Hydrophobic chains of different length were then introduced by acid-mediated esterification with fatty alcohols  leading to the desired alkyl glucuronates with moderate to good yields (49%–72%). The methodology was successfully applied to methyl α-d-mannopyranoside and methyl β-d-galactopyranoside. Physicochemical properties, such as critical micelle concentration (CMC), equilibrium surface tension at CMC (γcmc), solubility, and Krafft temperature were measured, and the effect of structural modifications on surface active properties and micelle formation was discussed.Large quantities (>3 g) of a new series of alkyl uronates were synthesized in two steps from commercial methyl hexopyranosides. Firstly, several tens of grams of free methyl α- Carbohydrate-based surfactants, including sugar esters, have recently become more and more attractive because of their renewable origin, biodegradability, non-toxicity, and physicochemical behaviors ,2,3. AmoHerein, we propose to synthesize a new series of sugar fatty esters with well-defined structures -catalyzed oxidation of methyl glycosides at primary position, followed by a H8 alkyl chain. The role of the hydrophobic tail length was also investigated using incrementally-increased hydrophobic chain lengths  with an identical headgroup of Glc. The effect of the β configuration was studied for one molecule, via the octyl (1-β-methyl)galacturonate.In order to measure the influence of the headgroup on the amphiphilic properties of this new series of sugar esters, the methodology was applied to different sugars  with the same tail lengths as the C6, C8, C10, and C12 alkyl chain, the case for commonly ionic/nonionic used surfactants. Their water solubility and adsorption behavior, including Krafft point, concentration limit of clear solution, surface-tension lowering, and critical micellar concentration in water were studied to explore the influence of some structural factors, i.e., alkyl chain and sugar head.It is well known that solubility and surface adsorption properties are critically important aspects for most amphiphilic molecules. These properties, as well as phase diagram, are always influenced by the sugar head for sugar-based surfactants . Amphiph2 (Zhao′s procedure) max, while hexyl, decyl, and dodecyl derivatives  have a CMC in the range of [C]max values, or CMC value slightly higher than [C]max, considering the standard deviation. In the same way, results show that octyl (1-α-methyl)mannuronate 7 has a CMC value in the range of [C]max values. This behavior implies that, above [C]max, multiple non-miscible phases, including soluble micelles or not, and dispersion of structures, such as hexagonal or lamellar phases, could co-exist [By comparing the CMC values with the solubility limit [C]co-exist . These rco-exist  and moreco-exist . Obvious3d and octyl (1-β-methyl)galacturonate 5, these results suggest a moderate ability of these alkyl sugar esters to form micelles, due to an area of limpid solution restricted and close to the CMC.To sum up, except for octyl (1-α-methyl)glucuronate D) values are given in 10−1 deg cm2 g−1 and concentrations (c) are given in g for 100 mL. Mass analyses were performed on a Waters spectrometer  using electrospray ionization (Z-Spray). NMR analysis were performed on a BRUKER DRX spectrometer  operating at 600 MHz for proton 1H and 151 MHz for 13C atoms or a spectrometer Bruker (Bruker) operating at 400 MHz for 1H and 100 MHz for 13C. Samples of oxidized products were prepared in deuterium oxide while samples of esters were dissolved in MeOD-d4.All chemicals were purchased from Fisher Scientific , and Sigma Aldrich  and used as received. Optical rotations were measured with a Perkin-Elmer Model 343 polarimeter  using a sodium lamp at 20 °C. Optical rotation  (4–15 g) was firstly dissolved in deionized water (10 mL/g) and acetonitrile (10 mL/g) was added. TEMPO (0.3 eq.), BAIB (2.2 eq.), and NaHCO3 (1 eq.) were subsequently added and the mixture was stirred at 0 °C for 2 h and at room temperature. The reaction was monitored by TLC. After completion, the reaction was quenched by the addition of ethanol, followed by evaporation and decantation with H2O/EtOAc. The aqueous phase was concentrated and the resultant syrup was evaporated to dryness overnight to quantitatively give the corresponding sodium uronate as a white crystalline solid. For methyl α-d-glucopyranoside, the reaction was finished after 15h; for the methyl β-d-galactopyranoside, a second addition of TEMPO (0.3 eq.) and BAIB (2.2 eq.) was necessary and the reaction was finished after 48 h; for methyl α-d-mannopyranoside, two supplementary additions of TEMPO (0.3 eq.) and BAIB (2.2 eq.) and 8 days were necessary to oxidize all of the mannoside.Methyl glycoside (methyl α-2SO4 (0.9 mL) were successively added and the mixture was stirred at 65 °C. The reaction was monitored by TLC. After completion (~24 h), the mixture was neutralized with triethylamine until pH ~ 7, filtered, and evaporated. The crude was purified by flash column chromatography to give the corresponding ester. When using octanol, decanol, and dodecanol, the excess of alcohol was firstly removed using cyclohexane, then CH2Cl2/MeOH eluent (from 100/0 to 93/7) was used. Sodium 1-methyluronate (5 g) was added to the desired fatty alcohol (100 mL) and a suspension was obtained. Sodium sulfate (5 g) and a catalytic amount of concentrated HAll the NMR spectra are available in the d-glucopyranosid)uronateSodium ; 1H-NMR  δ 4.83 , 3.90 , 3.69 , 3.61 , 3.51 , 3.43 . 13C-NMR  δ 176.6 (C=O), 99.3 (C-1), 72.8 (C-3), 71.9 , 71.0 (C-2), 55.1 (-O-CH3). HRMS (ESI) m/z calcd for C7H12O7 Na [M − Na]+ 231.0481, found 231.0489.d-glucopyranosid)uronateEthyl (methyl α-3a. Colorless syrup. Yield 58% from sodium (methyl α-d-glucopyranosid)uronate using the general procedure. Rf = 0.21 (CH2Cl2/MeOH = 93:7); c 0.1; MeOH); 1H-NMR  δ 4.70 , 4.22 , 4.00 , 3.61 , 3.52 , 3.43 , 1.29 . 13C-NMR  δ 171.5 (C=O), 101.9 (C-1), 74.5 (C-3), 73.3 (C-2), 73.1 (C-4), 72.9 (C-5), 62.5 (-O-CH2-), 56.0 (-O-CH3), 14.4 (-CH3). HRMS (ESI) m/z cald for C9H16O7Na [M + Na]+ 259.0794, found 259.0798.d-glucopyranosid)uronateButyl (methyl α-3b. White powder. Yield 72% from sodium (methyl α-d-glucopyranosid)uronate using the general procedure. Rf = 0.28 (EtOAc); c 0.1; MeOH); 1H-NMR  δ 4.71 , 4.19 , 4.01 , 3.62 , 3.53 , 3.42 , 1.66 , 1.42 , 0.96 . 13C-NMR  δ 171.6 (C=O), 101.9 (C-1), 74.6 (C-3), 73.3 (C-2), 73.1 (C-4), 72.9 (C-5), 66.2 (-O-CH2-), 56.0 (-O-CH3), 31.7 (-O-CH2-CH2-), 20.1 (-CH2-CH3), 14.0 (-CH3). HRMS (ESI) m/z cald for C11H20O7Na [M + Na]+ 287.1107, found 287.1118.Hexyl (methyl α-D-glucopyranosid)uronate3c. Colorless syrup. Yield 59% from sodium (methyl α-d-glucopyranosid)uronate using the general procedure. Rf = 0.23 (CH2Cl2/MeOH = 93:7); c 0.1; MeOH); 1H-NMR  δ 4.71 , 4.18 , 4.01 , 3.62 , 3.53 , 3.45 , 1.68 , 1.36 3-CH3), 0.91 . 13C-NMR  δ 171.6 (C=O), 101.9 (C-1), 74.6 (C-3), 73.3 (C-2), 73.1 (C-4), 72.9 (C-5), 66.5 (-O-CH2-), 56.0 (-O-CH3), 32.6, 29.5, 26.6, 23.6 (-O-CH2-(CH2)4-CH3), 14.3 (-CH3). HRMS (ESI) m/z cald for C13H25O7 [M + H]+ 293.1600, found 293.1608.Octyl (methyl α-D-glucopyranosid)uronate3d. White syrup. Yield 57% from sodium (methyl α-d-glucopyranosid)uronate using the general procedure. Rf = 0.24 (CH2Cl2/MeOH = 93:7); c 0.1; MeOH); 1H-NMR  δ 4.71 , 4.18 , 4.01 , 3.62 , 3.52 , 3.45 , 1.68 , 1.35 5-CH3), 0.91 . 13C-NMR  δ 171.6 (C=O), 101.9 (C-1), 74.5 (C-3), 73.3 (C-2), 73.1 (C-4), 72.9 (C-5), 66.5 (-O-CH2-), 56.0 (-O-CH3), 33.0, 30.3, 30.3, 29.9, 26.9, 23.7 (-O-CH2-(CH2)6-CH3), 14.4 (-CH3). HRMS (ESI) m/z cald for C15H28O7Na [M + Na]+ 343.1733, found 343.1744.d-glucopyranosid)uronateDecyl (methyl α-3e. White powder. Yield 63% from sodium (methyl α-d-glucopyranosid)uronate using the general procedure. Rf = 0.4 (EtOAc/MeOH = 95:5); c 0.1; MeOH); 1H-NMR  δ 4.71 , 4.18 , 4.01 , 3.62 , 3.52 , 3.44 , 1.68 , 1.35 7-CH3), 0.90 . 13C-NMR  δ 171.6 (C=O), 101.9 (C-1), 74.5 (C-3), 73.3 (C-2), 73.1 (C-4), 72.9 (C-5), 66.5 (-O-CH2-), 56.0 (-O-CH3), 33.1, 30.7 (2C), 30.5, 30.4, 29.6, 26.9, 23.7 (-O-CH2-(CH2)8-CH3), 14.5 (-CH3). HRMS (ESI) m/z cald C17H36NO7 [M + NH4]+ 366.2492, found 366.2499.-d-glucopyranosid)uronateDodecyl (methyl α3f. White powder. Yield 49% from sodium (methyl α-d-glucopyranosid)uronate using the general procedure. Rf = 0.35 (EtOAc); c 0.1; MeOH); 1H-NMR  δ 4.71 , 4.18 , 4.01 , 3.62 , 3.52 , 3.44 , 1.68 , 1.32 9-CH3), 0.89 . 13C-NMR  δ 171.6 (C=O), 101.9 (C-1), 74.6 (C-3), 73.3 (C-2), 73.1 (C-4), 72.9 (C-5), 66.5 (-O-CH2-), 56.0 (-O-CH3), 33.1, 30.8, 30.8, 30.7, 30.7, 30.5, 30.4, 29.6, 26.9, 23.8 (-O-CH2-(CH2)8-CH3), 14.5 (-CH3). HRMS (ESI) m/z cald C19H36O7Na [M + Na]+ 399.2359, found 399.2368.d-galactopyranosid)uronateSodium ; 1H-NMR  δ 4.27 , 4.18 , 4.01 , 3.66 , 3.56 , 3.48 . 13C-NMR  δ 175.0 (C=O), 103.2 (C-1), 75.4 (C-5), 72.8 (C-3), 70.3 (C-2), 70.3 (C-4), 57.1 (-O-CH3). HRMS (ESI) m/z calcd for C7H12O7Na [M + Na]+ 231.0481, found 231.0488.d-galactopyranosid)uronateOctyl uronate using the general procedure. Rf = 0.29 (CH2Cl2/MeOH = 93:7); c 0.1; MeOH); 1H-NMR  δ 4.26 , 4.18 , 4.13 , 3.54 , 1.68 , 1.35 5-CH3), 0.9 . 13C-NMR  δ 170.3 (C=O), 105.7 (C-1), 75.4 (C-5), 74.4 (C-3), 71.8 (C-2), 71.5 (C-4), 66.4 (-O-CH2-), 57.5 (-O-CH3), 33.0, 30.3 (2C), 29.7, 27.0, 23.7 (-O-CH2-(CH2)6-CH3), 14.4 (-CH3). HRMS (ESI) m/z cald for C15H29O7 [M + H]+ 321.1913, found 321.1898.d-mannopyranosid)uronateSodium ; 1H-NMR  δ 4.77 , 3.88 , 3.86–3.73 , 3.39 . 13C-NMR  δ 176.7 (C=O), 100.8 (C-1), 72.8 (C-5), 70.3 (C-3), 69.7 (C-2), 68.7 (C-4), 54.9 (-O-CH3). HRMS (ESI) m/z calcd for C7H12O7 Na [M + Na]+ 231.0481, found 231.0487.d-mannopyranosid)uronateOctyl (methyl α-7. Yellow syrup. Yield 44% from sodium (methyl α-d-mannopyranosid)uronate using the general procedure. Rf = 0.31 (CH2Cl2/MeOH = 93:7); c 0.1; MeOH); 1H-NMR  δ 4.71 , 4.17 , 3.98 , 3.92 , 3.78 , 3.67 , 3.40 , 1.68 , 1.34 5-CH3), 0.89 . 13C-NMR  δ 171.5 (C=O), 103.2 (C-1), 74.0 (C-5), 72.1 (C-3), 71.4 (C-2), 69.8 (C-4), 66.4 (-O-CH2-), 55.7 (-O-CH3), 32.9, 30.3 (2C), 29.6, 26.9, 23.7 (-O-CH2-(CH2)6-CH3), 14.4 (-CH3). HRMS (ESI) m/z cald for C15H28O7Na [M + Na]+ 343.1733, found 343.1746.max. When solubilization does not occur at room temperature, a slow heating to 50 °C was applied under magnetic agitation. This allows the examination of the temperature-driven solubilization behavior and verifies the presence of the Krafft point (Tk), above which the surfactant solubility in water increases abruptly abruptly . At Tk, abruptly . In otheabruptly . Thus, Tabruptly ,41. All cmc). The surface excess (Γmax) and minimal area per molecule at air-aqueous solution interface (Amin) can be calculated from Gibbs equation for non-ionic surfactants:−1K−1)’ T is the measuring temperature (K); γ is the surface tension (N·m−1), c is the concentration (mol·L−1); Amin is the minimal area per molecule (Å2); and NA is Avogadro′s number (6.02 × 1023 mol−1).The surface activity of the synthesized molecules was studied mainly in terms of their adsorption behavior on the air-aqueous solution interface. The equilibrium surface tensions γ were measured by means of a Wilhelmy plate using a K100 Processor Tensiometer . The equilibrium surface tension γ was plotted as a function of the log concentration for all molecules, from which we can derive various parameters, like critical micellar concentration (CMC) extrapolated from the break in the surface tension curve and surface tension at the CMC glucuronates. CMC values were shown to significantly decrease when the length of the chain increases, while γcmc slightly increases with the number of carbons of the alkyl chain. On the other hand, for a given alkyl chain length (C8), sugar head nature  does not influence the interfacial properties. So, as yet observed for others sugar-based surfactants, a small variation in the alkyl chain length has a greater effect on the surface activity than the nature of the sugar head. This study reveals that these sugar esters are of particular interest, resulting in a significant reduction of the effective amount of molecules required to give a minimum surface tension of pure water, similar to corresponding alkylpolyglycosides or common polyoxyethylene nonionic surfactants. However, the proposed molecules present a limited micellar phase area, whatever the alkyl chain length or the nature of the sugar head. Indeed, the solubility limits to obtain a clear solution significantly decrease when the length of the chain increases. This point can be problematic for industrial applications and can lead to a decrease in the ease of their handing and formulating. Nevertheless, this work illustrates that anticipating the solubility properties of new amphiphilic molecules is tricky and challenging. The improvement of molecular structures and synthesis of new sugar ester surfactants should be investigated to enhance sugar ester solubility properties and explore their potential applications in accordance with specific industrial needs. For example, additional results indicate that synthesized sugar esters with a galactose sugar head enhance the solubility.A new family of sugar-based surfactants was synthesized at the multigram scale using a simple and rapid two-step protocol from commercial methyl glycosides and characterized by physicochemical analyses. This methodology, which uses TEMPO-catalyzed oxidation and acid-catalyzed esterification, was applied to variable sugar heads  and fatty alcohols  and led to the corresponding alkyl (1-methyl)uronates with moderate to good yields (44%–72% for two steps). The role of the chain length on the interfacial properties was evaluated at 25 °C using C


