N--acetamide, 2-ethoxycarbonyl-N--acetamide or 2-phenylcarbamoyl-N--acetamide systems as precursors. The latter compounds were used to synthesize polyfunctional thiophene-, thiazole-, pyrazole, pyridine-, pyrimidine-, oxazine-, as well as acyclic moieties. The dyes and dye precursors were characterized by elemental analysis and spectral methods. All dyes and their precursors were screened in vitro and evaluated for both their antibacterial and antifungal activities. MIC data of the novel dye systems and their respective precursors showed significant antimicrobial activity against most tested organisms. Some compounds exhibited comparable or even higher efficiency than selected standards. Dyes were applied at 5% depth for disperse dyeing of nylon, acetate and polyester fabrics. Their spectral characteristics and fastness properties were measured and evaluated. A series of novel polyfunctionalized acyclic and heterocyclic dye precursors and their respective azo (hydrazone) counterpart dyes and dye precursors based on conjugate enaminones and/or enaminonitrile moieties were synthesized. The dyes and their precursors are based on 2-cyano- The growing interest in heterocyclic azo dye chemistry is focused on designing new synthetic approaches to these materials, theoretical calculations, and applications in various industrial fields. Besides having important-applications as textile colorants ,5,6,7,8,via treatment with either N-haloamine moieties or by quaternary ammonium salts (QAS) has been discussed thiophene (1)  ion peaks at m/z 292, 339 and 336, respectively. When 3 and 4 were condensed with benzaldehyde their respective benzylidene derivatives 6 [7 were obtained  along with other δ-1H signals and νmax absorption bands detected in the respective regions . Compounds 6 and 7 exhibited molecular ion peaks [M+] at m/z 380 and [M+−1] at m/z 426, respectively. The spectral data of 2, 3 and 4 by different reagents. Thus, subjecting the title precursors to electrophilic or nucleophilic attack on their respective cyano-, ethoxycarbonyl-, and phenylcarbamoyl acetamido moieties, a variety of highly functionalized acyclic or heterocyclic molecules were obtained. When the intermediates 2, 3 and 4 were subjected to coupling with diazotized aryl (heteraryl) amines, the corresponding cyano-, ethoxy-carbonyl-, or phenylcarbamoyl azo (hydrazone) dyes 8a-d, 9a-e and 10 were produced (A) and the hydrazo-keto form (B). The dyes revealed common features indicating their existence in the azo-hydrazone form, whereby they exhibited intense amidic C=O absorptions around 1640 cm−1 (IR), δ-1H singlets about 12.80 ppm corresponding to a tautomeric hydrazone NH, along with singlets at δ ~ 10.88-15.70 ppm assigned to an enolic OH (1H-NMR). Other νmax values due to CN, ethoxycarbonyl C=O, phenyl-carbamoyl C=O (IR) in addition to δ-1H signals exhibited for four cyclohexene CH2s, amidic NH, ester CH3 and CH2 as well as phenyl aromatic protons (1H-NMR) were detected in the respective spectral regions. The mass spectra of the dyes revealed molecular ion peaks [M+] in agreement with their molecular formulae.Aiming to produce novel azo dyes and dye precursors with biological activities for dyeing and/or finishing of textile fibers, we considered the regioselectivity for attack on the key precursors produced . The dye2, 3 and 4 with active methylene reagents  afforded the respective 2-oxopyridine derivatives 11a-d, 12a-b, 13a-b  for compounds 12b, 13b. Compounds 12a, 12b revealed δ-1H triplets about δ 1.13 ppm and δ-1H quartets about δ 3.56 ppm due to the ester CH3 and CH2 protons, respectively.All data for compounds 12a-b, 13a-b, in the 1673-1630 and 1663-1600 cm−1 regions, respectively. Mass spectral analysis displayed molecular ion peaks at m/z 357 [M+−1], 359 [M+] and 405 [M+] corresponding to the respective molecular formulae C17H18N4O3S for compound 12a, C17H17N3O4S for 12b and C21H19N5O2S for 13a.Additionally two carbonyl stretching modes due to a pyridine oxo function and an ethoxycarbonyl or phenylcarbamoyl C=O were observed for compounds 11a-d, 12a-b were coupled with diazotized aryl amines, the corresponding 2-oxo-5-pyridine azo (hydrazone) dyes 14a-d, 15a-b were obtained , the respective 2-oxo-3-pyridine azo (hydrazone) dyes 16a-b were obtained  (Other 2-oxopyridine systems reagents  (PhCH=C(ts  at m/z 262 for compound 21a, [M+] at m/z 336 for 21b, [M++1] m/z 365 for 22a and [M++2] at m/z 442 for 22b. The data corresponded to respective molecular formulae C12H12N4OS, C18H16N4OS, C18H16N6OS and C24H20N6OS.On the other hand, when produced . The reaechanism . Microan10 with methylene carbonitrile reagents  in the presence of a catalytic amount of triethylamine. The reaction afforded the functionalized 1,3-oxazine azo dyes 23a-b, respectively  followed by 1,6-dipolar cyclization. The reaction took place through the non-isolable intermediate A. The data obtained from the IR, 1H-NMR, and MS spectra for the oxazine systems 23a-b confirmed the proposed cyclization mechanism. Both dye systems displayed, in their respective 1H-NMR spectra oxazine C5-H protons, both at δ 6.92 ppm, as well as two D2O exchangeable NH2 singlets, both at δ 3.64. Compound 23a exhibited a δ-1H singlet at 8.27 ppm due to the oxazine C4-imino function, while 23b showed νmax at 1662 cm−1 due to the oxazine C4-oxo absorption. The mass spectra of 23a-b exhibited [M+] ion peaks at m/z 509 and [M++1] at 511, respectively, indicating their corresponding molecular formulae C27H23N7O2S and C27H22N6O3S. The proposed mechanism involves nucleophilic attack by the enolic tautomer of 9a and 10 with phenyl isothiocyanate in 1,4-dioxane containing a catalytic amount of triethylamine. The reaction involved a nucleophilic attack by the amidic NH function in 9a and 10 on the C=S terminal of the isocyanate reagent to produce the acyclic intermediate A. the latter then underwent 1,6-dipolar cyclization through elimination of EtOH or H-OH to afford the functionalized pyrimidine azo (hydrazone) dye systems 24a and 24b, respectively -acetamide, 2-ethoxycarbonyl-N--acetamide or 2-phenylcarbamoyl-N--acetamide. The dyes have their chromophoric system either attached to the acetamido terminus  are listed in max 1610-1600 cm−1). Additionally, the azo-hydrazone tautomer is evident in the 1H-NMR spectra where the H peak of the imine group characteristically appears at δ ~12.00 ppm. The UV/vis absorption maxima of the synthesized dyes were tested in the aqueous solution [after adding dimethyl sulphoxide (1.00/20.00 mL) to improve the water solubility]. From the analysis of the UV/vis absorption data, it was found that, for each inter-related system, none of the dyes changed significantly with respect to each other. The target dyes revealed analytical and spectral data in accordance to their molecular structures. Most dyes exist in the azo-hydrazone tautomeric structure, as revealed by spectral data. The possibility of H bond formation between the imine-H of the hydrazone tautomer and the carbonyl bond  as well max values of some dyes could be attributed to more extensive delocalization within each system configuration which in turn, is dependent on the presence of electron donating substituents. Suitable electronic absorptions were found in the blue violet region of 390-420 nm. An absorption band below 250 nm was detected for most dye systems which could be relatable to electron transition through a possible azo ligand resulting from intramolecular chelation by H-bond . Rubbing fastness of the samples, assessed in terms of dry and wet rubbing indicated good fastness to rubbing for both dry and wet (3-5). Perspiration fastness properties  of the dyed samples in terms of ratings for staining of adjacent fabrics and change are very good (4-5). The high ratings for change in colour at both acidic and alkaline conditions indicate that the sensitivity of the dyed samples are not related to PH. This may be due to the stability of the dyes towards degradation under either acidic or basic conditions. Light fastness of the dyeing was of a generally of good order (3-5). The lightness ratings were primarily influenced by the depth of the hue. The data for colour yield expressed as K/S indicated higher values on nylon in comparison to other fabrics. This is probably due to the high substantivity of the dyes on nylon. From the aforementioned data, it is noteworthy that the fastness properties for the dyes under investigation are inter-related since they depend, among other factors, on the rate of diffusion of the dye in the fabric. This rate is a function of the geometry of the dye molecule. Fastness to rubbing depends on the presence of loose dye particles on the fabric surface. The washing and perspiration fastness is, to a certain extent, dependent on the substantivity of the dye for the fabric which determines its tendency to partition in favour of the fabric.in vitro for their antibacterial and antifungal activities against selected microbial strains, namely Escherichia coli ECT 101 (Gram-negative bacteria), Bacillus subtilis CECT 498 and Bacillus cereus CECT 148 (Gram-positive bacteria) and Candida albicans 1394 .The newly synthesized dyes and dye precursor were screened The minimal inhibitory concentration (MIC) in μg/mL was determined using an adapted agar streak dilution method based on radial diffusion ,46. UndeThe antimicrobial screening results of the synthesized compounds and standard antibiotics are given in 8a-d, 9a-e, 15b, 16a-b, 22b, 23a and dye precursors 3, 13b, 17a, 21a) were found to be totally inactive towards the Gram-negative bacterial species E. coli. This could be related to the presence of electron withdrawing functions  conjugated with electron donating groups . The increase in electron attracting character of these compounds cause a relative decrease in their biological activity. Furthermore, the intricate nature of the cell wall of this Gram-negative bacterial strain compared with Gram-positive test bacteria B. subtilis and B.cereus may inhibit the adsorption of most compounds onto the surface and consequently revealed weaker susceptibility for these compounds.According to this study it was observed that the MIC values of the dyes and dye precursors are generally within the 0.04-30.00 μg/mL range against all tested microbial organisms, which reveals significant and potent antimicrobial activity . HoweverC. albicans  were chosen to study their biological activities towards E. coli and Pseudomonas aeruginosa (Gram-negative bacteria), B. subtilis,Staphylococcus aureus and B. cereus (Gram-positive bacteria) and C. albicans . Diameters of the inhibition zones in mm were measured. The data of the disc susceptibility tests showed significant and potent antimicrobial activity of the dyed fabrics against all tested organisms (Selected dyed fabrics (nylon 66 dyed with rganisms .1H-NMR spectra were recorded with Varian Gemini-200 (200 MHz) and Jeol AS 500 MHz instruments, 13C-NMR spectra were measured with the Jeol AS 500 MHz instrument. Both spectra were performed in DMSO-6d as solvent using TMS as internal standard and chemical shifts are expressed as δ ppm. MS (EI) spectra were recorded with Hewlett Packard 5988 A GC/MS system and GCMS-QP 1000 Ex Shimadzu instruments. UV/vis absorption maxima (λmax) were recorded on UV-16600 Series. Analytical data were obtained from the Micro-analytical Data Unit at Cairo University and were performed on Vario EL III Elemental analyzer. The dyeing operation was carried out using Wemer Mathis AG Textimaschine-Laborapparate CH-8155 Niederhasli/Zürich apparatus. Colour strength (K/S) of the dyed samples was measured by using OPTIMATCH 3100. The colour fastness to washing was determined using Launder-ometer. Colour fastness to rubbing was determined using Crock-Meter Type FD II and colour fastness to perspiration was determined using Prespiration Tester. The light fastness test was measured by using Mercury-Tungsten lamp. All melting points were determined on an Electrothermal digital melting point apparatus and are uncorrected. IR spectra (KBr discs) were recorded on a FTIR plus 460 or Pye Unicam SP-1000 spectrophotometer. 