Synthetic routes to 5'-azidoribonucleosides are reported for adenosine, cytidine, guanosine, and uridine, resulting in a widely applicable one-pot methodology for the synthesis of these and related compounds. The target compounds are appropriate as precursors in a variety of purposive syntheses, as the synthetic and therapeutic relevance of azido- and amino-modified nucleosides is expansive. Furthermore, in the conversion of alcohols to azides, these methods offer a tractable alternative to the Mitsunobu and other more difficult reactions. The vast utilization of nucleosides in most aspects of cellular function makes them a tantalizing target for a variety of therapies with potentially wide ranging physiological and pharmacological effects ,2,3,4. T in vivo ,5,6,7. T in vivo . The use in vivo ,10,11, iThe azide functional group is among the most useful in organic synthesis, owing to its superlative reactivity. A great body of work has been done on azidonucleotides in particular, and although their bioactivity has been found to be negligible in many instances, they remain an important intermediate in a variety of purposive syntheses . NucleosMost contemporary alcohol to azide syntheses rely on two-step protocols proceeding through a halogenated intermediate, Mitsunobu-type displacement reactions, or other tenuous methodologies employing toxic, complex and/or expensive reagents ,27,28. Hexo-cyclic amine moieties found in their respective nitrogenous bases prior to selective substitution of the 5'-hydroxyl group. Subsequent protection of the 2' and 3' hydroxyl groups of each nucleoside to produce starting material compounds 1–4  after creation of the initial Appel product and subsequent substitution by azide, no PPh3 remains to form the said intermediate; and (ii) product recovery is significantly improved due to a reduction in the amount of phosphonium oxide by-product formed. Applying this reduced-reagent one-pot technique to the protected starting material generated from adenosine, guanosine, cytidine, and uridine afforded 5'-azido-5'-deoxyribonucleosides in high yields for application as intermediates and precursors in a variety of nucleoside syntheses.Staudinger reduction of azides requires excess triphenylphosphine to form the iminophosphorane intermediate , which itraction . ReducinOur interest in 5'-azido- and 5'-aminonucleoside analogs stems primarily from their application as precursors in the synthesis of positively charged guanidine-linked ribooligonucleotides RNG in a and P3'1–4 in the minimum amount of anhydrous DMF and then allowed to react with PPh3, CBr4, and NaN3 added in order, allowing the previous reactant to completely dissolve before proceeding to the next. The reaction mixtures were stirred for 10 min at room temperature then stirred overnight at 90 °C. The products were purified by column chromatography to afford 5'-azidonucleotides 5–8 in excellent yields (81%–90%). Protection of the 2' and 3' hydroxyl groups seems to be vindicated as our one-pot yields exceeded those reported by Hata et al. [One pot reactions were instead prepared from a et al.  for two Although guanidinium linkages are of primary focus within our lab, further modification may reveal synthetic nucleoside analogues with the ability to treat or aid immune system response, whether through anti-viral or therapeutic methods. Nucleosides were of primary focus only several decades ago, and with advances in methodology and technology, therapeutic nucleosides will again move into the spotlight as agents of vast clinical potential.1H-NMR spectra were recorded on a 500 MHz Varian instrument, using CDCl3 or DMSO-d6 as solvents. Chemical shifts were reported in δ ppm and coupling constants (J) are given in Hz. Thin-layer chromatography (TLC) was carried out on Silica Gel 60 F-254 pre-coated plates  and visualization of the products was performed under UV light. Silica gel used for flash column chromatography was Scientific Silica Gel . ESI mass spectra were recorded on QSTAR Pulsar Quadrupole/time-of-flight mass spectrometer with Turbo Ion Spray Ionization Source . All reactions were performed using anhydrous solvents under N2 atmosphere. All flash column chromatography performed used 3:1 ethyl acetate to hexanes for product isolation.N6-Benzoyl-2',3'-O-isopropylideneadenosine (1). To a stirred solution of N6-benzoyladenosine  in anhydrous acetone (100 mL) was added 2,2-dimethoxypropane  and p-toluenesulfonic acid monohydrate . The reaction mixture was refluxed for 18 h, then filtered to remove any unreacted starting material. The filtrate was concentrated under vacuum and the residue was dissolved in EtOAc, washed with 5% NaHCO3, brine, and dried over sodium sulfate anhydrous. The solvent was removed under reduced pressure and the solid product was washed with diethyl ether to remove any residual 2,2-dimethoxypropane then filtered. The product was further purified by silica gel column chromatography (3:1 EtOAc to hexanes) to afford pure compound 1 . HRMS (ESI) m/z calcd. for C20H21N5O5 (M+Na)+ 434.1440, found 434.1441. NMR (DMSO-d6): 11.38 br s, 1H (NH); 8.91 s, 1H (H-8); 8.81 s, 1H (H-2); 8.18-7.67 m, 5H (H-Bz); 6.40 d, 1H, J = 11.8 (H-1'); 5.56 q, 1H, J = 8.3 (H-2'); 5.28 br s, 1H (OH-5'); 5.13 t, 1H, J = 5.9 (H-3'); 4.18 q, 1H, J = 6.3 (H-4'); 3.69–3.67 m, 2H (H-5'); 1.63 s, 3H (CH3); 1.25 s, 3H (CH3). N2-Isobutyryl-2',3'-O-isopropylideneguanosine (2). To a stirred solution of N2-isobutyrylguanosine  in anhydrous acetone (100 mL) was added 2,2-dimethoxypropane  and p-toluenesulfonic acid monohydrate . The reaction mixture was refluxed for 18 h. The solvent was removed under reduced pressure, and the residue was dissolved in EtOAc, washed with 5% NaHCO3, brine, and dried over sodium sulfate anhydrous. The solvent was removed under vacuum, and the residue was stirred for five minutes in ether then filtered to afford pure compound 2 . 1H-NMR (DMSO-d6): 12.10 s, 1H (NH); 11.54 s, 1H (NH); 7.83 s, 1H (H-8); 5.84 d, 1H, J = 2.8, (H-1'); 5.11 m, 1H (H-2'); 5.04 m, 1H (H-3'); 4.44 q, 1H, J = 3.9 (H-4'); 3.99–3.78 m, 2H, (H-5'); 2.78-2.76 m, 1H (CH); 1.62 s, 3H (CH3); 1.37 s, 3H (CH3); 1.28 d, 6H, J = 6.8 (C(CH3)2).N4-Benzoyl-2',3'-O-isopropylidenecytidine (3). To a stirred suspension of N4-benzoylcytidine  in anhydrous acetone (100 mL), p-toluenesulfonic acid monohydrate  and 2-2-dimethyoxypropane  were added and the reaction mixture was refluxed overnight under N2 atmosphere. To remove the unreacted starting material, the mixture was filtered through Celite, which was then washed with acetone and methylene chloride. The filtrate was concentrated under vacuum and the residue was dissolved in EtOAc, washed with 5% NaHCO3, brine, and dried over sodium sulfate anhydrous. The solvent was removed under reduced pressure, and the residue was stirred for five minutes in ether then filtered to afford pure compound 3 . 1H- NMR (DMSO-d6): 11.22 br s, 1H (NH); 8.29 d, 1H, J = 7.8 (H-6); 8.00-7.51 m, 5H (BzH); 7.32 d, 1H, J = 7.2 (H-5); 5.85 d, 1H, J = 2.0 (H-1'); 5.12 t, 1H, J = 5.4 (OH-5'); 4.90 dd, 1H, J = 4.0, 1.9 (H-2'); 4.76 q, 1H, J = 3.4 (H-3'); 4.21 q, 1H, J = 3.5 (H-4'); 3.64-3.55 m, 2H (H-5'); 1.48 s, 3H (CH3); 1.28 s, 3H (CH3).2',3'-O-Isopropylideneuridine (4). To a stirred solution of uridine  in anhydrous acetone (100 mL) was added 2,2-dimethoxypropane  and p-toluenesulfonic acid monohydrate . The reaction mixture was stirred overnight at room temperature. The solvent was removed under reduced pressure, and the residue was dissolved in EtOAc, washed with 5% NaHCO3, brine, and dried over sodium sulfate anhydrous. The solvent was removed under vacuum, and the crude was purified by silica gel column chromatography (3:1 ratio of EtOAc to hexanes) to afford compound 4 . 1H-NMR (DMSO-d6): 11.36 s 1H, (NH); 7.78 d, 1H, J = 8.3 (H-6); 5.81 d, 1H, J = 2.5 (H-1'); 5.62 d, 1H, J = 7.9 (H-5); 5.08 t, 1H, J = 5.4 (OH-5'); 4.88 q, 1H, J = 2.4 (H-2'); 4.73 q, 1H, J = 3.9 (H-3'); 4.06 q, 1H, J = 4.4 (H-4'); 3.57–3.52 m, 2H (H-5'); 1.46 s, 3H (CH3); 1.27 s, 3H (CH3).5'-Azido-N6-benzoyl-2',3'-O-isopropylidene-5'-deoxyadenosine (5). To a stirred solution of compound 1  in anhydrous DMF (30 mL) was added PPh3  and CBr4 . The mixture was stirred under N2 at room temperature for 5 min, then excess NaN3  was added. The reaction mixture was heated under N2 at 90 °C for 24 h. The reaction was quenched by addition of H2O (10 mL). After stirring for 5 min, the mixture was diluted with EtOAc (20 mL), washed with 5% NaHCO3, brine, and the organic layer was collected and dried over sodium sulfate anhydrous. The solvent was removed under reduced pressure and the crude product was purified by silica gel column chromatography (3:1 ratio of EtOAc to hexanes) to afford white solid of compound 5 . HRMS (ESI) m/z calcd. for C20H20N8O4 (M+Na)+ 459.1505, found 459.1490. 1H-NMR (CDCl3): 10.75 br s, 1H (NH); 8.99 s, 1H (H-8); 8.84 s, 1H (H-2); 8.16–7.26 m, 5H (H-Bz); 6.29 d, 1H, J = 11.8 (H-1'); 5.46 m, 1H (H-2'); 5.07 m, 1H (H-3'); 4.41 br s, 1H (OH-5'); 4.13 q, 1H, J = 6.9 (H-4'); 3.60 m, 2H (H-5'); 1.60 s, 3H (CH3); 1.25 s, 3H (CH3).5'-Azido-N2-isobutyryl-2',3'-O-isopropylidene-5'-deoxyguanosine (6). To a stirred solution of compound 2  in anhydrous DMF (30 mL) was added PPh3  and CBr4 . The mixture was stirred under N2 at room temperature for 5 min, then excess NaN3  was added. The reaction mixture was heated under N2 at 90 °C for 24 h. The reaction was quenched by addition of H2O (10 mL). After stirring for 5 min, the mixture was diluted with EtOAc (20 mL), washed with 5% NaHCO3, brine, and the organic layer was collected and dried over sodium sulfate anhydrous. The solvent was removed under reduced pressure and the crude product was purified by silica gel column chromatography (3:1 ratio of EtOAc to hexanes) to afford a pale yellow solid of compound 6 . HRMS (ESI) m/z calcd. for C17H22N8O5 (M+Na)+ 441.1611, found 441.1591. 1H-NMR (CDCl3): 12.02 s, 1H (NH); 7.83 s, 1H (H-8); 5.99 d, 1H, J = 2.9 (H-1'); 5.15 q, 1H, J = 2.9 (H-2'); 4.94 q, 1H, J = 3.9 (H-3'); 4.35 q, 1H, J = 4.4 (H-4'); 3.65–3.51 m, 2H, (H-5'); 2.62-2.58 m, 1H (CH); 1.62 s, 3H (CH3); 1.39 s, 3H (CH3); 1.29 d, 6H, J = 6.8 (C(CH3)2).5'-Azido-N4-benzoyl-2',3'-O-isopropylidene-5'-deoxycytidine (7). To a stirred solution of compound 3  in anhydrous DMF (30 mL) was added PPh3  and CBr4 . The mixture was stirred under N2 at room temperature for 5 min, then excess NaN3  was added. The reaction mixture was heated under N2 at 90 °C for 24 h. The reaction was quenched by addition of H2O (10 mL). After stirring for 5 min, the mixture was diluted with EtOAc (20 mL), washed with 5% NaHCO3, brine, and the organic layer was collected and dried over sodium sulfate anhydrous. The solvent was removed under reduced pressure and the crude product was purified by silica gel column chromatography (3:1 ratio of EtOAc to hexanes) to afford compound 7 . HRMS (ESI) m/z calcd. for C19H20N6O5 (M+Na)+ 435.1393, found 435.1375. 1H-NMR (CDCl3): 7.91 d, 1H, J = 7.3 (H-6); 7.63-7.51 m, 5H (BzH); 7.32 d, 1H, J = 7.2 (H-5); 5.70 d, 1H, J = 1.6 (H-1'); 4.91 dd, 1H, J = 4.4, 3.9 (H-2'); 4.34 m, 1H (H-3'); 4.14 q, 1H, J = 6.8 (H-4'); 3.77–3.63 m, 2H (H-5'); 1.58 s, 3H (CH3); 1.36 s, 3H (CH3).5'-Azido-2',3'-O-isopropylidene-5'-deoxyuridine (8). To a stirred solution of compound 4  in anhydrous DMF (30 mL) was added PPh3  and CBr4 . The mixture was stirred under N2 at room temperature for 5 min, then excess NaN3  was added. The reaction mixture was heated under N2 at 90 °C for 24 h. The reaction was quenched by addition of H2O (10 mL). After stirring for 5 min, the mixture was diluted with EtOAc (20 mL), washed with 5% NaHCO3, brine, and the organic layer was collected and dried over sodium sulfate anhydrous. The solvent was removed under reduced pressure and the crude product was purified by silica gel column chromatography (3:1 ratio of EtOAc to hexanes) to afford a white solid of compound 8 . HRMS (ESI) m/z calcd. for C12H15N5O5 (M+Na)+ 332.0971, found 332.0972. 1H-NMR (CDCl3): 9.30 s 1H, (NH); 7.30 d, 1H, J = 7.9 (H-6); 5.91 d, 1H (H-1'); 5.78 d, 1H, J = 7.8 (H-5); 5.00 d, 1H, J = 6.4 (H-2'); 4.82 t, 1H, J = 3.9 (H-3'); 4.24 q, 1H, J = 4.9 (H-4'); 4.13–3.62 m, 2H (H-5'); 1.56 s, 3H (CH3); 1.35 s, 3H (CH3).1H-NMR and high resolution mass spectrometry. Owing to the unique chemical characteristics of the azide functional group, these molecules have the potential to be used in applications even more diverse than their contemporary use. In particular, these molecules are available for solid-phase synthesis of ribonucleic acids with guanadium linkages.We have confirmed the synthesis of 5'-azido-5'-deoxyribonucleosides of the bases adenine, guanine, cytosine, and uracil by means of


