The synthesis, characterization, and properties of these new synthesized thiadiazole derivatives are discussed. A dimerization of 5-amino-3H-1,3,4-thiadiazole-2-thione (14) by sodium nitrite resulting in di- disulfide (19) is also reported. The preliminary in vitro evaluation of these newly synthesized compounds is discussed.The synthesis of six thiadiazole nucleoside analogs is reported: 5-diacetylamino-1,2,4-thiadiazol-3-one ( Since Heidelberger et al. discovered 5-fluorouracil, a pyrimidine antimetabolite, in 1957, pyrimidine antimetabolites have become one of the most important groups of anticancer agents. Some pyrimidine nucleosides have been proven to possess promising anticancer activity , such as1) and five thiadiazole-based nucleoside analogs: 5-diacetylamino-1,2,4-thiadiazol-3-one (1), 5-amino-2 (tetrahydrofuran-2-yl)-1,2,4-thiadiazol-3-one (2), 5-amino-3-[(2′-hydroxyethoxy)-methyl]-1,3,4-thiadiazol-2-one (5-amino-3-(4′-hydroxy-2′-hydroxymethyl-butyl)-1,3,4-thiadiazole-2-thione (4), (S)-5-amino-3--1,3,4-thiadiazole-2-thione (5), and (R)-5-amino-3--1,3,4-thiadiazole-2-thione (6). 5 and 6 are stereoisomers  and 5-amino-2-(tetrahydrofuran-2-yl)-1,2,4-thiadiazol-3-one (2) is shown in 10) was prepared first according to a known method, and then based on 10, 1,2,4-thiadiazole derivatives 1 and 2 were produced. The preparation of 10 was carried out using modified procedures based on an approach by Kurzer -1,3,4-thiadiazol-2-one  was successfully synthesized in an 80% yield. The hydrolysis of 12 with a trace of hydrochloric acid gave 13 as white crystals in 53% yield. The structures of compounds 11–13 were identified by their analytical and spectral data. These data were also compared with literature values for identification.The literature shows two other possible synthetic approaches. Rüfenacht  used potandquist  reported13), acyclonucleoside analog 3 was successfully synthesized by a convenient one-pot method. This synthetic method was based on modified reactions reported by Keyser et al. -1,3,4-thiadiazol-2-one (3).After the preparation of 5-amino-3H-1,3,4-thiadiazol-2-one  was determined on the basis of its analytical and spectral data, which are in agreement with the proposed structure.The structure of 5-amino-3-[(2′-hydroxyethoxy)methyl]-1,3,4-thiadiazol-2-one (14) plausible  contains a primary amino group, which needs to be protected in this synthetic approach. A very common protecting group, Boc (tert-butoxycarbonyl), was introduced, and it worked with this synthetic method very well. 5-Amino-3H-1,3,4-thiadiazole-2-thione (14) was treated with di-tert-butyl dicarbonate (Boc anhydride) to produce 5-tert-butylcarbamate-3H-1,3,4-thiadiazol-2-thione (15) as a light yellow solid in 65% yield. Nucleophile 15 reacted with Michael acceptor dimethyl itaconate at room temperature to afford dimethyl 2- succinate (16) in 81% yield. A strong base, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), was used in this Michael-type addition reaction. In the next step, two ester groups in 16 were reduced to two alcohol groups by Ca(BH4)2. Ca(BH4)2 was prepared in situ by treating NaBH4 with calcium chloride. A clear gel-like product obtained after this reduction reaction was 5-tert- butoxycarbonyl-3-[4′-hydroxy-2′-(hydroxymethyl)butyl]-1,3,4-thiadiazole-2-thione (17). Although attempts to produce 5-tert-butoxycarbonyl-3-[4′-hydroxy-2′-(hydroxymethyl)butyl]-1,3,4-thiadiazole-2-thione (17) with other reducing agents, such as NaBH4, DIBAL , or LiAlH4, were also carried out, Ca(BH4)2 was proven to afford the highest yield, 96%. It appears that DIBAL and LiAlH4can cleave the thiadiazole ring. A Boc deprotection of compound 17 by 60% trifluoroacetic acid (TFA) in methylene chloride at room temperature reproduced the primary amino group and afforded the final product 4 in 78% yield.5-Amino-3H-1,3,4-thiadiazole-2-thione (15), dimethyl2-succinate (16), 5-tert-butoxycarbonyl-3-[4′-hydroxy-2′-(hydroxymethyl)butyl]-1,3,4-thiadiazole-2-thione (17), and 5-amino-3-(4′-hydroxy-2′-hydroxymethyl-butyl)- 1,3,4-thiadiazole-2-thione (4) are in agreement with the proposed structure.The analytical and spectral data of 5-tert-butylcarbamate-3H-1,3,4-thiadiazole-2-thione (5) and (R)-5-amino-3--1,3,4-thiadiazole-2-thione (6) was prepared by using the synthetic method described in 14) using Boc anhydride was the same as the previous reactions in 15 was converted into a sodium salt with sodium hydride in anhydrous dioxane, followed by a nucleophilic substitution reaction with racemic 3-chloro-1,2-propanediol to obtain 5-tert-butoxycarbonyl-3--1,3,4-thiadiazole-2-thione (18). The experimental procedure for this nucleophilic substitution reaction was modified on the basis of a similar reaction reported by Párkányi et al. D20 = −33.0° , and specific rotation of the (R)-5-amino-3--1,3,4-thiadiazole-2-thione (6) was [α]D20 = +31.1° . These results proved that separate enantiomers 5 and 6 have been obtained. As enantiomers, in situ, which could react with 14 to form a diazonium salt. Then, this diazonium group could be replaced by a functional group such as OH. Various experimental conditions and acids have been used to study this reaction. After treating 14 with sodium nitrite and acetic acid in an ice bath, a yellow solid was produced in 73% yield. Surprisingly, the NMR and mass spectrometry have shown that this yellow product was not the compound we proposed from a diazotization reaction, but a disulfide, bis- disulfide (19) (4·5H2O) was also used with sodium nitrite to yield 19 successfully in 68% yield.In our attempts to carry out a diazotization reaction, we discovered an interesting dimerization reaction. The reaction was designed to use sodium nitrite and an acid to prepare nitrous acid ide (19) . The pri14 to produce disulfide 19. The preparation of 19 was reported by Mahieu et al. in 1986 with sulfonyl chloride as the oxidant -1,3,4-thiadiazol-2-one (3). 5-Amino-3H-1,3,4-thiadiazolin-3-one (13)  was suspended in 10 mL of anhydrous CH3CN. 2.76 mL (0.01 mol) of bis(trimethylsilyl)acetamide was added dropwise. The mixture turned clear after several minutes. To this solution, 0.7 mL (0.01 mol) 1,3-dioxolane, 1.7 g (0.01 mol) KI, and 1.38 mL (0.01 mol) chlorotrimethylsilane were added. The mixture was stirred for 16 hours at room temperature under argon. After the reaction, 20 mL of methanol was added to quench the reaction, and the reaction mixture changed from light yellow to dark brown. NaHCO3 was added to neutralize the mixture to pH 8. Then, the solvent was evaporated under reduced pressure, and the residue was loaded onto a silica gel column for purification. The final product was 0.52 g, yield 50%, mp 65–67°C. 1H NMR (DMSO-d6): 7.21 ppm , 5.37 ppm , 4.67 ppm , 3.60 ppm , 3.49 ppm . 13C NMR (DMSO-d6): 181.81 ppm, 157.75 ppm, 77.04 ppm, 71.64 ppm, 59.85 ppm. IR: 3410 , 3292 , 2925 , 1600 , 1568 , 1412 , 1350 , 1100  cm−1. HRMS: Anal. Calcd. for C5H9N3O3S: 214.0257 (M + Na)+. Found: 214.0274. 5-tert-Butylcarbamate-3H-1,3,4-thiadiazol-2-thione (15). 5-Amino-3H-1,3,4-thiadiazole-2-thione (14) succinate (16). 5-tert-Butylcarbamate-3H-1,3,4-thiadiazole-2-thione (15)  was dissolved in 30 mL DMF, followed by the addition of 1.8 g (12 mmol) DBU and 1.9 g (12 mmol) dimethyl itaconate. The mixture was stirred for 40 hours under room temperature. Then, the solvent DMF was evaporated under high vacuum. The residue was loaded onto a silica gel column for purification. After column chromatography, 3.82 g white solid was collected, yield 81%, mp 140–142°C. 1H NMR (CDCl3): 7.89 ppm , 4.42–4.62 ppm , 3.71 ppm , 3.66 ppm , 3.43–3.50 ppm , 2.56–2.78 ppm , 1.49 ppm . 13C NMR (CDCl3): 183.33 ppm, 172.26 ppm (2 C=O), 171.60 ppm, 151.00 ppm, 84.50 ppm, 52.57 ppm, 52.04 ppm, 50.17 ppm, 39.97 ppm, 32.82 ppm, 27.98 ppm. IR: 3238 , 2980 , 1708 , 1587 , 1480 , 1447 , 1317 , 1151  cm−1. HRMS: Anal. Calcd. for C14H21N3O6S2: 414.0769 (M + Na)+. Found: 414.0778.5-tert-Butoxycarbonyl-3-[4 ′-hydroxy-2 ′-(hydroxymethyl) butyl]-1,3,4-thiadiazole-2-thione (17). NaBH4  was stired with CaCl2  in 25 mL anhydrous THF for one hour. After the addition of 0.11 g (0.36 mmol) 2-succinate (16), the mixture was stirred under nitrogen for 24 hours. 0.5 mL of methanol was added to quench the reaction. Then, the solid in the mixture was eliminated by filtration. The solvent in the filtrate was evaporated under reduced pressure. The residue was loaded onto a silica gel column for separation. 0.12 g clear gel-like product was collected via column chromatography, yield 96%. 1H NMR (CD3OD): 4.42–4.62 ppm , 3.62–3.65 ppm , 3.49–3.59 ppm , 2.28–2.38 ppm , 1.55–1.68 ppm , 1.51 ppm . 13C NMR (CD3OD): 183.42 ppm, 154.88 ppm, 154.01 ppm, 83.87 ppm, 63.05 ppm, 60.66 ppm, 52.70 ppm, 38.83 ppm, 32.79 ppm, 28.33 ppm. IR: 3337 , 2926 , 1715 , 1578 , 1447 , 1370 , 1150  cm−1. HRMS: Anal. Calcd. for C12H21N3O4S2: 358.0865 (M + Na)+. Found: 358.0880. 5-Amino-3-(4 ′-hydroxy-2 ′-hydroxymethyl-butyl)-1,3,4-thiadiazole-2-thione (4). 3 mL chilled 60% TFA in CH2Cl2 was added to 0.18 g (0.5 mmol) 5-tert-butoxycarbonyl-3-[4′-hydroxy-2′-(hydroxymethyl)butyl]-1,3,4- thiadiazole-2-thione (17) in an ice bath. After stirring at room temperature for 3 hours, the reaction mixture was extracted with 2 mL H2O twice. The saturated NaHCO3 solution was added to the aqueous phase to neutralize the acid. The solvent in the aqueous phase was evaporated under high vacuum, and then, the residue was loaded onto a column for purification. After column chromatography, 0.092 g clear liquid was collected, yield 78%. 1H NMR (CD3OD): 4.15–4.17 ppm , 3.63–3.66 ppm , 3.50–3.59 ppm , 2.26–2.36 ppm , 1.55–1.71 ppm . 13C NMR (CD3OD): 183.42 ppm, 154.01 ppm, 63.05 ppm, 60.66 ppm, 52.70 ppm, 38.83 ppm, 32.79 ppm. IR: 3321 , 3145 , 2844 , 1616 , 1514 , 1386 , 1097  cm−1. HRMS: Anal. Calcd. for C7H13N3O2S2: 258.0341 (M + Na)+. Found: 258.0349. 5-Amino-3--1,3,4-thiadiazole-2-thione (7). 5-tert-butylcarbamate-3H-1,3,4-thiadiazole-2- thione (15)  was suspended in 20 mL anhydrous dioxane, followed by an addition of 0.028 g NaH. The mixture was stirred for one hour at room temperature under argon. Then, 0.10 g (1 mmol) of 3-chloro-1,2-propanediol was added dropwise. The mixture was refluxed for 24 hours under argon. After refluxing, the solvent was evaporated under reduced pressure. The residue was dissolved in 10 mL methanol, and then, the solid was filtered out. The solvent in the filtrate was evaporated to obtain the crude product of 5-tert-butoxycarbonyl-3--1,3,4- thiadiazole-2-thione (18). Next, this crude product was stirred with 20 mL chilled 60% TFA in CH2Cl2 at room temperature for 3 hours. Then, this mixture was extracted with water three times and all the aqueous layers were combined. The saturated NaHCO3 solution was added to the aqueous phase to neutralize the acid. The solvent in the aqueous phase was evaporated under reduced pressure, and then, the residue was loaded onto a column for purification. After column chromatography, 0.14 g white solid was collected, yield 69%, mp 139–142°C. 1H NMR (CD3OD): 3.92–3.98 ppm , 3.69–3.82 ppm , 3.49–3.57 ppm . 13C NMR (CD3OD): 169.90 ppm, 153.78 ppm, 71.07 ppm, 65.02 ppm, 50.13 ppm. IR: 3255 , 2900 , 1616 , 1514 , 1386 , 1097  cm−1. HRMS: Anal. Calcd. for C5H9N3O2S2: 208.0209 (M + H)+. Found: 208.0202. (S)-5-Amino-3--1,3,4-thiadiazole-2-thione (5). The preparation of 5 followed the procedure for the synthesis of racemic mixture 7, except that (R)-3-chloro-1,2-propanediol was used instead of the racemic 3-chloro-1,2-propanediol in the reaction. The melting point, IR, NMR, and MS results are identical with its enantiomer 6 and racemate 7, [α]D20 = −33.0° .(R)-5-Amino-3--1,3,4-thiadiazole-2-thione (6). The preparation of 6 followed the procedure for the synthesis of racemic mixture 7, except that (S)-3-chloro-1,2-propanediol was used instead of racemic 3-chloro-1,2-propanediol in the reaction. The melting point, IR, NMR, and MS results are identical with its enantiomer 5 and racemate 7, [α]D20 = +31.1° .Bis- Disulfide (19) [14)  was added to a solution of 1.5 mL H2O and 0.3 mL of glacial acetic acid. This mixture was cooled in an ice bath for 10 minutes. Then, a solution of 0.09 g (1 mmol) NaNO2 in 0.2 mL of H2O was added dropwise. After the mixture was stirred in an ice bath for 10 minutes, the dark yellow solid was filtered off and rinsed with two portions of 0.5 mL ethanol. After drying under reduced pressure, 0.098 g yellow solid was collected, yield 73%. (b) 5-Amino-3H-1,3,4- thiadiazole-2-thione (14)  was added into a solution of 3 mL 50/50 ethanol/H2O mixture and kept in an ice bath. After the addition of 0.08 g NaNO2 to the mixture, 0.64 g melted SnCl4 ·5H2O was added dropwise. Immediately after the addition of SnCl4, a dark yellow solid formed. After stirring at room temperature for almost 20 minutes, the dark yellow solid was filtered off and rinsed twice with 0.5 mL cold water. After drying under reduced pressure, 0.091 g, the yellow solid was collected, yield 68%; mp 235-236°C (lit. mp 235–238°C [1H NMR (DMSO-d6): 7.76  ppm. 13C NMR (DMSO-d6): 149.13 ppm, 172.68 ppm. IR: 3266 , 3069 , 1633 , 1494 , 1381 , 1135  cm−1. MS m/z: 264.9 M+. HRMS: Anal. Calcd. for C4H4N6S4: 264.9453 (M + H)+. Found: 264.9459. ide (19) . (a) 5-A35–238°C ). 1H NMR4H4N6S4: C, 18.17; H, 1.53; N, 31.79; S, 48.51. Found: C, 18.31; H, 1.44; N, 31.86; S, 48.10. Anal. Calcd. for C