2-Cyano-N--acetamide (2) was synthesized following the method previously described by our group  (8.00), 340 [M++1] (12.50), 339 [M+] (49.70), 178 (100.00), 93 (74.90), 77 (56.20).2-(1H-Benzoimidazol-2-yl)-N--acetamide (5): Equimolar amounts of 3  and o-phenylenediamine  were heated in an oil bath at 140 °C for 1h. The reaction mixture was then boiled in ethanol (30 mL) for few minutes, poured onto ice/water mixture and the formed product was crystallized from 1,4-dioxane. Pale brown crystals, m.p. 94-98 °C, yield: 2.35 g (70%); Anal. For C18H16N4OS (336.41), : 64.26/64.51 (C), 4.79/4.81 (H), 16.65/16.30 (N), 9.53/9.82 (S); IR : 3392 (2NH), 3050 (CH aromatic), 2924-2844 (CH2), 2196 (CN), 1607 (C=O), 1544, (C=N), 1502, 1431(C=C); 1H-NMR : 1.77-2.93 , 4.44 , 6.88-7.63 , 8.37, 10.54 ; 13C-NMR: 23.3, 24.4, 28.4, 29.6 (4 CH2), 40.7 (CH2), 93.3 (thiophene-C3), 115.9 (CN), 121.0, 121.4, 122.2, 122.5, 127.4 (C6H4), 131.0, 131.8, 146.4 (thiophene 3C), 145.8 (imidazole-C2), 162.5 (C=O), 175.7 (C=N); MS m/z (%): 337 [M++1] (55.42), 336 [M+] (100.00), 335 [M+–1] (41.43).3  or 4  in 1,4-dioxane (25 mL) containing piperidine (0.50 mL), benzaldehyde  was added. The reaction mixture was heated under reflux for 5h. The solid products formed upon pouring onto ice/water mixture were containing few drops of hydrochloric acid collected by filtration and crystallized from 1,4-dioxane.To a mixture of equimolar amounts of 2--3-phenyl-acryclic acid ethyl ester (6): The data for compound 6 has been published earlier by our group , 426 [M+–1] (36.83), 140 (74.67), 73 (100.00), 77 (1.52).2 , in ethanol (20 mL) containing sodium hydroxide (1.00 g) an equimolar amount of diazotized aniline, diazotized p-chloroaniline, diazotized p-toluidene and diazotized p-methoxyaniline were gradually added while stirring. The solid products formed upon cooling in an ice-bath were collected by filtration, washed with water and crystallized from 1,4-dioxane.To a cold solution (0-5 °C) of 2-Cyano-2-(2-phenylhydrazono)-N--acetamide(8a): the data of compound 8a has been published earlier by our group [ur group .2-[(4-Chlorophenyl)-hydrazono]-2-cyano-N--acetamide (8b): Reddish brown crystals, m.p. 133-138 °C, yield: 3.45 g (90%); Anal. For C18H14N5OSCl (383.85), : 56.32/56.54 (C), 3.68/3.90 (H), 18.24/18.00 (N), 8.35/8.60 (S); IR : 3332-3218 , 3090 (CH aromatic), 2932-2855 (CH2 cyclohexene), 2256, 2214 (2CN), 1627 (C=O), 1575, 1490 (C=C), 1535 (=N-NH); 1H-NMR : 1.74-2.89 , 6.92-7.83 , 11.80, 12.80 , 15.70  ; MS m/z (%): 385 [M++2] (18.90), 383 [M+], 220 (13.50), 178 (16.20), 127 (64.90), 65 (100.00).2-Cyano-N--2-(p-tolyl-hydrazono)-acetamide (8c): Dark reddish brown crystals, m.p. 116-120 °C, yield: 2.00 g (55%); Anal. For C19H17N5OS (363.44), : 62.79/63.16 (C), 4.71/4.99 (H), 19.27/18.90 (N), 8.82/9.04 (S); IR : 3330-3218 , 3090 (CH aromatic), 2929-2857 , 2253, 2218 (2CN), 1622 (C=O), 1574, 1443 (C=C), 1518 (=N-NH); 1H-NMR : 1.17 , 1.71-2.84 , 6.88  7.13-7.65 , 11.99, , 15.59  ; MS m/z (%): 363 [M+], 150 (71.40), 77 (54.80), 50 (100.00). 2-Cyano-N--2-[(4-methoxyphenyl)-hydrazono]acetamide (8d): Dark reddish brown crystals, m.p. 90-94 °C, yield: 2.31 g (61%); Anal. For C19H17N5O2S (379.44), : 60.14/59.80 (C), 4.52/4.82 (H), 18.46/18.10 (N), 8.45/8.82 (S); IR : 3325-3215 (2NH), 3090 (CH aromatic), 2932-2839 , 2254, 2211 (2CN), 1610 (C=O), 1576, 1459 (C=C), 1511 (=N-NH); 1H-NMR : 1.20 , 1.75-2.50 , 7.07-7.79 , 10.80, 11.90 , 12.43 ; MS m/z (%): 379 [M+] (54.49), 300 (62.55), 61 (100.00).3 , in ethanol (20 mL) containing sodium hydroxide (1.00 g) an equimolar amount of diazotized aniline, diazotized p-chloroaniline, diazotized p-toluidene or diazotized p-methoxyaniline was gradually added while stirring. The solid products formed upon cooling in an ice-bath were collected by filtration, washed with water and crystallized from 1,4-dioxane. To a cold solution (0-5 °C) of N--2-(phenylhydrazono)-malonamic acid ethyl ester (9a): the data of compound 9a has been published earlier by our group [ur group .2-[(4-Chlorophenyl)-hydrazono]-N-malonamic acid ethyl ester (9b): Orange crystals, m.p. 186-190 °C, yield: 4.09 g (95%); Anal. For C20H19ClN4O3S (430.91), : 55.75/55.42 (C), 4.44/4.11 (H), 13.00/13.34 (N), 7.44/7.73 (S); IR : 3439-3348 (2NH), 3159 (CH aromatic), 2932-2850 , 2208 (CN), 1664, 1627 (2C=O), 1590, 1453 (C=C), 1529 (=N-NH); 1H-NMR : 1.81 , 2.50-2.66 , 3.80 , 7.42-7.74 , 10.60, 12.50 , 13.60 . N--2-(p-tolyl-hydrazono)-malonamic acid ethyl ester (9c): Pale orange crystals, m.p. 256-260 °C, yield: 3.94 g (96%); Anal. For C21H22N4O3S (410.49), : 61.45/61.17 (C), 5.40/5.60 (H), 13.65/14.00 (N), 7.81/8.02 (S); IR : 3470-3163 , 3090 (CH aromatic), 2932-2852 , 2210 (CN), 1665, 1630 (2C=O), 1570, 1454 (C=C), 1525 (=N-NH); 1H-NMR : 1.20 , 1.75 , 1.88-2.85 , 3.30 , 7.20-7.62 , 10.70, 12.59 , 13.66 ; MS m/z (%): 410 [M+] (18.90), 233 (29.70), 204 (13.50), 178 (37.80), 106 (100.00), 77 (70.30).N--2-[(4-methoxy-phenyl)-hydrazono]-malonamic acid ethyl ester (9d): Deep orange crystals, m.p. 240-245 °C, yield: 3.54 g (83%); Anal. For C21H22N4O4S (426.49), : 59.14/58.90 (C), 5.20/5.43 (H), 13.14/13.30 (N), 7.52/7.82 (S); IR : 3457-3188 , 3078-3001 (CH aromatic), 2933-2841 , 2210 (CN), 1663, 1600 (2C=O), 1571, 1457 (C=C), 1530 (=N-NH) ; 1H-NMR : 1.01 , 1.71 , 1.87-2.85 , 3.70 , 6.91-7.92 , 11.78, 13.45 , 14.93 ; MS m/z (%): 426 [M+] (1.90), 249 (4.50), 204 (11.60), 178 (63.00), 150 (100.00).N--2-malonamic acid ethyl ester (9e): Compound 9e was synthesized according to the following method: A diazotized solution of 2-amino-3-cyano-4,5,6,7-tetrahydrobenzo[b]-thiophene (1) was prepared by dissolving 1  in a mixture of acetic acid (10 mL) and propionic acid (2 mL) and stirring while cooling (5 °C) for 20 min. Concentrated sulphuric acid (5 mL) was then added, followed by portion-wise addition of sodium nitrite . The diazotized solution was heated (60-65 °C) while stirring for 1 h until a clear solution was obtained, cooled to 5 °C, and then added to a solution of 3  in ethanol (35 mL) containing sodium hydroxide (1.00 g) while cooling in an ice bath. The reaction mixture was neutralized by pouring onto ice/water mixture containing few drops of hydrochloric acid. The solid product, thus formed, was collected by filtration and crystallized from ethanol. Brown crystals, m.p. 271-273 °C, yield: 3.85 g (80%); Anal. For C23H23N5O3S2 (481.59), : 57.36/57.01 (C), 4.81/5.02 (H), 14.54/14.32 (N), 13.32/13.60 (S); IR : 3450-3193 , 3085-3005 (CH aromatic), 2936-2859 , 2211, 2195 (2CN), 1678, 1628 (2C=O), 1573, 1464 (C=C), 1545 (=N-NH); 1H-NMR : 1.20 , 1.75-2.58 , 3.85 , 6.90, 11.50 , 11.77 ; MS m/z (%): 481 [M+] (0.02), 163 (27.63), 135 (100.00), 105 (31.48), 77 (17.45).\-phenyl-2-(phenylhydrazono)malonamideN--N (10): Method (A): To a cold solution (0-5 °C) of 4 , in ethanol (20 mL) containing sodium hydroxide (1.00 g) an equimolar amount of diazotized aniline was gradually added while stirring. The solid product formed upon cooling in an ice-bath was collected by filtration, washed with water and crystallized from 1,4-dioxane. Method (B): To a solution of 9a  in dimethylformamide (30 mL), aniline  was added. The reaction mixture was heated under reflux for 5 h. The solid product formed upon pouring onto ice/water mixture was collected by filtration, washed with water and crystallized from 1,4-dioxane. Pale orange crystals, m.p. 106-110 °C, yield: 3.41 g (77% in method A and B); Anal. For C24H21N5O2S (443.52), : 64.99/64.62 (C), 4.77/4.36 (H), 15.79/15.57 (N), 7.23/7.67 (S); IR : 3290-3209 , 3063 (CH aromatic), 2934-2857 (CH2 cyclohexene), 2210 (CN), 1667, 1598, (2C=O) 1527,1443 (C=C); 1H-NMR : 1.71-2.62 , 7.26-7.57 , 10.14, 11.76, 13.35 , 13.78 ; MS m/z (%): 443 [M+] (3.50), 339 (18.10), 178 (100.00), 93 (91.20), 77 (68.40).\-phenyl-2-(phenylhydrazono)malonamideN--N (10): Method (A): To a cold solution (0-5 °C) of 4 , in ethanol (20 mL) containing sodium hydroxide (1.00 g) an equimolar amount of diazotized aniline was gradually added while stirring. The solid product formed upon cooling in an ice-bath was collected by filtration, washed with water and crystallized from 1,4-dioxane. Method (B): To a solution of 9a  in dimethylformamide (30 mL), aniline  was added. The reaction mixture was heated under reflux for 5 h. The solid product formed upon pouring onto ice/water mixture was collected by filtration, washed with water and crystallized from 1,4-dioxane. Pale orange crystals, m.p. 106-110 °C, yield: 3.41 g (77% in method A and B); Anal. For C24H21N5O2S (443.52), : 64.99/64.62 (C), 4.77/4.36 (H), 15.79/15.57 (N), 7.23/7.67 (S); IR :3290-3209 , 3063 (CH aromatic), 2934-2857 (CH2 cyclohexene), 2210 (CN), 1667, 1598, (2C=O) 1527,1443 (C=C); 1H-NMR : 1.71-2.62 , 7.26-7.57 , 10.14, 11.76, 13.35 , 13.78 ; MS m/z (%): 443 [M+] (3.50), 339 (18.10), 178 (100.00), 93 (91.20), 77 (68.40).11a-d have been published earlier by our group  (12.37), 356 [M+–2] (18.51), 178 (100.00), 150 (99.10).4-Amino-1--6-hydroxy-2-oxo-1,2-dihydropyridine-3-carboxylic acid ethyl ester (12b): Pale yellow crystals, m.p. 268-270 °C, yield: 2.59 g (72%); Anal. For C17H17N3O4S (359.40), : 56.81/57.01 (C), 4.77/4.40 (H), 11.69/11.82 (N), 8.92/9.20 (S); IR : 3740-3191 , 3081 (CH aromatic), 2932-2852 , 2208 (CN), 1676, 1635 (2C=O), 1546, 1460 (C=C); 1H-NMR : 1.14 , 1.75-2.89 , 3.85 , 4.07 , 6.90 , 11.76  ; MS m/z (%): 360 [M++1] (0.11), 359 [M+] (0.09), 178 (100.00), 150 (76.57).4,6-Diamino-1--2-oxo-1,2-dihydropyridine-3-carboxylic acid phenylamide (13a): Yellowish white crystals, m.p. 210-212 °C, yield: 2.84 g (70%); Anal. For C21H19N5O2S (405.47), : 62.21/61.91 (C), 4.72/4.92 (H), 17.27/17.00 (N), 7.91/8.10 (S); IR : 3400-3282 , 3081 (CH aromatic), 2932 (CH2 cyclohexene), 2213 (CN), 1663, 1600, (2C=O) 1544,1442 (C=C); 1H-NMR : 1.72-2.94 , 3.44 , 3.64 , 6.92 , 7.27-7.56 , 10.18 , 11.77 ; MS m/z (%): 405 [M+], 407 [M++2] (41.83), 405 [M+] (42.89), 54 (100.00).4-Amino-1--6-hydroxy-2-oxo-1,2-dihydropyridine-3-carboxylic acid phenylamide (13b): Pale yellow crystals, m.p. 135-138 °C, yield: 2.48 g (61%); Anal. For C21H18N4O3S (406.46), : 62.05/61.72 (C), 4.46/4.72 (H), 13.78/13.40 (N), 7.89/8.21 (S); IR : 3426-3221 , 3082 (CH aromatic), 2932-2848 (CH2 cyclohexene), 2202 (CN), 1662, 1613, (2C=O) 1536,1441 (C=C); 1H-NMR : 1.70-2.59 , 3.68 , 6.90 , 7.03-7.61 , 10.13-10.17 , 11.75 .11a , 11b , 11c , 11d , 12a  or 12b , in ethanol (20 mL) containing sodium hydroxide (1.00 g) an equimolar