Gynura procumbens showed virucidal and antireplicative actions against herpes simplex virus HSV-1 and HSV-2. It was further chromatographed on MCI gel CHP20P column giving the extract fractions F1 (water), F2 (water-methanol) F3 (methanol), and F4 (ethyl acetate). All but F1 had virucidal action against both viral types. We reported here the active compounds from F2 and F3. The antiherpetic compounds of F2 was a mixture of dicaffeoylquinic acids with virucidal and antireplicative actions against HSV-2  Virucidal compounds of F3 were a mixture of β-sitosterol and stigmasterol (IC50 250.0 μg/mL against HSV-1), a mixture of β-sitosteryl and stigmasteryl glucosides (IC50 50.0 μg/mL against HSV-2) and 1, 2-bis-dodecanoyl-3-α-D-glucopyranosyl-sn-glycerol (IC50 of 40.0 μg/mL against HSV-2). Herbal products containing 1 and 2% of standardized ethanol extract were prepared. Double-blind randomized controlled clinical trial of the products was performed in patients with recurrent herpes labialis. Results showed that the number of patients, whose lesions healed within 7 days and the average healing time of both groups differed insignificantly. Viral culture on D7 indicated a decrease of infected patients from 48.7% to 7.69% in treated group whereas in placebo group the infected patients decreased from 31.25% to 20.00%. The viral reduction in treated group indicated the benefit of the product. Insignificant result might arise from a low number of participated patients and insufficient concentration of plant extract in herbal product. The ethanol extract of  Gynura procumbens (Lour.) Merr. is mentioned in traditional Chinese medicine as a topical anti-inflammatory remedy +  m/z 599 and 597 ; EI-MS (pos. ion mode) m/z 414 and 412 [M+H]+ (corresponding aglycones); m/z 396 and 394 (low intensity) [M+H-H2O]+; 1H-NMR  δ 5.35  5.23  5.08  5.04  4.55  4.40  4.26  4.04  3.96  2.72–2.47  2.14–1.74  1.93  1.7  1.4  1.0–1.8  1.28  1.09  1.0  0.94, 0.96  0.93  0.92  0.95  0.90–1.30  0.90  0.89  0.88  0.8–2.2  0.69  0.67 ; 13C-NMR  δ 140.955 (C-5) 138.833 (C-22) 129.521 (C-23) 121.921 (C-6) 102.607 (C-1′) 78.605  78.440 (C-5′) 75.331 (C-2′) 71.761 (C-4′) 62.894  56.971  56.889  56.297  56.132  51.460  50.404 (C-9) 46.097  42.527  42.396  40.783  40.010 (C-12) 39.368 (C-4) 37.526 (C-1) 36.967 (C-10) 36.424  34.269  32.196 (C-7) 32.114  30.290 (C-2) 29.531  29.317  28.560  25.714  24.579  24.546  23.444  21.502  21.305 (C-11) 19.446  19.216  19.051  12.191  12.010 .8:  α-D-Glucopyranosyl-sn-Glycerol 1,2-bis-Dodecanoyl-3- . Colorless waxy mass, Rf 0.15, silica gel 60, CHCl3/MeOH/H2O/(20 : 5 : 0.5); 1H-NMR (CD3OD), 300 MHz) δ 5.28  4.77  4.50  4.14  4.00  3.60  3.56  3.41  3.28  3.19  3.00  2.30  1.57  1.25  0.84 ; 13C-NMR : δ 192.31  99.08 (C-1′′′) 74.00  73.00 (C-2′′′) 71.50 (C-2) 70.00 (C-5′′′) 66.00  64.00  61.00  34.69  29.52–30.06  25.27  23.02 .9:β-Sitosterol  A Mixture of  and Stigmasterol . White needles, mp 145–148°C; Rf 0.75, silica gel 60, CHCl3/MeOH/H2O/(20 : 5 : 0.5); 1H-NMR  δ 5.34  5.15  5.01  3.52  2.26  2.08–1.78  1.3–0.8  1.7  1.28  1.2  1–1.18  1.0  0.95  0.84  0.82  0.80  0.8–2.2  0.68  0.70 ; 13C-NMR  δ 140.77 (C-5) 138.30  129.30  121.70 (C-6) 71.82 (C-3) 56.88  56.79  56.09  55.99  51.24  50.19  50.16  45.87  42.33 (C-4) 42.23 (C-13) 40.47 (C-20) 39.80  39.70  36.53  36.52  36.15  33.97  32.27 (C-1) 31.92  31.88  31.69 (C-2) 29.19  28.91  28.24  26.13  25.40  24.37  24.31  23.09  21.21  21.09  19.81  19.39 (C-19) 19.05  18.99  18.79  12.23 (C-29) 12.05  11.86 . ∗,∗∗Pairs interchangeable.1 and 4 were not determined because of the minute amount. The compounds 3, 5, and 6were inactive. The chemical structures of 1, 3, 4, 5, and 6 were identified as follows.The antiherpetic activities of 1: 5-O-Caffeoyl-D-Quinic Acid . Colorless amorphous powder; UV (MeOH) λmax 326 nm, shoulder at 299 nm; ESI MS (pos. ion mode) m/z 355 [M+H]+; ; 1H-NMR  δ 7.40 , 7.00 , 6.94 , 6.73 , 6.14 , 5.10 , 3.90 , 3.48 , 1.97 , 1.86 , 1.71 ; 13C-NMR : δ 175.00 (C-7), 165.90 (C-9′), 148.20 (C-3′), 145.47 (C-4′), 144.67 (C-7′), 125.56 (C-1′), 121.18 (C-6′), 115.68 (C-5′), 114.70 (C-8′), 114.45 (C-2′), 73.20 (C-1), 71.50 (C-3), 71.19 (C-5), 69.52 (C-4), 36.92 (C-2).3:α-L-Rhamnosyl  A Mixture of Kaempferyl-3-O-(1→6)β-D-Glucopyranoside -α-L-Rhamnosyl  and Kaempferyl-3-O-(1→6)β-D-Galactopyranoside- . Yellow amorphous powder, mp 188–190°C (Lit. mp 198–200°C) [λmax 265 nm (band II) and 349 nm (band I); 1H-NMR  δ 12.48  10.10 (OH) 8.04  7.96  6.87  6.85  6.18  6.40  6.38  5.31  5.27  5.16  5.05  5.03  4.88  4.55  4.52  4.38  4.37  3.68  3.58  3.10–3.58  1.05  0.97 . 8–200°C) ; UV  δ 177.00 (C-4) 165.00 (C-7) 161.00 (C-5) 160.00 (C-4′) 156.00  133.00 (C-3) 131.00  121.00 (C-1′) 115.01  115.00  103.00 (C-10) 101.00 (C-1′′) 100.00 (C-1′′′) 98.50 (C-6) 93.50 (C-8) 75.65  75.50  73.93  73.31  72.74  71.50  71.50  70.33  70.33 (C-3′′′) 70.02  67.66  67.66 (C-5′′′) 66.49 (C-6′′) 17.41  17.31  [4:β-D-Glucopyranoside  λmax 256 nm (band II) and 358 nm (band I); 1H-NMR  δ 7.72  7.52  6.89  6.36  6.21  5.15  3.71  3.56  3.48  3.42  3.34  3.22 ; 13C-NMR : δ 179.49 (C-4) 166.19 (C-7) 159.04 (C-3) 158.49  149.86 (C-4′) 145.92 (C-3′) 135.60 (C-3) 123.19 (C-1′) 123.09 (C-6′) 117.56 (C-5′) 116.03 (C-2′) 105.39 (C-10) 104.31 (C-1′′) 99.65 (C-6) 94.78 (C-8) 78.38 (C-3′′) 78.11 (C-5′′) 75.72 (C-2′′) 71.24 (C-4′′) 62.56 (C-6′′).5: Kaempferyl Glucopyranoside . Yellow amorphous powder, mp 165–170°C (with decomposition) [λmax 266 nm (band II) and 349 nm (band I); IR (KBr) νmax 3350–3450 (O–H), 2933 ), 1633 (C=O), 1611 (C=C), 1604 (C=O) cm−1; ESI MS: MR 448. 1H-NMR  δ 12.50 , 8.01 , 6.84 , 6.38 , 6.17 , 5.41 , 5.24 (2′′-OH), 4.96 (5′′-OH), 4.86 , 4.16 (6′′-OH), 3.55–3.30 , 3.18 (H-5′′), 3.16 , 3.06 ; 13C-NMR  177.13 (C-4), 165.10 (C-7), 161.09 (C-5), 156.43 (C-9), 155.88 (C-2), 133.08 (C-3), 130.75 , 120.88 (C-1′), 115.02 , 103.43 (C-10), 100.97 , 99.00 (C-6), 93.78 (C-8), 77.41 (C-3′′), 76.39 (C-5′′), 74.17 (C-2′′), 69.84 (C-4′′), 60.80 (C-6′′).osition) ; UV . Yellow amorphous powder; UV (MeOH) λmax 260 nm (shoulder) and 365 nm (band I); 1H-NMR (CD3OD 300 MHz) δ 8.07 , 6.91 , 6.40 , 6.19 ; 13C-NMR  δ 177.42 (C-4), 165.56 (C-7), 162.46 (C-5), 160.56 (C-4′), 158.24 (C-9), 148.10 (C-2), 137.16 (C-3), 130.68 , 123.76 (C-1′), 116.31 , 104.56 (C-10), 99.29 (C-6), 94.486 (C-8).G. procumbens, we performed the double-blind, randomized, controlled clinical trial in patients with recurrent herpes labialis of the herbal products, which contained 1 and 2% of the standardized plant extracts.To evaluate the antiherpetic effectiveness of A total of 65 patients participated in the study. Four patients  did not appear for the followup. One patient in the treated group did not record the date of full crusting and complete healing. One patient in the placebo group could not continue treatment due to allergic contact dermatitis. Therefore, only 59 patients were evaluated. They were divided into the treated group with 1% herbal gel (19 patients), the treated group with 2% herbal gel (22 patients), and the placebo group (18 patients). The antiherpetic results of the treated groups  were not different significantly. The data from both treated groups were thus combined  and compared to the placebo group which comprised 18 patients . Both groups were comparable for age, sex, pain, and itching scores, proportion of patient who are suffering from pain, and percentage of patients who had positive tests for Tzanck smear and viral culture. The only significant difference was the proportion of itching patients. It was greater in the treated group as shown in The positive results of Tzanck smear of the treated and control groups were 70.0 and 72.2%, respectively. The viral culture showed the presence of HSV-1 in the treated and control groups 46.2 and 31.3%, respectively. The patients in the treated and control groups were not significantly different, apart from the itching in the treated group, which was significantly different from the control .The first followup  indicated that the treated group still suffered the pain, but the number decreased from 27 patients (65.90%) to 20 patients (34.15%), the itching decreasing from 68.29 to 48.78%. The patients who are suffering from pain in the placebo group decreased from 11 in 18 patients (61.10%) to 6 in 18 patients (33.33%), but the proportion of patients with itching remain unchanged . The average pain scores of the treated and placebo groups were 1.11 and 1.41, respectively. The average itching scores of the treated and the placebo groups were 1.60 and 1.44, respectively. The results of both groups were not significantly different .Twenty-one of 41 patients in the treated group (51.22%) had full crusting within 4 days. It was comparable to 9 of 18 patients (50%) in the placebo group. Lesions completely healed within 7 days in twenty-six patients (63.41%) of the treated group whereas it was 8 in 18 patients (44.44%) for the placebo group. In the treated group, the average time for full crusting was 4.9 days and for complete healing was 8.7 days. The same figures for the placebo group were 5.1 and 9.4 days, respectively. The viral culture on D7 showed the number of patients in the treated group who had positive culture to be reduced from 19 to 3 whereas those in the placebo group reduced from 5 to 3 patients. The results differed from the placebo insignificantly .Side Effects. The blood chemistry  of the participated patients was normal after the treatment. There was one patient in the placebo group suffering from allergic contact dermatitis, so the treatment was discontinued. There were 10 patients (24.4%) in the treated group and 5 patients (27.8%) in the placebo group who had mild itching and irritation, but all patients could tolerate the symptoms without disruption of the treatment. The side effects in the treated and placebo groups were not significantly different.G. procumbens showed that the ethanol extract of the aerial plant parts, the drug material, had the virucidal action against HSV-1 and HSV-2  and prevented the viral replication with IC50 584.0 and 568.0 μg/mL, respectively. Several antiherpetic compounds were isolated from the extract. Some of them such as dicaffeoylquinic acids (2) were known for the antiviral activity [7, 8, and 9. The antiherpetic activity against HSV-2 of 8, the glycosphingolipid, was stronger than 2 (dicaffeoylquinic acids). In addition, several flavonoids were found in the extract. They probably exerted the activity at the concentration above the MNTD. The test concentrations should not exceed the MNTD because the cultured cells would be damaged. However, the presence of the flavonoids possibly imparted anti-inflammatory effect [The phytochemical work on activity . We founy effect , which cThe laboratory evidence directed us to carry on the clinical study. The double-blind randomized, controlled clinical trial of recurrent herpes labialis with herbal and placebo gels was conducted. The ethanol extract was standardized using a mixture of sitosteryl and stigmasteryl glucosides as a marker compound (standard) because it could be isolated in a sufficient amount without difficulty. The standardized extract was incorporated in gel base as 1 and 2% herbal gels. The placebo gel was prepared similarly, colored like herbal gel but without the extract. G. procumbens tended to reduce the suffering from HSV-1 infection and might have some antiherpetic action. We performed the stability test of the herbal gel and found that the appearance and the content of the marker compound remained unchanged when storing the gel at 25°C for 24 months and at 40°C for 6 months. To improve the efficacy result of this herbal gel for the treatment of recurrent herpes labialis, we recommend the change of gel base to reduce the irritation and increase the potency of it. For further clinical investigation, we recommend increasing patients number and high concentration of the plant extract in the herbal gel.We could not achieve the number of the participated patients as calculated (50 in each group), though the trials were conducted at three hospitals. There were only 65 patients participating in the trial, but only 59 patients completed the study. Thus, the power of this clinical trial reduced from 80% to only 50%. Though the antiherpetic result of herbal gel differed insignificantly from the placebo, it tended to have some benefits. These included the tendency of the decreasing number of patients who suffered from itches in the treated group, the greater number of patients healing within 7 days, and the lesser number of patients infected with HSV-1. From the traditional, laboratory, and clinical pieces of evidence, we could postulate that Gynura procumbens comprised antiherpetic compounds, that is, caffeoylquinic acid derivatives, phytosteryl glucosides, and glycoglycerolipids. The flavonoids in this plant possibly imparted anti-inflammatory effect which was advantageous to the herpetic patients. The laboratory evidence and the reduction of the infection incidence in patients supported the antiherpetic effect of G. procumbens. The insignificant result of the clinical study might arise from the low participated patient number and insufficient extract concentration in the herbal product.