These new type of chemical entities showed no significant activity on the broad spectrum of DNA and RNA viruses. Results of antiproliferative assays performed on a panel of selected human tumor cell lines revealed that only compounds 1 and 5 showed moderate and selective cytostatic effect against HeLa cells (IC50 = 24 and 32 µM) with no concomitant cytotoxic effects on human normal fibroblasts (BJ). Importantly, an imidazole derivative containing a pyrrolidine moiety linked via an ethylenic spacer (3) showed a selective cytostatic effect toward cervical carcinoma (HeLa) cells (IC50 = 9.5 µM) with no apparent cytotoxicity on human normal fibroblasts (BJ). This compound can be therefore considered as a potential anti-tumor lead compound for further synthetic structure optimization.In the present paper, we report on the synthesis, and  It has been found that infection with respiratory syncytial virus (RSV), which manifests primarily as bronchiolitis or viral pneumonia, is the leading cause of lower respiratory tract infections (LRTIs) in infants and young children . RibavirFurthermore, imidazole is an entity incorporated into many important biological molecules with a wide range of pharmacological activity. In the field of drug discovery the imidazole scaffold is widely used in the drug design strategy and imidazoles are generally well known as anticancer agents as well .in vitro activity at low micromolar concentrations against lung, breast, ovarian and prostate cancer cell lines diazepine-4,8-dione ring system have exhibited potent activities against HBV and HCV diazepine ring system have already shown potent ll lines . Also, i and HCV .H-imidazole-4,5-dicarboxylic acid/amide derivatives (1–8), imidazodiazepine-4,8-dione derivatives (9–11) and carbamoyl imino-ureido derivatives of imidazole (12 and 13) (In the light of these findings we efficiently synthesized a series of new 1 and 13)  and evalH-imidazole-4,5-dimethyl dicarboxylate (1–4), 1H-imidazole-4,5-diamide (5–8), imidazolediazepine-4,8-dione (9–11) and acyclic carbamoyl imino-ureido imidazole derivatives (12 and 13) is performed according to the The synthesis of new 1H-imidazole-dicarboxylate (dMIdC) with K2CO3 and different 2-chloroalkylamine hydrochlorides, that is 4-(2-chloroethyl)morpholine, 4-(2-chloroethyl)piperidine, 4-(2-chloroethyl)pyrrolidine and 3-dimethylaminopropyl chloride gave 4,5-dimethyl dicarboxylate derivatives (1–4) with alkylamino chains at the N-1 of the imidazole ring. 1H-imidazo-4,5-diamide derivatives (5–7) were obtained by ammonolysis of diesters 1–4 in polar protic solvent. Time-controlled reaction of compounds 1–3 with guanidine-hydrochloride and sodium-methoxide gave cyclized imidazodiazepine-4,8-dione derivatives (9–11) and a mixture of two regioisomeric guanidino carbamoyl-imidazole derivatives which in subsequent in situ reaction with 2-fluorophenyl isocyanate in DMF afforded carbamoyl imino-ureido derivatives 12 and 13. It is worth noting that the reaction of imidazodiazepine-4,8-dione derivatives (9–11) with 2-fluorophenyl isocyanate did not gave the desired ureido-imidazodiazepine-4,8-dione derivative. The main reason obviously lies in the fact that 6-amino-diazepine-4,8-dione molecules (9–11) are predominantly in solution in their imino tautomeric form as confirmed by NMR spectroscopy, which is inactive in the final step of the synthesis to create the ureido derivative of imidazodiazepine-4,8-dione. Morpholine derivative 12 for which we believed to have a diazepine-4,8-dione structure and whose NMR spectrum indicated next to all diagnostic signals for the assumed structure the presence of an unidentified signal at approximately 3.8 ppm attributable to a OMe group, which is consistent with the results of the determination of the molecular ion mass by high resolution mass spectrometry.Reaction of 4,5-dimethyl 11–13 have been confirmed by 1H and 13C-NMR spectra . The analysis of the spectra was achieved on the basis of the chemical shift, signal multiplicity and integral values. 19F NMR resonances of 12 and 13 were well resolved . 1H decoupled 13C-NMR showed C–F coupling constants that enabled straightforward identification of fluorinated carbon atoms and their neighbors. Two sets of signals were observed for 9 in the ratio 4:1 as was estimated from the integral values of 1H-NMR signals . Two species were most probably detected due to presence of dynamic equilibrium between amine and imine tautomers (C8–NH2 and C8=NH). C8 chemical shifts of the major species showed value of 149.99 ppm, which is characteristic for the imine form. Therefore, imine form of 9 is the predominant species in solution. Likewise, broad 13C-NMR signals suggested presence of tautomer forms for 10 and 11. Imine forms are favored according to C8 chemical shift .The structures of 1H-13C HSQC and HMBC spectra allowed assignment of C2, C3 and C5 atoms. Interestingly, major chemical shifts differences between 12 and 13 were observed for C2 and C3 atoms. The correlation signals between methylene protons CH2-1' and C2 and C5 in combination with chemical shift values of imidazole carbon atoms suggested that COOCH3 group is attached to C2 (δ 121.43 ppm) and fluoro-phenyl-ureido-imino-methyl-carbamoyl moiety to C3 (δ 145.53) in 12. On the other hand, COOCH3 in attached to C3 in 13, which is indicated by C2 and C3 chemical shift (δ 127.71 and 134.19 ppm).The correlation signals observed in 12 and 13 exist as regioisomers with respect to substitution at positions C-2 and C-3 of the imidazole moiety. This implies that both regioisomers are formed as indicated in in situ reaction gave compounds 12 and 13. Structural differences between 12 and 13, which were suggested by distinct NMR chemical shifts of C2 and C3, were assessed by NOESY experiments. Unfortunately, only trivial NOESY cross-peaks were observed for 12 and 13. COOCH3 group showed no NOESY signals with methylene protons and therefore no particular conformational preferences could be established for these compounds.These spectroscopic results indicate that compounds 1–3, 5–7 and 9–13 were evaluated for their antiviral activity against a wide variety of DNA and RNA viruses, including herpes simplex virus type 1 (HSV-1) (KOS), HSV-2 (G), vaccinia virus and vesicular stomatitis virus in HEL cells, parainfluenza-3, reovirus-1, Sindbis, Coxsackie B4, and Punta Toro virus in Vero cells, vesicular stomatitis virus, Coxsackie virus B4, and respiratory syncytial virus in HeLa cells and influenza A  and influenza B viruses in MDCK cells. Unfortunately, none of the compounds showed pronounced antiviral activity at subtoxic concentrations. No cytotoxicity for all evaluated compounds on HEL, Vero, HeLa and MDCK cell cultures was observed (data not shown).Compounds 1–12 were evaluated for their antiproliferative effect against several malignant tumor cell lines: cervical carcinoma (HeLa), colorectal adenocarcinoma, metastatic (SW 620), breast epithelial adenocarcinoma, metastatic (MCF-7) and human hepatocarcinoma (HepG2) cells and compared with their effects on the growth of normal human skin fibroblasts (BJ) (11) showed a modest cytostatic effect on colon cancer cells (SW620) (IC50 = 20 µM) while imidazole derivative containing a pyrrolidine moiety linked via an ethylenic spacer (3) showed specific antiproliferative effect on HeLa cells (IC50 = 9.5 µM). However, both compounds 11 and 3 exerted moderate growth inhibition only at the highest tested concentrations (10 and 100 µM). Other compounds exerted weak or no antproliferative effects on tested cell lines.Compounds sts (BJ) . The imi3OH) was stored over 3 Å molecular sieves. Dimethylformamide (DMF) was stored over 4 Å molecular sieves. Dichloromethane (CH2Cl2) was refluxed over phosphorus pentoxide (P2O5), distilled and stored over 4 Å molecular sieves. Merck silica gel 60 F254 plates were used for thin-layer chromatography. Column chromatography was performed with Merck silica gel (0.063−0.200 mm), with dichloromethane/methanol as eluent. 1H and 13C-NMR spectra were recorded on a Varian Gemini 300 spectrometer  and Varian NMR System 600 and Varian Unity Inova 300 and Agilent Technologies DD2 300 MHz NMR spectrometers  Samples were measured in CDCl3 and DMSO-d6 solutions at 25 °C in 5 mm NMR tubes. Chemical shifts (δ) in ppm were referred to TMS. High performance LC was performed on Agilent 1100 series system with UV detection (photodiode array detector) using Zorbax C18 reverse-phase analytical column . All compound used for biological evaluation showed >95% purity in this HPLC system. The electron impact mass spectra and the purity of compounds were assessed by using Agilent Technologies 6410 Triple Quad LC/MS instrument equipped with electrospray interface and triple quadrupole analyzer (LC-MS/MS).All commercially available chemicals were purchased from Sigma Aldrich  and used without purification. All solvents were analytical grade purity and dried. Methanol  and K2CO3  in CH3CN (30 mL) is added 4-(2-chloroethyl)morpholine hydrochloride (for cmpd 1), 4-(2-chloroethyl)piperidine hydrochloride (for cmpd 2), 4-(2-chloroethyl)pyrrolidine hydrochloride (for cmpd 3) or 3-dimethylaminopropyl chloride hydrochloride (for cmpd 4) (5.43 mmol). Reaction mixture is stirred at room temperature for 48 h. The solvent is evaporated and the crude product simply extracted with CH2Cl2 or purified by silica gel column chromatography (CH2Cl2/CH3OH = 20/1).To a solution of 4,5-dimethyl 11-(2-Morpholin-4-yl-ethyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester (1): Following general procedure compound 1 is obtained as a yellow oil ; MS m/z: 297.1 (M+1). 1H-NMR (DMSO-d6): δ 2.37 , 2.58 , 3.52 , 3.79 , 3.84 , 4.29 , 7.97 ppm . 13C-NMR (DMSO-d6): δ 43.63 (C1'), 52.29 (COOCH3-3), 52.86 (COOCH3-2), 53.63 (C4'), 58.72 (C2'), 66.57 (C5'), 124.99 (C2), 136.24 (C3), 141.58 (C5), 160.78 (COOCH3), 163.44 ppm (COOCH3).1-(2-Piperidin-1-yl-ethyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester (2): Following general procedure compound 2 is obtained as a pale yellow oil ; MS m/z: 296.2 (M+1). 1H-NMR (DMSO-d6): δ 1.25–1.45 , 2.30 , 2.51 , 3.77 , 3.83 , 4.25 , 7.92 ppm . 13C-NMR (DMSO-d6): δ 24.29 (C6'), 25.94 (C5'), 44.09 (C1'), 52.28 (COOCH3-3), 52.84 (COOCH3-2), 54.47 (C4'), 59.10 (C2'), 125.17 (C2), 136.05 (C3), 141.47 (C5), 160.79 (COOCH3), 163.42 ppm (COOCH3).1-(2-Pyrrolidin-1-yl-ethyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester (3): Following general procedure compound 3 is obtained as a pale yellow oil ; MS m/z: 282.1 (M+1). 1H-NMR (DMSO-d6): δ 1.69 , 2.43 , 2.70 , 3.78 , 3.83 , 4.27 , 7.96 ppm . 13C-NMR (DMSO-d6): δ 23.64 (C5'), 45.64 (C1'), 52.30 (COOCH3-3), 52.87 (COOCH3-2), 53.96 (C4'), 56.20 (C2'), 125.18 (C2), 136.06 (C3), 141.41 (C5), 160.78 (COOCH3), 163.39 ppm (COOCH3).1-(3-Dimethylamino-propyl)-1H-imidazole-4,5-dicarboxylic acid dimethyl ester (4): Following general procedure compound 4 is obtained as a pale yellow oil ; MS m/z: 270.1 (M+1). 