amount of diazotized aniline was gradually added while stirring. The solid products formed upon cooling in an ice-bath were collected by filtration, washed with water and crystallized from 1,4-dioxane.To a cold solution (0-5 °C) of 4,6-Diamino-1--2-oxo-5-phenylazo-1,2-dihydro-pyridine-3-carbonitrile (14a): Orange crystals, m.p. 255-260 °C, yield: 3.45 g (83%); Anal. For C21H17N7OS (415.47), : 60.71/60.41 (C), 4.12/4.30 (H), 23.60/23.21 (N), 7.72/8.11 (S); IR : 3446-3220 (2NH2), 3139-3067 (CH aromatic), 2930-2855 (CH2 cyclohexene), 2213, 2195 (2CN), 1679 (C=O), 1608, 1490 (C=C), 1545 (N=N); 1H-NMR : 1.77-2.64 , 3.90, 4.10 , 7.15-7.69 .4-Amino-1--6-hydroxy-2-oxo-5-phenylazo-1,2-dihydro-pyridine-3-carbonitrile (14b): Dark orange crystals, m.p. 230-235 °C, yield: 4.12 g (99%); Anal. For C21H16N6O2S (416.11), : 60.56/60.72 (C), 3.87/3.74 (H), 20.18/19.94 (N), 7.70/8.00 (S); IR : 3444 (OH), 3358-3217 (NH2), 3064 (CH aromatic), 2928-2850 (CH2 cyclohexene), 2210, 2195 (2CN), 1679 (C=O), 1600, 1452 (C=C), 1539 (N=N); 1H-NMR : 1.75-2.64 , 4.09 , 7.02-7.84 , 12.65 .1--4,6-dimethyl-2-oxo-5-phenylazo-1,2-dihydro-pyridine-3-carbonitrile (14c): Orange crystals, m.p. 140-145 °C, yield: 2.60 g (63%); Anal. For C23H19N5OS (413.13), : 66.81/66.43 (C), 4.63/4.42 (H), 16.94/16.60 (N), 7.75/8.10 (S); IR : 3100 (CH aromatic), 2933-2856 (CH2 cyclohexene), 2218, 2195 (2CN), 1671 (C=O), 1575, 1446 (C=C), 1541 (N=N); 1H-NMR : 1.81, 2.16 , 2.41-2.79 , 6.56-7.71 ; MS m/z (%): 413 [M+], 412 [M+–1] (5.90), 411 [M+–2] (23.50), 77 (41.20), 51 (100.00).1--6-hydroxy-4-methyl-2-oxo-5-phenylazo-1,2-dihydro-pyridine-3-carbonitrile (14d): Pale brown crystals, m.p. 165-170 °C, yield: 3.11 g (75%); Anal. For C22H17N5O2S (415.11), : 63.60/63.22 (C), 4.12/4.41 (H), 16.86/17.10 (N), 7.72/8.01 (S); IR : 3450-3219 (OH), 3083 (CH aromatic), 2933-2856 , 2214, 2195 (2CN), 1685 (C=O), 1630, 1492 (C=C), 1546 (N=N); 1H-NMR : 1.76 , 2.49-2.64 , 7.17-7.69 , 11.88 .4,6-Diamino-1--2-oxo-5-phenylazo-1,2-dihydro-pyridine-3-carboxylic acid ethyl ester (15a): Pale orange crystals, m.p. 217-222 °C, yield: 3.65 g (79%); Anal. For C23H22N6O3S (462.52), : 59.73/60.10 (C), 4.79/4.40 (H), 18.17/18.43 (N), 6.93/6.60 (S); IR : 3348-3191 (2NH2), 3079 (CH aromatic), 2933-2851 , 2211 (CN), 1671, 1630 (2C=O), 1580, 1455 (C=C), 1530 (N=N); 1H-NMR : 1.33 , 1.75-2.58 , 4.30 , 7.19-7.71 , 3.19, 3.80 .4-Amino-1--6-hydroxy-2-oxo-5-phenylazo-1,2-dihydro-pyridine-3-carboxylic acid ethyl ester (15b): Pale orange crystals, m.p. 180-185 °C, yield: 3.16 g (78%); Anal. For C23H21N5O4S (463.51), : 59.60/59.90 (C), 4.57/4.20 (H), 15.11/15.40 (N), 6.92/6.60 (S); IR : 3440-3202 , 3079 (CH aromatic), 2932-2851 , 2211 (CN), 1675, 1640 (2C=O), 1590, 1455 (C=C), 1533 (N=N); 1H-NMR : 1.32 , 1.75-2.64 , 3.85 , 4.31 , 7.22-7.74 , 11.77 .16a-b have been published earlier by our group  (22.07), 353 (100.00), 325 (56.14). 5-Benzoyl-2,4-bis-phenylamino-thiophene-3-carboxylic acid -amide (18b): Brown crystals, m.p. 78-82 °C, yield: 5.63 g (98%); Anal. For C33H26N4O2S2 (574.72), : 68.97/68.62 (C), 4.56/4.83 (H), 9.75/10.01 (N), 11.16/11.40 (S); IR : 3276 (3NH), 3059 (CH aromatic), 2928-2853 (CH2 cyclohexene), 2207 (CN), 1644, 1548, (2C=O) 1496,1443 (C=C); 1H-NMR : 1.71-2.85 , 6.96-7.98 , 9.20, 10.15, 11.51 ; 13C NMR: 22.8, 23.3, 23.8, 24.0 (4CH2), 92.6 (thiophene-C3), 114.8 (CN), 119.6, 126.3, 127.1, 127.4, 128.6, 128.8, 131.6, 131.9 147.2, 153.3, 157.3 , 129.3, 129.7 (thiophene-C5), 130.8, 131.0 (thiophene-C4), (thiophene-C2), 162.8, 165.9 (2C=O); MS m/z (%): 574 [M+] (12.97), 572 [M+–2] (20.01), 413 (100.00), 313 (58.64), 150 (15.81).\-phenylmalonamideN--2-(4-methyl-3-phenyl-3H-thiazol-2-ylidene)-N (19): Reddish brown crystals, m.p. 88-90 °C, yield: 4.41 g (86%); Anal. For C28H24N4O2S2 (512.65), : 65.60/65.51 (C), 4.72/4.60 (H), 10.93/11.21 (N), 12.51/12.20 (S); IR : 3415-3282 (2NH), 3062 (CH aromatic), 2926-2847 , 2204 (CN), 1655, 1600, (2C=O) 1545,1491 (C=C); 1H-NMR : 1.22 , 1.52-2.89 , 6.62 , 6.93-7.95 , 10.19, 11.48 ; 13C NMR: 18.8 (CH3), 22.8, 23.3, 23.8, 24.0 (4CH2), 97.5 (thiophene-C3), 96.8, 104.5 (C=C), 115.9 (CN), 119.6, 125.6, 128.1, 128.7, 129.0, 129.3, 129.7, 132.7, 137.1, 140.0, 159.0 , 161.3, 166.2 (2C=O); MS m/z (%): 512 [M+] (0.03), 351 (92.46), 323 (44.41), 77 (100.00).3  in ethanol (25 mL) and dimethylformamide (5 mL) containing triethylamine (1.00 mL), malononitrile  was added followed by the addition of elemental sulfur . The reaction mixture was heated under reflux for 5 h, then cooled and neutralized by pouring onto ice/water mixture containing few drops of hydrochloric acid. The solid product formed was collected by filtration and crystallized from dimethylformamide. Brown crystals, m.p. 277-280 °C, yield: 3.27 g (95%); Anal. For C15H12N4O2S2 (344.41), : 52.31/52.60 (C), 3.51/3.70 (H), 16.27/16.06 (N), 18.62/18.30 (S); IR : 3333-3196 , 3083 (CH aromatic), 2931-2852 (CH2 cyclohexene), 2250, 2209 (CN),1631 (C=O), 1546, 1459 (C=C); 1H-NMR : 1.72-2.85 , 3.82 , 6.92 , 11.79 ; 13C-NMR: 23.7, 24.0, 24.3, 24.7 (4CH2), 93.3 (thiophene C-3), 114.6, 116.8 (2 CN), 126.8, 129.0, 130.6, 131.3, 133.8, 146.9 (two thiophene C), 165.0 (C=O); MS m/z (%): 344 [M+] (1.26), 178 (62.51), 150 (100.00).To a solution of 3  or 9a  in 1,4-dioxane (25 mL) and dimethylformamide (10 mL), either hydrazine hydrate , or phenyl hydrazine  was added. The reaction mixture, in each case, was heated under reflux for 5 h. The solid product formed, in each case, upon pouring onto ice/water mixture was collected by filtration, and crystallized from 1,4-dioxane/dimethylformamide mixture.To a solution of compound 2--4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (21a): Creamy crystals, m.p. 290-292 °C, yield: 2.52 g (97%); Anal. For C12H12N4OS (260.31), : 55.37/55.60 (C), 4.65/4.32 (H), 21.52/21.22 (N), 12.32/11.94 (S); IR : 3431-3207 (2NH), 3087-3006 (CH aromatic), 2935-2857 (CH2 cyclohexene), 2211 (CN), 1680 (C=O), 1548, 1463 (C=C); 1H-NMR : 1.76-2.59 , 3.85 , 6.95 , 11.77 ; MS m/z (%): 262 [M++2] (0.51), 220 (4.15), 205 (8.39), 178 (100.00), 150 (75.36).2--4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (21b): Creamy crystals, m.p. 280-282 °C, yield: 2.66 g (79%); Anal. For C18H16N4OS (336.41), : 64.26/64.00 (C), 4.79/4.88 (H), 16.65/16.30 (N), 9.53/9.80 (S); IR : 3431-3201 (NH), 3086-3006 (CH aromatic), 2935-2852 (CH2 cyclohexene), 2213 (CN), 1681 (C=O), 1546, 1463 (C=C); 1H-NMR : 1.75-2.59 , 3.85 , 6.70-7.20 , 11.77 ; MS m/z (%): 337 [M++1] (4.49), 336 [M+] (4.25), 334 [M+–2] (4.82), 284 (43.09), 77 (100.00).2--4,5,6,7-tetrahydrobenzo[b]-thiophene-3-carbonitrile (22a): Orange crystals, m.p. 135-137 °C, yield: 3.21 g (88%); Anal. For C18H16N6OS (364.42), : 59.32/59.00 (C), 4.43/4.33 (H), 23.06/22.80 (N), 8.80/9.10 (S); IR : 3343-3152 , 3059 (CH aromatic), 2932-2848 (CH2 cyclohexene), 2208 (CN), 1665 (C=O), 1527,1450 (C=C); 1H-NMR : 1.74-2.85 , 6.92-7.70 , 8.83 , 10.73, 11.20 , 12.58 ; MS m/z (%): 366 [M++2] (4.00), 365 [M++1] (4.60), 178 (25.28), 150 (14.39), 92 (100.00), 77 (30.97).2--4,5,6,7-tetrahydro-benzo[b]thiophene-3-carbonitrile (22b): Pale orange crystals, m.p. 106-110 °C, yield: 3.17 g (72%); Anal. For C24H20N6OS (440.52), : 65.44/65.30 (C), 4.58/4.61 (H), 19.08/18.80 (N), 7.28/7.56 (S); IR : 3744-3339 , 3054 (CH aromatic), 2931-2850 (CH2 cyclohexene), 2208 (CN), 1663 (C=O), 1528,1452 (C=C); 1H-NMR : 1.73-2.94 , 6.22-7.65 , 8.83, 12.50 , 13.61 ; MS m/z (%): 442 [M++2] (0.48), 319 (18.95), 178 (43.94), 150 (37.51), 92 (100.00), 77 (97.99).10  in ethanol (25 mL) and dimethylformamide (5 mL) containing triethylamine (1.00 mL), either malononitrile  or ethyl cyanoacetate  was added. The reaction mixture, in each case, was heated under reflux for 5 h, then cooled and neutralized by pouring onto ice/water mixture containing few drops of hydrochloric acid. The solid product formed, in each case, was filtered off and crystallized from ethanol/dimethylformamide mixture. To a solution of 2--oxazin-2-ylidene]-N-phenyl-2-phenylazo-aceamide (23a): Greenish brown crystals, m.p. 115-120 °C, yield: 4.43 g (87%); Anal. For C27H23N7O2S (509.58), : 63.64/63.52 (C), 4.55/4.70 (H), 19.24/18.90 (N), 6.29/6.52 (S); IR : 3324-3208 , 3050 (CH aromatic), 2932 (CH2 cyclohexene), 2205 (CN), 1600 (C=O) 1525,1449 (C=C); 1H-NMR : 1.72-2.69 , 3.64 , 6.92 , 7.26-7.57 , 8.27, 10.14 ; MS m/z (%): 509 [M+] (15.97), 489 (14.39), 256 (48.14), 57 (100.00).2-oxazin-2-ylidene]-N-phenyl-2-phenylazo-aceamide (23b): Brown crystals, m.p. 100-105 °C, yield: 4.44 g (87%); Anal. For C27H22N6O3S (510.57), : 63.52/63.24 (C), 4.34/4.41 (H), 16.46/16.10 (N), 6.28/6.52 (S); IR : 3318 , 3060 (CH aromatic), 2930 (CH2 cyclohexene), 2207 (CN), 1662, 1600 (2C=O) 1523,1447 (C=C); 1H-NMR : 1.72-2.61 , 3.64 , 6.92 , 7.27-7.58 , 10.14 ; MS m/z (%): 511 [M++1] (20.89), 487 (26.56), 138 (46.17), 97 (69.73), 57 (100.00).9a  or 10  and phenyl isothiocyanate  in 1,4-dioxane (20 mL) containing triethylamine (1.0 mL) were heated under reflux for 5 h. After cooling, the reaction mixtures were acidified by hydrochloric acid and the crude products were precipitated, collected by filtration and crystallized from dimethylformamide.Equimolar amounts of 2--4,5,6,7-tetra-hydro-benzo[b]thiophene-3-carbonitrile (24a): Brown crystals, m.p. 288-290 °C, yield: 3.16 g (65%); Anal. For C25H19N5O2S2 (485.58), : 61.84/61.60 (C), 3.94/4.12 (H), 14.42/14.10 (N), 13.21/12.92 (S); IR : 3447-3343 , 3046 (CH aromatic), 2932-2853 (CH2 cyclohexene), 2206 (CN), 1665, 1620 (2C=O), 1527, 1451 (C=C); 1H-NMR : 1.75-2.85 , 7.19-7.72 , 12.58 , 13.62 ; MS m/z (%): 485 [M+], 484 [M+–1] (9.69), 436 (9.01), 178 (21.68), 150 (26.20), 92 (100.00), 77 (52.53).2-[6-Oxo-3-phenyl-5-(phenylhydrazono)-4-phenylimino-2-thioxotetrahydropyrimidin-1-yl]-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (24b): Dark green crystals, m.p. 68-70 °C, yield: 3.53 g (63%); Anal. For C31H24N6OS2 (560.69), : 66.41/66.10 (C), 4.31/4.31 (H), 14.99/14.63 (N), 11.44/11.51 (S); IR : 3212-3119 , 3047 (CH aromatic), 2930 (CH2 cyclohexene), 2206 (CN), 1664 (C=O) 1594,1443 (C=C), 1529 (=N-NH), 1326, 1290 (C=S); 1H-NMR : 1.73-2.85 , 7.14-7.65 , 11.03 , 13.36 ; MS m/z (%): 560 [M+], 561 [M++1] (35.20), 559 [M+–1] (29.13), 150 (86.53), 106 (100.00), 93 (70.49).Unless otherwise indicated, dyeing was performed using a solution containing 5% dye (based on weight of sample), 2 g/L dispersing agent and ammonium persulphate at 120 °C for 45 min. A material to liquor ratio 1:20 was used. The dye solution was adjusted at Ph = 4.5-5 using acetic acid. After the end of dyeing time, the fabric sample was washed in a solution containing 5 g/L detergent for several times until a clear solution was obtained. Finally the fabric sample was rinsed with water and dried at ambient conditions. The colour of the dyes on cellulose triacetate, nylon 66 and polyester fibers are indicated  Table 1Table 1.max = 400 nm (Colour strength of the dyed samples expressed as (K/S) was measured at λ= 400 nm .The colour fastness to washing, rubbing (dry and wet crocking) and perspiration was determined according to the standard method . Data arThe composite specimens were sewed between two pieces of bleached cotton fabric and then immersed into an aqueous solution containing 5g/L soap non-ionic detergents at liquor ratio 50:1 and 2g/L sodium carbonate. The bath was thermostatically