N-(adamantyloxy)formaldimine as a room-temperature-stable compound that exists in solution in monomeric form. This product was used for reactions with α-hydroxyiminoketones leading to a new class of 2-unsubstituted imidazole 3-oxides bearing the adamantyloxy substituent at N(1). Their reactions with 2,2,4,4-tetramethylcyclobutane-1,3-dithione or with acetic acid anhydride occurred analogously to those of 1-alkylimidazole 3-oxides to give imidazol-2-thiones and imidazol-2-ones, respectively. Treatment of 1-(adamantyloxy)imidazole 3-oxides with Raney-Ni afforded the corresponding imidazole derivatives without cleavage of the N(1)–O bond. Finally, the O-alkylation reactions of the new imidazole N-oxides with 1-bromopentane or 1-bromododecane open access to diversely substituted, non-symmetric 1,3-dialkoxyimidazolium salts. Adamantyloxyamine reacts with glyoxal and formaldehyde in the presence of hydrobromic acid yielding symmetric 1,3-di(adamantyloxy)-1H-imidazolium bromide in good yield. Deprotonation of the latter with triethylamine in the presence of elemental sulfur allows the in situ generation of the corresponding imidazol-2-ylidene, which traps elemental sulfur yielding a 1,3-dihydro-2H-imidazole-2-thione as the final product.Adamantyloxyamine reacts with formaldehyde to give  N-oxides constitute a practically valuable class of five-membered aromatic N-heterocycles -cycloadduct A is presented in 11b enters an analogous reaction with the starting imidazole N-oxide 7, leading to a second molecule of 10 and 2,2,4,4-tetramethylcyclobutane-1,3-dione.Sulfur-transfer reactions offer an attractive approach to imidazole-2-thiones starting with 2-unsubstituted imidazole N-oxides . The impN-oxides –23. Howe5% yield ; its strN-oxides is a known procedure for the preparation of alkoxyimidazolium salts +, 247.1810; found, 247.1811.Sulfur-transfer reactions; Preparation of 3H-imidazole-2-thiones 10 – general procedure: A solution of the corresponding imidazole N-oxide 7 (1 mmol) in 3 mL of CH2Cl2 was treated with 103 mg (0.6 mmol) of dithione 11a and the obtained solution was stirred magnetically at rt for 1 h. The solvent was evaporated and the obtained residue was triturated with petroleum ether. The solid obtained thereby was filtered off and dried in the air.1-Adamantyloxy-4,5-dimethyl-1H-imidazole-2(3H)-thione (10a): Yield: 103 mg (37%). Pale yellow crystals; mp 137–139 °C; 1H NMR δ 1.64 ), 2.06, 2.09 , 2.13 ), 2.23 ), 12.0  ppm; 13C NMR δ 9.3, 9.4 (2CH3), 31.5 (3CH(ad)), 35.9, 42.0 (6CH2(ad)), 88.8 (Cq(ad)-O), 117.4, 121.7 (2C=), 157.4 (C=S) ppm; IR υ: 3065 (m), 2907 (vs), 2853 (m), 1493 (vs), 1428 (m), 1399 (m), 1367 (m), 1248 (s), 1046 (vs), 889 (s), 900 (s), 773 (s), 743 (vs), 698 (vs), 577 (m) cm−1; HRMS (ESI+): calcd. for [C15H23N2OS]+, 279.1531; found, 279.1532.Preparation of non-symmetric 1,3-dialkoxyimidazolium bromides 13a–g – general procedure: The corresponding imidazole N-oxide 7 (1 mmol) dissolved in 2–3 mL of chloroform was treated with alkylating reagent 12a  or 12b , which were added drop-wise at rt. The obtained reaction mixtures were stirred magnetically at rt and the progress of the reaction was monitored by TLC . When the starting 7 was completely consumed, the solvent was evaporated and the residue was triturated with diethyl ether. In most cases, solid, colorless or beige colored products were formed. In the case of 13e, a semi-solid product was formed; no crystallization was observed even after several days at rt. Similar properties were displayed by product 13d, which did not crystallize at all and was analyzed as an oily material.1-Adamantyloxy-4,5-dimethyl-3-pentyloxy-1H-imidazolium bromide (13a): Yield: 325 mg (79%). Beige crystals; mp 73 °C (dec.); 1H NMR δ 0.94 ), 1.38–1.40 ), 1.46–1.50 ), 1.69 ), 1.85–1.87 ), 2.02 ), 2.27 , 2.30 , 2.35 ), 4.79 , 11.46 ) ppm; 13C NMR δ 7.3, 8.8, 13.9 (3CH3), 22.4, 27.5, 27.8 (3CH2(p)), 31.2 (3CH(ad)), 35.4, 40.7 (6CH2(ad)), 83.9 (CH2-O), 91.5 (Cq(ad)-O), 120.0, 123.8 (2C=), 131.3 (HC(2)) ppm; IR υ: 2952 (s), 2911 (s), 1628 (m), 1373 (m), 1356 (m), 1039 (s), 958 (s), 877 (s), 593 (s) cm−1; HRMS (ESI+): calcd. for [C20H33N2O2]+, 333.2542; found, 333.2552.Preparation of symmetric imidazolium salt 15: To a solution containing 145 mg of glyoxal (40% aqueous solution), adamantyloxyamine , and paraformaldehyde  in 4.5 mL of glacial acetic acid, hydrobromic acid ) was added in one portion and the mixture obtained thereby was stirred magnetically overnight at rt. Next day, the solution was evaporated to dryness and the residue obtained as a thick oil was purified chromatographically on a short silica gel column using CH2Cl2/MeOH (97:3 mixture) as an eluent. The required product was isolated as a crystalline material. Additional crystallization from diisopropyl ether/CH2Cl2 mixture afforded analytically pure imidazolium salt 15.1,3-Di(adamantyloxy)imidazolium bromide (15): Yield: 190 mg (41%). Colorless crystals; mp ca. 160 °C (dec.); 1H NMR δ 1.59, 1.63 ), 1.95 ), 2.29 ), 7.71 , 11.03 ) ppm; 13C NMR δ 31.0, 35.3, 40.2 (12CH2(ad)), 6CH(ad)), 90.8 (2C(ad)-O), 120.2 (2CH=), 133.7 (HC(2)) ppm; IR υ: 2903 (s), 2847 (m), 1546 (m), 1449 (m), 1358 (m), 1293 (m), 1045 (s), 1001 (s), 961 (s), 881 (s), 726 (s), 542 (vs), 449 (vs) cm−1; anal. calcd for C23H33BrN2O2∙H2O (467.44): C, 59.10; H, 7.55; N, 5.99; found: C, 59.08; H, 7.62; N, 5.79.File 1Experimental and analytical data and copies of NMR spectra.


Acaromyces ingoldii FS121, which was obtained from the China South Sea, yielded a new naphtha-pyrandione analogue, acaromycin A (1) and a new thiazole analogue, acaromyester A (2), as well as the known compound (+)-cryptosporin (3). Their structures, including absolute configurations, were determined by extensive spectroscopic analysis and electronic circular dichroism (ECD) spectra. Compounds 1–3 were evaluated for in vitro growth inhibitory activities against four tumor cell lines , wherein compounds 1 and 3 exhibited considerable growth inhibitory effects, with IC50 values less than 10 µM.Activity-guided isolation of the fermentation broth of the deep-sea derived fungus  Marine organisms live in a biologically competitive environment with unique environmental conditions, therefore marine-derived fungi have been proven to be a rich source of structurally unique and biologically active secondary metabolites ,7,8,9,10Acaromyces ingoldii FS121 showed potent cell growth inhibitory effects against SF-268, MCF-7, NCI-H460 and HepG-2 human tumor cell lines , five sp2 quaternary carbons δC 162.2 (C-7), 155.2 (C-10a), 132.1 (C-9a), 118.8 (C-4a), 114.1 (C-5a), three sp2 methines δC 137.2 (C-6), 124.7 (C-8), 119.3 (C-9), three sp3 methines δC 76.2 (C-2), 66.9 (C-3), 65.1 (C-4), and one methyl δC 16.7 (C-11). As six of nine degrees of unsaturation account for four double bonds (eight sp2 carbons), and two carbonyl groups, the remaining degrees of unsaturation requires that 1 be a tricyclic system. The HMBC correlations (H 11.68) to C-5a, C-7 and C-8. Furthermore, the 1H-1H COSY spectrum -cryptosporin (3), except for the OH at C-9 in 3 was locked at C-7 in 1. Therefore, the planar structure of 1 was confirmed.Acaromycin A , H-3 , H-4 ] with those of 3 . This indicated that these protons were cofacial and designated as β-oriented, whereas the Me-11 was α-oriented in 1. The absolute configuration of 1 was determined by the CD spectrum. The absolute configuration of (+)-cryptosporin (3) was established by synthesis and its CD spectra data was reported in the literature . This assumption was further confirmed by the HMBC correlations from H-2 to C-1, C-3 and C-8, H-5 to C-3 and C-7, and H-8 to C-6 and C-4. Besides, the 1H-NMR of 2 also exhibited a methyl signal at δH 2.24 , two methylene protons at δH 3.02  and 3.84 , and an olefinic proton due to the thiazole ring at δH 8.61 , suggesting the presence of a 4-methyl-5-thiazoleethanol substructure in 2, which has been reported in the literature + 299.0533 .Acaromyester A (2): light pink oil; UV (MeOH) λmax (log ε) 202 (4.24), 225 (4.08), 277 (3.44) nm; IR νmax 3309, 3178, 1732, 1716, 1516, 1236, 1145, 804, 783 cm−1; 1H- and 13C-NMR, see m/z [M + H]+ 278.0845 .1–3 were tested against four tumor cell lines, including MCF-7 (human breast adenocarcinoma cell line), NCI-H460  and SF-268 (human glioma cell line), HepG-2 (human hepatomacarcinoma cell line) by the SRB method pyran-5,10-quinone and a new heterodimeric compound combining a hydroxyphenylacetate and a 4-methyl-5-thiazoleethanol substructure, which were isolated from the fermentation broth of Acaromyces ingoldii FS121. It merits attention that compounds 1 and 3 contained a 3,4-dihydro-2H-naphtho-pyran-5,10-quinone core framework, which exhibited a wide range of biological activities [1–3 were evaluated, wherein compound 1 and 3 showed considerable activities against MCF-7, NCI-H460, SF-268 and HepG-2 tumor cell lines.In summary, we have described herein the isolation and structural elucidation of a new 3,4-dihydro-2


Their structures were established by a combination of spectroscopic data analysis, besides comparison with literature data. Compounds 1–4 were evaluated for their inhibitory activities against 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1).A new ursane-type triterpenoid, 3 Teucrium viscidum Blume (Lamiaceae), known as Shan-Huo-Xiang in China, is an annual herb, whose whole parts are used as a traditional Chinese medicine to treat hemoptysis, hematemesis, pulmonary abscesses, traumatic injuries, and bites of rabies-stricken dogs or venomous snakes + . The IR spectrum showed absorption bands for hydroxyl (3729 cm–1), carbonyl (1685 cm–1) and benzene ring (1602 cm–1) functions. The 1H-NMR spectrum of 1 , 1.05 , 1.08 , 1.23 , and 1.26 , one secondary methyl doublet protons at δH 1.09 , three protons attached to oxygenated carbons at δH 4.50 , 4.27 , 3.48 , three olefinic protons at δH 7.03 , 6.09 , 5.52 , and a p-disubstituted benzene ring at δH 8.09  and 7.22 . The 13C-NMR, DEPT, and HSQC spectra for 1 exhibited thirty nine carbon signals differentiated as six methyls, ten methylenes (including an oxygenated), thirteen methines (including seven olefins and an oxygenated), and ten quaternary carbons .Compound rum of 1  showed f1 were quite similar to those of 3β-hydroxy-urs-12-en-28-oic-acid (3), except for signals for a nine-carbon chain + .1–4 on human 11β-HSD1. Microsomes containing 11β-HSD1 were used according to the reported studies  cortisone. The product, [3H] cortisol, was specifically captured by monoclonal antibody coupling to protein A-coated SPA beads. IC50  values were calculated by using Prism Version 4 .The scintillation proximity assay (SPA) was used to analyze the inhibitory activities of compounds  studies . The fulβ-hydroxy-urs-30-p-Z-hydroxycinnamoyloxy-12-en-28-oic-acid (1), was isolated from the whole parts of T. viscidum together with three known triterpenoids, 3β-hydroxy-urs-30-p-E-hydroxycinnamoyloxy-12-en-28-oic-acid (2), 2α,3β,19α-trihydroxy-urs-12-en-28-oic-acid (3), ursolic acid (4), four known lignans, pinoresinol (5), 9α-hydroxypinoresinol (6), (+)-medioresinol (7), (+)-kobusin (8), and two steroids, β-sitosterol (9), daucosterol (10). Their structures were established by a combination of spectroscopic data analysis, and comparison with literature data. Compounds 2–10 were isolated from T. viscidum for the first time. Compounds 1–4 were tested for 11β-HSD1 inhibition activities in vitro, whereby compound 4 showed stronger inhibitory activity, while compounds 1–3 did not show obvious 11β-HSD1 inhibitory activities.A new ursane-type triterpenoid, 3