1H-NMR (DMSO-d6): δ 1.84 , 2.16 , 2.29 , 3.76 , 3.81 , 4.18 , 7.96 ppm . 13C-NMR (DMSO-d6): δ 28.21 (C2'), 44.74 (C1'), 45.04 (CH3) 52.34 (COOCH3-3), 52.99 (COOCH3-2), 55.73 (C3'), 124.89 (C2), 136.37 (C3), 141.29 (C5), 160.67 (COOCH3), 163.39 ppm (COOCH3).1–4  in anhydrous CH3OH (15 mL) ammonia is introduced at 0 °C. After saturation a reaction mixture is stirred overnight at room temperature. The solvent and excess ammonia are removed under reduced pressure and the crude product purified by silica gel column chromatography (CH2Cl2/CH3OH = 2/1).To a stirred solution of compounds 1-(2-Morpholin-4-yl-ethyl)-1H-imidazole-4,5-dicarboxylic acid diamide (5): According to the general procedure compound 5 is obtained as a white crystals ; MS m/z: 267.1 (M+1). 1H-NMR (DMSO-d6): δ 2.39 , 2.59 , 3.51 , 4.53 , 7.53 , 7.75 , 7.88 , 7.95 , 10.65 ppm . 13C-NMR (DMSO-d6): δ 43.93 (C1'), 53.25 (C4'), 58.51 (C2'), 66.18 (C5'), 126.16 (C2), 136.90 (C3), 140.53 (C5), 160.66 (CO-2), 165.80 ppm (CO-3).1-(2-Piperidin-1-il-etil)-1H-imidazol-4,5-diamide (6): According to the general procedure compound 6 is obtained as a white crystals ; MS m/z: 265.2 (M+1). 1H-NMR (DMSO-d6): δ 1.35 , 1.43 , 2.34 , 2.54 , 4.51 , 7.51 , 7.74 , 7.85 , 7.94 , 10.64 ppm . 13C-NMR (DMSO-d6): δ 24.41 (C6'), 26.08 (C5'), 44.90 (C1'), 54.55 (C4'), 59.30 (C2'), 126.64 (C2), 135.36 (C3), 140.98 (C5), 161.14 (CO-2), 166.31 ppm (CO-3).1-(2-Piperidin-1-yl-ethyl)-1H-imidazole-4,5-dicarboxylic acid diamide (7): According to the general procedure compound 7 is obtained as a white crystals ; MS m/z: 252.2 (M+1). 1H-NMR (DMSO-d6): δ 1.65 , 2.45 , 2.72 , 4.52 , 7.53 , 7.75 , 7.88 , 7.94 , 10.65 ppm . 13C-NMR (DMSO-d6): δ 23.16 (C5'), 46.03 (C1'), 53.52 (C4'), 55.97 (C2'), 126.11 (C2), 134.98 (C3), 140.42 (C5), 160.61 (CO-2), 165.80 ppm (CO-3).1-(3-Dimethylamino-propyl)-1H-imidazole-4,5-dicarboxylic acid diamide (8): According to the general procedure compound 8 is obtained as a white crystals ; MS m/z: 240.1 (M+1). 1H-NMR (DMSO-d6): δ 1.83 , 2.11 , 2.14 , 4.42 , 7.57 , 7.83 , 7.89 , 7.98 , 10.66 ppm . 13C-NMR (DMSO-d6): δ 29.09 (C2'), 45.50 (CH3), 45.93 (C3'), 56.31 (C1'), 126.71 (C2), 135.60 (C3), 140.61 (C5), 161.02 (CO-2), 166.25 ppm (CO-3).1–3 (1.35 mmol) in anhydrous methanol (5 mL). Reaction mixture is stirred for 72 h at room temperature. The solvent is evaporated and the crude product purified by silica gel column chromatography (CH2Cl2/CH3OH = 2/1).To a solution of guanidine hydrochloride (5.38 mmol) in anhydrous methanol (10 mL) cooled to 0 °C is added a solution of sodium methoxide . The reaction mixture is stirred for 30 min at 0 °C. The sodium salt formed is removed by filtration, and the filtrate thus obtained is added to a solution of compounds 6-Imino-1-(2-morpholin-4-yl-ethyl)-6,7-dihydro-1H,5H-1,3,5,7-tetraaza-azulene-4,8-dione (9): ; MS m/z: 292.1 (M+1). 1H-NMR (DMSO-d6): δ 2.38 , 2.58 , 3.51 , 4.48 , 6.64 , 7.37 , 8.04 , 10.66 ppm . 13C-NMR (DMSO-d6): δ 43.18 (C1'), 53.19 (C4'), 58.27 (C2'), 66.15 (C5'), 129.7 (C2), 135.2 (C3), 143.41 (C5), 149.99 (C8), 160.27 and 161.63 ppm (C6 and C10).6-Imino-1-(2-piperidin-1-yl-ethyl)-6,7-dihydro-1H,5H-1,3,5,7-tetraaza-azulene-4,8-dione (10): ; MS m/z: 290.2 (M+1). 1H-NMR (DMSO-d6): δ 1.36 , 1.43 , 2.35 , 2.55 , 4.46 , 6.48 , 7.55 , 8.02 , 10.60 ppm . 13C-NMR (DMSO-d6): δ 23.86 (C6'), 25.51 (C5'), 43.62 (C1'), 53.98 (C4'), 58.50 (C2'), 130.8 (C2), 133.8 (C3), 143.35 (C5), 149.70 (C8), 161.92 and 162.16 ppm (C6 and C10).6-Imino-1-(2-pyrrolidin-1-yl-ethyl)-6,7-dihydro-1H,5H-1,3,5,7-tetraaza-azulene-4,8-dione (11): ; MS m/z: 292.1 (M+1). 1H-NMR (DMSO-d6): δ 1.90 , 3.24 , 3.54 , 4.68 , 6.57 , 7.68 , 8.19 , 10.74 ppm . 13C-NMR (DMSO-d6): δ 22.65 (C5'), 43.06 (C1'), 53.79 (C4'), 54.14 (C2'), 130.7 (C2), 134.4 (C3), 143.5 (C5), 150.12 (C8), 159.15 and 161.97 ppm (C6 and C10).1 or 2  in anhydrous methanol (5 mL). Reaction mixture is stirred for 8 h at room temperature, solvent evaporated and in reaction mixture in situ dissolved in DMF (3 mL) is added 2-fluorophenyl isocyanate . Reaction mixture is stirred for 24 h at room temperature. The solvent is evaporated and the crude product purified by silica gel column chromatography (CH2Cl2/CH3OH = 60/1) to give:To a solution of guanidine hydrochloride  in anhydrous methanol (8 mL) cooled to 0 °C is added a solution of sodium methoxide . The reaction mixture is stirred for 30 min at 0 °C. The sodium salt formed is removed by filtration, and the filtrate thus obtained is added to a solution of compound 5-({[3-(2-Fluoro-phenyl)-ureido]-imino-methyl}-carbamoyl)-1-(2-morpholin-4-yl-ethyl)-1H-imidazole-4-carboxylic acid methyl ester (12) as a white crystals ; MS m/z 462.1915 (M+1). 1H-NMR (DMSO-d6): δ 2.48 , 2.73 , 3.66 , 4.10 , 4.55 , 6.97 , 7.06 , 7.11 , 7.37 , 7.71 , 8.29 , 8.83 , 9.57 , 12.48 ppm . 13C-NMR (DMSO-d6): δ 24.11 (C6'), 25.71 (C5'), 43.49 (C1'), 54.29 (C4'), 59.05 (C2'), 115.10 , 116.5 (F-C6H4), 121.43 (C2), 124.80 , 136.3 (F-C6H4), 139.97 (C5), 145.53 (C3), 150.95 , 158.20, 161.73 and 163.60 (2×C=O and C=NH), 167.55 (COOCH3). 19F NMR (DMSO-d6): δ −125.27 .5-({[3-(2-Fluoro-phenyl)-ureido]-imino-methyl}-carbamoyl)-3-(2-piperidin-1-yl-ethyl)-3H-imidazole-4-carboxylic acid methyl ester (13) as a yellow oil ; MS m/z 460.2 (M+1). 1H-NMR (CDCl3): δ 1.34 , 1.42 , 2.30 , 2.5 , 3.73 , 4.17 , 6.5–7.0 , 7.69 , 7.7–8.6, 12.3 ppm . 13C-NMR (CDCl3): δ 46.15 (C1'), 53.79 (C4'), 53.86 (COO3CH), 58.60 (C2'), 66.90 (C5'), 114.70 , 120.47 (F-C6H4), 122.66 , 124.36 , 127.69 , 127.71 (C2), 134.19 (C3), 142.33 (C5), 152.25 , 158.04, 160.43 and 162.98 (2×C=O and C=NH), 165.70 (COOCH3). 19F NMR (CDCl3): δ −124.37 .2 at 37 °C.The cell lines HeLa , SW620 , MCF-7 , HepG2 (hepatocellular carcinoma) and BJ , were cultured as monolayers and maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin in a humidified atmosphere with 5% CO50 values for each compound were calculated from dose-response curves using linear regression analysis by fitting the mean test concentrations that give PG values above and below the reference value. If, however, all of the tested concentrations produce PGs exceeding the respective reference level of effect  for a given cell line, the highest tested concentration is assigned as the default value . Each test point was performed in quadruplicate in three individual experiments. The results were statistically analyzed . Finally, the effects of the tested substances were evaluated by plotting the mean percentage growth for each cell type in comparison to control on dose response graphs.The panel cell lines were inoculated onto a series of standard 96-well microtiter plates on day 0, at 3,000 to 5,000 cells per well according to the doubling times of specific cell line. Test agents were then added in 10-fold dilutions  and incubated for further 72 h. Working dilutions were freshly prepared on the day of testing in the growth medium. The solvent (DMSO) was also tested for eventual inhibitory activity by adjusting its concentration to be the same as in the working concentrations (DMSO concentration never exceeded 0.1%). After 72 h of incubation, the cell growth rate was evaluated by performing the MTT assay: experimentally determined absorbance values were transformed into a cell percentage growth (PG) using the formulas proposed by NIH and described previously , Vero , HeLa  or MDCK [influenza A  and influenza B] cell cultures. Confluent cell cultures (or nearly confluent for MDCK cells) in microtiter 96-well plates were inoculated with 100 CCIDThe main objective of this study was synthesis of a molecule that would be closely related to RSV604. None of the compounds showed pronounced antiviral activity at subtoxic concentrations. No cytotoxicity for all evaluated compounds on HEL, Vero, HeLa and MDCK cell cultures was observed.N-1 of imidazole or a closer resemblance to the active compound RSV604, that was chemically caused and constrained by the formation of the inactive imino form of the cyclized product instead of the targeted amino form. The 1H-imidazole-4,5-dicarboxylic acid dimethyl ester derivative having an ethyl morpholino ligand (compound 1) and the 1H-imidazole-4,5-diamide derivative with morpholino moiety bound to an imidazole ring (compound 5) have also exerted selective but modest cytostatic effect towards human cervix carcinoma (HeLa) cells (IC50 = 24 and 32 µM), while moderate non-selective antiproliferative activity was observed for compound 10. The imidazolediazepine-4,8-dione derivative linked with a pyrrolidine ligand (11) showed only a modest cytostatic effect on colon cancer cells (SW620) (IC50 = 20 µM). These compounds showed no cytotoxic effects on human normal fibroblasts.From the structure-activity point of view, we believe that the main reason for lack of antiviral activity is probably the absence of either sugar moiety at 12 and 13. Moreover, 1H-imidazole derivative containing a pyrrolidine moiety linked via an ethylenic spacer to N-1 of the imidazole ring (compound 3) exerted a more pronounced selective cytostatic effect towards human cervix cancer (HeLa) cells (IC50 = 9.5 µM) in comparison with other tested compounds with no apparent cytotoxicity on normal human skin fibroblasts (BJ) as well. This compound might therefore be suited for further exploration as a potential lead compound and chemical modifications.Synthetic chemistry is often full of surprises and presented compounds containing a carbamoyl imino-ureido moiety are structurally very interesting, e.g., compounds


H-benzo[h]chromenes 4, 6–11, 13, 14; 7H-benzo[h]chromenopyrimidines 15–18, 20, and 14H-benzo[h]chromenotriazolopyrimidine derivatives 19a–e, 24 was prepared. The structures of the synthesized compounds were characterized on the basis of their spectral data. Some of the target compounds were examined for their antiproliferative activity against three cell lines; breast carcinoma (MCF-7), human colon carcinoma (HCT-116) and hepatocellular carcinoma (HepG-2). The cytotoxic behavior has been tested using MTT assay and the inhibitory activity was referenced to three standard anticancer drugs: vinblastine, colchicine and doxorubicin. The bioassays demonstrated that some of the new compounds exerted remarkable inhibitory effects as compared to the standard drugs on the growth of the three tested human tumor cell lines. The structure–activity relationships (SAR) study highlights that the antitumor activity of the target compounds was significantly affected by the lipophilicity of the substituent at 2- or 3- and fused rings at the 2,3-positions.A series of novel 4 In cancer research, multidrug resistance (MDR) is one of the major aspects that causes failure in therapeutic treatment. This phenomenon could occur either via inherited or acquired approaches, which refers to the initial resistance to a specific drug and the development of resistance after successful treatment, respectively. There have been numerous attempts to overcome these obstacles, including applying drug treatment in combination protocols. In the meantime, the development of new materials for drug design continues to be crucial in addressing this phenomenon.Heterocyclic compounds, in particular oxygen-containing molecules, represent an indispensable class due to their physicochemical properties. The literature reveals that chromenes and benzochromenes are important pharmacophores associated with a broad range of pharmacological activities, such as antimicrobial ,2,3,4, aBenzochromene compounds have been synthesized using various strategies such as multicomponent reactions (MCRs) ,15,16,17A) is currently in Phase I/II clinical trials for the treatment of advanced solid tumors chromene-3-carbonitrile (F) serves as a blood anticoagulant analogue of warfarin [N-succinimido)-4H-benzo[h]-chromene-3-carbonitriles (G) display anti-rheumatic activity chromene-3-carbonitrile (4) and ethyl 2-amino-4-(4-methoxyphenyl)-6-methoxy-4H-benzo- [h]chromene-3-carboxylate (6), respectively. The optical activities of the target compounds 4 and 6 were measured using a Carl Zeiss polarimeter. The results indicated that compounds 4 and 6 have zero rotation  and thus are in the form of a racemic (±) mixture as illustrated in The 4H-benzo[h]chromene-3-carbonitrile (4) with benzaldehyde in ethanolic piperidine solution under reflux afforded the Schiff base product 7. Acylation of compound 