adjusted to 90 °C for 30 min; then samples were removed, rinsed twice with occasional hand squeezing, then dried. Evaluation of the wash fastness was established using the Grey-scale for colour change .The test is designed for determining the degree of colour, which may transfer from the surface of the coloured fabric to another surface, by rubbing.Dry crocking test: The test specimen was placed flat on the base of the crock-meter. A white testing cloth was mounted. The covered finger was lowered on to the test specimen and caused to slide 20 times back and forth by making ten complete turns at a rate of one turn/sec. The white test sample was then removed for evaluation using the Grey-scale for staining.Wet crocking test: The white test sample was thoroughly wetted out in water to a 65% and then picked up. The procedure was run as above. The white test samples were air dried before evaluation.Two artificial perspiration solutions were prepared according to the following: Acidic solution: L-Histidine monohydrochloride monohydrate (0.5 g), sodium chloride (5 g) and sodium dihydrogen phosphate-1-hydrate (2.2 g) were dissolved in one liter distilled water. The pH was adjusted to 5.5 by 0.1N sodium hydroxide solution.Alkaline solution: L-Histidine monohydrochloride monohydrate (0.5 g), sodium chloride (5 g) and di-sodium hydrogen phosphate-2-hydrate (2.5 g) were dissolved in one liter distilled water. The pH was adjusted to 8 by 0.1N sodium hydroxide solution.The coloured specimen was sewed between two pieces of bleached cotton specimen. The composite sample was then immersed for 30 min. in the acidic perspiration solution at 37 °C (±2) with occasional agitation and squeezing to insure complete wetting. The test specimen was placed between two plastic plates under a force of about 5 Kg. The plates containing the composite specimens were left for about 6-8 hours. The same experiment was followed with another composite sample using the alkaline perspiration solution. The effect on the colour of the test specimen was expressed and defined by reference to Grey-scale for color change.The light fastness test was assessed in accordance with test method (ISO 105-A03). Using Mercury-Tungsten lamp, (continuous light) for 40 hours. The effect on the colour of the test samples was expressed and defined by reference to Grey-scale for colour change.in vitro antimicrobial activity evaluation, microorganism suspensions were prepared to contain approximately 108 cuf/mL and the plates were inoculated. A stock solution of each of the synthesized compounds (1.0 mg/mL) in DMSO was prepared and graded dilutions of the tested compounds were incorporated in a cavity  made in the center of the Petri dish (nutrient agar for bacteria and Sabouraud vs. dextrose agar medium for fungi). The plates were incubated in duplicates for 24 h at 37 °C (for bacteria) and at 30 °C (for fungi). A positive control using only inoculation and a negative control using only DMSO in the cavity were carried out. The results of antimicrobial screening of the synthesized and standard antibiotics are given in Microorganisms used were obtained from Microbial Chemistry Department, National Research Center, Cairo, Egypt. For the Nutrient broth/agar medium: contains (g/L), (5) peptone, (3) beef extract. For solid medium 15 g/L agar was added. Malt broth/agar medium: contains (g/L), (5) peptone, (24) malt extract. For solid medium 15 g/L agar was added. 2.7.3. Growth conditions an inoculum of each bacterial strain was suspended in 25 mL of nutrient broth medium and shaken for 24 h at 37 °C. For yeast, malt broth was inoculated with test organism and incubated at 28 °C for 24 h.Disc diffusion method with some modifications was used for screening the nylon 66, acetate and polyester fabric samples for antimicrobial activity . NutrienOur synthetic strategy provides a simple protocol for producing dyes and dye precursors based on conjugate enaminones and/or enaminonitriles with potent dyeing and/or antimicrobial finishing capabilities. The novel compounds could be lead for the development of new functional materials with special finish properties for textile fabrics. Moreover, the results of the present study may point that the novel products could be useful as synthetic precursors for azo- and azomethine ligands or polymethine dyes which may be suitable for both electronic and optical applications. It is clear that fabrics dyed or treated with the synthesized novel systems exhibit significant antimicrobial activity. This is due to the inherent antimicrobial character of the dyes or dye precursors. The presence of an oxo moiety which could be responsible to complex or bind with nucleophilic amino acids in proteins leads to inactivation of the protein and loss of function. Moreover, formation of H-bonds with water molecules enable these compounds to more readily form positive ions, thereby inhibition of microbial growth by adsorption onto bacterial surface. As a consequence, we can conclude that the newly synthesized systems could be accepted as promising to develop new antibacterial compounds.


H-thienopyrrolizinones, were efficiently synthesized in four steps with fair overall yields (34–57%). All tripentone derivatives were tested in the range of 0.1–100 μM for cytotoxicity against two human tumor cell lines, HCT-116  and MCF-7 (human breast cancer). The most active derivative, with GI50 values of 4.25 µM and 20.73 µM for HCT-116 and MCF-7 cells, respectively, did not affect the viability of Caco-2 differentiated in normal intestinal-like cells, suggesting tumor cells as the main target of its cytotoxic action. The same compound was further investigated in order to study its mode of action. Results showed that it did not exert necrotic effects, while induced a clear shift of viable cells towards early apoptosis. Flow cytometric analysis demonstrated that this compound caused cell cycle alteration, inhibiting its progression in S and G2/M phases.Tripentones represent an interesting class of compounds due to their significant cytotoxicity against different human tumor cells in the submicro-nanomolar range. New tripentone analogs, in which a pyridine moiety replaces the thiophene ring originating the fused azaindole system endowed with anticancer activity viz 8 Cancer is one of the most common causes of morbidity representing an important social problem supposed to maintain its primacy after heart and circulatory disorders, with many new cases in the world per year ,2. In moH-thienopyrrolizinones, also known as “tripentones” , showed high cytotoxicity pyrrolizin-9-one derivative, showed significant antitumor activity in different human tumor cell lines with IC50 values in the submicromolar or nanomolar range. Biological studies performed on this compound demonstrated that it acts as a pro-apoptotic agent, causing cell cycle arrest at the G2/M phase and increasing caspase 3 activity. Further studies, performed in order to clarify its mechanism of action, showed that it causes the inhibition of tubulin polymerization with an IC50 value of 2.9 µM, as well as the activation of several kinases including FLT3-ITD pyrrolizin-9-one 9a  in toluene (4 mL), the opportune heteroaryl carbohydrazide (0.29 mmol) was added. The resulting suspension was refluxed, using Dean-Stark apparatus, for 24–32 h and then chilled overnight. The product was collected by filtration, washed with toluene, and dried under vacuum to afford compound 9e. In the case of derivatives 9b,d,h, the reaction mixture was quenched with a small amount of water, then extracted with DCM (3 × 10 mL), dried over anhydrous Na2SO4, and evaporated in vacuo. It was purified by silica gel column chromatography eluting by DCM:MeOH, 97:3 (9b), DCM (9d), and DCM:ethyl acetate, 90:10 (9h) to give the desired compound.To a solution of 9N′-pyrrolizin-9-ylidene]pyridine-4-carbohydrazide (9b): Conditions: reflux for 24 h. Yield: 69%, light brown powder; m.p.: 244.0–244.4 °C; IR: 3402 (NH), 1683 (CO) cm−1; 1H-NMR  δ: 6.41 , 6.89 , 7.10 , 7.38 , 7.57 , 7.88 , 8.38 , 8.85 , 14.15 ; 13C-NMR  δ: 109.8 (d), 115.9 (d), 116.3 (d), 117.5 (d), 121.3 (d × 2), 125.1 (d), 130.6 (s), 136.2 (s), 139.7 (s), 140.1 (s), 143.5 (d), 147.5 (s), 150.8 (d × 2), 162.0 (s). Anal. Calculated for C16H11N5O (MW: 289.29): C, 66.43; H, 3.83; N, 24.21%. Found: C, 66.21; H, 3.68; N, 24.36%.N′-pyrrolizin-9-ylidene]furan-2-carbohydrazide (9d): Conditions: reflux for 32 h. Yield: 72%, yellow oil; IR: 3317 (NH), 1680 (CO) cm−1; 1H-NMR  δ: 6.39 , 6.60 , 6.85 , 7.08 , 7.36 , 7.55 , 7.63 , 8.40 , 13.95 ; 13C-NMR  δ: 109.0 (d), 112.3 (d), 115.4 (d), 115.9 (d), 116.1 (d), 117.1 (d), 124.6 (d), 130.9 (s), 135.9 (s), 138.6 (s), 143.5 (d), 145.0 (d), 147.1 (s), 147.4 (s), 155.2 (s). Anal. Calculated for C15H10N4O2 (MW: 278.27): C, 64.74; H, 3.62; N, 20.13%. Found: C, 64.89; H, 3.58; N, 20.07%.N′-pyrrolizin-9-ylidene]thiophene-2-carbohydrazide (9e): Conditions: reflux for 24 h. Yield: 70%, dark yellow powder; m.p.: 163.0–163.7 °C; IR: 3428 (NH), 1656 (CO) cm−1; 1H-NMR  δ: 6.44 , 6.75 , 7.30 , 7.64–7.58 , 7.90–7.84 , 8.00 , 8.12 , 8.50 , 13.93 ; 13C-NMR  δ: 107.5 (d), 115.4 (d), 117.7 (d), 119.1 (d), 125.3 (s), 125.8 (d), 128.2 (d), 129.0 (d), 129.6 (d), 132.8 (s), 135.7 (s), 137.2 (s), 143.9 (d), 146.1 (s), 164.7 (s). Anal. Calculated for C15H10N4OS (MW: 294.33): C, 61.21; H, 3.42; N, 19.04%. Found: C, 61.35; H, 3.28; N, 19.14%.N′-pyrrolizin-9-ylidene]benzohydrazide (9h): Conditions: reflux for 27 h. Yield: 76%, yellow powder; m.p.: 177.2–178.0 °C; IR: 3393 (NH), 1681 (CO) cm−1; 1H-NMR  δ: 6.44 , 6.74 , 7.70–7.53 , 7.98 , 8.11 , 8.50 , 13.90 ; 13C-NMR  δ: 107.7 (d), 115.4 (d), 117.7 (d), 119.1 (d), 125.7 (d × 2), 127.2 (d), 129.1 (d × 2), 129.7 (s), 132.6 (d), 133.2 (s), 135.7 (s), 138.3 (s), 143.9 (d), 146.2 (s), 162.3 (s). Anal. Calculated for C17H12N4O (MW: 288.30): C, 70.82; H, 4.20; N, 19.43%. Found: C, 70.75; H, 4.31; N, 19.29%.H-pyridopyrrolizin-9-one 9a  in anhydrous ethanol (5 mL), the opportune heteroaryl carbohydrazide (0.38 mmol) was added. The resulting solution was heated to reflux for 24 h and then chilled overnight. The product was collected by filtration, washed with cold ethanol, and dried under vacuum to afford the desired compound 9f,g, or purified by silica gel column chromatography using DCM:ethyl acetate, 75:25, as an eluent to give product 9c.To a solution of 9N′-pyrrolizin-9-ylidene]pyridine-3-carbohydrazide (9c): Yield: 93%, yellow powder; m.p.: 182.5–182.7 °C; IR: 3385 (NH), 1684 (CO) cm−1; 1H-NMR  δ: 6.44 , 6.77 , 7.70–7.57 , 8.11 , 8.32 , 8.49 , 8.85 , 9.13 , 13.92 ; 13C-NMR  δ: 108.2 (d), 115.3 (d), 115.5 (d), 117.9 (d), 118.0 (d), 119.2 (d), 123.9 (d), 125.9 (d), 128.5 (s), 129.6 (s), 135.8 (s), 144.0 (d), 145.5 (s), 146.4 (s), 148.8 (d), 161.0 (s). Anal. Calculated for C16H11N5O (MW: 289.29): C, 66.43; H, 3.83; N, 24.21%. Found: C, 66.28; H, 3.76; N, 24.11%.4-Amino-N′-pyrrolizin-9-ylidene]benzohydrazide (9f): Yield: 60%, yellow powder; m.p.: 276.1–277.0 °C; IR: 3447–3337 (NH2), 3227 (NH), 1662 (CO) cm−1; 1H-NMR  δ: 6.07 , 6.48 , 6.72 , 7.64 , 7.74 , 8.15 , 8.56 , 13.73 ; 13C-NMR  δ: 106.7 (d), 113.1 (d × 2), 115.3 (d), 117.0 (d × 2), 118.5 (s), 118.9 (d), 125.3 (d), 129.1 (d), 130.1 (s), 135.4 (s), 136.5 (s), 143.9 (d), 146.2 (s), 152.9 (s), 