.Compound ral data  of 2 wit3 was isolated as white amorphous powder. The molecular formula was established as C55H88O23 by positive HR-ESI-MS  and NMR spectral data, indicating 12 degrees of unsaturation. In the 1H NMR spectrum of 3, four anomeric H-atom at δH 6.45 (br. s), 6.15 , 5.13  and 4.89  correlated with anomeric carbon signals at δC 101.2, 94.5, 105.5 and 107.1 in the HSQC spectrum, respectively, suggesting the presence of four sugar residues: one rhamnopyranosyl (Rha), one xylopyranosyl (Xyl), and two glucopyranosyl (Glc and Glc’) units. Detailed comparison of the 1H and 13C NMR spectra of 3  to C-28, from HRha-1 (δH 6.45) to CGlc-2 (δc 76.3), from HXyl-1 (δH 5.13) to CGlc-6 (δc 68.3), and from HGlc’-1 (δH 4.89) to CRha-4 (δc 85.7), adequately illustrated the structure of 3 as 3β-hydroxy-23-acetyloxy-olean-12-en-28-oic acid 28-O-β-D-xylopyranosyl(1→6)-[β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside, named Bodinioside U.Compound tra of 3  with tho4 was obtained as white amorphous powder. It exhibited a quasi-molecular ion peak at m/z 1205.5950 [M − H]− in the negative HR-ESI-MS spectrum, suggesting the molecular formula C58H94O26, indicating 12 degrees of unsaturation. Besides one hydroxyl taking the place of an acetoxy group substituent on C-23, most NMR signals (1 and 2) of 4 were nearly identical to those of Bodinioside H -β-D- glucopyranoside, and named Bodinioside V.Compound 5) was established as C52H84O21 on the basis of the negative HR-ESI-MS from the quasi molecular ion peak at m/z 1089.5454 [M + COOH]−, indicating 11 degrees of unsaturation. In the 1H NMR spectrum of 5, four anomeric H-atom at δH 6.51 (br. s), 6.11 , 5.01  and 4.85  correlated with anomeric carbon signals at δC 101.3, 94.6, 106.7 and 105.4 in the HSQC spectrum, respectively. Acid hydrolysis of 5 yielded two D-xylose (Xyl), L-rhamnose (Rha) and D-glucose (Glc) as sugar residues by GC chromatography. Interpretation of its NMR data -[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside, and named Bodinioside W.The molecular formula of Bodinioside W  and 6.23 , which correlated with anomeric carbon signals at δC 101.6 and 95.1 in the HSQC spectrum, respectively, suggesting the presence of two sugar moieties. Acid hydrolysis of 6 yielded L-rhamnose (Rha) and D-glucose (Glc) as sugar residues by GC chromatography. The NMR data of 6 were highly analogous to the sericoside + in the positive HR-ESI-MS spectrum, suggesting a molecular formula C42H68O15, indicating nine degrees of unsaturation. The 1H NMR spectrum , 1.39 , 1.03 , 1.19 , 1.62 , and 1.06  in correlation with carbons at δC 14.2 (C-24), 17.5 (C-25), 17.4 (C-26), 24.1 (C-27), 26.9 (C-29), and 16.6 (C-30) in the HSQC spectrum, respectively. The signal at δH 5.55 , corresponding to the carbon at δC 128.2 (C-12), coupled with δC 139.2 (C-13) in the 13C NMR spectrum. On the basis of the above spectroscopic data, compound 7 was suggested to possess an ursane-12-ene skeleton −, HR-ESI-MS (neg.) m/z: 1277.6148 [M − H]−, . 1H and 13C NMR: White amorphous powder. c = 0.15, MeOH), ESI-MS (neg.) m/z: 1247 [M − H]−, HR-ESI-MS (neg.) m/z: 1247.6070 [M − H]−, . 1H and 13C NMR: White amorphous powder. c = 0.34, MeOH), ESI-MS (pos.) m/z: 1140 [M + Na]+, HR-ESI-MS (pos.) m/z: 1139.5614 [M + Na]+, . 1H and 13C NMR: White amorphous powder. c = 0.12, MeOH), ESI-MS (pos.) m/z: 1230 [M + Na]+, HR-ESI-MS (neg.) m/z: 1205.5950 [M − H]−, . 1H and 13C NMR: White amorphous powder. c = 0.23, MeOH), ESI-MS (neg.) m/z: 1044 [M − H]−, HR-ESI-MS (pos.) m/z: 1089.5454 [M + COOH]−, . 1H and 13C NMR: White amorphous powder. c = 0.16, MeOH), ESI-MS (pos.) m/z: 835 [M + Na]+, HR-ESI-MS (pos.) m/z: 835.4459 [M + Na]+, . 