Aspergillus terreus (No. GX7-3B) was cultivated in potato dextrose liquid medium, and one rare thiophene compound (1), together with anhydrojavanicin (2), 8-O-methylbostrycoidin (3), 8-O-methyljavanicin (4), botryosphaerone D (5), 6-ethyl-5-hydroxy-3,7-dimethoxynaphthoquinone (6), 3β,5α-dihydroxy--ergosta-7,22-dien-6-one (7), 3β,5α,14α-trihydroxy--ergosta-7,22-dien-6-one (8), NGA0187 (9) and beauvericin (10), were isolated. Their structures were elucidated by analysis of spectroscopic data. This is the first report of a natural origin for compound 6. Moreover, compounds 3, 4, 5, 7, 8 and 10 were obtained from marine microorganism for the first time. In the bioactive assays in vitro, compounds 2, 3, 9 and 10 displayed remarkable inhibiting actions against α-acetylcholinesterase (AChE) with IC50 values 2.01, 6.71, 1.89, and 3.09 μM, respectively. Furthermore, in the cytotoxicity assays, compounds 7 and 10 exhibited strong or moderate cytotoxic activities against MCF-7, A549, Hela and KB cell lines with IC50 values 4.98 and 2.02 (MCF-7), 1.95 and 0.82 (A549), 0.68 and 1.14 (Hela), and 1.50 and 1.10 μM (KB), respectively; compound 8 had weak inhibitory activities against these tumor cell lines; compounds 1, 2, 3, 4, 5, 6 and 9 exhibited no inhibitory activities against them.The mangrove endophytic fungus  Aspergillus terreus (No. GX7-3B) is a mangrove endophytic fungal strain from the South China Sea. We have previously reported that four sesquiterpenes together with cyclo [IIe–IIe] dipeptide, ergosterol and ergosterol peroxide were isolated from this strain using glucose yeast-extract peptone (GYP) as the cultivation medium. The sesquiterpenes included botryosphaerin F, a new bioactive compound -5,7-dimethoxynaphtho thiophene-4,9-dione.Compound ound see . This wand 1 see . The cou2–10 by spectral analysis and comparison with reported literature data. Isolated of compound 6 from natural sources hasn’t previously been reported, although it has been synthesized  = +271.80 . 1H NMR : δ 1.71 , δ 4.03 , δ 4.04 , δ 5.65 , δ 6.84 , δ 8.09 , δ 3.49 , δ 13.53 . 13C NMR : δ 23.3 (C-11), 56.6 (C-13), 57.4 (C-12), 65.6 (C-10), 104.2 (C-6), 112.0 (C-4a), 117.2 (C-8a), 128.2 (C-3), 129.4 (C-3a), 140.2 (C-9a), 149.6 (C-8), 155.6 (C-7), 156.5 (C-5), 162.7 (C-2), 177.5 (C-4), 187.3 (C-9). EI-MS at m/z = 334, ESI-MS at m/z = 335 [M + H]+; HR-EI-MS m/z: 334.0501 [M]+, . UV (MeOH): λmax (log ε) = 259 (0.32) nm. IR (KBr): νmax = 3428, 2921, 2852, 1652, 1623, 1544, 1515, 1508, 1460, 1434, 1371, 1316, 1216, 1028, 896, 758, 581, 469 cm−1.Compound 1–10 were dissolved in DMSO to different concentrations. 10 μL of sample solution and 10 μL of α-acetylcholinesterase solution  were added to 960 μL of phosphate buffer and incubated at 37 °C for 30 min, and then 20 μL of substrate were added to initiate the enzyme reaction. The enzyme reaction was carried out at 37 °C for 30 min. Product was monitored spectrophotometrically by measuring the absorbance (λ = 400 nm). Huperzine A was used as reference standard inhibitor for comparison. Dose-response curves were obtained by performing assays in the presence of increasing concentrations of inhibitors. IC50 value was determined by interpolation of the dose-response curves. For all tests, the inhibition assays were performed in triplicate.α-Acetylcholinesterase inhibitory activity was assayed using 50 mM phosphate buffer (pH 7.0). Compounds 2) for 24 h, followed by exposure to various concentrations of compounds tested for 48 h. Subsequently 20 μL of MTT solution (5 mg/mL) were added to each well and mixed, the cells were then incubated for an additional 4 h. Culture supernatant were removed, 150 μL of DMSO were added to each well to fully dissolve the MTT—formazan crystals. Cell growth inhibition was determined by measuring the absorbance (Abs) at λ = 570 nm using a microplate reader and calculated according to the following equation:The cytotoxic activities were determined using MCF-7, A549, Hela, KB human tumor cells by the MTT colorimetric method. Cells were harvested during logarithmic growth phase and seeded in 96-well plates at a density of 1 × 104 cells/mL, and cultured at 37 °C in a humidified incubator (5% CO50) were obtained from linear regression analysis of the concentration-response curves plotted for each tested compound (Bliss’s software). Results were expressed as the mean value of triplicate data points.The half maximal inhibitory concentrations (ICAspergillus terreus (No. GX7-3B) could produce a rich variety of secondary metabolites, and small changes in the cultivation conditions could completely shift the metabolic profile of this strain. Its metabolites obtained on PDB medium were very different from those of on GYP medium. When the strain was cultivated on PDB medium, one novel sulfur-containing compound, as well as nine known compounds were isolated, which had exhibited antitumor or AChE inhibition activities.This work showed that


The synthesis and properties of a series of bis-tetrathiafulvalenes (bis-TTFs) containing nitrophenyl, aminophenyl or dimethylaminophenyl is reported. The synthesis was carried out by using routes involving Wittig-type, cross-coupling, reduction and alkylation reactions. The electron donor ability of these new compounds has been measured by cyclic voltammetry (CV). Charge transfer complexes with tetracyanoquinodimethane (TCNQ) were prepared by chemical redox reactions. The complexes have been proven to give conducting materials. Tetrathiafulvalene (TTF) and its derivatives have attracted attention for many years because of their electron donor ability and the electrical conductivity of their charge transfer salts, which was started in 1973 with the synthesis of the tetrathiafulvalene-tetracyanoquinodimethane complex (TTF-TCNQ) by Cowan and coworkers  exhibiti60 + 739 and [M + H]+ 767, respectively, and by their 1H RMN spectra, the aromatic protons for 12a as two doublets at 7.55 ppm and at 8.25 ppm, with the same coupling constants of 9.37 Hz, as well as another characteristic triplet at 3.59 ppm for the two (CH2)2 groups protons. On the other hand, the spectrum of 12b exhibited a multiplet at 2.47 ppm for the two CH2CH2CH2 groups. Moreover, the spectra of 13a,b showed amino group protons as multiplet around 3.50–3.85 ppm. The final products 14a,b showed the absence of the amino group proton signals and the presence of dimethylamino group proton signals as singlets at 3.35 ppm and at 3.20 ppm, respectively.In the n-Bu4NClO4, 0.1 M) in dry acetonitrile, as supporting electrolyte. A scan rate of 100 m·Vs−1 was used. The CV measurements showed reversible redox waves for all the compounds studied and the corresponding oxidation potentials Eox were determined by the SQW technique. The results are summarized in The redox behavior of these new functional mono- and bis-TTF was studied in solution by cyclic voltammetry (CV) and by square wave voltammetry (SQW). Measurements were performed under nitrogen at room temperature using a glassy carbon working electrode, a Pt counter electrode and a standard calomel electrode (SCE) as reference, with tetrabutylammonium perchlorate (I (a) SQW curve shows two sharp oxidation waves each with one electron process for the mono-TTF  SQW curve is observed for compounds 11a,b, 12a,b, 13a,b and 14a,b , ,  and . The first oxidation potentials in each pair are shifted cathodically by 20 mV. This may be due to the alkyl linked group. The oxidation potentials of compounds 14a,b are slightly higher than that of compounds 13a,b, on the other hand, the compounds 12a,b are slightly higher than that of compounds 14a,b. This should be attributable to the electronic properties of the nitrophenyl, aminophenyl and dimethylaminophenyl groups. All the oxidation potential values measured for mono-TTFs were found higher than the oxidation potentials of bis-TTFs. These results showed the good donor ability of this new series of mono-TTFs derivatives, which consequently should lead to conducting materials.The oxidation potentials are almost identical for each pair of bis-donors  increases with a decrease of the temperature to the value of 6000 S cm−1 at 60 K where a metal-insulator transition occurs. Since then, great interest has been devoted to this type of material, and a great number of new organic donors and acceptors have been synthesized as well as their charge-transfer salts.In the 1970s, the organic donor TTF  led to t9–14 with 7,7,8,8-tetracyanoquinodimethane (TCNQ) in hot acetonitrile solution gave the corresponding charge transfer complexes (CTC). Most of the solids were isolated in powder forms. Electrical conductivity was only measured on compressed pellets at room temperature using a two probe technique. The results are reported in Therefore, the complexation of the donors 9-TCNQ, 10a-TCNQ and 10b-TCNQ resulting from mono-TTFs, can be classified in the area of conductors. In fact, they have a conductivity measured on powder compressed pellets of 8.60 10−1 to 1.30 10−2 S cm−1, that allows a conductivity ten times greater on single crystal.For this family of materials, only CTC −3 to 10−6 S cm−1. This can be due to a structural disorder and/or a full charge transfer of an electron for each molecule.Other, CTC resulting from bis-TTFs can be classified in the category of semi-conductors materials with conductivities from 10NMR spectra were recorded on a Brucker AC 250 instrument. Microanalyses were performed in the Microanalysis Laboratory of ENSCM (Montpellier). FAB mass spectra were recorded on a JOEL JMS-DX 300 spectrometer. Uncorrected melting points were measured on a 510 Buchi apparatus. Cyclic voltammetry measurements were carried out on a PAR-273 potentiostat/galvanostat. All solvents were dried by standard methods and all commercial reagents used without purification. All reactions were performed under an inert atmosphere of nitrogen.p-nitrophenyl)-1,3-dithiole-2-thione 1a  in dry methylene chloride (25 mL) was treated with methyl triflate  at 0 °C. The mixture was stirred under nitrogen for 4 h and dry ether (150 mL) was added. The violet-orange salt 2 was obtained by filtration after 24 h, washed with more dry ether, and dried. Yield = 95%; orange powder, mp = 237 °C; 1H NMR (CDCl3) δ ppm: 3.10 ; 7.37 ; 7.53 ; 8.24 ; M.S: : 420 [M + H]+; M = 419; Anal. Calcd for C11H8S4NO5F3: C, 31.49; H, 1.92; S, 30.57; found: C, 31.70; H, 2.14; S, 30.60.A suspension of 4- in dry ethanol (10 mL) was treated with NaBH4  at 0 °C under nitrogen. After the reaction mixture was stirred for 2 h, the solvent was evaporated under vacuum. The residue was dissolved in dichloromethane (100 mL), washed three times with water and dried over magnesium sulphate; the product was obtained after evaporation. Yield = 75%; TLC: Rf = 0.85 (CH2Cl2); dark red oil; 1H NMR (CDCl3) δ ppm: 2.84 ; 5.63 ; 7.15 ; 7.33 ; 8.00 ; M.S: : 272 [M + H]+; M = 271; Anal. Calcd for C10H9S3NO2: C, 44.25; H, 3.34; S, 35.44; found: C, 44.00; H, 3.07; S, 35.75.A suspension of 3  in 30 mL of acetic anhydride at 0 °C. The reaction mixture was stirred for 15 min, then anhydrous ether was added. The 1,3-dithilium salt was precipitated, collected by filtration and washed with ether. Yield = 45%; white powder, mp = 253 °C; 1H NMR (CDCl3) δ ppm: 7.50 ; 7.85 ; 8.20 ; 9.35 ; M.S: : 312 [M + H]+; M = 311; Anal. Calcd for C9H6S2NO2BF4: C, 34.74; H, 1.94; S, 20.61; found: C, 34.98; H, 2.16; S, 20.96.Tetrafluoroboric acid  was added dropwise under nitrogen to a solution of p-Nitrophenyl)-1,3-dithiole-2-thione 1a +; M = 495; Anal. Calcd for C18H13S6N3O2: C, 43.61; H, 2.64; S, 38.81; found: C, 43.40; H, 2.40; S, 39.06.TLC: Rf = 0.53  dissolved in dry and degassed DMF (30 mL). The reaction mixture was stirred for 10 min, the colour becoming dark violet. Then, an excess of 1,2-dibromoethane or 1,3-dibromopropane (10 equiv.) was added in one portion. The colour of the reaction mixture became clear, and the reaction mixture was stirred at room temperature for 1 h. The solvent was removed in vacuo, the residue was dissolved in dichloromethane (100 mL), washed three times with water and dried over magnesium sulphate. The mixture was concentrated in vacuo and the residue was