Each of these derivatives bears a well-defined number of cationic charges aimed to enhance and control their ability to target pathogenic Gram-positive and Gram-negative bacterial cells for allowing photodynamic inactivation. The synthesis was achieved by the use of a common synthon of pentacationic N,N′,N,N,N,N-hexapropyl-hexa(aminoethyl)amine arm (C3N6+) having six attached propyl groups, instead of methyl or ethyl groups, to provide a well-balanced hydrophobicity–hydrophilicity character to pentacationic precursor intermediates and better compatibility with the highly hydrophobic C60 cage moiety. We demonstrated two plausible synthetic routes for the preparation of 1 and 2 with the product characterization via various spectroscopic methods.We designed and synthesized two analogous pentacationic [60]fullerenyl monoadducts, C However, since only one addend group is able to be attached to the cage in a monoadducts, sufficient hydrophilicity is required to allow compatibility of the resulting derivative with water. Broad-spectrum one-photon based photodynamic therapy 1γ-PDT-medbacteria ,3,4,5. Tstate of 0fullerenen (1O2) , which iγ-PDT agents for targeting and photokilling against both Gram-positive and Gram-negative bacterial species fullerenyl monoadducts bearing a well-defined high number of cationic charges that hitherto had remained challenging and has rarely been reported to date. In this paper, we describe a rational linkage of water-soluble quaternary alkylammonium multi-salts and ester-amide functional groups to a well-defined pentacationic arm together with an efficient synthetic method for its attachment on a C60 nanocage. The synthesis led to the preparation of new pentacationic [60]fullerene-based nano-photosensitizers 1 and 2, as shown in Accordingly, we considered the structural modification of CIn these structures, arm moieties each bearing a high number of cationic charges and an amide moiety are capable of inducing the H-bonding in the vicinity of the fullerene cage. 60 cage. The resulting amphiphilic derivatives have been reported to undergo different forms of solid aggregation in aqueous solution if the hydrophilic moiety of the addend is insufficiently large to overcome the high hydrophobicity of the fullerene cage. To circumvent this solid aggregation problem, we undertook the effort to synthesize a well-defined water-soluble pentacationic N,N′,N,N,N,N-hexapropyl-hexa(aminoethyl)amine arm moiety C3N6+, as a charged C3N6 (6), with the number of charge being fixed at five per arm and used as the common synthon in the preparation of [60]fullerene monoadducts, as shown in 3N6+ arm to a single addend, such as the arm precursors ME1N6+C3 (9) and ME3N6+C3 (10), to enhance the water-solubility. Synthesis of 9 and 10 began with the reaction of either 2-methoxyethanol or triethylene glycol monomethyl ether with 2,2-dimethyl-1,3-dioxane-4,6-dione  at 90–95 °C for a period of 12 h to afford malonic acid methoxyethyleneglycol ester, ME1 (4), or malonic acid methoxytriethyleneglycol ester, ME3 (5), in 95 or 90% yield, respectively. Amidation reaction of 4 and 5 was carried out by the treatment with N-hydroxysuccinamide and N,N′-dicyclohexyl carbodiimide (DCC) in anyhydrous THF at ambient temperature over a period of 12 h, followed by the removal of insoluble byproduct of N,N′-dicyclohexyl urea and the further treatment with N,N′,N,N,N,N-hexapropyl-hexa(aminoethyl)amine, C3N6 (6), for an additional period of 12 h. These reactions resulted in the corresponding products of methoxyethyleneglycol-malonamide ester, ME1N6C3 (7), and methoxy-tri(ethyleneglycol)-malonamide ester, ME3N6C3 (8), in 82 and 80% yield, respectively. Quaternization reaction of mono- and tri(ethoxylated) hexaaminomalonamide precursors 7 and 8 using methyl iodide as the methylation agent at 45–50 °C for a period of 3.0 days afforded the corresponding methoxyethyleneglycol-malonamide ester methyl quaternary ammonium salt, ME1N6+C3 (9), and its tri(ethyleneglycolated) analogue ME3N6+C3 (10) in 94 and 92% yield, respectively. Attachment of a C60 cage on the quaternary ammonium salts of malonamide was accomplished by the treatment of 9 and 10 in DMF with predissolved C60 in toluene in the presence of 1.8-diazabicyclo[5.4.0]-undec-7-ene (DBU) at ambient temperature for a period of 8.0 h. In this reaction, carbon tetrabromide was applied as the bromination agent for the replacement of malonyl α-proton in situ. To minimize the possible formation of partial fullerenyl byproducts containing multiaddends, an excess amount (2.5 equiv.) of C60 was applied. At the end of fullerenation, an excessive amount of C60 molecules was recovered and removed by repeatedly washing the crude products with toluene until the observation of a clear toluene solution in washings. The reaction procedure led to the isolation of methoxyethyleneglycol-[60]fullerenyl malonate quaternary methyl ammonium salt, C60(>ME1N6+C3) (1), and its tri(ethyleneglycolated) analogue C60(>ME3N6+C3) (2) in 55 and 50% yield, respectively.Enhancement of the hydrophilicity of fullerene derivatives can be achieved by incorporation of the oligo(ethylene glycol) unit ,14,15, a4 and 5 were converted to their corresponding tert-butyl(2-methoxyethyl)malonate ester, ME1(t-C4) (11), and its tri(ethyleneglycolated) analogue ME3(t-C4) (12) in 74 and 77% yield, respectively, byan esterification reaction with t-butyl alcohol in anyhydrous dichloromethane (DCM) in the presence of N,N'-dicyclohexylcarbodiimide (DCC) at ambient temperature for a period of 8.0 h. Subsequent fullerenyl cyclopropanation reaction of 11 and 12 was carried out by treatment with [60]fullerene solution in a mixture of toluene and 1,2-dichlorobenzene in the presence of carbon tetrabromide and 1.8-diazabicyclo[5.4.0]-undec-7-ene (DBU) over a period of 12 h at room temperature. The products tert-butyl(2-methoxyethyl)[60]fullerenyl malonate, C60[>ME1(t-C4)] (13), and tert-butyl(methoxy-triethyleneglycol)[60]fullerenyl malonate, C60[>ME3(t-C4)] (14), were purified by column chromatography  as brown solids in 78 and 75% yield, respectively. Direct transamidation reaction of 13 and 14 with C3N6 was not successful owing to the complicated side-reaction arising from the attack of C3N6 on C60 itself at elevated temperatures which is required for the effective transamidation. Therefore, we carried out the transformation of 13 and 14 to the corresponding methoxyethyleneglycol-[60]fullerenyl malonate 17, C60(>ME1N6C3), and its methoxytriethyleneglycol ester analogous 18, C60(>ME3N6C3), respectively, by the acid hydrolysis of 13 and 14 first to 2-[60]fullerenyl-3-(2-methoxyethoxy)-3-oxopropanoic acid 15, C60(>ME1H), and its methoxy-triethyleneglycol ester analogous 16, C60(>ME3H), respectively. The latter products were obtained as brown solids in yields of 89% and 87%, respectively. Conversion of 15 and 16 to their acid chloride intermediates was carried out by the reaction with thionyl chloride in THF. To minimize the amination of C3N6 on C60, subsequent reaction with the acid chloride intermediate was performed at 0 °C to room temperature over a period of 3.0 h. A relatively pure C60(>ME1N6C3) 17 was obtained in 47% yield. It was accompanied with a small quantity of byproduct as a decarboxylated fullerene malonate-monoadduct that is removable. The stability of 17 and 18 in either solution or solid phase was found to be low owing to possible intermolecular complex formation between the hexaamine moiety and C60 cage giving insoluble aggregate solids. Therefore, compound 17 and 18 was quaternized to the protonated ammonium–trifluoroacetate salt C60(>ME1N6+C3) (1′) and C60(>ME3N6+C3) (2′), respectively, with trifluoroacetic acid in a short period of time immediately after the workup procedure and purification. Final synthesis of the products pentacationic methoxyethyleneglycol-[60]fullerenyl malonate quaternary methyl ammonium salt C60(>ME1N6+C3) (1), and its analogous methoxy-triethyleneglycol ester C60(>ME3N6+C3) (2), was carried out by the neutralization of 1′ and 2′ first with aqueous potassium carbonate in CHCl3 as a biphase solution. Resulting [60]fullerenyl malonates 17 and 18, respectively, were then transferred to a solvent mixture of anhydrous CHCl3 and DMF (2:1) for methyl quaternization with an excess amount of iodomethane added portionwise over a period of 3.0 days at 45 °C to afford 1 and 2. Successful amidation conversion from malonic acid 5 to ME3N6C3 was verified by the FT-IR spectrum of 8 –] and ester carbonyl (–C=O) moieties, respectively. In the same spectrum, IR peaks located at 2952, 2928, 2862, and 2805 cm−1 were assigned to the stretching absorption bands of aliphatic –C-H. Anti-symmetric deformations of –CH3 groups and scissor vibrations of –CH2 groups appeared as medium intensity bands centered around 1456 cm−1, while symmetric deformations of CH3 groups exhibited the absorption around 1379 cm−1 that overlaps with the –N-C− stretching vibration of amide [–N-(C=O)–] bands at 1400 cm−1. A strong broad band centered at 1104 cm−1 was assigned to the stretching vibrations of –C-O-C– and –C-N– moieties. Methyl quaternization of 8, having tertiary penta(ethylamino) arms, to ME3N6+C310 with iodide salts showed retention of some of IR absorption bands  2 at 524 cm−1 in the spectrum of 2. Similar infrared absorption bands of C60(>ME1N6+C3) 1 were observed, except a relatively smaller –C-O-C– band than that of 2 centered at 1000–1200 cm−1.Alternatively, as shown in rum of 8 a showingon bands b, except1 and 2 was proven to be difficult due to their polycationic nature and facile fragmentations occurring at the conjunction of the C60 cage and the pentacationic malonate arm, giving mainly the highly detectable C60 ion mass fragment at m/z 721, as displayed in MALDI−TOF mass spectra using sinapic acid as the matrix. All fragmented high mass ions were very weak in intensity including peaks at m/z 1930 (M+–I−) (+–I−) as the molecular ion of 1 and 2, respectively. The most pronounced mass fragmentation ions at m/z 874 and 876 in both spectra of 1 and 2 were assigned to the substructure of C60[>H(C=O)NHCH2CH2N+-PrMe2], indicating a main malonylamide moiety being attached on a C60 cage, consistent with the [60]fullerenyl monoadduct structure. Mass spectroscopic data collection of both  (M+–I−)  and 20191H-NMR spectroscopic analyses, we first well-characterized the arm structures ME1(t-C4)  (12) by identification of all protons ] (13) was evident by the disappearance of a Hα proton peak with the chemical shift at δ 3.33. It was also accompanied with up-fielded shifts of two proton peaks to δ 3.06 and 3.26, corresponding to the chemical shift of terminal methoxy protons Hg and ethylene oxide protons Hf, in toluene-d8 from δ 3.34 and 3.56 of 11 in CDCl3, respectively, reflecting partly the solvent effect. Hydrolysis of 13 to C60(>ME1H) (15) was also apparent by the loss of t-butyl protons at δ 1.55, as shown in g, Hf, and He) to δ 3.34, 3.71, and 4.59, respectively, in THF-d8 showing a larger solvent effect. Monoadduct structures of 13 and 14 were also