The pharmacological screening showed that many of these obtained compounds have good anti-inflammatory activities, comparable to the reference drug. The toxicity of the compounds was also assayed via the determination of their LD50 values. The structures of newly synthesized compounds were confirmed by IR, 1H-NMR, MS spectral data and elemental analysis.A new series of fused triazolo- and tetrazolopyrimidine derivatives  On the H3 group .2 with phenyl isothiocyanate in boiling ethanol yielded compound 11, which was treated with methanolic sodium hydroxide to give the cyclized compound 12. The 1H-NMR spectrum of compound 12 showed a singlet at δ = 10.23 ppm due to the NH group. Finally, compound 2 was reacted with benzaldehyde to give arylidenehydrazine derivative 13, which was cyclized with bromine and sodium carbonate to give triazolopyrimldine 14 .Two pharmacological activities namely; anti-inflammatory and ulcerogenic activities were tested despite their different biological receptors. Eleven representative compounds—3, 6, 9 and 10 possess strong anti-inflammatory activity , in comparison to that of indomethacin (44.7 ± 1.2%). Compounds 7, 8 and 12 possess moderate anti-inflammatory activity , in comparison to that of indomethacin (44.7 ± 1.2%). Compounds 4, 5 and 14 possess weak anti-inflammatory activity from , in comparison to that of indomethacin (44.7 ± 1.2%). The anti-inflammatory assay results (percent inhibition of edema obtained by the reference drug and tested compounds) after 2 h show that compounds 2, 3, 6, 9 and 10 possess strong anti-inflammatory activity , in comparison to that of indomethacin (52.4 ± 1.2%). Compounds 7, 8 and 12 possess moderate anti-inflammatory activity  in comparison to that of indomethacin (52.4 ± 1.2%). Compounds 4, 5 and 14 possess weak anti-inflammatory activity from  in comparison to that of indomethacin (52.4 ± 1.2%). The results of anti-inflammatory after 3 h show that compounds 2, 3, 6, 9 and 10 possess strong anti-inflammatory activity  in comparison to that of indomethacin (61.2 ± 1.3%). Compounds 7, 8 and 12 possess moderate anti-inflammatory activity  in comparison to that of indomethacin (61.2 ± 1.3%). Compounds 4, 5 and 14 possess weak anti-inflammatory activity  in comparison to that of indomethacin (61.2 ± 1.3%)  show that compounds 2,  ± 1.3%) .s2 and 10 showed higher anti-inflammatory activities than the other compounds so they were also screened for their ulcerogenic activity at dose levels of 10, 50 and 100 mg/kg b.m.  were recorded on a FT IR-8201 PC spectrophotometer. The 1H-NMR spectra were measured with a Jeol FTGNM-EX 270, 270 MHz instrument in DMSO-d6 and the chemical shifts were recorded in  relative to TMS. The mass spectra were run at 70 eV with a Finnigan SSQ 7000 spectrometer using EI and the m/z values are indicated in Dalton. TLC  was used to follow the reactions.Melting points were determined on open glass capillaries using an Electrothermal IA 9000 SERIES digital melting point apparatus  and are uncorrected. Elemental analyses were performed with all final compounds on an Elementar Vario EL, located at the Microanalytical Unit, National Research Centre, Cairo, Egypt and were found within 2-(4-Methoxyphenyl)-4-hydrazinocycloocteno[4.5]thieno[2.3-d]pyrimidine (2). A mixture of 1  and hydrazine hydrate  in n-butanol (20 mL) was heated under reflux for 6 h, the formed solid was filtered off and crystallized from ethanol to give 2 as a green powder (76%); mp 235–237 °C; IR : 3336–3420 ; 1H-NMR : 1.28–1.93 , 2.74–3.12 , 3.52 , 6.45 , 7.14–7.56 , 9.12 ; MS: m/z (%): 354  and 216 . Anal. calcd for C19H22N4OS (354.43): C, 64.40; H, 6.21; N, 15.81; Found: C, 64.13; H, 6.02; N, 15.59.5-(4-Methoxyphenyl)-3-cyanomethylcycloocteno[4.5]thieno[2.3-d]triazolo-pyrimidine (3). A mixture of 1  and cyanoacetyl hydrazine  in n-butanol (30 mL) was refluxed for 8 h, the product obtained after cooling was crystallized from pet. ether (b.p. 40–60 °C) to give 3 as a reddish-brown powder (72%); mp 289–291°C; IR : 2219 (CN); 1H-NMR : 1.25–1.84 , 2.36–2.87 , 3.18 , 3.46 , 7.21–7.63 ; MS: m/z (%): 403  and 256 . Anal. calcd. for C22H21N5OS (403.45): C, 65.50; H, 5.21; N, 17.36; Found: C, 65.24; H, 5.01; N, 17.12.1  in ethanol (20 mL). The reaction mixture was refluxed for 5 h then, the solid separated was refluxed with acetic anhydride (15 mL) for 3 h and the product obtained after cooling was crystallized from a proper solvent to give 4 and 5, respectively.Amino acids, namely glycine and anthranilic acid (0.01 mol), respectively, were poured into a solution of 5-(4-Methoxyphenyl)-3-oxo-cycloocteno[4.5]thino-2H-pyrazolo-pyrimidine (4). From ethanol, a brown powder (59%); mp over 300 °C; IR : 1730 (C=O); 1H-NMR : 1.36–1.78 , 2.23–2.75 , 2.91 , 3.18  and 7.12–7.58 ; MS: m/z (%): 379  and 216 . Anal. calcd. for C21H21N3O2S (379.43): C, 66.49; H, 5.54; N, 11.08; Found: C, 66.21; H, 5.19; N, 10.82.8-(4-Methoxyphenyl)-6-oxo-cycloocteno[4.5]thinopyrimidino[4.3-b]quinazoline (5). From methanol, a yellow powder (71%); mp over 250 °C; IR : 1732 (C=O); 1H-NMR : 1.31–1.54 , 1.91–2.14, 2.91  and 7.16–7.64 ; MS: m/z (%): 455 ; 264 . Anal. calcd. for C26H23N4O2S (455.51): C, 70.74; H, 5.21; N, 9.52; Found: C, 70.51; H, 5.01; N, 9.26.5-(4-Methoxyphenyl)-cycloocteno[4.5]thieno[2.3-d]tetrazolo[4.5-e]pyrimidine (6). A solution of 1  and sodium azide  in glacial acetic acid (30 mL) was refluxed for 3 h. The product obtained after cooling was crystallized from benzene to give 6 as a green powder (59%); mp 254–256 °C; IR : 1621–1435 , 1281 (N-N=N), 1136 (tetrazole ring); 1H-NMR : 1.43–1.85 , 2.87–2.92 , 3.15  and 7.26–7.38 ; MS: m/z (%) ; 365  and 216 . Anal. calcd. for C19H19N5OS(365.41): C, 62.46; H, 5.20; N, 19.17; Found: C, 62.14; H, 5.04; N, 19.03.6-(4-Methoxyphenyl)-3,4-dioxo-2H-cycloocteno[4.5]thienotriazino-pyrimidine (7). A solution of 2  and diethyl oxalate  in ethanol (30 mL) was refluxed for 8 h.; The solid obtained after cooling was filtered and crystallized from ethanol to give 7 as a reddish brown solid (64%); mp over 300 °C; IR : 3315 (NH), 1730 , 1728 (C=O amide of C-4 triazine); 1H-NMR spectrum : 1.32–1.91 , 2.67–2.82 , 3.25 , 7.24–7.38  and 10.17 ; MS: m/z (%); 408  and 337 . Anal. calcd. for C21H20N4O3S (408.43): C, 61.76; H, 4.90; N, 13.72; Found: C, 61.32; H, 4.68; N, 13.43.6-(4-Methoxyphenyl)-3-oxo-2,4-dihydrocyclooctenothienotriazino-pyrimidine (8). A mixture of 2  and chloroacetyl chloride  in pyridine (20 mL) was refluxed for 10 h, then, poured on dil. HCl. The solid product formed was crystallized from ethanol to give 8 as a brown powder (73%); mp 284–286 °C; IR : 3315 (NH), 1725 (C=O); 1H-NMR : 1.15–1.82 , 1.98–2.65 , 3.52 , 3.81 , 7.21–7.59  and 9.01 ; MS: m/z (%): 394  and 230 . Anal. calcd. for C21H22N4O2S (394.45): C, 63.95; H, 5.58; N, 14.21; Found: C, 63.63; H, 5.32; N, 14.03.5-(4-Methoxy)phenyl-3-oxo-2H-cyclooctenothienotriazolopyrimidine (9). A mixture of 2  and ethyl chloroformate  in pyridine (20 mL) was refluxed for 12 h then, poured on dil. HCl. The solid obtained was crystallized from methanol to give 9 as a green powder (54%); mp > 300 °C; IR : 3320 (NH), 1731 (C=O); 1H-NMR : 1.32–1.72 , 2.19–2.41 , 2.84 , 6.24 , 7.08–7.31 ,; MS : m/z (%) ; 380  and 216 . Anal. calcd. for C20H20N4O2S (380.42): C, 63.15; H, 5.26; N, 14.73; Found: C, 62.89; H, 5.04; N, 14.49.5-(4-Methoxyphenyl)-3-methyl-2H-cyclooctenothienotriazolo- pyrimidine (10) A solution of 2  and acetic anhydride (20 mL) was heated under reflux for 2 h. The solid obtained after cooling was crystallized from pet. ether to give 10 as a yellow powder (54%); mp 272–274 °C; IR : 1418–1432 ; 1H-NMR : 1.17–1.68 ,1.93–2.81, 2.16 , 2.21 and 7.22–7.38 ; MS: m/z (%); 379  and 231 . Anal. calcd. for C21 H22N4OS (379.49): C, 66.67; H, 5.82; N, 14.81; Found: C, 66.39; H, 5.57; N, 14.53.N-thienopyrimidino-4-yl]-N-phenylthio-semicarbazide (11). A mixture of 2  and phenyl isothiocyanate (2.50 mL) in ethanol (30 mL) was refluxed for 5 h, the product formed after cooling was collected and crystallized from methanol to give 11 as a reddish-brown solid (68%); mp 189–191 °C; IR : 3,342–3,415 (3 NH), 1,310 (C=S); 1H-NMR : 1.26–1.91 , 2.05–3.18 , 3.42 , 7.21–7.46 , 10.22 , 10.54  and 11.15 ; MS: m/z (%); 489  and 216 . Anal. calcd. for C26 H27N5OS2 (489.59); C, 63.80; H, 5.52; N, 14.31; Found: C, 63.61; H, 5.25; N, 14.19.5-(4-Methoxyphenyl)-3-anilinocyclooctenothienotriazolopyrimidine (12) A mixture of 11  and sod.hydroxide  in methanol (20 mL) was refluxed for 4 h. The product formed after cooling was collected and crystallized from acetic acid to give 12 as white powder (69%); mp 257–259 °C; IR : 3316–3350 ; 1H-NMR : 1.32–1.74 , 1.89–2.61 , 2.95 , 7.31–7.75  and 10.23 ; MS: m/z (%); 455  and 228 . Anal. calcd for C26H25N5OS (455.52): C, 68.57; H, 5.49; N, 15.38; Found: C, 68.21; H, 5.16; N, 15.14.2-(4-Methoxyphenyl)-4-(N-benzylidenehydrazino)cyclooctenothieno2,3-d]pyrimidine (13) A mixture of 2  and benzaldehyde (2.50 mL) in ethanol (20 mL) was heated under reflux for 2 h then cooled, The separated solid was filtered, dried and crystallized from pet. ether (b.p. 40–60 °C) to afford 13 as a green powder (48%); mp 174–176 °C; IR : 3350 (NH); 1H-NMR : 1.25–1.82 , 3.06–3.14 , 3.32  ,3.65 , 7.21–7.78  and 10.42 ; MS: m/z (%); 442  and 323 . Anal. calcd. for C26H26N4OS (442.51): C, 70.58; H, 5.88; N, 12.67; Found: C, 70.31; H,5.64; N, 12.39.5-(4-Methoxyphenyl)-4-phenylcyclooctenothienotriazolopyrimidine (14) To a stirred mixture of 13  and anhydrous sodium bicarbonate  in chloroform (20 mL) bromine (1.50 mL) was added. The mixture was stirred at room temperature for 5 h, then left to stand overnight, and evaporated under vacuum. The residue was triturated with ice-cold water, and the product formed was filtered, washed with water, dried and crystallized from ethanol to give 14 as a reddish brown solid (63%); mp over 300 °C; IR : 1620‒1435 ; 1H-NMR : 1.19–1.72 , 2.94–3.22 , 3.41  and 7.25–7.64 ; MS: m/z (%); 440  and 256 . Anal. calcd. for C26H24N4OS (440.50); C, 70.91; H, 5.45; N, 12.72; Found: C, 70.65; H, 5.21; N, 12.42.Female albino mice (16–18 g) and Sprague Dawley mice (100 g) obtained from the Theodor Bilharz Research Institute  were used. Approval of the institutional animal ethical committee for the animal studies was obtained from the Office of Environmental Health and Radiation Safety, ACUC Protocol 1096-5. The animals were maintained according to accepted standards of animal care.Newly synthesized thienopyrimidine derivatives were dissolved in 0.5% carboxymethyl cellulose (CMC) as a homogeneous solution and administered intraperitonneally (i.p.). One hundred and eight rats were divided into eighteen groups, each group consisting of six animals. Anti-inflammatory activity of the compounds was studied in mice using carrageenan induced edema. A suspension of the tested compound and the reference drug, indomethacin in aqueous solution was administered orally at a dose 5 mg/kg. Control animals were treated with 0.5% CMC only. After 30 min, 0.1 mL of freshly prepared 1.0% carrageenan solution  was injected into the sub-plantar region of the right hind paw according to Hernandez-Perez . The rigSeventy-two mice were divided into twelve groups. Ulcerogenic activity was evaluated after oral administration of the tested compounds or indomethacin at doses of 10, 50, and 100 mg/kg. Control mice received 0.5% CMC. Food but not water was removed 24 h before administration of the tested compounds. After 6 h, the mice were sacrificed; the stomach was removed and opened along the greater curvature, washed with distilled water and cleaned gently by dipping in saline. The mucosa damage for each stomach was examined using a stereoscopic microscope and compared with the reference drug indomethacin according to the reported procedure .50) of the most active compounds 2, 3, 6, 9 and 10 were determined in mice [50 was processed by a graphical method.The median lethal doses . One-way analysis of variance (ANOVA) followed by Tukey’s post test was used to assess the presence of significant differences. Differences were considered statistically significant at 1 was used to synthesize the following compounds: hydrazine thinopyrimidine 2, triazolothinopyrimidine 3, benzopyrimidinothieno pyrimidine 5 and tetrazolothienopyrimidine 6. Compound 2 was used to synthesize the triazino- and triazolothinopyrimidine derivatives 7, 8, 9 and 10, respectively. Also, compound 2 was carried out to synthesize of triazolothinopyrimidine derivatives 12 and 14. The newly synthesized compounds 2–10, 12 and 14 were screened for their anti-inflammatory activity compared to indomethacin which was used as reference drug. Compounds 3, 6, 9 and 10 possess strong anti-inflammatory activity, and compounds 8, 7 and 12 possess moderate anti-inflammatory activity, while compounds 4, 5 and 14 possess weak anti-inflammatory activity.The objective of the present study was to synthesize and investigate the anti-inflammatory activities of some new thienopyrimidine derivatives. The starting material


