Klebsiella pneumoniae and cyclodextrin glucanotransferase (CGTase) was investigated. K. pneumoniae converted α- and δ-tocopherols into the corresponding β-glucosides in 10 and 8% yield, respectively. CGTase glycosylated α-tocopheryl β-glucoside to α-tocopheryl β-maltoside (51%) and α-tocopheryl β-maltotrioside (35%). On the other hand, δ-tocopheryl β-glucoside was converted into the corresponding β-maltoside (45%) and β-maltotrioside (29%) by CGTase. The β-glucoside of α-tocopherol, and β-glucoside and β-maltoside of δ-tocopherol showed inhibitory effects on IgE antibody production and on histamine release from rat peritoneal mast cells.The glycosylation of α- and δ-tocopherols using  Tocopherols have been known to be essential nutrients for reproduction since 1922 . RecentlKlebsiella pneumoniae and cyclodextrin glucanotransferase (CGTase), into the corresponding β-glucosides and β-malto-oligosaccharides as tocopherol derivatives which have potent anti-allergic activity. The tocopheryl glycosides synthesized here could be useful as food additives with anti-allergic activity.Glycosylation is a characteristic reaction which converts water-insoluble and unstable aromatic compounds into the corresponding water-soluble and stable compounds to improve their bioavailability and pharmacological properties. Therefore, tocopheryl glycosides (tocopherol derivatives) are of importance for the food and drug industries. Considerable effort has been made so far to synthesize tocopheryl glycosides by chemical methods to enhance the water-solublility of tocopherols ,11. Addii.e., K. pneumoniae, Xanthomonas campestris, and Lactobacillus delbrueckii, are known to produce exopolysaccharides which are excreted into the culture medium + ion peak at m/z 911.4851 (HRFABMS), consistent with a molecular formula of C45H76O17 . The 1H-NMR spectrum of 8 showed three proton signals at δ 4.37 , 4.59 , and 5.10 , which indicated the presence of one β-anomer and two α-anomers in the sugar moiety. The sugar component in 8 was shown to be maltotriose based on the coupling pattern of the sugar proton signals and the chemical shifts of the sugar carbon signals . Substrates, α- and δ-tocopherols which were : 4 g Bacto-peptone, 4 g yeast extract, and 25 g glycerol. The culture medium for X. campestris was composed of 10 g of maltose, 6 g of Bacto-peptone, 0.8 g of yeast extract, and 0.4 g of MgSO4, and that for L. delbrueckii consisted of 20 g of lactose, 5 g of yeast nitrogen base, 20 g of Bacto-casitone, 1 g of sorbitan monooleate, 2 g of K2HPO4, 0.1 g of MgSO4, 0.05 g of MnSO4, 2 g of ammonium citrate, and 5 g of sodium acetate. The cells were grown with continuous shaking on a rotary shaker (120 rpm) at 30 °C.n-butanol. The n-butanol fraction was purified by preparative HPLC to give tocopheryl β-glucoside.Each culture of three bacteria was individually grown in 500-mL conical flasks containing 200 mL of culture medium at 30 °C. Prior to use for the experiments, the cells were harvested by centrifugation at 8,000 g for 15 min. Tocopheryl β-glucosides were prepared as follows. A total of 2 mmol of tocopherol (0.2 mmol/flask) was added to ten 300-mL conical flasks containing 5 g of bacterium cells and 1 g of glucose in 100 mL of freshly prepared culture medium. The mixture was incubated with continuous shaking on a rotary shaker (120 rpm) for 48 h at 30 °C. The reaction mixture was centrifuged at 8000 g for 15 min to remove the cells and the supernatant was extracted with i.e., tocopheryl β-maltoside and tocopheryl β-maltotrioside. Spectral data of new compounds are as follows:To a solution containing 0.1 mmol of tocopheryl β-glucoside and 5 g of starch in 25 mM of sodium phosphate buffer (pH 7.0) was added 100 U of CGTase. After stirring of the reaction mixture at 40 °C for 24 h, the mixture was centrifuged at 3,000 g for 10 min. The supernatant was subjected on to a Sephadex G-25 column equilibrated with water to remove CGTase. The fractions containing glycosides were purified by preparative HPLC to give two tocopheryl β-glucooligosaccharides, δ-Tocopheryl β-maltoside (7): White amorphous powder; FABMS m/z: 749.4455 [M+Na]+; 1H-NMR : δ 0.86 , 0.87 , 1.01-1.51 , 1.25 , 1.50-1.56 , 2.09 , 2.73 , 2.76 , 3.09-3.99 , 4.41 , 5.11 , 6.69 , 6.73 ; 13C-NMR : δ 16.3 (8-CH3), 20.4 , 22.0 (C-11), 23.0 , 23.7 (C-3), 24.5 (1-CH3), 25.4 (C-15), 25.7 (C-19), 28.8 (C-21), 32.6 (C-2), 33.9 , 38.5 , 40.5 (C-20), 40.8 (C-10), 61.5 (C-6''), 62.8 (C-6'), 71.5 (C-4''), 74.1 (C-2''), 74.2 (C-5''), 74.8 (C-2'), 75.1 (C-3''), 76.7 (C-1), 76.8 (C-5'), 77.6 (C-3'), 80.7 (C-4'), 101.5 (C-1'), 102.9 (C-1''), 116.3 (C-5), 118.7 (C-7), 122.2 (C-4), 127.6 (C-8), 148.5 (C-9), 151.5 (C-6).δ-Tocopheryl β-maltotrioside (8): White amorphous powder; FABMS m/z: 911.4851 [M+Na]+; 1H- NMR (CD3OD): δ 0.86 , 0.87 , 1.00-1.55 , 1.25 , 1.51-1.57 , 2.09 , 2.73 , 2.77 , 3.05-3.99 , 4.37 , 4.59 , 5.10  6.69 , 6.73 ; 13C- NMR (CD3OD): δ 16.3 (8-CH3), 20.5 , 22.0 (C-11), 22.9 , 23.7 (C-3), 24.5 (1-CH3), 25.4 (C-15), 25.5 (C-19), 28.8 (C-21), 32.6 (C-2), 33.9 , 38.5 , 40.5 (C-20), 40.8 (C-10), 61.9 (C-6''), 62.2 (C-6'''), 62.5 (C-6'), 70.8 (C-4'''), 73.5 (C-5''), 73.7 (C-2''), 74.1 (C-2'''), 74.5 , 75.1 , 76.7 (C-1), 76.8 (C-5'), 77.7 (C-3'), 80.5 (C-4'), 81.5 (C-4''), 102.0 (C-1'), 102.5 (C-1''), 102.8 (C-1'''), 116.3 (C-5), 118.9 (C-7), 122.2 (C-4), 127.6 (C-8), 148.5 (C-9), 151.5 (C-6).3 and pertussis toxin were used as the adjuvants . Sensitization was made by injection of a mixture (0.6 mL) of the antigen and the adjuvant into the paws of each rat . Paw edema was measured 24 h after injection and the treated rats were divided in groups with an equal average swelling volume. Each sample was dissolved in physiological saline containing 10% Nikkol and the solution containing 0.4 mg of sample was injected daily into each rat for 11 d starting on the day of grouping. Hydrocortisone was used as the positive control. The amount of IgE was measured by the passive cutaneous anaphylaxis method on the 15th day [The inhibitory action of tocopheryl β-glycosides on IgE antibody formation was examined as follows. Glutenin (1 mg/rat) was used as the antigen, and Al(OH)15th day . The res2, 5.6 mM glucose, and 20 mM HEPES (pH 7.4) to give approximately 104 mast cells/mL. Cell viability was always greater than 90% as judged by the trypan blue exclusion test. Mast cells were preincubated with the test compound (1 μM) for 15 min at 37 °C, and subsequently exposed to compound 48/80 at 0.35 μg/mL. Histamine release was determined by a fluorometric assay according to the previously reported method [Effects of tocopheryl β-glycosides on histamine release from rat peritoneal mast cells were determned according to the previously reported method . Peritond method , and wasi.e., β-glucosides, β-maltosides, and β-maltotriosides of α- and δ-tocopherols, were successfully synthesized through sequential biocatalytic glycosylation by K. pneumoniae and CGTase. Three tocopheryl β-glycosides showed suppressive action on IgE antibody formation and exhibited inhibitory effects on histamine release from rat peritoneal mast cells. Particularly, the β-glucosides of both α- and δ-tocopherols showed significant anti-allergic activity among the tocopheryl β-glycosides synthesized here. It should be emphasized that the two biocatalysts, i.e., K. pneumoniae and CGTase, are potentially useful for practical production of tocopheryl glycosides as food additives.Tocopheryl β-glycosides (tocopherol derivatives),


The simple and caged xanthones from Clusiaceae showed significant antineoplastic activity. This study aims to identify structural diverse xanthones and search for novel antitumor natural products from this family plants.1a and 1b were elucidated mainly through comprehensive NMR and MS spectroscopic data, and their absolute configurations were determined by the comparison of the experimental and calculated electronic circular dichroism.The structures of new compounds 1a and 1b), along with thirty known analogues (2–31), were isolated from extracts of the stems and leaves of C. cochinchinense. Preliminary biological assay of some isolates against HL-60, PC-3, and MDA-MB-231 cancer cell lines.A pair of new xanthone enantiomers, (+)- and (−)-cracochinxanthone A  contains supplementary material, which is available to authorized users. Cratoxylum cochinchinense Blume (Clusiaceae) is a deciduous shrub tree growing abundantly in southeast Asian countries + .Cracochinxanthone A : yellow 1a). Yellow needles; c 0.06 MeOH); ECD (MeOH 0.58) λmax (Δε) 241 (+ 3.65), 270 (− 4.22), 317 (− 1.91) nm.(+) Cracochinxanthone A (1b). Yellow needles; c 0.07 MeOH); ECD (MeOH 0.70) λmax (Δε) 242 (− 4.06), 273 (+ 3.72), 316 (+ 1.45) nm.(−) Cracochinxanthone A  was used as a positive control. Detailed methodology for the cell growth inhibition test has been described in a previous report . The IC51) was obtained as a yellow needle, and its molecular formula was determined as C23H22O5 with 13° of unsaturation from the HRESIMS data of [M + H]+ ion at m/z 379.1541 . The UV bands observed at λmax 319, 268 and 235 nm suggested a xanthone skeleton [1H NMR data showed signals for a hydrogen bond hydroxy proton at δH 13.35 , a free phenolic hydroxy proton at δH 9.95 , a set of ABX coupling system aromatic protons at δH 7.40 , 7.46  and 7.28 , along with the typical signals of a 3-methylbut-2-enyl (prenyl) moiety at δH 3.43 , 5.19 , 1.82  and 1.62 . The remaining proton signals were assigned to a dihydrofuran ring with an isopropenyl group at δH 2.95 , 2.82 , 4.24 , 4.89 and 4.75 , 1.76 , and the corresponding carbon signals at δC 147.1 (C-3′), 110.0 (C-4′), 74.8 (C-2′), 28.9 (C-1′) and 18.1 (C-5′) were assigned through HSQC correlations [13C NMR displayed 23 carbon resonances including one conjugated carbonyl carbon, sixteen aromatic/olefinic carbons, three methyl, two methylene and one oxygenated methine (Table δH 2.95) and Hb-1′ (2.82) to C-1 (δC 158.0), C-2 (108.0) and C-3 (163.5), as well as from H-2′ (δH 4.24) to C-2. The cross peaks between H-1″ (δH 3.43) and C-3, C-4a (δC 152.9) and C-4 (106.3) confirmed the location of the prenyl group at C-4. The correlations of H-5 (δH 7.46) with C-7 (δC 153.8), C-8a (120.1) and C-10a (149.0), H-8 (δH 7.40) with C-6 (124.4), C-9 (δC 179.9) and C-10a, H-6 (δH 7.28) with C-8 (δC 107.9) and C-10a, and OH-7 (δ 9.95) with C-6, C-7 and C-8 indicated that the free phenolic hydroxy located at C-7  and − 11.3 (c 0.07 MeOH), respectively. Therefore, the structures of 1a and 1b were named as (+) and (−)-cracochinxanthone A.Cracochinxanthone A might be a racemic mixture due to the smooth ECD curve as well as close to zero optical rotation. Subsequent chiral HPLC separation of  1b Fig. . And the2) [3) [4) [5) [6) [7) [8) [9) [10) [11) [12) [13) [14) [15) [16) [17) [18) [19) [20) [21) [22) [23) [24) [25) [H-pyranoxanthone (26) [27) [1 (28) [29) [30) [31) [By comparison with those data from the literatures, the known analogues were identified as cochinchinoxanthone (2) , 1,4,7-t2) [3) , gentise [3) [4) , 1,6-dih [4) [5) , 1,7-dih [5) [6) , 1,7-dih [6) [7) , 1,7-dih [7) [8) , 1,7-dih [8) [9) , 1,5,6-t[9) [10) , 1,4,7-t10) [11) , 1,5,6-t11) [12) , 1,3,5,612) [13) , 1,3,6,713) [14) , 1,3,6-t14) [15) , cratoxa15) [16) , 1,2,4-t16) [17) , 1,3,7-t17) [18) , dulcisx18) [19) , cudratr19) [20) , γ-mango20) [21) , 1,3,7-t21) [22) , cochinc22) [23) , cochinc23) [24) , prunifl24) [25) , 1,3,5-tone (26) , prunifl26) [27) , xanthon [1 (28) , osajaxa28) [29) , cochinc29) [30) , 1,7-dih30) [31) .1a, 1b, 3, 4, 6, 10, 12, 19–25, 27 and 28 displayed antiproliferative effect against HL-60 cells with IC50 values ranging from 1.00 to 19.78 μM, especially 23 bearing one prenyl and one geranyl groups with an IC50 value of 1.0 μM and 27 possessing a pyran ring with 1-hydroxy-4,4-dimethyl with an IC50 value of 1.89 μM. Compounds 19–25, 27 and 28 exhibited potent inhibitory activity against PC-3 cells with IC50 values ranging from 11.77 to 27.11 μM and compounds 1b, 19–25 and 28 displayed significant cytotoxicity against MDA-MB-231 cells with IC50 values ranging from 7.94 to 18.46 μM, respectively. It is worth to mention that compounds 23–25 possessing one prenyl and one geranyl and 19–22 with two prenyl groups showed high sensitivity against three human cancer cell lines than others without prenyl unit.The antiproliferative activities of some xanthones were evaluated against HL-60, PC-3, and MDA-MB-231 cancer cell lines (Table 1a) and (−)-cracochinxanthone A (1b), along with 30 known analogues (2–31) were isolated from the stems and leaves of C. cochinchinense. The antiproliferative activities of some selected compounds against human HL-60, PC-3, and MDA-MB-231 cancer cell lines were screened by the trypan blue and MTT methods. The polyprenylated or geranylated xanthones exhibited potent cytotoxicity against three human malignant cell lines, which could be further developed as potential lead compounds in the design for the treatment of cancer.A pair of new racemic mixture of xanthones, (+)-cracochinxanthone A (Additional file 1. Minimum standards of reporting checklist.Additional file 2. Supporting information.