162.3 (s). Anal. Calculated for C17H13N5O (MW: 303.32): C, 67.32; H, 4.32; N, 23.09%. Found: C, 67.45; H, 4.15; N, 23.31%.4-Hydroxy-N′-pyrrolizin-9-ylidene]benzohydrazide (9g): Yield: 90%, light yellow solid; m.p.: 294.5–295.3 °C; IR: 3415 (NH), 3201 (OH), 1675 (CO) cm−1; 1H-NMR  δ: 6.43 , 6.70 , 6.95 , 7.60 , 7.85 , 8.08 , 8.51 , 10.35 ,13.78 ; 13C-NMR  δ: 107.2 (d) 115.3 (d), 115.7 (d × 2), 117.3 (d), 118.9 (d), 123.0 (s), 125.5 (d × 2), 129.4 (d), 129.9 (s), 135.5 (s), 137.4 (s), 143.9 (d), 146.2 (s), 161.3 (s), 162.0 (s). Anal. Calculated for C17H12N4O2 (MW: 304.30): C, 67.10; H, 3.97; N, 18.41%. Found: C, 67.43; H, 3.85; N, 18.08%.8 cells/L in a humidified 5% CO2 atmosphere at 37 °C. In all experiments, HCT-116 and MCF-7 cells were left to incubate overnight to allow adhesion before treatment with the compounds or vehicle alone (control cells), while Caco-2 cells were treated 15 days after confluence, at which time the cells were differentiated in normal intestinal-like cells [Tripentone derivatives, prepared as described above, were dissolved in dimethyl sulfoxide (DMSO) and then diluted in culture medium to obtain a DMSO concentration not exceeding 0.1%. HCT-116 , MCF-7 (human breast cancer), and Caco-2  cell lines were purchased from American Type Culture Collection, Rockville, MD, USA and grown in Dulbecco’s Modified Eagle’s Medium(DMEM) supplemented with 10% fetal, 10% fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 μg/mL), and gentamicin (5 μg/mL). Cells were maintained in log phase by seeding twice a week at a density of 3 × 10ke cells .No differences were found between cells treated with DMSO 0.1% and untreated cells in terms of cell number and viability.H-tetrazolium (MTT) to purple formazan by mitochondrial dehydrogenases [4 cells/well in 96-well plates containing 200 μL DMEM. When appropriated, monolayer cultures were treated for 72 h with various concentrations (0.1–100 μM) of the tested compounds. Then cells were washed with fresh medium and 50 μL FBS-free medium containing 5 mg/mL MTT. Cells were incubated for 2 h at 37 °C, then the medium was discarded by centrifugation, formazan blue formed in the cells dissolved in DMSO, and absorbance was measured at 570 nm in a microplate reader . Formazan of control cells was taken as 100% viability. The growth inhibition activity of compounds was defined as the GI50 value, which represents the log of the molar concentration of the compound that inhibits 50% cell growth. Each experiment was repeated three times in triplicate.Cytotoxic activity of the tripentone derivatives was determined by the colorimetric assay based on the reduction of 3-bromide-2,5-diphenyl-2ogenases . Briefly6 cells/ mL with combining buffer according to the manufacturer’ instructions . One hundred microliters of cell suspensions were added to a new tube, and incubated with Annexin V-FITC and PI solution at room temperature in the dark for 15 min. Then samples of at least 1.0 × 104 cells were subjected to fluorescence-activated cell sorting (FACS) analysis by an Epics XL™ flow cytometer  using Expo 32 ACD software , using the appropriate bi-dimensional gating method.The apoptosis-induced PS externalization to the cell surface was measured using flow cytometry by double staining with Annexin V-Fluorescein isothiocyanate (Annexin V-FITC)/propidium iodide (PI). Annexin V binding to phosphatidylserine was used to identify the earliest stage of apoptosis. PI, which does not enter cells with intact membranes, was used to distinguish between early apoptotic cells (annexin V-FITC positive and PI negative), late apoptotic cells (annexin V-FITC/PI-double positive), and necrotic cells (annexin V-FITC negative and PI positive). After 24 h treatment, HCT-116 and MCF-7 cells were harvested by trypsinization and adjusted at 1.0 × 104 cells/cm2) were seeded in triplicate in 24-wells culture plates. After an overnight incubation, the cells were washed with fresh medium and incubated with compound 9h in DMEM for 24 h. Then cells were harvested by trypsinization. Aliquots of 1.0 × 106 cells were washed with PBS and incubated in the dark in a PBS solution containing 20 μg/mL PI and 200 μg/mL RNase, for 30 min at room temperature. Then samples of at least 1.0 × 104 cells were subjected to FACS analysis.The cell cycle stage was analyzed by flow cytometry. HCT-116 and MCF-7 cells . All synthesized derivatives were tested for cytotoxicity against two human tumor cell lines, HCT-116  and MCF-7 (human breast cancer), by MTT assay. On the basis of the obtained results, the most active derivative was further investigated to study its mode of action. Flow cytometric analysis showed that it did not exert necrotic effects, but induced a clear shift of viable cells towards early apoptosis, causing the inhibition of the cell cycle progression in S and G2/M phases.


Holostylis reniformis (Aristolochiaceae) yielded three new natural sesquiterpenes, a sesquiterpene with an unusual carbon skeleton, 4,5-seco-guaiane , a nine-membered lactone with new carbon skeleton , and a new megastigmane , together with bulnesol and sitosterol-3-O-β-D-glucopyranoside. The structures of these compounds were determined by spectroscopic analyses and B3LYP/STO-3G** theoretical studies. Root extracts of  Holostylis reniformis Duch. (Aristolochiaceae) has been used in traditional Brazilian medicine as an antirheumatic, stomachic, and depurative +. The 13C-NMR spectra of 1 showed signals for 15 carbons, including two carbonyl groups at δ 213.4 and 208.8, the latter of which corresponded to a methyl ketone , and a quaternary carbinolic carbon (δ 73.0) , and gHMQC experiments. The gHMBC spectrum showed correlations between the carbonyl carbon signal at δC 208.8 (C-4) and the methyl hydrogens at δH 2.04 (3H-15) and the methylene hydrogens at δH 2.37 and 2.19 (2H-3), whereas, the carbonyl carbon signal at δC 213.4 (C-5) was correlated to H-6β (δH 2.13). Both C-7 (δC 46.4) and C-11 (δC 73.0) were shown to be correlated to the hydrogens of two methyl groups (δH 1.13 and 1.10). 1H–1H COSY experiment showed couplings between H-7 (δH 1.84) and H-6α (δH 2.52), H-6β (δH 2.13), H-8α (δH 1.75), and H-8β (δH 1.10), as well as between H-3a (δH 2.37) and H-3b, (δH 2.19), H-2a (δH 1.41), and H-2b (δH 1.13). The latter  and H-8β were correlated to C-10 (δC 34.9), as well as 3H-14 (δH 0.70) were correlated to C-1 (δC 53.8) and C-9 (δC 37.0) by gHMBC experiments. Moreover, selective irradiation at δH 2.84 (H-1α) simplified the signals at δH 1.41 (H-2a), 1.13 (H-2b), and 1.93 (H-10), and evidenced a cis relationship between H-1 and H-10 (J = 2.5 Hz). Long-range coupling between H-6α (δH 2.52) and H-8α (δH 1.75) was also observed (J = 1.7 Hz) by the selective irradiation of these hydrogen frequencies. Based on all these data a 4,5-seco-guaiane structure can be suggested for 1, i.e., a bulnesol-type structure that was oxidized at C-4 and C-5. Spatial interactions between H-1 and H-2a, H-6α, H-7, H-9α, and H-10, as well as between H-8β and H-6β and 3H-14 observed by 1D-NOESY experiments allowed us to determine the conformation and the relative configuration as 1S,7R,10S (H 1.10) is consistent with an endocyclic hydrogen protected by the carbonyl group at C-5. The spatial interactions between the methyl hydrogens 3H-14 and H-2a, H-8β, and H-9β confirmed this relative configuration.HRMS of compound (δ 73.0) . The preS,7R,10S . The higepi isomer  and m/z. 269.1745 [M+H]+ . From these data, a total of four hydrogen deficiencies were determined for the structure, one of which was a ring, since no additional sp2 carbon was observed in the 13C-NMR spectrum. In addition, a carbinolic carbon (δC 85.0), five methylenes , two methine groups , and four methyl groups  were observed by an analysis of the 1H and 13C-NMR spectra, DEPT 135°, gHSQC, and gHMQC experiments , and gHMBC experiments allowed us to determine the chemical shifts of the remaining hydrogens  (gHMBC spectrum showed correlations between the carbonyl carbon at δC 208.0 (C-4) and the methyl hydrogens at δH 2.10 (3H-15) and the methylene hydrogens at δH 2.51 (2H-5). These latter were also correlated to the methine carbon at δC 49.2 (C-7). The carbinolic carbon and C-7 correlated with methyl hydrogens at δH 1.57 (3H-12) and 1.26 (3H-13) as well as with H-8α (δH 1.96). Selective irradiation at δH 2.26 (H-7) led to the simplification of the multiplets at δH 2.53 (H-8β), 1.96 (H-8α), 1.57 (H-6a), and 1.23 (H-6b). 1H–1H COSY experiment showed couplings between 2H-5 (δH 2.51) and H-6a and H-6b, as well as between H-10 (δH 2.71) and 3H-14 (δH 0.97), H-1α (δH 2.06), and H-1β (δH 1.51). 1D-TOCSY experiments evidenced long-distance coupling between H-10 and 2H-2 (δH ~2.2). The gHMBC spectra also showed correlations between the carbonyl carbon at δ 215.4 (C-9) and H-8α and 3H-14, which, in turn, were correlated to C-1 and C-10. The chemical shifts of the carbinolic carbon and the acyl group suggested the presence of a lactone. Based on all of these data, and with the aid of 1D-TOCSY experiments, a nine-membered lactone structure, with a 3-oxobutyl substituent, was established for 2. Moreover, the coupling constant values (J) for the hydrogens that showed virtual couplings  . The gHM and C-3 .3 showed a quasi-molecular ion [M+Na]+ at m/z 307.1525 (C15H24O5+Na). The 1H and 13C-NMR spectra of 3 , a methyl group  linked at the β position of an α,β-unsaturated carbonyl group , and a trans olefin  linked to carbinolic carbons . These data, together with the results of gCOSY, gHMQC, gHMBC, and gNOESY experiments, led to a megastigmane carbon skeleton for 3. Indeed, the 1H and 13C-NMR data of 3 . The absolute configuration of cucumegastigmane I was determined by using a modified Mosher’s method and by application of the circular dichroism (CD) helicity rule on analogous compounds for determination of C-6 configuration . The residues were extracted with ethanol in a Soxhlet apparatus and the extracts were individually concentrated. The crude hexane extract (6.17 g) was subjected to CC  to give 28 fractions (3CN and the soluble-portion was subjected to HPLC (MeOH-H2O 7:3) to give 3 (11.6 mg). Fraction 23 (9.0 mg) was washed with MeOH and the resulting soluble-portion gave sitosterol-3-O-β-D-glucopyranoside (3.0 mg).The acetone extract from roots (3.73 g) was also subjected to CC to give 26 fractions, as previously described . Fractiorel.--6-(2-Hydroxypropan-2-yl)-3-methyl-2-(3-oxobutyl)cycloheptanone : Colorless oil; + (C15H26O3+Na) (94) , 237.18 [M+H−H2O]+ (100); ESIMS 20 eV, positive mode, m/z (rel. int.): 237 (100), 235 (35).rel.--5,9,9-Trimethyl-8-(3-oxobutyl)oxonane-2,6-dione : Colorless oil; +  (60); ESIMS 20 eV, positive mode, m/z (rel. int.): 269 (100), 251 (55), 233, [M+Na−H3CCOCH3] (18).-6,9-Dihydroxy-10-(2'-hydroxyethoxy)-4,7-megastigmadien-3-one : Yellow oil; + (100) ; ESIMS 20 eV, positive mode, m/z (rel. int.): 307 (38), 277 (87), 275 (100), 237 (43), 205 (47), 130 (24). CD : [θ]221 0, [θ]243 + 7590, [θ]271 0, [θ]312−1815.H. reniformis can synthesize a variety of seco compounds, including lignans and sesquiterpenes. Among them, bulnesol and two unusual 4,5-seco-guainanes, which may be biogenetic derivatives of hedycaryol, were isolated from the roots of this species. In addition, a new megastigmadienone was isolated.