confirmed by the detection of well-defined characteristic fullerenyl sp2 carbon peaks in their 13C-NMR spectra  and 4 single carbon peaks for 14 in the region of δ 135–150, consistent with a Cv molecular symmetry for both 13 and 14. It also displayed two clear carbonyl carbon peaks at δ 163.45 (O=C-O-tert-butyl) and 161.86 (O=C-O–) in toluene-d8, for 13 and the corresponding peaks at δ 163.95 and 162.16 in CDCl3 for 14. Evidence of the acid formation in the structure of 15 and C60(>ME3H) (16) was given by the solubility change and up-fielded shifts of the carbonyl chemical-shift to δ 161.13 (O=C-OH) and 161.62 (O=C-O–) in THF-d8–CS2 (2:1) for 15 (16 showing down-fielded shifts to δ 163.79 (O=C-OH) and 164.12 (O=C-O–)  11, a and ME3 spectra b showing) for 15 c, partly(O=C-O–) d from th15 and 16 displayed a group of 27 peaks each accounted for two sp2 fullerenyl carbons and four single sp2 carbon peaks in the region of δ 135–145 provided the further confirmation of a Cv molecular symmetry of C60(>ME1H) and C60(>ME3H), respectively, as monoadducts without change during the chemical reaction. In addition, the spectra of both compounds 3N6+ arm to the malonic acid moiety of C60(>ME1H) leading to the structure of C60(>ME1N6+C3) 1′ and subsequently 1 was confirmed by the analyses of both infrared and 1H-NMR spectra. The latter of 1′ showed proton peaks corresponding to the C3N6+ arm moiety with overlap of a number of terminal ammonium-propyl α-protons (Hc), α-protons to amide group (Hd), glycol ether protons (Hf), and terminal methoxy protons (Hg) giving broad multiplet peaks in the region of δ 3.0–4.25 in CDCl3–toluene-d8 (δ 4.25–4.75 were assigned to ammonium-ethyl protons (Hc′) and α-protons to the ester group (He). In the case of 1, chemical shifts of similar groups of protons  and  were assigned to those of broad multiplet peaks at δ 3.0–3.75 and 3.75–4.5, respectively, in DMSO-d6–toluene-d8 (2:1), as shown in 13C-NMR spectrum of C60(>ME1N6+C3) 1′  in the region of δ 135−150 that provided a clear confirmation of a Cv molecular symmetry of C60(>ME1N6+C3) monoadduct.Attachment of hexa(aminoethyl)amine Cluene-d8 d. The se6+C3) 1′ e showed N-hydroxysuccinamide (NHS), N,N′-dicyclohexyl carbodiimide (DCC), iodomethane, carbon tetrabromide (CBr4), 1,8-diazabicyclo-undec-7-ene (DBU), 2-methyl-2-propanol, trifluoroacetic acid (TFA), thionyl chloride (SOCl2), K2CO3, γ-butyrolactone (GBL), BF3·Et2O, triethylamine, and pyridine were purchased from Aldrich Chemicals and used without further purification. Malonyl chloride was purchased from TCI America. A C60 sample with a purity of 99.0% was used. Sodium sulfate was employed as a drying agent. Solvents were routinely distilled before use. The reagents 2-methoxyethanol, triethylene glycol monomethyl ether, 2,2-dimethyl-1,3-dioxane-4,6-dione (Meldrum’s acid), 1H-NMR and 13C-NMR spectra were recorded on a Bruker Avance Spectrospin-500 spectrometer. UV-Vis spectra were recorded on a Perkin Elmer Lambda 750 UV-Vis-NIR Spectrometer. MALDI-mass spectra were recorded on a WATERS Micromass MALDI-TOF mass spectrometer. Elemental analysis was taken by Galbraith Laboratories, Inc. .Infrared spectra were recorded as KBr pellets on a Thermo Nicolet Avatar 370 FT-IR spectrometer. 3, Meldrum’s acid, 1.9 g, 13.2 mmol) was stirred under an Ar atmosphere over a period of 12 h at 95 °C. The reaction mixture was cooled to room temperature, treated with aqueous sodium carbonate solution (5%), and washed with diethyl ether. The resulting aqueous layer was subsequently treated with dil. HCl (2.0 N) and extracted with ethyl acetate (50 mL). The ethyl acetate solution was dried over Na2SO4 and concentrated on rotary evaporator to give the product ME1 (4) in 95% yield (2.0 g). Spectroscopic data: FT-IR (KBr) υmax 3060 (w), 2880 (w), 2814 (w), 1727 (vs), 1458 (w), 1399 (m), 1318 (s), 1249 (s), 1134 (s), 1100 (s), 1039 (m), 952 (w), 848 (s), and 755 (m) cm−1; 1H-NMR  δ 10.60 , 4.31 , 3.62 , 3.45 , and 3.38 .A mixture of 2-methoxyethanol  and 2,2-dimethyl-1,3-dioxane-4,6-dione  was stirred under Ar atmosphere over a period of 12 h at 90 °C. The reaction mixture was cooled to room temperature, treated with aqueous sodium carbonate solution (5%), and washed with diethyl ether. The resulting aqueous layer was subsequently treated with dil. HCl (2.0 N) and extracted with ethyl acetate (50 mL). The ethyl acetate solution was dried over Na2SO4 and concentrated on rotary evaporator to give the product ME3 (5) in 90% yield (1.37 g). Spectroscopic data: FT-IR (KBr) υmax 3056 (w), 2880 (w), 2817 (w), 1728 (vs), 1455 (w), 1394 (m), 1318 (s), 1251 (s), 1134 (vs), 1098 (vs), 1034 (m), 952 (w), 847 (s), and 753 (m) cm−1; 1H-NMR  δ 9.65 , 4.32 , 3.70 , 3.65–3.55 , 3.42 , and 3.41 .A mixture of triethylene glycol monomethyl ether  and 2,2-dimethyl-1,3-dioxane-4,6-dione , N-hydroxysuccinamide , and N,N′-dicyclohexyl carbodiimide  in anyhydrous tetrahydrofuran (20 mL) were stirred under N2 atmosphere over a period of 12 h at ambient temperature. The resulting white solids of N,N′-dicyclohexyl urea byproduct were filtered off and the filtrate was taken into a second round-bottom flask containing N,N′,N,N,N,N-hexapropyl-hexa(aminoethyl)amine 6 . The mixture was stirred under N2 atmosphere for an additional period of 12 h. At the end of the reaction, the solvent was removed on rotavap. To this residue, ice-cold hexane–dichloromethane  was added followed by filtration to remove further white solids of N-hydroxysuccinamide. The filtrate was washed with aqueous sodium carbonate (5%) solution (10 mL). The organic layer was then dried and concentrated to give ME1N6C3 (7) as yellow liquid in 82% yield (1.59 g). Spectroscopic data: ESI–MS (rel. intensity) m/z 568, 570 , 571, 629 (M+), 630 , 631, 646, 666, 670, 688, 700, 715 , 716, 755, 757, 772, 773, 775, 800 (MH+2[CH2CH2N(CH2CH2CH3)], 15%), 801, 832, 860, 1150, 1210, 1212 (the dimer mass–CH3OCH2), 1236, and 1252 (from the dimer ion mass); FT-IR (KBr) υmax 3259 (w), 2956 (vs), 2932 (s), 2872 (m), 2808 (m), 1742 (s), 1660 (s), 1550 (w), 1459 s), 1273 (m), 1128 (m), 1074 (s), 1031 (m), and 742 (w) cm−1; 1H-NMR  δ 4.29 , 3.61 , 3.40 , 3.34–3.32 , 2.38–2.57 , 1.47–1.44 , and 0.90–0.87 ; 13C-NMR  δ 168.78 (–C=O), 164.65 (O=C-NH–), 70.06, 64.15, 58.90, 57.35, 57.29, 57.13, 56.83 (4C), 56.72, 56.55, 53.65, 53.00, 52.81, 52.58, 52.46, 52.00, 41.60, 37.58, 20.41, 20.39, 20.37, 20.32 (2C), 20.18, 11.90 (3C), 11.87 (2C), and 11.74. A mixture of malonic acid methoxyethyleneglycol ester 5 , N-hydroxysuccinamide , and N,N′-dicyclohexyl carbodiimide  in anyhydrous tetrahydrofuran (30 mL) were stirred under N2 atmosphere for a period of 12 h at ambient temperature. The resulting white solids of N,N′-dicyclohexyl urea byproduct were filtered off and the filtrate was taken into a second round-bottom flask containing N,N′,N,N,N,N-hexapropyl-hexa(aminoethyl)amine 6 . The mixture was stirred under N2 atmosphere for an additional period of 12 h. At the end of the reaction, the solvent was removed on rotavap. To this residue, ice-cold hexane–dichloromethane  was added followed by filtration to remove white solids of N-hydroxysuccinamide. The filtrate was washed with aqueous sodium carbonate (5%) solution (10 mL). The organic phase was then dried and concentrated to give ME3N6C3 (8) as yellow liquid in 80% yield (2.29 g). Spectroscopic data: ESI–MS (rel. intensity) m/z 512, 568 , 660 , 716, 718 , 734, 776, 803 , 819, 860, 888 (MH++2[CH2CH2N(CH2CH2CH3)] from the dimer mass, 8%), 944, 951, 1008, 1090, 1226 (the dimer mass–[MeO(EG)3-CO], 3%), 1238, 1282, 1340, 1430, and 1434 (the dimer ion); FT-IR (KBr) υmax 3328 (w), 2952 (m), 2928 (m), 2862 (m), 2805 (m), 1736 (vs), 1667 (s), 1533 (m), 1456 (m), 1400 (m), 1379 (m), 1245 (m), 1104 (vs), 1032 (w), 938 (w), and 732 (m) cm−1; 1H-NMR  δ 4.22 , 3.65 , 3.58–3.60 , 3.48 , 3.32 , 3.26 , 2.31−2.48 , 1.40–1.38 , and 0.82−0.79 ; 13C-NMR  δ 168.83 (–C=O), 164.70 (O=C-NH–), 71.86 (2C), 70.53, 70.51, 68.74, 64.30, 58.95, 57.32 (2C), 57.14, 56.83 (4C), 56.72, 56.53, 53.64, 53.41, 52.98 (2C), 52.80, 52.45, 41.54, 37.55, 20.40, 20.37, 20.31 (3C), 20.24, 11.91 (3C), 11.88 (2C), and 11.76.A mixture of malonic acid methoxytriethyleneglycol ester 7  in anhydrous chloroform–DMF (10:1) was added iodomethane  and stirred at 45 °C for a period of 3.0 days. At the end of quaternization, the solvent was evaporated to afford ME1N6+C3 (9) in 94% yield (2.0 g). Spectroscopic data: FT-IR (KBr) υmax 3444 (s), 3236 (m), 2968 (vs), 2937 (vs), 2877 (m), 1735 (s), 1667 (s), 1536 (m), 1459 (vs), 1331 (w), 1126 (m), 1032 (m), 948 (m), 872 (w), 750 (vs), and 661 (m) cm−1; 1H-NMR  δ 3.80–4.25 , 3.22–3.80 , 1.50–1.85 , and 0.88-0.99 ; 13C-NMR  δ 168.09 (-C=O), 166.69 (O=C-NH–), 79.90, 79.70, 70.01, 64.28 (4C), 63.59 (5C), 58.54, 55.54 (4C), 53.96 (2C), 49.37 (3C), 48.85 (2C), 42.62, 16.27 (2C), 15.95 (4C), 10.94 (2C), 10.82 (2C), and 10.72 (2C) (one peak is covered by DMSO peaks). Anal. Calcd for C34H72N6O4∙4.5CH3I∙2H2O (based on 90% quarternization on average): C, 35.47; H, 6.92; N, 6.45; I, 43.80; O, 7.36%. Found: C, 34.72; H, 6.82; N, 6.46; I, 42.31%.A solution of malonamide ester 8  in anhydrous chloroform–DMF (10:1) was added iodomethane  and stirred at 45 °C for a period of 3.0 days. At the end of quaternization, the solvent was evaporated to afford ME3N6+C3 (10) in 92% yield (0.92 g). Spectroscopic data: ESI–MS (rel. intensity) m/z 448 (100%), 503, 588 (40%), 731 , 760, 815, 872, 873 , 930, 1014, 1016 , 1074, 1099, 1157 , 1198, 1216, 1299, 1300 , 1385, 1391, 1392, 1442, 1443, and 1444 (M++H2O); FT-IR (KBr) υmax 3436 (vs), 3255 (m), 3006 (m), 2968 (s), 2935 (s), 2877 (m), 1736 (s), 1665 (s), 1634 (s), 1545 (w), 1459 (s), 1383 (w), 1351 (w), 1332 (w), 1271 (w), 1200 (m), 1110 (s), 1034 (m), 950 (m), 877 (w), 757 (w), and 600 (m) cm−1; 1H-NMR  δ 3.80–4.25 , 2.90–3.80 , 1.50–1.80 , and 0.85–0.99 ; 13C-NMR  δ 168.13 (–C=O), 166.69 (O=C-NH–), 71.72, 70.23, 70.18, 70.03, 68.57, 63.56 (9C), 54.79 (6C), 49.05 (5C), 42.62, 16.18 (2C), 15.97 (4C), 10.95 (2C), 10.82 (3C), and 10.70 (one peak is covered by DMSO peaks). Anal. Calcd for C38H80N6O6∙4CH3I∙3H2O (based on 80% quarternization on average): C, 37.67; H, 7.38; N, 6.28; I, 37.91; O, 10.76%. Found: C, 36.10; H, 7.03; N, 6.81; I, 37.90%.A solution of malonamide ester 60. To the resulting purple-colored solution added carbon tetrabromide  followed by a solution of the compound 9  in anhydrous DMF (100 mL). The solution mixture was stirred for an additional 30 min and added slowly 1.8-diazabicyclo[5.4.0]-undec-7-ene  over a period of 45 min. The color of solution slowly turns into brown in a reaction period of 8.0 h. The solution was then concentrated on rotavap to roughly 100 mL. Upon the addition of methanol to this concentrated solution, the crude product was precipitated as brown solids which were collected via centrifugation. Unreacted C60 in the crude solids was removed by repeated washings with toluene (5 × 100 mL) until no color in the washing solution or filtrate. The remaining product of C60(>ME1N6+C3) (1) was obtained as brown solids in 55% yield . Spectroscopic data: FT-IR (KBr) υmax 3383 (vs), 2963 (m), 2932 (m), 2870 (m), 2814 (w), 1739 (s), 1686 (s), 1625 (s), 1455 (vs), 1426 (s), 1373 (w), 1240 (w), 1067 (s), 1031 (s), 947 (m), 728 (m), 575 (m), and 525  cm−1; UV-Vis  λmax 323 nm (shoulder peak); 1H-NMR  δ 3.80–4.25 , 2.90–3.80 , 1.50–1.80 , and 0.88–0.99 . We found that electronic interferences of iodide anions in a high quantity with the fullerene cage or possible partial electron-transfer events prohibited the detection of fullerenyl carbon peaks .Finely divided [60]fullerene  was taken into a round bottom flask and added anhydrous toluene (700 mL) under nitrogen. The solution was stirred for 12 h at ambient temperature to ensure complete dissolution of C60. To the resulting purple-colored solution added carbon tetrabromide  followed by a solution of compound 10  in anhydrous DMF (100 mL). The solution mixture was stirred for an additional 30 min of stirring and added slowly 1.8-diazabicyclo[5.4.0]-undec-7-ene  over a period of 45 min. The color of solution slowly turns into brown in a reaction period of 8 h. The solution was then concentrated on rotavap to roughly 100 