4 with acetic anhydride under reflux for 1/2 h gave the N-acetyl derivative 2-acetylamino-4-(4-methoxyphenyl)-6-methoxy-4H-benzo[h]chromene-3-carbonitrile (8a), while applying the same conditions for 6 h gave the corresponding N,N-diacetyl derivative 2-diacetylamino-4-(4-methoxyphenyl)-6-methoxy-4H-benzo[h]chromene-3-carbonitrile (8b). Furthermore, treatment of 4 with triethyl orthoformate in acetic anhydride or dimethylformamide-dipentylacetal (DMF-DPA) in benzene under reflux gave the corresponding desired products 2-ethoxymethyleneamino-4-(4-methoxyphenyl)-6-methoxy-4H-benzo[h]chromene-3-carbonitrile (9) and 2-dimethylaminomethyleneamino-4-(4-methoxy-phenyl)-6-methoxy-4H-benzo[h]chromene-3-carbonitrile (10), respectively. Reaction of the imidate 9 with dimethylamine in methanol at room temperature under stirring for 1 h yielded the imidine 10, which can be obtained as described before from the reaction of 4 and (DMF-DPA), while ammonolysis of compound 9 with NH3 gas bubbled in methanol at room temperature under stirring for 1 h afforded the open chain product 2-aminomethyleneamino-4-(4-methoxyphenyl)-6-methoxy-4H-benzo[h]-chromene-3-carbonitrile (11). These reactions are depicted in Condensation of 2-amino-4-(4-methoxyphenyl)-6-methoxy-46 with triethyl orthoformate afforded ethyl 4-(4-methoxphenyl)-2-formamido-6-methoxy-4H-benzo[h]chromene-3-carboxylate (13), instead of the desired product, ethyl 2-ethoxymethyleneamino-4-(4-methoxphenyl)-6-methoxy-4H-benzo[h]-[h]chromene-3-carboxylate (12), while reaction of 6 with (DMF-DPA) produced ethyl 2-dimethyl-aminomethyleneamino-4-(4-methoxyphenyl)-6-methoxy-4H-benzo[h]chromene-3-carboxylate (14). The formation of compound 13 can be rationalized through initial formation of a product by adding H2O to the ethoxymethyleneamino group (-N=CHOEt) of 12, which then loses ethanol to give the 2-formamido derivative 13 chromenopyrimidin-8-one (15), while reaction of 4 with formamide under reflux was unsuccessful, as 8-amino-7-(4-methoxy-phenyl)-5-methoxy-7H-benzo[h]chromenopyrimidine (16) was not formed. In contrast, compound 16 could be synthesized by cyclization of 11 in ethanolic piperidine solution under reflux [The β-enaminonitrile r reflux  (Scheme 9 with methylamine in methanol at room temperature under stirring for 1 h afforded the cycloaddition product 7-(4-methoxyphenyl)-5-methoxy-8-imino-9-methyl-7H-benzo[h]chromenopyrimidine (17), while hydrazinolysis of 9 afforded the corresponding cycloaddition product 9-amino-7-(4-methoxyphenyl)-5-methoxy-8-imino-7H-benzo[h]chromeno-pyrimidine (18). These results are depicted in Interaction of the imidate 18 proved to be a useful intermediate for the synthesis of a variety of 2-substituted 14H-benzo[h]chromenotriazolopyrimidines. Consequently, condensation of the aminoimino compound 18 with either formic acid or methyl formate in benzene at reflux yielded 14-(4-methoxyphenyl)-12-methoxy-14H-benzo[h]chromenotriazolo-pyrimidine (19a), while acylation of 18 with acetyl chloride or acetic anhydride gave the pentacyclic 2-methyltriazolopyrimidine derivative 19b. Moreover, condensation of 18 with diethyl oxalate and ethyl cyanoacetate afforded the desired products, 2-ethoxycarbonyltriazolopyrimidine 19c and 2-cyanomethyltriazolopyrimidine derivatives 19d, respectively. Finally, aroylation of 18 with benzoyl chloride in refluxing dry benzene proceeded readily to give the 2-phenyltriazolopyrimidine derivative 19e. These results are depicted in The aminoimino compound 18 with benzaldehyde in ethanolic piperidine solution under reflux gave the open chain molecule 9-benzylideneamino-8-imino-7-(4-methox-phenyl)-5-methoxy-7H-benzo[h]chromenopyrimidine (20) -chromenopyrimidine nucleus is more valuable than the 4H-benzo[h]chromene nucleus.The corresponding partition coefficients  at the 2-position, exhibited an increase in the activity against HCT-116 cancer cells compared to vinblastine and colchicine, whereas compounds 7, 9, 8b, 11, 10 and 4 with hydrophobic substituents  at the 2-position displayed a remarkable enhancement in the antitumor activity against HCT-116 cancer cells compared to colchicine. This behavior suggests that the substitution at 2-position could be tolerated and the incorporation of hydrophobic substituents is beneficial for increasing the value of Log P as shown in 6 compared to compound 4. The blocking of the 2-(-NH2) group in compound 6 with hydrophilic or hydrophobic groups such as 2(-NHCHO) or 2-(-N=CHNMe2) caused a remarkable enhancement in the antitumor activity for compounds 13 and 14 against HCT-116 cancer cells compared to colchicine. The introduction of a pyrimidine ring at the 2,3-postions of compound 4 with a hydrophobic group 8-(=NH), 9-(-NH2) for compound 18 and 8-(=NH), 9-(-Me) for compound 17 resulted in a remarkable enhancement of potency against HCT-116 cancer cells as compared to vinblastine and colchicine. In contrast, the presence of a hydrophilic 8-(-C=O) group in compound 15 resulted in a partial loss of the activity. The same behavior has been observed after incorporation of hydrophobic groups  into compounds 16, 17 and 20, hinting that grafting a pyrimidine ring at the 2,3-postions with a lipophilic substituent (hydrophobic group) like imino/amino or imino/methyl groups is more beneficial than other lipophilic substituenta (hydrophilic or hydrophobic groups) like carbonyl, imino or benzylideneamino moieties for the activity by increasing the value of Log P as shown in Further investigation of the impact of the substitution pattern at the previous positions of the synthesized compounds on the antitumor activities was then conducted. Compound 7, 18, 17 and 8a,b were the most active analogs through this study with IC50 values of 0.7 ± 0.11, 0.8 ± 0.08, 0.9 ± 0.11, 0.9 ± 0.1 and 3.9 ± 0.3, respectively, in comparison to the reference drugs vinblastine and colchicine (IC50 = 4.6 ± 0.01 and 10.6 ± 0.04 µg/mL). Additionally, compounds 15, 11, 10 and 4 displayed good activity against HepG-2 in comparison to colchicine and the other compounds 9, 6, 14, 16, 13 and 20. These results imply that the introduction of a hydrophobic group (-N=CHPh or -NHAc) at the 2-position of the chromene nucleus and the incorporation of a pyrimidine nucleus at the 2,3-positions with a hydrophobic group  were indispensable for the activities against HepG-2 by decreasing the value of log P as shown in 17 was found to be the most potent derivative against MCF-7 as compared to doxorubicin, as it was almost equipotent as doxorubicin, while compounds 18 and 17 were almost equipotent as doxorubicin against HCT-116. Besides, compounds 7 and 18 with IC50 = 0.7 ± 0.11 and 0.8 ± 0.08 µg/mL displayed significant growth inhibitory activity against HepG-2 in comparison to doxorubicin, and compounds 17 and 8a were equipotent as doxorubicin, indicating that hydrophobic groups like benzylideneamino and acetylamino moieties at 2-postion is preferred for antitumor activity more than other hydrophobic groups with decreasing value of log P as shown in H-benzo[h]chromene nucleus more significantly than the 7H-benzo[h]chromenopyrimidine nucleus. The other compounds showed moderate to fair cytotoxic activities as compared to doxorubicin.Concerning the activity against HepG-2, compounds H-chromene and pyrimidine moieties are crucial elements for the antitumor activity. The incorporation of pyrimidine rings at the 2,3-postions with groups  or (-N=CHPh and -NHAc) at the 2-postion of the chromene nucleus is favorable and greatly enriches the activity more than the other hydrophobic and hydrophilic groups tested.Finally, we can deduce that the substitution pattern at the 2,3-positions or fused rings at the 2,3-positions on the synthesized 41H-NMR (500 MHz) and 13C-NMR spectra (125 MHz) were recorded using an AV 500 MHz spectrometer . Chemical shifts (δ) are expressed in parts per million (ppm). The 1H-NMR and 13C-NMR spectra of the compounds are provided in the 254  plates was used to check the purity of the compounds.Commercial-grade solvents and reagents were purchased from Sigma-Aldrich  and used without further purification. Melting points were measured with a Stuart Scientific  apparatus and are uncorrected. IR spectra were determined as KBr pellets on a FT/IR 460 plus spectrophotometer . 2-Amino-4-(4-methoxyphenyl)-6-methoxy-4H-benzo[h]chromene-3-carbonitrile (4). A reaction mixture of 4-methoxy-1-naphthol , 4-methoxybenzaldehyde , malononitrile  and piperidine (0.5 mL) in absolute ethanol (30 mL) was heated under microwave irradiation conditions for 2 min. at 140 °C. After the completion of the reaction, the mixture was cooled at room temperature and the precipitated solid was filtered off, washed with methanol and recrystallized from ethanol to give the desired compound 4 as a colorless solid, yield: 87%, m.p. 180–181 °C; IR : 3443, 3332, 3207 (NH2), 3079, 3029 (CH-arom.), 2995, 2895 , 2193 (CN); 1H-NMR ; 3.72 , 3.81 , 4.80 , 7.07 , 6.52–8.11 ; 13C-NMR ; 40.70 (C-4), 54.96 (CH3), 55.64 (CH3), 56.43 (C-3), 103.44 (C-5), 114.00 (Ar-C), 118.09 (CN), 121.61 (C-4a), 123.66 (C-10), 124.33 (C-7), 126.10 (C-8), 127.17 (C-6a), 128.30 (C-9), 128.56 (C-10a), 128.77 (Ar-C), 136.74 (Ar-C), 137.64 (C-10b), 151.10 (C-6), 158.12 (Ar-C), 160.25 (C-2); its MS (m/z), 358  with a base peak at 251 (100); C22H18N2O3 (358.39); calcd. % C: 73.73, % H: 5.06, % N: 7.82; found; % C: 73.79, % H: 5.11, % N: 7.89.Ethyl 2-amino-4-(4-methoxyphenyl)-6-methoxy-4H-benzo[h]chromene-3-carboxylate (6). A reaction mixture of 4-methoxy-1-naphthol , 4-methoxybenzaldehyde , ethyl cyanoacetate  and piperidine (0.5 mL) in absolute ethanol (30 mL) was heated under microwave irradiation conditions for 2 min. at 140 °C. After the completion of the reaction, the mixture was cooled at room temperature and the precipitated solid was filtered off, washed with methanol and recrystallized from ethanol to give the desired compound 6 as a colorless solid, yield: 72%, m.p. 159–60 °C; IR : 3414, 3300 (NH2), 3014 (CH-arom.), 2997, 2963, 2875 , 1682 (CO); 1H-NMR ; 1.12 , 3.77 , 3.91 , 4.11 , 4.99 , 7.76 , 6.45–8.30 ; 13C-NMR ; 14.31 (CH3), 40.11 (C-4), 55.43 (CH3), 55.63 (CH3), 58.60 (CH2), 99.78 (C-3), 103.36 (C-5), 114.89 (Ar-C), 115.18 (CN), 121.98 (C-4a), 122.61 (C-10), 122.97 (C-7), 124.78 (C-8), 126.59 (C-6a), 127.81, (C-9), 128.52 (C-10a), 130.54 (Ar-C), 142.17 (C-10b), 142.61(Ar-C), 148.69 (C-6), 151.75 (Ar-C), 161.60 (C-2); its MS (m/z), 405  with a base peak at 299 (100); C24H23NO5 (405.44); calcd. % C: 71.10, % H: 5.72, % N: 3.45; found; % C: 71.15, % H: 5.73, % N: 3.49.2-Benzylideneamino-4-(4-methoxyphenyl)-6-methoxy-4H-benzo[h]chromene-3-carbonitrile (7). A mixture of compound 4 , benzaldehyde  and piperidine (0.5 mL) was refluxed in ethanol (20 mL) for 2 h. (TLC monitoring). The formed precipitate was filtered, washed with cooled methanol, dried and recrystallized from ethanol to afford 7 as a yellow solid, yield: 75%, m.p. 203–204 °C; IR : 3043, 3000 (CH-arom.), 2980, 2896 , 2227 (CN); 1H-NMR ; 3.78 , 3.90 , 4.86 , 6.45–8.18 , 8.40 ; 13C-NMR ; 40.01 (C-4), 55.33 (CH3), 55.59 (CH3), 99.70 (C-3), 103.30 (C-5), 114.33 (Ar-C), 115.12 (CN), 121.96 (C-4a), 122.01 (C-10), 122.87 (C-7), 124.73 (C-8), 126.50 (C-6a), 127.01, (C-9), 127.52 (C-10a), 128.25 (Ar-C), 128.30 (Ar-C), 129.21 (Ar-C), 130.50 (Ar-C), 140.15 (C-10b), 144.60 (Ar-C), 146.66 (C-6), 150.70 (Ar-C), 155.73 (N=CH ), 160.62 (C-2); its MS (m/z), 446  with a base peak at 101 (100); C29H22N2O3 (446.50); calcd. % C: 78.01, % H: 4.97, % N: 6.27; found; % C: 78.10, % H: 5.00, % N: 6.31.2-Acetylamino-4-(4-methoxyphenyl)-6-methoxy-4H-benzo[h]chromene-3-carbonitrile (8a). A solution of compound 4  was refluxed in acetic anhydride (20 mL) for 1/2 h. The solvent was removed under reduced pressure and the resulting solid was collected and washed with cooled methanol, filtered, dried and recrystallized from ethanol to afford 8a as a yellow solid, yield: 52%, m.p. 150–151 °C; IR : 3272 (NH), 3021 (CH-arom.), 2995, 2945, 2903, 2845 , 2217 (CN), 1699 (CO); 1H-NMR ; 2.13 , 3.76 , 3.84 , 5.29 , 6.53–8.14 , 10.70 ; 13C-NMR ; 24.50 (CH3), 41.84 (C-4), 55.07 (CH3), 55.89 (CH3), 92.18 (C-3), 102.93 (C-5), 114.37 (Ar-C), 115.51 (CN), 116.39 (C-4a), 120.62 (C-10), 121.72 (C-7), 123.53 (C-6a), 124.57 (C-8), 126.76 (C-9), 127.91 (C-10a), 129.41 (Ar-C), 134.44 (Ar-C), 136.94 (C-10b), 