purified by chromatography on a silica gel column (eluent: dichloromethane).Cesium hydroxide monohydrate  in dry methanol (10 mL) was added to tetrathiafulvalene dicyano derivative 2,3-Bis(2-bromoethylthio)-6-p-nitrophenyl tetrathiafulvalene10a: Yield = 45%; TLC: Rf = 0.85 (CH2Cl2); dark-violet powder, mp = 93 °C; 1H NMR (CDCl3) δ ppm: 3.30 ; 3.85 ; 6.80 ; 7.55 ; 8.17 ; M.S: : 604 [M + H]+; M = 603; Anal. Calcd for C15H14S6NO2Br: C, 31.84; H, 2.17; S, 31.88; found: C, 32.09; H, 2.37; S, 31.73.2,3-Bis(3-bromopropylthio)-6-p-nitrophenyl tetrathiafulvalene10b: Yield = 44%; TLC: Rf = 0.85 (CH2Cl2); dark-violet powder, mp = 84 °C; 1H NMR (CDCl3) δ ppm: 2.15 ; 2.93 ; 3.55 ; 6.76 ; 7.50 ; 8.20 ; M.S: : 632 [M + H]+; M = 631; Anal. Calcd for C16H16S6NO2Br: C, 34.23; H, 2.71; S, 30.46; found: C, 34.03; H, 2.56; S, 30.76.11a and 11b were synthesized by employing the same experimental process as 10 from 1 equiv. of 10a or 10b, 1 equiv. of 2,3-bis(2-cyanoethylthio)-6,7-di(methyl)tetrathiafulvalene 8 and 1 equiv. of cesium hydroxide.Compounds -Nitrophenyl bis-tetrathiafulvalene11a: Yield = 40%; TLC: Rf = 0.74 (CH2Cl2); violet powder, mp = 104 °C; 1H NMR (CDCl3) δ ppm: 1.84 ; 2.84 ; 3.19 ; 3.37 ; 3.89 ; 3.62 ; 6.85 ; 7.56 ; 8.26 ; M.S: : 869 [M + H]+; M = 868; Anal. Calcd for C27H19S12N2O2Br: C, 37.35; H, 2.21; S, 44.32; found: C, 37.60; H, 2.46; S, 44.12.p-Nitrophenyl bis-tetrathiafulvalene11b: Yield = 38%; TLC: Rf = 0.74 (CH2Cl2); violet powder, m.p. = 96 °C; 1H NMR (CDCl3) δ ppm: 1.80 ; 2.81 ; 3.16 ; 2.09 ; 2.88 ; 3.52 ; 2.45 ; 2.95 ; 6.82 ; 7.54 ; 8.24 ; M.S: : 897 [M + H]+; M = 896; Anal. Calcd for C29H23S12N2O2Br: C, 38.87; H, 2.59; S, 42.93; found: C, 39.07; H, 2.80; S, 42.74.p12a and 12b were synthesized by employing the same experimental process as 10 from 1 equiv. of 11a or 11b and 1 equiv. cesium hydroxide.Compounds -Nitrophenyl bis-tetrathiafulvalene12a: Yield = 88%; TLC: Rf = 0.70 (CH2Cl2); violet powder, mp = 163 °C; 1H NMR (CDCl3) δ ppm: 1.55 ; 3.59 ; 6.83 ; 7.55 ; 8.25 ; M.S: : 739 [M + H]+; M = 738; Anal. Calcd for C24H21S12NO2: C, 39.05; H, 2.59; S, 52.12; found: C, 39.30; H, 2.69; S, 51.92.p-Nitrophenyl bis-tetrathiafulvalene12b: Yield = 94%; TLC: Rf = 0.70 (CH2Cl2); violet powder, m.p. = 155 °C; 1H NMR (CDCl3) δ ppm: 1.55 ; 2.47 ; 2.99 ; 6.83 ; 7.55 ; 8.25 ; M.S: : 767 [M + H]+; M = 766; Anal. Calcd for C25H23S12NO2: C, 40.75; H, 3.02; S, 50.21; found: C, 40.95; H, 3.17; S, 49.96.pp-nitrophenyl-bis-TTFs derivatives 12a or 12b (4 mmol), tin , and aqueous solution of HCl (35%)  in ethanol (30 mL) was refluxed for 4 h under nitrogen. During this time the initial black solution turned light yellow. The solution was then concentrated in vacuo and treated with an aqueous solution (100 mL) of sodium hydroxide (0.1 M) and extracted with ether. The organic phase was washed with water, dried (MgSO4), and concentrated in vacuo. The product was subjected to column chromatography on silica gel (CH2Cl2), affording the expected compounds 13a and 13b as powder.A stirred mixture of 4--Aminophenyl bis-tetrathiafulvalene13a: Yield = 52%; TLC: Rf = 0.62 (CH2Cl2); dark-orange powder, mp = 138 °C; 1H NMR (CDCl3) δ ppm: 1.53 ; 3.50 ; 3.55–3.85 ; 6.72 ; 6.60 ; 7.09 ; M.S: : 709 [M + H]+; M = 708; Anal. Calcd for C24H23S12N: C, 40.70; H, 2.98; S, 54.33; found: C, 40.97; H, 3.09; S, 54.04.p-Aminophenyl bis-tetrathiafulvalene13b: Yield = 48%; TLC: Rf = 0.62 (CH2Cl2); dark-orange powder, m.p. = 132 °C; 1H NMR (CDCl3) δ ppm: 1.54 ; 2.44 ; 2.95 ; 3.50–3.80 ; 6.70 ; 6.60 ; 7.09 ; M.S: : 737 [M + H]+; M = 736; Anal. Calcd for C25H25S12N: C, 42.41; H, 3.42; S, 52.26; found: C, 42.70; H, 3.54; S, 52.10.p13a or 13b (3 mmol) and of iodomethane  in acetone (15 mL) under nitrogen, K2CO3  was added. After 4 days of stirring at room temperature, the precipitate obtained was filtered, washed with acetone, and then extracted with CH2Cl2. The organic phase was dried (MgSO4) and concentrated in vacuo, providing the expected compounds 14a and 14b as dark orange powder.To a stirred solution of 4-aminophenyl-bis-TTF -Dimethylaminophenyl bis-tetrathiafulvalene14a: Yield = 76%; TLC: Rf = 0.78 (CH2Cl2); orange powder, mp = 182 °C; 1H NMR (CDCl3) δ ppm: 1.53 ; 3.35 ; 3.51 ; 6.61 ; 7.71 ; 8.00 ; M.S: : 737 [M + H]+; M = 736; Anal. Calcd for C26H27S12N: C, 42.41; H, 3.42; S, 52.26; found: C, 42.13; H, 3.28; S, 52.57.p-Dimethylaminophenyl bis-tetrathiafulvalene14b: Yield = 75%; TLC: Rf = 0.78 (CH2Cl2); orange powder, mp = 176 °C; 1H NMR (CDCl3) δ ppm: 1.54 ; 2.43 ; 2.96 ; 3.35 ; 6.60 ; 7.71 ; 8.00 ; M.S: : 765 [M + H]+; M = 764; Anal. Calcd for C27H29S12N: C, 44.00; H, 3.82; S, 50.34; found: C, 43.76; H, 3.63; S, 50.71.pIn summary, we have successfully prepared via Wittig-type, cross-coupling, reduction and alkylation synthetic strategies some new mono-TTFs and bis-TTFs containing nitrophenyl, aminophenyl or dimethylaminophenyl groups. All donors synthesized during the course of this work have been characterized by cyclic voltammetry and their oxidation potentials were determined by cyclic voltammetry. Charge transfer complexes of the donors with TCNQ were prepared and the electrical conductivity of these materials was measured, some CTC are conductive.


As showed by HPLC/MS analysis, the yield in indolizines was higher in the presence of the biocatalyst than in absence of enzyme. Lipase A, from Candida antarctica, showed high catalytic activity and selectivity for the cycloaddition reactions. When the reactions were performed under ultrasound irradiation, the Candida antarctica lipase catalyzed reactions yielded pure indolozines, in good yields and in very short time.Marine microorganisms are of considerable interest as a promising source of enzymes with unsuspected potentials as catalysts for chemical synthesis. We describe here an efficient method for  The indolizine ring is an important structural system frequently found in natural products and has been used as an important skeleton in pharmaceutics because of its interesting and promising biological properties ,2,3. BotWe are interested in the synthesis of a variety of indolizine derivatives, and in the study of their biological, physical, and spectroscopic properties ,12,13. MCandida antarctica is probably one of the most useful microorganism, which has been cited in numerous patents, and has also been used in a variety of commercial applications. The lipases A and B isolated from Candida antarctica (and their immobilized forms CAL A and CAL B) catalyze a variety of chemical reactions including ester hydrolysis, transesterification, and amide synthesis ; 7.42–7.73 ; 4,4 ; 1,43 ; 13C-NMR : 185.67 (2C=O), 163.97 (2C=Oester), 139.78 (2C), 139.70 (2C), 136.61 (2C), 131.74 (2CH), 129.53 (2CH), 129.33 (2CH), 129.05 (4CH), 128.52 (4CH), 122.90 (2C), 116.96 (2C), 113.53 (2CH), 107.55 (2C), 60.37 (2CH2), 14.59 (2CH3); MS (APCI+): m/z: 585 [M + H]+; anal. C 73.72; H 5.14; N 4.76%, calcd. for C36H28N2O6, C 73.96, H 4.83, N 4.79%. 1,1′-diethyldicarboxylate-3,3′-bis(p-methoxy-benzoyl)-7,7′-bis(indolizine) (1b): yellow crystals, mp 290–291 °C; IR : 3140 ; 2977 ; 1700 ; 1665 ; 1597, 1508, 1463 ; 1339  1249, 1220, 1167 ; 1077 ; 1H-NMR  δ/ppm: 9.98 ; 8.80; 7.79 ; 7.76 ; 7.33 ; 7.03 ; 4.43 ; 3,93  1,43 ; 13C-NMR : 186.06 (2C=O), 165.28 (2C=Oester), 163.71 (2C), 138.57 (2C), 136.57 (4CH), 132.42 (2C), 131.81 (2CH), 129.04 (2CH), 122.2 (4CH), 122 (2CH), 117.72 (2CH), 114 (4CH), 113.98 (2CH), 107 (2C), 61.09 (CH2), 56,03 (OCH3), 14,73 (CH3); MS (APCI+): m/z: 645 [M + H]+; anal. C 70.92; H 4.88; N 4.28%, calcd. for C38H32N2O8, C 70.80, H 5.00, N 4.35%.1,1′-diethyldicarboxylate-3,3′-bis(p-nitro-benzoyl)-7,7′-bis(indolizine) (1c): yellow crystals, mp 271–272 °C; IR : 3359 ; 2978 ; 1700 ; 1656 ; 1464, 1428 ; 1521, 1338 ; 1208, 1169 ; 1077 ; 1H-NMR  δ/ppm: 10.13 ; 8.99; 8.03 ; 7.8 ; 7.48 ; 7.42 ; 4.4 ; 1.43 ; 13C-NMR : 184.15 (2C=O), 164.52 (2C=Oester), 150.16 (2C), 143.06 (2C); 138.14 (2C); 132.60 (2C); 129.86 (4CH); 129.14 (2CH); 127.46 (2CH); 123.52 (4CH); 122.74 (2C); 118.28 (2CH); 113.07 (2CH); 108.16 (2Cquat); 60.53 (2CH2); 14.29 (2CH3); MS (APCI+): m/z: 675 [M + H]+; anal. C 64.15; H 4.11; N 8.26%, calcd. for C36H26N4O10, C 64.09, H 3.88, N 8.31%. one-pot synthesis for indolizines, which involves cycloadditions of ethyl propiolate with the in situ generated ylides, in aqueous media.Our synthetic approach is the first example for biocatalyzed Candida antarctica lipase catalyzed the biotransformation of 4,4′-bipyridine, halide derivatives, and ethyl propiolate to form indolizine compounds. A better yield of indolizine was obtained when CAL A was used as a biocatalyst. The catalytic reactions with lipase from Candida antarctica produced almost pure indolizines with good yields and in very short time under ultrasound activation. As mentioned above, the lipase catalyzed synthesis was performed in water, a green solvent compared to the organic solvents usually required for the classical methodologies.The best results were obtained when the bis-indolizines of biological interest.The increasing importance of safety, health, and environmental issues as well as initiatives in green and sustainable chemistry, white and industrial biotechnology, and the new perspectives of molecular economy plainly justify the work done to develop this biocatalytic approach using enzymes for indolizine synthesis. Further developments of this methodology are under investigations. In particular, the process will be extended to the synthesis of variously substituted mono- and


All newly synthesized compounds were evaluated for their antimycobacterial activities against Mycobacterium tuberculosis H37Rv using the microplatealamar blue assay (MABA). Compounds 4f  and 4g  showed antimycobacterial activity with IC90, 7.57 and 2.96 μg/mL, respectively.A series of new 2-(phenylthio) benzoylarylhydrazones were synthesized by acid-catalyzed condensation of hydrazide 3 with corresponding aldehydes. The chemical structures of the compounds were elucidated by FT-IR,  Mycobacterium tuberculosis strains to antimycobacterial agents is an increasing problem world wide (Tuberculosis (TB) is one of the first identified infectious diseases and remains a major health problem with two million deaths and eight million new cases annually . Resistarld wide .Excessive use of antibiotics is generally accepted to be the main reason for increased antibiotic resistance among bacteria -5. Over The hydrazone type and nitroheteroaryl-containing compounds constitute important classes of biologically active drug molecules which have attractive attention of medicinal chemists due to their antituberculosis and antibacterial activity -13. HoweChemistry1H-NMR and Mass spectra.The synthesis of hydrazone derivatives was performed following the steps shown in scheme 1. According to our previously described method, methyl 2-(phenylthio)benzoate 2 was prepared via esterification of 2-(Phenylthio)benzoic acid 1 and the key intermediate 2-(phenylthio)benzoic acid hydrazide 3 was prepared from the reaction of hydrazine hydrate with compound 2 . Target 1H-NMR spectra were obtained using a Bruker FT-80 spectrometer . Mass spectra were obtained using a Finnigan-MAT TSQ-70 spectrometer at 70 eV . The IR spectra were obtained using Nicolet FT-IR Magna 550 spectrographs (KBr disks) . Elemental analyses were carried out with a Perkin Elmer Model 240-c apparatus .The results of the elemental analyses  were within ± 0.4% of the calculated amounts.Chemicals were purchased from Merck chemical company . 