mL. Upon the addition of methanol to this concentrated solution, the crude product was precipitated as brown solids which were collected via centrifugation. Unreacted C60 in the crude solids was removed by repeated washings with toluene (5 × 100 mL) until no color in the washing solution or filtrate. The remaining product of C60(>ME3N6+C3) (2) was obtained as brown solids in 50% yield . Spectroscopic data: FT-IR (KBr) υmax 3433 (vs), 3262 (s), 2963 (m), 2925 (m), 2868 (m), 2809 (w), 1736 (s), 1695 (m), 1630 (s), 1459 (s), 1384 (w), 1197 (m), 1107 (s), 1071 (s), 938 (m), 757 (w), and 525  cm−1; UV-Vis  λmax 323 nm (shoulder peak); 1H-NMR  δ 3.80–4.20 , 2.90–3.80 , 1.50–1.75 , and 0.88–0.99 . We found that electronic interferences of iodide anions in a high quantity with the fullerene cage or possible partial electron-transfer events prohibited the detection of fullerenyl carbon peaks . Finely divided [60]fullerene  was taken into a round bottom flask and added anhydrous toluene (700 mL) under nitrogen. The solution was stirred for 12 h at ambient temperature to ensure complete dissolution of C4 , 2-methyl-2-propanol , and N,N′-dicyclohexyl carbodiimide  in anyhydrous dichloromethane (20 mL) were stirred under atmospheric pressure of N2 over a period of 8.0 h at ambient temperature. The resulting white solid of N,N′-dicyclohexyl urea was filtered and the filtrate was washed with aqueous sodium carbonate solution . The organic layer was then dried over sodium sulfate and concentrated on rotavap to give ME1(t-C4) (11) in 74% yield (1.0 g) as light yellow liquid. Spectroscopic data: FT-IR (KBr) υmax 2980 (w), 2930 (w), 2880 (w), 2824 (w), 1748 (s), 1726 (vs), 1455 (w), 1406 (w), 1393 (w), 1368 (m), 1330 (m), 1281 (m), 1250 (m), 1199 (m), 1127 (vs), 1100 (m), 1037 (s), 966 (m), 864 (m), 838 (m), 759 (w), and 738 (w) cm−1; 1H-NMR  δ 4.27 , 3.56 , 3.34 , 3.29 , and 1.43 .A mixture of malonic acid methoxyethyleneglycol ester 5 , 2-methyl-2-propanol , and N,N′-dicyclohexyl carbodiimide  in anyhydrous dichloromethane (20 mL) were stirred under atmospheric pressure of N2 over a period of 8.0 h at ambient temperature. The resulting white solid of N,N′-dicyclohexyl urea was filtered and the filtrate was washed with aqueous sodium carbonate solution . The organic layer was then dried over sodium sulfate and concentrated on rotavap to give ME3(t-C4) (12) in 77% yield (2.85 g) as light yellow liquid. Spectroscopic data: FT-IR (KBr) υmax 2973 (w), 2930 (w), 2876 (w), 2817 (w), 1748 (s), 1727 (vs), 1455 (w), 1393 (w), 1368 (m), 1330 (m), 1282 (m), 1250 (m), 1198 (m), 1134 (vs), 1104 (vs), 1040 (m), 966 (m), 864 (m), 759 (w), and 735 (w) cm−1; 1H-NMR  δ 4.26 , 3.68 , 3.63 , 3.52 , 3.35 , 3.29 , and 1.44 .A mixture of malonic acid methoxytriethyleneglycol ester 60. To the resulting purple-colored solution was added carbon tetrabromide  followed by a solution of tert-butyl(2-methoxyethyl)malonate  in anhydrous toluene (10 mL). The solution mixture was stirred for an additional 30 min and added slowly 1.8-diazabicyclo[5.4.0]-undec-7-ene  over a period of 1.0 h. The color of solution slowly turned into brown in a reaction period of 12 h. The solution was then concentrated on a rotavap. The resulting crude product was purified using column chromatography with silica gel as the stationary phase and toluene–ethyl acetate (20:1) as eluent, giving the isolation of tert-butyl(2-methoxyethyl)[60]fullerenyl malonate (13), C60[>ME1(t-C4)], as brown solids in 78% yield (1.00 g). Spectroscopic data: FT-IR (KBr) υmax 3442 , 2972 (w), 2917 (w), 2873 (w), 2814 (w), 1739 (vs), 1645 (m), 1426 (m), 1390 (w), 1366 (m), 1268 (s), 1232 (vs), 1179 (m), 1151 (s), 1110 (m), 1059 (m) 1026 (m), 828 (w), 738 (w), 704 (w), 577 (m), 551 (m), and 525  cm−1; 1H-NMR  δ 4.28 , 3.26 , 3.06 , and 1.55 ; 13C-NMR  δ 163.45 (O=C-O-tert-butyl), 161.86 (O=C-O–), 145.89 (2C), 145.53 (2C), 145.22 (2C), 145.13 (2C), 145.06 (3C), 144.95 (2C), 144.93 (2C), 144.60 (2C), 144.59 (2C), 144.46 (3C), 144.45 (4C), 143.71 (2C), 143.68 (2C), 142.91 (C), 142.89 (C), 142.83 (2C), 142.81 (3C), 142.80 (3C), 142.74 (2C), 142.03 (2C), 142.02 (2C), 141.76 (2C), 141.74 (2C), 140.74 (2C), 140.68 (2C), 139.37 (2C), 138.93 (2C), 84.13, 72.07 , 69.74, 65.49, 58.00, 53.45, and 27.49 (3C).Finely divided [60]fullerene  was taken into a round bottom flask and added anhydrous toluene (850 mL) and 1,2-dichlorobenzene (30 mL) under nitrogen. The solution was stirred for 1.0 h at ambient temperature to ensure complete dissolution of C60. To the resulting purple-colored solution was added carbon tetrabromide  followed by a solution of tert-butyl(methoxy-triethyleneglycol)malonate 12  in anhydrous toluene (10 mL). The solution mixture was stirred for an additional 30 min and added slowly 1.8-diazabicyclo[5.4.0]-undec-7-ene  over a period of 1.0 h. The color of solution slowly turned into brown in a reaction period of 12 h. The solution was then concentrated on rotavap and the resulting crude product was purified by column chromatography with silica gel as the stationary phase and toluene−ethyl acetate (9:1) as eluent to afford C60[>ME3(t-C4)] (14) as brown solids in 75% yield (1.60 g). Spectroscopic data: FT-IR (KBr) υmax 3421 , 2967 (w), 2914 (w), 2864 (w), 2814 (w), 1740 (s), 1634 (m), 1456 (m), 1426 (m), 1392 (m), 1368 (m), 1269 (s), 1253 (s), 1226 (s), 1180 (m), 1154 (s), 1108 (m), 1029 (m), 845 (m), 737 (m), 702 (m), 669 (m), 576 (m), 550 (m), and 525 (vs) cm−1; 1H-NMR  δ 4.65 , 3.90 , 3.73 , 3.65–3.72 , 3.56 , 3.39 , and 1.70 ; 13C-NMR  δ 163.95 (O=C-O-tert-butyl), 162.16 (O=C-O–), 145.61 (2C), 145.41 (2C), 145.29 (2C), 145.25 (4C), 145.22 (2C), 145.16 (3C), 144.85 (2C), 144.71 (C), 144.69 (3C), 144.66 (2C), 144.57 (4C), 143.89 (3C), 143.09 (2C), 143.08 (2C), 143.01 (4C), 142.99 (2C), 142.97 (2C), 142.23 (2C), 142.21 (2C), 141.91 (2C), 141.90 (2C), 140.94 (2C), 140.89 (2C), 139.11 (2C), 138.92 (2C), 85.16, 71.95 , 71.84, 70.68, 70.67, 70.65, 68.84, 66.07, 59.10, 53.06, and 28.07 (3C).Finely divided [60]fullerene  was taken into a round bottom flask and added anhydrous toluene (850 mL) and 1,2-dichlorobenzene (50 mL) under nitrogen. The solution was stirred for 1.0 h at ambient temperature to ensure complete dissolution of C13  was taken into a round bottom flask containing anhydrous dichloromethane (50 mL) and purged with N2 for 15 minutes at ambient temperature. To this reaction mixture was added trifluoroacetic acid  and stirred for overnight at room temperature. At the end of the reaction, dichloromethane was removed on rotavap. Additional dichloromethane (3 × 15 mL) was added and removed on rotavap in order to fully eliminate an excessive amount of trifluoroacetic acid. The resulting residue was then washed with diethyl ether (2 × 15 mL) to afford C60(>ME1H) (15) as brown solids in 89% yield (0.50 g). Spectroscopic data: FT-IR (KBr) υmax 3670 , 2977 (w), 2917 (w), 2878 (w), 2814 (w), 1792 (w), 1750 (s), 1708 (s), 1681 (m), 1541 (w), 1427 (m), 1390 (w), 1366 (m), 1267 (s), 1250 (s), 1231 (s), 1186 (m), 1114 (w), 1059 (w) 1020 (w), 864 (w), 743 (w), 701 (m), 572 (m), 552 (m), and 525  cm−1; 1H-NMR  δ 4.59 , 3.71 , and 3.34 ; 13C-NMR  δ 161.62 (O=C-O–), 161.13 (O=C-OH), 144.54 (2C), 144.06 (2C), 143.53 (2C), 143.39 (2C), 143.26 (2C), 143.25 (2C), 143.19 (2C), 143.16 (2C), 142.91 (C), 142.85 (2C), 142.78 (C), 142.73 (2C), 142.70 (2C), 142.53 (4C), 141.99 (2C), 141.95 (2C), 141.14 (C), 141.12 (C), 141.10 (2C), 141.04 (4C), 140.98 (2C), 140.31 (4C), 140.07 (2C), 139.95 (2C), 138.89 (2C) , 138.87 (2C), 137.87 (2C), 136.76 (2C), 70.48 , 68.14, 63.98, 56.30, and 51.46The compound of [60]fullerenyl malonate 14  was taken into a round bottom flask containing anhydrous dichloromethane (50 mL) and purged with N2 for a period of 15 minutes at ambient temperature. To this reaction mixture was added trifluoroacetic acid (50 mL) and stirred for overnight at room temperature. At the end of the reaction, dichloromethane was removed on rotavap. Additional dichloromethane (3 × 20 mL) was added and removed on rotavap in ordered to fully eliminate an excessive amount of trifluoroacetic acid. The resulting residue was then washed with diethyl ether (3 × 20 mL) to afford C60(>ME3H) (16) as brown solids in 87% yield (0.62 g). Spectroscopic data: FT-IR (KBr) υmax 3673 , 2917 (w), 2894 (w), 2870 (w), 2814 (w), 1788 (w), 1741 (vs), 1578 (w), 1532 (w), 1426 (m), 1383 (w), 1266 (m), 1230 (s), 1203 (m), 1180 (w), 1095 (m), 1060 (w) 845 (w), 698 (m), 579 (m), 549 (m), and 525 (vs) cm−1; 1H-NMR  δ 4.70 , 3.95 , 3.80 , 3.76 , and 3.59 ; 13C-NMR  δ 164.12 (O=C-O–), 163.79 (O=C-OH), 145.57 (2C), 145.52 (2C), 145.33 (3C), 145.32 (3C), 145.22 (4C), 145.15 (3C), 144.84 (2C), 144.75 (C), 144.68 (4C), 144.57 (2C), 144.54 (2C), 143.90 (2C), 143.88 (2C), 143.04 (3C), 142.99 (4C), 142.96 (2C), 142.23 (3C), 142.01 (2C), 141.97 (2C), 140.93 (3C), 140.92 (3C), 139.36 (2C), 138.90 (2C), 72.19, 71.88 , 70.89, 70.64, 70.33, 69.70, 68.34, 65.99, 58.78, and 52.51.The compound of [60]fullerenyl malonate 15  was taken into a round bottom flask containing anhydrous tetrahydrofuran (20 mL). To this reaction mixture was added thionyl chloride  under N2 atmosphere and refluxed for a period of 2.0 h. An excessive amount of thionyl chloride was removed on rotavap. Fresh anhydrous tetrahydrofuran (20 mL) was added. To this reaction solution was added slowly N,N′,N,N,N,N-hexapropyl-hexa(aminoethyl)amine 6  at 0 °C. It was warmed gradually to room temperature and stirred at this temperature for a period of 3.0 h. The resulting solution was concentrated on rotavap with the residue washed sequentially with hexane (10 mL), methanol (2 × 10 mL), and toluene (3 × 10 mL) to fully remove unreacted N,N′,N,N,N,N-hexapropyl-hexa(aminoethyl)amine and decarboxylated C60 byproducts. A relatively pure C60(>ME1N6C3) (17) was obtained in 47% yield (0.11 g). It was subsequently treated with CF3COOH to result in pentacationic quaternary ammonium−trifluoroacetate salt, C60(>ME1N6+C3) (1′), specifically, for NMR measurements. Spectroscopic data of the compound 17: FT-IR (KBr) υmax 3670 , 2977 (w), 2917 (w), 2878 (w), 2814 (w), 1792 (w), 1750 (s), 1708 (s), 1681 (m), 1541 (w), 1427 (m), 1390 (w), 1366 (m), 1267 (s), 1250 (s), 1231 (s), 1186 (m), 1114 (w), 1059 (w), 1020 (w), 864 (w), 743 (w), 701 (m), 572 (m), 552 (m), and 525  cm−1. Spectroscopic data of the compound 1′:1H-NMR  δ 4.22–4.68 , 3.90–4.05 , 3.22–3.75 , 1.28–1.36 , and 0.97 ; 13C-NMR  d 167.72 (O=C-O–), 166.46 (O=C-NH–), 148.00 (2C), 145.54 (2C), 145.30 (2C), 145.29 (2C), 145.24 (2C), 145.14 (2C), 145.02 (2C), 144.73 (4C), 144.68 (4C), 144.53 (2C), 143.98 (2C), 143.77 (2C), 143.27 (2C), 143.11 (C), 143.04 (2C), 143.01 (2C), 142.98 (C), 142.87 (2C), 142.44 (2C), 142.22 (2C), 142.12 (2C), 142.01 (2C), 141.17 (2C), 140.98 (2C), 140.51 (2C), 137.92 (4C), 136.50 (2C), 70.47, 70.23 , 70.08, 69.73, 64.65, 64.40, 58.94, 58.68, 38.43, 31.84, 22.04, 21.40, 16.66, and 10.10 (quaternary aminocarbon peaks were low in intensity). The compound of 2-[60]fullerenyl-3-(2-methoxyethoxy)-3-oxopropanoic acid 1′ (100 mg) in chloroform (50 mL) was neutralized with aqueous potassium carbonate . The resulting [60]fullerenyl malonate 17 was then dissolved in a mixture of anhydrous chloroform (30 mL) and dimethylformamide (15 mL). To this reaction, an excess amount of iodomethane was added in several portions over the reaction period and stirred at 45 °C for 3.0 days. At the end of the reaction, the solvent was removed on rotavap to yield pentacationic C60(>ME1N6+C3) (1). Spectroscopic data: MALDI–TOF–MS  m/z 673 (20%), 697 (50%), 721 , 734 (80%), 746 (15%), 761 (10%), 772 (10%), 874 , 1442 [(C60H)2+ cluster], 1565, 1634, 1709, 1769, 1851, 1930 (M+–I−); FT-IR (KBr) υmax 3688 , 2918 (s), 2870 (m), 2840 (m), 2807 (w), 1784 (w), 1736 (vs), 1663 (s), 1574 (w), 1433 (m), 1383 (w), 1252 (w), 1187 (m), 1163 (s), 1126 (m), 1090 (m), 1031 (s), 842 (w), 762 (w), 704 (w), 661 (w), 569 (w), and 524  cm−1; UV-Vis  λmax 323 nm (shoulder peak); 1H-NMR  δ 3.80–4.25 , 2.90–3.80 , 1.50–1.80 , and 0.88–0.99 . We found that electronic interferences of iodide anions in a high quantity with the fullerene cage or possible partial electron-transfer events prohibited the detection of fullerenyl carbon peaks .A solution of [60]fullerenyl malonate quaternary ammonium−trifluoroacetate salt 2 was carried out by using a similar procedure as that of 1 except methoxytriethyleneglycol ester was applied instead. Spectroscopic data: MALDI–TOF–MS m/z 698 (10%), 721 , 735 , 749, 773, 782, 789, 809, 874 [C60(>H(C=O)NHCH2CH2N+-propylMe2)], 914, 940, 995, 1027, 1054, 1278 (w), 1395 (w), 1417 (vw), 1499 (vw), 1792 (vw), and 2019 ; FT-IR (KBr) υmax 3424 (vs), 2967 (m), 2925 (m), 2874 (m), 2824 (w), 1738 (s), 1681 (s), 1628 (s), 1454 (vs), 1429 (s), 1383 (m), 1064 (s), 1028 (s), 941 (m), 727 (m), 572 (m), and 524  cm−1; UV-vis  λmax 323 nm (shoulder peak); 1H-NMR  δ 3.80–4.20 , 2.90–3.80 , 1.50–1.75 , and 0.88–0.99 . We found that electronic interferences of iodide anions in a high quantity with the fullerene cage or possible partial electron-transfer events prohibited the detection of fullerenyl carbon peaks . Synthesis of the compound 60(>ME1N6+C3) (1) and C60(>ME3N6+C3) (2), with variation of the methoxyethyleneglycol length. Each of these derivatives bears a well-defined number of cationic charges aimed to enhance and control their ability to target pathogenic Gram-positive and Gram-negative bacterial cells. The intrinsic nature of the high charge number and increased water-solubility of the precursor arm intermediates hindered the efficiency of their reactions with a highly hydrophobic C60 cage having low compatibility in polar solvents. Furthermore, consecutive ethylamino group linkage in a structure of N,N′,N,N,N,N-hexapropyl-hexa(aminoethyl)amine (C3N6) largely increased its electron-donating capability that gave complication in forming an insoluble partial charge-transfer complex with C60 during the reaction and workup procedures. After many attempts, we found a circumventive solution by the use of partially quaternized C3N6 prior to the reaction with C60 coupled with the modification of C3N6 arm using propyl groups, instead of methyl or ethyl groups, to provide a well-balanced hydrophobicity– hydrophilicity character of pentacationic precursor intermediates and better compatibility with the C60 cage moiety.We have designed and synthesized two analogous pentacationic [60]fullerenyl monoadducts, C


Moringa peregrina is a wild plant that grown in the eastern desert mountains in Egypt. Although, this plant is native to Egypt, no details studies were traced on its chemical composition and biological activity.The different fractions of the ethanolic extract of the dried aerial parts of the plants were subjected to fractionation and purification on various silica and sephadex columns for the isolation of the major compounds which were tested for there anticancer activity. The aqueous and ethanolic extract as well as its different fractions were tested for antihyperglycemic effect on Streptozitocin-induced diabetes in rats.M. peregrina yielded lupeol acetate (1), β-amyrin (2), α-amyrin (3), β-sitosterol (4), β-sitosterol-3-O-glucoside (5), apigenin (6), rhamnetin (7), neochlorogenic acid (10), rhamnetin-3-O-rutinoside (12), and 6-methoxy-acacetin-8-C-β-glucoside (13) which were isolated for the first time from the plant. Compound (13) was isolated for the first time from genus Moringa. In addition, quercetin (8), chryseriol-7-O-rhamnoside (9) and quercetin-3-O-rutinoside (11) were also isolated. Identification has been established by spectral data . The major isolated compounds were found to have valuable cytotoxic activities against breast (MCF 7) and colon (HCT 116) cancer cell lines and their activities were comparable to the reference drug doxorubicin. On the other hand, the aqueous and ethanolic extracts as well as the n-hexane fraction were found to have potent antihyperglycemic effect on Streptozitocin-induced diabetes in rats.Investigation of the different fractions of the ethanolic extract of the aerial parts of M. peregrina is rich in biologically active ingredients which showed potent cytotoxic activity and also its ethanolic extraxt exert a significant antihyperglycemic effect.The Egyptian plant  Moringa peregrina (Forssk.) Fiori, a single report was found about the antihyperglycemic effect of the ethanolic extract of the defatted aerial parts from which quercetin, quercetin-3-O-rutinoside, chryseriol-7-O-rhamnoside, and 6, 8, 3’,5’-tetramethoxy apigenin were isolated.+ (7.9), 408 [M-CH3 COO]+ (40), 218 (56), 203 (77), 189 (100).MS : Compound 2White needle crystals (n-hexane).mp: 195-197°C.Rf: 0.56 (n-hexane-ethyl acetate 9:1).IR (KBr): 3400, 3242, 1690.m/z (%) = 426.1 [M]+ (10), 218 (100), 203 (79), and 189 (60).MS : Compound 3White needle crystals (n-hexane).mp:185-186°C.Rf: 0.4 (n-hexane-ethyl acetate 9:1).IR (KBr): 3400, 3242, 1690.m/z (%) = 426 [M]+ (12.3), 218 (100), 203 (38.46), and 189 (34.61).MS : Compound 4White needle crystals (n-hexane).mp: 140-141°C.Rf: 0.25 (n-hexane-ethyl acetate 9:1).IR (KBr): 3400, 3242, 1690, 1212, 1051, 1022, 953.m/z (%) = 414 [M]+ (100), 396 (51), 329 (42), 303 (44), 273 (60), and 255 (80).MS : Compound 5White microcrystalline powder.mp: 290°C.Rf: 0.37 (chloroform-methanol 9.5:0.5).IR (KBr): 3400, 3242, 1690, 1212, 1051, 1022, 953.1H-NMR : 0.66 , 0.78 , 0.83 , 0.90 , 0.92 , 0.96 , 3.03 , 4.21 , 5.33  ppm.Compound 6Yellow microcrystalline powder.mp: 348-350°C.Rf: 0.45 (chloroform-methanol 9.5:0.5).IR (KBr): 3300, 3050, 2920, 1660, 1620, 1510, 1360, 1060, 910.max nm: (MeOH) 266, 296sh, 336 (NaOMe) 274, 324, 390 (AlCl3) 274, 299, 386 (AlCl3 /HCl) 274, 299, 382 (NaOAc) 271, 300, 376 (NaOAc-H3 BO3) 269, 301sh, 338.UV-Vis λCompound 7Yellow microcrystalline powder.mp: 294-296°C.Rf: 0.42 (chloroform-methanol 9.5:0.5).IR (KBr): 3295, 3050, 2920, 1640, 1620, 1510, 1360, 1060, 910.max nm: (MeOH) 256, 286sh, 371 (NaOMe) 286, 432 (AlCl3)272, 302sh, 451 (AlCl3 /HCl) 268, 299sh, 356 (NaOAc) 255, 292sh, 387 (NaOAc-H3 BO3) 267, 389.UV-Vis λCompound 8Yellow microcrystalline powder.mp: 317-319°C.Rf: 0.5 (chloroform-methanol 9:1).IR (KBr): 3300, 3050, 2920, 1600, 1640, 1510, 1360, 1295, 1060, 910.max nm: (MeOH) 260, 368 (NaOMe) 272, 326sh, 406 (AlCl3)272, 446 (AlCl3 /HCl) 266, 430 (NaOAc) 272, 404 (NaOAc-H3 BO3) 260, 384.UV-Vis λCompound 9Yellow microcrystalline powder.mp: 230-232°C.Rf: 0.6 (chloroform-methanol 8:2).IR (KBr): 3300, 3050, 2920, 1600, 1640, 1510, 1360,1295, 1060, 910.max nm: (MeOH) 268, 267sh, 344 (NaOMe) 262, 404 (AlCl3)276, 330sh, 354sh, 384 (AlCl3 -HCl) 274, 300sh, 352sh, 382 (NaOAc) 268, 352sh 406 (NaOAc-H3 BO3) 264, 338.UV-Vis λCompound 10Yellowish-white amorphous powder.Rf: 0.23 [ethyl acetate-methanol-water-formic acid (100:16:12:1:0.1 v/v/v/v)].IR (KBr): 2800, 1620, 1420, 1300, 1310, 1200, 1180, 1150.max nm: (MeOH) 290, 326UV-Vis λ1H-NMR : 1.59 , 1.78 , 1.94 , 3.79 , 3.94 , 5.14 , 6.18 , 6.73 , 6.94 , 7.04 , 7.40 .Compound 11Yellow amorphous powder.mp: 190-192°C.Rf: 0.3 [ethyl acetate-methanol-water-formic acid (100:16:12:1:0.1 v/v/v/v)].IR (KBr): 3300, 3050, 2920, 1600, 1640, 1510, 1360, 1295, 1060, 910.max nm: (MeOH) 258, 300sh, 358. (NaOMe) 268, 328sh, 410 (AlCl3)270, 306sh, 426 (AlCl3 /HCl) 268, 298sh, 366, 400 (NaOAc) 264, 300sh, 382 (NaOAc-H3 BO3) 262, 308sh, 378.UV-Vis λCompound 12Yellow amorphous powder.Rf: 0.34 [ethyl acetate-methanol-water-formic acid (100:16:12:1:0.1 v/v/v/v)].IR (KBr): 3300, 3050, 2920, 1600, 1640, 1510, 1360, 1295, 1060, 910.max nm: (MeOH) 256, 270sh, 358. (NaOMe) 273, 328, 415 (AlCl3)270, 299sh, 407 (AlCl3 /HCl) 267, 298sh, 360 (NaOAc) 274, 316, 387 (NaOAc-H3 BO3) 257, 390.UV-Vis λ1H-NMR : 0.97 , 3.84 , 4.39 , 5.42 , 6.18 , 6.37 , 6.90 , 7.50 , 7.53 .13C-NMR : 66.84 (C-6’’), 70.09 (C-4’’),74.27 (C-2’’),75.91 (C-5’’), 76.39 (C-3’’), 101.22 (C-1’’), 17.68 (C-6’’), 68.26 (C-5’’’) 70.29 (C-2’’’), 70.58 (C-3’’’), 71.79 (C-4’’’), 100.86 (C-1’’’), 93.60 (C-8), 98.78 (C-6), 103.88 (C-10), 115.07 (C-2’), 116.22 (C-5’), 121.03 (C-6’), 122.26 (C-1’), 133.00 (C-3), 144.73 (C-3’), 146.87 (C-2), 148.43 (C-4’), 156.37 (C-9), 161.14 (C-5), 164.44 (C-7), 178.07 (C-4).Compound 13Yellow amorphous powder.Rf: 0.5 [ethyl acetate-methanol-water-formic acid (100:16:12:1:0.1 v/v/v/v)].IR (KBr): 3300, 3050, 2920, 1602, 1640, 1510, 1360, 1295, 1064, 915.max nm: (MeOH) 269, 302sh, 327, (NaOMe) 276, 295sh, 364 (AlCl3)259sh, 277, 292sh, 380 (AlCl3 /HCl) 260sh, 279, 296sh, 352, 384 (NaOAc) 278, 297sh, 358 (NaOAc-H3 BO3)267, 309sh, 331.UV-Vis λ1H-NMR : 3.70 , 3.74 , 4.66 , 6.75 , 6.89 , 7.98 .13C-NMR : 61.96 (C-6’’), 70.85(C-4’’), 70.86 (C-2’’), 73.45 (C-1’’), 78.83 (C-3’’), 82.01 (C-5’’), 56.61 (4’-OCH3), 61.7 (6-OCH3), 102.41 (C-3), 103.96 (C-8), 104.60 (C-10), 116.14 , 121.95 (C-1’), 129.36 , 133.07 (C-6), 156.05 (C-9), 160.43 , 162.88 (C-2), 164.11 (C-7), 181.99 (C-4). Structures of the isolated compounds are shown in ®), regular, soluble human insulin injection, Lilly Company, USA.Insulin  and IC10  for each tested sample were calculated and recorded in Cytotoxicity was tested using the method of Skehan et al. on 2 hum50 of the aqueous and ethanolic extracts was calculated according to Karber (1931).[The LDKarber 191.[3]ad libitum throughout the treatment period. The rats were allowed 1 week to acclimatize to pharmacology departmental animal house.Healthy albino Wistar rats  obtained from the animal house were housed throughout the experiment in polycarbonated cages and the housing facility was maintained at standard conditions: temperature (28°C ± 2°C), relative humidity (50% ± 5%), and a 12:12 h light:dark cycle. Water and commercial palletized diet were available to the animals Rats were rendered diabetic by injecting a freshly prepared streptozotocin . by comparing its IR, UV spectral data,1H-NMR spectrum, and1H-1H COSY with the published data.[12 was identified as rhamnetin-3-O-rhamnoglucoside through its IR, UV spectral data,1H-NMR, and13C-NMR spectra.[11 was identified as quercetin-3-O-rutinoside from its IR, UV data, and by direct comparison with an authentic sample (co-TLC).Four compounds 10, 11, 12, and 13) were isolated from the , and 13 hed data. Compound spectra.15 Compou1H and13C-NMR spectrum of compound 13 indicated the presence of an acacetin nucleus[1H-NMR spectrum indicated a β-linked sugar and glycosylation was at C-8.[13C-NMR spectrum through the upfield shift of the anomeric carbon C-1’’, which appeared at δ 78.83 ppm and the downfield shift of C-8 (by about 9 ppm), which appeared at δ 102.41 ppm.[13C-NMR spectrum at δ 61.7 ppm suggested the presence of a methoxy group at C-6. This was confirmed through the downfield shift of C-6 (by about 35 ppm), which appeared at 133.07 ppm.[The IR, UV spectral data, andn nucleus–17 with 2.41 ppm. The abse3.07 ppm. From theM. peregrina had a potent cytotoxic activity against the 2 tested human cell lines Colon cancer cell line (HCT116) and breast cancer cell line (MCF-7) [50 of the different fractions, which were comparable to that of doxorubicin. The major isolated compounds were also tested for their cytotoxic activity, and they showed a potent activity against the 2 cancer cell lines.On measuring the acute toxicity, the plant showed no toxic symptoms and no deaths after oral and intraperitoneal administration of the tested doses. The different fractions of the ethanolic extracts of the aerial parts of  (MCF-7) . This isM. peregrina significantly decreased blood glucose level of streptozitocin diabetic rats [®. Accordingly, the n-hexane (HF), chloroform (CF), ethyl acetate (CF) and n-butanol (BF) fractions of the ethanolic extract were also tested for their antihyperglycemic effect through intraperitoneal injection of a dose of 50 mg/kg b.wt. using insulin as a standard antidiabetic [On the other hand, the aqueous (A) and ethanolic (E) extracts of the aerial parts of tic rats  and theidiabetic . The n-hdiabetic


