Synthesis Antiviral and Cytostatic Evaluation of Unsaturated Exomethylene and Keto span class=smallCapsDspan-Lyxopyranonucleoside Analogues.
код для вставкиСкачатьArch. Pharm. Chem. Life Sci. 2009, 342, 353 – 360 N. Tzioumaki et al. 353 Full Paper Synthesis, Antiviral and Cytostatic Evaluation of Unsaturated Exomethylene and Keto D-Lyxopyranonucleoside Analogues Niki Tzioumaki1, Evangelia Tsoukala1, Stella Manta1, George Agelis2, Jan Balzarini3, and Dimitri Komiotis1 1 Department of Biochemistry and Biotechnology, Laboratory of Organic Chemistry, University of Thessaly, Larissa, Greece 2 Department of Chemistry, Laboratory of Organic Chemistry, University of Patras, Patras, Greece 3 Rega Institute for Medical Research, Katholieke Universtiteit Leuven, Leuven, Belgium This report describes the synthesis of unsaturated exomethylene lyxopyranonucleoside analogues as potential biologically active agents. Commercially available 1,2,3,4-tetra-O-acetyl-a-Dlyxopyranose 1 was condensed with silylated thymine and uracil, respectively, deacetylated and acetalated to afford 1-(2,3-O-isopropylidene-a-D-lyxopyranosyl)thymine 4a and 1-(2,3-O-isopropylidene-a-D-lyxopyranosyl)uracil 4b. The new derivatives 1-(2,3,4-trideoxy-4-methylene-a-pent-2-enopyranosyl)thymine 8a and 1-(2,3,4-trideoxy-4-methylene-a-pent-2-enopyranosyl)uracil 8b were prepared via two different key intermediates, 7a, b and 13a, b in order to elucidate the influence of 29,39-unsaturation and to clarify the difference between the keto and exomethylene group on the biological activity of the target molecules. Compounds 7a, b, 8a, b, and 13a, b were evaluated for their antiviral and cytostatic activity using several virus strains and cell lines. Whereas no marked antiviral activity was noticed, 13a and 13b showed a cytostatic activity that ranged between 7 and 23 lM for 13a and 26 and 38 lM for 13b against murine leukemia L1210, human lymphocyte Molt4/C8 and CEM cells, and human breast carcinoma MCF7 cells. Keywords: Cytotoxicity / Exomethylene nucleosides / Ketonucleosides / Unsaturated nucleosides / Received: January 8, 2009; accepted: April 17, 2009 DOI 10.1002/ardp.200900004 Introduction Nucleosides and their analogues have a very important place in medicinal chemistry as structural basis for the development of antitumor and antiviral agents [1 – 6]. A variety of strategies have been devised to design nucleoside analogues, involving several structural modifications on the sugar moiety of naturally occurring nucleosides [7]. Consequently, over the past two decades, extensive work has been conducted to search for new types of nucleosides with high activity and less toxicity, and those that are less prone to trigger drug resistance [8]. Correspondence: Dr. Dimitri Komiotis, Department of Biochemistry & Biotechnology, Laboratory of Organic Chemistry, University of Thessaly, Ploutonos Str. 26, 41221 Larissa, Greece. E-mail: dkom@bio.uth.gr Fax: +30 2410 565-290 i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Lately, nucleosides bearing a six-membered carbohydrate moiety have been evaluated for their potential antiviral [9 – 11], antioxidant [12], and antibiotic [13] properties and as building blocks in nucleic acid synthesis [14, 15]. Insertion of an unsaturation in the 29,39-position of the sugar moiety of the aforementioned nucleosides, distinguishes this ring system from common six-membered rings [16] and allows to categorize the cyclohexene ring as a bioisoster of a saturated furanose ring. Thus, unsaturated nucleosides are recognized as an important class of biologically active molecules [7]. Among them, the unsaturated keto and exocyclic methylene nucleosides exhibit interesting antitumor and antiviral properties [17 – 22] and a number of them proved to be key intermediates in synthetic and biosynthetic processes. In an attempt to find new antitumor and antiviral agents, we have previously synthesized a series of unsaturated keto [23, 24] as well as exomethylene pyranonucleo- 354 N. Tzioumaki et al. side analogues [25, 26], which proved to be efficient as tumor cell growth inhibitors and showed to have a promising potential in combating rotaviral infections. Our studies demonstrated that the presence of a primary hydroxyl group did not seem to be a prerequisite for biological activity, contrary to the 69-protected analogues that exhibited the most promising cytotoxic and antiviral properties. In view of these