5-Nitrothiophen-2-carboxaldehyde was purchased from Alfa Aesar Company. Melting points were taken of a Kofler hot-stage apparatus  and are uncorrected. General procedure for the synthesis of 2-(Phenylthio)benzoylarylhydrazones (4a-4h)A mixture of hydrazide 3 (4.1mmol) and corresponding aldehyde (4.3 mmol) in absolute ethanol (40 mL) was stirred at room temperature for 2 to 3 h in the presence of hydrochloric acid (2 drops) as a catalyst. The end of the reaction was observed with TLC and the hydrazones 4a-4h were isolated by concentration of the reaction mixture under reduced pressure, followed by neutralization with a 10% aqueous solution of sodium bicarbonate. The resulting precipitate was filtered, washed with water (20 mL) and crystallized from a suitable solvent.2-(Phenylthio)benzoic acid (Benzylidene) hydrazide (4a)1H-NMR (CDCl3) 11.81 , 7.37-7.25 ; MS: m/z (%) 332 , 213 (92), 181 (57), 92 (100), 63 (71); Anal. Calcd.for C20H16N2OS: C, 72.26; H, 4.85; N, 8.43. Found: C, 72.57; H, 4.76; N, 8.58.Yield 71% , m.p 225-226°C (ethanol ); IR (KBr): 3206 (NH), 3047 , 1644 (C=O); 2-(Phenylthio)benzoic acid (4-Nitrobenzylidene) hydrazide (4b)1H-NMR (CDCl3): 12.01 , 8.20 , 8.14 , 7.70-7.27 ; MS: m/z (%) 376 , 211 (100), 182 (80); Anal. Calcd.for C20H15N3O3S: C, 63.65; H, 4.01; N, 11.13. Found: C, 63.86; H, 3.94; N, 11.04.Yield 95%, m.p 152-154°C (ethanol); IR (KBr): 3277 (NH), 3075 , 1664 (C=O), 1511, 1342 (NO2) ; 2-(Phenylthio)benzoic acid (4-Pyridinylidene) hydrazide (4c)1H-NMR (DMSO-d6): 12.15 , 8.65 , 8.31 , 7.61-7.19 . MS: m/z (%) 333 , 213 (64), 184 (37), 96 (86); Anal. Calcd, for C19H15N3OS: C, 68.45; H, 4.53; N, 12.6. Found: C, 68.57; H, 4.35; N, 12.68.Yield 86%, m.p 165-167°C (ethyl acetate); IR (KBr): 3277 (NH), 3047 , 1669 (C=O); 2-(Phenylthio)benzoic acid (2-Furylidene) hydrazide (4d)1H–NMR (DMSO-d6): 11.7 , 8.20 , 7.84 -6.91 , 6.64 ; MS: m/z (%) 322 , 212 (100), 183 (72), 92 (20); Anal. Calcd.for C18H14N2O2S: C, 67.06; H, 4.38; N, 8.69. Found: C, 67.32; H, 4.29; N, 8.89.Yield 88%, m.p 209-212°C (ethanol); IR (KBr): 3201 (NH), 3042 , 1639 (C=O); 2-(Phenylthio)benzoic acid (2-Thienylidene) hydrazide (4e)1H-NMR (DMSO-d6): 11.61 , 8.25 , 7.65-7.13 ; Ms: m/z (%) 338 , 211 (100), 182 (86), 92 (58); Anal. Calcd.for C18H14N2OS2: C, 63.88; H, 4.17; N, 8.28. Found: C, 64.02; H, 4.33, N, 8.49.Yield 97%, m.p 197-199°C (ethanol); IR (KBr): 3200 (NH), 3042 , 1639 (C=O); 2-(Phenylthio) benzoic acid (5-Nitro-2-Furyliden) hydrazide (4f)2); 1H-NMR (DMSO-d6): 12.30 , 8.25 , 7.71 , 7.55-7.02 . MS: m/z (%) 367 , 284 (6), 213 (100), 193 (12), 93 (14); Anal. Calcd.for C18H13N3O4S: C, 58.85; H, 3.57; N, 11.44. Found: C, 58.70; H, 3.68; N, 11.51.Yield 84%, m.p 208-210°C (ethanol); IR (KBr): 3175 (NH), 3010, 2 970 , 1649 (C=O), 1526, 1337 (NO2-(Phenylthio) benzoic acid (5-Nitro-2-Thienyliden) hydrazide (4g)2); 1H-NMR(DMSO-d6): 12.25 , 8.51 , 8.07 , 7.61-7.13 .Yield 91% , m.p 178-180°C (ethanol); IR (KBr): 3180 (NH), 3010, 2975 , 1649 (C=O), 1531, 1337 , 213 (100), 184 (11), 93 (7); Anal. Calcd.for C18H13N3O3S2: C, 56.38; H, 3.42; N, 10.96. Found: C, 56.29; H, 3.37; N, 11.12.MS: m/z (%) 383 , in BACTEC 12B medium, using the MicroplateAlamar Blue Assay (MABA)  by the US National Institute of health. Primary screening was conducted at a single concentration of 10 μg/mL against y (MABA) .90 ≤ 10 μg/mL) were retested at lower concentrations by serial dilution against mycobacterium tuberculosis H37Rv to determine the actual MIC, using the MABA method. The VERO cell cytotoxicity assay (50% inhibitory concentration IC50) was done in parallel with TB dose response assay to determine the selectivity index (SI), defined as the ratio of the measured IC50  to the IC90 (H37Rv) Mycobacterium tuberculosis.Compounds demonstrating at least 90% inhibition in the primary screening (IC90 ≤ 10 μg/mL). Compound 4g showed an IC90 value of 2.68, IC50 value of 3.11 and the selectivity index (SI) of 1.05. Compound 4f showed IC90 value of 7.57, IC50 value of 2.92 and the selectivity index (SI) of 0.39  benzoylarylhydrazone derivatives were synthesized and evaluated against Mycobacterium tuberculosis. From eight tested compounds, two (4f and 4g) displayed significant inhibitory effects (IC of 0.39 . Despite the good activity of compounds 4f and 4g, their imidazole analogue, 4h, was weakly active. The results are in agreement with another research in which the comparison between nitroimidazole and nitrofuryl containing heteroaryl derivatives showed that the nitroimidazole ring resulted in compounds devoid of antimycobacterial activity . SimilarThese effective derivatives are ideally suited for further modification to obtain more active and less cytotoxic antimycobacterial compounds.


The ribosomal P70 S6 kinases play a crucial role in PI3K/mTOR regulated signalling pathways and are therefore potential targets for the treatment of a variety of diseases including diabetes and cancer. In this study we describe the identification of three series of chemically distinct S6K1 inhibitors. In addition, we report a novel PKA-S6K1 chimeric protein with five mutations in or near its ATP-binding site, which was used to determine the binding mode of two of the three inhibitor series, and provided a robust system to aid the optimisation of the oxadiazole-substituted benzimidazole inhibitor series. We show that the resulting oxadiazole-substituted aza-benzimidazole is a potent and ligand efficient S6 kinase inhibitor, which blocks the phosphorylation of RPS6 at Ser235/236 in TSC negative HCV29 human bladder cancer cells by inhibiting S6 kinase activity and thus provides a useful tool compound to investigate the function of S6 kinases. RPS6KB1 (S6K1) and RPS6KB2 (S6K2) are key effectors of PI3K/mTOR-regulated signalling, and have been implicated in a variety of human diseases including diabetes and cancer +, Rt = 4.39 min; HR LC-MS: m/z calcd for C10H10N5O: 216.0879 [M+H]+; found: 216.0878.Benzimidazole 14a  was diluted in MeOH (2 mL) and aqueous HCl . Solid NaNO2  was added portionwise over 10 min. After the addition, the reaction mixture was made basic with aqueous NaOH  and hydroxylamine hydrochloride  was added. The reaction mixture was heated to reflux for 2 h, cooled and filtered (H2O and MeOH). Purification of the resulting solid by chromatography  afforded the title compound as a yellow solid . 1H NMR : δ=7.82 , 7.79 , 7.43 , 7.36 , 6.97 , 4.70 , 1.39 ppm ; 13C NMR : δ=156.1 (s), 141.7 (s), 140.1 (s), 138.1 (s), 134.7 (s), 124.5 (d), 123.1 (d), 120.0 (d), 111.0 (d), 40.1 (t), 14.8 ppm (q); LC-MS: m/z: 230 [M+H]+, Rt = 4.65 min; HR LC-MS: m/z calcd for C11H12N5O: 230.1036 [M+H]+; found: 230.1036.The benzimidazole 14b  was dissolved in MeOH (2.5 mL) and aqueous HCl . Solid NaNO2  was added portionwise over 10 min. After the addition, the reaction mixture was made basic with aqueous NaOH  and hydroxylamine  was added. The reaction mixture was heated to reflux for 3 h, cooled and filtered (H2O and MeOH). Purification of the resulting solid by chromatography  afforded the title compound as a green solid . 1H NMR : δ=7.81 , 7.76 , 7.43 , 7.35 , 6.91 , 3.66 , 1.31-1.27 , 1.04-1.01 ppm ; 13C NMR : δ=156.1 (s), 142.3 (s), 141.4 (s), 138.1 (s), 135.9 (s), 124.4 (d), 123.1 (d), 120.1 (d), 112.0 (d), 26.7 (d), 8.3 ppm (t); LC-MS: m/z: 242 [M+H]+, Rt = 4.67 min; HR LC-MS: m/z calcd for C12H12N5O: 242.1036 [M+H]+; found: 242.1038.The benzimidazole 14c  was diluted in MeOH (1 mL) and aqueous HCl . Solid NaNO2  was added portionwise over 10 min. After the addition, the reaction mixture was made basic with aqueous NaOH  and hydroxylamine  was added. The reaction mixture was heated to reflux for 5 h, cooled and filtered (H2O and MeOH). Purification of the resulting solid by chromatography  afforded the title compound as a white solid . 1H NMR : δ=7.87 , 7.72 , 7.41 , 7.37 , 7.30 , 7.24 , 7.15 , 7.03 , 5.97 ppm ; 13C NMR : δ=156.1 (s), 141.7 (s), 140.6 (s), 138.1 (s), 136.4 (s), 135.3 (s), 128.7 (2d), 127.5 (d), 126.5 (2d), 124.8 (d), 123.4 (d), 120.1 (d), 111.5 (d), 48.0 ppm (t); LC-MS: m/z: 292 [M+H]+, Rt = 5.07 min; HR LC-MS: m/z calcd for C16H14N5O: 292.1193 [M+H]+; found: 292.1199.The benzimidazole (14d)  was diluted in MeOH (3 mL) and aqueous HCl . Solid NaNO4. The excess solvent was removed under reduced pressure and the residue purified by flash chromatography  to afford title compound 15e  as a white solid. 1H NMR : δ=7.82 , 7.43 , 7.36 , 6.99 , 4.59 , 1.36 , 0.48 ppm ; 13C NMR : δ=156.2, 141.7, 140.2, 138.2, 135.4, 124.6, 123.1, 119.9, 111.5, 48.8, 11.3, 3.4 ppm; LC-MS: m/z: 256.11 [M+H]+, Rt = 2.90 min; HR LC-MS: m/z calcd for C13H13N5O: 256.2752 [M+H]+; found: 256.1194.NaH  was added to a stirred solution of 2-aminoxadiazole benzimidazole , in DMF (10 mL) at room temperature. The mixture was stirred at room temperature for 45 min, followed by the drop-wise addition of (bromomethyl)cyclopropane  dissolved in DMF (1 mL). This mixture was heated at 65 °C for 18 h. After cooling to room temperature, the excess DMF was removed under reduced pressure and the resulting mixture was diluted with diethyl ether (50 mL). The organic mixture was washed with water and brine before drying over MgSO4 and evaporated under vacuum. The crude product was purified by flash chromatography  to give 41 mg (70%) of the title compound as a white solid. 1H NMR : δ=11.07 , 7.84 , 7.72 , 7.60 , 7.42 , 3.77–3.45 , 2.46 , 1.45 , 1.20–1.04 ppm ; 13C NMR : δ=168.3 (s), 150.1 (s), 141.6 (s), 141.2 (s), 135.9 (s), 124.4 (d), 123.1 (d), 120.2 (d), 111.9 (d), 99.5 (s), 26.0 (d), 23.3 (t), 7.7 ppm (t); LC-MS: m/z: 242 [M–(CH3C=O)+H]+, Rt = 2.41 min; HR LC-MS: m/z calcd for C14H14N5O2: 284.1142 [M+H]+; found: 284.1143.A mixture of amino-oxadiazole (15c)  and acetyl chloride  was stirred at room temperature for 2 h. The reaction mixture was partitioned between EtOAc and saturated aqueous sodium bicarbonate solution (200 mL). After separation, the organic phase was washed with brine, dried over MgSO3 , Cu powder , methyl-(3-hydroxyphenyl)sulfone , and Cs2CO3 . Bromoazabenzimidazole 21a  was added under Ar followed by anhydrous DMF (1.5 mL). The tube was sealed under Ar, and the mixture was heated to 150 °C under microwave irradiation for 30 min. After cooling to room temperature, the mixture was diluted with dichloromethane and filtered. The filtrate was concentrated in vacuo and partitioned between water and EtOAc. After separation, the water phase was extracted with EtOAc and the organics were combined, washed twice with water, dried over MgSO4 and concentrated. Purification of the residue by flash chromatography on silica gel  afforded 21b  as a white solid . 1H NMR : δ=8.79 , 7.75 , 7.70 , 7.67 , 7.63 , 7.50 , 6.92 , 4.70 , 3.25 , 1.41 ppm ; 13C NMR : δ=158.2 (s), 156.1 (s), 155.6 (s), 143.1 (s), 143.0 (s), 142.2 (s), 140.0 (d), 137.8 (s), 137.1 (s), 130.9 (d), 125.3 (d), 122.1 (d), 118.2 (d), 93.7 (d), 43.3 (q), 40.6 (t), 14.6 ppm (q); LC-MS: m/z: 401 [M+H]+, Rt = 2.27 min; HR LC-MS: m/z calcd for C17H17N6O4S: 401.1026 [M+H]+; found: 401.1026.A glass tube (evacuated and back-filled with Ar) was charged with Fe(acac)2 , 1 M aqueous NaOH  and Pd(PPh3)4  were added to a solution of 21a  in dimethoxyethane . The reaction mixture was heated in a microwave reactor at 140 °C for 1.5 h. The reaction mixture was then partitioned between saturated aqueous NH4Cl solution and EtOAc. After separation, the organic phase was washed with brine, dried over MgSO4 and evaporated under vacuum. The residue was first purified by ion exchange chromatography on SCX-II acidic resin (2 g) eluting with MeOH then a 2 M solution of NH3 in MeOH. After concentration, the resulting solid was purified on a small pad of silica gel  to afford 21c  as a white solid. 1H NMR : δ=9.20 , 8.46 , 8.25 , 7.53 , 7.44 , 6.96 , 4.79 , 1.45 ppm ; 13C NMR : δ=156.2 (s), 150.5 (s), 142.4 (s), 142.3 (d), 140.8 (s), 139.1 (s), 138.5 (s), 137.9 (s), 128.8 (d), 128.6 (d), 128.5 (d), 126.8 (d), 102.4 (d), 40.5 (t), 14.9 ppm (q); LC-MS: m/z: 307 [M+H]+, Rt = 2.23 min; HR LC-MS: m/z calcd for C16H15N6O: 308.1329 [M+H]+; found: 308.1337.PhB(OH)N-methylpyrrolidinone (0.5 mL). The reaction mixture was heated in a microwave reactor at 160 °C for 3 h. The mixture was cooled and concentrated. Purification of the crude by preparative TLC, eluting with EtOAc afforded 21d  as a yellow solid. 1H NMR : δ=8.73 , 7.04 , 6.90 , 4.61 , 3.87-3.67 , 3.61-3.42 , 1.36 ppm ; 13C NMR : δ=156.7 (s), 156.0 (s), 142.5 (s), 140.6 (s), 140.3 (d), 137.9 (s), 133.7 (s), 86.1 (d), 66.0 (t), 46.2 (t), 40.0 (t), 14.6 ppm (q); LC-MS: m/z: 316 [M+H]+, Rt = 2.50 min; HR LC-MS: m/z calcd for C14H18N7O2: 316.1516 [M+H]+; found: 316.1515.Morpholine  was added to a solution of 21a  in 2 , 1 M aqueous NaOH  and Pd(PPh3)4  were added to a solution of 21a  in DME (2 mL). The reaction mixture was heated in a microwave reactor at 140 °C for 2 h. The reaction mixture was then partitioned between saturated aqueous NH4Cl solution and EtOAc. After separation, the organic phase was washed with brine, dried over MgSO4 and evaporated under vacuum. Purification of the residue by flash chromatography  afforded 21e  as a white solid. 