FIN219-OE, cry1, FIN219-OE/cry1, cry2, FIN219-OE/cry2, cry1cry2, FIN219-OE/cry1cry2’, not ‘Col-0, FIN219-OE, cry1, cry2, FIN219-OE/cry1, cry1cry2, FIN219-OE/cry2, FIN219-OE/cry1cry2’. The authors have provided a corrected version here.There is an error in panel A of


The transformation of the 4,4′-diaryl- and 4,4′-di(thien-2-yl)-5,5′-bi occurs at lower temperatures in the presence of the thiophiles triphenylphosphine or tetraethylammonium iodide. Optimized reaction conditions and a mechanistic rationale for the thiophile-mediated ring transformation are presented.Thermolysis of 4,4′-dichloro-, 4,4′-diaryl-, and 4,4′-di(thien-2-yl)-5,5′-bi affords the respective 3,6-dichloro-, 3,6-diaryl- and 3,6-di(thien-2-yl)isothiazolo[5,4- Since the first thiazolothiazole (1) was reported in 1960 [d]thiazole (2) which, like thiazolothiazole (1), shares a common C-C bond between the two thiazoles is less well known; only 14 analogues are known [d]isothiazole (3) [d]-isothiazole (4) isothiazoles: (1) 3,6-bis(acylimino)-3H,6H-dithiolodithioles 5 react with hydroxylamine to undergo an Assisted Nucleophilic Ring Opening Ring Closure (ANRORC) style exchange of S for NH to give 3,6-bis(acylamino)isothiazoloisothiazoles 6 isothiazole (8b) isothiazole (8b) [d]isothiazoles like 3 in the materials sciences, we were interested in developing a new complementary route to this ring system.Three strategies are reported for the preparation of isothiazolothiophenes 13 isothiazole (8a) as well as several unstable (2D TLC), unidentified yellow and orange products (Early studies on (one (14) , 3,4-dic14) isothiazole (8a) is a new dihaloisothiazoloisothiazole and a potentially useful biheterole building block. To support its structure, single crystal XRD crystallography was carried out -4,4′-dichloro-5,5′-bi (11a) (0.10 mmol) at ca. 300 °C under argon atmosphere led to the formation of the dichloroisothiazoloisothiazole 8a in a low yield (8%) together with elemental sulfur (S8) (TLC) arylisothiazoloisothiazoles 8b–f in 82–96% yields  (TLC) , entry 1E)-4,4′-dichloro-5,5′-bi 11a into the isothiazoloisothiazole 8a we then investigated the use of thiophilic agents triphenylphosphine, BnEt3NCl or Et4NI, but obtained in each case only an intractable tarry mass. Not surprisingly, dichloroisothiazoloisothiazole 8a, which hosts a variety of highly electrophilic sites  was unstable to the thiophiles. This was partially attributed to the excellent nucleofuge ability of the C3/6 chlorine substituents. This lability was also evident during efforts to carry out substitution of the chlorides by methoxide or pyrrolidine nucleophiles, the former leading to intractable baseline and the latter to a mixture of monocyclic oligosulfides  that could not be isolated pure. Moreover our efforts to carry out Suzuki-Miyaura , Stille  or Sonogashira couplings  led to only traces of product and mainly gave degradation of the ring system. The difficulty in displacing isothiazole C3 chlorides has been previously reported isothiazole (8e) in 92% yield  (2 equiv) in a mix of chloroform/acetic acid (50:50) heated at ca. 70 °C for 5 h afforded the useful 3,6-diisothiazoloisothiazole (16) in 63% yield isothiazoloisothiazole 3% yield . Efforts17 that then collapses to the 14π 4,8-disubstituted [1–3]dithiazinodithiazine 18 which collapses to the thermodynamically more stable 10π 3,6-disubstituted isothiazolo-isothiazole 8  thiophenes could also involve homolytic pathways + .To a stirred solution of  under an argon atmosphere was immersed into a preheated (~230–300 °C) Wood′s metal bath for 0.5 min. On cooling to ca. 20 °C the residue was triturated with n-hexane, filtered and recrystallized to give isothiazolo-isothiazole 8 as colorless crystals  tals see .8 (0.05 mmol) in anhydrous toluene (5 mL) at ca. 20 °C was added Et4NI (0.2 equiv). The reaction mixture was then heated at reflux (ca. 110 °C) until complete consumption of the starting dithiazole 8 (by TLC). The reaction mixture was allowed to cool to ca. 20 °C, the volatiles removed in vacuo, and then the residue was triturated with n-hexane (1.5 mL), the precipitate was filtered and washed (acetone 2 × 1.5 mL) and dried. Recrystallisation of the residue gave the isothiazoloisothiazole 8 as colorless crystals  tals see .4Cl2N2S2 requires C, 22.76; N, 13.27%); δC  155.9, 138.4; νmax (KBr) 1596, 1332, 1188, 1076, 792, 768 cm−1; λmax (CH2Cl2)/nm 236 (log ε 3.31), 304 (3.98); m/z (EI) 214 , 212 , 210 , 175 (11), 114 (22), 88 (50), 70 (100). as colorless prisms, m.p. 210–212 °C (decomp.) (acetone). ; δH  8.00 , 7.59–7.48 ; δC  156.3, 156.1, 132.9, 130.2, 129.3, 127.3; νmax (KBr) 1620, 1420, 1330, 1280, 1160, 978, 802, 763, 692 cm−1; λmax (CH2Cl2)/nm 242 (log ε 4.59), 332 (4.32); m/z (EI) 294 , 191 (77), 146 (37), 88 (100); HRMS m/z (ESI) 295.0336 [M + Na]+ . as colorless blocks, m.p. 203–204 °C ; δH  8.06 , 7.49 ; δC  165.4 , 157.2, 155.9, 130.7 , 130.1, 117.9 ; νmax (KBr) 1600, 1515, 1440, 1420, 1315, 1238, 1180, 1100, 980, 840, 805, 720 cm−1; λmax (CH2Cl2)/nm 242 (log ε 4.05), 330 (3.76); m/z (EI) 330 , 235 (8), 209 (32), 164 (30), 121 (35), 88 (100); HRMS m/z (ESI) 330.0091 [M]+ ., as colorless crystals, m.p. 268–269 °C (acetone); ; δH  7.92 , 7.22 , 3.89 ; δC  162.4, 157.7, 156.7, 129.8, 126.4, 116.3, 56.8 (OMe); νmax (KBr) 1608, 1576, 1532, 1416, 1312, 1296, 1268, 1176, 1024, 832, 800 cm−1; λmax (CH2Cl2)/nm 249 (log ε 4.35), 344 (4.17); m/z (EI) 354 , 311 (2), 221 (22), 177 (27), 134 (30), 88 (100); HRMS m/z (ESI) 355.0546 [M + H]+ ., as colorless prisms, m.p. 238–239 °C (acetone); ; δH  7.52–7.48 , 7.21–7.18 ; δC  155.3, 150.9, 136.7, 129.0, 128.2, 127.7; νmax (KBr) 3095 and 3031 (aryl С-H), 1652, 1536, 1448, 1424, 1344, 1256, 1224, 1048, 852, 800, 760, 704, 680, 664, 624 cm−1; λmax (CH2Cl2)/nm 257 (log ε 4.08), 348 (3.98); m/z (EI) 306 , 223 (3), 197 (20), 165 (9), 153 (20), 109 (25), 88 (100); HRMS m/z (ESI) 306.9463 [M + H]+ ., as colorless crystals, m.p. 250–251 °C (acetone); ; δH  7.93 , 7.76 ; δC  157.5, 154.7, 132.6, 131.7, 128.9, 122.7; νmax (KBr) 1588, 1429, 1401, 1322, 1289, 1184, 1075, 1008, 972, 830, 805, 710, 652 cm−1; λmax (CH2Cl2)/nm 254 (log ε 4.77), 337 (4.44); m/z (EI) 454 , 452 , 450 , 295 (5), 271 (17), 190 (43), 155 (17), 102 (32), 88 (75); HRMS m/z (ESI) 452.8541 [M + H]+ ., as colorless crystals, m.p. 234–235 °C (acetone);   in CHCl3/AcOH (50:50) (12 mL) at ca. 20 °C was added NBS . The mixture was then heated to ca. 70 °C for 7 h then allowed to cool to ca. 20 °C. The precipitate was filtered, washed with diethyl ether (2 × 2 mL), dried to afford the title compound16  as colorless crystals. The combined solvents were evaporated under reduced pressure and separated by column chromatography on silica gel to afford addition quantity of the title compound 16 . Combined yield of 3,6-diisothiazoloisothiazole (16) ; m.p. 255–257 °C (acetone); ; δH 6.25 , 6.06 ; δC  150.0, 141.9, 137.2, 133.8, 131.1, 125.6; νmax (KBr) 3091, 3051, 1656, 1598, 1543, 1452, 1398, 1349, 1299, 1212, 1120, 976, 934, 805, 782, 723, 660 cm−1; λmax (CH2Cl2)/nm 279 (log ε 4.32), 354 (4.32); m/z (EI) 466 , 464 , 462 , 384 (63), 306 (12), 222 (6), 88 (25); HRMS m/z (ESI) 464.7670 (MH)+ .To a stirred solution of 3,6-di(thien-2-yl)isothiazoloisothiazoles in low to high yields. The transformation of the di(het)arylbi(dithiazolylidenes) into di(het)arylisothiazoloisothiazoles can be achieved under milder conditions via thiophile-mediated ANRORC-type reactions with the use of Et4NI (0.2 equiv) as thiophile. The transformation provides access to 3,6-di(thien-2-yl)-substituted isothiazoloisothiazole which can be valued scaffolds in molecular electronic materials.4,4′-Dichloro- and 4,4′-di(het)aryl-5,5′-bi undergo thermolysis to afford the respective 3,6-dichloro- and 3,6-di(het)arylisothiazolo[5,4-


Spongia sp., has led to the isolation of four new scalarane sesterterpenes, scalalactams A–D (1–4). Their chemical structures were elucidated from the analysis of spectroscopic data including 1D-and 2D-NMR as well as MS data. Scalalactams A–D (1–4) possess a scalarane carbon skeleton with a rare structural feature of a γ-lactam moiety within the molecules. Scalalactams A and B (1 and 2) have an extended isopropanyl chain at the lactam ring, and scalalactams C and D (3 and 4) possess a phenethyl group at the lactam ring moiety. Scalalactams A–D (1–4) did not show FXR antagonistic activity nor cytotoxicity up to 100 μM.Intensive study on the chemical components of a Korean marine sponge,  The 1H-NMR spectrum of 1 revealed the presence of two downfield methine protons , together with two methylene systems . The 1H-NMR spectrum of 1 also featured one acetyl group 12-OAc (δH 2.18), one methoxy group 16-OMe (δH 3.44) and seven methyl groups with all singlets . Interpretation of HSQC spectroscopic data of 1  and H-2′ (δH 1.68), and long-range HMBC correlations from H3-4′ (δH 1.24) to C-2′ (δC 43.0), C-3′ (δC 69.9), and C-5′ (δC 28.9) provided the assignment of the 3′-hydroxy isopropanyl unit. Three-bond HMBC correlations from H-15 to C-17 (δC 138.8) and from H3-25 to C-18 (δC 150.9), and from H-1′ (δH 3.56) to C-19 and C-20 (both δC 169.1 not separable), suggested the presence of a pyrrole-2,5-dione moiety in the molecule. Lastly, the 3′-hydroxy isopropanyl unit which was connected through a nitrogen atom in the pyrrole-2,5-dione moiety, was determined from the chemical shifts of H-1′ (δH 3.56)/C-1′ (δC 33.5) and from the observation of three-bond HMBC correlations from H-1′ (δH 3.56) to C-19 and C-20 (both δC 169.1 not separable).A 3′-hydroxyisopropanyl unit was established from the analysis of the 1 were established from the analysis of coupling constants and NOESY spectra. NOESY cross-peaks [H-5/H-9/H-12/H-14/H-16] with large coupling constant of H-9 (J = 13.1 Hz) indicated the axial orientation of C-5, C-9, C-12, C-14, and C-16. The β-configuration of the acetyl group at C-12 was assigned by a coupling constant of H-12  and NOESY correlations between H-12 and H-9, and between H-12 and H-14 + in HRFABMS. The IR spectrum showed the presence of an ester at 1737 cm−1 and 1238 cm−1. The 1H-NMR spectrum of 3 revealed the presence of five aromatic protons , three downfield methine protons , together with two downfield methylene systems . The 1H-NMR spectrum of 3 also displayed one acetyl group 12-OAc , and two methoxy protons [16-OMe  and 20-OMe ], five methyl singlets . Analysis of HSQC spectroscopic data of 3 indicated eight methyl, nine methylene, eleven methine, and nine fully-substituted carbons. The structure of 3 was established from the interpretation of 1H-1H COSY and HMBC spectroscopic data. 1H-1H COSY cross-peaks  provided two sets of three-carbon and three sets of two-carbon units. 