151.74 (C-6), 152.48, (Ar-C), 158.79 (C-2), 170.60 (CO); its MS (m/z), 400  with a base peak at 64 (100); C24H20N2O4 (400.43); calcd. % C: 71.99, % H: 5.03, % N: 7.00; found; % C: 71.88, % H: 4.90, % N: 6.97.2-Diacetylamino-4-(4-methoxyphenyl)-6-methoxy-4H-benzo[h]chromene-3-carbonitrile (8b). Compound 8b was obtained via refluxing of 4  with acetic anhydride (20 mL) for 6 h. Reaction work up was performed according to the procedure described for 8a. 8b was collected as a yellow solid, yield: 50%, m.p. 172–173 °C; IR : 3012 (CH-arom.), 2945, 2912, 2845 , 2219 (CN), 1735 (CO); 1H-NMR ; 2.47 , 3.75 , 3.84 , 5.29 , 6.53–8.14 ; 13C-NMR ; 23.06 (CH3), 41.84 (C-4), 55.06 (CH3), 55.88 (CH3), 92.18 (C-3), 102.92 (C-5), 114.37 (Ar-C), 115.51 (CN), 116.93 (C-4a), 120.61 (C-10), 121.72 (C-7), 123.54 (C-8), 124.57 (C-6a), 126.75 (C-9), 127.90 (C-10a), 129.41 (Ar-C), 134.44 (Ar-C), 136.94 (C-10b), 151.74 (C-6), 152.48 (Ar-C), 158.79 (C-2), 170.50 (CO); its MS (m/z), 442  with a base peak at 294 (100); C26H22N2O5 (442.15); calcd. % C: 70.58, % H: 5.01, % N: 6.33; found; % C: 70.61, % H: 5.03, % N: 6.36.2-Ethoxymethyleneamino-4-(4-methoxyphenyl)-6-methoxy-4H-benzo[h]chromene-3-carbonitrile (9). A mixture of 4  with triethyl orthoformate  and acetic anhydride (30 mL) was refluxed for 2 h. The solvent was removed under reduced pressure and the resulting solid was washed with methanol and recrystallized from benzene to give 9 as a yellow solid, yield: 86%, m.p. 145–146 °C; IR : 3012 (CH-arom.), 2962, 2928, 2870, 2836 , 2206 (CN); 1H-NMR ; 1.36 , 3.73 , 3.79 , 4.39 , 4.97 , 6.43–8.13 , 8.89 ; 13C-NMR ; 13.89 (CH3), 41.77 (C-4), 55.03 (CH3), 55.67 (CH3), 63.82 (CH2), 80.07 (C-3), 103.12 (C-5), 114.20 (Ar-C), 116.62 (CN), 118.16 (C-4a), 121.55 (C-10), 123.87 (C-7), 124.55 (C-8), 126.38 (C-6a), 127.45 (C-9), 128.29 (C-10a), 129.14 (Ar-C), 135.93 (Ar-C), 136.86 (C-10b), 151.64 (C-6), 157.08 (Ar-C),158.50 (C-2), 161.45 (N=CH); its MS (m/z), 414  with a base peak at 308 (100); C25H22N2O4 (414.45); calcd. % C: 72.45, % H: 5.35, % N: 6.76; found; % C: 72.43, % H: 5.33, % N: 6.73.2-Dimethylaminomethyleneamino-4-(4-methoxyphenyl)-6-methoxy-4H-benzo[h]chromene-3-carbonitrile (10) Method (a): A mixture of 4  with dimethylformaide-dipentylacetal (DMF-DPA)  and benzene (30 mL) was refluxed for 3 h. The solvent was removed under reduced pressure and the resulting solid was recrystallized from benzene to give 10 as a colorless solid, yield: 90%, m.p. 215–216 °C; IR : 3062 (CH-arom.), 2995, 2962, 2934, 2878, 2836 , 2189 (CN); 1H-NMR ; 3.32, 3.34 , 3.73 , 3.81 , 4.90 , 6.50–8.39 , 8.62 ; 13C-NMR ; 34.25 (CH3), 42.01 (C-4), 55.01 (CH3), 55.67 (CH3), 72.45 (C-3), 103.46 (C-5), 114.07 (Ar-C), 117.40 (CN), 120.35 (C-4a), 121.27 (C-10), 121.39 (C-7), 124.03 (C-8), 124.44 (C-6a), 126.17 (C-9), 127.25 (C-10a), 128.79 (Ar-C), 137.12 (Ar-C), 137.21 (C-10b), 151.14 (C-6), 154.41 (Ar-C), 158.21 (C-2), 159.45 (N=CH); its MS (m/z), 413  with a base peak at 77 (100); C25H23N3O3 (413.47); calcd. % C: 72.62, % H: 5.61, % N: 10.16; found; % C: 72.58, % H: 5.58, % N: 10.20. Method (b): A mixture of imadate 9  and dimethylamine  in methanol (30 mL), was stirred at room temperature for 1 h then left overnight to precipitate. The solid product was collected by filtration, washed with methanol and recrystallized from proper solvent to afford 10.2-Aminomethyleneamino-4-(4-methoxyphenyl)-6-methoxy-4H-benzo[h]chromene-3-carbonitrile (11). A mixture of imidate 9  and NH3 gas bubbled in methanol (30 mL) was stirred at room temperature for 1 h. (TLC monitoring) and the mixture was left overnight. The solid product was collected by filtration, washed with methanol and recrystallized from dioxane to give 11 as a colourless solid, yield: 81%, m.p. 245–246 °C; IR : 3468, 3309, 3165 (NH2), 3004 (CH-arom.), 2962, 2920, 2828 , 2203 (CN); its MS (m/z), 385  with a base peak at 279 (100); C23H19N3O3 (385.42); calcd. % C: 71.67, % H: 4.97, % N: 10.90; found; % C: 71.59, % H: 4.94, % N: 10.85.Ethyl 4-(4–4-methoxphenyl)-2-formamido-6-methoxy-4H-benzo[h]chromene-3-carboxylate (13). Compound 13 was prepared from 6 , triethyl orthoformate  and acetic anhydride (30 mL) according to the procedure described for compound 9. Compound 13 was recrystallized from benzene as a pale yellow solid, yield: 70%, m.p. 150–151 °C; IR : 3405 (NH), 3071 (CH-arom.), 2937, 2825 , 1682 (CO), 1709 (CO); 1H-NMR ; 1.18 , 3.69 , 3.88 , 4.11 , 5.11 , 6.73–8.28 , 10.59 , 10.70 ; 13C-NMR ; 13.99 (CH3), 40.33 (C-4), 54.95 (CH3), 55.84 (CH3), 60.40 (CH2), 92.14 (C-3), 103.55 (C-5), 113.55 (Ar-C), 120.32 (C-4a), 121.57 (C-10), 124.50 (C-7), 126.19 (C-8), 127.57 (C-6a), 128.18 (C-9), 128.39 (C-10a), 128.55 (Ar-C), 137.96 (C-10b), 139.70 (Ar-C), 151.97 (C-6), 152.28 (C-2), 157.55 (Ar-C), 166.45 (CO), 167.50 (CO); its MS (m/z), 433  with a base peak at 68 (100); C25H23NO6 (433.45); calcd. % C: 69.27, % H: 5.35, % N: 3.23; found; % C: 69.19, % H: 5.39, % N: 3.18.Ethyl 2-dimethylaminomethyleneamino-4-(4-methoxyphenyl)-6-methoxy-4H-benzo[h]chromene-3-carboxylate (14). Compound 14 was prepared from 6 , dimethylformamide-dipentylacetal (DMF-DPA)  and benzene (30 mL) according to the procedure described for compound 10 (Method (a)). Compound 14 was recrystallized from benzene as a pale yellow solid, yield: 68%, m.p. 120–121 °C; IR : 3062, 3029 (CH-arom.), 2932, 2878, 2828 , 1660 (CO); 1H-NMR ; 1.16 , 3.34, 3.17 , 3.67 , 3.87 , 4.00 , 5.11 , 6.76–8.25 , 8.22 ; 13C-NMR ; 14.14 (CH3), 34.23 (CH3), 42.12 (C-4), 54.89 (CH3), 55.68 (CH3), 58.82 (CH2), 90.00 (C-3), 103.76 (C-5), 113.58 (Ar-C), 120.81 (C-4a), 121.53 (C-10), 123.96 (C-7), 124.04 (C-8), 125.54 (C-6a), 126.92 (C-9), 128.27 (C-10a), 128.33 (Ar-C), 137.89 (C-10b), 139.32 (Ar-C), 150.97 (C-6), 155.72 (Ar-C), 157.58 (C-2), 160.73 (N=CH), 167.11 (CO); its MS (m/z), 460  with a base peak at 336 (100); C27H28N2O5 (460.52); calcd. % C: 70.42, % H: 6.13, % N: 6.08; found; % C: 70.37, % H: 6.08, % N: 6.03.7-(4-Methoxyphenyl)-5-methoxy-7H,9H-benzo[h]chromenopyrimidin-8-one (15). A mixture of compound 4  and formic acid  was refluxed for 3–5 h. (TLC monitoring). The excess of formic acid was removed under reduced pressure and the resulting solid was recrystallized from ethanol/dioxan to give 15 as a yellow solid, yield: 87%, m.p. 170–171 °C; IR : 3501 (NH), 3077, 3001 (CH-arom.), 2958, 2932, 2891 , 1763 (CO); its MS (m/z), 386  with a base peak at 75; C23H18N2O4 (386.40); calcd. % C: 71.49, % H: 4.70, % N: 7.25; found; % C: 71.52, % H: 4.81, % N: 7.34.8-Amino-7-(4-methoxyphenyl)-5-methoxy-7H-benzo[h]chromenopyrimidine (16). Compound 11  was refluxed in ethanol in the presence of pipridine (0.5 mL) for 2 h. The formed precipitate was collected by filtration, washed with methanol and recrystallized from ethanol to give 16 as a colorless solid, yield: 80%, m.p. 277–278 °C; IR : 3468, 3414, 3305 (NH2), 3097 (CH-arom.), 2995, 2970, 2928, 2836 ; 1H-NMR ; 3.68 , 3.89 , 5.29 , 7.29 , 8.26 , 6.59–8.25 ; 13C-NMR ; 38.00 (C-7), 54.97 (CH3), 55.74 (CH3), 95.86 (C-7a), 103.36 (C-6), 114.00 (Ar-C), 119.47 (C-6a), 120.68 (C-1), 121.64 (C-4), 124.20 (C-3), 124.38 (C-4a), 126.17 (C-2), 127.40 (C-1a), 128.63 (Ar-C), 136.16 (C-1b), 137.63 (Ar-C), 151.26 (C-5), 156.39 (C-10), 158.10 (Ar-C), 162.23 (C-11a), 162.63 (C-8); its MS (m/z), 385  with a base peak at 278 (100); C23H19N3O3 (385.42); calcd. % C: 71.67, % H: 4.97, % N: 10.90; found; % C: 71.58, % H: 4.89, % N: 10.81.7-(4-Methoxyphenyl)-5-methoxy-8-imino-9-methyl-7H-benzo[h]chromenopyrimidine (17). This compound was prepared from the imidate 9 , methylamine  and methanol (30 mL) according to the procedure described for compound 11. Compound 17 was recrystallized from dioxane as a colorless solid, yield: 79%, m.p. 199–200 °C; IR : 3331 (NH), 3062 (CH-arom.), 2995, 2965, 2924, 2836 ; its MS (m/z), 399  with a base peak at 76 (100); C24H21N3O3 (399.44); calcd. % C: 72.16, % H: 5.30, % N: 10.52; found; % C: 72.21, % H: 5.36, % N: 10.58.9-Amino-7-(4-methoxyphenyl)-5-methoxy-8-imino-7H-benzo[h]chromenopyrimidine (18). Prepared from the imidate 9 , hydrazine hydrate  and methanol (30 mL) according to the procedure described for compound 11. Compound 18 was recrystallized from benzene as a colorless solid, yield: 88%, m.p. 210–211 °C; IR : 3347, 3301, 3263 (NH and NH2), 2994, 2965, 2932, 2838 ; 1H-NMR ; 3.68 , 3.86 , 5.23 , 5.70 , 6.84 , 7.58 , 8.21–6.60 ; 13C-NMR ; 40.04 (C-7), 55.97 (CH3), 55.69 (CH3), 98.50 (C-7a), 103.75 (C-6), 114.12 (Ar-C), 118.50 (C-6a), 121.62 (C-1), 124.02 (C-4), 124.30 (C-3), 126.09 (C-4a), 127.31 (C-2), 128.29 (C-1a), 128.83 (Ar-C), 136.38 (C-1b), 137.18 (Ar-C), 149.10 (C-10), 151.29 (C-5), 155.30 (C-11a), 157.50 (Ar-C), 159.00 (C-8); its MS (m/z), 400  with a base peak at 76 (100); C23H20N4O3 (400.43); calcd. % C: 68.99, % H: 5.03, % N: 13.99; found; % C: 68.91, % H: 4.99, % N: 13.89.14-(4-Methoxyphenyl)-12-methoxy-14H-benzo[h]chromenotriazolopyrimidine (19a). A mixture of the aminoimino compound 18  and formic acid or methyl formate  in dry benzene (30 mL) was refluxed for 5 h (TLC monitoring). The solvent was removed under reduced pressure and the resulting solid was recrystallized from dioxane to give (19a) as a colorless solid, yield: 88%, m.p. 225–226 °C; IR : 3006 (CH-arom.), 2975, 2932, 2837 , 1653 (C=N); its MS (m/z), 410  with a base peak at 76 (100); C24H18N4O3 (410.42); calcd. % C: 70.23, % H: 4.42, % N: 13.65; found; % C: 70.17, % H: 4.38, % N: 13.61.14-(4-Methoxyphenyl)-12-methoxy-2-methyl-14H-benzo[h]chromenotriazolopyrimidine (19b). A mixture of the aminoimino compound 18  and acetyl chloride or acetic anhydride (30 mL) was refluxed for 2 h (TLC monitoring). The solvent was removed under reduced pressure and the resulting solid was recrystallized from dioxane to give 19b as a colorless solid, yield: 95%, m.p. 225–226 °C; IR : 3010 (CH-arom.), 2970, 2912, 2837 , 1622 (C=N); its MS (m/z), 424  with a base peak at 318 (100); C25H20N4O3 (424.45); calcd. % C: 70.74, % H: 4.75, % N: 13.20; found; % C: 70.80, % H: 4.81, % N: 13.27.2-Ethoxycarbony-14-(4-methoxyphenyl)-12-methoxy-14H-benzo[h]chromenotriazolopyri-midine (19c). A mixture of the aminoimino compound 18  and diethyl oxalate  in ethanol (30 mL) was refluxed for 2 h (TLC monitoring). The solvent was removed under reduced pressure and the resulting solid was recrystallized from ethanol/benzene to give 19c as a colorless solid, yield: 79%, m.p. 225–226 °C; IR : 3071 (CH-arom.), 2970, 2937, 2836 , 1745 (CO), 1623 (C=N); 1H-NMR ; 1.36 , 3.66 , 3.92 , 4.42 , 5.87 , 6.80–8.34 , 9.80 ,; 13C-NMR ; 13.98 (CH3), 40.04 (C-14), 54.96 (CH3), 55.89 (CH3), 61.91 (CH2), 102.52 (C-13), 103.67 (Ar-C), 114.03 (C-13a), 118.00 (C-14a), 120.58 (C-8), 121.76 (C-11), 124.03 (C-10), 124.54 (C-11a), 126.45 (C-9), 127.68 (C-8a), 128.96 (Ar-C), 136.00 (Ar-C), 137.74 (C-5), 141.09 (C-8b), 151.93 (C-14b), 152.94 (C-12), 154.49 (C-2), 157.42 (Ar-C), 158.18 (C-6a), 159.48 (CO); its MS (m/z), 482  with a base peak at 76 (100); C27H22N4O5 (482.49); calcd. % C: 67.21, % H: 4.60, % N: 11.61; found; % C: 67.28, % H: 4.67, % N: 11.67.2-Cyanomethyl-14-(4-methoxyphenyl)-12-methoxy-14H-benzo[h]chromenotriazolopyri-midine (19d). Compound 19d was prepared from the aminoimino compound 18 , ethyl cyanoacetate  and ethanol (30 mL) according to the procedure described for compound 19c. Compound 19d was recrystallized from ethanol/benzene as colorless solid, yield: 85%, m.p. 235–236 °C; IR : 3097, 3000 (CH-arom.), 2975, 2928, 2836 , 2258 (CN), 1652 (C=N); 1H-NMR ; 3.45 , 3.68 , 3.92 , 5.53 , 6.60–8.26 , 8.53 ; 13C-NMR ; 25.05 (CH2), 37.95 (C-14), 55.00 (CH3), 55.89 (CH3), 97.65 (C-13), 102.78 (C-13a), 114.21 (Ar-C), 117.83 (CN), 118.28 (C-14a), 120.46 (C-8), 121.74 (C-11), 123.88 (C-10), 124.47 (C-11a), 126.52 (C-9), 127.84 (C-8a), 128.71 (Ar-C), 134.55 (Ar-C), 136.86 (C-5), 151.08 (C-8b), 152.21 (C-14b), 153.32 (C-2), 158.48 (C-12), 158.99 (Ar-C), 167.35 (C-6a); its MS (m/z), 449 ; C26H19N5O3 (449.46); calcd. % C: 69.48, % H: 4.26, % N: 15.58; found; % C: 69.51, % H: 4.29, % N: 15.62.14-(4-Methoxyphenyl)-12-methoxy-2-phenyl-14H-benzo[h]chromenotriazolopyrimidine (19e). Method (a): Compound 19e was prepared from the aminoimino compound 18 , benzoyl chloride (0.01 mol) according to the procedure described for compound 19a. Compound 19e was recrystallized from benzene as a colourless solid, yield: 88%, m.p. 289–290 °C; IR : 3017, 3020 (CH-arom.), 