1H NMR : δ=8.99 , 7.70 , 6.93 , 4.66 , 2.63 , 1.39 ppm ; 13C NMR : δ=156.1 (s), 151.7 (s), 141.8 (d), 141.5 (s), 140.2 (s), 137.9 (s), 137.3 (s), 104.5 (d), 40.3 (t), 24.3 (t), 14.7 ppm (q); LC-MS: m/z: 245 [M+H]+, Rt = 1.73 min; HR LC-MS: m/z calcd for C11H13N6O: 245.1145 [M+H]+; found: 245.1151.MeB(OH)N-methylglucamide and 1 mM DTT and containing 1 mM PKA-inhibitor peptide PKI  or an in-house synthesised peptide corresponding to residues 5-22 of the PKA-inhibitor peptide, but terminating with an amide at the C-terminus. Crystals of the PKA-S6K1/PKI staurosporine ternary complex were obtained by streak-seeding into fresh hanging drops containing the same protein and reservoir solutions as used for the crystallisation of the binary PKA-S6K1/PKI complex, with the addition of 0.5 mM staurosporine. All other protein-inhibitor complexes were obtained by soaking experiments of selected inhibitors at a final concentration of 1 mM as previously described for PKA [v/v) 2-methyl-2,4-pentanediol, 25 mM MES-Bis/Tris pH 6.5, 0.1 mM EDTA and 1 mM DTT, prior to flash-cooling in liquid nitrogen. The data for the binary PKA-S6K1/PKI complex were collected at beamline ID14EH2 at the ESRF . Data for the ternary PKA-S6K1/PKI/inhibitor complexes were collected at the following beamlines: staurosporine- and compound 1-bound complexes were collected at beamline I02 of the Diamond Light Source  and the data for the protein-inhibitor complexes with compounds 15e, 21a, and 21e were collected at beamline ID14EH4 at the ESRF. All data sets were integrated, merged and scaled using the programs iMosflm [Crystallisation of the PKA-S6K1 chimera was performed using the same conditions as previously reported for the PKA/PKB chimera . Prior t for PKA . Crystal iMosflm , Pointle iMosflm , 38 from iMosflm   causing 50% inhibition of proliferation for tumour cells were determined using a sulforhodamine B assay. Electrochemiluminescent immunoassays  measuring the inhibition of phosphorylation of RPS6-ser235/236, AKT-Thr308, AKT-Ser473, GSK3b-Ser9 and S6K-Ser421/424 were carried out as described in Raynaud et al. [The human bladder carcinoma cell line HCV29 was obtained from Dr Richard Lamb . This cell line has elevated mTORC1 and S6K activity through loss of TSC1  as a res


Twelve-membered macrocyclic lactones 2a,b with Ci symmetry including two oxetane-rings, which are the Paternò–Büchi dimerization products, were isolated in ca. 20% yield. The intramolecular cyclization products, such as 3-alkoxyoxetane and 2,7-dioxabicyclo[2.2.1]hept-5-ene derivatives, were not detected in the photolysate.Furan-2-ylmethyl 2-oxoacetates  Alternatively, 2,7-dioxabicyclo[2.2.1]hept-5-ene OBH would be formed from the 1,6-biradical form. The regioisomeric oxetane 3OX should be formed via the regioisomeric biradical BR’. Biradical BR is energetically more stable than BR’, because BR can undergo radical delocalization. The electrophilic oxygen of the excited carbonyl should preferably interact with more nucleophilic C1 carbon to give selectively the biradical BR. Thus, only the 2-alkoxyoxetane 2OX has been observed in the Paternò–Büchi reactions reported so far hept-5-ene derivative B, or intermolecularly to give the 2-alkoxyoxetane derivative C  was irradiated in degassed benzene with a high-pressure Hg lamp with a Pyrex filter (2a (R = Ph), which possesses Ci symmetry, was obtained as the major product and contains the biologically important macrocyclic lactone structure hept-5-ene , Hiroshima University.1a–c  were dissolved in benzene (7.0 ml) and the degassed reaction mixture was irradiated with a high-pressure Hg lamp  with a Pyrex filter. After 13 h, the solvent was removed in vacuo and dimethyl fumarate added as an internal standard. 1H NMR  was measured to determine the ratio of products. After the photoreaction, the residue was purified by repeated column chromatography and PTLC (hexane–EtOAc = 2:1) to give 2a,b as colorless crystals.The furan derivatives 2a: 1H NMR  δ 7.55–7.51 , 7.40–7.31 , 6.35 , 5.28 , 4.82 , 4.67 , 3.71 ; 13C NMR : δ 171.6 (C), 148.7 (CH), 136.0 (CH), 128.6 (CH), 128.4 (CH), 125.7 (CH), 112.6 (C), 102.1 (CH), 90.7 (C), 61.4 (CH2), 55.2 (CH); HRMS (ESI) m/z calcd for C26H20O8Na (M + Na)+ 483.10504, found 483.10526.2b: 1H NMR : δ 6.66 , 5.23 , 5.14 , 4.27 , 3.77 , 1.57 ; 13C NMR : δ 173.3 (C), 149.8 (CH), 112.7 (C), 101.3 (CH), 88.1 (C), 61.5 (CH2), 53.1 (CH), 21.2 (CH3); HRMS (ESI) m/z calcd for C16H16O8Na (M + Na)+ 359.07374, found 359.07391.File 11a–c, the detail of the X-ray structure of compound 2a, and 1H NMR and 13C NMR spectra for compounds 2a,b.Experimental section for preparation of compounds File 22a.X-Ray crystallographic data for compound


A facile synthesis of azabicycloadducts is described by 1,3-dipolar cycloaddition reactions of thioisatin with thiazolidine-2-carboxylic acid in the presence of various electron rich and electron deficient dipolarophiles. Theoritical calculations have been performed to study the regioselectivity of products. The geometrical and energetic properties have been analyzed for the different reactants, transition states and cycloadducts formed. The construction of sophisticated molecules requires viable, selective and highly reliable reactions as potent synthetic tools -3. The 1The 1,3-dipolar cycloaddition reaction of an azomethine ylide with an alkene leads to the formation of pyrrolidine ,14 deriv1 with thiazolidine-2-carboxylic 2 acid was carried out in equimolar ratio in refluxing dry acetonitrile in the presence of diphenylacetylene as dipolarophile to afford a diastereomeric mixture of {-spiro-6,7-diphenyl-1aza-4thia-bicyclo -6-octene-8,3'}-benzo[b]thiophene-2'-one} 8 as cis/trans isomers. Analogous reactions of thioisatin with other dipolarophiles viz methyl acrylate, phenylacetylene, phenylpropyne and ethyl phenyl propiolate produced diasteroisomeric mixtures of cycloadducts 5-9 in 75%- 63% yield. The mechanism for the formation of the cycloadducts 5-9 involve the initial formation of an iminium species 3 followed by the loss of CO2 via stereospecific 1,3-cycloreversion [4. Subsequent [3 + 2] cycloaddition with various dipolarophiles then produce novel azabicycloadducts  for 3H protons, another triplet at δ 2.60 (J = 3.0Hz) was associated with 2H protons, a singlet at δ 4.15 appeared for 5H and the aromatic protons resonated between δ 6.79- δ 8.12 ppm. Its 13C NMR spectrum showed a signal at 183.54 for C-2' carbonyl carbon, aromatic carbons appeared in the range δ 146.41-δ131.32 ppm, the olefenic carbons  resonated at δ 127.32 and 126.54, spiro carbon (C-8) was noticed at δ 86.23, C-5 at δ 46.72, C-2 at δ 45.43, and C-3 at 34.56 ppm respectively. Additional evidence was gathered from the mass spectrum of cycloadduct 8. The molecular ion and the base peaks were present at m/z 413(32%) and 235(100%) respectively; another peak at m/z 385(39%) corresponded to [M-CO].+ whereas the peak at m/z 108(35%) was assigned to [C6H4S].+.The structure of all the cycloadducts has been ascertained from their spectral data. Thus the IR spectrum of a typical diphenylacetylene cycloadducts amy) 4 derived from thioisatin and thiazolidine-2-carboxylic acid has been optimized on Gaussian 03 program at AM1 level.The stereochemical course of the cycloaddition was examined by AM1 calculations. To calculate the relevant activation and stabilization energies, minimized geometries of the reactants, products and transition states are required. The molecular geometry of the simplest azomethine ylide . The favoured path involves HOMOdipole-LUMOdipolarophile energy gap is lower than the LUMOdipole-HOMOdipolarophile gap and therefore the dominant FMO approach is HOMOdipole-LUMOdipolarophile. Both the HOMO and the LUMO of the dipole show uneven distribution of the electron density along the C-N-C dipole. In the HOMO case, the orbital coefficient is larger at C1 (0.288) than at C2 (-0.163). Similarly in the LUMO of methyl acrylate the atomic orbital coefficient are (0.611) and (-0.521) respectively  with the formation of products having two chiral centers. Therefore a total of 4 + 4 = 8 isomers (4 pairs of enantiomers) could be possible 8a-8h  and stabilization energy of amy with different dipolarophiles have been tabulated in Table ΔHFrom the above discussions, it may be concluded that:4syn and anti4.a.The azomethine ylide exits in two conformations; dipole-LUMOdipolarophile as this energy gap is lower than the LUMOdipole-HOMOdipolarophile gap.b. The dominant FMO approach is HOMOc. Azomethine ylide is stabilized by the delocalization of dipolar charge into thioisatin nucleus, thus increasing the activation energy for the reaction path.-1). The 1H NMR spectra and 13C NMR spectra have been recorded on a Bruker DRX-300 MHz and 75.47 MHz model respectively in CDCl3 and DMSO using tetramethylsilane as an internal standard. The chemical shifts are given in δ ppm values. The mass spectra were recorded on a JEOL-SX 102 (FAB). Most of the spectra were recorded at CDRI, Lucknow, India. Elemental analyses were performed on a Perkin Elmer Series C, H, N, S Analyzer 2400. The solvents were purified by standard procedures [The uncorrected melting points were taken in open glass capillaries. The IR spectra were recorded on a Nicolet Magna IR Spectrometer Model 550 in KBr pellets and band positions are reported in wave numbers , thiazolidine-2-carboxylic acid 2  and methyl acrylate  in dry acetonitrile (50 ml) was refluxed for 22 h. The reaction was monitored by TLC until the consumption of the reactants. The reaction mixture was filtered, solvent evaporated and the residue was subjected to column chromatography. The petroleum ether/chloroform (4:1) fraction afforded the desired azabicycloadduct 5 as pale brown solid.An equimolar mixture of thiosatin -1. 1H NMR : = 2.29 , 2.46, 2.50, 2.68, 3.10, 4.15, 7.36-7.54. 13C NMR : 35.43(CH2S), 44.58(CH2), 46.34(CH), 85.99(C-N), 125.86-124.32(C = C), 142.58-131.36(CHaro), 175.67(O = C-O), 184.32(C = O). EI-MS: m/z (%) = 321(M+ 38), 275(100), 261(20), 284(15), 234(18). Anal. Calcd. For C15H15NO3S2: C, 56.05%; H, 4.70%; N, 4.36%. Found: C, 56.45%; H, 4.78%; N, 4.76%.Pale brown solid, yield: 0.33g (70%), mp: 105-107°C. IR (KBr): 1710(C = O),1450(C-N), 715(C-S) cm-1. 1H NMR : = 2.50, 2.68, 4.12, 4.32, 7.32-7.54. 13C NMR : 31.51(CH2S), 36.54(CH2), 45.46(CH), 85.95(C-N), 128.81-124.49(C = C), 143.87-132.36(CHaro), 180.79(C = O). EI-MS: m/z (%) = 337(M+ 36), 203(100), 309(20), 108(34). Anal.Calcd for C19H15NOS2: C, 67.62%; H, 4.48%; N, 4.15%. Found: C, 67.78%; H, 4.53%; N, 4.32%.Coffee brown powder, yield: 0.31g (65%), mp: 95-97°C. IR (KBr): 1720(C = O), 1445(C-N), 690(C-S) cm-1.1H NMR : 2.14, 2.50, 2.68, 2.84, 7.59-8.45. 13C NMR: 32.33(CH2S), 35.43(CH2), 44.45(CH), 86.54(C-N), 124.23-122.86(C = C), 143.41-130.63(CHaro), 184.34(C = O). Anal.Calcd for C20H17NOS2: C, 68.34%; H, 4.87%; N, 3.98%. Found: C, 68.55%; H, 4.93%; N, 4.13%.Brownish solid, yield: 0.32g (68%), mp: 120-122°C. IR (KBr): 1725(C = O), 1420(C-N), 678(C-S) cm-1. 1H NMR : 2.51, 2.60, 4.15 6.79-8.12. 13C NMR : 34.54(CH2S), 45.43(CH2), 46.72(CH), 88.23(C-N), 127.32-126.54(C = C), 146.41-132.32(Caro), 183.54(C = O). EI-MS: m/z (%) = 413(M+ 32), 385(39), 235(100), 108(35). Anal. Calcd for C25H19NOS2: C, 72.61%; H, 4.63%; N, 3.39%. Found: C, 72.73%; H, 4.78%; N, 3.86%.Shiny brown powder, yield: 0.29g (63%), mp: 135-133°C. IR (KBr): 1715(C = O), 1420(C-N), 6905(C-S) cm-1. 1H NMR : 1.25, 2.61, 2.69, 2.67, 3.75,4.12  7.49-7.94. 13C NMR : 33.85(CH2), 44.96(CH), 46.32(CH2), 84.36(C-N), 124.32-122.93(C = C), 140.14-130.12(CHaro), 180.25(O = C-O), 185.44(C = O). EI-MS: m/z (%) = 321(M+ 38), 275(100), 261(20), 284(15), 234(18). Anal. Calcd for C22H19NO3S2: C, 64.52%; H, 4.68%; N, 3.42%. Found: C, 64.68%; H, 4.79%; N, 3.54%.Dark brown power, yield: 0.31g (69%), mp: 110-112°C. IR (KBr): 1710(C = O), 1410(C-N), 690(C-S) cmThe authors declare that they have no competing interests.