1H-1H COSY correlations of H-4′/H-4′′and H-6′ to H-5′/H-5′′ also permitted a phenyl group. The phenyl group was further extended with two carbon units from the observation of long-range HMBC correlations from the methylene protons H-2′ to carbons C-3′, and C-4′/C-4′′. The scalarane moiety was established from the interpretation of HMBC spectroscopic data. In particular, the long-range HMBC correlations from two methyl singlets H3-21 and H3-22 to C-3, C-4, and C-5; from the methyl singlet H3-23 to C-1, C-5, C-9, and C-10; from the methyl singlet H3-24 to C-7, C-8, C-9, and C-14; from the methyl singlet H3-25 to C-12, C-13, C-14, and C-18; from a methine proton H-20 to 20-OMe, C-17, C-18, and C-19 allowed the scalarane carbon skeleton to be established. Additionally, the chemical shifts difference of C-17 (δC 148.0) and C-18 (δC 146.1) secure the position of the carbonyl at C-19  with large coupling constant of H-9 (J = 12.3 Hz) indicated the axial orientation of C-5, C-9, C-12, C-14, and C-16. NOESY cross-peaks  suggested the β-congifuration of C-22, C-23, C-24, and C-25 [J = 11.1 Hz) also indicated that H-12 had an axial orientation. The β-configuration of the methoxy group at C-20 was determined from the observation of a NOESY correlation between H-16 (δH 3.75) and H-20 (δH 5.10).Relative configurations of 4 was deduced as C36H51NO5 based on the protonated peak at m/z 578.3855 [M + H]+ in HRFABMS. The 1H-NMR spectrum of 4 was almost identical to that of 3 except for the absence of one methyl singlet and the presence of an exchangeable proton. The observation of 1H-1H COSY correlations between 20-OH (δH 1.49) and H-20 (δH 5.05) indicated that 4 had a hydroxy group at C-20 instead of a methoxy group. Interpretation of 2D-NMR spectroscopic data allowed the planar structure of 4 to be assigned as shown.The molecular formula of 4 was established by analysis of coupling constants and NOESY spectroscopic data. Similar NOESY cross-peaks were observed with compound 3 which suggested the same orientation of the carbons. A large-magnitude coupling constant for H-12  suggested the β-configuration of the acetyl group at C-12, while a NOESY correlation between H-14 and H-16 indicated the β-configuration of the methoxy group at C-16, respectively. The β-configuration of the hydroxy group at C-20 was also assigned from the observation of NOESY correlations between 20-OH (δH 1.47) and 16-OMe, and between H-20 and H-16. There is possibility that these new isolates are methylated artifact based on the presence of O-methoxy group at C-16 and C-19. It wasn’t possible to address this as the supply of the raw sponge material was limited.Relative stereochemistry of Spongia sp. showed farnesoid X-activated receptor (FXR) antagonistic activity and cytotoxicity against a CV-1 monkey kidney cell line + .2):Scalalactam B ; UV λmax (log ε) 210 (3.83), 232 (2.09); IR (KBr) ν max: 3391, 1763, 1681, 1233 cm−1; 1H-, 13C-, and 2D-NMR data, m/z 540.3690 [M + H − H2O]+ .3):Scalalactam C ; UV λmax (log ε) 210 (3.83), 254 (2.12) nm; IR (KBr) ν max: 3392, 1760, 1683, 1238 cm−1; 1H- and 13C-NMR data, m/z 592.3995 [M + H]+ .4):Scalalactam D ; UV λmax (log ε) 210 (3.83), 254 (2.11) nm; IR (KBr) ν max: 3392, 1765, 1681, 1235 cm−1; 1H-and 13C-NMR data, m/z 578.3855 [M + H]+ .2 at 37 °C for 24 h. Transient cotransfection with pCMX-hFXR, pCMX-β-GAL and Tk-(EcRE)6-LUC were carried out using SuperFect , according to the manufacturer’s instructions. After 24 h incubation, cotransfected cells were treated with a control vehicle (DMSO), or the indicated compounds, for the FXR antagonist test in the presence of 50 μM chenodeoxycholic acid . Cells were harvested at 24 h, and luciferase activities were assayed as described [CV-1 cells were seeded in 96-well plates in Dulbecco’s modified Eagle’s medium (GIBCO) supplemented with 10% lipid-depleted fetal bovine serum in humidified air containing 5% COescribed ,33. Luci2 at 37 °C. After 24 h incubation, indicated compounds were administrated at various concentrations up to 100 μM. Cells were harvested at 24 h and were incubated with 1 mg/mL 3-4,5-dimethylthiazol-(2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution for 1 h at 37 °C. DMSO was added to each well in order to dissolve the produced purple formazan crystals, and the absorbance of each well was measured at 450 nm using an ELISA reader. The experiment was carried out in triplicate and repeated three times.CV-1 cells were seeded in 96-well plates in DMEM supplemented with 10% fetal bovine serum in humidified air containing 5% CO


The structures of these compounds were determined by spectroscopic and chemical transformation methods. Graviquinone (1) showed the strongest cytotoxicity against MCF-7, NCI-H460, and SF-268 cell lines. Methyl 2,5-dihydroxycinnamate (4), graviphane (13), and dehydrograviphane (14) exhibited very potent DPPH scavenging activity compared with α-tocopherol. Methyl 2,5-dihydroxycinnamate (4) and bis-norstriatol (17) demonstrated strong inhibition of L-DOPA oxidation by mushroom tyrosinase compared with kojic acid.Three new compounds: Graviquinone ( Grevillea robusta A. CUNN (Proteaceae), commonly known as “silky oak”, is native to Australia  implied that a hydroxyl group was attached to C-3 (δ 69.2). The long chain substituent that was apparently attached to C-5 (δ 33.0) was assigned based on the HMBC correlations of H-5 [δ 1.60 ] with C-1' (δ 36.5). The NOE correlations between H-3 and H-5 suggested the cis relative configuration of the two substituents in the cyclohexanone , 6.53 , 6.75 , 6.82 , 6.98 , and 7.96  were very closely related to methyl 2,5-dihydroxycinnamate 4 benzene-1,3-diol (18) benzene-1,3-diol (18), and gravirobustol A (19) (545 mg), which unfortunately could not be obtained as a pure compound. Using the same elution conditions as for fraction 6, fraction 7 produced a mixture of gravicycle (5) and dehydrogravicycle (6) (52 mg). However, extensive efforts were made through repeated CC to purify these components for identification.The 3 extract was subjected to silica gel CC, eluting with CHCl3-MeOH (20:1) in a step gradient that gradually increased the polarity with pure MeOH to afford five fractions. Fraction 2 was separated using silica gel CC, eluting with a gradient of (i-Pr)2O-MeOH (50:1 to pure MeOH), to give p-nitrophenol (6 mg), methyl p-hydroxybenzoate (4 mg), 4-hydroxybenzaldehyde (6 mg), cinnamic acid (1 mg), and graviquinone (1) (1.8 g), successively. Fraction 3 was purified by silica gel CC, eluting with (i-Pr)2O-MeOH (50:1 to pure MeOH), to obtain itaconic acid 4-methyl ester (2 mg), 4-hydroxyacetophenone (2 mg), methyl 3,4-dihydroxybenzoate (2 mg), and rhamnocitrin (4 mg). Fraction 4 was separated using silica gel CC, eluting with (i-Pr)2O-Me2CO (5:1 to pure Me2CO), to afford methyl coumarate (4 mg) and 5-ethoxy-2-hydroxycinnamate (3) (5 mg). Using the same elution conditions as for fraction 4, fraction 5 produced hydroquinone (2 mg), p-hydroxybenzoic acid (3 mg), and grasshopperketone (5 mg).The CHCl3-MeOH (20:1 to pure MeOH) to yield twelve fractions. Fractions 1 and 2 were further purified by silica gel CC, eluting with a gradient of CHCl3-MeOH (6:1 to pure MeOH), to give quercetin (30 mg) and kaempferol (4 mg), respectively. Fraction 3 was separated using silica gel CC, eluting with CHCl3-Me2CO (10:1 to pure Me2CO), to afford methyl 2,5-dihydroxycinnamate (4) (1.1 g), eriodictyol-7-methyl ether (24 mg) and p-coumaric acid (18 mg). Fraction 5 was chromatographed using the same elution conditions as for fraction 3 to give sakranetin (10 mg), rhamnetin (18 mg), and 7-O-methylrutin (5 mg).The EtOAc extract was subjected to silica gel CC, eluting with a gradient of CHClThe cytotoxicity assay was carried out according to procedures that have been previously described in the literature .1, 4, 5, 7, 8, 11−18, and 20 was measured with DPPH• using the method of Chiu et al. + . 1H-NMR (acetone-d6) δ 3.64 , 5.49 , 6.13 , 6.19 , 6.63 , 6.82 ; 13C NMR (acetone-d6) δ 50.2 (9-OCH3), 68.1 (C-4), 120.3 (C-8), 126.5 (C-2 and -6), 145.5 (C-7), 147.9 (C-3 and -5), 164.8 (C-9), 183.6 (C-1).cis-3-Hydroxy-5-pentadecylcyclohexanone (2). White amorphous powder mp 62−64 °C [α]D −101°  UV (CHCl3) maxλ (log ε) 241 (3.1), 280 (2.9) nm IR (KBr) maxν 3424, 1709, 1595, 1469 cm−1 EIMS m/z (rel. int.) 324 , 306 (6), 113 (100), 95 (19); HR-EIMS m/z 324.3206 [M]+ . 1H NMR (CDCl3) δ 0.88 , 1.25 , 1.28 , 1.31–1.45 , 1.60 , 1.94 , 2.21 , 2.33 , 2.37 , 2.72 , 3.91 H, H; 13C-NMR (CDCl3) δ 14.1 (C-15'), 22.7 (C-14'), 26.6 (C-2'), 29.3–29.7 (C-3'−12'), 31.9 (C-13'), 33.0 (C-5), 36.5 (C-1'), 41.0 (C-4), 47.0 (C-6), 50.9 (C-2), 69.2 (C-3), 208.8 (C-1).Methyl 5-ethoxy-2-hydroxycinnamate (3). Yellow amorphous powder. mp 98−100 °C. UV (MeOH) maxλ (log ε) 249 (3.65), 278 (3.77), 351 (3.41) nm. IR (KBr) maxν 3404, 1705, 1630, 1455 cm−1. EIMS m/z (rel. int.) 222 , 162 (32), 149 (24), 134 (100), 107 (33), 105 (38), 77 (81); HR-EIMS m/z 222.0891 [M]+ . 1H NMR (CDCl3) δ 1.39 , 3.81 , 3.98 , 5.68 , 6.53 , 6.75 , 6.82 , 6.98 , 7.96 ; 13C NMR (CDCl3) δ 14.9 (5-OCH23CH), 51.7 (9-OCH3), 64.2 (5-O2CHCH3), 113.5 (C-6), 117.3 (C-3), 118.4 (C-8), 118.5 (C-4), 122.1 (C-1), 140.1 (C-7), 149.2 (C-2), 152.9 (C-5), 168.1 (C-9).1), cis-3-hydroxy-5-pentadecylcyclohexanone (2), and methyl 5-ethoxy-2-hydroxycinnamate (3), and thirty-eight known compounds were isolated from the leaves of G. robusta. Graviquinone (1) showed the strongest cytotoxicity against the tested tumor cell lines. In addition, our results also indicated that the alkyl chain structures, either cyclic or straight chain, had no impact on the anti-cancer activity of alkylresorcinols. Subsequently, methyl 2,5-dihydroxycinnamate (4) showed very potent DPPH inhibition activity with IC50 value of 0.53 μM, as compared with α-tocopherol. Moreover, methyl 2,5-dihydroxycinnamate (4) and bis-norstriatol (17) demonstrated strong inhibition activities for L-DOPA oxidation by a mushroom tyrosinase, with IC50 values of 69.22 and 65.54 μM, respectively.Three new compounds, graviquinone (