2975, 2918, 2826 , 1620 (C=N); 1H-NMR ; 3.65 , 3.91 , 5.90 , 6.83–8.36 , 9.69 ; 13C-NMR ; 40.03 (C-14), 54.93 (CH3), 55.87 (CH3), 101.81 (C-13), 103.84 (C-13a), 113.94 (Ar-C), 117.88 (C-14a), 120.63 (C-8), 121.76 (C-11), 124.07 (C-10), 124.59 (C-11a), 126.41 (C-9), 127.16 (C-8a), 127.62 (Ar-C), 129.05 (Ar-C), 129.14 (Ar-C), 129.68 (Ar-C), 136.10 (Ar-C), 138.12 (C-5), 140.06 (C-8b), 151.82 (C-14b), 153.19 (C-12), 153.98 (Ar-C) 158.12 (C-2), 165.47 (C-6a); its MS (m/z), 486  with a base peak at 380 (100); C30H22N4O3 (486.52); calcd. % C: 74.06, % H: 4.56, % N: 11.52; found; % C: 74.00, % H: 4.61, % N: 11.58. Method (b): A mixture of compound 20 , 1,4-dioxine (20 mL) and piperidine (0.5 mL) was refluxed for 2 h (TLC monitoring). The solvent was removed under reduced pressure and the resulting solid was recrystallized from ethanol/benzene to give (19e), yield: 88%.9-Benzylideneamino-7-(4-methoxyphenyl)-5-methoxy-8-imino-7H-benzo[h]chromenopyrimidine (20). A mixture of aminoimino compound 18  and benzaldehyde  in ethanol (30 mL) and piperidine (0.5 mL) was refluxed for 2 h. The solvent was removed under reduced pressure and the resulting solid was recrystallized from dioxane to give the open chain product 20 as a pale yellow solid, yield: 98%, m.p. 218–219 °C; IR : 3209 (NH), 3054, 3009, 3000 (CH-arom.), 2937, 2894 , 1649 (C=N); its MS (m/z), 488  with a base peak at 385 (100); C30H24N4O3 (488.54); calcd. % C: 73.76, % H: 4.95, % N: 11.47; found; % C: 73.69, % H: 4.89, % N: 11.41.3-Ethoxycarbonyl-14-(4-Methoxyphenyl)-12-methoxy-14H-benzo[h]chromenotriazolopy-rimidine-2-one (24). A mixture of the aminoimino compound 18  and ethyl chloroformate (0.015 mol) in dry benzene (30 mL) was refluxed for 2 hr (TLC monitoring). The solid product was collected by filtration and recrystallized from benzene to give 24 as a colourless solid, yield: 60%, m.p. >300 °C; IR : 3062 (CH-arom.), 2975, 2962, 2920, 2839 , 1763 (CO ester), 1730 (CO); 1H-NMR ; 1.34 , 3.67 , 3.89 , 4.44 , 5.44 , 6.81–8.28 , 9.33 ; 13C-NMR ; 13.98 (CH3), 37.78 (C-14), 55.02 (CH3), 55.92 (CH3), 64.48 (CH2), 98.18 (C-14a), 103.96 (C-13), 113.98 (Ar-C), 118.14 (C-13a), 120.50 (C-8), 121.75 (C-11), 123.91 (C-10), 124.46 (C-11a), 126.42 (C-9), 127.80 (C-8a), 129.24 (Ar-C), 137.58 (C-8b), 141.52 (Ar-C), 145.40 (C-5), 148.28 (C-12), 151.99 (C-6a), 152.17 (Ar-C), 154.83 (C-14b), 158.24 (CO), 158.28 (CO); its MS (m/z), 498  with a base peak at 93 (100); C27H22N4O6 (486.52); calcd. % C: 65.05, % H: 4.45, % N: 11.24; found; % C: 65.00, % H: 4.41, % N: 11.21.2 and were subculture two to three times a week.The tumor cell lines breast adenocarcinoma (MCF-7), human colon carcinoma (HCT-116) and hepatocellular carcinoma (HepG-2) were obtained from the American Type Culture Collection . The cells were grown on RPMI-1640 medium supplemented with 10% inactivated fetal calf serum and 50 µg/mL gentamycin. The cells were maintained at 37 °C in a humidified atmosphere with 5% CO4 cell/well in Corning 96-well tissue culture plates and then incubated for 24 h. The tested compounds with concentrations ranging from 0 to 50 μg/mL were then added into 96-well plates (six replicates) to achieve different conc. for each compound. Six vehicle controls with media or 0.5% DMSO were run for each 96 well plate as a control. After incubating for 24 h, the numbers of viable cells were determined by the MTT test. Briefly, the media was removed from the 96 well plates and replaced with 100 μL of fresh culture RPMI 1640 medium without phenol red then 10 µL of the 12 mM MTT stock solution (5 mg of MTT in 1 mL of PBS) to each well including the untreated controls. The 96-well plates were then incubated at 37 °C and 5% CO2 for 4 h. An 85-μL aliquot of the media was removed from the wells, and 50 µL of DMSO was added to each well and mixed thoroughly with the pipette and incubated at 37 °C for 10 min. Then, the optical density was measured at 590 nm with the microplate reader  to determine the number of viable cells and the percentage of viability was calculated as [1 − (ODt/ODc)] × 100% where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of untreated cells. The relation between surviving cells and drug concentration is plotted to get the survival curve of each tumor cell line after treatment with the specified compound. The 50% inhibitory concentration (IC50), the concentration required to cause toxic effects in 50% of intact cells, was estimated from graphic plots of the dose response curve for each conc. using GraphPad Prism software chromene-3-carbonitrile and ethyl 2-amino-4-(4-methoxy-phenyl)-6-methoxy-4H-benzo[h]chromene-3-carboxylate. The new molecules have been evaluated for their antitumor activities against three cancer cell lines: breast adenocarcinoma (MCF-7), human colon carcinoma (HCT-116) and hepatocellular carcinoma (HepG-2). This pharmacological study was also undertaken to evaluate the effects of the substituents and the pyrimidine rings on the antitumor activities. The obtained results have shown that most of these synthesized compounds exhibited good antitumor activities towards the tested cell lines. The SAR study along with the biological assay confirmed that the incorporation of pyrimidine rings at the 2,3-positions with 8-(=NH), 9-(-NH2) and 8-(=NH), 9-(-Me) or (-N=CHPh and -NHAc) at the 2-position of the chromene nucleus enhances the activity more than other groups.The synthesis of new compounds with potential applications as drug replacements is an area of high interest in literature in order to overcome the drug resistance issue. For that reason, novel 4


Isaria fumosorosea KCh J2) towards selected steroid compounds  was investigated. All tested substrates were efficiently transformed. The structure of the substrate has a crucial impact on regio- and stereoselectivity of hydroxylation since it affects binding to the active site of the enzyme. Androstenedione was hydroxylated in the 7α-position to give a key intermediate in the synthesis of the diuretic-7α-hydroxyandrost-4-ene-3,17-dione with 82% conversion. Adrenosterone and 17α-methyltestosterone were hydroxylated in the 6β-position. Hydroxylated derivatives such as 15β-hydroxy-17α-methyltestosterone and 6β,12β-dihydroxy-17α-methyltestosterone were also observed. In the culture of Isaria fumosorosea KCh J2, DHEA was effectively hydroxylated in the C-7 position and then oxidized to give 7-oxo-DHEA, 3β,7α- and 3β,7β-dihydroxy-17a-oxa-d-homo-androst-5-ene-17-one. We obtained 7β-OH-DHEA lactone with 82% yield during 3 days transformation of highly concentrated (5 g/L) DHEA.The catalytic activity of enzymes produced by an entomopathogenic filamentous fungus ( Curvularia lanata significantly decreased the number of chemical steps (from 36 to 11) and the price of hydrocortisone to less than 1 $ [Steroid compounds are very common in pharmacy and medicine because of their variety of biological activities ,2,3. Hydthan 1 $  An additthan 1 $ . In manyAbsidia coerulea AM93, Fusarium culmorum, Mortierella isabellina AM212, Gelasinospora retispora, Mucor silvaticus as well as 7β-hydroxy-DHEA in the cultures of Cephalosporium aphidicola, Aspergillus wentii MRC 200316, and Mortierella isabellina AM212 are described in the literature [Chaetomium sp. KCH 6651, Didymosphearia igniaria KCH 6670, Paecilomyces victoriae or Absidia glauca [Penicillium, e.g., P. simplicissimum, P. lanosocoeruleum, P. commune or P. chrysogenum [Dehydroepiandrosterone (DHEA) is a natural steroid hormone produced by the adrenal cortex. Its hydroxylated derivatives have immunomodulatory, antiproliferative, neuroprotective and anti-inflammatory properties. They are responsible for anti-glucocorticoid action ,14,15,16terature ,25,26,27a glauca ,29,30. 7a glauca ,33,34. Ma glauca . The fira glauca . High acysogenum ,38.Isaria fumosorosea strain, belongs to an interesting group of about 700 species (from >100 genera) of entomopathogenic (insect-pathogenic) fungi that constitute a unique, highly specialized trophic subgroup [Isaria fumosorosea (previously Paecilomyces fumosoroseus [Plutella xylostella), the Asian citrus psyllid (Diaphorina citri), whiteflies and other pest insects [I. fumosorosea directly penetrates the host through the exoskeleton, with the help of a range of hydrolytic enzymes secreted by growing hyphae. These enzymes include chitinases, proteases, and lipases, which destroy the complex and variable structure of the insect cuticle [Beauveria bassiana, have been used as a biocatalyst. The species is able to provide highly effective glucosylation of aromatic compounds, as well as polyphenols [B. bassiana strains to multiple metabolites [The biocatalyst used in this work, an subgroup ,40. Fungsubgroup ,42. Isarsoroseus ,44) is a insects ,46,47,48 cuticle ,51,52,53yphenols ,58,59,60yphenols ,63,64,65yphenols ,67. Therabolites ,67.Isaria fumosorosea KCh J2 strain against selected steroid compounds. The strain was isolated from a spider cadaver. Substrates in this study were androstenedione, adrenosterone, progesterone, 17α-methyltestosterone and dehydroepiandrosterone. All substrates were transformed with high conversion in a short period of time. The effect of molecular structure on regio- and stereoselectivity of hydroxylation was observed. An additional goal was to obtain lactone derivatives of DHEA and to optimize the process. The course of biotransformation was established by a screening procedure. The structure of products was determined by analysis of pure fractions obtained from preparative biotransformations.The purpose of this study was to investigate the catalytic ability of entomopathogenic filamentous fungus of the 1H-NMR), carbon-13 nuclear magnetic resonance (13C-NMR) (Product structures were determined by proton nuclear magnetic resonance (13C-NMR) , heteronIsaria fumosorosea KCh J2 culture gave only one monohydroxylation product, 7α-hydroxyandrost-4-ene-3,17-dione (7α-OH-AD), in high yield  in gh yield . All thegh yield . Prolongstantial ,71,72. Tstantial .Cunninghamella elegant, Aspergillus tamarii Kita, Cephalosporium aphidicola, Fusarium lini or Trichothecium roseum [Isaria fumosorosea KCh J2.In contrast, the transformation of adrenosterone (Adr), another substrate with a 3-on-4-ene moiety and a carbonyl group attached to C-17, did not result in the formation of the corresponding 7α-hydroxy derivative . Probablm roseum ,75,76, dIsaria fumosorosea KCh J2 17α-methyltestosterone (17mT) was transformed into two monohydroxylated derivatives: 6β-OH-17mT and 15β-OH-17mT, in less than 24 h  used in this study.Ten mg of progesterone was transformed in the culture of Isaria fumosorosea KCh J2 gave an analogous result. Surprisingly, in the transformation of both alcohols, we did not observe any amount of 7-oxo-DHEA. This compound as a substrate was not reduced to any 7-hydroxy derivatives after 3 days of transformation. The total amount of 7-alcohol came from hydroxylation of DHEA, not from the reduction of 7-oxo-DHEA. Milecka-Tronina et al. and Kołek et al. also observed, in cultures of different genera—Absidia coerulea AM93 and Mortierella isabellina AM212— the oxidation of stereoisomers of 7OH-DHEA to 7-oxo-DHEA but not a stereoselective reduction to the opposite 7-alcohols [Saccharomyces cerevisiae [Dehydroepiandrosterone (DHEA) was transformed in less than 24 h in the culture of Isaria fumosorosea KCh J2 strain . To estaalcohols . Howeverrevisiae ,79,80,81revisiae . Thus, irevisiae . In our Isaria fumosorosea KCh J2 to give a final medium concentration of DHEA in the range of 0.1 to 5.0 g/L. Analysis of the composition of the reaction mixtures indicates that the 7β-OH product is formed in the largest amount  were purchased from Sigma-Aldrich . 