A series of novel isatine-sulphonamide derivatives have been synthesized by combining isatin derivatives with sulphonamides. The structure of the synthesized compounds were elucidated by spectral analysis . Investigation of anti-HIV activity was done against HIV-1(IIIB) in MT-4 cells and HIV integrase inhibitory activity. 4-(1-acetyl-5-methyl-2-oxoindolin-3-ylideneamino)-N-benzenesulfonamide (SPIII-5ME-AC) inhibits the HIV Integrase enzymatic activity as both over all and strand transfer reaction and 4-(1-benzoyl-5-chloro-2-oxoindolin-3-ylideneamino)-N-benzene sulfonamide (SPIII-5Cl-BZ) exhibits 36 percent maximum protection against HIV-1 at sub toxic concentration. Isatin , a versatile lead molecule for potential bioactive agents and its derivatives were reported to possess wide spectrum of activity. Methisazone (N-methylisatin-ß-thiosemicarbazone) was one of the first clinically used synthetic antiviral agents11Melting points were determined using Thomas melting point apparatus and are uncorrected. The purity was checked by TLC using silica gel G as stationary phase. The structure of the synthesized compounds was elucidated using a Perkin Elmer FT-IR in KBr disc and PMR was taken on a Bruker AMX-(400 MHz) FT-NMR. Mass spectra were obtained on a Varian Atlas CH-7 Mass spectrometer at 70 eV.−1: 3300 (NH), 1510 (C=N), 1674 (C=0), 1583 (C=C), PMR (DMSO-d6) δ ppm: 2 , 7.1-7.9 , 8.1 , EI- MS (m/e): 301.32. SPIII-SMe yield: 72%, mp: 128°, IR (KBr) cm−1: 3320 (NH), 1590 (C=N), 1650 (C=0), 1522 (C=C), PMR (DMSO-d6) δ ppm: 2.35 , 2.1 , 7.0-7.9 , 8.2 , EI-MS (m/e): 315.35. SPIII-SCl yield: 82%, mp: 181°, IR (KBr) cm−1: 3370 (NH), 1577 (C=N), 1680 (C=0), 1526 (C=C), PMR (DMSO-d6) δ ppm: 2.1 , 7.0-7.9 , 8.0 , EI-MS (m/e): 355.77.SPIII-SM yield: 65%, mp: 192°, IR (KBr) cm−1: 3340 (NH), 1670 (C=N), 1695 (C=0), 1520 (C=C), PMR (DMSO-d6) δ ppm: 2.10 , 4.1 , 7.0-7.92 , 8.0 , EI-MS (m/e): 396.42Isatine-sulphonamide derivatives were synthesized by refluxing an equimolar (0.01 mol) mixture of isatins  and sulphonamides (sulphonilamide and sulphamoxazole) for 6 h in 10 ml of glacial acetic acid. The mixture was cooled to room temperature and poured into crushed ice; the solid thus obtained was recrystallized from ethanol. SPIII-S yield: 68%, mp: 116°, IR (KBr) cm−1: 3310 (NH), 1690 (C=N), 1705 (C=0), 1510 (C=C), PMR (DMSO-d6) δ ppm: 2.30 , 2.42 , 4.1 , 6.1 , 7.0-7.92 , EI-MS (m/e):528.38. SPIII-5Br-BZ yield: 72%, mp: 285°, IR (KBr) cm−1: 3320 (NH), 1695 (C=N), 1707 (C=0), 1520 (C=C), PMR (DMSO-d6) δ ppm: 2.32 , 4.1 , 6.1 , 7.1-8.0 , EI-MS (m/e):590.04. SPIII-5Me-AC yield: 72%, mp: 228°, IR (KBr) cm−1: 3360 (NH), 1685 (C=N), 1705 (C=0), 1530 (C=C), PMR (DMSO-d6) δ ppm: 2.10 , 2.32 , 2.45 , 4.1 , 6.1 , 7.0-7.92 , 8.0 , EI-MS (m/e): 463.51. SPIII-5Cl-BZ yield: 83%, mp: 179°, IR (KBr) cm−1: 3335 (NH), 1662 (C=N), 1710 (C=0), 1530 (C=C), PMR (DMSO-d6) δ ppm: 2.35 , 4.1 , 6.1 , 7.1-8.0 , EI-MS (m/e): 446.02N-acyl-isatinesulphonamide derivatives were synthesized by refluxing an equimolar mixture of (0.01 mol) isatins  and sulphadimidine for 6 h in 10 ml of glacial acetic acid. The mixture was cooled to room temperature and poured into crushed ice; the solid thus obtained was recrystallized from ethanol. SPIII-5Br-AC yield: 67%, mp: 221°, IR (KBr) cmB) in MT-4 cells50) were added to a flat-bottomed microtiter tray with 50 μl of medium containing various concentrations of the test compounds. MT-4 cells were added at a final concentration of 6×105 cells/ml. After 5 d of incubation, at 37° the numbers of viable cells were determined by the 3--2,5-diphenyltetrazolium bromide (MTT) method. Cytotoxicity of the compounds for mock-infected MT-4 cells was also assessed by the MTT method.The compounds were tested for antiHIV activity against the replication of HIV-1  is labeled with biotin on the 3' end and the other oligo is labeled with digoxigenin at the 5' end. For the overall integration assay the second 5'-digoxigenin labeled oligo is (5'-GACCCTTTTAGTC AGTGTGGAAAATCTCTAGCAGT-3'). For the Strand Transfer assay the second oligo is missing the GT oligonucleotides at the 3'. The integrase enzyme was diluted to the same specific activity in 750 mM NaCl, 10 mM Tris pH 7.6, 10% glycerol and 1 mM β-mercaptoethanol. To perform the reaction 4 μl diluted integrase (corresponds to a concentration of WT integrase of 1.6 μM) and 4 μl annealed oligos (7 nM) was added in a final reaction volume of 40 μl containing 10 mM MgCl50) values of the synthesized compounds against the replication of HIV-1(IIIB) in acutely infected MT-4 cells were higher than the cytotoxic concentration (CC50). Lead molecules isatins  and sulphadimidine not active against HIV-1(IIIB) in MT-4 cells, but the combined product isatinesulphadimidine  lead molecule is essential for antiHIV activity. Substitution in NH group of isatin (N-acylation) abolishes the antiHIV activity of the lead molecule SPIII (4-(1-acetyl-5-methyl-2-oxoindolin-3-ylideneamino)-N-benzene sulfonamide (SPIII-5ME-AC) inhibits the HIV Integrase enzymatic activity and 4-(1-benzoyl-5-chloro-2-oxoindolin-3-ylideneamino)-N-benzenesulfonamide (SPIII-5Cl-BZ) exhibits 36 percent maximum protection against HIV-1 at subtoxic concentration. None of the compounds exhibited antiHIV effect and all the compounds displayed cytotoxic properties in the lymphocyte cell line (MT-4 cells). The 50% effective concentration (ECe SPIII,  were actle SPIII .


Vigna genus are important food resources and there have already been many reports regarding their bioactivities. In our preliminary bioassay, the chloroform layer of methanol extracts of V. vexillata demonstrated significant anti-inflammatory bioactivity. Therefore, the present research is aimed to purify and identify the anti-inflammatory principles of V. vexillata. One new sterol (1) and two new isoflavones  were reported from the natural sources for the first time and their chemical structures were determined by the spectroscopic and mass spectrometric analyses. In addition, 37 known compounds were identified by comparison of their physical and spectroscopic data with those reported in the literature. Among the isolates, daidzein (23), abscisic acid (25), and quercetin (40) displayed the most significant inhibition of superoxide anion generation and elastase release.The seeds of  Glycine soja and their biological activity −) in HR-ESI-MS analysis. The UV absorption maxima at 280 and 229 nm were characteristic of a benzene moiety D25 −300.0 . UV (MeOH) λmax (log ɛ): 280 (2.29), 229  nm. IR (Neat) νmax: 3438, 2956, 2868, 1721, 1642, 1516, 1461, 1377, 1261, 1207, 1172, 1103, 1063, 1024 cm−1. ESI-MS (rel. int. %): m/z 575 , 559 (100). HR-ESI-MS: m/z 575.4093 [M−H]− . 1H-NMR : δ 6.76 , 6.57 , 5.36 , 4.66 , 4.23 , 3.68 , 2.34 , 2.00 , 1.85–1.87 , 1.70 , 1.57 , 1.46 , 1.36 , 1.26 , 1.18 , 1.07 , 1.01 , 1.01 , 0.92 , 0.85 , 0.84 , 0.83 , 0.82 , 0.68 . 13C-NMR : δ 172.0 (C-1′), 154.0 (C-7′), 139.6 (C-5), 131.1 (C-4′), 129.1 , 122.8 (C-6), 115.0 , 74.7 (C-3), 56.7 (C-14), 56.1 (C-17), 50.0 (C-9), 45.9 (C-24), 44.3 (C-3′), 43.9 (C-2′), 42.3 (C-13), 39.7 (C-12), 38.1 (C-4), 37.0 (C-1), 36.6 (C-10), 36.2 (C-20), 34.0 (C-22), 31.9 (C-7), 31.9 (C-8), 29.2 (C-25), 28.2 (C-16), 27.8 (C-2), 26.2 (C-23), 24.3 (C-15), 23.1 (C-28), 21.0 (C-11), 19.8 (C-26), 19.3 (C-19), 19.0 (C-27), 18.8 (C-21), 12.0 (C-29), 11.9 (C-18).White powder, mp 145–147 °C . UV (MeOH) λmax (log ɛ): 340 (1.86), 296 , 265 (2.84), 222 , 213 (2.53) nm. IR (Neat) νmax: 3418, 2928, 1656, 1614, 1515, 1480, 1433, 1402, 1320, 1286, 1251, 1205, 1169, 1131, 1065, 1027 cm−1. ESI-MS (rel. int. %): m/z 335 , 321 (76), 305 (88). HR-ESI-MS: m/z 335.0905 [M−H]− . 1H-NMR : δ 13.26 , 8.77 , 8.18 , 7.43 , 6.90 , 6.27 , 5.46 , 5.12 , 4.95 , 3.51 , 3.09 , 1.78 . 13C-NMR : δ 181.8 (C-4), 167.4 (C-7), 164.7 (C-5), 158.6 (C-4′), 154.0 (C-2), 153.8 (C-9), 144.6 (C-3″), 131.3 , 124.2 (C-3), 122.9 (C-1′), 116.0 , 112.9 (C-4″), 106.5 (C-10), 104.0 (C-8), 94.5 (C-6), 88.8 (C-2″), 31.3 (C-1″), 17.1 (C-5″).White powder, mp > 300 °C (MeOH); [α]D25 −80.0 . UV (MeOH) λmax (log ɛ): 307 (2.25), 250 (2.71), 243 , 216 (2.38) nm. IR (Neat) νmax: 3292, 2928, 2858, 1627, 1594, 1515, 1449, 1389, 1318, 1270, 1208, 1175, 1105, 1066, 1021 cm-1. ESI-MS (rel. int. %): m/z 343 , 321 . HR-ESI-MS: m/z 343.0944 [M+Na]+ , 321.1128 [M+H]+ . 1H-NMR : δ 8.17 , 8.05 , 7.46 , 6.95 , 6.88 , 5.52 , 5.15 , 4.97 , 3.64 , 3.23 , 1.81 . 13C-NMR : δ 175.8 (C-4), 165.7 (C-7), 158.3 (C-4′), 154.5 (C-9), 153.0 (C-2), 144.6 (C-3″), 131.2 , 128.4 (C-5), 125.3 (C-3), 124.3 (C-1′), 119.8 (C-10), 115.9 , 114.3 (C-8), 112.8 (C-4″), 109.0 (C-6), 88.7 (C-2″), 32.0 (C-1″), 17.1 (C-5″).White plates, mp 227–229 °C (MeOH); [α]2+)-free Hank’s balanced salt solution (HBSS) buffer at pH 7.4, and were maintained at 4 °C before use.Neutrophils were isolated with a standard method of dextran sedimentation prior to centrifugation in a Ficoll Hypaque gradient and hypotonic lysis of erythrocytes. Blood was drawn from healthy human donors (20–30 years old) by venipuncture into heparin-coated vacutainer tubes, using a protocol approved by the institutional review board at Chang Gung Memorial Hospital. Blood samples were mixed gently with an equal volume of 3% dextran solution. The leukocyte-rich plasma was collected after sedimentation of the red cells for 30 min at room temperature. The leukocyte-rich plasma was transferred on top of 20 mL Ficoll solution (1.077 g/mL) and spun down at 400 g for 40 min at 20 °C. The granulocyte/erythrocyte pellets were resuspended in ice-cold 0.2% NaCl to lyse erythrocytes. After 30 sec, the same volume of 1.6% NaCl solution was added to reconstitute the isotonic condition. Purified neutrophils were pelleted and then resuspended in a calcium  were equilibrated at 37 °C for 2 min and incubated with drugs or an equal volume of vehicle  for 5 min. Cells were activated with 100 nM FMLP during the preincubation of 1 μg/mL cytochalasin B (FMLP/CB) for 3 min. Changes in the absorbance with a reduction in ferricytochrome c at 550 nm were continuously monitored in a double-beam, six-cell positioner spectrophotometer with constant stirring . Calculations were based on differences in the reactions with and without SOD (100 U/mL) divided by the extinction coefficient for the reduction of ferricytochrome c (ɛ = 21.1/mM/10 mm).The assay of the generation of superoxide anion was based on the SOD-inhibitable reduction of ferricytochrome c . In briep-nitroanilide as the elastase substrate. Briefly, after supplementation with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 μM), neutrophils (6 × 105/mL) were equilibrated at 37 °C for 2 min and incubated with drugs or an equal volume of vehicle  for 5 min. Cells were activated by 100 nM FMLP and 0.5 μg/mL cytochalasin B, and changes in absorbance at 405 nm were continuously monitored to assay elastase release. The results were expressed as the percent of elastase release in the FMLP/CB-activated, drug-free control system.Degranulation of azurophilic granules was determined by elastase release as described previously . Experim50) was computer-assisted (PHARM/PCS v.4.2). Statistical comparisons were made between groups using Student’s t test. Values of P less than 0.05 were considered to be statistically significant.Results were expressed as mean ± S.D. Computation of 50% inhibitory concentration  along with 37 known constituents (4–40). In the screening of their bioactivity, three compounds, daidzein (23), abscisic acid (25), and quercetin (40) from the active chloroform fraction demonstrated significant anti-inflammatory potentials compared with the PI3K inhibitor LY294002 and a NADPH oxidase inhibitor DPI. It is well known that the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) pathway plays an important role in neutrophil activation. The NADPH oxidase is also important in the inflammatory mechanism. Therefore, the purified principles of V. vexillata were potential to be developed as new anti-inflammatory drugs through the inhibition of PI3K or NADPH oxidase. This is the first report of complete chemical compositions of V. vexillata and it would provide the comprehensive knowledge related to the further discovery of phytochemical lead compounds from natural food sources.The present investigation on the methanol extracts of