Application of this powerful chemical tool at the last stage of the synthesis of kinase inhibitors allowed the synthesis of arrays of molecules inspired by fragment-growing studies generated by molecular modeling calculations. Among the potentially active compounds designed through this strategy, FC162 (4c) exhibits nanomolar IC50 values against some kinases, and is the best candidate for the development as a DYRK kinase inhibitor.Efficient metal catalyzed C–H arylation of 8-alkyl-thiazolo[5,4- CDKs), mitogen-activated protein kinases (MAP kinases), glycogen synthase kinases (GSK), and Ccd2-like kinases (CLKs) quinazolin-4-amines has been demonstrated, aiming at the improved treatment of Down syndrome (DS), early Alzheimer’s disease (AD), and cancers quinazolin-9(8H)-one analogues slightly reduced the activity, but the key effects of the carbimidate functional group in C2 were highlighted.At the same time, novel thiazoloquinazolin-9(8H)-ones, a fragment-growing approach was performed using a novel in silico tool that drills down through, to evaluate hundreds of thousands fragments extracted from co-crystallized kinase/inhibitor complexes. This innovative and appealing tool quinazolin-9(8H)-ones was envisioned by addition of (hetero)-aromatic fragments -ones (series 4) which were evaluated as potential kinase inhibitors. According to our recent experience in carbon–carbon bond formation quinazolin-9(8H)-ones are substituted at N8 position by an aliphatic chain and modified in C2 (N8-benzylated-thiazolo-quinazolin-9(8H)-one (1) was chosen as model substrate.The target thiazoloquinazoline-2-carbonitrile, which was decyanated (CN group hydrolysis and decarboxylation) by heating in HBr. This route has the advantage to be scalable, allowing an easy synthesis of the key intermediate 1 at a multigram scale  Cu-am scale .f]quinazolin-9(8H)-one 1 with aryl halides were previously determined [f]quinazolin-9(8H)-one 1 was heated in a sealed tube at 120 °C for 10 min in the presence of copper iodide  and 1,8-diazabicyclo[5.4.0]undec-7-ene  as a base, in dry DMF. After addition of Pd(OAc)2 (10 mol%) and the appropriate aryl halide (2.0 equiv), the reaction mixture was heated again at 120 °C for 5 h  was explained by difficult work-up and purification. A better yield of 43% was obtained by using 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) quinazolin-9(8H)-ones tested, only two molecules of series 3 (3a and 3b) exhibited micromolar IC50 values against kinases CLK1 and GSK3, and nanomolar range inhibition against DYRK1A -ones. The envisioned modifications depicted in Taking these preliminary results into account, series 4) were 2-(pyridin-3-yl)thiazoloquinazolin-9(8H)-ones, substituted in position N8 by an aliphatic fragment  were synthesized via the polyfunctionalized methyl 6-amino-2-cyanobenzo[d]thiazole-7-carboxylate (5), a versatile precursor already described in previous studies amino)benzo[d]-thiazole-7-carboxylate (6) in excellent yield (90%). This formimidamide was heated at 100 °C under microwave irradiation in the presence of 1.05 equivalents of appropriate amines, in acetic acid. After an irradiation time of 15 min, the corresponding N8-substituted-9-oxo-8,9-dihydrothiazoloquinazoline-2-carbonitriles (series 7a–f) were obtained in moderate to good yields (43–86%) quinazolin-9(8H)-one with alkyl groups, such as methyl, iso-propyl, or cycloakyl containing at least 4 carbons, resulted in complete loss of activity -one skeleton thus provides an efficient and simplified route towards these valuable sulfur-containing bioactive heterocycles.Microwave-assisted regioselective C–H bond arylation of the thiazoloquinazolin-9(8H)-one (4c)  exhibited nanomolar IC50 values .In series 3a–c, two molecules exhibited nanomolar to submicromolar IC50 values against DYRK1A . It is remarkable that these two derivatives were poor inhibitors of CLK1 and GSK3 compared with 4c, suggesting a high selectivity for DYRK1A. Except for kinase GSK3, the inhibition profile of 4c for CLK1 and DYRK1A was close to that of Harmine.In series FC162 with DYRK1A. Two main binding modes (f]quinazolin-9(8H)-one skeleton, in which an interaction with the catalytic lysine is retrieved. The pyridine moiety interacts with the hinge region by forming a hydrogen bond with the backbone NH of Leu231. The second possible binding mode showed a complete flip of the molecule in the cavity. Indeed, the pyridine moiety interacted with the catalytic lysine, while the thiazoloquinazolin-9(8H)-one skeleton interacted with the hinge region through hydrogen bond. The docking score of the two poses was quite similar, thus, both binding modes are equally possible for this compound.Docking calculations were next performed in order to predict the molecular interactions of ng modes  were obt3a and 4c with a 3-pyridinyl group in position 2 had a higher activity than the series of phenylated derivatives 2 and 9. These results are notably in agreement with the fragment-growing experiments, which suggested replacement of the imidate group by a more stable heteroaromatic substituent  were acquired at 295 K using an AVANCE 300 MHz spectrometer  at 300 and 75.4 MHz, using TMS as an internal standard. Coupling constants J are in Hz, and chemical shifts are given in ppm. Signals in 13C spectra were assigned based on the result of 13CDEPT135 experiments (see v/v); starting condition is 90% A and 10% B, in which the solvent B changed to 10% to 90% in 4% by min. Flow rate was 0.5 mL/min, and 5 µL were injected. The percentage of purity of all products was more than 98%.All reagents were purchased from commercial suppliers and were used without further purification, except for DMF, which was stored under argon and activated molecular sieves. All reactions were monitored by thin-layer chromatography with silica gel 60 F254  precoated aluminium plates (0.25 mm). Visualization was performed with a UV light at wavelengths of 254 nm. Purifications were conducted with a flash column chromatography system (Puriflash) equipped with a dual UV/vis spectrophotometer (200–600 nm), a fraction collector (176 tubes), a dual piston pump , which allowed quaternary gradients, and an additional inlet for air purge . Melting points of solid compounds were measured with a SMP3 Melting Point instrument  with a precision of 1.5 °C. IR spectra were recorded with a Spectrum 100 Series FTIR spectrometer . Liquids and solids were investigated with a single-reflection attenuated total reflectance (ATR) accessory; the absorption bands are given in cments see . Mass sp1 and 7a–f were described in ref quinazolin-9(8H)-one (8a): Reaction time: 55 min; Yield: 98%; 112 mg; white solid; Rf: 0.47 ; mp 221–224 °C; IR (neat) νmax 3308, 3149, 3077, 1902, 1698, 1664, 1589, 1386 cm−1; 1H-NMR : Hδ 9.22 , 8.48 , 8.20 , 7.87 , 3.73 ; 13C{1H}-NMR : Cδ 160.2 (C), 157.8 (CH), 152.5 (C), 147.1 (C), 146.5 (CH), 130.2 (C), 129.3 (CH), 126.1 (CH), 116.3 (C), 34.3 (CH3). HRMS (ESI+): Calcd for C10H7N3OS [M + H]+: 218.0388; Found: 218.03386.8-Isopropylthiazoloquinazolin-9(8H)-one (8b): Reaction time: 45 min; Yield: 99%; 145 mg; beige solid; Rf: 0.69 ; mp 237–240 °C; IR (neat) νmax 3053, 2981, 2920, 1897, 1651, 1583, 1445, 1350, 1265, 1172, 827 cm−1; 1H-NMR : δH 9.19 , 8.44 , 8.25 , 7.84 , 5.37–5.12 , 1.55 , 1.53 ; 13C{1H}-NMR : δC 159.6 (C), 157.7 (CH), 152.5 (C), 146.6 (C), 143.4 (CH), 130.6 (C), 129.4 (CH), 126.0 (CH), 116.4 (C), 47.0 (CH), 22.2(2 × CH3). HRMS (ESI+): Calcd for C12H11N3OS [M + H]+: 246.0701; Found: 246.0708.8-Cyclopropylthiazoloquinazolin-9(8H)-one (8c), was described in ref quinazolin-9(8H)-one (8d): Reaction time: 45 min; Yield: 97%; 148 mg; white solid; Rf: 0.76 ; mp 242–245 °C; IR (neat) νmax 3045, 2992, 2957, 2879, 1655, 1601, 1585, 1349, 1277, 1163, 856 cm−1; 1H-NMR : δH 9.20 , 8.46 , 8.31 , 7.86 , 5.21–5.06 , 2.71–2.55 , 2.54–2.35 , 2.05–1.90 ; 13C-NMR : δC 159.0 (C), 157.8 (CH), 152.6 (C), 146.8 (C), 143.8 (CH), 130.5 (C), 129.5 (CH), 126.1 (CH), 116.3 (C), 50.9 (CH), 29.9 (2 × CH2), 15.5 (CH2). HRMS (ESI+): Calcd for C13H11N3OS [M + H]+: 258.0701; Found: 258.0701.8-Cyclopentylthiazoloquinazolin-9(8H)-one (8e): Reaction time: 45 min; Yield: 98%; 160 mg; white solid; Rf: 0.69 ; mp 214–217 °C; IR (neat) νmax 2960, 2874, 1661, 1585, 1348, 1259, 1154, 841, 818 cm−1; 1H-NMR : δH 9.22 , 8.49 , 8.27 , 7.87 , 5.36–5.20 , 2.38–2.21 , 2.12–1.74 ; 13C{1H}-NMR : δC 159.8 (C), 157.7 (CH), 152.3 (C), 146.4 (C), 144.1 (CH), 130.4 (C), 129.2 (CH), 125.8 (CH), 116.1 (C), 56.4 (CH), 32.1 (2 × CH2), 24.5 (2 × CH2). HRMS (ESI+): Calcd for C14H13N3OS [M + H]+: 272.0858; Found: 272.0855.8-Cyclohexylthiazoloquinazolin-9(8H)-one (8f): Reaction time: 90 min; Yield: 98%; 168 mg; white solid; Rf: 0.75 ; mp 227–230 °C; IR (neat) νmax 3082, 3053, 2932, 2850, 1895, 1665, 1588, 1466, 1448, 1273, 1135, 845, 814 cm−1; 1H-NMR : δH 9.19 , 8.44 , 8.25 , 7.83 , 5.03–4.71 , 2.20–1.87 , 1.84–1.62 , 1.62–1.41 , 1.36–1.17 ; 13C{1H}-NMR : δC 159.6 (C), 157.7 (CH), 152.5 (C), 146.5 (C), 143.7 (CH), 129.4 (CH), 126.0 (CH), 116.4 (C), 54.3 (CH), 32.7 (2 × CH2), 26.0 (2 × CH2), 25.3 (CH2). HRMS (ESI+): Calcd for C15H15N3OS [M + H]+: 286.114; Found: 286.1021.f]quinazolin-9(8H)-one 8a–f (0.341 mmol), copper iodide , and TBD  in dry DMF (850 µL) were added to a 2 mL glass vial, which was sealed under argon atmosphere. The mixture was stirred under microwave irradiation at 120 °C for 10 min. Then, Pd(OAc)2  and 3-iodopyridine  were added to the mixture and purged with argon. The reaction was then stirred under microwave irradiation at 120 °C for 5 h. The resulting solution was diluted with dichloromethane, and washed three times with a 5% aqueous ammonia solution, then with water and brine. The organic layer was dried over Na2SO4 and concentrated under vacuum. The crude product was purified by flash chromatography on silica gel with MeOH/CH2Cl2 as eluent , to afford the corresponding product.Thiazoloquinazolin-9(8H)-one (4a): Yield: 14%; 14 mg; Yellow solid; Rf: 0.36 ; mp 272–275 °C; IR (neat) νmax 3068, 1651, 1588, 1446, 1346, 699 cm−1; 1H-NMR : Hδ 9.39 , 8.73 , 8.58–8.36 , 8.19 , 7.86 , 7.47 , 3.73 ; 13C{1H}-NMR : Cδ 168.36 (C), 160.40 (C), 153.29 (C), 151.82 (CH), 148.66 (CH), 147.20 (C), 146.60 (CH), 134.71 (CH), 131.54 (C), 129.77 (C), 129.25 (CH), 126.60 (CH), 124.06 (CH), 116.48 (C), 34.37 (CH3). HRMS (ESI+): Calcd for C15H10N4OS [M + H]+: 295.0654; Found: 295.0668.8-Isopropyl-2-(pyridin-3-yl)thiazoloquinazolin-9(8H)-one (4b): Yield: 64%; 70 mg; Yellow solid; Rf: 0.46 ; mp 202–205 °C; IR (neat) νmax 3540, (C), 151.82 (CH), 148.66 (CH), 147.20 (C), 146.60 (CH), 134.71 (CH), 131.54 (C), 129.77 (C), 129.25 (CH), 126.60 (CH), 124.06 (CH), 116.48 (C), 34.37 (CH3). HRMS (ESI+): Calcd for C15H10N4OS [M + H]+: 295.0654; Found: 295.0668.8-Cyclopropyl-2-(pyridin-3-yl)thiazoloquinazolin-9(8H)-one (4corFC162) +: 321.0807; Found: 321.0810.rFC162) : yield: 8-Cyclobutyl-2-(pyridin-3-yl)thiazoloquinazolin-9(8H)-one (4d): Yield: 57%; 64 mg; Yellow solid; Rf: 0.45 ; mp 251–254 °C; IR (neat) νmax 3072, 2992, 2948, 2865, 1906, 1655, 1584, 1347, 848 cm−1; 1H-NMR : Hδ 9.39 , 8.72 , 8.45–8.29 , 8.30 , 7.86 , 7.46 , 5.18–5.09 , 2.70–2.60 , 2.54–2.40 , 2.04–1.94 ; 13C{1H}-NMR : Cδ 168.2 (C), 160.0 (C), 153.2 (C), 151.8 (CH), 148.6 (CH), 146.7 (C), 143.8 (CH), 134.7 (CH), 131.6 (C), 129.8 (C), 129.2 (CH), 126.5 (CH), 124.1 (CH), 116.3 (C), 51.1 (CH), 29.8 (2 × CH2), 15.5 (CH2). HRMS (ESI+): Calcd for C18H14N4OS [M + H]+: 335.0967; Found: 335.0974.8-Cyclopentyl-2-(pyridin-3-yl)thiazoloquinazolin-9(8H)-one (4e): Yield: 65%; 77 mg; Yellow solid; Rf: 0.43 ; mp 212–215 °C; IR (neat) νmax 3064, 3964, 2872; 1902, 1645, 1584, 1451, 828 cm−1; 1H-NMR : Hδ 9.40 , 8.73 , 8.50–8.38 , 8.25 , 7.86 , 7.46 , 5.39–5.08 , 2.46–2.16 , 2.14–1.67 ; 13C{1H}-NMR : Cδ 168.0 (C), 159.8 (C), 152.9 (C), 151.5 (CH), 148.4 (CH), 146.3, 144.1 (CH), 134.4 (CH), 131.5 (C), 129.8 (C), 128.9 (CH), 126.2 (CH), 124.1 (CH), 116.2 (C), 56.8 (CH), 32.1 (2 × CH2), 24.6 (2 × CH2). HRMS (ESI+): Calcd for C19H16N4OS [M + H]+: 349.1123; Found: 349.1115.8-Cyclohexyl-2-(pyridin-3-yl)thiazoloquinazolin-9(8H)-one (4f): Yield: 54%; 67 mg; Yellow solid; Rf: 0.43 ; mp 201–204 °C; IR (neat) νmax 3309, 3146, 1076, 2920, 1902, 1661, 1587, 1350, 1333, 826 cm−1; 1H-NMR : Hδ 9.38 , 8.72 , 8.56–8.35 , 8.25 , 7.84 , 7.45 , 4.96–4.79 , 2.16–1.89 , 1.86–1.65 , 1.65–1.43 , 1.38–1.16 ; 13C{1H}-NMR : Cδ 168.1 (C), 159.6 (C), 153.0 (C), 151.6 (CH), 148.5 (CH), 146.3 (C), 143.7 (CH), 134.5 (CH), 131.7 (C), 129.8 (C), 129.1 (CH), 126.3 (CH), 124.0 (CH), 116.3 (C), 54.5 (CH), 32.6 (2 × CH2), 25.9 (2 × CH2), 25.2 (CH2). HRMS (ESI+): Calcd for C20H18N4OS [M + H]+: 363.1280; Found: 363.1295.2; 60 mM β-glycerophosphate; 15 mM p-nitrophenylphosphate; 2 mM dithiothreitol (DTT); 1 mM Na3VO4; 1 mM NaF; 1 mM di-sodium phenylphosphate; 1× protease inhibitor cocktail; 0.2% Nonidet P-40 substitute.Homogenization buffer: 25 mM MOPS; 15 mM EGTA; 15 mM MgCl2; 1 mM EGTA; 1 mM DTT; 25 mM Tris/HCl, and 50 μg/mL heparin.Buffer A: 10 mM MgClp-nitrophenylphosphate; 25 mM MOPS pH 7.0; 5 mM EGTA; 15 mM MgCl2; 1 mM DTT; and 0.1 mM sodium vanadate.Buffer B: 60 mM β-glycerophosphate; 30 mM All chemicals were purchased from Sigma-Aldrich , unless otherwise stated, and the protease inhibitor cocktail was from Roche .Kinase activities were assayed in triplicates, in buffer A or B, at 30 °C, at a final adenosine triphosphate (ATP) concentration of 15 μmol/L. Blank values were subtracted, and activities were expressed in percent (%) of the maximal activity, i.e., in the absence of inhibitors. Controls were performed with appropriate dilutions of dimethyl sulfoxide (DMSO).CDK5/p25  was prepared as previously described [33P] ATP  in a final volume of 30 μL. After 30 min incubation at 30 °C, 25 μL aliquots of supernatant were spotted onto sheets of Whatman P81 phosphocellulose paper, and 20 s later, the filters were washed eight times (for at least 5 min each time) in a solution of 10 mL phosphoric acid/L of water. The wet filters were counted in the presence of 1 mL ACS (Amersham) scintillation fluid.escribed . Its kinGSK-3α/β  was assayed, in buffer A, with 0.5 mg BSA/mL + 1 mM DTT, using GS-1 (YRRAAVPPSPSLSRHSSPHQSpEDEEE) (pS stands for phosphorylated serine), a GSK-3 specific substrate [33P] ATP  in a final volume of 30 μL. After 30 min incubation at 30 °C, the reaction was stopped by harvesting onto P81 phosphocellulose supernatant  using a FilterMate harvester , and were washed in 1% phosphoric acid. Scintillation fluid was added, and the radioactivity measured in a Packard counter.ubstrate , in the CLK1  was assayed in buffer A (+0.15 mg BSA/mL) with RS peptide (GRSRSRSRSRSR) (1 μg/assay).CK1δ/ε  was assayed as described for CDK1, but in buffer B, and using 25 μM CKS peptide (RRKHAAIGpSAYSITA), a CK1-specific substrate [ubstrate .DYRK1A  was purified by affinity chromatography on glutathione-agarose and assayed as described for CDK1/cyclin B, with with 0.5 mg BSA/mL + 1 mM DTT and using Woodtide (KKISGRLSPIMTEQ) (1.5 μg/assay) as a substrate, a residue of transcription factor FKHR.FC162 (4c) was found to be the best candidate for development as a DYRK inhibitor.This work demonstrates the efficacy of synthetic methodologies, such as C–H arylation of arenes and hetero-arenes for SAR studies. The application of this powerful tool at the last stage of the synthesis of kinase inhibitors allowed the synthesis of arrays of molecules inspired by fragment-growing studies generated by molecular modeling calculations. Among the potential active compounds generated through this strategy,


Docking studies revealed that although the pharmacophoric substitute of the compound 5b, methylsulfonyl group, has been directly attached to the central ring, it is in the same direction of the sulfonamide group of Celecoxib, a known selective cyclooxygenase-2 inhibitor. Therefore effective hydrogen binding with Arg513 is established. Also, additional hydrogen binding could form between NH of anilino moiety of the 5b and Arg120. All of the compounds had selective inhibitory activity against cyclooxygenase-2 in micromolar concentrations comparable with the reference, Celecoxibe. Finally, compound 5b with the selectivity index 115 and IC The twoe market , 22. Thue market . The maje market -26. Accoyl rings -29. In cyl rings -37, in tyl rings . The docking study and biological evaluation were performed to clear the orientation of the synthesized compounds in the COX-2 active site and inhibitory activity of all compounds respectively.Molecular Modeling StudiesDocking simulation was performed to predict interaction of compounds (5a–f) with COX-2 binding site. The high resolution crystal structure of COX with Celecoxib as a cognate ligand was retrieved from RCSB Protein Data Bank (PDB code: 6COX). The structures of the compounds were investigated using the Lamarckian genetic algorithm search method implemented in AutoDock 4.0 software. The receptor was kept rigid and ligands were allowed to be flexible. Polar hydrogens and Kollman united atom partial charges were added to the individual protein atoms. The HyperChem 8 software was used for energy minimization of each structure under MM+ method and AutoDockTools 4.0 version 1.5.6rc3 for conversion of file formats to pdbqt. A docking grid box was built with 40, 40, and 40 points in 24.4370, 22.8660, and 48.5210 directions and the number of generations and maximum number of energy evaluations was set to 100 and 2,700,000, respectively. The docking results were clustered with a root mean square deviation (RMSD) of 0.5 Å and evaluated by Pymol software -40.Chemistry 1HNMR spectra; DMSO-d6 was used as solvents. LC Mass spectra and elemental analysis were achieved by HPLC Agilent system and Costech  elemental analyzer respectively.All the chemicals and solvents were purchased from Merck or Aldrich Company and were used without further purification. Thin layer chromatography (TLC) was performed on commercially available Merck precoated plates . Melting points were obtained using the Electrothermal 9100 apparatus and are uncorrected. Infrared spectra were acquired on a Perkin Elmer 843 spectrometer. A Bruker FT-400 MHz instrument  was applied to obtain (1-benzyl-2-mercapto-1H-imidazol-5-yl)methanol (1)1. mp: 127 °C; IR: (KBr) ν (cm-1) 2739-3200 (O-H). 1HNMR  δppm: 4.14 , 5.29 , 5.32 , 6.87 , 7.22 , 7.26 , 7.32 , 12.04 . LC-MS [M+1]+: m/z 221, [M+23]+: m/z 243, [M+39]+: m/z 259. Anal. Calcd for C11H12N2OS: C, 59.97; H, 5.49; N, 12.72. Found: C, 59.85; H, 5.51; N, 12.75. Potassium thiocyanate  , dihydroxyacetone  and benzylamine  were refluxed at 55 °C for 18 h in 136 mL of water and glacial acetic acid (7/93). The mixture was alkalinized with NaOH 10% and extracted with chloroform. The resulting precipitates were formed after acidifying of the aqueous phase with hydrochloric acid and recrystallized from ethanol 96% to give 6.66 g (54.1%) of (1-benzyl-2-(methylthio)-1H-imidazol-5-yl)methanol (2)1 and 1.1 mL (5 mmol) sodium iodide in presence of 10 mL of NaOH 10% in ethanol was stirred at room temperature for one hour. The solvent was evaporated and the remnant was acidified and extracted with chloroform. The Aqueous phase was alkalinized with NaOH 10% to participate 0.81 g (69%) of 2. mp: 106 °C; IR: (KBr) ν (cm-1) 2850-3200 (O-H). 1HNMR  δppm: 2.44 , 4.33 , 5.21 , 6.93 , 7.07 , 7.26 , 7.34 . LC-MS [M+1]+: m/z 235, [M+23]+: m/z 257. Anal. Calcd for C12H14N2OS: C, 61.51; H, 6.02; N, 11.96. Found: C, 61.75; H, 5.96; N, 11.93.A solution of 1.1 g (5 mmol) intermediate (1-benzyl-2-(methylsulfonyl)-1H-imidazol-5-yl)methanol (3)2  in THF was added to a solution of Oxon  in water. The mixture was stirred at room temperature overnight and after evaporating THF, 0.62 g (54.1%) of compound 3 was participated. mp: 127 °C; IR: (KBr) ν (cm-1) 2848-3538 (O-H), 1144, 1327 (SO2). 1HNMR  δppm: 3.28 , 4.35 , 5.47 , 5.62 , 7.10 , 7.30 , 7.36 . LC-MS [M+1]+: m/z 267, [M+23]+: m/z 289. Anal. Calcd for C12H14N2O3S: C, 54.12; H, 5.30; N, 10.52. Found: C, 53.96; H, 5.34; N, 10.60.Compound 1-benzyl-5-(chloromethyl)-2-(methylsulfonyl)-1H-imidazole(4)3  was refluxed in 2 mL (16.8 mmol) of thionyl chloride for 4 h. After evaporating thionyl chloride, 0.21 g of compound 4 (57%) was obtained. mp: 127 °C; IR: (KBr) ν (cm-1) 1154, 1347 (SO2). 1HNMR  δppm: 3.33 , 4.78 , 5.66 , 7.10 , 7.31 , 7.34 , 7.40 . LC-MS [M+1]+: m/z 285, [M+23]+: m/z 307. Anal. Calcd for C12H13ClN2O2S: C, 50.61; H, 4.60; N, 9.84. Found: C, 50.74; H, 4.56; N, 9.79.Compound General procedure for the synthesis of the compounds 5a-5e4 (1.5 mmol) and proper amine (1.5 mmol) in 5 mL ACN, in presence of catalytic amount of potassium iodide and potassium carbonate was reflux overnight. The solvent was evaporated and the residue was purified with column chromatography to give final products 5a-5e.A solution of compound N-((1-benzyl-2-(methylsulfonyl)-1H-imidazol-5-yl)methyl)benzamine (5a)-1): 3317 (N-H), 1127, 1308 (SO2). 1HNMR  δppm: 3.32 , 4.11 , 5.66 , 6.10 , 6.50 , 6.56 , 7.04 , 7.11 , 7.22 , 7.40 . LC-MS [M+1]+: m/z 342, [M+23]+: m/z 364. Anal. Calcd for C18H19N3O2S: C, 63.32; H, 5.61; N, 12.31. Found: C, 64.00; H, 5.56; N, 12.25.Yield: 65%, mp: 114-115°C; IR: (KBr) ν (cmN-((1-benzyl-2-(methylsulfonyl)-1H-imidazol-5-yl)methyl)-4-methoxybenzamine (5b) -1): 3250 (N-H), 1150, 1360 (SO2), 1HNMR  δppm: 3.27 , 3.76 , 4.10 , 5.70 , 6.50 , 6.78 , 7.12 , 7.14 , 7.36 . LC-MS [M+1]+: m/z 372, [M+23]+: m/z 410. Anal. Calcd for C19H21N3O3S: C, 61.44; H, 5.70; N, 11.31. Found: C, 61.52; H, 5.72; N, 11.23.Yield: 10%, mp: 152 °C; IR: (KBr) ν (cmN-((1-benzyl-2-(methylsulfonyl)-1H-imidazol-5-yl)methyl)-4-bromobenzamine (5c)-1): 3288 (N-H), 1136, 1329 (SO2). 1HNMR  δppm: 3.32 , 4.10 , 5.64 , 6.37 , 6.43 , 7.10 , 7.17 , 7.32 , 7.38 . LC-MS [M+1]+: m/z 420, [M+3]+: m/z 423, [M+23]+: m/z 442, [M+25]+: m/z 444. Anal. Calcd for C18H18BrN3O2S: C, 51.43; H, 4.32; N, 10.00. Found: C, 51.48; H, 4.30; N, 9.76.Yield: 16%, mp: 155 °C; IR: (KBr) ν (cmN-((1-benzyl-2-(methylsulfonyl)-1H-imidazol-5-yl)methyl)-4-nitrobenzamine (5d)-1): 3310 (N-H), 1110-1308 (SO2), 1324-1528 (NO2). 1HNMR  δppm: 3.32 , 4.30 , 5.65 , 6.56 , 7.07 , 7.17 , 7.31 , 7.36 , 7.66 , 7.96 . LC-MS [M+23]+: m/z 409. Anal. Calcd for C18H18N4O4S: C, 55.95; H, 4.70; N, 14.50. Found: C, 55.91; H, 4.74; N, 14.43.Yield: 10%, mp: 177 °C; IR: (KBr) ν (cmN-((1-benzyl-2-(methylsulfonyl)-1H-imidazol-5-yl)methyl)-4-chlorobenzamine (5e)-1): 3298 (N-H), 1140, 1333 (SO2). 