Their structures were elucidated on the basis of physical and spectral techniques, besides comparison with literature data. Compounds 1–3 showed inhibitory activities against the A549, HEPG2, and B16F10 cell lines.Three new ursane-type triterpenoids, 3 Saprosma belongs to the family Rubiaceous which has about 50 species found around the world, and has been used as a traditional medicine for the treatment of fever and lumbocrural pain −, 513.3 [M+Na]+; HRESIMS m/z: 513.3559 [M+Na]+ .Compound 2: white amorphous solid;  −20.73 ; IR (KBr) υmax 3440, 2948, 2873, 1704, 1636, 1458, 1387, 1174, 1070, 1032, 986, 928 cm–1; 1H- and 13C-NMR data see m/z: 487.6 [M−H]−, 511.5 [M+Na]+; HRESIMS m/z: 511.3389 [M+Na]+ .Compound 3: white amorphous solid;  –37.84 ; IR (KBr) υmax 3442, 2951, 2875, 1704, 1647, 1458, 1384, 1229, 1176, 1110, 1035, 920 cm–1; 1H- and 13C-NMR data see m/z: 505.7 [M−H]−, 529.5 [M+Na]+; HRESIMS m/z: 529.3501 [M+Na]+ .Compound 1–3 were dissolved in DMSO . The cytotoxicity of compounds 1–3 against A549, MDA-MB-231, HEPG2 and B16F10 was determined by standard MTS assay  reagent and incubated for 3 h. Cell viability was determined by measuring the optical density at 490 nm using a Biotek microplate reader . Untreated cells in medium were used as control. Corresponding groups without cells were used as blanks. All experiments were carried out with four replicates.Test compounds S. merrillii afforded three new triterpenoids: 3α,6α,30-trihydroxy-ursan-28-oic acid (1), 3α,30-dihydroxy-6-oxo-ursan-28-oic acid (2), 3α,6α,7α,30-tetrahydroxy-ursan-28-oic acid (3), together with one known triterpenoid, betulinic acid (4) [5) [6) [p-hydroxybenzoic acid (7) [8) [9) [10) and daucosterol (11). Compound 1 exhibited weak cytotoxicity against the B16F10 cell line with an IC50 value of 72.72 μM, while 2 inhibited the proliferation of the A549 cell line with an IC50 value of 24.66 μM  , one kno (4) [5) , four kn [5) [6) , p-hydroacid (7) , syringi (7) [8) , isovani [8) [9) , two ste24.66 μM .


O-ethylmanoalide (3), was isolated from the Indian Ocean sponge Luffariella cf. variabilis, together with the known compounds manoalide (1), seco-manoalide, manoalide monoacetate and 24-O-methyl-manoalide (2). The structure of compound 3 was elucidated by interpretation of its spectroscopic data.A new manoalide-related sesterterpene, 24- Luffariella . The ether linkage between C-24 and C-26 was suggested by the 13C-NMR chemical shift of C-24 which resonated at a lower field  than the C-24 of (1) bearing an hydroxyl group . These data suggested structure 3 for 24-O-ethylmanoalide  25D + 63° ; IR (CHCl3) νmax 3410, 2925, 1790, 1762, 1098, 1040 cm-1; 1H- and 13C-NMR, see m/z 444 [M] + (22), 426 (3), 398 (9), 380 (2), 261 (5), 203 (4), 177 (6), 137 (100), 123 (12), 121 (12), 107 (9), 95 (26), 81 (22).


Their ability to inhibit inflammatory responses elicited in peritoneal macrophages by TLR ligands was investigated. Compounds 5 and 6 showed significant anti-inflammatory effects, as they inhibited the protein expression of nitric oxide synthase (NOS-2), cyclooxygenase-2 (COX-2), and cytokine production  induced by the ligand of TLR4, lipopolysaccharide (LPS), acting at the transcriptional level. Some structure–activity relationships were outlined. Compound 5 was selected as a representative compound and molecular mechanisms involved in its biological activity were investigated. Inhibition of NF-κB and p38 signaling seems to be involved in the mechanism of action of compound 5. In addition, this compound also inhibited inflammatory responses mediated by ligands of TLR2 and TLR3 receptors. To rationalize the obtained results, molecular docking and molecular dynamic studies were carried out on TLR4. All these data indicate that solidagenone derivative 5 might be used for the design of new anti-inflammatory agents.A series of nine derivatives ( Inflammation is a protective physiological response of body tissues to harmful stimuli such as microbial infection or tissue injury. Nevertheless, an imbalance in the mechanisms that govern this process lead to the development of several pathologies ,2. The tThe inflammatory response is initiated by the cellular sensing of either pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) through pattern recognition receptors (PRRs), such as Toll-like receptors (TLR), which trigger specific signaling pathways. During the inflammatory process, macrophages, being critical role players, dominate in the context of tissue injury and in the subsequent tissue repair ,4. Upon Solidago chilensis, a plant traditionally used to treat symptomatologies related to inflammation [Traditionally, natural products have represented an excellent resource for drug discovery and development. Natural product chemistry has been an effective strategy for the identification of bioactive lead candidates. An interesting group of natural products are the diterpenes, as they possess a plethora of biological activities and display many pharmacological properties with therapeutic potential ,12,13,14ammation .2−10) in peritoneal macrophages.Therefore, as part of our ongoing screening program to evaluate the potential of diterpenes as a source of novel bioactive compounds in inflammation-based diseases, a series of diterpenes were semisynthesized from the natural diterpene solidagenone. Herein, we report the preparation and anti-inflammatory effects of these solidagenone derivatives  phosphorylation.Among the derivatives evaluated, solidagenone derivative 1), aiming to obtain new derivatives and to evaluate the role of the different functional groups and moieties present in the structure of 1 . Compound 2 has three fewer carbons than 1, and it is probably formed by rupture of the furan ring followed by reductive cyclization. The existence of the lactone moiety was supported by the presence of a carbonyl group at 176.0 ppm, a quaternary oxygenated carbon at δ 90.0, and two methylene carbons at δ 30.9 and 23.9, respectively. The HSQC and HMBC (2) in the corresponding spectra ratified its structure.Thus, the treatment of solidagenone with NaBHand HMBC  correlat1 was treated with AcCl/Py, the two compounds (3) and (4) were formed. A plausible mechanism of formation involves the acetylation of the hydroxyl group and elimination of the corresponding acetyl group via 1,2-methyl shift to afford (3). An intramolecular Michael addition of the furan ring on the α,β-unsaturated carbonyl system could explain the formation of the tetracyclic compound (4) (When ound (4) .3 were analyzed in order to corroborate this plausible mechanism. One of them  and (6) were formed. The regiosubstitution of the furan ring was determined by an HMBC experiment. Thus, the following HMBC correlations: H-14 with C-13, C-15, and C-16; and H-12 with C-13, C-14, and C-16 located the two bromine atoms at the carbons C-15 and C-16. The third bromine was located at C-7 in compound 6, since a singlet was observed at δ 4.37, which showed HMBC correlations with the carbonyl carbon at δ 203.3 (C-6) and with the olefinic carbons C-8 and C-9. The relative orientation was determined as β on the basis of the ROEs detected in the ROESY spectrum. The reaction of 1 with BBr3 led to the monobrominated derivative (7), which showed similar NMR data to compound 6, but corresponding to the furan ring. A similar chlorine derivative (8) was obtained when 1 was reacted with thionyl chloride in DCM. The treatment of 1 with NaH in THF led to derivative (9). The hydrogenation of 1 yielded the corresponding derivative (10) with the furan ring reduced to tetrahydrofuran. The orientation α of the methyl group at C-8 was established on the basis of the NOE effect between the multiplet at δ 2.34  and the singlet at δ 1.03 .When 1–10) regulate the inflammatory response, we investigated the anti-inflammatory effects and underlying mechanisms of action of these derivatives in peritoneal macrophages. We first identified compounds which can inhibit the inflammatory response induced by bacterial lipopolysaccharide (LPS), a well-characterized ligand of TLR4 in murine peritoneal macrophages. For this, cells were preincubated with diterpenes for 30 min before their stimulation with LPS (200 ng/mL), and NO production was assayed. Initial testing of the compounds at a fixed concentration of 20 µM revealed that all the derivatives exhibited significant inhibitory effects on NO release. Four compounds  were able to inhibit NO release by more than 50% .To discard the possibility that the inhibitory effect on NO release was due to cytotoxicity, we analyzed the percentage of cell viability by MTT -2,5-diphenyltetrazolium bromide) assay. As shown in 4 with that of compound 5, the presence of bromines in the furan ring is associated with a lower percentage of NO release. The replacement of a bromine group at C-7 (7) by a chlorine group (8) led to a lower inhibition of NO production.From the obtained results, some structure–activity relationships were outlined. It was thus found that the presence of bromine groups is important for the activity in the bicyclic derivatives. If we compare the inhibition of NO release of compound 5 and 6 were selected for further evaluation. Studies of the dose–response effects of compounds 5 and 6 on NO release showed NO inhibitory effects with IC50 values of 9.1 ± 1.51 and 14.52 ± 1.03 µM, respectively , interleukin (IL)-6, and IL-12. All cytokines were downregulated at the mRNA level in the presence of compound 5 after stimulation with LPS  proteins, leading to the translocation of p65/p50 heterodimers to the nucleus. Interestingly, this transcription factor has been a common target in the mechanism of action of some diterpenes ,17,18,19rylation , whereas5 exhibits inhibitory effects against other TLRs, we examined the response of macrophages to the TLR3 ligand polyriboinosinic–polyribocytidylic acid (polyI:C) and to the TLR2 ligand lipoteichoic acid (LTA).Pharmacological regulation of TLR signaling is a promising therapeutic strategy for various inflammatory and autoimmune diseases ,24,25. O5. COX-2 expression induced by LPS and LTA was also inhibited by compound 5. These results suggest that this diterpene has a broad suppressive effect on TLR-mediated inflammatory responses. Considering the pathological role of TLRs in inflammatory diseases, inhibitors targeting TLR signaling may emerge as novel therapeutics to treat these disorders [The accumulation of the proinflammatory mediator NO in the culture medium was also dose-dependently (5–20 µM) inhibited, regardless of ligand treatment A. The doisorders .5 and 6, we carried out a molecular docking study using the Glide software [5 and 6, we simulated docking of the compounds into the large hydrophobic binding pocket of MD-2 to see if these compounds could interact with this target, and consequently, to understand the possible binding mode and key active site interactions. An analysis of the docking results showed that the compounds fit very well and could be inserted into the large hydrophobic binding pocket of MD-2 and easily occupy a large portion of the LPS binding site. In addition, these docking results strongly suggested that the solidagenone derivatives 5 and 6 share a common binding mode into the hydrophobic binding pocket of MD-2. The best docking scores were found in the range from −6.83 to −8.51 kcal mol−1. According to the predicted binding modes, the most active compounds showed a π–π stacking interaction between the furan ring present in these compounds and some amino acid residues. Hydrophobic interactions therefore probably play a dominant role in the interaction. In the favored docking conformation, many hydrophobic side-chain residues of the MD-2 pocket are in close proximity to the solidagenone derivatives.In order to propose a mode of action of the significant anti-inflammatory effects of the solidagenone derivatives software  on repor5), the furan ring showed a key π–π interaction with Phe 76 (5 forms a bulky hindrance that blocks the interaction of LPS with MD-2.In the predicted pose of the most active compound (h Phe 76 . There ah Phe 76 . Taken t5 with molecular dynamics (MD) simulation, and the protein–ligand interactions were investigated throughout the course of the MD simulations. The simulations provide minute detail about the movement of every particle/atom over time. This will help in answering the questions arising about the deviation and fluctuation patterns of the proteins.The docking protocols can be combined with accurate molecular dynamics (MD) simulation techniques to predict more reliable protein–ligand complex structures and confirm system stability. We explored the stability of the best docking pose of 5 with MD-2 was performed over 50 ns in an explicitly aqueous solution environment employing the Desmond Software , the OPL-2005 force field, and the TIP3P solvent model. Molecular dynamic simulations were performed to confirm the orientation of compound 5 in the binding site. The MD simulation results indicated that the compound does not leave the binding cavity of the protein and remains in a similar orientation during the entire simulation  parts per million and coupling constants (J) in hertz (Hz). 