Five of the newly synthesized compounds were selected by the National Cancer Institute  for a primary in vitro antitumor assay. 7-Chloro-3,5-diphenyl-thiazolopyrimidin-2-one (5a) proved to be the most active one among the screened derivatives and was further evaluated in the full panel of 60 cell lines at five different concentrations. The structures of compounds were determined by IR, 1H-NMR, 13C-NMR, X-ray, and elemental analysis.The 2-oxo analogs of thiazolo[4,5- In contrast with related 2-thioxo-thiazolopyrimidine derivatives, the 2-oxo analogs have not been very well explored in medicinal chemistry. The synthesis and biological evaluation of the substituted 2-oxo-thiazolopyrimidines have been the subject of several review articles. They were reported to possess antibacterial, antifungal  and 7-chloro-3,5-diaryl-thiazolopyrimidine-2-thiones (3) with diethyl sulfate and water for the replacement of the 2-thioxo group by an oxygen function   to evaluate their growth inhibitory effects on 60 human cancer cell lines, derived from nine neoplasmatic diseases. Five derivatives 4a, 4b, 5a, 5b, and 5d were selected for a primary in vitro antitumor assay, at 10−5 M concentration. Results were expressed as percent growth of the treated cells, compound 5a showing mean percent growth =71.26 was further tested at five different concentrations.In this study, in continuation of our work on thiazolopyrimidin-2-ones 4a–4f and 5a–5f were synthesized as shown in Scheme H-thiazolopyrimidin-7-ones (2) and 7-chloro-3,5-diaryl-thiazolopyrimidine-2-thiones (3)  and 4a was slightly active against renal cancer CAKI-1 cell line (26.76 % growth).All synthesized compounds were submitted for testing at the NCI to evaluate the growth inhibitory effect. Five compounds 5a, 5b, and 5d which possess electron-withdrawing 7-chloro substituent showed variable antitumor activity, reported as the percentage of growth of treated cells; the preliminary screening results are shown in Table 5a, 5b, and 5d exhibited antiproliferative effect against cell lines of leukemia, non-small cell lung cancer, colon cancer, melanoma, ovarian cancer, and renal cancer. It is worth to mention that substance 5a showed noticeable cytotoxic activity against renal cancer (UO-31), melanoma (MALME-3M), and non-small cell lung cancer (NCI-H522) while compound 5b was most effective against the last one. This limited data indicate that the replacement of the 7-oxo group with the small, non-polar chloro substituent substantially increased anticancer activity. Remarkable low growth percent values against a minimum number of cell lines (mean growth) was obtained only for compound 5a which was approved for the further screening test to evaluate the growth inhibition (GI), and cytostatic and cytotoxic effects. The selected compound was additionally evaluated at tenfold dilution of five different concentrations, from 10−4 to 10−8 M on approximately 60 human tumor cell lines panels. Three different dose–response parameters, GI50, TGI, and LC50, were calculated for each cell line. GI50 is the molar concentration of the compound required for half GI. Total growth inhibition (TGI) is the molar concentration of the compound resulting in TGI; TGI signifies the cytostatic effect. LC50 is the molar concentration of the compound resulting in a 50 % death of the initial cells; LC50 signifies the cytotoxic effect. The overview of these parameters of compound 5a is reported in Table 50 values of thioguanine (TG), the NCI standard anticancer agent. The log GI50 values lower than −5 showed a notable activity level. It can be noticed that compound 5a proved to be very sensitive toward non-small cell lung cancer NCI-H522 and renal cancer UO-31 log GI50 −5.91 and −5.88, respectively, . GI of most cell lines of standard TG is higher than that showed by investigated compound 5a; but against the following cell lines: K-562, NCI-H322M, NCI-H522, SW-620, U251, SK-MEL-28, IGROV1, A498, and HS 578T, compound 5a was more active than TG. TG is a guanine analog and thiazolopyrimidines can be considered as 7-thio analogs of the purine bases guanine and adenine. Thiazolopyrimidine derivatives may interfere with the synthesis of guanine nucleotides as antimetabolites.Compounds 1H-NMR and 13C-NMR spectra were acquired in d6-DMSO on a Bruker ARX 300 MHz  instrument. Tetramethylsilane was used as the internal standard and all chemical shift values were expressed in parts per million . IR spectra were recorded on a Specord M80 spectrometer using KBr pellets. X-Ray Crystallography: the data were collected using the Bruker KAPPA APEXII ULTRA controlled by APEXII software. Reaction progress and the purity of the obtained compounds were monitored by thin-layer chromatography on Merck silica gel plates  using the solvent system dichloromethane: 1-propanol (10:1) for elution. Iodine was used as a developing agent. The chemicals and reagents for syntheses were obtained from Alfa Aesar , Chempur (Piekary Sl. Poland), and Sigma-Aldrich . Starting compounds are synthesized according to the literature  analyzer, and results within ±0.4 % of the theoretical values were obtained for the new compounds. 2 or 3 (10 mmol) in acetonitrile (20 ml), diethyl sulfate  was added, and the reaction mixture was heated under reflux for 1 h at 130 °C. After cooling, 100 ml of water was added and the reaction mixture was refluxed with stirring for 2 h during which the product was precipitated. The solid was filtered and suspended in a hot mixture of methanol and 5 % NaHCO3. The reaction mixture was allowed to cool, and the crude product was filtered and crystallized from appropriate solvent.To a solution of appropriate compound −1: 3450, 3080 (NH), 1680 (C=O), 1530 (C=N), 1260 (C–S–C), 760 (phenyl). 1H-NMR (d6-DMSO) δ: 7.42–7.93 , 13.19 . Anal. Calcd for C17H11N3O2S: C, 63.54; H, 3.45; N, 13.08. Found: C, 63.44; H, 3.52; N, 13.27.IR (KBr) cm−1: 3450, 3090 (NH), 1670 (C=O), 1590 (C=N), 1230 (C–S–C), 760 (phenyl). 1H-NMR (d6-DMSO) δ: 7.51–7.94 , 13.22 . Anal. Calcd for C17H10ClN3O2S: C, 57.39; H, 2.83; N, 11.81. Found: C, 57.56; H, 3.01; N, 11.97.IR (KBr) cm−1: 3470, 3080 (NH), 1680 (C=O), 1590 (C=N), 1260 (C–S–C), 760 (phenyl). 1H-NMR (d6-DMSO) δ: 7.34–7.99 , 13.27 . Anal. Calcd for C17H10ClN3O2S: C, 57.39; H, 2.83; N, 11.81. Found: C, 57.59; H, 2.87; N, 11.85.IR (KBr) cm−1: 3450, 3090 (NH), 1680 (C=O), 1610 (C=N), 1240 (C–S–C), 770 (phenyl). 1H-NMR (d6-DMSO) δ: 7.31–8.20 , 13.20 . Anal. Calcd for C17H10FN3O2S: C, 60.17; H, 2.97; N, 12.38. Found: C, 59.98; H, 3.03; N, 12.41.IR (KBr) cm−1: 3470, 3090 (NH), 1690 (C=O), 1570 (C=N), 1240 (C–S–C), 780 (phenyl). 1H-NMR (d6-DMSO) δ: 7.22–8.03 , 13.21 . Anal. Calcd for C17H9FN3O2S: C, 57.14; H, 2.54; N, 11.76. Found: C, 57.31; H, 2.55; N, 11.94.IR (KBr) cm−1: 3450, 3080 (NH), 1680 (C=O), 1590 (C=N), 1260 (C–S–C), 760 (phenyl). 1H-NMR (d6-DMSO) δ: 7.45–8.16 , 13.19 . Anal. Calcd for C17H10BrN3O2S: C, 51.01; H, 2.52; N, 10.50. Found: C, 51.14; H, 2.60; N, 10.66.IR (KBr) cm−1: 1680 (C=O), 1590 (C=N), 1260 (C–S–C), 760 (phenyl). 1H-NMR (d6-DMSO) δ: 7.46–8.13 . Anal. Calcd for C17H10ClN3OS: C, 60.09; H, 2.97; N, 12.37. Found: C, 59.98; H, 3.01; N, 12.38.IR (KBr) cm−1: 1680 (C=O), 1560 (C=N), 1230 (C–S–C), 760 (phenyl). 1H-NMR (d6-DMSO) δ: 7.52–8.11 . Anal. Calcd for C17H9Cl2N3OS: C, 54.56; H, 2.42; N, 11.23. Found: C, 54.60; H, 2.49; N, 11.29.IR (KBr) cm−1: 1690 (C=O), 1570 (C=N), 1250 (C–S–C), 760 (phenyl). 1H-NMR (d6-DMSO) δ: 7.52–8.11 . Anal. Calcd for C17H9Cl2N3OS: C, 54.56; H, 2.42; N, 11.23. Found: C, 54.65; H, 2.50; N, 11.33.IR (KBr) cm−1: 1690 (C=O), 1600 (C=N), 1240 (C–S–C), 760 (phenyl). 1H-NMR (d6-DMSO) δ: 7.27–8.15 . Anal. Calcd for C17H9ClN3OS: C, 57.10; H, 2.73; N, 11.75. Found: C, 57.21; H, 2.86; N, 11.83.IR (KBr) cm−1: 1690 (C = O), 1590 (C = N), 1250 (C–S–C), 770 (phenyl). 1H-NMR (d6-DMSO) δ: 7.26–8.22 . Anal. Calcd for C17H8ClF2N3OS: C, 54.34; H, 2.15; N, 11.18. Found: C, 54.42; H, 2.20; N, 11.26.IR (KBr) cm−1: 1680 (C=O), 1560 (C=N), 1250 (C–S–C), 770 (phenyl). 1H-NMR (d6-DMSO) δ: 7.33–8.16 . Anal. Calcd for C17H9BrClN3OS: C, 48.77; H, 2.17; N, 10.04. Found: C, 48.91; H, 2.25; N, 10.12.IR (KBr) cmCharacteristic data of the new compounds are depicted in Table −5 M concentration. Compounds with significant GI are evaluated at five different concentrations ranging from 10−4 to 10−8 M. The percent growth was evaluated versus controls not treated with tested compounds. Preparation of the tested compounds and the sulforhodamine B (SRB) protein assay which was used to estimate cell viability of growth were described previously . The test agents were preliminarily evaluated in the full panel of 60 different human tumor cell lines derived from nine cancer diseases, namely leukemia, melanoma, non-small cell lung c., colon c., brain c., breast c., ovarian c., prostate c., and renal c., at single 10


Wolfiporia extensa (Polyporaceae) is used to invigorate the spleen and to tranquilize the mind in Chinese herbal medicine. Lanostane-type triterpene acids were regard as major secondary metabolites from dried sclerotia of W. extensa.Dried sclerotia of epi-benzoyloxyl-dehydrotumulosic acid (1), 3-epi--dehydrotumulosic acid (2) and 3-epi-(3′-hydroxy-3′-methylglutaryloxyl)-dehydrotumulosic acid (3), were isolated from the sclerotia of W. extensa, together with 3 known lanostane derivatives (4–6). Their structures were elucidated on the basis of spectroscopic analysis, including 1D and 2D-NMR techniques.Three new lanostane-type triterpene acids, 3-W. extensa in this paper.Six lanostane derivatives including three new triterpene acids and three known compounds were reported from the sclerotia of  Wolfiporia extensa (Polyporaceae), well known as ‘Fu-Ling’ in China, is used to invigorate the spleen and to tranquilize the mind in Chinese herb medicine , one terminal methylene group at δ 4.84  and 4.97 , two olefinic methylenes at δ 5.39  and δ 5.64 ], together with signals from typical benzoyl group δ 8.18 , 7.35 , 7.46] ], two carbons from terminal methylene group δ 107.0 (C-31) and 156.1 (C-24)], four olefinic carbons δ 116.7 (C-11), 120.8 (C-7), 142.9 (C-8) and 146.0 (C-9)], two oxygenated methylenes δ 79.0 (C-3) and 76.4 (C-16)], seven methyl carbons δ 17.6 (C-18), 21.9 (C-27), 22.0 (C-26), 22.4 (C-29), 22.7 (C-19), 26.6 (C-30) and 28.1 (C-28)], signals from benzoyl group δ 165.9 (C-7′), 133.2 (C-4′), 131.4 (C-1′), 129.8 , and 128.9 ], and signals from other fifteen carbons (see Table epi-dehydrotumulosic acid (6), except for the existence of an additional set of signals arising from the benzoyl group in 1 and a methoxyl group δ 52.2 (−OCH3)]. HMBC experiment showed correlations between methoxyl proton (δ 3.63) with 3′-C  indicated the methyl malonate group  along with 13 C-NMR showed signals δ 171.4 (C-1′), 46.3 (C-2′), 69.9 (C-3′), 46.4 (C-4′), 174.6 (C-5′), and 28.4 (−CH3)]. Those signals were assigned to 3-hydroxy-3-methylglutaryl group based on HMQC and HMBC spectra data. It was further confirmed from ESI-MS experiment, which showed fragment ions at m/z 525.4 [M-H-102 (CH (CH3) (OH)-CH2-COOH)]-. The HMBC correlations of H-3 (δ 4.94 br s) with C-1′ (δ 171.4) confirmed that the 3-hydroxy-3-methylglutaryloxyl group was at C-3 in 3  in 3 was deduced by comparing of the compound 3 specific rotation features with those of (+)-3-epi-dehydrotumulosic acid, and (3′ S)-(+)-3-hydroxy-3-methylglutaric acid, which are dextrorotatory +, calcd for C38H53O5, 589.3893. ESI-MS (m/z) (rel. int.): 587.3 [M - 1]- (100), 417.0 (23), 338.9 (4), 208.8 (13).Colourless needles; 1 H-NMR (in pyridine-d5): see Table 13 C-NMR (in pyridine-d5): see Table -1: 3416, 2960, 1736, 1707, 1641, 1254, 1152, 891, 800. UV λMeOH max nm (logϵ): 243 (4.16). HRESI-MS (m/z): 607.3605 [M + Na]+, calcd for C35H52O7Na, 607.3611.Colourless needles; c = 0.1705, pyridine); 1 H-NMR (in pyridine-d5): see Table 13 C-NMR (in pyridine-d5): see Table -1: 3389, 2962, 1707, 1642, 1205, 1176, 891, 802, 780, 770. UV λMeOH max nm (logϵ): 244 (4.13); HRESI-MS (m/z): 651.3880 [M + Na]+, calcd for C37H56O8Na, 651.3873. ESI-MS (m/z) (rel. int.): 627.5 [M - 1]- (100), 525.4 (5).Colourless needles; [α] = − 7.6 (The authors declare that they have no competing interests.GS carried out the chemical analysis-structure elucidation and drafted the Manuscript; NZ carried out the chemical studies; SW employed in the several chemical assays of extraction and isolation; YL worked at the part of experimental design; YB engaged in the part of chemical analysis-structure elucidation; CS carried out the part of chemical assays of extraction and isolation; SR conceived of the study and its design and coordination of the scientific teams. All authors have read and approved the final manuscript.