1H and 13C NMR: White amorphous powder. c = 0.26, MeOH), ESI-MS (pos.) m/z: 835 [M + Na]+, HR-ESI-MS (pos.) m/z: 835.4456 [M + Na]+, . 1H and 13C NMR: White amorphous powder. 1–7 (1.0 mg for each compound) were hydrolyzed with 1 M HCl (0.4 mL) and heated at 90–100 °C for 5 h. The mixture was neutralized by the addition of Amberlite IRA400 (OH− form) and then filtered. The filtrate was dried in vacuo, dissolved in 0.2 mL of pyridine containing L-cysteine methyl ester (10 mg/mL) and reacted at 60 °C for 1 h. To this mixture, a solution (0.2 mL) of trimethylsilyl imidazole in pyridine (10 mg/mL) was added, and then heated at 60 °C for 1 h. The final mixture was directly analyzed by GC . The authentic samples D- and L-glucose, D- and L-xylose, L-arabinose, and L-rhamnose were treated in the same manner. Under these conditions, the retention times of authentic samples D- and L-glucose, D- and L-xylose, L-arabinose and L-rhamnose were 18.29, 18.87, 13.35, 14.01, 14.30 and 14.97 min, respectively. During our studies, identical retention times observed between the different hydrolysates and authentic standards.Compounds 1–7 were evaluated, using influenza strain A/WSN/33/2009 (H1N1). For the inhibitory activity assays, compounds 1–7 were dissolved and then diluted with DMSO, using Oseltamivir as a positive control. MDCK cells were seeded into 96-well plates, incubated overnight and infected with influenza virus (MOI ¼ 0.1). The cells were suspended in DMEM supplemented with 1% fetal bovine serum (FBS), containing test compound and 2 mg/mL TPCK-treated trypsin, and a final DMSO concentration of 1% was added in each well. After 40 h of incubation, Cell Titer-Glo reagent was added, and the plates were read, using a plate reader [The anti-influenza virus activities of compounds e reader . The inhe reader .


Goniobranchus aureopurpureus and Goniobranchus sp. 1 afforded 11 new diterpenoids (1–11), all of which possess a tetracyclic spongian-16-one scaffold with extensive oxidation at C-6, C-7, C-11, C-12, C-13, and/or C-20. The structures and relative configuration were investigated by NMR experiments, while X-ray crystallography provided the absolute configuration of 1, including a 2′S configuration for the 2-methylbutanoate substituent located at C-7. Dissection of animal tissue revealed that the mantle and viscera tissues differed in their metabolite composition with diterpenes 1–11 present in the mantle tissue of the two nudibranch species.Extracts of the mantle and viscera of the Indo-Pacific nudibranchs  Dictyoceratida and Dendroceratida, and nudibranchs (shell-less mollusc) predators +). Examination of the 1H and 13C NMR spectroscopic data  and a methyl triplet at δH 0.91 (J = 7.4 Hz) for the methyl groups of a 2-methylbutanoate ester; there was also an acetate methyl singlet at δH 2.03. HMBC correlations from the signals at δH 4.87  and 1.15  to the signal at δC 174.9 confirmed the 2-methylbutanoate group at C-7. The configuration of the 2′-methyl in the ester sidechain could not be established further, owing to the small sample size, but was selected as identical to that in 1 on biogenetic considerations. HMBC correlations from H-20a (δH 4.79) and H-20b (δH 4.73) to the signal at δC 170.6 confirmed the position of the acetoxy group at C-20; there were NOESY correlations between H2-20/Me-17 and H-20b/Me-19. The doublet appearance of H-7 (J = 3.2 Hz) was initially considered consistent with an equatorial OH group at C-6; however, the signal for H-5 was a broadened singlet rather than the doublet with a large J value anticipated if H-6 was axial, (cf. aplyroseol-19 from Chromodoris reticulata + in HRESIMS analysis, which established the molecular formula