Our results support that compounds 1–5 potently inhibited LPS (lipopolysaccharide)-induced nitric oxide (NO) production in a dose-dependent manner. The IC50 values of compounds 1, 3 and 5 were 1.8 ± 0.2, 18.8 ± 0.6 and 0.8 ± 0.3 μg/mL, respectively.Three new benzenoids, 3-isopropenyl-2-methoxy-6-methyl-4,5-methylenedioxyphenol ( Antrodia camphorata Wu, Ryvarden and Chang  (Polyporaceae) is a parasitic fungus on the inner wall of the heartwood of Cinnamomun kanehiria Hay (Lauraceae). The fruiting bodies of A. camphorata are called “chang-chih” or “niu-chang-chih” in Taiwan. Traditionally, the fungus has been used for the treatment of food and drug intoxication, diarrhea, abdominal pain, hypertension and liver cancer , a Me group  and a MeO group  on the phenol. The presence of an isopropenyl group was revealed by a Me group , two olefinic protons of CH2  and a quaternary C-atom [δC 127.2 (C-7)]. On the basis of HMBC (H 5.93 (OCH2O) coupled to δC 133.0 (C-5) and 136.0 (C-4); δH 2.27 (Me) correlated to δC 110.0 (C-6), 132.0 (C-1) and 133.0 (C-5); δH 4.64 (OH) coupled to δC 132.0 (C-1); δH 3.94 (MeO) coupled to δC 138.5 (C-2); δH 1.99 (Me-7) coupled to δC 97.1 (C-3)] and a combination with the Nuclear Overhauser Effect Spectroscopy (NOESY) experiment (H 3.94) correlation with isopropenyl group ] corroborated the locations of the functional groups on the benzene ring. On the basis of the 1H- and 13C-NMR . The presence of phenolic moiety was revealed by IR spectral data . The above data combined with the NMR data , 5.98 ], two MeO groups , two Me groups  and a single aromatic proton [δH 5.94 ]. The 13C-NMR , 15.8 (Me-3′)], two MeO , two OCH2O , one aromatic CH [δC 109.5 (C-2′)] and 11 aromatic quaternary C-atoms . These data also indicated a biphenyl skeleton. Assignment of chemical shifts of all protonated C-atoms and their associated H-atoms in the molecule can be finished according to HMQC data. On the basis of HMBC . The presence of phenolic moiety was revealed by IR spectral data . According to the molecular formula, IR spectrum combined with nine 13C-NMR signals indicated that compound 3 is a symmetrical biphenolic derivative. These data with the NMR data ], a MeO group [δH 3.88 ], a Me group [δH 1.82 ] and a hydroxy group [δH 4.56 ]. The 13C-NMR ], a MeO [δC 60.1 (MeO-3)], a OCH2O [δC 101.7 (5-OCH2O-6)] and six aromatic quaternary C-atoms (δC 114.6 (C-2), 123.7 (C-1), 133.2 (C-4), 133.3 (C-6), 136.0 (C-3), 138.9 (C-5). Because HR-FABMS showed that the molecular formula is C18H18O8, 3 was suggested to be a symmetrical biphenolic compound. The HMBC data  + .Colorless oil. UV (MeOH): 280 (3.92). IR (neat): 3440, 1618, 1510, 1470, 1230, 1061, 1026. 1H- and 13C-NMR: see m/z: 347.1128 [M + 1]+ .Amorphous solid. UV (MeOH): 276 (3.86). IR (KBr): 3481, 1615, 1512, 1468, 1240, 1155. 1H- and 13C-NMR: see m/z: 363.1076 [M + 1]+ .Amorphous solid. UV (MeOH): 270 (3.74). IR (KBr): 3421, 1605, 1508, 1472, 1233, 1130, 1089. Escherichia coli, serotype 0127:B8), Carr (type IV), indomethacin, MTT  and other chemicals were purchased from Sigma-Aldrich Chemical Co. .LPS (endotoxin from 2 incubator (5% CO2 in air) at 37 °C and subcultured every 3 days at a dilution of 1:5 using 0.05% trypsin-0.02% EDTA in Ca2+-, Mg2+-free phosphate-buffered saline (DPBS).A murine macrophage cell line, RAW264.7 (BCRC No. 60001), was purchased from the Bioresources Collection and Research Center  of the Food Industry Research and Development Institute . Cells were cultured in plastic dishes containing Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum  in a CO5) were cultured in 96-well plate containing DMEM supplemented with 10% FBS for 1 day to become nearly confluent. Then, cells were cultured with compounds 1–5 in the presence of 100 ng/mL LPS (lipopolysaccharide)  for 24 h. After that, the cells were washed twice with DPBS and incubated with 100 μL of 0.5 mg/mL MTT for 2 h at 37 °C testing for cell viability. The medium was then discarded, and 100 μL dimethyl sulfoxide (DMSO) was added. After 30-min incubation, absorbance at 570 nm was read using a microplate reader .Cells (2 × 10NO production was indirectly assessed by measuring the nitrite levels in the cultured media and serum determined by a colorimetric method based on the Griess reaction. The cells were incubated with different concentrations of samples in the presence of LPS (100 ng/mL) at 37 °C for 24 h. Then, cells were dispensed into 96-well plates, and 100 μL of each supernatant was mixed with the same volume of Griess reagent  and incubated at room temperature for 10 min; the absorbance was measured at 540 nm with a Micro-Reader (Molecular Devices).50 values were estimated using a non-linear regression algorithm . Statistical evaluation was carried out by one-way analysis of variance .IC1), 2-hydroxy-4,4′-dimethoxy-3,3′- dimethyl-5,6,5′,6′-bimethylenedioxybiphenyl (2) and 4,4′-dihydroxy-3,3′-dimethoxy-2,2′-dimethyl- 5,6,5′,6′-bimethylenedioxybiphenyl (3) are new compounds from A. camphorata. Compounds 1, 3 and 5 displayed a significant concentration-dependent inhibition of NO production with IC50 values 1.8 ± 0.2, 18.8 ± 0.6 and 0.8 ± 0.3 μg/mL, respectively.3-Isopropenyl-2-methoxy-6-methyl-4,5-methylenedioxyphenol (


Synthetic sialic acid analogues with multiple modifications at different positions(C-1/C-2/C-4/C-8/C-9) are investigated by molecular mechanics and molecular dynamics to determine their conformational preferences and structural stability to interact with their natural receptors. Sialic acids with multiple modifications are soaked in a periodic box of water as solvent. Molecular mechanics and a 2 nanosecond molecular dynamics are done using amber force fields with 30 picosecond equilibrium. Direct and water mediated hydrogen bonds existing in the sialic acid analogues, aiding for their structural stabilization are identified in this study. The accessible conformations of side chain linkages of sialic acid analogues holding multiple substituents are determined from molecular dynamics trajectory at every 1ps interval. Transitions between different minimum energy regions in conformational maps are also noticed in C-1, C-2, C-4, C-8 and C-9 substituents. Docking studies were done to find the binding mode of the sialic acid analogues with Influenza hemagglutinin. This finding provides stereo chemical explanation and conformational preference of sialic acid analogues which may be crucial for the design of sialic acid analogues as inhibitors for different sialic acid specific pathogenic proteins such as influenza toxins and neuraminidases. The binding of a virus particle to the surface receptors of a host cell is mediated by viral proteins, which specifically recognize receptor determinants, such as peptides, lipids or carbohydrates. The recBased on this knowledge, it should, in principle be possible to find a neuraminic acid analogue that mimics the cell receptor and thus preferentially binds to the virus, thereby blocking attachment. One approach in the design of high-affinity inhibitors is to use Neu5Ac (or its 2α-O-methyl derivative) as a scaffold and to modify its functional groups in order to increase its affinity for the HA. The configuration of neuraminic acid places the carboxylate in the axial position is the alpha-anomer of neuraminic acid.Present work was initiated with the modeling of the alpha-anomer of neuraminic acid and its derivatives having multiple substitutions at C-1, C-2, C-4, C-8 and C-9 positions. Molecular mechanics and molecular dynamics calculations were performed. The conformational behaviour of varying substituent holding side chains of neuraminic acid in aqueous environment were studied. The direct and water-mediated hydrogen bonds, which played a major role in the structural stability of neuraminic acid were also analyzed. Docking studies were done to study the binding mode of neuraminic acid derivatives into the binding pocket of Influenza HA.et al., 1992[et al., 1998[1=0 when C3-C2 cis to C1-O1A; γ2=0 when C2-C1 cis to O1A-C11, where C11 is the carbonyl carbon atom from the substitution group. For C-2 substitution, θ1=0 when C4-C3 cis to C2-O2; θ2=0 when C3-C2 cis to O2-C12, where C12 is the carbon attached to the substituent group. For C-4 substitution, β1=0 when C2-C3 cis to C4-O4; β2=0 when C3-C4 cis to O4-C13, where C13 is the carbon atom attached to the substituent group. For C-8 substitution, χ1=0 when C5-C6 cis to C7-C8; χ3=0 when C6-C7 cis to C8-O8. For C-9 substitution, χ1=0 when C5-C6 cis to C7-C8; χ2=0 when C6-C7 cis to C8-C9.The modeled neuraminic acid derivatives with multiple substituents are shown in al., 1992 and Bianal., 1998. The iniMolecular mechanics calculations were carried out in the Pentium IV workstation using SANDER module of software AMBER10. The for-1. The energy minimized structures for all the neuraminic acid derivatives are intensively analyzed through graphics software VEGA ZZ to work out the direct and solvent mediated hydrogen bonds.A periodic box enclosing the neuraminic acid analogues in solution is constructed to turn it into a periodic system for the simulation programs and periodic boundary conditions are applied on constant volume . The nonTo understand the conformational dynamics of the neuraminic acid analogues in aqueous environment, molecular dynamics calculations were performed over a period of 30ps equilibration followed by a 2 ns production run with explicit inclusion of water molecules. The width of integration step of the MD simulation was 1fs. The history of information was recorded for every 1000 steps of trajectory which resulted in 2000 structures. The temperature was maintained to be 300K. The total simulation time was around 17 h for each molecule. The MD trajectory information collected for every 1ps were analyzed using PTRAJ  module of AMBER10 package.Docking studies were done for all the 18 sialic acid analogues using Schrodinger (maestro). The protein is prepared by optimizing and minimizing the structure using Protein Preparation Wizard. The grid is generated using Receptor Grid Generation by picking the reference ligand which is already present in the PDB structure. HTVS is performed by importing 18 minimized sialic acid analogues using GLIDE module of Schrodinger software8. InducedThe relative energy is calculated for all the 10 neuraminic acid derivatives with respect to the absolute minimum energy of neuraminic acid . The minimum energy conformations of neuraminic acid derivatives with respect to their relative energy are displayed in 1, χ2) prefer values around . For Analogue 6 (2α-O-methyl-4-O-capriloyl-5-N-acetyl neuraminic acid), the substituent dwelling side chain torsion angles  and  prefer the values  and , respectively.It is noted from 2 group contributed to both water mediated and direct hydrogen bond. It is also observed that analogue 6 (2α-O-methyl-4-O-capriloyl-5-N-acetyl neuraminic acid) has three water mediated hydrogen bonds and three direct hydrogen bonds. It is inferred that the maximum number of direct and water mediated hydrogen bonds present in each neuraminic acid derivatives are responsible for the minimum energy and its stabilization of the molecule.Figs. To study the conformational dynamics of the neuraminic acid derivatives, a 2ns molecular dynamics simulation was carried out. An in-depth analysis on the conformational features of all the 10 neuraminic acid analogues was done by collecting the frames for every 1ps.1 and γ2 exhibit a bifurcation throughout the molecular dynamics simulation. The torsional angle γ1 of analogue 2 (methyl 2α-O-methyl-5-N-acetyl neuraminate), analogue 3 , analogue 4 , analogue 5 (benzyl 2α-O-methyl-4-O-capryloil-5-N-acetyl neuraminate) and analogue 8 (benzyl-2α-O-methyl-4-O-(8-morpholin)-capriyloyl-5-N-acetyl-neuraminate) shows good distribution throughout the MD simulation, however γ2 is rigid in +180° and -180° regions. The shift of γ1 from -180° region to +70° region results in the energy decrease of 5 kcal/mol. 1 and γ2 along with  distribution plot of analogue 5 (benzyl 2α-O-methyl-4-O-capryloil-5-N-acetyl neuraminate). In the case of analogue 7 (benzyl-2α-O-methyl-4-O-(8-morpholin)-capriloyl-5-N-acetyl-8,9-O-isopropylidene-neuraminate), γ1 bifurcates in negative regions such as -180°, -120° and -60°, whereas γ2 prefers +180° and -180° region.For analogue 1 (methyl 5-N-acetyl neuraminate), the dihedral angles γ1 is rigid in -60° region and θ2 prefers +180° and -180° regions. 