The applicability of the supports to oligonucleotide synthesis by the phosphoramidite strategy has been demonstrated by assembling a 3′-TTT-5′ trimer from commercially available 5′-O-thymidine 3′-phosphoramidite. To simplify the coupling cycle, the 5′-O- protecting group has been replaced with an acetal that upon acidolytic removal yields volatile products. For this purpose, 5′-O-(1-methoxy-1-methylethyl)-protected 3′-s of thymidine (5a), N4-benzoyl-2′-deoxycytidine (5b) and N6-benzoyl-2′-deoxyadenosine (5c) have been synthesized and utilized in synthesis of a pentameric oligonucleotide 3′-TTCAT-5′ on the permethylated cyclodextrin support 11c.Novel soluble supports for oligonucleotide synthesis  This protocol has been applied to the synthesis of both oligonucleotides and their phosphoromonothioate analogs by the phosphoramidite − signal at m/z 2335.71 referring to the deprotected product appeared, no sign of the [M−H]− signal of 2407.75 corresponding to the protected support was observed.The synthesis of 3′-TTT-5′ was then repeated using 11a was then subjected to coupling with 1.5 equiv. of 5a in MeCN under nitrogen, using 4,5-dicyanoimidazole as an activator (6 h at r.t.), and the phosphite triester obtained was oxidized by addition of I2 in aq. lutidine/THF (30 min at r.t.). Upon DCM/HSO3−-workup, lutidine was partly transferred to the organic phase and, hence, the soluble-supported product obtained by evaporation of DCM still contained traces of lutidine. The residue was dissolved in a 2:1 mixture of dioxane and MeOH and the pH of the solution was adjusted between 4 and 5 to remove the 5′-acetal protection and the reaction was allowed to proceed for 1 h. To remove the traces of reagents and the products formed from the unreacted building block upon oxidation, the support was purified by flash chromatography on a short silica gel column before the next coupling. The overall yield after two couplings, determined by UV-spectroscopy, was 87%, corresponding to 93% coupling yield. The trimer released by ammonolysis was homogeneous by RP-HPLC  mixture of aq. THF and 2,6-lutidine was carried out (45 min). After aq. NaHSO3/DCM work-up, the organic phase was dried over Na2SO4, filtered and evaporated to dryness under reduced pressure. The residue was dissolved in a 2:1 (v/v) mixture of dioxane and MeOH and HCl in dioxane (0.1 mol·L−1) was added to adjust the pH to 4. After 2 h agitation, the mixture was evaporated to dryness and coevaporated with pyridine and MeCN. A rapid column chromatography on a short column was applied to remove the excess of the unreacted building block. Fractions containing the desired product were mixed, evaporated by under reduced pressure and dried under high vacuum. The identity of the support bound dimer was verified by ESI-MS. The same coupling cycle was then repeated by using 5b, 5c and again 5a as building blocks. However, the coupling mixture contained about 20% DCM to ensure complete dissolution of the cyclodextrin-bound starting material. Finally, conventional ammonolysis was carried out to release and deprotect the pentameric oligonucleotide and the support was removed by equilibration between water and DCM. The gravimetrically determined overall yield of the pentamer was 52%, corresponding to 85% average coupling yield. Finally a hetero-sequence containing 2′-deoxyadenosine and 2′-deoxycytidine in addition to thymidine was assembled on support 254), using short wavelength UV, KMnO4 staining or burning with 10% aq. H2SO4 for detection. 1D and 2D NMR spectra were recorded on a Bruker Avence 500 MHz or 400 MHz at 25 °C. The chemical shifts are given in ppm. Mass spectra were recorded on a Bruker Daltonics MicrOTOF-Q spectrometer using ESI ionization. RP HPLC was performed on a Thermo ODS Hypersil C18  column using UV detection at 260 nm.Reactions were monitored by TLC thymidine (1a) was synthesized as described previously +, 493.27 [M+Na]+, 509.24 [M+K]+; calcd. 471.27 [M+H]+, 493.25 [M+Na]+, 509.23 [M+K]+.eviously . Thymidi) in DMF  mL were terature . Positiv4-Benzoyl-5′-O--3′-O-(tert-butyldimethylsilyl)-2′-deoxycytidineN (1b). Commercially available N6-benzoyl-5′-O--2′-deoxycytidine  was dissolved in DMF (5 mL). tert-Butyldimethylsilyl chloride  and imidazole  were added and the reaction was allowed to proceed for 15 h at r.t. The mixture was equilibrated between water and EtOAc. The organic phase was dried on Na2SO4 and concentrated to dryness, giving 1b in 92% yield . The 1H- and 13C-NMR spectra were identical with those reported in literature [m/z obsd. 746.32 [M−H]−; calcd. 746.33 [M−H]−.terature . Negativ6-Benzoyl-5′-O--3′-O-(tertN-butyldimethylsilyl)-2′-deoxyadenosine (1c) was obtained in 89% yield  from commercial N6-benzoyl-5′-O--2′-deoxyadenosine as described above for compd. 1b. 1H-NMR : δ = 0.00 , 0.84 , 2.24–2.29 , 2.35–2.44 , 3.18 , 3.29 , 3.68 , 3.96–4.00 , 4.71–4.77 , 6.38 , 6.74 , 7.20–7.40 , 7.60–8.00 , 8.24 , 8.56 , 9.52 . 13C-NMR  δ = −4.6 (SiMe), −3.9 (SiMe), 17.3 (SiCMe3), 24.8 (SiCMe3), 38.8 (C2′), 54.6 (OMe), 62.9 (C5′), 71.7 (C3′), 84.1 (C4′), 85.7 (DMTr), 86.0 (C1′), 112.7 (DMTr), 116.9 (DMTr), 126.4 (C5), 127.4 (DMTr), 127.6 (Bz), 127.8 (DMTr), 128.3 (Bz), 129.6 (DMTr), 132.1 (Bz), 133.6 (Bz), 135.3 (DMTr), 142.4 (C8), 144.7 (DMTr), 149.5 (C4), 151.4 (C2), 151.5 (C6), 158.3 (DMTr), 164. 9 (C=O). Negative ion ESI-MS: m/z obsd. 770.3509 [M−H]−; calcd. 770.3374 [M−H]−.3′-O-(tert-Butyldimethylsilyl)thymidine (2a). A 1:1:4 (v/v/v) mixture of TFA, water and THF (30 mL) was added dropwise on an ice-bath to compd. 1a dissolved in a minimal volume of THF. After 1.5 h, another portion (30 mL) of the same mixture was added. The progress of desilylation was monitored by TLC and the reaction was quenched after 5.5 h by equilibration between diethyl ether and aq. NaHCO3 (sat). The organic phase was washed with aq. NaHCO3, dried over Na2SO4 and concentrated to white solid foam . The 1H-NMR spectrum  was identical to that reported previously −, 391.13 [M+Cl]−, calcd. 355.1689 [M−H]−, 391.15 [M+Cl]−.eviously . 13C-NMR4-Benzoyl-3′-O-(tert-butyldimethylsilyl)-N2′-deoxycytidine (2b). Compd. 1b  was dissolved in DCM (5 mL) containing 3% dichloroacetic acid (150 μL) and MeOH (2 mL) was added. After 2 h stirring at r.t., DCM/aq. NaHCO3 work-up was carried out and the organic phase was dried over Na2SO4. The solvent was removed by evaporation, and the residue was purified by a silica gel column chromatography (a stepwise gradient of 1–10% MeOH in DCM) to obtain 2b in 90% yield as white solid foam . The 1H- and 13C-NMR spectra were identical with those reported in literature [m/z obsd. 444.20 [M−H]−; calcd. 444.20 [M−H]−. terature . Negativ6-Benzoyl-3′-O-(tert-butyldimethylsilylN)-2′-deoxyadenosine (2c) was obtained in 84% yield  from 1c as described above for 2b. The 1H-NMR spectrum was identical with that reported in literature −; calcd. 468.2067 [M−H]−.terature . 13C-NMR5′-O-(1-Methoxy-1-methylethyl)-3′-O-(tert-butyldimethylsilyl)thymidine (3a). Compound 2a  was dissolved in 1,4-dioxane (5 mL) and 11 equiv. of 2-methoxypropene  and 0.02 equiv. of p-toluenesulfonic acid monohydrate  in dioxane (4 mL) were added. In 1 h, all the starting material had disappeared. The crude product was purified by silica gel column chromatography using EtOAc containing 1% petroleum ether and Et3N as an eluent. The yield was 91%. 1H-NMR : δ = −0.01 , 0.81 , 1.31 Me2), 1.84 , 1.95–2.06 , 2.15–2.25 , 3.14 Me2), 3.47–3.92 , 4.32–4.36 , 6.24–6.26 , 7.46 , 10.00 ; 13C NMR : δ = −4.9 and −4.8 (SiMe2), 12.4 (C5-Me), 17.9 (Si-CMe3), 24.3 (C5′-OC(OMe)Me2), 25.6 (Si-CMe3), 41.2 (C2′), 48.6 (C5′-OC(OMe)Me2), 60.3 (C5′), 72.2 (C3′), 84.7 (C4′), 86.2 (C1′), 100.2 (C5′-OC(OMe)Me2), 110.6 (C5),135.3 (C6), 150.6 (C2), 164.2 (C4). High resolution positive ion ESI-MS: m/z obsd. 451.2241 [M+Na]+; calcd. 451.2240 [M+Na]+. 4-Benzoyl-5′-ON-(1-methoxy-1-methylethyl)-3′-O-(tert-butyldimethylsilyl)-2′-deoxycytidine (3b) was obtained in 91% yield  from compd. 2b, as described above for compd. 3a. 1H-NMR : δ = −0.07 , 0.82 , 1.33 and 1.35 Me2), 2.11–2.17 , 2.43–2.50 , 3.17 Me2), 3.53 , 3.70 , 3.97–4.01 , 4.30–4.34 , 6.17–6.20 , 7.44 , 7.53 , 7.85 ; 8.45 , 9.22 ; 13C-NMR : δ = −4.9 and −4.6 (SiMe2), 17.9 (Si-CMe3), 24.3 (C5′-OC(OMe)Me2), 25.6 (Si-CMe3), 42.1 (C2′), 48.7 (C5′-OC(OMe)Me2), 59.1 (C5′), 70.3 (C3′), 86.3 (C4′), 86.9 (C1′), 96.0 (C5), 100.3 (C5′-OC(OMe)Me2), 127.7 (Bz), 128.8 (Bz), 133.0 (Bz), 133.2 (Bz), 144.7 (C6), 154.7 (C2), 162.3 (C4), 167.0 (Bz). High resolution negative ion ESI-MS: m/z obsd. 516.2564 [M−H]−; calcd. 516.2530 [M−H]−. 6-Benzoyl-5′-O-(1-methoxy-1-methylethyl)-3′-ON-(tert-butyldimethylsilyl)-2′-deoxyadenosine (3c) was obtained in 81% yield  from compd. 2c, as described above for compd. 3a. 1H-NMR : δ = 0.00 , 0.80 , 1.14 Me2, 2.27–2.37 , 2.67–2.73 , 2.92 Me2), 3.36–4.03 , 4.56–4.62 , 6.32 , 7.37 , 7.47 , 7.86 , 8.24 , 8.50 , 9.44 ; 13C-NMR : δ = −6.0 (SiMe2), 17.3 (Si-CMe3), 23.2 and 23.4 (C5′-OC(OMe)Me2), 24.8 (Si-CMe3), 39.7 (C2′), 47.5 (C5′-OC(OMe)Me2), 60.0 (C5′), 72.0 (C3′), 83.9 (C4′), 86.1 (C1′), 99.6 (C5′-OC(OMe)Me2), 124.2 (C5), 127.8 (Bz), 128.2 (Bz), 132.1 (Bz), 133.5 (Bz), 141.9 (C8), 149.4 (C4), 151.5 (C2&C6), 164.9 (Bz). Hugh resolution negative ion ESI-MS: m/z obsd. 540.2663 [M−H]−, calcd. 540.2642 [M−H]−. 5′-O-(1-Methoxy-1-methylethyl)thymidine (4a). Compound 3a  was treated with 2 equiv. of tetrabutylammonium fluoride  in THF (4 mL). After stirring for 4.5 h at r.t., the mixture was equilibrated between EtOAc and aq. NaHCO3, the organic phase was evaporated to dryness and the residue was purified by silica gel column chromatography using DCM containing 1-5% MeOH as eluent. Yield 91% (111 mg). 1H-NMR : δ = 1.39 and 1.41 Me2), 1.93 , 2.14–2.19 , 2.38–2.41 , 3.23 Me2), 3.64 , 3.69 , 4.15 , 4.48 , 6.41 , 7.61 , 9.50 ; 13C-NMR : δ = 12.3 (C5-Me), 24.2 (C5′-OC(OMe)Me2), 40.7 (C2′), 48.6 (C5′-OC(OMe)Me2), 60.8 (C5′), 72.2 (C3′), 84.8 (C4′), 85.8 (C1′), 100.2 (C5′-OC(OMe)Me2), 110.6 (C5), 135.6 (C6), 150.4 (C2), 163.8 (C4). High resolution negative ion ESI-MS: m/z obsd. 313.1424 [M−H]−, calcd. 313.1400 [M−H]−.4-Benzoyl-5′-ON-(1-methoxy-1-methylethyl)-2′-deoxycytidine (4b) was obtained in 81% yield  from 3b as described above for compd. 4a. 1H-NMR : δ = 1.28 and 1.31 Me2), 2.21–2.28 , 2.65–2.73 , 3.23 Me2), 3.67 , 3.76 , 4.20–4.24 , 4.46–4.49 , 4.30–4.41 , 6.31–6.34 , 7.40–7.56 , 7.61  7.93 , 8.48 ; 9.60 ; 13C-NMR : δ = 24.3 (C5′-OC(OMe)Me2), 42.1 (C2′), 48.8 (C5′-OC(OMe)Me2), 60.1 (C5′), 70.6 (C3′), 86.4 (C4′), 87.3 (C1′), 98.2 (C5), 100.3 (C5′-OC(OMe)Me2), 127.7 (Bz), 128.9 (Bz), 133.1 (Bz), 133.6 (Bz), 145.0 (C6), 155.1 (C2), 162.4 (C4), 167.0 (Bz). High resolution negative ion ESI-MS: m/z obsd. 402.1692 [M−H]−; calcd. 402.1665 [M−H]−.6-Benzoyl-5′-ON-(1-methoxy-1-methylethyl)-2′-deoxyadenosine (4c) was obtained in 89% yield  from 3c as described above for compd. 