D-xylofuranosyl)-5-iodo-pyrrolopyrimidine 1, an unusual naturally occurring marine nucleoside isolated from an ascidan, Diplosoma sp., was synthesized from D-xylose in seven steps with 28% overall yield on 10 g scale. The key step was Vorbrüggen glycosylation of 5-iodo-pyrrolopyrimidine with 5-deoxy-1,2-O-diacetyl-3-O-benzoyl-D-xylofuranose. Its absolute configuration was confirmed.4-Amino-7-(5′-deoxy-β- A and B [d]pyrimidine nucleosides have triggered the continued interest of medicinal chemists in the past decades pyrimidine 1 was first isolated by Japanese scientists from an ascidan, Diplosoma sp. It was found that nucleoside 1 causes complete inhibition of cell division in fertilized sea urchin eggs at 1 µg/mL concentration pyrimidines (7-deazapurine) were seldom used as donors in Vorbrüggen glycosylation. The reason might be possibly ascribed to the nonreactive nature of N-7 in pyrrolopyrimidines pyrimidine nucleosides with good yields [d]pyrimidine nucleosides has not been reported. It is important to further prove this protocol’s generality and reproducibility. We herein report a practical and efficient synthesis of 1 from D-xylose with application of Vorbrüggen glycosylation as the key step  on 50 g scale pyrimidin-4-ol 11 with phosphoryl chloride gave 4-chloro-7H-pyrrolopyrimidine 12 in 93% yield pyrimidine 4 was first treated with 1.2 equiv. of BSA, followed by 1.2 equiv. of 5′-deoxy-1,2,3-tri-O-acetyl-β-D-xylofuranose 3 and 2 equiv. of TMSOTf at room temperature. The resulting mixture was heated to 80 °C for 12 h. After workup, column chromatography afforded 4-chloro-7-(5′-deoxy-β-D-xylofuranosyl)-5-iodo-pyrrolopyrimidine 10 in 56% yield as pure β anomer on 20 g scale. The glycosylation yield was lower than that of the corresponding 5′-deoxy-1,2,3-tri-O-acetyl-D-ribofuranose acceptor, which typically gave more than 80% yield . Furthermore, because the starting material is D-xylose, the absolute configuration of nucleoside 1 is undoubtedly D. All other spectral data are in agreement with that of the reported natural nucleoside 1 pyrimidine was synthesized in our lab on 500 g scale. BSA and TMSOTf were purchased from Sigma Aldrich. MeCN was dried over CaH2 and distilled prior to use. Thin layer chromatography was performed using silica gel GF-254 plates  with detection by UV (254 nm), or charting with 10% sulfuric acid in ethanol. Column chromatography was performed on silica gel . NMR spectra were recorded on a Bruker AV400 spectrometer and chemical shifts (δ) are reported in ppm. 1H NMR and 13C NMR spectra were calibrated with TMS as internal standard and coupling constants (J) are reported in Hz. The ESI-HRMS were obtained on a Bruker Dalton microTOFQ II spectrometer in positive ion mode.4-Chloro-5-iodo-7D-Xylose  was suspended in acetone (500 mL) containing concentrated H2SO4 . The mixture was stirred for 30 min at room temperature. Then a solution of Na2CO3  in water (224 mL) was added carefully with cooling to 0 °C. After addition, the mixture was stirred for a further 3 h. Then, solid Na2CO3  was added in 3 portions over 30 min. The resulted Na2SO4 was filtered off and washed with acetone. The combined filtrates were evaporated to give crude 6, which was purified by a silica gel column  to afford crystallized 6 . mp 42–43 °C; 1H NMR  δ: 5.97 , 4.51 , 4.31 , 4.27 , 4.16 , 4.09 , 4.03 , 2.04 , 1.47 , 1.31 ; 13C NMR  δ: 111.8, 104.8, 85.6, 78.8, 78.7, 61.0, 26.7, 26.2.O-isopropylidene-α-D-xylofuranose 6  in THF (400 mL) and Et3N (300 mL) at 0 °C. After addition, the solution was stirred at room temperature overnight. CH3OH (10 mL) was added to quench the reaction. After filtration, the resulting solution was evaporated to dryness. The residue was dissolved in EtOAc (500 mL) and washed with H2O (100 mL), sat. NaHCO3 (100 mL) and brine (100 mL), then dried with anhydrous Na2SO4. After filtration, the solution was evaporated under reduced pressure. The residue was purified over a silica gel column (EtOAc:Hexane = 1:1) to afford 7 . mp 134–135 °C; 1H NMR  δ: 7.80 , 7.36 , 5.88 , 4.52 , 4.34~4.31 , 4.13 , 2.46 , 2.27 , 1.59 , 1.47 , 1.30 ; 13C NMR  δ: 145.3, 132.4, 130.0, 128.0, 112.2, 104.9, 85.0, 76.7, 74.3, 66.0, 26.8, 26.2, 21.7; MS (ESI) m/z 345.2 [M + H]+.A solution of TsCl  in THF (500 mL) was added slowly to 1,2-4  was added in portions to 5-O-tosylate-1,2-O-isopropylidene-α-D-xylofuranose 7  in anhydrous THF (800 mL). The resulting suspension was refluxed for 6 h. EtOAc (50 mL) was added slowly to quench the reaction. Then H2O (100 mL) was added and the suspension was stirred for 2 h at room temperature. After filtration over Celite, the filtrate was evaporated to remove THF. EtOAc (500 mL) was added. The solution was washed with H2O (100 mL), sat. NaHCO3 (100 mL) and brine (100 mL), then dried with anhydrous Na2SO4. After filtration, the solution was evaporated under reduced pressure. The residue was purified over a silica gel column  to afford 8  as white solid. mp 81–83 °C; 1H NMR  δ: 5.83 , 4.47 , 4.25 , 3.93 , 2.78 , 1.44 , 1.24 ; 13C NMR  δ: 111.4, 104.3, 85.5, 76.2, 76.1, 26.5, 26.1, 12.8; MS (ESI) m/z 175.3 [M + H]+.LiAlHO-isopropylidene-α-D-xylofuranose 8  in anhydrous pyridine (100 mL) at 0 °C. The resulting solution was stirred for 2 h at room temperature. Ethanol (0.5 mL) was added to quench the reaction. It was evaporated under reduced pressure to dryness. The residue was dissolved in DCM (300 mL) and washed with H2O (50 mL), sat. NaHCO3 (50 mL) and brine (50 mL), then dried with anhydrous Na2SO4. After filtration, the resulting solution was evaporated under reduced pressure. The residue was purified over a silica gel column  to afford 9  as white solid. 1H NMR  δ: 8.02 , 7.57 , 7.41 , 5.95 , 5.32 , 4.64 , 4.54~4.49 ,1.52 , 1.29 ; 13C NMR  δ: 165.3, 133.3, 129.7, 129.4, 129.4, 128.5, 128.5, 111.6, 104.6, 83.9, 78.0, 76.9, 75.2, 26.6, 26.1, 13.3; MS (ESI) m/z 279.2 [M + H]+.Benzoyl chloride  was added slowly to a solution of 5-deoxy-1,2-O-benzoyl-1,2-O-isopropylidene-α-D-xylofuranose 9  was dissolved in AcOH (130 mL) and Ac2O (150 mL). Concentrated H2SO4 (7.5 mL) was added dropwise. The solution was stirred at room temperature for 26 h. The solution was poured into H2O (500 mL) and extracted with DCM (3 × 100 mL). The combined organic layer was washed with H2O (3 × 50 mL), sat. NaHCO3 (3 × 50 mL) and brine (50 mL), then dried with anhydrous Na2SO4. After filtration, the resulting solution was evaporated under reduced pressure to afford 10  as anomers. It was used directly without further purification. 5-Deoxy-3-H-pyrrolopyrimidine 4  in dry MeCN (350 mL) was added BSA  and stirred for 10 min at room temperature. After addition of 5-deoxy-3-O-benzoyl-1,2-O-diacetate-D-xylofuranose 3 , TMSOTf  was added to the mixture. The mixture was stirred for 15 min before heating to 80 °C for 12 h. The reaction was cooled to room temperature. Water (400 mL) was added to quench the reaction. The solution was extracted with EtOAc (300 mL × 3). The combined organic layer was washed with sat. NaHCO3 (350 mL × 1) and brine (350 mL × 2), and dried with anhydrous Na2SO4, filtered and evaporated under reduced pressure. The residue was purified over a silica gel column  to afford 10 . mp 136–137 °C;1H NMR : δ 8.61 , 8.01 , 7.79 , 7.63 , 7.55 , 6.44 , 5.56 , 5.47 , 4.63 , 2.19 , 1.43 . 13C NMR : δ 169.1, 165.0, 152.8, 151.2, 150.5, 133.9, 133.4, 132.0, 129.8, 129.0, 128.5, 117.4, 87.8, 81.3, 78.4, 76.8, 76.6, 52.8, 20.6, 14.6; MS (ESI) m/z 542.2 [M + H]+; HRMS (ESI) m/z calcd for [M + H]+ C20H18IClN3O5: 541.9974, found: 541.9968.To a suspension of 4-chloro-5-iodo-710  in methanolic ammonia  was placed in an autoclave and stirred at 130 °C for 12 h. After cooling, the mixture was concentrated to dryness and the residue was purified over a silica gel column  to afford 1  as yellow powder. mp 190-191 °C; [α]25D −68 ; CD: λext 240 nm (∆ε −2.6) and λext 209 nm (∆ε −4.4);1H NMR : δ 8.11 , 7.60 , 6.73 , 5.96 , 5.82 , 5.69 , 4.21 , 4.12 , 3.81, 1.21 ; 13C NMR : δ 157.6 (C-4), 152.3 (C-2), 150.0 (C-8), 128.6 (C-6), 103.4 (C-9), 89.8 (C-1′), 82.4 (C-2′), 78.4 (C-4′), 77.0 (C-3′), 51.8 (C-5), 14.4 (C-5′); HRMS (ESI) m/z calcd for [M + H]+ C11H14IN4O3: 377.0105, found: 377.0098.A solution of D-xylofuranosyl)-5-iodo-pyrrolopyrimidine 1 was developed on the basis of Vorbrüggen glycosylation. It has the merits of cost efficiency, mild reaction conditions, and easy access to diversity-oriented derivatives. We are currently in the process of applying this approach to other 4-amino-7-(5′-deoxy-β-D-xylofuranosyl)-pyrrolopyrimidine derivatives and studying their biological activities, such as insecticides, which will be reported in due course. In conclusion, a practical and efficient approach for 10 g scale synthesis of marine nucleoside 4-amino-7-(5′-deoxy-β-