1HNMR  δppm: 3.32 , 4.10 , 5.64 , 6.32 , 6.47 , 7.06 , 7.10 , 7.32 , 7.38 . LC-MS [M+23]+: m/z 398, [M+25]+: m/z 400. Anal. Calcd for C18H18ClN3O2S: C, 57.52; H, 4.83; N, 11.18. Found: C, 57.80; H, 4.79; N, 11.11.Yield: 25%, mp: 142 °C; IR: (KBr) ν .The inhibitory activity of the synthesized compounds was evaluated against COX-1 and COX-2 enzymes with Cayman colorimetric-based human cyclooxygenase assay kit (item number 701050). The enzyme was incubated with inhibitors for 2 min in 0.1 M Bis-Tris/HCl buffer (pH 8.0) at 25 °C. Arachidonic acid and Celecoxib were used as substrate and reference drug respectively. All test samples were dissolved in DMSO and absorbance was read at 590 nm. The ICMolecular Modeling Studies5b, N-((1-benzyl-2-(methylsulfonyl)-1H-imidazol-5-yl)methyl)-4-methoxyaniline, along with Celecoxib in the active site of the COX-2 enzyme examined by a flexible docking experiment using AutoDock 4.0 software was observed in 5b and Celecoxib were in the same direction for hydrogen binding with Arg513 and also the phenyl ring of the 5b may bind in a lipophilic pocket formed by Trp387, Tyr385, and Val349 of the active site. Additional hydrogen binding could form between NH of anilino moiety of the 5b and Arg120. Isobutyl moiety of Leu359 may form hydrophobic interaction with methyl group of methoxy substituent of the 5b.To predict interaction of compounds .The designed compounds were synthesized according to In-vitro Biological activity50 values in the range of 0.7-3.6 µM, while IC50 values of COX-1 inhibition were 78-138µM. The rank order for the contribution of substituents to the COX-2 inhibitory activity of the synthesized compounds is: OCH3 > Br > NO2 > H > Cl. Results reveal that the compound bearing methoxy (5b), shows the best inhibitory activity and selectivity against COX-2 with IC50 of 0.71 µM and selectivity index of 115 due to additional hydrophobic interaction of methoxy with Leu359. The electron withdrawing substitutes and Hydrogen at the para position of anilino ring, despite selectivity to COX-2, show no considerable priority with each other.As shown in 5b was found to be the most potent inhibitor with IC50 of 0.71 µM and selectivity index of 115 in targeting COX-2 enzyme. Finally, these structures seem to be valuable leading scaffold to design and develop novel selective COX-2 inhibitors.New imidazole-based compounds as non-classical selective cyclooxygenase-2 inhibitors were investigated by attaching the suitable pharmacophore directly to the central cyclic ring. The docking study shows the compounds fitted in the COX-2 catalytic pocket and interacted well with the active site residues. The synthesized compounds had comparable inhibitory activity to Celecoxib. Compound


Aspergillus flocculosus, one aspyrone-related polyketide aspilactonol G (2), one meroterpenoid 12-epi-aspertetranone D (4), two drimane derivatives , together with five known metabolites . The structures of compounds 1–10 were established by NMR and MS techniques. The absolute stereoconfigurations of compounds 1 and 2 were determined by a modified Mosher’s method. The absolute configurations of compounds 4 and 7 were established by a combination of analysis of ROESY data and coupling constants as well as biogenetic considerations. Compounds 7 and 8 exhibited cytotoxic activity toward human prostate cancer 22Rv1, human breast cancer MCF-7, and murine neuroblastoma Neuro-2a cells.Four new compounds were isolated from the Vietnamese marine sediment-derived fungus  Aspergillus, section Circumdati  +, and was supported by the 13C NMR spectrum.The molecular formula of compound 4 , one sp3-methylene group , two sp3-methines , two oxygen-bearing ones , one quaternary sp3-carbon (δC 55.5), three oxygen-bearing quaternary sp3-carbons , two quaternary sp2-carbons , three oxygen-bearing quaternary sp2-carbons , and two ketone groups .An analysis of NMR data of 4  revealed4 (13C NMR spectrum of 4 (5) , wit, wit4 , three 3sp-methylene groups , 1.38, 1.24), two oxygen-bearing sp3-methylene groups , two sp3-methine groups , including one oxygen-bearing, one sp2-methine group , three quaternary sp3-carbons , including one oxygen-bearing, and two quaternary sp2-carbons .A close inspection of the ata of 7  revealed13C NMR data of 7 were similar to those of the drimane moiety of insulicolide A (8) +, which was supported by the 13C NMR spectrum.The molecular formula of compound 1H and 13C NMR data of 9 , three sp3-methylene groups (δC 37.8 (2C), 18.0; δH 1.71, 1.59, 1.54, 1.45, 1.32, 1.10), two oxygen-bearing 3sp-methylene groups , three sp3-methine groups , including two oxygen-bearing, two quaternary sp3-carbons , and three quaternary sp2-carbons .A close inspection of the ata of 9  revealedH 3.99) to C-5 (δC 48.6), C-7 (δC 64.1), C-8 (δC 122.1), C-9 (δC 173.1), and C-10 (δC 36.3), from H-7 (δH 4.00) to C-12 (δC 173.4), from H2-11  to C-8, C-9, and C-12, from H3-13 (δH 0.97) to C-3 (δC 37.8), C-4 (δC 38.3), C-5, and C-14 (δC 65.6), from H3-15 (δH 1.40) to C-1 (δC 37.8), C-5, C-9, and C-10 indicated the drimane moiety in 9 being the same as in 7α,14-dihydroxy-6β-p-nitrobenzoylconfertifolin + .Aspilactonol F (2): white powder; c 0.49, MeOH); UV (MeOH) λmax (log ε) 214 (4.05) nm; ECD  λmax (Δε) 216 (–11.51) nm; 1H and 13C NMR data see m/z 209.0782 [M + Na]+ .Aspilactonol G (Epi-aspertetranone D (4): white powder;c 0.07, MeOH); UV (MeOH) λmax (log ε) 290 (3.93), 208 (4.53) nm; ECD  λmax (Δε) 209 (+25.54), 284 (+1.86) nm; 1H and 13C NMR data see m/z 459.1628 [M + Na]+ .12-7): white crystals;c 0.15, MeOH); UV (MeOH) λmax (log ε) 206 (3.61) nm; ECD  λmax (Δε) 224 (–2.33) nm; 1H and 13C NMR data see m/z 305.1361 [M + Na]+ .6β,9α,14-trihydroxycinnamolide (9): white crystals;c 0.36, MeOH); UV (MeOH) λmax (log ε) 214 (4.00) nm; ECD  λmax (Δε) 217 (+3.68), 243 (+1.51) nm; 1H and 13C NMR data see m/z 305.1361 [M + Na]+ .6β,7β,14-trihydroxyconfertifolin (R)-MTPA-Cl (4 μL) were added to a solution of 1 (1.0 mg) in pyridine at room temperature and stirred for 5 h. After evaporation of the solvent, the residue was purified by HPLC on a YMC SIL column  to afford the (S)-MTPA ester (0.5 mg). The (R)-MTPA ester (0.5 mg) was prepared in a similar manner using (S)-MTPA-Cl.The compounds 4-dimethylaminopyridine  and (S)-MTPA ester of 1: 1H NMR  δ: 6.88 , 5.28-5.34 , 4.84 , 3.48 , 3.43 , 2.56-2.60 , 1.26 , 1.24 , 7.39–7.48 . HRESIMS m/z 641.1576 [M + Na]+ .(R)-MTPA ester of 1: 1H NMR  δ: 6.52 , 5.25 , 5.20 , 4.56 , 3.56 , 3.50 , 2.48-2.51 , 1.35 , 1.29 , 7.38–7.52 . HRESIMS m/z 641.1577 [M + Na]+ .(R)-MTPA-Cl (9 μL) was added to a solution of 2 (1.9 mg) in pyridine at room temperature and stirred for 2 h. After evaporation of the solvent, the residue was purified by HPLC on a YMC SIL column  to afford the (S)-MTPA ester (1.4 mg). The (R)-MTPA ester (1.5 mg) was prepared in a similar manner using (S)-MTPA-Cl.(S)-MTPA ester of 2: 1H NMR  δ: 6.86 , 5.32 , 5.23 , 4.81 , 3.52 , 3.47 , 2.65 , 2.48 , 1.39 , 1.29 , 7.38–7.50 . HRESIMS m/z 641.1576 [M + Na]+ .(R)-MTPA ester of 2: 1H NMR  δ: 6.68 , 5.30 , 5.26 , 4.82 , 3.53 , 3.48 , 2.61 , 2.46 , 1.33 , 1.25 , 7.37–7.52 . HRESIMS m/z 641.1576 [M + Na]+ . and 1% penicillin/streptomycin .The human prostate cancer cell line 22Rv1 was cultured according to the manufacturer’s instructions in 10% FBS/RPMI medium (Invitrogen). Cells were continuously kept in culture for a maximum of 3 months, were routinely inspected microscopically for stable phenotype, and regularly checked for contamination with mycoplasma. Cell line authentication was performed by DSMZ  using highly polymorphic short tandem repeat loci [v/v) CO2.All cells were incubated at 37 °C in a humidified atmosphere containing 5% , a new meroterpenoid, 12-epi-aspertetranone D (4), two new drimane derivatives , together with six known metabolites were isolated from the Vietnamese marine sediment-derived fungus A. flocculosus. The structures of compounds 1–10 were established using spectroscopic methods. The absolute configurations of chiral centers were determined using either a modified Mosher’s method (for compounds 1 and 2) or a combination of ROESY data, coupling constants analysis and biogenetic considerations for compounds 4, 7 and 9. Drimane sesquiterpenoid derivatives 7 and 8 showed cytotoxicity toward human prostate cancer 22Rv1, human breast cancer MCF-7, and murine neuroblastoma Neuro-2a cells. The analysis of structure–activity relationships of compounds 7–10 together with literature data showed that these compounds have three sites in their structures related to cytotoxicity, i.e., a double bond at C7=C8, a hydroxyl group at C-9, and a p-nitrobenzoyl moiety.A new aspyrone-related polyketide, aspilactonol G (


Gentianella acuta has been widely used for the treatment of diarrhea, hepatitis, arrhythmia, and coronary heart disease. In the course of investigating efficacy compounds to treat diarrhea using a mouse isolated intestine tissue model, we found 70% EtOH extract of G. acuta whole plants had an inhibitory effect on intestine contraction tension. Here, nineteen constituents, including five new compounds, named as gentiiridosides A (1), B (2), gentilignanoside A (3), (1R)-2,2,3-trimethyl-4-hydroxymethylcyclopent-3-ene-1-methyl-O-β-d-glucopyranoside (4), and (3Z)-3-hexene-1,5-diol 1-O-α-l-arabinopyranosyl(1→6)-β-d-glucopyranoside (5) were obtained from it. The structures of them were elucidated by chemical and spectroscopic methods. Furthermore, the inhibitory effects on motility of mouse isolated intestine tissue of the above mentioned compounds and other thirteen iridoid- and secoiridoid-type monoterpenes  previously obtained in the plant were analyzed. As results, new compound 5, some secoiridoid-type monoterpenes 7, 10, 12–14, 16, and 17, as well as 7-O-9′-type lignans 31 and 32 displayed significant inhibitory effect on contraction tension at 40 μM.As a Mongolian native medicine and Ewenki folk medicinal plant,  Gentianella acuta (Michx.) Hulten belongs to the family Gentianaceae, distributed mainly in the north of China, Mongolia plateau, Siberia, and Far East areas of Russia . Negative high resolution electrospray ionization-time of flight-mass spectra (HRESI-TOF-MS) afforded [M – H]− at m/z 777.2261 , supporting a molecular formula of C36H42O19 for 1. The absorption bands showed in the infrared (IR) spectrum suggested the presence of hydroxyl (3372 cm−1), α,β-unsaturated carbonyl (1712 cm−1), aromatic ring , and O-glycosidic linkage (1078 cm−1). The sugars in 1 were found to be d-glucose by acid hydrolysis with 1 M HCl ; }, and two β-d-glucopyranosyl . On the other hand, thirty-six carbon signals were shown in its 13C-NMR spectrum, in addition to twenty-six carbon signals occupied by the abovementioned fragments, the other 10 carbon signals, together with the relative proton signals  indicated the aglycon of 1 was iridoid. As shown in 1H 1H COSY experiment on 1 suggested the existence of five partial structures. Furthermore, in the HMBC experiment, long-range correlations from δH 5.50 (H-1) to δC 152.4 (C-3); δH 7.46 (H-3) to δC 32.5 (C-5), 96.2 (C-1), 112.8 (C-4), 170.9 (C-11); δH 4.96 (H-7) to δC 166.1 (C-7′′); δH 4.69 (H-1′) to δC 96.2 (C-1); δH 7.89 (H-2′′), 7.84 (H-6′′) to δC 166.1 (C-7′′); δH 5.02 (H-1′′′) to δC 159.2 (C-3′′); δH 3.51 (H-2′′′) to δC 166.8 (C-7′′′′); δH 7.82 (H-2′′′′), 7.88 (H-6′′′′) to δC 166.8 (C-7′′′′) were observed. Therefore, the planar structure of 1 was constructed. The relative configuration of 1 was determined by a nuclear Overhauser effect spectroscopy (NOESY) experiment, and NOE correlations were observed between H-1 and H-8; H-5 and H-7; H3-10 and H-7, H-9. The 1H and 13C-NMR sepctra of 1 were found very similar to those of swertiaside (26) .  