1H and 13C spectra were referenced using the solvent signal as the internal standard. HREIMS were recorded using a high-resolution magnetic trisector (EBE) mass analyzer. Analytical thin-layer chromatography plates used were POLYGRAM-SILG/UV. Preparative thin-layer chromatography was carried out with Analtech GF plates  using appropriate mixtures of ethyl acetate and hexanes. All solvents and reagents were purified by standard techniques as reported previously [1) was isolated from the air-dried rhizomes of Solidago chilensis Meyen (Asteraceae) as previously reported [NMR spectra were recorded in CDCleviously  or used reported .1HNMR (400 MHz) δ 1.00 , 1.14 , 1.16 , 1.19 , 1.34 , 1.52 , 1.57 , 1.77 , 1,90 , 2.01 , 2.65 , 2.70 , 5.70 , 6.30 , 7.25 , 7.37 ; 13C-NMR (100 MHz) δ 200.1 (C-6), 155.6 (C-8), 143.1 (C-15), 138.5 (C-16), 129.2 (C-7), 125.1 (C-13), 110.7 (C-14), 76.5 (C-9), 55.8 (C-5), 46.4 (C-10), 42.6 (C-3), 33.8 (C-19), 33.2 (C-11), 32.2 (C-4), 31.6 (C-1), 21.7 (C-18), 21.2 (C-12), 20.0 (C-20), 18.3 (C-17), 17.8 (C-2); HR-ESMS m/z 316.2047 .1 in 2 mL ethanol, a solution of 15 mg (2 equiv.) of NaBH4 in 2 mL of EtOH was added dropwise at rt. After 2 h stirring, the mixture was acidified with 10% HCl and extracted with DCM. The organic phase was dried with MgSO4, filtered, and concentrated. The residue was purified by preparative TLC using hexane/EtOAc 4:1 as the eluent to yield 8 mg (17% yield) of 2.To a solution of 55 mg (0.17 mmol) of 1H-NMR (400 MHz) δ 5.76 , 2.68 , 2.64 , 2.58 , 2.28 , 2.14 , 1.84 , 1.52 , 1.45 , 1.31 , 1.16 , 0.96 ; 13C-NMR (100 MHz) δ 198.9 (C-6), 176.0 (C-13), 149.3 (C-8), 130.9 (C-7), 90.0 (C-9), 56.5 (C-5), 44.5 (C-10), 42.1 (C-3), 33.1 (C-16), 32.2 (C-4), 31.5 (C-1), 30.9 (C-12), 23.9 (C-11), 21.6 (C-15), 18.8 (C-17), 18.1 (C-2), 17.5 (C-14); HR-ESMS m/z 299.1623 .1 in 2 mL of N,N-dimethylaniline was treated with an excess of AcCl (2 mL) and stirred at rt for 2 h. Then, ice was added and the reaction mixture was extracted with DCM. The solvent was evaporated and the residue was purified by preparative TLC, using hexane/EtOAc 4:1 as the eluent, to obtain 7 mg (15%) of compound 3 and 17 mg (37%) of compound 4.A solution of 50 mg (0.16 mmol) of Compound3: 1H-NMR (400 MHz) δ 7.33 , 7.14 , 6.18 , 6.14 , 2.34 , 2.20 , 1.95 , 1.92 , 1.85 , 1.63 , 1.47 , 1.32 , 1.30 , 1.20 ; 13C-NMR (100 MHz) δ 185.8 (C-6), 159.8 (C-8), 158.1 (C-10), 142.8 (C-15), 141.1 (C-5), 138.5 (C-16), 130.6 (C-7), 124.2 (C-13), 110.6 (C-14), 46.4 (C-9), 41.5 (C-11), 37.4 (C-3), 33.7 (C-4), 28.4 (C-19), 28.0 (C-18), 27.4 (C-1), 25.4 (C-20), 19.9 (C-12), 19.0 (C-2), 18.9 (C-17); HR-ESIMS m/z 321.1829 .Compound4: 1H-NMR (400 MHz) δ 7.21 , 6.10 , 2.73 , 2.52 , 2.42 , 2.32 , 1.95 , 1.84 , 1.58 , 1.42 , 1.24 , 1.21 , 1.14 , 1.06 ; 13C-NMR (100 MHz) δ 196.5 (C-6), 159.2 (C-10), 154.6 (C-5), 154.6 (C-16), 141.1 (C-15), 114.5 (C-13), 109.6 (C-14), 43.5 (C-9), 41.5 (C-3), 41.4 (C-7), 39.7 (C-8), 33.6 (C-4), 30.4 (C-11), 28.8 (C-19), 28.6 (C-18), 28.5 (C-1), 28.5 (C-20), 19.4 (C-12), 19.3 (C-2), 19.2 (C-17); HR-ESIMS m/z 321.1833 .1 in 3 mL of dry DCM was stirred with 32 μL (2 equiv.) of Br2 at rt for 15 h. Then, the solvent was evaporated and the residue was purified by preparative TLC, using hexane/EtOAc 9:1 as the eluent. Two products were obtained: 22 mg (15%) of 5 and 17 mg (11%) of 6.A solution of 100 mg (0.32 mmol) of 5: 1H-NMR (400 MHz) δ 6.15 , 2.32 , 2.22 , 1.96 , 1.82 , 1.67 , 1.51 , 1.32 , 1.31 , 1.23 ; 13C-NMR (100 MHz) δ 185.7 (C-6), 159.2 (C-8), 157.7 (C-10), 141.4 (C-5), 130.7 (C-7), 126.2 (C-15), 121.6 (C-13), 119.6 (C-16), 114.5 (C-14), 46.2 (C-9), 41.5 (C-11), 36.8 (C-3), 33.7 (C-4), 28.5 (C-19), 28.0 (C-18), 27.4 (C-1), 25.1 (C-20), 20.6 (C-12), 19.0 (C-2), 18.9 (C-17); HR-ESIMS m/z 477.0034 .Compound 6: 1H-NMR (400 MHz) δ 6.28 , 4.37 , 3.22 , 2.45 , 2.28 , 2.18 , 1.95 , 1.86 , 1.69 , 1.56 , 1.42 , 1.32 , 1.17 , 1.04 , 0.95 ; 13C-NMR (100 MHz) δ 203.3 (C-6), 147.7 (C-8), 126.4 (C-9), 126.3 (C-13), 121.8 (C-15), 119.6 (C-16), 114.4 (C-14), 57.7 (C-5), 54.9 (C-7), 46.3 (C-10), 41.8 (C-3), 36.1 (C-11), 31.9 (C-4), 31.6 (C-19), 28.8 (C-1), 25.0 (C-12), 22.0 (C-18), 21.5 (C-20), 18.6 (C-2), 17.4 (C-17); HR-ESIMS m/z 556.9216 .Compound 1 in 5 mL of dry DCM at 0 °C was treated with 0.63 mL (2 equiv.) of a 1 M solution of BBr3 in DCM. The mixture was stirred for 4 h. Then, the solvent was evaporated and the residue purified by preparative TLC using hexane/EtOAc 9:1 as the eluent to yield 24 mg (21%) of compound 7. 1H-NMR (400 MHz) δ 7.37 , 7.26 , 6.30 , 4.38 , 3.23 , 2.55 , 2.35 , 2.23 , 1.91 , 1.72 , 1.65 , 1.49 , 1.41 , 1.32 , 1.17 , 1.01 , 0.96 ; 13C-NMR (100 MHz) δ 203.5 (C-6), 148.8 (C-8), 142.9 (C-15), 138.5 (C-16), 125.7 (C-9), 124.4 (C-13), 110.6 (C-14), 57.8 (C-5), 55.3 (C-7), 46.3 (C-10), 41.8 (C-3), 36.1 (C-11), 31.9 (C-4), 31.7 (C-19), 29.6 (C-1), 24.6 (C-12), 22.0 (C-18), 21.5 (C-20), 18.6 (C-2), 17.4 (C-17); HR-EIMS m/z 378.1122 .A solution of 100 mg (0.32 mmol) of 1 in 5 mL of dry DCM, 92 μL (4 equiv.) of SOCl2 was added under a dry N2 atmosphere. The mixture was refluxed for 15 h until disappearance of the starting diterpene. At this point, the mixture was cooled, the solvent was evaporated, and the residue was purified by preparative TLC using hexane/EtOAc 9:1 as the eluent to obtain 24 mg (25%) of compound 8. 1H-NMR (400 MHz) δ 7.37 , 7.26 , 6.30 , 4.10 , 3.04 , 2.55 , 2.37 , 2.25 , 1.95 , 1.82 , 1.65 , 1.57 , 1.40 , 1.32 , 1.17 , 1.02 , 0.97 ; 13C-NMR (100 MHz) δ 203.9 (C-6), 148.5 (C-8), 142.9 (C-15), 138.5 (C-16), 125.5 (C-9), 124.5 (C-13), 110.6 (C-14), 62.6 (C-7), 58.2 (C-5), 46.2 (C-10), 41.8 (C-3), 36.3 (C-11), 31.9 (C-4), 31.8 (C-19), 29.4 (C-1), 24.9 (C-12), 21.9 (C-18), 21.1 (C-18), 18.6 (C-2), 16.6 (C-17); HR-EIMS m/z 334.1694 .To a solution of 100 mg (0.316 mmol) of 1 in 5 mL of dry THF, 26 mg (2 equiv.) of NaH was added under a N2 atmosphere. The mixture was refluxed for 15 h, then MeOH was added and the solvent was evaporated under vacuum. The residue was purified by preparative TLC, using hexane/EtOAc 9:1 as the eluent, to yield 19 mg (20%) of compound 9. 1H-NMR (400 MHz) δ 7.39 , 7.29 , 6.99 , 6.33 , 2.65 , 2.57 , 2.05 , 1.99 , 1.87 , 1.72 , 1.56 , 1.43 , 1.39 ; 13C-NMR (100 MHz) δ: 181.6 (C-6), 165.8 (C-7), 143.1 (C-15), 140.4 (C-5), 140.4 (C-10), 138.6 (C-16), 127.3 (C-8), 124.3 (C-13), 110.5 (C-14), 43.8 (C-9), 37.2 , 35.6 (C-4), 31.5 (C-1), 29.4 (C-3), 28.0 (C-19), 27.9 (C-18), 27.6 (C-20), 23.7 (C-12), 17.2 (C-2), 11.5 (C-17); HR-ESMIS m/z 337.1776 .To a solution of 100 mg (0.32 mmol) of 1 in 5 mL of DCM/MeOH 1:1 was hydrogenated in the presence of a catalytic amount of Pd/C 10% for 15 h. Then, the mixture was filtered and the solvent was evaporated under vacuum and purified by preparative TLC, using hexane/EtOAc 4:1 as the eluent, to yield 26 mg (25%) of 10.A mixture of 100 mg (0.32 mmol) of 1H-NMR (400 MHz), δ (ppm): 3.92 , 3.87 , 3.76 , 3.36 , 2.99 , 2.96 , 2.34 , 2.14 , 2.08 , 1.95 , 1.77 , 1.52 , 1.25 , 1.25 , 1.08 , 1.03 , 1.00 , 0.95 ; 13C-NMR (100 MHz) δ: 212.8 (C-6), 77.9 (C-9), 73.3 (C-16), 67.8 (C-15), 57.9 (C-5), 48.7 (C-10), 46.9 (C-7), 42.0 (C-3), 40.9 (C-8), 39.9 (C-13), 32.8 (C-19), 32.6 (C-11), 32.4 (C-14), 32.1 (C-4), 31.1 (C-1), 26.6 (C-12), 21.8 (C-18), 18.8 (C-20), 18.5 (C-17), 17.8 (C-2); HR-EIMS m/z 322.2514 .Peritoneal macrophages were obtained from Male BALB/c mice free of pathogens (8–10 weeks old) from Charles River Laboratories. All experimental protocols were reviewed and approved by the Institutional Committee for Research Ethics and were in accordance with Spanish and European guidelines legislation.Four days prior to the assay, mice were injected intraperitoneally (i.p.) with 2.5 mL of thioglycolate broth. Elicited peritoneal macrophages were obtained as previously described . Cells w5 cells/well), stimulated with LPS (200 ng/mL), and incubated with the compounds for 24 h. Nitrite levels in supernatants were measured using the Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphtylethylenediamine dihydrochloride). Briefly, 100 µL of supernatant was mixed with an equal volume of Griess reagent and incubated at room temperature for 10 min. Absorbance was then measured at 540 nm using a microplate reader  and compared with a sodium nitrite standard curve.Peritoneal macrophages were seeded on a 96-well plate  was added to the cells and the cells were incubated at 37 °C for 4 h. The reaction product, formazan, was extracted with dimethyl sulfoxide (DMSO) and the absorbance was read at 540 nm. Results are expressed as the percent reduction in cell viability compared to untreated control cultures for at least three independent experiments.Cytosolic extracts were prepared as previously described . ProteinTotal RNA was isolated from cells with Trizol reagent (Invitrogen). Quantitative PCR (SYBRgreen) analysis was performed with an ABI7700 sequence analyzer as described . Each sa3 with default parameters and without constrains. The three-dimensional structures of the ligands to be docked were generated and prepared using LigPrep as implemented in Maestro 11.5  to generate the most probable ionization states at pH 7 ± 1 . These conformations were used as the initial input structures for the docking study. In this stage, a series of treatments were applied to the structures. Finally, the geometries were optimized using the OPLS3 force field. These conformations were used as the initial input structures for the docking study. The ligands were docked using the extra precision mode (XP) [The X-ray coordinates of human TLR4 in complex with MD-2 and LPS were extracted from the Protein Data Bank (PDB code 4G8A). The PDB structures were prepared for docking using the Protein Preparation Workflow  accessible from the Maestro program . The substrate and water molecules were removed beyond 5 Å, bond corrections were applied to the cocrystallized ligands, and an exhaustive sampling of the orientations of groups was performed. Finally, the receptors were optimized in Maestro 11.5 by using the OPLS3 force field before the docking study. In the final stage, the optimization and minimization of the ligand–protein complexes were carried out with the OPLS3 force field and the default value for RMSD of 0.30 Å for nonhydrogen atoms were used. The receptor grids were generated using the prepared proteins, with the docking grids centered on the center of the bound ligand for each receptor. A receptor grid was generated using a 1.00 van der Waals (vdW) radius scaling factor and 0.25 partial charge cutoff. The binding sites were enclosed in a grid box of 20 Åode (XP)  without ode (XP) ; GlideScode (XP) . The XP The Optimized Potentials for Liquid Simulations-2005 (OPLS2005)  force fit-test for comparison between two groups. For comparison between two or more groups, one-way ANOVA followed by Bonferroni’s post-hoc comparisons were used. Differences were considered significant at * p < 0.05. All statistical analyses were conducted using GraphPad Prism 5.0 . For Western blots, a linear correlation was observed between increasing amounts of input protein and signal intensity.All numerical data were presented as means ± SD for at least three independent experiments. Statistical significance was estimated by the Student’s 5 and 6) were promising diterpenes. Accordingly, their mechanism of action was studied in peritoneal macrophages. Compound 5 suppressed the expression of the inflammatory enzymes NOS-2 and COX-2 and cytokine production induced by LPS in macrophages through inhibition of NF-κB and p38 signaling. In addition, this compound also inhibited inflammatory responses mediated by ligands of TLR2 and TLR3. Docking studies revealed that derivatives 5 and 6 fit very well into the large hydrophobic binding pocket of MD-2 of TLR-4. Furthermore, molecular dynamics (MD) simulations showed that 5 was additionally stabilized by hydrogen bonds between the carbonyl group present in the solidagenone scaffold and the critical polar amino acid residues Ser 120 and Arg 90, which could explain the potency of this derivative. Taken together, our results suggest that diterpene 5 might be a promising therapeutic agent in drug development for inflammatory diseases.A series of solidagenone derivatives were semisynthesized. Initial screening for anti-inflammatory potential in terms of inhibition of NO production and cytotoxicity showed that two compounds (namely