3β,7β-Dihydroxyandrost-5-ene-17-one (7β-OH-DHEA), 3β,7β-dihydroxyandrost-5-ene-17-one (7β-OH-DHEA) and 3β-hydroxyandrost-5-ene-7,17-dione (7-oxo-DHEA) were isolated as a products of DHEA transformation in the culture of Isaria fumosorosea KCh J2 was obtained from the collection of the Department of Chemistry, Wrocław University of Environmental and Life Sciences . Isolation and identification procedures were described in our previous paper [The microorganism us paper . The strI. fumosorosea KCh J2 strain and then incubated for 3 days at 24 °C on a rotary shaker. Then 10 mg of a substrate dissolved in 1 mL of tetrahydrofuran (THF) was added. Samples were taken on the 1st, 3rd and 7th day of the process and products were subsequently extracted using chloroform and analyzed using TLC and GC.Erlenmeyer flasks (300 mL), each containing 100 mL of the cultivation medium , were inoculated with a suspension of Incubation of DHEA was carried out in Erlenmeyer flasks (300 mL) containing 100 mL of the cultivation medium. Ten mg of DHEA was added in THF (1 mL) to a 3-day-old culture of the investigated strain. The transformation conditions were the same as in the standard experiment . SamplesTo the 3-day-old culture, cultivated as described in Isaria fumosorosea KCh J2, cultivated as described in To each flask containing 100 mL of the 3-day-old culture of I. fumosorosea KCh J2 was incubated under the same conditions as in the screening procedure and then 100 mg of substrate dissolved in 2 mL of THF was added to the 3-day-old culture. After the complete transformation of the substrate, the mixture was extracted with CHCl3 (3 × 300 mL), dried (MgSO4) and concentrated in vacuo. The crude mixture obtained this way was separated by preparative TLC and analyzed .The same transformations were performed on the preparative scale in 2000 mL flasks, each containing 500 mL of the cultivation medium. The culture of v/v) as an eluent. After elution products were detected under UV light (365 nm) then scraped from the plate and eluted with acetone to give fractions. Analytical TLC was carried out on silica gel G . Compounds were detected by spraying the plates with a H2SO4/CH3OH mixture  and visualized under UV light (254 nm). GC analysis was performed using a Hewlett-Packard 5890A (Series II) GC instrument fitted with a flame ionization detector (FID) . The DB-5MS (cross-linked phenyl- methylsiloxane) capillary column (30 m × 0.32 mm × 0.25 μm) was used to determine the composition of product mixtures. The following temperature program was used: 220 °C (1 min)/4 °C/min/260 °C (1 min)/30 °C/min/300 °C (5 min). For gas chromatography – mass spectrometry GC-MS analysis, a GCMS-SATURN 2000 instrument  was used with a ZB-1 (crosslinked phenyl-methylsiloxane) capillary column (30 m × 0.25 mm × 0.25 μm). The following temperature programme was used: 220 °C (1 min)/5 °C/1 min/300 °C (7 min) .The course of the biotransformation was monitored by means of TLC. The composition of product mixtures was established by GC. The crude mixture was separated by preparative TLC  and hexane/acetone mixture  . The NMRIsaria fumosorosea KCh J2 culture 42.3 mg (42%) of 7α-hydroxyandrost-4-ene-3,17-dione (7α-OH-AD) was isolated as the sole product . 1H-NMR (600 MHz) (ppm) (CDCl3) δ: 0.89 ; 1.20 ; 1.26 ; 1.45 ; 1.50–1.60 ; 1.68–1.79 ; 1.82 ; 2.01–2.13 ; 2.35 ; 2.37–2.49 ; 2.65 ; 4.09 ; 5.78 .Isaria fumosorosea KCh J2 culture 33.1 mg (33%) of 6β-hydroxyandrost-4-ene-3,11,17-trione (6β-OH-Adr) was isolated as the sole product . 1H-NMR (600 MHz) (ppm) (CDCl3) δ: 0.89 ; 1.50 ; 1.60 ; 1.62 ; 1.73 ; 1.86 ; 1.89 ; 2.12–2.18 ; 2.20–2.37 ; 2.44–2.60 ; 2.80 ; 4.39 ; 5.79 .Isaria fumosorosea KCh J2 culture 8.4 mg (8%) of 6β-hydroxy-17α-methyltestosterone (6β-OH-17mT), 5.1 mg (5%) of 15β-hydroxy-17α-methyltestosterone (15β-OH-17mT) and 34.3 mg (34%) of 6β,12β-dihydroxy-17α-methyltestosterone  were isolated (After 7 days transformation of 100 mg of 17α-methyltestosterone in isolated . 6β-Hydroxy-17α-methyltestosterone (6β-OH-17mT). 1H-NMR (600 MHz) (ppm) (CDCl3) δ: 0.89 ; 0.94 ; 1.19–1.23 ; 1.22 ; 1.28–1.38 ; 1.40 ; 1.50 ; 1.55–1.64 ; 1.69 ; 1.76 ; 1.84 ; 2.01 ; 2.03–2.07 ; 2.39 ; 2.52 ; 4.35 ; 5.82 . 3, the compound was dissolved in the deuterated THF(THF-d8) and NMR analysis was performed again (Due to the low solubility of the resulting product in CDCled again . 6β-Hydroxy-17α-methyltestosterone (6β-OH-17mT). 1H-NMR (600 MHz) (ppm) (THF-8d) δ: 0.87 ; 0.90 ; 1.13 ; 1.13 ; 1.21 ; 1.25–1.33 ; 1.38 ; 1.48–1.55 ; 1.56–1.62 ; 1.66 ; 1.80–1.86 ; 1.93 ; 2.01 ; 2.08 ; 2.22 ; 2.44 ; 3.29 ; 4.17 ; 4.29 ; 5.66 .15β-Hydroxy-17α-methyltestosterone (15β-OH-17mT). 1H-NMR (600 MHz) (ppm) (CDCl3) δ: 0.99 ; 1.06–1.13 ; 1.15 ; 1.18 ; 1.24 ; 1.29 ; 1.46 ; 1.52 ; 1.63 ; 1.74 ; 2.00 ; 2.05 ; 2.10 ; 2.27 ; 2.30-2.37 ; 2.43 ; 2.48 ; 4.20 ; 5.74 . 3, the compound was dissolved in the deuterated DMSO (DMSO-d6) and NMR analysis was performed again . 1H-NMR (600 MHz) (ppm) (DMSO-d6) δ: 0.81 ; 0.90 ; 0.98–1.09 ; 1.21 ; 1.22–1.27 ; 1.28 ; 1.32 ; 1.48–1.55 ; 1.60 ; 1.73 ; 1.78 ; 1.82 ; 1.90 ; 2.20 ; 2.45 ; 3.52–3.56 ; 3.57 ; 4.00 ; 5.11 ; 5.11 ; 5.66 .Isaria fumosorosea KCh J2 culture in 48 h gave many products. Eight fractions from crude mixture were separated by preparative TLC. NMR analysis of each fraction exposed a mixture of products. Analysis of NMR, TLC and GC data ensured that the biotransformation of progesterone was effective although the quantity of products makes identification impossible.The complete transformation of 100 mg of progesterone by Isaria fumosorosea KCh J2 culture 10.2 mg (5%) of 3β,7α-dihydroxyandrost-5-ene-17-one (7α-OH-DHEA), 86.4 mg (43%) of 3β,7β-dihydroxyandrost-5-ene-17-one (7β-OH-DHEA), 5.8 mg (3%) of 3β-hydroxyandrost-5-ene-7,17-dione (7-oxo-DHEA) and 31.7 mg (16%) of 3β,7β-dihydroxy-17a-oxa-d-homo-androst-5-ene-17-one (7β-OH-DHEA lactone) were isolated. After 1 day transformation of 200 mg of dehydroepiandrosterone in Isaria fumosorosea KCh J2 culture 30.1 mg (15%) 3β,7α-dihydroxy-17a-oxa-d-homo-androst-5-ene-17-one (7α-OH-DHEA lactone) and 119.8 mg (60%) of 3β,7β-dihydroxy-17a-oxa-d-homo-androst-5-ene-17-one (7β-OH-DHEA lactone) were isolated . 1H-NMR (600 MHz) (ppm) (CDCl3) δ: 0.87 ; 1.01 ; 1.11 ; 1.23–1.31 ; 1.49 ; 1.50–1.60 ; 1.64–1.72 ; 1.78 ; 1.80–1.89 ; 2.07–2.17 ; 2.29 ; 2.35 ; 2.33 ; 3.56 ; 3.96 ; 5.63 .3β,7β-Dihydroxyandrost-5-ene-17-one (7β-OH-DHEA). 1H-NMR (600 MHz) (ppm) (CDCl3) δ: 0.88 ; 1.05 ; 1.06 ; 1.10 ; 1.25 ; 1.44 ; 1.47–1.55 ; 1.57 ; 1.70 ; 1.81–1.89 ; 2.11 ; 2.24 ; 2.26 ; 2.35 ; 2.45 ; 3.55 ; 3.95 ; 5.31 .3β-Hydroxyandrost-5-ene-7,17-dione (7-oxo-DHEA). 1H-NMR (600 MHz) (ppm) (CDCl3) δ: 0.89 ; 1.22 ; 1.23–1.34 ; 1.54–1.68 ; 1.70–1.91 ; 1.91–2.00 ; 2.14 ; 2.35–2.45 ; 2.46 ; 2.54 ; 2.81 ; 3.68 ; 5.74 d-homo-androst-5-ene-17-one3β,7α-Dihydroxy-17a-oxa- (7α-OH-DHEA lactone). 1H-NMR (600 MHz) (ppm) (CDCl3) δ: 0.97 ; 1.13 ; 1.33 ; 1.34–1.40 ; 1.50–1.56 ; 1.67 ; 1.81 ; 1.86-1.91 ; 1.94–2.02 ; 2.11 ; 2.24 ; 2.37 ; 2.63 ; 2.69 ; 3.59 ; 3.99 ; 5.62 .d-homo-androst-5-ene-17-one3β,7β-Dihydroxy-17a-oxa- (7β-OH-DHEA lactone). 1H-NMR (600 MHz) (ppm) (CDCl3) δ: 1.03 ; 1.10 ; 1.25 ; 1.32 ; 1.34 ; 1.51 ; 1.59 ; 1.65 ; 1.74–1.83 ; 1.85–1.91 ; 1.98 ; 2.24 ; 2.37 ; 2.44 ; 2.57 ; 2.69 ; 3.57 ; 3.95 ; 5.27 .Isaria fumosorosea KCh J2 has broad ability to transform steroid substrates into the corresponding hydroxylated derivatives. Transformations of androstenedione gave 7α-OH-AD as the sole product in high yield after a short period of time. Transformation of DHEA gave hydroxylated D ring lactones. The strain is able to transform the substrate in a concentration of 5.0 g/L in less than 72 h. With such a large amount of substrate, we observed higher specificity of hydroxylation and faster transformation to the corresponding lactones. The high substrate specificity and acceptance of a high substrate concentration by this strain could be utilized in the production of steroid compounds on an industrial scale. These results encourage further investigation of metabolic activity of Isaria fumosorosea KCh J2 and other entomopathogenic fungi. The entomopathogenic fungus


N-arylhydrazine-1-carbothioamide (5a–b) intermediate, through simple variation of the basic cyclisation conditions. Some of the quinoline-based oxadiazole analogues (e.g. compound 6i) were found to exhibit sub-micromolar anti-proliferative activity in Bcl-2-expressing cancer cell lines, and sub-micromolar IC50 activity within a Bcl2-Bim peptide ELISA assay. The Bcl-2 targeted anticancer activity of 6i was further rationalised via computational molecular modelling, offering possibilities to extend this work into the design of further potent and selective Bcl-2 inhibitory heteroaromatics with therapeutic potential.The Bcl-2 protein has been studied as an anticancer drug target in recent years, due to its gatekeeper role in resisting programmed cancer cell death (apoptosis), and the design of BH3 domain mimetics has led to the clinical approval of Venetoclax (ABT-199) for the treatment of chronic lymphocytic leukaemia. In this work we extend our previous studies on the discovery of indole-based heterocycles as Bcl-2 inhibitors, to the identification of quinolin-4-yl based oxadiazole and triazole analogues. Target compounds were readily synthesized via a common aryl-substituted quinolin-4-carbonyl- Resistance to apoptosis (programmed cell death) is an extensively studied cancer hallmark  that hasN-aryl--oxadiazol-2-amines and 5-(indol-3-yl)-2-[(2-nitrophenyl)amino]-oxadiazole as Bcl-2 inhibitory anticancer agents -5-(indol-3-yl)-1,2,4-triazol-4-amines, 5-(indol-3-yl)-r agents  5,6,7].,7.N-aryl1a–b were first reacted with 4-bromoacetophenone (2) under basic conditions in refluxing ethanol to provide the intermediate 2-(4-bromophenyl)quinoline-4-carboxylic acids 3a–b according to the standard Pfitzinger reaction protocol phenyl]-5-methyl-pyrazole-3-carboxamide) and the Bcl-2 binding pocket oxadiazol-2-amine  in the Bcl-2 binding pocket indicate a number of similarities to the native co-crystallised sulfonamide ligand, showing five H-bonds with key Bcl-2 binding site residues. The following interactions are notable: (i) the oxygen atom of the NO2 group was a H-bond acceptor from the OH of Tyr161 (distance 3.4Å); (ii) the NH group bridging the oxadiazole and nitrophenyl formed H-bonds with the NH2 of Gln58 (distance 3.4Å); (iii) similarly to the co-crystalized ligand the oxadiazole moiety of 6i formed two H-bonds with key residue Tyr67; (iv) the nitrogen and oxygen atoms of the oxadiazole formed two H-bonds with the OH of Tyr67 (distance 3.5Å and 3.3Å respectively); (v) the O atom of the OCH3 group was a H-bond acceptor with the NH2 of Arg105 (distance 2.6Å). 6i in more detail and binding modes between the two ligands.Our docking results for {5-oxadiazol-2-amine (6i) showed a similar binding mode within the Bcl-2 pocket compared to the native sulfonamide ligand, noting that the nitrophenylamino-oxadiazole component of 6i occupied the same position as the iodonaphthalene sulfonamide moiety of the native ligand  to form either quinolin-4-yl-N-aryl-oxadiazol-2-amines (6a–k) or quinolin-4-yl-benzylthiotriazoles (8a–b) by simple variation of the basic reaction conditions. From the compounds active in Bcl-2 expressing cancer cell lines and selected for further study, compound 6i exhibited potent inhibitory activity in the Bcl-2-Bim peptide ELISA assay. On the other hand, compound 6c, overall one of the most potent compounds against Bcl-2 expressing cell lines, gave a moderate ELISA IC50 value. This mismatch between cellular and target-based activity is likely indicative of additional molecular targets for these molecules as yet undiscovered, as would be expected to be the case for small heterocyclic molecules of this type. The quinoline-oxadiazole based compound 6i (R1 = 4-nitrophenyl) in particular was found to exhibit potency within Bcl-2 expressing cancer cells and the ELISA assay and will be the subject of further investigation.This new series of quinoline-based heterocycles further supports our concept that rationally designed Bcl-2 inhibitory aromatic heterocycles can effectively interfere with Bcl-2–Bim peptide binding and exhibit potent anti-proliferative activity against Bcl-2 expressing human cancer cell lines. Access to the target compounds was achieved via a straightforward and efficient synthetic route. In particular, our research demonstrated complete regiochemical control of cyclisation from a common 2-(2-(4-aryl)quinoline-4-carbonyl)-4Si with coupling constants  in Hz. Mass spectrometry was run in electrospray positive ionization mode . Elemental analysis (% CHN) was run by combustion analysis through an outsourced service . Commercial compounds were used as received; 2-(4-bromophenyl)quinoline-4-carboxylic acid (3a) oxadiazol-2-amine (6a-k) in 40%–78% isolated yield.A suspension of quinoline-4-carbonyl-{5-oxadiazol-2-amine (6a). Yield 76%, m.p. 254–257 °C. 1H-NMR (DMSO-d6): 7.49 , 7.72 , 7.83 , 7.95 , 8.25 , 8.5 , 9.12 , 11.20 . m/z (ES+) 478.10 (M+). Anal. (C23H14BrClN4O): % CHN required: C 57.82, H 2.95, N 11.73; found C 57.82, H 2.82, N 11.74.