spectra  and vari0.0° c 0., MeOH]. Gentiiridoside B (2) was obtained as a white powder with negative optical rotation . HRESI-TOF-MS exhibited a molecular ion peak at m/z 679.1023 [M – H]−, and revealed the molecular formula C28H40O18  for it. Acid hydrolysis of 2 with 1 M HCl afforded d-glucose, whose absolute configuration was determined by HPLC analysis , and one α-d-glucopyranosyl [δ 4.91 ]. Twenty-eight carbon signals were displayed in its 13C-NMR spectrum, except for the above mentiond moieties, the other ten signals as well as their relative 1H-NMR signals  suggested the aglycon of 2 was the same as that of gentiopicroside (20) . The molecular formula, C27H34O13, of 3 was determined from Q-TOF-ESI-MS analysis . Its IR spectrum showed absorption bands due to hydroxyl (3368 cm−1), aromatic ring , and O-glycosidic linkage (1073 cm−1). The 1H, 13C-NMR spectra  showed signals ascribable to one ABX-type aromatic protons , one 1,3,4,5-symmetrical substituted phenyl group [δ 6.64 ], two methylene bearing oxygen funtion {δ , ]}, three methoxyl , and one β-d-glucopyranosyl [δ 4.88 ]. The planar structure of 3 was constructed by the assignment of the 1H 1H COSY and HMBC experiments as shown in 1H 1H COSY experiment indicated the presence of three partial moieties. On the other hand, in the HMBC experiment, long-range correlations were found from the following proton and carbon pairs: δH 6.64  to δC 129.9 (C-1), 136.2 (C-4), 148.9 ; δH 4.84 (H-7) to δC 51.8 (C-8′), 64.6 (C-9), 106.2 , 129.9 (C-1); δH 3.64, 3.80 (H2-9) to δC 51.8 (C-8′), 83.3 (C-8), 85.8 (C-7); δH 6.90 (H-2′) to δC 35.1 (C-7′), 122.4 (C-6′), 146.5 (C-4′), 150.9 (C-3′); δH 7.10 (H-5′) to δC 136.9 (C-1′), 146.5 (C-4′), 150.9 (C-3′); δH 6.77 (H-6′) to δC 35.1 (C-7′), 114.4 (C-2′), 146.5 (C-4′); δH 2.54, 3.13 (H2-7′) to δC 72.0 (C-9′), 114.4 (C-2′), 122.4 (C-6′), 136.9 (C-1′); δH 2.59 (H-8′) to δC 136.9 (C-1′); δH 3.64, 4.06 (H2-9′) to δC 35.1 (C-7′), 83.3 (C-8), 85.8 (C-7); δH 3.84  to δC 148.9 ; δH 3.86 (3′-OCH3) to δC 150.9 (C-3′); δH 4.88 (H-1′′) to δC 146.5 (C-4′). Furthermore, the relative configuration of 3 was determined by the NOE correlations between δH 4.84 (H-7) and δH 3.64, 3.80 (H2-9); δH 3.64, 3.80 (H2-9) and δH 2.54, 3.13 (H2-7′) observed in its NOESY spectrum. Finally, 3 showed negative Cotton effect at 278 and 232 nm, which indicated the absolute configuration of it was 7R,8S,8′S , two methylene with oxygen function {, 4.07 }, one β-d-glucopyranosyl [δ 4.26 ], one methylene , together with one methine [δ 2.15 ]. The 1H 1H COSY experiment suggested the presence of two partial fragments shown in bold lines (4 was further elucidated by the long-range correlations from δH 0.87 (2β-CH3) to δC 27.2 (2α-CH3), 49.0 (C-1), 49.3 (C-2), 143.5 (C-3); δH 1.09 (2α-CH3) to δC 20.1 (2β-CH3), 49.0 (C-1), 49.3 (C-2), 143.5 (C-3); δH 1.56 (3-CH3) to δC 49.3 (C-2), 133.1 (C-4), 143.5 (C-3); δH 4.07 (4-CH2OH) to δC 36.6 (C-5), 133.1 (C-4), 143.5 (C-3); δH 2.15, 2.48 (H2-5) to δC 49.3 (C-2), 72.1 (1-CH2OH), 133.1 (C-4), 143.5 (C-3); δH 4.26 (H-1′) to δC 72.1 (1-CH2OH) observed in the HMBC spectrum. To determine the sterostructure of it, 4 was treated with β-glucosidase, to give the aglycon, (1R)-2,2,3-trimethyl-4-hydroxymethylcyclopent-3-ene-1-methanol (4a), which was obtained with negative optical rotation  and had only one chiral carbon. Using the the same method reported in literatures . Its molecular formula, C17H30O11 , was recorded by Q-TOF-ESI-MS. Furthermore, using acid hydrolysis and HPLC analysis, the presence of d-glucose and l-arabinose in 5 was revealed , one methoxyl [δ 1.20 ], one β-d-glucopyranosyl [δ 4.27 ], along with one α-l-arabinopyranosyl [δ 4.31 ]. The planar structure of 5 was constructed on the basis of 1H 1H COSY and HMBC experiments. Namely, the 1H 1H COSY experiment suggested the existence of three partial structures, as shown as bold lines in H 4.27 (H-1′) to δC 70.4 (C-1); δH 4.31 (H-1′′) to δC 69.6 (C-6′) were observed. Finally, the NOE correlation between δH 2.39, 2.46 (H2-2), and δH 4.61 (H-5); 5.45 (H-3) and δH 5.47 (H-5) found in the NOESY spectrum indicated the configuration in the 3-position was Z. Consequently, the structure of 5 was elucidated to be (3Z)-3-hexene-1,5-diol 1-O-α-l-arabinopyranosyl(1→6)-β-d-glucopyranoside.(3Z)-3-Hexene-1,5-diol 1-O-α- spectra  and 2D NG. acuta and the abovementioned isolates on motility of mouse isolated intestine tissue were determined by using the same method as reported previously  to give 16 fractions (Fr. 1–Fr. 16). Fraction 7 (25.7 g) was centrifugated (MeOH), and two fractions (Fr. 7-1–Fr. 7-2) were yielded. Fraction 7-2 (9.8 g) was separated by PHPLC [CH3CN–H2O  + 1% HAc] to give 27 fractions (Fr. 7-2-1–Fr. 7-2-27). Fraction 7-2-2 (70.6 mg) was purified by PHPLC [MeOH–H2O ], and 2,3-dihydroxy-1-(4-hydroxy-3-methoxyphenyl)-propan-1-one  was given. Fraction 7-2-8 (46.3 mg) was isolated by PHPLC [CH3CN-H2O  + 1% HAc] to gain -erythro-4,7,9,9′-tetrahydroxy-3,3′-dimethoxy-8-O-4′-neolignan . Fraction 9 (15.0 g) was subjected to Sephadex LH-20 CC [CHCl3–MeOH ] to yield seven fractions (Fr. 9-1–Fr. 9-7). Fraction 9-4 (7.3 g) was separated by PHPLC [CH3CN–H2O  + 1% HAc], as a result, 12 fractions (Fr. 9-4-1–Fr. 9-4-12) were obtained. Fraction 9-4-2 (962.8 mg) was purified by PHPLC [MeOH–H2O  + 1% HAc] to give gentiiridoside B . Fraction 9-4-9 (277.1 mg) was centrifuged (MeOH), and two fractions (Fr. 9-4-9-1–Fr. 9-4-9-2) were gained. Fraction 9-4-9-2 (199.7 mg) was isolated by PHPLC [MeOH–H2O  + 1% HAc] to yield plucheoside D3 . Fraction 11 (20.0 g) was subjected to PHPLC [CH3CN–H2O  + 1% HAc], and 29 fractions (Fr. 11-1–Fr. 11-29) were given. Fraction 11-5 (631.3 mg) was separated by PHPLC [CH3CN–H2O  + 1% HAc], and 8-epiloganin  was yielded. Fraction 11-6 (388.0 mg) was isolated by PHPLC [CH3CN–H2O  + 1% HAc] to yield gentilignanoside A , loganin , and berchemol-4′-O-β-d-glucoside . Fraction 11-12 (660.5 mg) was centrifugated (MeOH) and further purified by PHPLC [MeOH–H2O  + 1% HAc] to give (1R)-2,2,3-trimethyl-4-hydroxymethylcyclopent-3-ene-1-methyl-O-β-d-glucopyranoside . Fraction 11-17 (790.7 mg) was isolated by PHPLC [MeOH–H2O  + 1% HAc] to obtain six fractions (Fr. 11-17-1–Fr. 11-17-6). Fraction 11-17-1 (97.1 mg) was purified by PHPLC [CH3CN-H2O  + 1% HAc] to gain swertiaside . Fraction 11-21 (376.1 mg) was separated by PHPLC [MeOH–H2O  + 1% HAc] to afford -9′-methoxy-dehydrodiconiferyl alcohol 4-O-β-d-glucopyranoside . Fraction 13 (20.0 g) was subjected to PHPLC [MeOH-H2O  + 1% HAc], and 20 fractions (Fr. 13-1–Fr. 13-20) were yielded. Fraction 13-11 (1.9 g) was centrifuged (MeOH) to obtain two fractions (Fr. 13-11-1–Fr. 13-11-2). Fraction 13-11-1 (1.0 g) was isolated by PHPLC [CH3CN–H2O  + 1% HAc], as a result, eleven fractions (Fr. 13-11-1-1–Fr. 13-11-1-11) were given. Fraction 13-11-1-9 (117.1 mg) were purified by Sephadex LH-20 CC (MeOH) and PHPLC [MeOH–H2O  + 1% HAc] to afford 5α-carboxystrictosidine . Fraction 13-12 (501.4 mg) was isolated by PHPLC [CH3CN–H2O  + 1% HAc] and to yield (E)-aldosecologanin . Fraction 13-17 (504.0 mg) was purified by PHPLC [CH3CN–H2O  + 1% HAc] to yield gentiiridoside A . Fraction 14 (15.3 g) was subjected to PHPLC [CH3CN–H2O  + 1% HAc], and 20 fractions (Fr. 14-1–Fr. 14-20) were obtained. Fraction 14-1 (1.4 g) was separated by PHPLC [CH3CN–H2O  + 1% HAc] to gain 14 fractions (Fr. 14-1-1–Fr. 14-1-14). Fraction 14-1-2 (32.1 mg) was purified by Sephadex LH-20 CC (MeOH) and finally by PHPLC [CH3CN–H2O ] to give (3Z)-3-hexene-1,5-diol 1-O-α-l-arabinopyranosyl(1→6)-β-d-glucopyranoside .The 95% EtOH eluate (200.0 g) was subjected to silica gel CC [CHCl3 layer  was subjected to Silica gel CC [CHCl3–MeOH  → CHCl3–MeOH–H2O  → MeOH], and eight fractions (Fr. C-1–Fr. C-8) were yielded. Fraction C-5 (1.1 g) was separated by Sephadex LH-20 CC [MeOH–CH2Cl2 ] to gain three fractions (Fr. C-5-1–Fr. C-5-3). Fraction C-5-2 (110.0 mg) was purified by PHPLC [MeOH-H2O ] to afford -dehydrodiconiferyl alcohol  and (–)-berchemol . Fraction C-7 (9.0 g) was isolated by ODS CC [MeOH–H2O  → MeOH], and eight fractions (Fr. C-7-1–Fr. C-7-8) were obtained. Fraction C-7-2 (351.4 mg) was purified by PHPLC [MeOH–H2O  + 1% HAc] to give gentiopicroside . Fraction C-7-6 (486.4 g) was subjected to Sephadex LH-20 CC [MeOH–CH2Cl2 ] and PHPLC [MeOH–H2O  + 1% HAc] to afford trifloroside .The CHCl7–10, 13–16, 18, 19, 21, 22 and 25 were obtained and identified by using the method reported previously − .Gentiiridoside B (2): White powder; – .Gentilignanoside A (3): White powder; −m/z 567.2083 [M – H]− .(1R)-2,2,d-glucopyranoside3-Trimethyl-4-hydroxymethylcyclopent-3-ene-1-methyl-O-β- (4): White powder; – .(3Z)-3-Hexene-1,5-diol 1-O-αl-arabinopyranosyl(1→6)-β-d-glucopyranoside- (5): White powder; – .Enzymatic Hydrolysis of4 A solution of 4 (6.0 mg) in H2O (2.0 mL) was reacted with β-glucosidase  at 37 °C for 2.5 h. Then the reaction mixture was extracted with EtOAc. And the residue from EtOAc solvent was subjected to Silica gel CC [CHCl3–MeOH ], as a result, the aglycon, (1R)-2,2,3-trimethyl-4-hydroxymethylcyclopent-3-ene-1-methanol  was yielded.(1R)-2,2,3-Trimethyl-4-hydroxymethylcyclopent-3-ene-1-methanol (4a): White powder; − .Acid Hydrolysis of1–5 The solution of compounds 1–5 (each 2.0 mg) in 1 M HCl (1.0 mL) was treated by using the same method as described in reference [3CN–H2O . As results, d-glucose was detected from the aqueous phase of 1–5, and l-arabinose was found from that of 5 by comparison of its retention time and optical rotation with those of the authentic sample, d-glucose (tR 16.8 min (positive)) and l-arabinose (tR 13.1 min (positive)), respectively.eference : They we1–32 on motility of mice isolated intestine tissue were determined by using the similar method as we reported previously [2 0.2 g, KCl 0.2 g, MgCl2 0.1 g, NaHCO3 1.0 g, KH2PO4 0.05 g, glucose 1.0 g, pH 7.4) and maintained at a constant temperature (37.0 ± 0.5°C), and bubbled with 95% O2 and 5% CO2 gas. The intestinal tissue was fixed on bottom hook in and the other end was connected to an isometric tension transducer. Samples in DMSO solution were added after 15 min to equilibrate incubation, the final DMSO concentration was 0.1%, and the final concentration of fractions and compounds was 100 μg/mL and 40 μM, respectively. The mean tension and frequency of intestine muscle contractions were recorded for 1 min before and 4 min after drug additions using isolated tissue bath systems . Loperamide hydrochloride  was used as a positive control, and the final concentration was 10 μM.Inhibitory effects of fractions and compounds eviously ,28: Micep < 0.05 was considered to indicate statistical significance.Values are expressed as mean ± S.D. All the grouped data were statistically performed with SPSS 11.0. Significant differences between means were evaluated by one-way analysis of variance (ANOVA) and Tukey’s Studentized range test was used for post hoc evaluations. 1), B (2), gentilignanoside A (3), (1R)-2,2,3-trimethyl-4-hydroxymethylcyclopent-3-ene-1-methyl-O-β-d-glucopyranoside (4), and (3Z)-3-hexene-1,5-diol 1-O-α-l-arabinopyranosyl(1→6)-β-d-glucopyranoside (5) were obtained from the whole plants of G. acuta in our on-going program of screening the phytochemical and bioactive constituents. Among the known isolates, 6, 11, 12, 17, 23, and 27–32 were isolated from the genus firstly. The structures of them were elucidated by chemical and spectroscopic methods. In summary, nineteen constituents, including five new ones, gentiiridosides A  previously obtained in the plant were analyzed. Resultingly, 5, 7, 10, 12, 14, 16, 17, 31, and 32 displayed significant inhibitory effects on contraction tension. On the other hand, structure-activity relationship analysis revealed that the inhibitory effect of secoiridoid-type monoterpenes was stronger than that of iridoid-type monoterpenes, and 7-O-9′-type lignan displayed stronger inhibitory bioactivity than 8-O-4′- and 7-O-4′-type lignan.Furthermore, the inhibitory effects on motility of mouse isolated intestine tissue of the above mentioned compounds and other thirteen isolates (G. acuta were xanthones, monoterpenes, lignans, and phenolic acids. Among them, xanthones and part of the monoterpenes were major bioactivity substances of G. acuta. These results suggested that G. acuta and its constituents have potential value in discovering new medicines for abnormal intestinal motility.Our previous ,5,6 and