4a. 1H-NMR : δ = 1.26 Me2), 2.40–2.50 , 2.70–2.79 , 3.04 Me2), 3.40–4.10 , 4.56 , 6.45–6.48 , 7.50 , 7.60 , 7.99 , 8.42 , 8.63 ; 9.69 ; 13C-NMR : δ = 23.3 and 23.5 (C5′-OC(OMe)Me2), 39.6 (C2′), 47.5 (C5′-OC(OMe)Me2), 60.3 (C5′), 70.8 (C3′), 83.8 (C4′), 85.9 (C1′), 100.3 (C5′-OC(OMe)Me2), 124.0 (C5), 127.8 (Bz), 128.3 (Bz), 132.2 (Bz), 133.2 (Bz), 141.8 (C8 ), 149.4 (C4), 151.5 (C2), 151.6 (C6), 165.1 (Bz). High resolution negative ion ESI-MS: m/z obsd. 426.1783 [M−H]−; calcd. 426.1777 [M−H]−. 5′-O-(1-Methoxy-1-methylethyl)thymidine 3′-phosphoramidite (5a). Glassware and stirrer bar were dried for 15 hours in an oven (120 °C). All chemicals and reagents were placed in a box under nitrogen. Compd. 4a  was dissolved in dry DCM (20 mL). Triethylamine  and 1-chloro-1-(2-cyanoethoxy)-N,N-diisopropylphosphinamine  were added and the mixture was stirred for 2.5 h at r.t. under nitrogen. The mixture was then loaded onto a silica gel column and the product was eluted with a 50:49:1 mixture of EtOAc, petroleum ether and Et3N. Compd. 5a was obtained as a white foam (yield 85%), which was stored at −18 °C. For the mixture of RP and SP diastereomers: 1H-NMR : δ = 1.17 , 1.33 and 1.34 Me2), 1.84 , 2.20–2.45 , 2.67 , 3.15 and 3.17 Me2), 3.53–4.25 , 4.48–4.59 , 6.24 , 7.52 and 7.54 , 9.80 ; 13C-NMR : δ = 11.3 (C5-Me), 19.7 (OCH2CH2CN), 23.4 (C5′-OC(OMe)Me2), 23.6 (NCHMe2), 38.7 (C2′), 42.6 (C5′-OC(OMe)Me2), 47.8 (NCHMe2), 57.9 (OCH2CH2CN), 60.2 (C5′), 73.4 (C3′), 81.7 (C4′), 84.1 (C1′), 99.8 (C5′-OC(OMe)Me2), 109.9 (C5), 118.1 (OCH2CH2CN), 135.6 (C6), 150.6 (C2), 163.8 (C4); 31P-NMR (CD3CN): δ = 149.3 and 148.9. Positive ion ESI-MS: m/z obsd. 537.25 [M+Na]+; calcd. 537.25 [M+Na]+.4-Benzoyl-5′-ON-(1-methoxy-1-methylethyl)-2′-deoxycytidine 3′--phosphoramidite (5b) was obtained in 86% yield  from 4b as described above for compd. 5a. For the mixture of RP and SP diastereomers: 1H-NMR : δ = 1.20 , 1.40 Me2), 2.20–2.45 , 2.64 , 3.22 Me2), 3.45–4.45 , 4.51–4.60 , 6.25–6.35 , 7.46 , 7.57 , 7.95 , 8.42 , 9.60 ; 13C-NMR : 18.5 (OCH2CH2CN), 22.4 (C5′-OC(OMe)Me2), 22.6 (NCHMe2), 38.9 (C2′), 41.4 (C5′-OC(OMe)Me2), 47.0 (NCHMe2), 57.0 (OCH2CH2CN), 58.2 (C5′), 71.1 (C3′), 85.2 (C4′), 86.0 (C1′), 95.0 (C5), 98.5 (C5′-OC(OMe)Me2), 116.2 (OCH2CH2CN), 126.2 (Bz), 127.2 (Bz), 131.3 (Bz), 131.4 (Bz), 143.3 (C6), 152.8 (C2), 156.6 (C4), 150.8 (C2), 165.5 (Bz); 31P-NMR (CDCl3): δ = 148.5 and 149.0. Negative ion ESI-MS: m/z obsd. 602.28 [M−H]−; calcd. 602.27 [M−H]−.6-Benzoyl-5′-ON-(1-methoxy-1-methylethyl)-2′-deoxyadenosine 3′--phosphoramidite (5c) was obtained in 83% yield  from 4c as described above for compd. 5a. For the mixture of RP and SP diastereomers: 1H-NMR : δ = 1.18 , 1.39 and 1.41 Me2), 2.20–2.45 , 2.66 , 3.22 and 3.24 Me2), 3.43–4.25 , 4.53–4.59 , 6.28 , 7.46 , 7.58 , 7.96 , 8.28 , 8.46 , 9.30 ; 13C-NMR : 18.6 (OCH2CH2CN), 22.5 (C5′-OC(OMe)Me2), 22.7 (NCHMe2), 39.3 (C2′), 41.4 (C5′-OC(OMe)Me2), 47.0 (NCHMe2), 56.9 (OCH2CH2CN), 58.0 (C5′), 70.9 (C3′), 83.9 (C4′), 85.3 (C1′), 98.5 (C5′-OC(OMe)Me2), 115.9 (OCH2CH2CN), 122.0 (C5), 126.1 (Bz), 127.0 (Bz), 131.1 (Bz), 131.4 (Bz), 142.8 (C8), 148.2 (C4), 150.2 (C6), 150.8 (C2), 163.8 (Bz); 31P-NMR (CDCl3): δ = 148.5 and 148.9. Negative ion ESI-MS: m/z obsd. 626.30 [M−H]−, calcd. 626.29 [M−H]−.5′-O-(1-Methoxy-1-methylethyl)-3′-O-(hex-5-ynoyl)thymidine (6). Dicyclohexylcarbodiimide  was dissolved in dry dioxane (2 mL) and the solution obtained was added dropwise on an ice-bath to 5-hexynoic acid  in dioxane (1 mL). After stirring for 2 h at r.t., the precipitated dicyclohexylurea was filtrated off and the volatiles were removed under reduced pressure, giving hex-5-ynoic anhydride as a slightly yellow liquid. Compd. 4a  was dissolved in dry pyridine (5 mL), hex-5-ynoic anhydride and a catalytic amount of dimethylaminopyridine were added, and the reaction was allowed to proceed 17 h at rt. The mixture was concentrated under reduced pressure and the residue was purified by silica gel chromatography applying gradient elution with 1–5% MeOH in DCM containing 1% triethylamine. Compound 6 was obtained as white foam in 92% yield . 1H-NMR : δ = 1.41 and 1.42 Me2), 1.84–1.89 , 1.95 , 2.02–2.04 , 2.18–2.24 , 2.28–2.31 , 2.39–2.43 , 2.52 , 3.25 Me2), 3.70–3.99 , 4.21 , 5.30–5.32 , 6.43 , 7.62 , 9.50 ; 13C-NMR : δ = 12.5 (C5-Me), 17.7 (C4 of hex-5-ynoyl), 23.3 (C3 of hex-5-ynoyl), 24.4 (C5′-OC(OMe)Me2), 32.7 (C2 of hex-5-ynoyl), 37.9 (C2’), 48.8 (C5′-OC(OMe)Me2), 61.1 (C5′), 69.5 (C6 of hex-5-ynoyl), 75.4 (C3′), 82.9 (C5 of hex-5-ynoyl), 83.9 (C4′), 84.6 (C1′), 100.4 (C5′-OC(OMe)Me2), 111.2 (C5), 135.2 (C6), 150.8 (C2), 164.0 (C4), 172.7 (C=O of hex-5-ynoyl). High resolution negative ion ESI-MS: m/z obsd. 407.1841 [M−H]−, calcd. 407.1818 [M−H]−.5′-O--3′-O-(hex-5-ynoyl)thymidine (7). Thymidine was converted to 5′-O-thymidine as described in literature [6. The yield of acylation was 99%. 1H- and 13C-NMR spectroscopic data was identical with that given by Oyelere et al. [m/z obsd. 637.2534 [M−H]−, calcd. 637.2550 [M−H]−.terature  and acyle et al. . High re6-O-(p-Toluenesulfonyl)-β-cyclodextrin (8). Monotosylated β-cyclodextrin was prepared by the method of Kuzuya et al. [1H- and 13C-NMR spectra of the product were identical to those reported previously in literature [m/z obsd 1287.41 [M−H]−, calcd. 1287.37 [M−H]−.a et al.  The 1H- terature . Negativ6-Azido-6-deoxy-β-cyclodextrin (9) was obtained by displacing the p-toluenesulfonyloxy group from compd. 8 with azide ion as reported by Petter et al. [1H- and 13C-NMR chemical shifts were identical with those reported previously [m/z obsd. 1158.36 [M−H]−, calcd. 1158.37 [M−H]−.r et al. . The 1H-eviously . NegativPeracetylated 6-azido-6-deoxy-β-cyclodextrin (10a). The azido-functionalized β-cyclodextrin (9) was peracetylated with acetic anhydride in pyridine at r.t., using 4 quiv. of acetic anhydride compared to the amount of free OH groups. The product was purified by silica gel chromatography applying a stepwise gradient elution with 1–5% MeOH in DCM containing 1% Et3N. The yield was 73%. 1H-NMR  2.05 , 3.65 , 3.77 , 3.85–3.90 , 4.03–4.12 , 4,18–4.28 , 4.40–4.50 , 4.65–4.75 , 5.00–5.15 , 5.18–5.23 ; 13C-NMR : 169.5 (Ac), 97.2 (C1), 77.3 (C4), 71.0 (C5), 70.8 (C3), 70.0 (C2), 62.6 (C6), 50.8 (CH2N3), 21.0 (Ac). Positive ion ESI-MS: m/z obsd. 2022.56 [M+Na]+, calcd. 2022.58 [M+Na]+.Permethylated 6-azido-6-deoxy-β-cyclodextrin (10b). Permethylation of the azido-functionalized β-cyclodextrin was achieved by treating 9 over a weekend with a modest excess of methyl iodide (1.25 equiv.) and sodium hydroxide (1.25 equiv.) compared to the amount of free hydroxyl functions, as described previously by Schomburg et al. −, calcd. 1475.66 [M+Cl]−.g et al. ,21. The  earlier . 13C-NMRImmobilization of 3′-O-(hex-5-ynoyl)thymidines to cyclodextrin supports. 3′-O-(hex-5-ynoyl)thymidines 6 and 7 were immobilized to the azido-functionalized supports 10a,b by Cu+-promoted 1,3-dipolar cyclo addition. Accordingly, the appropriate nucleoside, either 6  or 7  in dioxane (15 mL) was mixed with either 10a or 10b in the same solvent. Aq. CuSO4 (50 mmol·L−1) and sodium ascorbate (0.1 mmol·L−1) were added and the reaction was allowed to proceed for 3 d at r.t. Aq. CuSO4.5H2O and aq. sodium ascorbate were added daily to ensure quantitative formation of the 1,2,3-triazole linkages. The products 11a–c were obtained in about 50% yield as white foams by silica gel chromatography applying first a stepwise gradient from 4% to 34% cyclohexane in EtOAc containing 1% triethylamine, and then a stepwise gradient of 5% to 10% MeOH in DCM containing 1% triethylamine. The identity and homogeneity of the products were verified by ESI-MS and HPLC, respectively. 11b  was dissolved in DCM containing 3% dichloroacetic acid (5 mL) and stirred for 1 h. The mixture was diluted with DCM (25 mL) and washed with saturated aq. sodium bicarbonate (3 × 10 mL). Combined aqueous phases were back-extracted with DCM (20 mL). The combined organic phases were dried over anhydrous sodium sulfate and evaporated to dryness, giving 0.177 g deprotected material that still contained the DMTr alcohol. The product was subjected to coupling without purification. Support 11b (0.177 g) and commercially available 5′-O-DMTr-thymidine 3′- were dissolved in anhydrous MeCN (0.775 mL) under nitrogen and 0.45 mol·L−1 solution of tetrazole in MeCN  was added. The mixture was stirred under nitrogen for 1 h, iodine oxidation solution  was added and stirred for additional 30 min. The mixture was concentrated under reduced pressure, the residue was dissolved in DCM (20 mL) and washed with saturated aq sodium bisulfite (3 × 10 mL) and brine (3 × 10 mL). Combined aqueous phases were back-extracted with DCM (20 mL). The organic phases were combined, dried over anhydrous sodium sulfate and evaporated to dryness to give 0.196 g of white foam. The product was used for the next coupling cycle without purification. According to UV-spectrophotometric assay, the trimer was obtained in 85% yield, which corresponds to 92% coupling yield. The homogeneity of product was verified by RP-HPLC (see Deprotected HPLC see A. Glassware and stirrer bar were dried in the oven (120 °C). All chemicals and reagents were placed in a box under Nitrogen atmosphere. The solvents were dried over 3 Å molecular sieves.11a and 11c were treated with a mixture of MeOH (2 mL) and 0.1 mol·L−1 HCl in 1,4-dioxane (6 mL) for 30 min at r.t. The volatiles were removed under reduced pressure and traces of MeOH were removed by coevaporation with dry pyridine and MeCN. Support 11c was additionally purified chromatographically to remove the 4,4′-dimethoxytrityl alcohol released. Deprotected support 11a was used to assemble 3′-TTT-5′ and support 11c to assemble 3′-TTT-5′ and 3′-TTCAT-5′. Acetylated and methylated cyclodextrin supports 5a–c) in MeCN (1.5 equiv. in 1 mL) and 4,5-dicyanoimidazole (1.5 equiv. in 0.52 mL MeCN) were added under nitrogen. The mixture was stirred for 6 h and the oxidation solution  was added. After 30 min stirring, the mixture was diluted with DCM (20 mL) and washed with an equal volume of aq. NaHSO3 to remove the traces of iodine. The organic phase was dried over Na2SO4, filtered and evaporated to dryness under reduced pressure. The residue that still contained traces of 2,6-lutidine, was dissolved in MeOH (2 mL) and a sufficient volume of 0.1 mol L−1 HCl in dioxane was added to neutralize lutidine and adjust pH slightly below 4. After 30 min at r.t., the mixture was evaporated to dryness and the residue was