1 and θ2 along with  distribution plot of 2α-O-methyl-4-O-capriloyl-5-N-acetyl neuraminic acid  in aqueous environment.For analogue 2 (methyl 2α-O-methyl-5-N-acetyl neuraminate), 3 , 4 , 5 (benzyl 2α-O-methyl-4-O-capryloil-5-N-acetyl neuraminate), 6 (2α-O-methyl-4-O-capriloyl-5-N-acetyl neuraminic acid) and 7 (benzyl-2α-O-methyl-4-O-(8-morpholin)-capriloyl-5-N-acetyl-8,9-O-isopropylidene-neuraminate), θ2 of analogue 3 , analogue 5 (benzyl 2α-O-methyl-4-O-capryloil-5-N-acetyl neuraminate) and analogue 6 (2α-O-methyl-4-O-capriloyl-5-N-acetyl neuraminic acid) exhibits an additional bifurcation in -60° region. In the case of analogue 8 (benzyl-2α-O-methyl-4-O-(8-morpholin)-capriyloyl-5-N-acetyl-neuraminate), θ1 prefers -70° region and θ2 prefers +180°, -180° and -70° regions.However the dihedral angle θ1 of analogue 4 , 5 (benzyl 2α-O-methyl-4-O-capryloil-5-N-acetyl neuraminate), 7 (benzyl-2α-O-methyl-4-O-(8-morpholin)-capriloyl-5-N-acetyl-8,9-O-isopropylidene-neuraminate), and 8 (benzyl-2α-O-methyl-4-O-(8-morpholin)-capriyloyl-5-N-acetyl-neuraminate) prefers +180° and -180° regions. β2 of analogue 5 (benzyl 2α-O-methyl-4-O-capryloil-5-N-acetyl neuraminate), 7 (benzyl-2α-O-methyl-4-O-(8-morpholin)-capriloyl-5-N-acetyl-8,9-O-isopropylidene-neuraminate) and 8 (benzyl-2 α-O-methyl-4-O-(8-morpholin)-capriyloyl-5-N-acetyl-neuraminate) prefers -180° and -70° regions. β2 of analogue 4  prefers -180°, -120°, +180° and -60° regions.β2 shows good distribution in -180°, +180°, -70° and +60° regions. β1 prefers +180° and -180° regions. 1 and β2 along with  distribution plot of 2α -O-methyl-4-O-capriloyl-5-N-acetyl neuraminic acid  in aqueous environment.For analogue 7 (benzyl-2α-O-methyl-4-O-(8-morpholin)-capriloyl-5-N-acetyl-8,9-O-isopropylidene-neuraminate), the shift of β 2 from -70° to -180° region results in the energy decrease of up to 6 kcal/mol. In the case of analogue 6 (2α-O-methyl-4-O-capriloyl-5-N-acetyl neuraminic acid), β1 prefers +180° and -180° regions and χ3 is rigid in +70° region. 1 and χ3 along with  distribution plot of analogue 3 .For analogue 3 , analogue 4  and analogue 7 (benzyl-2α-O-methyl-4-O-(8-morpholin)-capriloyl-5-N-acetyl-8,9-O-isopropylidene-neuraminate), χ1 analogue 3 , 4  and 7 (benzyl-2α-O-methyl-4-O-(8-morpholin)-capriloyl-5-N-acetyl-8,9-O-isopropylidene neuraminate) prefers +180° and -180° regions and χ2 prefers -70° region. In the case of analogue 10 (5-N-acetyl-9-amino-9-deoxy neuraminic acid), χ1 prefers -180° +60° and +180° regions and χ2 shows bifurcation in -70°, +70°, +180° and -180° regions. The shift of χ1 from -180° region to +60° region resulted in the energy decrease of up to 10kcal/mol. 1 and χ2 along with  distribution plot of neuraminic acid analogues benzyl 2α-O-methyl-4-O-capriloyl-5-N-acetyl-8,9-O-isopropylidene neuraminate  and 5-N-acetyl-9-amino-9-deoxy neuraminic acid  in aqueous environment.χHigh Throughput Virtual Screening was done for ten neuraminic acid analogues to find out the structures (ligands) most likely to bind to the Influenza hemagglutinin. The top five ligands with best docking score and minimum energy are subjected to induced fit docking. The glide score along with the glide energy is displayed in 1 of analogue 10 (5-N-acetyl-9-amino-9-deoxy neuraminic acid) at the cost of 10 kcal/mol , 5-N-acetyl-9-amino-9-deoxy neuraminic acid , 5-N-acetyl-9-amino-9-deoxy neuraminic acid , benzyl 2α-O-methyl-4-O-capriloyl-5-N-acetyl-8,9-O-isopropylidene neuraminate  and benzyl 2α-O-methyl-5-N-acetyl-8,9-O-isopropylidene neuraminate  have greater docking score than the co-crystal ligand (PDB reference ligand) such as -8.64, -7.73, -7.71, -6.71 and -5.34, respectively and minimum energy such as -54.64 kcal/mol, -36.70 kcal/mol, -42.87 kcal/mol, -45.91 kcal/mol and -42.16 kcal/mol, respectively.The current study reveals the probable conformational models for neuraminic acid derivatives with multiple substitutions at positions C-1/C-2/C-4/C-8/C-9 in aqueous environment. Water mediated hydrogen bonding interaction plays a dominant role in stabilizing the conformational structures of these neuraminic acid derivatives. The accessible conformations for neuraminic acid analogues with multiple substituents holding side chain linkages observed by the present MD study correlate well with those reported for similar linkages in various Neu5Ac-α2→8-Neu5Ac moiety present in all the di- and tri-sialogangliosides by earlier studies12. Presekcal/mol , which iThe present study provides accessible conformational models for synthetic neuraminic acid analogues with multiple substituents at positions C-1, C-2, C-4, C-8 or C-1 in aqueous environment. Direct and water mediated hydrogen bonding schemes greatly involve in stabilizing the three dimensional conformational structures of these neuraminic acid analogues. This study also shows the dynamics trajectory and distribution plot for the substituent holding side chain linkages of the neuraminic acid analogues. The high affinity inhibitors modeled in this study saturate the hemagglutinin (HA) receptor and can


The structures of 1 to 3 were established by spectroscopic and physicochemical analysis, and chemical investigation including the total synthesis of 1. Treatment with novo-benzomalvins A (1), B (2), C (3), and N-methylnovobenzomalvin A (5) increased the expression of fibronectin in normal human neonatal dermal fibroblast cells.Three new quinazolinobenzodiazepine derivatives, novobenzomalvins A ( Aspergillus fumigatus is known as an important human pathogen, which produces many secondary metabolites. Recently, Hong et al. re-identified the fungus Aspergillus novofumigatus CBS11520 as the new Aspergillus sp., which is closely related to A. fumigatus  of the N-methylated product (5) derived from 1 [+136° ] was clearly different from that of benzomalvin A (4) [−106° ]  . Thus, 12 was found to be C23H17N3O3 by HREIMS. The 1H and 13C NMR spectra of 2 was similar to those of 1, expect for the downfield shift of the carbon at C-20 from δ 35.2 in 1 to δ 72.9 in 2 and the new appearance of a methine signal (δ 5.32) in 2 instead of a methylene signal (δ 3.29 and δ 3.59) in 1 (1H 1H COSY and HMBC spectra supported the planar structure of 2 being the 20-hydroxy derivative of 1 (2) was performed to confirm the structure, as the crystal of 2 was suitable for X-ray analysis. The result established the absolute configuration of 2 as shown in The molecular formula of 59) in 1 . Analysiive of 1 . X-ray c23H15N3O3) of 3 suggested the loss of two protons from that of 2 (C23H17N3O3) and thus indicated the replacement of a hydroxyl group in 2 with a carbonyl group in 3. The 1H and 13C NMR spectra of 3 and 2 showed only the difference of a carbonyl sp2 carbon signal (δ 188.9) in 3 instead of a secondary hydroxyl group  in 2  was prepared by treatment of 2 with pyridinium chlorochromate (PCC). The optical rotation of 3 derived from 2 was −329° , whereas that of naturally occurring 3 was −256° . Therefore, the absolute structure of 3 was determined to be the one shown in The molecular formula (C32) in 2 . The anaive of 1 . NovobenPenicillium sclerotigenum  to yield five fractions. Fraction 2 [CHCl3/acetone (3:2) eluate] was re-chromatographed by MPLC on silica gel [n-hexane/acetone (2:1) to acetone] followed by HPLC [CHCl3/acetone (15:1)] to give novobenzomalvins A (1: 75 mg), B (2: 100 mg), and C (3: 21 mg), along with epi-aszonalenins A and C, and helvolic acid. The spectral data of all compounds were identical to those in the literature D20 + 26° ; UV (MeOH) λmax (log ɛ) 218 (4.7), 228 (4.7), 270 (4.0), 280 (4.0), 311 (3.8), 317 (0.3) nm; IR (KBr) νmax 3420 (br), 1692, 1673, 1614, 1597 cm−1; CD  λmax (Δɛ) 210 (−27.8), 232 (14.7), 253 (−4.8), 278 (2.9) nm. The 1H and 13C NMR data, see 3) 1-NH to C-19, H-4 to C-2, C-3, C-5, H-5 to C-3, H-12 to C-10, C-14, C-16, H-15 to C-11, C-14, C-16, H-19 to C-2, C-18, C-20, C-21, H-20 to C-18, C-19, C-21, C-22, H-22(26) to C-20, H-23(25) to C-21, H-24 to C-22, C-26. EIMS m/z 383[M]+ (20), 365 (12), 277 (92), 249 (100), 234 (39), 220 (27), 192 (16), 174 (15), 130 (27), 77 (57); HREIMS m/z [M]+ 383.1248 .Colorless prisms (acetone/MeOH); mp 228–231°C; [α]D20 −256° ; UV(MeOH) λmax (log ɛ) 211 (4.5), 229 (4.5), 281 (3.8), 310 (3.6) nm; IR (KBr) νmax 3437 (br), 1697, 1672, 1615, 1599 cm−1; CD  λmax (Δɛ) 212 (0.5), 218 (−0.8), 229 (1.0), 250 (2.2), 296 (−0.3), 317 (0.2) nm. The 1H and 13C NMR data, see 3) 1-NH to C-19, H-4 to C-2, C-3, C-5, H-5 to C-3, H-12 to C-10, C-14, C-16, H-15 to C-11, C-14, C-16, H-19 to C-2, C-18, C-20, C-21, H-20 to C-18, C-19, C-21, C-22, H-22(26) to C-20, H-23(25) to C-21, H-24 to C-22, C-26. EIMS m/z 381[M]+ (73), 105 (100); HREIMS m/z [M]+ 381.1094 .Colorless amorphous solid; [α] 1 (8 mg) and KOH (10 mg) in DMSO (2 mL) and the mixture was stirred at room temperature (rt) for 1 h. The reaction mixture was added to H2O (3 mL) and extracted three times with CHCl3. The obtained CHCl3 solution was washed several times with H2O. After addition of anhydrous Na2SO4, the filtered solution was evaporated in vacuo. The resulting residue (9.1 mg) was purified by HPLC [benzene/acetone (18:1)] to give N-methylnovobenzomalvin A (5: 3.8 mg).Methyl iodide (2 mL) was added to a solution of D20+145° ; UV(MeOH) λmax (log ɛ) 212 (4.4), 229 (4.4), 230 (4.4), 270 (3.9), 309 (3.5) nm; CD  λmax (Δɛ) −77.9 (209), 22.2 (233), −11.6 (255), 8.4 (278), 4.7 (309), 4.0 (322) nm. 1H NMR  δ 8.33 , 7.96 ,7.84 , 7.62 , 7.56 , 7.27 , 7.22 , 4.90 , 3.82 , 3.44 , 3.11 . 13C NMR  δ 167.3 (C-2), 161.2 (C-10), 151.8 (C-18), 145.95 (C-16), 136.7 (C-21), 134.8 (C-14), 132.9 (C-8), 131.4 (C-6), 130.9 (C-3), 129.9 (C-4), 129.0 , 128.95 (C-5), 128.88 (C-7), 128.7 , 127.72 (C-15), 127.68 (C-13), 127.6 (C-12), 126.9 (C-24), 121.7 (C-11), 58.3 (C-19), 33.2 (C-20), and 27.9 (N-CH3). EIMS m/z 381[M]+ (33), 290 (92), 249 (100), 130 (27), 91 (22); HREIMS m/z [M]+ 381.1475 .Colorless amorphous solid; [α]1) was accomplished via triflate-catalyzed dehydrative cyclization promoted by microwave irradiation, following the report of Chu et al.  was a6: 5 g, 0.024 mol) and isatoic acid  was added to H2O (80 mL) at rt, and the mixture was stirred at reflux for 2 h. Then, the H2O layer was evaporated to give a crude residue. The residue was added to a solution of H2SO4/MeOH , and the mixture was stirred at reflux for 2 d. The reaction was quenched by the addition of saturated NaHCO3 solution. The aqueous layer was extracted with CHCl3. The combined organic layer was evaporated to give a crude residue, which was purified by MPLC [benzene/acetone (4:1)] to afford 7  as a colorless amorphous solid. 1H-NMR(CDCl3): δ 3.87 , 6.61 , 6.71 , 7.06 , 7.16 , 7.51 , 7.59 , 8.01 , 8.66 . 13C-NMR (CDCl3): δ 52.4, 115.2, 115.6, 116.9, 117.6, 120.4, 122.3, 127.7, 131.0, 132.9, 134.6, 142.0, 149.9, 168.1, 168.9. HREIMS: 270.0979 (M+), calcd for C15H14N2O3, 270.1004.Anthranilic acid (N-[(benzyloxy)carbonyl]-d-phenylalanine (Cbz-d-Phe), 1-ethyl-3-[3-(dimethyl-amino)propyl)]carbodiimide (EDC) , and N,N-di-iso-propyl-ethylamine (DIEA)  in CH2Cl2 (15 mL) was added dropwise to a solution of 7  in CH2Cl2 (5 mL) at rt, and the mixture was stirred for 5.5 h at rt. The reaction was quenched by the addition of saturated NaHCO3 solution. The aqueous layer was extracted with CHCl3. The combined organic layer was evaporated to give a crude residue, which was purified by MPLC [n-hexane/CHCl3/acetone (2:1:0.4)] to afford 8-Z  as a colorless amorphous solid. 1H NMR (CDCl3): δ 3.06 , 3.14 , 3.78 , 4.56 , 4.95 , 5.01 , 5.58 , 6.92-7.00 , 7.01-7.10 , 7.11-7.23 , 7.29 , 7.36 , 7.69 , 7.92 , 8.51-8.61 , 11.54 , 11.86 .13C NMR (CDCl3): δ 38.3, 52.4, 57.2, 66.8, 115.3, 120.4, 120.6, 121.3, 122.9, 123.4, 126.7, 126.9, 127.6 (2C), 127.8, 128.3 (2C), 128.4 (2C), 129.1 (2C), 130.8, 132.8, 134.4, 136.1, 136.2, 139.4, 140.9, 155.7, 167.1, 168.7, 169.7. ESIMS: 574.4 (M+Na)+.A solution of 8-Z  in MeOH (2 mL), 20% palladium on carbon (Pd/C) was added at rt, the mixture was stirred for 75 min at rt in a hydrogen atmosphere. The reaction was filtered and concentrated in vacuo to give 8 . 