as C25H40O5 with an additional five carbons and three oxygens compared with spongian-16-one. Due to the small sample quantity (<0.1 mg), a Shigemi tube was employed to increase the sensitivity of NMR signal detection + in the HRESIMS, corresponding to a molecular formula of C27H42O7, which was the same molecular formula observed for 1 and 2. The 1H and 13C NMR spectroscopic data revealed an acetate methyl singlet at δH 2.04 as well as oxymethylene signals at δH 4.04  and 3.91  for H2-20, similar to comparable signals for 1, and suggesting a C-20 hydroxy group. The major difference compared to the data for 1 was the presence of two methyl doublets at δH 0.95  and 0.96  suggesting a 3-methylbutanoate substituent. There were gCOSY correlations from the H-3′ methine  to both Me-4′ and Me-5′ and H2-2′ . HMBC correlations from the signals at δH 4.86 , 2.11 (7-OCOCH2CH(CH3)2, m, H-3′) and the methylene signals at δH 2.22 (7-OCO2CHCH(CH3)2, m, H2-2′) to the carbon at δC 171.9 confirmed the 3-methylbutanoate group was attached at C-7. These 1H chemical shifts and HMBC correlations were comparable to those of 7α-11α-dioxyspongian-16-one-7α-isopentanoate-11α-propionate +, corresponding to a molecular formula of C22H34O5. The 1H and 13C NMR spectroscopic data  to the carbonyl at δC 169.7 confirmed the position of the acetoxy group at C-7. The doublet appearance of H-7 suggested hydroxy substitution at C-6. The relative configuration of 5 was identical to that of 2 from the NOESY correlations between H-6/Me-18, H-7/Me-17, H-5/H-9, and H-9/H-14. Compound 5 was assigned the systematic name (-)-7α-acetoxy-6β-hydroxyspongian-16-one.Diterpene pic data  showed an-16-one . HMBC coGoniobranchus sp. 1 were collected from Mudjimba and Gneerings Reefs, South East Queensland, Australia. The extraction and chemical profile of the metabolites from the mantle and viscera tissue were carried out based on the previously described procedures. A total of fifteen spongian diterpene metabolites were isolated from Goniobranchus sp 1, including the new spongian-16-one analogues 6–11. From the mantle isoagatholactone + from HRESIMS, which corresponded to the same molecular formula as 5. The 1H and 13C NMR spectroscopic data again revealed an acetate group  while oxymethylene signals at δH 4.55  and 4.14  were consistent with those of H2-20 in 6. HMBC correlations from the signals at δH 4.55 and 4.14 as well as from δH 2.02 to the carbon at δC 170.8 and to C-10 (δC 40.4) confirmed the position of the acetate group at C-20. NOE correlations between H-20a/Me-19 and H-20b/Me-17 again placed the C-20 acetate on the same face as Me-17. The upfield chemical shift for H-14 , together with HMBC correlations from signals at δH 1.94 (H-14) and 4.13 (H-15ax) to the quaternary carbon at δC 83.7 (C-13), confirmed a hydroxy group at C-13. The configuration at C-13 was not explored further, owing to the small quantity (0.2 mg) of the sample, and was provisionally assigned by comparison with 1 and 3. NOESY correlations between H-5/H-9 and H-9/H-14 confirmed the remaining stereochemistry. Compound 7 was assigned the systematic name (-)-20-acetoxy-13-hydroxyspongian-16-one.Metabolite 8 was isolated as a colourless oil and displayed a sodiated ion at m/z 343.2245 [M + Na]+ from HRESIMS for C20H32O3 suggesting an extra hydroxy group compared to spongian-16-one. gCOSY correlations from the oxygenated methine proton signal at δH 4.52  to δH 1.63 (H2-11) and 2.66 (H-13) established the hydroxy group at the C-12 position, further confirmed by HMBC correlations. NOESY correlations observed between H-5/H-9, H-9/H-14, and H-12/Me-17 confirmed the overall stereochemistry. Compound 8 was named as (-)-12α-hydroxyspongian-16-one.Metabolite 9 was isolated as a colourless oil and produced an adduct ion at m/z 415.2458 [M+Na]+ from HRESIMS for C23H36O5. The 1H NMR spectrum indicated a quartet (2H) at δH 2.31 and a triplet at δH 1.13, corresponding to propionate methylene and methyl signals. Oxymethylene signals at δH 4.59  and 4.17  corresponded to those of H2-20 in 20-oxyspongian-16-one-propionate + in the HRESIMS, corresponding to a molecular formula of C26H40O7, and 16 mass units larger than that of 12α, 20-dioxyspongian-16-one-dipropionate +, giving the same molecular formula as 10, implying two propionate groups and a hydroxy group. The 1H and 13C NMR spectroscopic data revealed signals for two propionate groups. Similar to 10, two ester carbonyls (δC 172.6 and 175.5) and a lactone carbonyl (δC 178.2) were identified. HMBC correlations from the signals at δH 4.61  and 4.02  as well as from δH 2.50, 2.45 and 1.12 to the carbon at δC 175.5 and C-10 (δC 40.7) confirmed a propionate group at C-20. HMBC correlations from the signal at δH 5.54  to the signal at δC 172.6 located the second propionate group at C-12. Lastly, the occurrence of a signal at δH 1.34  for H-9, together with gCOSY correlations from H-12 and H-9 to H-11 (δH 4.46), established a hydroxy group at C-11. The 9.2 Hz coupling between H-12ax and H-13 again established a boat conformation for ring C + . (–)-13-Acetoxy-20-hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)-butanoate (2): colourless oil (0.07 mg); c 0.007, CHCl3); 1H NMR and 13C NMR , m/z 501.2824 [M + Na]+ . (–)-20-Acetoxy-6β-hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)-butanoate (3): colourless oil (0.06 mg); c 0.006, CHCl3); 1H NMR and 13C NMR , m/z 443.2779 [M + Na]+ . (–)-6β-Hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)-butanoate (4): colourless oil (0.8 mg); c 0.08, CHCl3); 1H NMR and 13C NMR , m/z 501.2831 [M + Na]+ . (–)-13-Acetoxy-20-hydroxy-7α-oxyspongian-16-one-7α-(3-methyl)-butanoate (5): colourless oil (0.22 mg); c 0.022, CHCl3); 1H NMR and 13C NMR , m/z 401.2293 [M + Na]+ . (–)-7α-Acetoxy-6β-hydroxyspongian-16-one (6): colourless oil (0.21 mg); c 0.021, CHCl3); 1H NMR and 13C NMR , m/z 457.2566 [M + Na]+ . (–)-20-Acetoxy-12α-oxyspongian-16-one-12α-propionate (7): colourless oil (0.17 mg); c 0.017, CHCl3); 1H NMR and 13C NMR , m/z 401.2291 [M + Na]+ . (–)-20-Acetoxy-13-hydroxyspongian-16-one (8): colourless oil (0.08 mg); c 0.01, CHCl3); 1H NMR and 13C NMR , m/z 343.2245 [M + Na]+ . (–)-12-Hydroxyspongian-16-one (9): colourless oil (0.97 mg); c 0.097, CHCl3); 1H NMR and 13C NMR , m/z 415.2458 [M + Na]+ . (–)-12-Hydroxy-20-oxyspongian-16-one-20-propionate (10): colourless oil (0.17 mg); c 0.017, CHCl3); 1H NMR and 13C NMR , m/z 487.2668 [M + Na]+ . (–)-12-Hydroxy-11,20-dioxyspongian-16-one-11,20-dipropionate (11): colourless oil (0.11 mg); c 0.011, CHCl3); 1H NMR and 13C NMR , m/z 487.2667 [M + Na]+ .(–)-11-Hydroxy-12,20-dioxyspongian-16-one-12,20-dipropionate : orthorhombic, space group P212121 (no. 19), a = 7.9081(2) Å, b = 11.0710(3) Å, c = 30.2192(8) Å, V = 2645.73(12) Å3, Z = 4, T = 100.01(10) K, μ(MoKα) = 0.085 mm-1, Dcalc = 1.202 g/cm3, 34647 reflections measured (4.562° ≤ 2Θ ≤ 56.56°), 6562 unique  which were used in all calculations. The final R1 was 0.0416 (I > 2σ(I)) and wR2 was 0.1191 .Crystallographic data for4: C27H42O7 (M =478.60 g/mol): orthorhombic, space group P212121 (no. 19), a = 7.9438(3) Å, b = 11.2958(8) Å, c = 28.897(2) Å, V = 2593.0(3) Å3, Z = 4, T = 99.99(10) K, μ(Mo Kα) = 0.087 mm−1, Dcalc = 1.226 g/cm3, 24810 reflections measured (4.578° ≤ 2Θ ≤ 50.246°), 4642 unique  which were used in all calculations. The final R1 was 0.0804 (I > 2σ(I)) and wR2 was 0.2256 .