coevaporated with pyridine and MeCN, and subjected to flash chromatographic separation with 1–15% MeOH in DCM to remove the remnants of the first coupling reaction. The subsequent couplings were then carried out in a similar manner, except that upon the two last couplings the support was dissolved in a 4:1 (v/v) mixture of MeCN and DCM instead of MeCN. The identitiy of the product was checked by negative ion ESI-MS after each coupling and deprotection.The deprotected support was dissolved in MeCN (2mL) and the desired 5′-acetal protected building block : CD-T 2334.7 (2334.7) [M−H]−, CD-TT 2691.7 (2691.8) [M−H]−, CD-TTT 3048.8 (3048.9) [M−H]−. The spectrophotometrically determined yield was 87%, corresponding to 93% average coupling yield. On assembling the same trimer on the methylated support 11c, the negative ion ESI -MS m/z values for the support-bound phosphate protected trimer were 2489.0 . In both cases, the m/z value for the released and deprotected 3′-TTT-5′ was 849.15 . The homogeneity of the products was verified by RP-HPLC , the identity of the product was checked by negative ion ESI-MS after each coupling and deprotection : CD-T 1774.8 (1774.8) [M−H]−, CD-TT 2131.8 (2131.9) [M−H]−, CD-TTC 2578.1 (2578.0) [M−H]−, CD-TTCA 1523.6 (1523.5) [M−2H]2−, CD-TTCAT 1702.0 (1702.1) [M−2H]2−. The overall yields after each coupling and subsequent removal of the 5′-protecting group were (CD refers to the cyclodextrin support): CD-TT-5′ , CD-TTC-5′ , CD-TTCA-5′  and CD-TTCAT-5′ . The identity of the released and deprotected pentamer was verified by negative ion ESI−-MS: m/z obsd. 1451.31 [M−H]−, calcd. 1451.28 [M−H]−. The homogeneity of the product was verified by reversed phase HPLC (see On preparing the pentamer on methylated cyclodextrin support HPLC see . The masO protecting group and permethylated β-cyclodextrin as a soluble support. Acid-catalyzed methanolysis of this 5′-acetal protection gives easily removable volatile products and hydrophobic cyclodextrin support allows efficient coupling on using only 50% excess of the monomeric building block, rapid flash chromatographic purification after each coupling cycle and extractive removal of the support after ammonolytic release/deprotection of the oligonucleotide. A useful method for the preparation of short oligodeoxyribonucleotides in solution has been developed. The essential features of the method include use of 1-methoxy-1-methylethyl group as the 5′-


ML3403 (4-(5-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)-N-(1-phenylethyl)pyridin-2-amine) undergoes rapid oxidation to the sulfoxide. Replacing the sulfur atom present in the two potent p38α mitogen-activated protein (MAP) kinase inhibitors ML3403 and LN950 (2-((5-(4-fluorophenyl)-4-(2-((3-methylbutan-2-yl)amino)pyridin-4-yl)-1H-imidazol-2-yl)thio)ethan-1-ol) by a methylene group resulted in 2-alkylimidazole derivatives 1 and 2, respectively, having a remarkably improved metabolic stability. The 2-alkylimidazole analogs 1 and 2 showed 20% and 10% biotransformation after 4 h of incubation with human liver microsomes, respectively. They display a 4-fold increased binding affinity towards the target kinase as well as similar in vitro potency and ex vivo efficacy relative to their 2-alkylsulfanylimidazole counterparts ML3403 and LN950. For example, 2-alkylimidazole 2, the analog of LN950, inhibits both the p38α MAP kinase as well as the LPS-stimulated tumor necrosis factor-α release from human whole blood in the low double-digit nanomolar range.In vitro and in vivo metabolism studies revealed that 2-alkylsulfanylimidazole  VX-745 (The p38α mitogen-activated protein (MAP) kinase is a ubiquitously expressed serine/threonine kinase, which is implicated in various cellular processes such as cell survival, proliferation and differentiation. Because it promotes the expression of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), the p38α MAP kinase has received a lot of attention as a target for drug discovery programs since the mid-1990s. Several p38α MAP kinase inhibitors were tested in clinical trials against cancer and chronic inflammatory diseases like rheumatoid arthritis or chronic obstructive pulmonary disease. Despite these major efforts, up to date there is still no p38α MAP kinase inhibitor on the market, as most of the trials were terminated due to adverse events or lack of efficacy. However, recent studies suggest an important role of the kinase in the pathogenesis of neurodegenerative diseases like multiple sclerosis and Alzheimer’s disease ,3. RecenVX-745  to treatSB203580 is a trisubstituted imidazole representing one of the first prototypical p38α MAP kinase inhibitors  +; HPLC: tR = 5.73 min.ione (4) . 1-(4-Fl4-(2-Ethyl-4-(4-fluorophenyl)-1H-imidazol-5-yl)-2-fluoropyridine (5). Compound 4  was dissolved in methanol (5 mL). Subsequently, 7 M ammonia in methanol solution  and propionaldehyde  were added successively. The reaction mixture was heated to 80 °C for 4 h. After removing the solvent under reduced pressure, the crude product was directly purified by flash chromatography  to give a white solid . 1H-NMR  δ 1.28 , 2.64–2.77 , 7.09 , 7.17–7.40 , 7.47–7.56 , 8.06 , 12.41 ; MS-ESI (m/z) 286.0 [M + H]+, 284.0 [M − H]−; HPLC: tR = 3.68 min. 4-(2-Ethyl-4-(4-fluorophenyl)-1H-imidazol-5-yl)-N-(1-phenylethyl)pyridin-2-amine (1). Compound 5  was dissolved in α-methylbenzylamine (1 mL) and heated for 72 h to 160 °C. After cooling to rt, the crude mixture was purified by flash chromatography  to give a white solid . 1H-NMR  δ 1.18–1.25 , 1.38–1.44 , 2.63 , 4.44 , 5.03 , 6.3 , 6.65 , 6.91–6.99 , 7.10–7.25 , 7.33 , 7.90 ; 13C-NMR  δ 12.6, 21.7, 24.3, 51.8, 104.0, 111.4, 115.5 , 125.6, 126.9, 128.5, 130.0 , 144.3, 147.8, 150.2, 158.1, 162.3 ; MS-ESI (m/z) 387.1 [M + H]+; 385.1 [M − H]−; HPLC: tR = 7.23 min (100% purity).4-(Triisopropylsilyl)oxy)butanal (9). 4-(Triisopropylsilyl)oxy)butan-1-ol (8) +; 313.1 [M − H]−; HPLC: tR = 5.07 min.4-(4-(4-Fluorophenyl)-2-(3-((triisopropylsilyl)oxy)propyl)-1H-imidazol-5-yl)-N-(3-methylbutan-2-yl)pyridin-2-amine (10). Compound 7  was dissolved in methanol (10 mL). Ammonium acetate  and aldehyde 9  were added. The reaction was heated to 80 °C for 4 h. After cooling to rt, saturated NaHCO3 solution was added. The aqueous phase was adjusted to pH 10 with 1 M aq. NaOH and extracted thrice with ethyl acetate. The combined organic layers were dried over anhydrous Na2SO4, the solvent was removed under reduced pressure and the residue was purified by flash chromatography  to give a colorless oil . 1H-NMR  δ 0.87 , 1.03–1.05 , 1.71 , 1.93–2.06 , 2.96 , 3.34–3.51 , 3.84 , 4.39 , 6.19–6.54 , 6.57–6.70 , 6.94–7.15 , 7.31–7.44 , 7.57 , 7.89 , 9.96–10.24 ; MS-ESI (m/z) 539.2 [M + H]+, 537.1 [M − H]−; HPLC: tR = 5.12 min.3-(4-(4-Fluorophenyl)-5-(2-((3-methylbutan-2-yl)amino)pyridin-4-yl)-1H-imidazol-2-yl)propan-1-ol (2). Compound 10  was dissolved in methanol (10 mL) and 2 M aq. HCl solution was added. The reaction was stirred at rt for 1 h. The organic solvent was removed under reduced pressure and the aqueous residue was treated with saturated NaHCO3 solution. A white precipitate was formed, collected by filtration and taken up in ethyl acetate. The organic solution was dried over anhydrous Na2SO4 and the compound was purified by flash chromatography  to give an off-white solid . 1H-NMR  δ 0.85 , 1.02 , 1.63–1.75 , 1.98 , 2.86–2.96 , 3.32–3.43 , 3.79 , 4.53 , 6.45 , 6.57 , 7.03 , 7.46 , 7.90 ; 13C-NMR  δ 16.9, 17.7, 18.8, 26.5, 29.8, 32.5, 52.2, 62.3, 103.8, 110.5, 115.6 , 130.1 , 147.8, 148.9, 158.5, 162.4 ; MS-ESI (m/z) 383.1 [M + H]+, 381.1 [M − H]−; HPLC: tR = 6.53 min (100% purity).+. The substrate (100 μM), the NADPH regenerating system and 4 mM MgCl2 × 6 H2O in 0.1 M Tris buffer (pH 7.4 at 37 °C) were preincubated for 5 min in an incubator at 37 °C and 750 rpm. The incubation mix was split into 50 μL aliquots and the reaction was started by addition of the HLM. Thereby the microsomal protein content was standardized to 1 mg/mL. To follow the course of metabolism, the reaction tubes were quenched at selected time points  by adding 100 μL internal standard at a concentration of 22.5 µM in MeCN. The samples were vortexed for 30 s and centrifuged . The supernatant was directly used for LC-MS analysis  were purchased from Merck . These microsomes were characterized in protein and CYP content. All incubations  were made in the presence of an NADPH-regenerating system, consisting of 5 mM Glucose-6-phosphate, 5 U/mL Glucose-6-phosphate dehydrogenase and 1 mM NADPCYP inhibition (fluorimetric detection) assay was performed by Eurofins Panlabs Inc.  with human recombinant CYP enzyme and the appropriate CYP substrate. ML3403 and LN950 by a methylene group results in 2-alkylimidazole derivatives 1 and 2, showing remarkably improved microsomal stability. Both compounds undergo minimal hydroxylation and N-dealkylation upon incubation with HLM. Trisubstituted imidazoles 1 and 2 display a 4-fold increase in binding affinity toward the kinase and possess a similar inhibition profile of p38α MAP kinase and the LPS-stimulated TNF-α release from HWB like their 2-alkylsulfanylimidazole counterparts ML3403 and LN950. However, the exchange of the sulfur atom present in ML3403 and LN950 by a methylene group is accompanied with a slightly higher CYP inhibition profile of 1 and 2. The most potent inhibitor, 2-alkylimidazole 2, inhibits both the p38α MAP kinase as well as the LPS-stimulated TNF-α release from human whole blood in the low double-digit nanomolar range. Moreover, the excellent metabolic profile of 2 gives advantage over the mixed pharmacokinetics of the p38α MAP kinase reference inhibitor ML3403 and its active metabolite. Therefore, 2-alkylimidazole 2 is a good alternative to evaluate the role of this kinase in in vitro and in vivo studies.Replacement of the sulfur atom present in both known p38α MAP kinase inhibitors