1H-NMR(CDCl3): δ 2.82 , 3.33 , 3.77 , 3.95 , 7.11-7.32 , 7.49-7.64 , 7.84 , 8.09 , 8.71 , 8.79 , 11.85 , 11.91 . ESIMS (%): 440.3 , 418.3 .To a solution of 8 (18.3 mg) in DMF (0.5 mL) was added tin(II) trifluoromethane sulfonate [Sn(OTf)2] , and the mixture was treated by microwave irradiation at a controlled temperature of 140 °C for 10 min. The reaction was evaporated in vacuo, and was purified by HPLC [n-hexane/acetone (2:1)] to afford 1  as a colorless amorphous solid.To a solution of the 2) was grown slowly from acetone/MeOH to give colorless prisms. Diffraction intensities were collected from a single crystal having approximate dimensions of 0.51 × 0.16 × 0.10 mm on a Rigaku RAXIS RAPID imaging plate area detector with graphite monochromated Cu-Kα radiation. Of 9116 reflections, 2974 were unique reflections. The data were corrected for Lorenz and polarization effects.Novobenzomalvin B , b= 7.80258 (15), c= 11.3914 (2) Å, β= 104.4659 (12)°, V = 867.07 (3) Å3, Z= 2, Dc= 1.468 g·cm−3, F(000)= 400, μ(Cu-Kα) = 8.109 cm−1.C2 was based on 8714 observed reflections and 280 variable parameters. The final R and Rw values were 0.0337 and 0.0940, respectively. The absolute structure was deduced based on the Flack parameter D20, HREIMS, UV, CD, TLC, and HPLC data.The CHl-glutamine and penicillin/streptomycin . Cells were plated at the density of 5 × 105 cells on 24-well plate . After 24 h incubation with serum-free DMEM, cells were treated with complete medium containing various compounds. At the end of 24 h incubation, cells were washed with cold PBS and lysed on ice in lysis buffer . The samples were resolved on 7.5% Tris-Glycine polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membrane . The membranes were blocked for 1 h in TBS containing 0.05% Tween-20 (TBS-T) and 5% fat-free milk and then incubated overnight at 4 °C with the primary antibody. The membranes were washed with TBS-T four times followed by incubation for 1 h at rt with HRP-conjugated anti-mouse IgG and the immunoreactivity was assessed by chemiluminescence.The antibodies used for Western blot analyses including mouse anti-fibronectin antibody  and anti-GAPDH antibody . Horseradish peroxidase (HRP)-conjugated anti-mouse and -rabbit IgG  were used as secondary antibodies. NHDF-neo cells  were grown in complete medium: Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% (v/v) fetal bovine serum (FBS) (Sigma), Densitometry analysis was performed using ImageJ 1.43 on immunoblots from three independent experiments. A t-test was performed with SYSTAT software .A. niger IFM 41398, A. fumigatus IFM 41362, C. albicans IFM 40009 and C. neoformans ATCC 90112 as test organisms. Novobenzomalvin A (1) to C (3) were applied to the paper disk (diameter: 8 mm) at 100 μg/disk, and the disks were placed on the assay plates. The test organisms were cultivated in potato dextrose agar  at 25 °C. After 48–72 h of incubation, zones of inhibition (the diameter measured in millimeters) were recorded.The antifungal assay was performed by using a previously reported method . The antH-tetrazolium) and 1-methoxy PMS (1-methoxy-phenazine methosulfate). Briefly, after the medium was removed, 10 μL of Cell Counting Kit-8 solution was added to each well, and the plates were incubated for 4 h, then cell numbers were obtained by scanning with a Bio-Rad Model Q4 550 microplate reader at 450 nm.The cytotoxicity assay was performed by a modified method . Cell we


The complex metabolic syndrome, diabetes mellitus, is a major human health concern in the world and is estimated to affect 300 million people by the year 2025. Several drugs such as sulfonylureas and biguanides are presently available to reduce hyperglycemia in diabetes mellitus. These drugs have side effects and thus searching for a new class of compounds is essential to overcome this problems.N-(4-phenylthiazol-2-yl)benzenesulfonamides derivatives were synthesized and assayed in-vivo to investigate their antidiabetic activities by streptozotocin-induced model in rat. These derivatives showed considerable biological efficacy when compared to glibenclamide, a potent and well-known antidiabetic agent, as a reference drug. Four of the compounds were A series of seven novel effective, amongst which 13 show more prominent activity at 100 mg/Kg p.o. The experimental results are statistically significant at p < 0.05 level. Resistance to the biological actions of insulin in the liver and peripheral tissues, together with pancreatic cell defects, is a major feature of the pathophysiology of human NIDDM , 4.Pharmaceutical intervention of hyperglycemia induced diabetic complications is actively pursued since it is very difficult to maintain normoglycemia by any means in patients with diabetes mellitus , 6.Several drugs such as sulfonylureas and biguanides are presently available to reduce hyperglycemia in diabetes mellitus. These drugs demonstrated significant side effects and thus searching for a new class of compounds is essential to overcome these problems . Therefo2NH2) is an active pharmacophore, exhibiting a wide variety of pharmacological activities such as antimicrobial, antimalarial, insulin-releasing antidiabetic, anti-HIV, high ceiling diuretic, antithyroid, and antitumor ethanone (1) was reacted with thiourea in refluxing ethanol to yield 4-(4-methoxyphenyl)thiazol-2-amine . In addition 4-(4-chlorophenyl) thiazol-2-amine  was produced through the reaction of 1-(4-chlorophenyl) ethanone (2) with thiourea in the presence of iodine in refluxing ethanol .The target compounds were synthesized by simple and facile condensation reaction of equimolar quantities of 2-amino thiazol  with appropriate sulfonyl chloride (compounds 5-11). The reactions were stirred at room temperature in pyridine for 4 days. The solid products was obtained by filtration and purified by recrystallization.1H NMR, IR and Mass spectroscopy. The hydrogen of amine in compounds 12-19 was detected at 8.6-9.0 ppm as a broad peak which was deshielded by an adjacent sulfonyl group. The feature of the benzenesulfonamides in the solid state is also supported by the IR spectral data (NH group band at ~ 3300 cm-1 and S=O band at ~ 1281-1157 cm-1) for the majority of the compounds.The synthesized compounds 12-19 were characterized by Synthesis of 4-(4-methoxyphenyl) thiazol-2-amine (3)The experimental protocol is based on a previously described methodology . To a soSynthesis of 4-(4-chlorophenyl) thiazol-2-amine (4)The mixture of thiourea  and iodine  in 10 mL of ethanol was added to the solution of 1-(4-chlorophenyl) ethanone  in 5 mL of ethanol. The mixture was heated under reflux for 1 h and stirred at room temperature for 24 h.After cooling, the precipitate was filtered, washed with water and the resulted crude product was purified by recrystallization from diethyl ether .General procedure for the synthesis of N-(4-(4-methoxyphenyl or 4-chlorophenyl)thiazol-2-yl) benzenesulfonamid (12-19)A mixture of 4-(4-methoxyphenyl or 4-chlorophenyl) thiazol-2-amine (1 mmol) and appropriate sulfonyl chloride (1 mmol) in pyridine (2 mL) was stirred at room temperature for 4 days. The mixture was evaporated under reduced pressure and the mixture was neutralized with dilute hydrochloric acid. The precipitate was filtered and washed with water and the resulting crude product was purified by recrystallization from methanol .N-(4-(4-Methoxyphenyl)thiazol-2-yl)benzenesulfonamid (12)-1): 3289 (NH), 1173 and 1255 (S=O), 1646 (C=N). 1H NMR (DMSO-d6) δ: 3.80 , 7.04 , 7.09 , 7.73 , 8.06 , 8.59 , 8.9 , 8.92 . MS: m/z (%) 346 , 206 (100), 191 (30), 164 (11), 149 (27), 94 (22), 77 (30). Anal. Calcd for C16H14N2O3S2: C, 55.47; H, 4.07; N, 8.09. Found: C, 55.26; H, 3.79; N, 7.79.Yield: 53 %; mp: 258-260°C; IR  thiazol-2-yl)benzenesulfonamide (13)-1): 3302 (NH), 1148 and 1296 (S=O), 1638 (C=N). 1H NMR (DMSO-d6) δ: 3.79 , 7.04 , 7.07 , 7.41 , 7.64 , 7.84 , 8.9 . MS: m/z (%) 414 , 236 (4), 226 (13), 206 (100), 191 (16), 164 (10), 149 (14), 109 (5), 77 (5). Anal. Calcd for C16H12Cl2N2O3S2: C, 46.27; H, 2.91; N, 6.75. Found: C, 45.99; H, 2.60; N, 6.45.Yield: 43 %; mp: 233-235°C; IR  thiazol-2-yl)benzenesulfonamide (14)-1): 3370 (NH), 1118 and 1322 (S=O), 1653 (C=N). 1H NMR (DMSO-d6) δ: 3.79 , 7.04 , 7.06 , 7.64 , 7.76 , 7.96 , 8.8 . MS: m/z (%) 450 , 448 , 238 (19), 206 (100), 191 (29), 171 (9), 164 (12), 149 (57), 121 (12), 107 (5). Anal. Calcd for C16H11Cl3N2O3S2: C, 42.73; H, 2.47; N, 6.23. Found: C, 42.42; H, 2.18; N, 5.94.Yield: 39 %; mp: 213-215°C; IR thiazol-2-yl)benzenesulfonamide (15)-1): 3326 (NH), 1187 and 1302 (S=O), 1640 (C=N). 1H NMR (DMSO-d6) δ: 3.74 , 3.79 , 6.86 , 7.03 , 7.05 , 7.53 , 7.65 , 8.8 . MS: m/z (%) 376, 238 (17), 206 (100), 191 (29), 188 (21), 164 (12), 149 (23), 123 (12), 77 (13).Anal. Calcd for C17H16N2O4S2: C, 54.24; H, 4.28; N, 7.44. Found: C, 53.96; H, 4.00; N, 7.13.Yield: 48 %; mp: 233-235°C; IR  thiazol-2-yl)benzenesulfonamide (16)-1): 3289 (NH),1119 and 1302 (S=O), 1651 (C=N). 1H NMR (DMSO-d6) δ: 3.74 , 6.95, 6.99 , 7.52 , 7.56 , 8.7 . MS: m/z (%) 426 , 424 , 238 (10), 206 (100), 191 (29), 164 (11), 149 (23), 121 (11), 77 (7). Anal. Calcd for C16H13BrN2O3S2: C, 45.18; H, 3.08; N, 6.59. Found: C, 44.87; H, 2.80; N, 6.30.Yield: 61 %; mp: 250-252°C; IR thiazol-2-yl)-4-methylbenzenesulfonamide (17)-1): 3268 (NH), 1178 and 1244 (S=O), 1625 (C=N). 1H NMR (DMSO-d6) δ: 2.29 , 3.80 , 7.03 , 7.04 , 7.12 , 7.48 , 7.68 , 8.6 . MS: m/z (%) 360, 206 (100), 191 (28), 164 (12), 149 (24), 121 (12), 77 (7). Anal. Calcd for C17H16N2O3S2: C, 56.65; H, 4.47; N, 7.77. Found: C, 56.35; H, 4.16; N, 7.47.Yield: 44 %; mp: 209-212°C; IR thiazol-2-yl)benzenesulfonamide (18)-1): 3292 (NH), 1169 and 1299 (S=O), 1645 (C=N). 1H NMR (DMSO-d6) δ: 3.79 , 7.03 , 7.06 , 7.38 , 7.61 , 7.64 . MS: m/z (%) 382 , 380, 206 (100), 191 (29), 164 (10), 149 (24), 128 (15), 111 (9), 75 (10). Anal. Calcd for C16H13ClN2O3S2: C, 50.46; H, 3.44; N, 7.36. Found: C, 50.16; H, 3.13; N, 7.07.Yield: 50 %; mp: 224-226°C; IR thiazol-2-yl)-4-methylbenzenesulfonamide (19)-1): 3330 (NH), 1165 and 1228 (S=O), 1644 (C=N). 1H NMR (DMSO-d6) δ: 2.28 , 7.11 , 7.21 , 7.48 , 7.51 , 7.75 , 8.6 . MS: m/z (%) 366 , 364 , 292 (7), 210 (100), 172 (32), 168 (51), 91 (68). Anal. Calcd for C16H13ClN2O2S2: C, 52.67; H, 3.59; N, 7.68. Found: C, 52.36; H, 3.30; N, 7.39.Yield: 47%; mp: 288-290°C; IR  and a 12 h light/dark cycle in animal house of PSRC/TUMS. They were fed by normal laboratory chow and water. Acute diabetes was induced by intravenous administration of streptozotocin-alloxan (40 mg/Kg of each) dissolved in 0.05 M citrate buffer with pH of 4.5 to 24-h fast rats. Blood glucose changes were measured by a glucometer from the rats’ tail veins every hour post administration of diabetes. Synthesized compounds were administered at a single dose of 100 mg/Kg. Glibenclamide (5 mg/Kg) as the standard was administered orally 48 h post administration of streptozotocin-alloxan. The basis of comparison was the blood glucose level 2 h post administration of drugs to diabetic rats.1H NMR, and elemental analysis. All the compounds were screened in-vivo for their oral hypoglycemic activity by streptozotocin-induced diabetic model in rat. Four of the compounds demonstrated remarkable hypoglycemic property, however with a degree of variation. The results of changes in blood glucose in diabetic rats treated with 100 mg/Kg p.o. of the synthesized benzenesulfonamides derivatives are presented in (A series of N-(4-phenylthiazol-2-yl) benzenesulfonamides derivatives were synthesized by reacting of equimolar quantities of 2-amino thiazol (compound 3 and 4) with appropriate aromatic sulfonyl chloride. The structures of these compounds were established by means of IR, ented in . A signiThe results indicated that the introduction of 2,5-dichloro group on phenyl sulfonyl group potentiated the antidiabetic activity of studied compounds. On the other hand, replacement of para hydrogen atom of phenyl sulfonyl group with other substitutes resulted in reduced antidiabetic potential of benzensulfanamide derivatives. It could be deduced that the presence of any group at 4-position of the phenylsulfonyl might sterically hinder the effective interaction of studied compounds with their receptor.N-(4-phenylthiazol-2-yl) benzenesulfonamides derivatives. Some of the synthesized compounds showed hypoglycemic activity. These results indicated that benzenesulfonamide could be served as potential antidiabetic agents in the same manner as sulfonylurea derivatives. It seems that structural modification of this scaffold will resulted in more potent oral antidibetic derivatives as it will be followed in our future projects.This study reports the synthesis and antidiabetic activity of novel