The synthesis of a monosilylated Grubbs-Hoveyda ruthenium alkylidene complex is described, as well as the preparation and characterization of the corresponding material by sol-gel cogelification with tetraethoxysilane (TEOS) and the assay of this recyclable supported catalyst in ring-closing diene and enyne metathesis reactions under thermal and microwave conditions.  The efficiency of boomerang supported catalysts increases notably when Hoveyda-type ligands are involved (release-return mechanism) +: 723.1652; Found: 723.1668.Complex 4  in anhydrous dichloromethane (0.5 mL). The mixture was stirred under argon at room temperature for 4 days. The solvent was removed under vacuum. The residue was washed with anhydrous pentane to afford 5  as a green solid. 1H-NMR  δ (ppm): 16.44 , 7.06 , 6.67 , 6.45 , 4.80 2, J = 6.0 Hz), 4.17 , 4.03-3.92 , 3.81 , 3.17 3), 2.46 , 2.39 , 2.06-2.00 , 1.63 3), 1.22 2 (CH+ -O-CH2-3CH), 0.63 3). 13C-NMR  δ (ppm): 296.3 (Ru=CH), 211.4 (Ru-NHC), 154.3 (aromatic C), 146.8 (aromatic C), 145.6 (aromatic C), 138.9 (mesityl C), 129.5 (mesityl CH), 115.8 (aromatic CH), 113.2 (aromatic CH), 108.1 (aromatic CH), 75.0 (-CH(CH3)2), 66.7 (-O-2CH-CH2-CH2-O- or -O-CH2-CH2-2CH-O-), 60.5 (-O-CH2-CH2-2CH-O- or -O-2CH-CH2-CH2-O ), 58.6 (-O-2CH-CH3), 51.6 (-N-2-CH2CH-N-), 43.1 (-NH-2CH-CH2-CH2-Si(OEt)3), 32.2 (-O-CH2-2CH-CH2-O-), 22.4 (-NH-CH2-2CH-CH2-Si(OEt)3), 21.1 (mesityl CH3+ -CH3)2(CH), 18.4 (-O-CH2-3CH). 7.7 (-NH-CH2-CH2-2CH-Si(OEt)3). ESI-MS m/z (rel. int. %): [M–Cl]+ 912.3 (100), [M–2Cl]+ 876.4 (33.5). HR-FAB: Calcd for [C44H65Cl2N3O7RuSi]+: 947.3012; Found: 947.3004.Freshly distilled 3-isocyanatopropyltriethoxysilane  was added under argon to 2O, 0.08 mmol NH4F) and distilled and deionized water  in anhydrous EtOH (2.8 mL) was added to a solution of 5  and TEOS 98%  in anhydrous EtOH (5.2 mL). The mixture was manually shaken for a minute to get a homogeneous solution and was left at room temperature without stirring. During the night gelification occurred and the gel was aged for 5 days. It was then powdered and washed successively several times with ethanol and then with dichloromethane. The solid was dried under vacuum , yielding M1 as a dark green powder (603 mg). 29Si-NMR  δ (ppm): -57 (T2), -64.1 (T3), -92.9 (Q2), -102.6 (Q3), -111.7 (Q4). EA Calcd. for C38H50Cl2N3O4RuSiO1.5·40SiO2 (considering complete condensation): 14.09% C, 1.56% H, 1.30% N, 2.19% Cl, 3.12% Ru, 35.54% Si. Found: 9.60% C, 2.55% H, 0.75% N, 1.00% Cl, 31.19% Si. ICP: 0.86%Ru . SBET: 332 m²/g; pore diameter: 103–111 Å; pore volume: 0.93–0.89 cm³/g.A solution of ammonium fluoride  in anhydrous and degassed dichloromethane (2.1 mL) was added under nitrogen to M1  placed in a Schlenk tube and the mixture was stirred under inert atmosphere at room temperature for 20 h (GC monitoring). The mixture was filtered under nitrogen atmosphere with a cannula and the solid was washed several times with 2 mL portions of anhydrous dichloromethane. The combined filtrates were evaporated to give pure 9 , whose spectroscopic data were coincident with that reported in the literature [1H-NMR  δ (ppm): 5.60 , 4.19 , 3.00 , 1.24 . A solution of 6  in anhydrous and degassed dichloromethane (2.1 mL) was added under nitrogen to M1  placed in a microwave vessel which was sealed, placed in the microwave cavity and irradiated to 60 ºC (PowerMax option enabled) for 20 min (GC monitoring). The mixture was filtered under nitrogen atmosphere with a cannula and the solid was washed several times with 2 mL portions of anhydrous dichloromethane. The combined filtrates were evaporated to give pure 9 . The catalyst M1 was dried and reused in a next cycle. Compounds 10 [11 [7 and 8, respectively, by analogous procedures under the conditions described in A solution of ounds 10  and 11 [s 10 [11  were obtM1 made by sol-gel cogelification with tetraethoxysilane (TEOS). This material has been assayed as a recyclable catalyst in the ring-closing metathesis reactions of some selected dienes and enynes. Good results have been obtained for the first cycle in dichloromethane at room temperature for dienes 6 and 7 and under refluxing conditions for enyne 8. Notably, considerable improvement has been achieved under microwave irradiation conditions. Thus, drastic reduction of the reaction time favoured the recyclability of the material. Under these conditions up to five cycles have been performed on diene 7 to afford 10 in a very fast and clean reaction. Supported catalyst M1 is, to our knowledge, the first case described in the literature of a heterogeneous metathesis catalyst prepared by sol-gel co-gelification from a silylated ruthenium alkylidene. We have synthesized and characterized a monosilylated Grubbs-Hoveyda ruthenium alkylidene complex, as well as the corresponding material


The structures of the synthesized compounds were assigned on the basis of FT-IR, 1H-NMR, 13C-NMR and mass spectroscopic data. The in vivo anti-inflammatory activity of the synthesized compounds was evaluated using the carrageenan-induced hind paw edema method and was compared with that of ibuprofen. Some of the newly synthesized compounds showed promising anti-inflammatory activity. The tertiary alcohols 1-10 were also screened for antibacterial activity against ten bacterial strains using seven Gram-positive and three Gram-negative bacteria and for antifungal activity against Aspergillus Flavus, Aspergillus Niger and Aspergillus pterus. Tertiary alcohols 1-10 were found to exhibit good to excellent antimicrobial activities compared to levofloxacin and fluconazole used as standard drugs.The synthesis of some novel alkyl/aryl substituted tertiary alcohols was accomplished in two steps. The synthetic route involves preparation of Grignard reagents by treating alkyl/aryl bromides with magnesium turnings in dry ether. Then substituted chalcones were reacted with the Grignard reagents to afford alkyl/aryl substituted tertiary alcohols  Staphylococcus aureus and vancomycin resistant Enterococci, has created a major concern in the medical field and an urgent need for new antibacterial agents  peaks, which were in agreement with their molecular formulae.Typically, tertiary alcohols in vivo antiinflammatory activity using the carrageenan-induced hind paw edema method -phenol (7). Prepared from 4-chloro-2′-hydroxychalcone  and 4-methyl-3-nitrophenyl magnesium bromide . Yield 61%; m.p. 192–193 °C; FTIR υmax: 3,332 (O-H), 1,619 (C=C) cm−1; 1H-NMR δ 3.65 , 6.68 , 6.48 , 9.1 , 2.31 , 6.76–7.87 ; 13C-NMR δ 21.3 (CH3), 76.1 (C-OH), 129.3 (β-C), 130.2 (α-C), 114–139 (aromatic-C); MS: m/z (%) 395.10 .(E)-2-phenol (8). Prepared from 2′-hydroxychalcone  and phenylmagnesium bromide . Yield 59%; m.p. 211–212 °C; FTIR υmax: 3,322 (O-H), 1,609 (C=C) cm−1; 1H-NMR δ 3.65 , 6.67 , 6.47 , 9.0 , 6.96–7.89 ; 13C-NMR δ 79.3 (C-OH), 129.1 (β-C), 130.4 (α-C), 118–133 (aromatic-C); MS: m/z (%) 302.10 .(E)-2-(1-(4-Chlorophenyl)-3(hydroxyhexa-1-en-3-yl)phenol (9). Prepared from 4-chloro-2′-hydroxy-chalcone  and phenylmagnesium bromide . Yield 63%; m.p. 197–198 °C; FTIR υmax: 3,325 (O-H), 1,605 (C=C) cm−1; 1H-NMR δ 3.66 , 6.67 , 6.47 , 9.1 , 1.77–1.33 , 0.89 , 6.76–7.85 ; 13C-NMR δ 21.4 (CH3) 78.1 (C-OH), 129.3 (β-C), 130.4 (α-C), 115–130 (aromatic-C); MS: m/z (%) 346.12 , 344.10 .(E)-2-(3-(4-Chlorophenyl)-1-(hydroxy-1-(4-methoxyphenyl)allyl)phenol (10). Prepared from 4-chloro-2′-hydroxychalcone  and anisyl magnesium bromide . Yield 71%; m.p. 211–212 °C; FTIR υmax: 3,320 (O-H), 1,617 (C=C) cm−1; 1H-NMR δ 3.66 , 6.67 , 6.39 , 9.1 , 3.85 , 6.76–7.88 ; 13C-NMR δ 58.5 (OCH3) 70.2 (C-OH), 129.6 (β-C), 125.3 (α-C), 114–139 (aromatic-C); MS m/z (%) 368.12 , 366.10 .We have investigated the effect of different substituents on growth inhibition of different bacterial and fungal strains and edema inhibition by some novel aryl/alkyl tertiary alcohols. The antifungal potential of the tertiary alcohols was high than the antibacterial activity. One of the tertiary alcohols showed higher antifungal activity than the standard. This derivative possesses a polar hydroxyl group, a hydrophobic alkyl chain and an electronegative chloro-substituent, which all play important role in antifungal activity.


Pseuduvaria trimera a new aporphine alkaloid; 8-hydroxy-1,4,5-trimethoxy-7-oxoaporphine or 8-hydroxyartabonatine C (1) was isolated, together with the known 1,2,3-trimethoxy-4,5-dioxo-6a,7-dehydroaporphine . Their structures were elucidated by a combination of spectral methods; mainly 2D NMR; IR and MS. Compounds 1 and 2 exhibited cytotoxic activity with IC50 values of 26.36 ± 5.18 μM and 12.88 ± 2.49 μM, respectively, for human hepatocellular carcinoma HepG2 cells, and 64.75 ± 4.45 and 67.06 ± 3.5 μM, respectively, for human breast cancer MDA-MB231 cells. Both compounds displayed anti-cancer activity but less than that of doxorubicin; a conventional chemotherapeutic drug, the IC50 levels of which were 2.21 ± 1.72 and 1.83 ± 0.09 μM for HepG2 and MDA-MB231 cells, respectively.From ethyl acetate-methanol extracts of leaves and twigs of  P. trimera belongs to the family Annonaceae, which includes about eight species in Thailand  +(26), 322(100), 307(6), 294(21), 297(16), 264(17), 91(15). COSY correlations H/H: 2/3, 3/2, 9/10, 10/9, 11, 11/10. HMBC correlations H/C:2/1a, 3; 3/1a, 2; 9/7a, 10, 10a; 10/9, 11, 11a; 11/1a, 7a, 11a; 1OCH3/1; 4OCH3/4; 5OCH3/5; 8OH/ 8. HR-ESI-MS (pos.) m/z: 360.0846 : Yellow crystals, m.p. 242–243 °C. UV (MeOH) λmax (log ε): 416(4.15), 317(4.34), 214(4.70), 210(4.66) nm. IR (KBr) vmax 3420, 2853, 1689, 1658, 1559, 1458, 1211 cm−1. 1H-NMR  data see 13C-NMR  data see m/z: 337 [M]+(48), 322(100), 306(3), 294(29), 279(23), 264(15), 251(52), 236(21), 180(25), 152(23). COSY correlations H/H: 8/9, 9/10, 10/11. HMBC correlations H/C:7/1b, 8; 8/7, 10; 9/11; 10/9; 11/7a, 9; 1OCH3/1; 2OCH3/2; 3OCH3/3; NH/1b, 4. HR-ESI-MS (pos.) m/z: 360.0846  were treated with each compound at indicated concentrations for 24 h. The compound 1 and 2 was dissolved in dimethyl sulfoxide (DMSO) as a vehicle and the maximum volume used did not exceed 10 μL/mL of media to avoid the cytotoxicity of DMSO.The human hepatocellular carcinoma HepG2 and breast cancer MDA-MB231 cells were cultured in Leibovitz’s L-15 and Dulbecco’s modified eagle media (DMEM), respectively, with 25 mM NaHCO1 and 2 at various concentrations for 24 h at 37 °C in a humidified 5% CO2 atmosphere. Doxorubicin, a chemotherapeutic drug, was used as positive control. Non-treated cells were used as negative control. Then 15 μL (sterile stock solution of 5 mg/mL MTT dye) were added to each well and the solution was incubated for 4 h at 37 °C in a humidified 5% CO2 atmosphere. The medium was removed and 100 μL of DMSO were added to each well, and mixed to dissolve the blue crystal formazan. The plate was read at 540 nm, with a reference wavelength of 630 nm, using a microplate reader  The percentage of cell viability was determined as follows:Cell viability was measured by MTT assay -2,5-diphenyltetrazolium bromide) . In brie1 and 2 displayed anti-cancer activity and have potential for use as anti-cancer drugs in human liver and breast cancer. but the anti-cancer activities of both compounds were less than those of doxorubicin in both cell lines. However, further research in an in vivo model is needed before any clinical usage.Compound