A thermal shift analysis of SIRT2 in the presence of tenovin-6, -43, a control tenovin and the known SIRT2 inhibitor AGK2 is also presented.The search for potent and selective sirtuin inhibitors continues as chemical tools of this type are of use in helping to assign the function of this interesting class of deacetylases. Here we describe SAR studies starting from the unselective sirtuin inhibitor tenovin-6. These studies identify a sub-micromolar inhibitor that has increased selectivity for SIRT2 over SIRT1 compared to tenovin-6. In addition, a  The. The9].  >200 µM . 600 reached 0.7–0.8. Cultures were cooled on ice before the addition of IPTG (0.1 mM) and Zn(OAc)2 (40 mM) and the culture grown at 18 °C overnight. The cells were harvested by centrifugation at 7500 rpm for 35 min before storing the resulting pellets at −20 °C. SIRT2 containing pellets were resuspended in lysis buffer  and incubated on ice for 30 min. The suspension was sonicated for 10 × 1 min. The soluble fraction was collected after centrifugation at 15,000 rpm for 30 min. The resulting supernatant was filtered (0.45 µm), and loaded onto a pre-equilibrated 5mL HisTrap HP column . The tagged protein eluted at 100 mM imidazole concentration. The fractions containing the enzyme were combined and dialysed in buffer  for 2 h. The protein was placed in fresh dialysis buffer and biotinylated thrombin added before leaving to cleave overnight at 4 °C. The enzyme was transferred into a tube where streptavidin agarose was added and the mixture gently mixed on a rocking table for 2 h at 4 °C. The suspension was centrifuged at 3,000 rpm for 10 min and the supernatant containing the purified enzyme decanted. The cleaved SIRT2 was further purified by Ni affinity chromatography as before, with cleaved protein eluting at 5 mM imidazole, followed by gel filtration chromatography on S-200 sephacryl column. The protein was concentrated by centrifugation using a spin concentrator. SIRT2 was concentrated to >12 mg/mL to ensure minimal amount of water in the NMR experiments.SIRT2 cDNA (residues 50-356) was expressed from Ncol and Xhol restriction sites of the pET32a vector  with an N-terminal His tag. Protein was overexpressed in BL21(DE3) pLysS bacterial cells . Competent bacteria were transformed with the SIRT2 plasmid and starter cultures (10 mL) grown overnight at 37 °C in LB medium with ampicillin (100 mg/L) and chloramphenicol (100 mg/L). The overnight starter culture was added to 1 L of LB media containing ampicillin and chloramphenicol which was grown at 37 °C with 200 rpm shaking, until the OD® fluorescence-based assay kit  and SirT2 expressed and purified in house. All other required reagents were provided in the kit which was stored at −78 °C. Before use, small aliquots of each enzyme (2–3 μL) were prepared, snap frozen in liquid nitrogen and stored at −78 °C. Fresh dilutions of compounds were prepared in DMSO and further diluted in assay buffer. NAD+ (12.5 μL at 4 mM) and Fluor de Lys® SIRT1 or SIRT2 (12.5 μL at 100 μM) in assay buffer were added to a white 96 well plate, followed by compound (10 μL) and lastly enzyme . After incubation for 1 h at 37 °C a developer solution (50 μL) was added to each reaction. The developer solution contained 38 μL buffer, 10 μL developer and 2 μL nicotinamide per reaction. The plate was then incubated for 45 min at rt and then read using a Spectra Max Gemini fluorimeter with an excitation wavelength of 355 nm and an emission wavelength of 460 nm. SigmaPlot software was used to generate fit curves for raw plots and the equation for each fit curve was used to calculate IC50 data.All compounds were tested for inhibition of SIRT1 and SIRT2 using human recombinant enzymes; SirT1 available in the Fluor de Lys2O buffer containing 50 mM Tris (pH 8), 150 mM NaCl, 2 mM KCl and 1 mM MgCl2 was added NAD+ (1 mM), peptide (200 μM) and inhibitor (varying concentration). The inhibitor was dissolved in 6d-DMSO before addition to the buffer ensuring that the final NMR sample contained only 0.5% DMSO for each experiment. The initial data was collected before addition of in house SIRT2 (10 μM) directly to the NMR tube. After the incubation was complete, the NMR tube was shaken and replaced in the spectrometer for data collection. 1D 1H-NMR with double solvent suppression (for H2O and TRIS) were recorded on a 500 MHz Bruker spectrometer at 37 °C.To a DThe thermal shift assay was performed using Corbett Rotor-Gene 6000  spectrofluorimeter. The prepared protein concentration was usually 5 µM and the ligand concentrations 50 µM. Reaction volume was usually 10 µL. Unfolding of the protein was monitored by measuring the fluorescence of the 1,8-anilinonaphthalene sulfonate (ANS), at 50–100 µM. The samples were heated at a rate of 1 °C/min. The samples were excited with 365 nm UV light and ANS fluorescence emission was registered at 460 nm light. Data analysis was performed as previously described .2; pyridine was dried over KOH pellets; all the other solvents were dried on a MBRAWN SPS-800 apparatus.All chemicals and solvents were purchased from Aldrich  or Alfa-Aesar  and used without further purification. All reactions were carried out under a positive pressure of nitrogen or argon in flame or oven-dried glassware. Ethanol was dried over Mg/I254 sheets . Compounds were visualized by UV light  and stained with potassium permanganate. Flash column chromatography was carried out on silica gel  or, where indicated, on basic alumina . Melting points were measured with an Electrothermal 9100 capillary melting point apparatus and are uncorrected.Thin layer chromatography (TLC) analysis was performed on silica pre-coated SIL G-25 UV−1).Fourier Transform infra-red spectra (FT-IR) were acquired on a Perkin Elmer Paragon 1000 FT spectrometer. Absorption maxima are reported in wavenumbers (cm1H-NMR spectra were measured at room temperature (298 K) on a Bruker DPX 400 (1H = 400 MHz) and Bruker Avance 300 (1H = 300.1 MHz) instruments. Deuterated solvents were used and 1H-NMR chemical shifts were internally referenced to CHCl3 (7.26 ppm) in chloroform-d1 solution. Chemical shifts are expressed as δ in unit of ppm. Unless otherwise stated, 13C-NMR spectra were recorded in the same conditions and in the same solvents using the PENDANT sequence mode on a Bruker DPX 400 (13C = 100 MHz). Data processing was carried out using TOPSPIN 2 NMR version . In 1H-NMR assignment the multiplicity used is indicated by the following abbreviations: s = singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quartet, m = multiplet, brs = broad singlet. Signals of protons and carbons were assigned, as far as possible, by using the following two-dimensional NMR spectroscopy techniques: [1H-1H] COSY, [1H-13C] COSY (HSQC: Heteronuclear Single Quantum Coherence) and long range [1H-13C] COSY (HMBC: Heteronuclear Multiple Bond Connectivity). Mass spectrometry  were recorded on a high performance orthogonal acceleration reflecting TOF mass spectrometer operating in positive and negative mode, coupled to a Waters 2975 HPLC.3 (1 equiv.) in dry DMF (1 vol.) under N2 was added K2CO3 (2 equiv.) and the alkyl halide (1.1 equiv.). The resulting solution was stirred for 16 h at room temperature before being partitioned between ethyl acetate (1 vol.) and water (0.5 vol.). The organic layer was washed with water (0.5 vol.), brine (0.5 vol.), dried (MgSO4), filtered and the solvent removed in vacuo to give the desired product which was used without further purification.To a stirred solution of Methyl 3,5-dichloro-4-ethoxybenzoate (4f). Prepared from compound 2 , K2CO3  and iodoethane  in DMF (10 mL). The product was obtained as a brown oil . νmax cm−1  2994, 1701, 1652, 1556, 1147, 854; δH  7.91 , 4.09 , 3.85  and 1.41 ; δC  164.7 (C), 155.5 (C), 130.2 (CH), 129.8 (C), 126.9 (C), 70.0 (CH2), 52.6 (CH3) and 15.5 (CH3); m/z (ES)+: 249.35 . Methyl 4-propoxy-3,5-dichlorobenzoate (4g). Prepared from 2 , K2CO3  and iodopropane  in DMF (10 mL). The product was obtained as a yellow-brown oil . νmax cm−1  2954 2880, 1733, 1690, 1592, 1556, 1462, 1288, 1138, 986; δH  7.90 , 3.97 , 3.84 , 1.82  and 1.02 ; δC  164.2 (C), 155.0 (C), 130.6 (CH), 130.1 (C), 127.3 (C), 76.0 (CH2), 53.6 (CH3), 23.8 (CH2) and 10.8 (CH3); m/z (ES)+: 263.24 .Methyl 4-butoxy-3,5-dichlorobenzoate (4h). Prepared from 2 , K2CO3  and iodobutane  in DMF (10 mL). The product was obtained as a brown oil . νmax cm−1  2959 (ArC-H), 1729, 1642, 1556, 1435, 1284, 1137, 987; δH  7.90 , 4.01 , 3.84 , 1.81–1.76 , 1.50  and 0.93 ; δC  164.7 (C), 155.6 (C), 130.3 (CH), 129.7 (C), 126.9 (C), 73.8 (CH2), 52.6 (CH3), 32.11 (CH2), 19.04 (CH2) and 13.8 (CH3); m/z (ES)+: 277.06 . in vacuo and the aqueous fraction acidified with 2 M HCl. The resulting precipitate was extracted with ethyl acetate (3 × 1 vol.) and the organic layers combined and washed with brine (0.5 vol.), dried (MgSO4), filtered and the solvent removed to yield the desired acid.The ester (1 equiv.) and sodium hydroxide (1.2 equiv.) were heated at reflux in a solution of methanol (1 vol.) and water (1 vol.) until the methyl ester was consumed by TLC (4–6 h). The methanol was removed 3,5-Dichloro-4-ethoxybenzoic acid (5f). Prepared from methyl 4-ethoxy-3,5-dichlorobenzoate  in MeOH/water (10 mL) and NaOH . The desired product was obtained as an off-white solid . Mp 179–180 °C; νmax cm−1 (KBr) 3225, 2104, 1635, 1206; δH  8.12 , 4.28 , 1.57 ; δC  169.6 (C), 156.3 (C), 130.8 (CH), 130.0 (C), 125.9 (C), 70.2 (CH2), 15.5 (CH3); m/z (ES)− 232.97 ; HRMS (ES−) .4-Propoxy-3,5-dichlorobenzoic acid (5g). Prepared from methyl 4-propoxy-3,5-dichlorobenzoate  in MeOH/water (10 mL) and NaOH . The product was obtained as a white solid . Mp 125–126 °C; νmax cm−1 (KBr) 2937, 1699, 1558, 1493, 1385, 1077, 906, 768; δH  7.97 , 4.00 , 1.83 , 1.03 ; δC  169.4, 156.4, 129.9, 125.9, 75.7, 23.4, 10.4; m/z (ES)− 247.23 ; HRMS (ES−)  4-Butoxy-3,5-dichlorobenzoic acid (5h). Prepared from methyl 4-butoxy-3,5-dichlorobenzoate  in MeOH/water (10 mL) and NaOH . The product was obtained as a yellow solid . Mp 98–99 °C; νmax cm−1 (KBr) 2955, 1682, 1214, 1557, 1388, 1056, 810; δH  7.91 , 4.04 , 1.8–1.7 , 1.49 , 0.94 ; δC  169.3 (C), 156.4 (C), 130.8 (CH), 130.2 (C), 125.9 (C), 73.8 (CH2), 32.1 (CH2), 19.03 (CH2), 13.8 (CH3); m/z (ES)− 261.02 ; HRMS (ES−) .2, was added a solution of oxalyl chloride (2 equiv.) in DCM (1 vol.). A drop of dry DMF was added and the resulting solution stirred at room temperature for 90 min. The solvent was removed in vacuo and the resulting acid chloride used immediately without purification or characterisation.To a stirred solution of the benzoic acid  (1 equiv.) in DCM (1 vol.), under N2, was added sodium thiocyanate (1 equiv.). The resulting suspension stirred at room temperature for 30 min before being cooled to 0 °C. A solution of the amine (1 equiv.) in dry acetone (1 vol.) was added and the resulting suspension allowed to warm to room temperature and stirred for 16 h. The reaction was filtered through Celite and the filtrate concentrated to give the crude product. The product was purified by column chromatography (1–20% methanol-DCM). The purified product