The new triterpene diglycosides 1-5 were identified as cimiacerol-3-O-β-d-xylopyranosyl-(1''→3')-β-d-xylopyranoside, 12β-hydroxycimigenol-3-O-β-d-xylopyranosyl-(1''→3')-β-d-xylopyranoside, 25-O-acetylcimig- enol-3-O-β-d-xylopyranosyl-(1''→3')-β-d-xylopyranoside, 24-acetylhydroshengmanol-3-O-β-d-xylopyranosyl-(1''→3')-β-d-xylopyranoside and 26-deoxyacetylacteol-3-O-β-d-xylo- pyranosyl-(1''→3')-β-d-xylopyranoside, respectively, based on analysis of their spectral data and chemical reactions.Five new 9,19-cycloartane triterpene diglycosides, which have been named cimiaceroside C ( Cimicifugafoetida (family Ranunculaceae) is a popular Chinese Traditional Medicine. Under the trivial name of “Shengma”, it has been used as an antipyretic and analgesic agent since ancient times -) as C40H64O13, and this was confirmed by negative HRFABMS: m/z 751.7352 ([M-H]-) . The IR spectrum showed strong hydroxyl bonds at 3376 cm-1. The 13C-NMR spectrum displayed 40 carbons, which included two sugar moieties at δC 106.3 (C-1'), 74.6 (C-2'), 87.4 (C-3'), 69.4 (C-4'), 67.5 (C-5'), and at δC 107.2 (C-1''), 75.4 (C-2''), 78.3 (C-3''), 71.0 (C-4''), 66.6 (C-5'') , seven methyls at δH1.19 , 1.76 , 1.67 , 0.83 , 1.31 , 1.02  and 1.21 , and two sugar anomeric protons at δH5.30  and 4.81   to 88.6 (C-3), and δH 4.81 (H-1'') to 87.4 (C-3') determined the inner xylose to be connected at C-3, and the terminal xylose to be at C-3' of the inner xylose. The substituent of the xylose sugar unit at C-3' led to significant downfield shift for the 13C-NMR signal at C-3' and upfield shifts for signals at C-2' and C-4'.Cimiaceroside C ( (C-5'') . The 1H-, H-1'') . The aboroside B , except 1 was determined on the basis of the ROESY experiments  and H-2'' (J = 7.6 Hz) suggested 1 had a β-d-xylopyranosyl-(1''→3')-β-d-xylopyranoside moiety. Thus, cimiaceroside C (1) was determined to be cimiacerol-3-O-β-d-xylopyranosyl-(1''→3')-β-d-xylopyranosideThe relative stereochemistry of eriments . A signi2) was isolated as a white powder. The negative HR-FAB-MS of 2 showed a quasi-molecular ion at m/z 767.4356, corresponding to the molecular formula of C40H64O14. Its 1H-NMR spectrum , seven methyls at δH1.41 , 1.49 , 1.50 , 1.21 , 1.29 , 1.02  and 1.38 , and two sugar anomeric protons at δH5.29  and 4.81 . The 13C-NMR spectrum exhibited 40 resonances, of which 30 were attributed to a triterpene skeleton, ten to two sugar units. A comparison of the 1H- and 13C-NMR spectra of 2 with those of cimiside A - and HRFABMS 793.4512 . The IR spectrum of 3 showed an absorption at 1739 cm-1 due to a carbonyl group. The 1H-NMR spectrum exhibited the cyclopropane methylene signals at δH 0.27 and 0.53 , two anomeric proton signals at δH5.32  and 4.83  and a methyl singlet at δH 2.01 (3H). The 13C-NMR spectrum of 3 showed 30 carbon signals for the triterpene skeleton, 10 for two sugar units and two for a carbonyl group. All this evidence suggested that 3 was a 9,19-cycloartane triterpenoid diglycoside. The structure of 3 resembled that of the known compound cimiside B 27D -31.3° ; negative FABMS m/z (%) 751 , 679 (8), 129 (24); negative HRFABMS: m/z 751.7352 ([M-H]-) ; IR (KBr) νmax 3376, 2972, 2940, 2872, 1732, 1639, 1462, 1442, 1382, 1368, 1251, 1209, 1164, 1081, 1052, 1001, 972, 894, 703, 616, 531, 429 cm-1; 13C-  and 1H-NMR (500 MHz) data, see CimifosideA (2): white powder; m.p. 287-289 oC; [α]27D +16.7° ; negative HRFABMS: m/z 767.4366 ; IR (KBr) νmax 3431, 2928, 2870, 1631, 1455, 1383, 1363, 1170, 1077, 1040, 563, 470 cm-1; 13C-  and 1H-NMR (500 MHz) data, see Cimifoside B (3): white powder; m.p. 165-166 oC; [α]27D -24.2° ; negative HRFABMS: m/z 793.4512, ; IR (KBr) νmax 3443, 2963, 2934, 2870, 1739, 1735, 1457, 1239, 1070 cm-1; 13C-  and 1H-NMR (500 MHz) data, see CimifosideC (4): white powder; m.p. 224-225 oC; [α]27D -60.0° ; negative HRFABMS: m/z 811.4678, ; IR (KBr) νmax 3425, 2940, 2870, 1712, 1632, 1457, 1379, 1266, 1159, 1107, 1072, 1042, 996, 969, 898, 797, 738, 630, 610, 591, 496, 449 cm-1; 13C-  and 1H-NMR (500 MHz) data, see CimifosideD (5): white powder; m.p. 185-186 oC; [α]27D 56.2° ; negative HRFABMS: m/z 791.4531([M-H]-) ; IR (KBr) νmax 3442, 2969, 2937, 2872, 1731, 1633, 1458, 1382, 1366, 1252, 1165, 1042, 984, 898, 841, 804, 677, 629, 602 cm-1; 13C-  and 1H-NMR (500 MHz) data, see 1-5 (6 mg of each) were refluxed with 5% HCl in MeOH (7 mL) for 8 h. Each mixture was diluted with H2O and neutralized with NaHCO3. The neutral hydrolysate revealed the presence of only xylose by TLC  upon comparison with the authentic sample.Compounds


Sabina gaussenii led to the isolation of two new cinnamyl isovalerate derivatives (1–2) and eighteen known compounds (3–20). Their structures were determined mainly by means of MS, 1D- and 2D-NMR data, and this is the first time these compounds have been reported from this plant. The biological activity test results indicated that the 90% acetone extract showed cytotoxicity against the human lung adenocarcinoma (A549) cell line (IC50 = 0.98 ± 0.1 μg/mL), compound 6 showed cytotoxicities against human cervical carcinoma (HeLa) (IC50 = 0.4 ± 0.1 μM ) and human gastric carcinoma (BGC-823) (IC50 = 0.9 ± 0.2 μM) cancer cell lines, and compound 19 showed cytotoxicities against HeLa (IC50 = 1.5 ± 0.4 μM), BGC-823 (IC50 = 7.0 ± 0.8 μM ), and A549 (IC50 = 10.6 ± 1.5 μM ) cancer cell lines.Chemical investigation of the 90% acetone extract of the branches and leaves of  Sabina gaussenii is endemic to China and is usually used as a hedge plant. The genus Sabina, which used to belong to genus Juniperus, has about 50 species and spread widely throughout the northern hemisphere [Sabina plants have been reported to be a rich source of bioactive terpenoids [S. gaussenii [S. gaussenii [1–2) together with eighteen other known compounds (3–20) from the branches and leaves of S. gaussenii, in addition to a screening of their cytotoxicities.misphere . Accordirpenoids . Up to naussenii . As partaussenii ,5,6,7. IS. gaussenii was extracted with 90% acetone at room temperature to give a crude extract, which was suspended in H2O and successively partitioned with petroleum ether, ethyl acetate (EtOAc), and n-butyl alcohol (n-BuOH). Column chromatographic separations of these extracts afforded compounds 1–20 (1–2) were identified by spectroscopic analyses and physicochemical properties, while the known compounds were identified as 3’,4’,5’-dimethoxycinnamyl isovalerate (3) [4) [5) [O-demethylepipodophyllotoxin (6) [7) [d-glucosyloxy coumarin (8) [d-glucosyloxy-2--propane-l,3-diol (9) [10) [11) [12) [epi-4-eudesmene-1β, 11-diol (13) [14) [15) [16) [17) [18) [19) [20) [The air-dried powder of the branches and leaves of nds 1–20 . The tworate (3) , 3’,4’,5 [4) [5) , 4’-O-deoxin (6) , 7-hydro (6) [7) , 7-β-d-garin (8) , 1-β-d-gdiol (9) , lβ,6α-d(9) [10) , selin-410) [11) , 4-eudes11) [12) , 7-epi-4iol (13) , 3-eudes13) [14) , 8α,11-e14) [15) , hinokii15) [16) , corchoi17) [18) , isocupr18) [19) , and sit19) [20)  by compa1 was obtained as a colorless oil. Its molecular was assigned as C18H26O5 on the basis of positive HRESIMS  and NMR spectra data , 3.84 ). The 13C- and DEPT-NMR spectra of 1 revealed 18 carbon signals: a carbonyl (δC 173.8 (C-1”)), a symmetrical benzene , 138.0 (C-4’), 132.0 (C-1’), 103.6 ), a double bond (δC 134.2 (C-3), 122.9 (C-2)), five methylenes (δC 64.9 (C-1), 34.3 (C-2”), 31.4 (C-4”), 24.7 (C-3”), 22.4 (C-5”)), and four methyls (δC 56.1 (C-2*OMe), 61.0 (C-OMe), 14.0 (C-6”)). The NMR data indicated that 1 was a phenylpropanoid, which was very similar with those of 3 [3, the only difference is a hexanoyl (δC 173.8 (C-1”), 34.3 (C-2”), 24.7 (C-3”), 31.4 (C-4”), 22.4 (C-5”), 14.0 (C-6”)) in 1 replaced the isovaleryl (δC 173.0 (C-1”), 43.4 (C-2”), 25.7 (C-3”), 22.4 ) in 3. The 1H-1H COSY correlations  and NMR spectra data. The NMR data of 2 was closely similar with those of 1, which suggested that 2 was also a phenylpropanoid. The only difference is that a 4”-methyl-hexanoyl (δC 174.0 (C-1”), 32.1 (C-2”), 31.4 (C-3”), 34.0 (C-4”), 29.1 (C-5”), 18.8 (C-6”)) in 2 replaced the hexanoyl (δC 173.8 (C-1”), 34.3 (C-2”), 24.7 (C-3”), 31.4 (C-4”), 22.4 (C-5”), 14.0 (C-6”), 11.4 (C-7”)) in 1. The 1H-1H COSY correlations between H-2” and H-3”, H-3” and H-4”, H-4” and H-5”, H-4” and H-7”, H-5” and H-6”, and the HMBC cross-peaks of H-2” with C-1”, confirmed the presence of the 4”-methyl-hexanoyl portion in 2. In the HMBC spectrum, the cross-peak of H-1 with C-1” suggested that the 4”-methyl-hexanoyl located at C-1 , human gastric carcinoma (BGC-823), and human lung adenocarcinoma (A549). The results indicated that the 90% acetone extract showed cytotoxicity against the A549 cell line (IC50 = 0.98 ± 0.1 μg/mL), compound 6 showed cytotoxicities against HeLa (IC50 = 0.4 ± 0.1 μM) and BGC-823 (IC50 = 0.9 ± 0.2 μM) cancer cell lines, and compound 19 showed cytotoxicities against HeLa (IC50 = 1.5 ± 0.4 μM), BGC-823 (IC50 = 7.0 ± 0.8 μM) and A549 (IC50 = 10.6 ± 1.5 μM) cancer cell lines.The −1. UV spectra were measured on SHIMADZU UV-2401PC spectrometer . NMR spectra were conducted on Bruker ARX-600 spectrometers  with TMS as internal standard, chemical shift (δ) was expressed in ppm, and coupling constants (J) in Hz. ESI and HR-ESI-MS were taken on an API Qstar-Pulsar-1 mass spectrometer .Spectra were recorded on a Bio-Rad FTS-135 spectrometer  with KBr pellets, ν in cmSabina gaussenii (Cheng) Cheng et W. T. Wang were collected from Kunming Botany Garden, Yunnan Province, People’s Republic of China, in August 2010. It was identified by Prof. Wei-bang Sun at Kunming Institute of Botany, Chinese Academy of Sciences.Branches and leaves of S. gaussenii were extracted with 90% acetone (3 × 40 L) at room temperature and then concentrated under reduced pressure. The concentrated acetone extract (910 g) was dissolved in 60 °C water and partitioned with petroleum ether, EtOAc, and n-BuOH, respectively, to afford petroleum ether fraction (170 g), EtOAc fraction (130 g), and n-BuOH fraction (250 g).The powdered air-dried branches and leaves (13 kg) of 2O  to produce thirteen subfractions A−M. Fraction C (41 g) was separated on a silica gel column and eluted with gradient mixtures of petroleum ether-acetone (from 20:1 to 1:1) and then separated on a column of RP-C18 silica gel  to yield five major components, with each purified by semipreparative HPLC  to afford 1 (2.9 mg), 2 (2.3 mg), 3 (16 mg), 10 (11 mg), and 20 (26 mg), respectively. Fraction E was chromatographed on a RP-C18 silica gel column  and then purified by semipreparative HPLC with CH3CN–H2O  as the mobile phase to give compounds 4 (29 mg), 11 (21 mg), 12 (17 mg), 13 (35 mg), 14 (11 mg), 16 (13 mg), and 18 (11 mg), respectively. The EtOAc fraction was subjected to silica gel column  to yield five subfractions N−R. Fraction P was chromatographed on a RP-C18 silica gel column  to give 5 (44 mg), 6 (28 mg), 7 (25 mg), 15 (27 mg), and 19 (81 mg), respectively. The n-BuOH fraction was subjected to silica gel column , and then subjected to RP-C18 column and eluted with MeOH–H2O (65:35) to obtain compounds 8 (32 mg), 9 (99 mg), and 17 (28 mg).The petroleum ether fraction (170 g) was separated on an MCI gel column eluted with MeOH–H3’,4’,5’-Trimethoxycinnamyl caproate (1): colorless oil. UV λmax (CH3OH) nm (log ε): 270 (4.32), 221 (4.64). IR (KBr) νmax (cm−1): 2957, 2935, 1735, 1583, 1507, 1462, 1419, 1242, 1128. 1H- and 13C-NMR: m/z 345.1674 .3’,4’,5’-Trimethoxycinnamyl 4”-methyl-caproate (2): colorless oil. UV λmax (CH3OH) nm (logε): 270 (3.52), 220 (3.86). IR (KBr) νmax (cm−1): 2959, 2928, 1735, 1584, 1508, 1462, 1420, 1242, 1128. 1H- and 13C-NMR: m/z 359.1842 .1–20) against the HeLa, BGC-823, and A549 cancer cell lines were measured using a sulforhodamine B  assay as described in the literature [2 at 37 °C, cells were fixed with 25 μL of ice-cold 50% trichloroacetic acid and incubated at 4 °C for 1 h. After washing with distilled water and air-drying, the plate was stained for 15 min with 100 μL of 0.4% SRB in 1% glacial acetic acid. The plates were washed with 1% acetic acid and air-dried. For reading the plate, the protein-bound dye was dissolved in 100 μL of 10 mM Tris base. The absorbance was measured at 560 nm on a microplate spectrophotometer . All tests were performed in triplicate, and results are expressed as IC50 values.The cytotoxicities of the 90% acetone extract and compounds . The next bioassay guided isolation led to the discovery of two cytotoxic compounds, compound 6 showed cytotoxicities against HeLa (IC50 = 0.4 ± 0.1 μM) and BGC-823 (IC50 = 0.9 ± 0.2 μM) cancer cell lines, and compound 19 showed cytotoxicities against HeLa (IC50 = 1.5 ± 0.4 μM), BGC-823 (IC50 = 7.0 ± 0.8 μM), and A549 (IC50 = 10.6 ± 1.5 μM) cancer cell lines. The result indicated that the podophyllotoxin type and the diterpene type compounds were the major cytotoxic constituents in this species, which might be worthy of more extensive investigation so that more novel bioactive compounds can be discovered in the future.This work was part of a series of investigations on anti-tumor compounds from Gymnospermae plants. Compounds