{5-oxadiazol-2-amine (6b). Yield 78%, m.p. 264–266 °C. 1H-NMR (DMSO-d6): 7.61 , 7.68 , 7.83 , 7.88 , 7.94 , 8.04 , 8.22 , 8.24 , 8.47 , 9.13 , 11.38 . m/z (ES+) 512.25 (M+). Anal. (C23H13BrCl2N4O): % CHN required: C 53.93, H 2.56, N 10.94; found C 53.59, H 2.68, N 10.67.{5-oxadiazol-2-amine (6c). Yield 40%, m.p. 265–267 °C. 1H-NMR (DMSO-d6): 2.96 , 7.23 , 7.6 , 7.70 , 7.95 , 8.21 , 8.31 , 8.35 , 8.46 , 9.15 , 10.86 . 13C-NMR (DMSO-d6): 20.32 (CH3), 116.85, 117.36, 122.16, 123.86, 125.85, 128.37, 129.00, 129.10, 129.56, 129.99, 130.76, 131.24, 132.04, 135.81, 137.11, 148.32, 154.72, 156.20, 160.28. HRMS m/z (ES+) required 457.0586 (M++1), found 457.0589.{5-oxadiazol-2-amine (6d). Yield 59%, m.p. 237–239 °C. 1H-NMR (DMSO-d6): 7.86 , 7.98 , 8.27 , 8.35 , 8.50 , 9.15 , 11.84 . m/z (ES+) 489.25 (M+). Anal. (C23H14BrN5O3): % CHN required: C 56.57, H 2.89, N 14.34; found C 56.59, H 2.79, N 14.37.{5-oxadiazol-2-amine (6e). Yield 65%, m.p. 229–231 °C. 1H-NMR (DMSO-d6): 3.79 , 7.00 , 7.56 , 7.97 , 7.92 , 8.20 , 8.50 , 9.17 , 10.78 . m/z (ES+) 474.05 (M+). Anal. (C24H17BrN4O2): % CHN required: C 60.90, H 3.62, N 11.84; found C 60.69, H 3.33, N 11.72.{5-oxadiazol-2-amine (6f). Yield 48%, m.p. 261–263 °C. 1H-NMR (DMSO-d6): 3.97 , 7.41 , 7.54 , 7.63 , 7.75 , 8.15 , 8.24 , 8.40 , 8.7 . m/z (ES+) 508.02 (M+). Anal. (C24H16BrClN4O2): % CHN required: C 56.77, H 3.18, N 11.03; found C 56.83, H 3.12, N 11.13.{5-oxadiazol-2-amine (6g). Yield 59%, m.p. 227–229 °C. 1H-NMR (DMSO-d6): 3.95 , 7.18 , 7.52 , 7.78 , 7.89 , 8.09 , 8.29 , 8.31 , 8.90 . m/z (ES+) 541.98 (M+). Anal. (C24H15BrCl2N4O2): % CHN required: C 53.16, H 2.79, N 10.33; found C 52.92, H 2.86, N 10.12.{5-oxadiazol-2-amine (6h). Yield 54%, m.p. 239–241 °C. 1H-NMR (DMSO-d6): 2.2 , 4.0 , 7.20 , 7.62 , 7.75 , 8.09 , 8.2 , 8.40 , 8.65 , 10.85 . HRMS m/z (ES+) required 487.0691 (M++1), found 487.0693.{5-oxadiazol-2-amine (6i). Yield 45%, m.p. 272–274 °C. 1H-NMR (DMSO-d6): 3.99 , 7.55 , 7.64 , 7.78 , 7.96 , 8.10 , 8.24 , 8.39 , 8.79 . HRMS (C24H16BrN5O4) m/z (ES+) required 517.0380 (M++1), found 517.0428. Anal. (C24H16BrN5O4): % CHN required: C 56.10, H 3.27; found C 55.90, H 3.11.{5-oxadiazol-2-amine (6j). Yield 57%, m.p. 232–234 °C. 1H-NMR (DMSO-d6): 3.69 , 3.95 , 6.90 , 7.58 , 7.80 , 8.10 , 8.19 , 8.40 , 8.54 . m/z (ES+) 505.07 (M+). Anal. (C25H19BrN4O3): % CHN required: C 59.65, H 3.80, N 11.13; found C 59.35, H 3.68, N 10.91.{5-oxadiazol-2-amine (6k). Yield 47%, m.p. 230–232 °C. 1H-NMR (DMSO-d6): 3.98 , 7.12 , 7.54 , 7.62 , 7.78 , 8.08 , 8.19 , 8.31 , 8.75 . m/z (ES+) 492.07 (M+). Anal. (C24H16BrFN4O2): % CHN required: C 59.01, H 3.68, N 10.90; found C 58.67, H 3.28, N 11.20.N-(4-aryl)hydrazine-1-carbothioamide (10 mmol) in 2N NaOH was heated under reflux for 3 h. After cooling to room temperature, water was added and the mixture carefully neutralized with 0.1M HCl. The precipitate formed was filtered, dried and recrystallized from ethanol to give the corresponding triazole thiol product (7a–b).A solution of 2-(2-(4-bromophenyl)-6-methoxyquinoline-4-carbonyl)-5-[2-(4-Bromophenyl)-6-methoxyquinolin-4-yl]-4-(4-methoxyphenyl)-4H-,2,4triaz (7a). Yield 58%, m.p. 199–201 °C. 1H-NMR (DMSO-d6): 3.70 , 3.87 , 6.86 , 7.14 , 7.41 , 7.51 , 7.65 , 7.83 , 7.95 .5-[2-(4-Bromophenyl)-6-methoxyquinolin-4-yl]-4--4H-,2,4triaz (7b). Yield 64%, m.p. 170–172 °C. 1H-NMR (DMSO-d6): 3.86 , 7.08 , 7.42 , 7.49 , 7.61 , 7.67 , 7.82 , 7.93 , 7.97 .H--4-aryl-4H-,2,4triaz5-[2-(4-Bromophenyl)-6-methoxyquinolin-4-yl]-4-(4-methoxyphenyl)-4H-,2,4triaz (8a). Yield 63%, m.p. 240–242 °C. 1H-NMR (DMSO-d6): 3.70 , 3.87 , 5.42 , 6.86 , 7.14 , 7.46 , 7.51 , 7.62 , 7.83 , 8.15 . m/z (ES+) 639.08 (M+).5-[2-(4-Bromophenyl)-6-methoxyquinolin-4-yl]-4--4H-,2,4triaz (8b). Yield 63%, m.p. 247–249 °C. 1H-NMR (DMSO-d6): 3.70 , 5.12 , 7.32 , 7.54 , 7.62 , 7.76 , 8.10 , 8.16 . Anal. (C32H21BrCl2N4O2S): % CHN required: C 56.82, H 3.13, N 8.28; found C 57.31, H 3.03, N 8.43.2. For each experiment, 3000 cells in 0.2 mL of medium were seeded into each well of a clear flat-bottomed 96-well plate and allowed to adhere for 24 h. The cells were then incubated with test compound (from 10 mM stock in DMSO) over a 10 fold dilution series with concentrations ranging from 0.00001 mM to 100 μM, each diluent being performed in triplicate. Control experiments were conducted using DMSO vehicle control with volumes equivalent to test compound concentrations. Cells were incubated with test compound for 72 h., followed by treatment of wells with 20 μL of 3--2,5-diphenyl tetrazolium bromide (MTT) solution . After 4 hours the solutions from the wells were removed and the resulting formazan crystals were dissolved in DMSO (200 μL) and incubated for 30 minutes at 37 °C. Absorbance was measured at 550 nm using an automated microplate reader. Mean IC50 values were obtained from plots of absorbance versus test compound concentration using GraphPad Prism 5 software . For each concentration, three independent repeat experiments were carried out to establish reproducibility.Human breast cancer MDA-MB-231 cells were cultured in RPMI 1640 medium  supplemented with 10% foetal bovine serum, 100 IU/mL pencillin, and 100 μg/mL streptomycin (Life Technologies). Human cervical cancer HeLa cells were cultured in D-MEM (Life Technologies) supplemented with 10% foetal bovine serum, 100 IU/mL pencillin, and 100 μg/mL streptomycin (Life Technologies). Cells were passaged routinely and maintained at 37 °C and 5% CO2. For each experiment 15,000 cells in 0.1 mL of medium were seeded into each well of a solid black fluorescence 96-well plate and incubated for 24 h. The cells were then incubated with test compound (from 10 mM stock in DMSO) over a 10-fold dilution series with concentrations ranging from 0.00001 mM to 100 μM, each diluent being performed in triplicate. Control experiments were conducted using DMSO vehicle control with volumes equivalent to test compound concentrations. Cells were incubated with test compound for 24 h, followed by treatment with 20 μL of CellTiter-Blue® . Fluorescence was measured using 544 nm excitation and 590 nm emission filters on a fluorescent plate reader. Mean IC50 values were obtained from plots of absorbance versus test compound concentration using GraphPad Prism 5 software . For each concentration, three independent repeat experiments were carried out to establish reproducibility.Human acute myeloid leukaemia KG1a cells and acute T-cell lymphocytic Jurkat cells were cultured in RPMI 1640 medium  supplemented with 10% foetal bovine serum, 100 IU/mL pencillin, and 100 μg/mL streptomycin (Life Technologies). Cells were passaged routinely and maintained at 37 °C and 5% COo-phenylenediamine  was freshly prepared at pH5 using phosphate-citrate buffer (Sigma-Aldrich), and hydrogen peroxide was added to the solution to give a final concentration of 0.004 %. Aliquots (200 μL) of the prepared solution were added to the wells and the plate incubated in the dark for 30 minutes at room temperature. The optical density was then read using a plate reader at 450 nm. The experiments were carried on three separate occasions, including both negative and positive controls, where the negative control contains all the components except the Bcl-2 protein, whereas the positive control contains all the components except the inhibitors.A streptavidin-coated 96-well microtitre plate  was washed three times with PBS containing 0.05% Tween-20. Biotinylated Bim peptide (residues 81–106) was diluted to 0.09 μg/mL (20 nM) in Superblock blocking buffer in PBS (Thermo Scientific), and aliquots (200 μL) were transferred to each well. Following incubation (1.5 h) at room temperature to allow immobilization of the Bim peptide on to the solid surface (via streptavidin-biotin interaction), the plate was washed three times with 0.5% BSA in PBS containing 0.05% Tween-20. Test compounds were dissolved in DMSO to obtain a 50 mM stock solution, and different concentrations of tested compound (10 fold dilution series from stock solution) were incubated with 20 nM His-tagged Bcl-2 protein  in PBS for one hour. The inhibitor and protein mixture (100 μL) were then transferred to the wells containing bound Bim peptide and incubated at room temperature for two hours. Following further washing of the plate in triplicate (0.5% BSA in PBS containing 0.05% Tween-20), anti-His antibody containing horseradish peroxidase enzyme  was diluted in 0.5% BSA in PBS (1:1000), and aliquots of 100 μL were added to each well. After incubation (one hour) at room temperature, the plate was washed three times as previously to remove any unbound antibody. A 200 μM solution of The reduction of affinity of the Bim peptide for Bcl-2 was calculated as:450 of treated wells (with inhibitor)/l450 of positive control × 100% Reduction in Bim affinity = l50 value for the Bcl-2 inhibition.A plot of log μM concentration for each inhibitor against the percentage reduction in affinity for the Bim peptide was created using non-linear regression curve analysis (GraphPad Prism 5), and the software used to generate the IChttp://www.rcsb.org/pdb code: 4AQ3). Hydrogen atoms were added to the protein, using the protonate 3D option in MOE (Molecular Operating Environment). Ligand structures were built within MOE and energy minimized using the MMFF94x force field until a RMSD gradient of 0.05 Kcal mol-1 A°-1 was reached. The defined pocket was taken as the active site; Alpha triangle was chosen as the replacement methodology, London dG as the scoring function and ten conformations were retained for each compound.All molecular modeling studies were performed on a RM Innovator Pentium IV (2.4 GHz) running Linux Fedora Core 3. The protein crystal structure of Bcl-2 was downloaded from (


Aspergillus wentii EN-48, was found to be a potent antioxidant against α,α-diphenyl-picrylhydrazyl (DPPH) radical. The structure of wentiquinone C was originally assigned as an anthraquinone derivative -8-methoxydibenzo oxepine-6,11-dione, 1) by 1D and 2D NMR experiments. However, the minor differences of the chemical shifts between xanthones and anthraquinones were queried, leading to the structure of 1 to be revised as a xanthone analog (8-hydroxy-6-(hydroxymethyl)-3-methoxy-9-oxo-9H-xanthene-1-carboxylic acid, 2) on the basis of a methylation and subsequent NMR measurements, and was confirmed by X-ray crystallographic analysis. The method established in this paper could be applied to the structural re-examination or revision for some of the reported seco-anthraquinone derivatives.Wentiquinone C, which was previously isolated from the marine brown alga-derived endophytic fungus  Aspergillus wentii EN-48, and the compound showed potent antioxidant activity against α,α-diphenyl-picrylhydrazyl (DPPH) radicals oxepine-6,11-dione , which contained a seven-membered lactone ring in the structure  as a xanthone (2). Meanwhile, Yenesew and co-workers proposed the revision for a group of seco-anthraquinones to xanthones +) .Yellow crystals; m.p. 287–289 °C; UV (MeOH) λmax (log ε) 235 (4.53), 248 (4.45), 303 (4.15), 350 (3.86) nm; 1H NMR  δ 6.94 , 7.06 , 7.17 , 6.94 , 4.63 , 3.92 , 3.85 , 3.88 , 5.54 ; 13C NMR δ 159.8 , 104.1 , 151.5 , 106.0 , 157.0 , 156.2 , 101.4 , 163.4 , 111.6 , 135.0 , 113.1 , 109.9 , 172.7 , 168.7 , 62.3 , 56.4 , 52.4 , 56.1 . HRESIMS m/z 345.0964 ([M + H]+) .Yellow amorphous powder; UV (MeOH) α radiation (λ = 1.54178 Å) at 293(2) K. The data were corrected for absorption by using the program SADABS . Crystallographic data of wentiquinone C (2) have been deposited in the Cambridge Crystallographic Data Centre as CCDC 1406785. The data can be obtained free of charge via http://www.ccdc.cam.ac.uk/data_request/cif .C2) by a methylation method and confirmed by an X-ray diffraction study. The characteristic differences between aromatic methoxy group and methyl ester were discussed here based on the literatures data, and the methylation method has proven to be useful to rectify such a misassignment for structurally related compounds.In conclusion, the structure of wentiquinone C was revised to be 8-hydroxy-6-(hydroxymethyl)-3-methoxy-9-oxo-9H-xanthene-1-carboxylic acid (