was dissolved in acetone and 2 M HCl in diethyl ether (1 equiv.) added slowly. The resulting precipitate was filtered and recrystallised from ethanol to afford the pure HCl salts.To a solution of the acid chloride (1 equiv.) in dry acetone (1 vol.) under N3,5-Dibromo-N-((4-(5-(dimethylamino)pentanamido)phenyl)carbamothioyl)-4-methoxybenzamide hydrochloride (Tenovin-36). Prepared from 3,5-dibromo-4-methoxybenzoyl chloride  and sodium thiocyanate  in dry acetone (3 mL) followed by addition of N-(4-aminophenyl)-5-(dimethylamino)pentanamide  in acetone (3 mL). The crude material was purified by column chromatography (1–20% MeOH-DCM) and this material subsequently converted to the HCl salt to afford the product as a yellow solid . Mp 159–160 °C; νmax cm−1 (KBr) 2924, 2857, 1669, 1606, 1541, 1495, 1405, 1262, 1094, 824; δH  12.41 , 11.85 , 10.21 , 9.75 , 8.34 , 7.69 , 3.96 , 3.14 , 2.83 2), 2.48 , 1.74 2); δC  178.5 (C), 170.8 (C), 164.9 (C), 156.9 (C), 137.5 (C), 133.2 (CH), 132.7 (C), 130.7 (C), 124.7 (CH), 119.0 (CH), 117.4 (C), 60.6 (CH3), 56.1 (CH2), 41.9 (CH3), 35.5 (CH2), 23.2 (CH2) and 22.0 (CH2); m/z (ES)+ 586.98 .N-((4-(5-(Dimethylamino)pentanamido)phenyl)carbamothioyl)-3,4,5-trimethoxybenzamide hydro-chloride (Tenovin-37). Prepared from 3,4,5-trimethoxybenzoyl chloride  and sodium thiocyanate  in dry acetone (3 mL) followed by addition of N-(4-aminophenyl)-5-(dimethylamino)pentanamide  in acetone (3 mL). The crude material was purified by column chromatography (1–20% MeOH-DCM) and this material subsequently converted to the HCl salt to afford the product as a yellow solid . Mp 115–117 °C; νmax cm−1 (KBr) 2925, 2858, 1670, 1642, 1495, 1405, 1340, 1261, 1125, 1050, 824; δH  12.65 , 11.56 , 10.15 , 9.82 , 7.62 , 7.38 , 3.88 2), 3.75 , 3.06 , 2.75 2), 2.39 , 1.65 2); δC  178.9 (C), 170.7 (C), 167.4 (C), 152.5 (C), 141.5 (C), 137.4 (C), 132.8 (C), 126.7 (C), 124.7 (CH), 119.0 (CH), 106.5 (CH), 60.1 (CH3), 56.6 (CH2), 56.1 (CH3), 41.9 (CH3), 35.5 (CH2), 23.2 (CH2), 23.2 (CH2), 21.9 (CH2); m/z (ES)+ 489.18, ; HRMS (ES)+ .N-(4-(5-(dimethylamino)pentanamido)phenylcarbamothioyl)-4-methoxy-3,5-dimethylbenzamide hydrochloride (Tenovin-38). Prepared from 4-methoxy-3,5-dimethylbenzoyl chloride  and sodium thiocyanate  in dry acetone (3 mL) followed by addition of N-(4-aminophenyl)-5-(dimethylamino)pentanamide  in acetone (3 mL). The crude material was purified by column chromatography (1–20% MeOH-DCM) and this material subsequently converted to the HCl salt. The product was obtained as a brown solid . Mp 190–192 °C; νmax cm−1 (KBr) 2925, 2854, 1667, 1604, 1515, 1337, 1307, 1166, 1007, 740; δH  12.64 , 11.36 , 10.20 , 9.91 , 7.79 , 7.69 , 7.63 , 3.77 , 3.15–3.08 , 2.79 2), 2.47–2.41 , 2.33 2), 1.76–1.69 2); δC  178.9 (C), 170.7 (C), 167.7 (C), 160.7 (C), 137.3 (C), 132.8 (C), 130.6 (C), 129.5 (CH), 126.9 (C), 124.7 (CH), 118.9 (CH), 59.4 (CH3), 56.2 (CH2), 42.0 (CH3), 35.5 (CH2), 23.3 (CH2), 21.9 (CH2), 15.8 (CH3); m/z (ES)+ 457.03 ; HRMS (ES)+ .N-((4-(5-(dimethylamino)pentanamido)phenyl)carbamothioyl)-3,5-difluoro-4-methoxybenzamide hydrochloride (Tenovin-39). Prepared from 3,5-difluoro-4-methoxybenzoyl chloride  and sodium thiocyanate  in dry acetone (3 mL) followed by addition of N-(4-aminophenyl)-5-(dimethylamino)pentanamide  in acetone (3 mL). The crude material was purified by column chromatography (1–20% MeOH-DCM) and this material subsequently converted to the HCl salt to afford the product as an off-white solid . Mp 153–154 °C; νmax cm−1 (KBr) 2924, 2859, 1659, 1608, 1544, 1495, 1451, 1404, 1262, 1180, 1049, 824; δH  12.33 , 11.54, 10.19 2), 7.81 , 7.62 , 7.53 , 4.08 , 3.00 , 2.68 2), 2.35  and 1.62 2); δC  180.5 (C), 173.7 (C), 167.0 (C), 157.2 (C), 155.2 (C), 138.3 (C), 135.2 (C), 127.8 (C), 125.9 (CH), 121.1 (CH), 114.1 (CH), 58.7 (CH2), 53.6 (CH3), 43.5 (CH3), 36.7 (CH2), 25.2 (CH2), 23.1 (CH2); m/z (ES)+ 465.03 ; HRMS (ES)+ .N-((4-(5-(dimethylamino)pentanamido)phenyl)carbamothioyl)-3,5-difluoro-4-ethoxybenzamide hydrochloride (Tenovin-40). Prepared from 3,5-difluoro-4-ethoxybenzoyl chloride  and sodium thiocyanate  in dry acetone (3 mL) followed by addition of N-(4-aminophenyl)-5-(dimethylamino)pentanamide  in acetone (3 mL). The crude material was purified by column chromatography (1–20% MeOH-DCM) and this material subsequently converted to the HCl salt to afford the product as an off-white solid . Mp 206–207 °C; νmax cm−1 (KBr) 2926, 2860, 1674, 1606, 1512, 1495, 1436, 1405, 1263, 1135, 1049, 874; δH  12.34 , 11.56 , 10.11 , 9.70 , 7.83 , 7.64 , 7.56 , 4.31 , 3.08-2.99 , 2.72 2), 2.38 , 1.71–1.56 2) and 1.32 ; δC  180.4 (C), 173.5 (C), 167.0 (C), 157.6 (C), 155.7 (C), 138.3 (C), 135.2 (C), 128.0 (C), 125.9 (CH), 121.1 (CH), 113.9 (CH), 58.7 (CH2), 43.5 (CH3), 36.7 (CH2), 30.8 (CH2), 25.2 (CH2), 23.1 (CH2), 15.8 (CH3); m/z (ES)+ 478.90 ; HRMS (ES)+ .3,5-Dichloro-N-(4-(5-(dimethylamino)pentanamido)phenylcarbamothioyl)-4-ethoxybenzamide hydrochloride (Tenovin-41). Prepared by the reaction of 3,5-dichloro-4-ethoxybenzoyl chloride  and sodium thiocyanate  in dry acetone (6 mL) followed by addition of a solution of N-(4-aminophenyl)-5-(dimethylamino)pentanamide  in dry acetone (6 mL) using general method F. The product was further purified by conversion to the HCl salt by the addition of 2M HCl in diethyl ether. The desired product was obtained as a brown sticky oil . νmax cm−1  2965, 2359.3, 1651, 1048, 1025, 998, 765; δH  12.29 , 11.68 , 10.21 , 8.08 , 7.79–7.46 , 4.19–4.06 , 3.06–2.93 , 2.69 2), 2.36–2.24 , 1.71–1.65 , 1.41–1.28 , 1.23–1.17 ; δC  180.4 (C), 173.5 (C), 167.0 (C), 156.7 (C), 138.2 (C), 135.2 (C), 131.1 (C), 130.9 (C), 130.4 (CH), 125.9 (CH), 121.2 (CH), 71.2 (CH2), 58.8 (CH2), 43.5 (CH3), 36.6 (CH2), 25.3 (CH2), 23.1 (CH2), 15.8 (CH3); m/z (ES)+ 510.94 ; HRMS (ES)+ .3,5-Dichloro-N-((4-(5-(dimethylamino)pentanamido)phenyl)carbamothioyl)-4-propoxybenzamide hydrochloride (Tenovin-42). Prepared from 3,5-dichloro-4-propoxybenzoyl chloride  and sodium thiocyanate  in dry acetone (3 mL) followed by addition of N-(4-aminophenyl)-5-(dimethylamino)pentanamide  in acetone (3 mL). The crude material was purified by column chromatography (1–20% MeOH-DCM) and this material subsequently converted to the HCl salt to afford the product as an off-white solid . Mp 167–168 °C; νmax cm−1 (KBr) 2927, 2861, 1674, 1636, 1606, 1539, 1473, 1452, 1265, 1148, 1051, 873; δH  12.31 , 11.70 , 10.23 , 10.13 , 8.09 , 7.65 , 7.56 , 4.04 , 3.02–2.96 , 2.72 2), 2.38–2.32 , 1.86–1.75 , 1.70–1.60 2), 1.03 ; δC  170.5 (C), 165.5 (C), 154.4 (C), 150.5 (C), 135.4 (C), 132.7 (C), 129.5 (C), 129.2 (CH), 128.6 (C), 120.2 (CH), 119.6 (CH), 75.4 (CH2), 56.2 (CH2), 42.0 (CH3), 35.4 (CH2), 23.3 (CH2), 22.9 (CH2), 21.9 (CH2), 10.6 (CH3); m/z (ES)+ 525.09 ; HRMS (ES)+ . 4-Butoxy-3,5-dichloro-N-((4-(5-(dimethylamino)pentanamido)phenyl)carbamothioyl)benzamide hydrochloride (Tenovin-43). Prepared from 3,5-dichloro-4-butoxybenzoyl chloride  and sodium thiocyanate  in dry acetone (2 mL) followed by addition of N-(4-aminophenyl)-5-(dimethylamino)pentanamide  in acetone (2 mL). The crude material was purified by column chromatography (1–20% MeOH-DCM) and this material subsequently converted to the HCl salt to afford the product as a cream solid . Mp 174–176 °C; νmax cm−1 (KBr) 2959, 2927, 2858, 1674, 1606, 1545, 1495, 1405, 1264, 1151, 1050, 876; δH  12.25 , 11.64 , 10.08 , 9.66 , 8.03 , 7.59 , 7.50 , 4.01 , 3.04–2.93 , 2.69 2), 2.32 , 1.71 , 1.66–1.50 2), 1.45 , 0.89 ; δC  179.5 (C) 170.5 (C), 165.6 (C), 154.4 (C), 135.3 (C), 132.5 (C), 129.5 (C), 129.2 (CH), 128.6 (C), 120.3 (CH), 119.6 (CH), 73.6 (CH2), 56.2 (CH2), 42.1 (CH3), 35.4 (CH2), 31.5 (CH2), 23.3 (CH2), 21.8 (CH2), 18.5 (CH2), 13.6 (CH3); m/z (ES)+ 539.09 ; HRMS (ES)+ .4-Butoxy-N-((4-(5-(dimethylamino)pentanamido)phenyl)carbamothioyl)benzamide hydrochloride (Tenovin-44). Prepared from 4-butoxybenzoyl chloride  and sodium thiocyanate  in dry acetone (3 mL) followed by addition of N-(4-aminophenyl)-5-(dimethylamino)pentanamide  in acetone (3 mL). The crude material was purified by column chromatography (1–20% MeOH-DCM) and this material subsequently converted to the HCl salt to afford the product as a brown solid . Mp 160–161 °C; νmax cm−1(KBr) 2956, 2927, 2861, 1670, 1606, 1546, 1495, 1408, 1251, 1190, 1050, 875; δH  12.63 , 11.33 , 10.43 , 10.27 , 7.98 , 7.66 , 7.57 , 7.04 , 4.06 , 3.09-3.00 , 2.70 2), 2.38 , 1.75–1.57 3), 1.43 , 0.93 ; δC  180.8 (C), 173.6 (C), 169.1 (C), 165.0 (C), 138.1 (C), 135.3 (C), 131.5 (CH), 125.8 (CH), 125.2 (C), 121.4 (CH), 115.6 (CH), 69.2 (CH2), 58.7 (CH2), 43.5 (CH3), 36.7 (CH2), 32.3 (CH2), 25.2 (CH2), 23.2 (CH2), 20.2 (CH2), 14.2 (CH3); m/z (ES)+ 471.04 ; HRMS (ES)+ . 1H and 13C-NMR spectra of all tenovin analogues are included in the electronic 1H-NMR methods can be used to follow the deacetylation of a histone H4-based N-acetylated substrate. By monitoring the signal corresponding to the methyl group of the N-acetyl functional group, reaction of the substrate can be followed. Our previously reported sirtuin inhibitor, tenovin-6, was shown to inhibit this reaction. Detailed studies showed that inhibition by tenovin-6 was dose dependent and that using this system tenovin-6 had an IC50 value of 139.2 ± 9.5 µM. Further studies led to the identification of a tenovin-6 analogue that has a high nanomolar IC50 value against SIRT2 (tenovin-43). Compounds with this level of potency are rare in the sirtuin inhibitor literature to date [1H NMR method and the commercially available assay, were in agreement. Tenovin-43 was also shown to inhibit SIRT2 at concentrations where no inhibition of SIRT2 by the current state of the art inhibitor AGK2 was observed. Whilst use of the NMR method is not suitable for the assessment of numerous analogues, the relative activity of inhibitors can be tested. In addition, researchers can use this new method as another means of reassuring themselves that their sirtuin modulators target the relevant enzyme activity. A thermal shift assay was also used with SIRT2 to measure the binding constants of tenovins-6, -43 and AGK2. Whilst the calculated binding constants of the tenovin analogues were in agreement with the order of potencies calculated by the commercially available assay kit and the NMR method, the binding constant for AGK2 was determined to be much larger than expected.There remains a need for novel approaches to assess the function of the sirtuin family of deacetylases. This is driven by the continuing interest in this important class of proteins. Here we show that  to date . Importa