observations, it was of interest to synthesize and evaluate a new class of sugar-modified pyranonucleosides, devoid of the primary hydroxymethyl function. Therefore, we present herein the synthesis of 29,39-unsaturated-49-exomethylene and 49-keto lyxopyranonucleoside analogues, containing a thymine or an uracil base moiety. This conjugation, reported for the first time, appeared to us as a challenge and the biological results confirmed our hypothesis. Results and discussion Chemistry Our synthetic strategy to the target unsaturated exomethylene lyxopyranonucleoside analogues 8a, b was to design and synthesize the saturated exomethylene compounds 7a, b, as key intermediates in order to elucidate the influence of the presence of a carbon-carbon double bond on the biological activity of the target molecules. Therefore, condensation of commercially available 1,2,3,4-tetra-O-acetyl-D-lyxopyranose 1 with silylated thymine and uracil in the presence of tin (IV) chloride afforded 1-(2,3,4-tri-O-acetyl-a-D-lyxopyranosyl)thymine 2a and 1-(2,3,4-tri-O-acetyl-a-D-lyxopyranosyl)uracil 2b, respectively. Removal of all O-acetyl protecting groups of 2a and 2b, with saturated methanolic ammonia [27], gave 3a, b in excellent yields. Specific acetalation of 3a, b using 2,2-dimethoxypropane (2,2-dmp) [27] in acetone led to the 29,39-O-isopropylidene derivatives 4a and 4b, respectively. Oxidation of the free hydroxyl group with pyridinium dichromate (PDC) / acetic anhydride (Ac2O) [28] led to the formation of the desired 49-ketonucleosides 5a, b. Wittig reaction [29] of the keto intermediates 5a and 5b resulted in 6a and 6b, respectively. Deisopropylidenation of the resulted derivatives 6a and 6b in CH2Cl2 / HCOOH, 1 : 1, led to the vicinal diol derivatives 7a and 7b, respectively. Olefination of 7a, b with iodine-triphenylphosphine (Ph3P)-imidazole [30-33] afforded 1-(2,3,4-trideoxy-4-methylene-a-pent-2-enopyranosyl)thymine 8a in 55% yield and 1-(2,3,4-trideoxy-4-methylene-a-pent-2-enopyranosyl)uracil 8b in 52% yield. In order to provide further information regarding the correlation between exomethylene unsaturated and keto i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Arch. Pharm. Chem. Life Sci. 2009, 342, 353 – 360 unsaturated nucleosides and their biological activity, we also conducted the synthesis of 49-keto-29,39-unsaturated analogues 13a and 13b. The isolated intermediates 4a, b were transformed into 9a, b by standard acetylation using Ac2O in pyridine. Deisopropylidenation of the resulted derivatives 9a, b, in CH2Cl2 / HCOOH, 1 : 1, led to the vicinal diol derivatives 10a, b in high yields (88% and 89%, respectively). Olefination of 1-(4-O-acetyl-a-D-lyxopyranosyl)thymine 10a and 1(4-O-acetyl-a-D-lyxopyranosyl)uracil 10b with iodoformPh3P-imidazole [33 – 36] afforded 11a, b, which after deacetylation with saturated methanolic ammonia [27] furnished the unprotected derivatives 12a, b. Oxidation of the free hydroxyl group in 4'-position of the sugar moiety of 12a, b with PDC / Ac2O [28] led to the formation of the desired unsaturated 49-ketonucleosides 13a, b, of which compound 13a has been previously described through an alternative approach starting from diacetylD-xylal [37]. Wittig condensation [29] of the obtained 49ketonucleosides 13a, b, with NaH and methyl triphenylphosphonium bromide (Ph3PCH3Br) at 08C, in the presence of t-amyl alcohol [37] in tetrahydrofuran (THF), afforded 1-(2,3,4-trideoxy-4-methylene-a-pent-2-enopyranosyl)thymine 8a and 1-(2,3,4-trideoxy-4-methylene-apent-2-enopyranosyl)uracil 8b, respectively. Biological activity The newly synthesized compounds 8a, b and the key intermediates 7a, b and 13a, b were evaluated for their potential antiviral activity. However, none of the compounds were inhibitory against a wide variety of DNA and RNA viruses. The test compounds 7a, b, 8a, b and 13a, b were also evaluated for their cytostatic activity against murine leukemia L1210, human lymphocyte Molt4/C8 and CEM, human breast cancer MCF7, and human colon carcinoma CACO-2 cells in cell culture. Whereas the exomethylene and the unsaturated exomethylene lyxopyranonucleoside analogues showed marginal, if any, significant cytostatic activity at 147 to A500 lM, the unsaturated keto lyxopyranonucleoside analogues were endowed with significantly more pronounced cytostatic activity. In particular, the thymine derivative 13a showed 50%-inhibitory concentrations in the lower micromolar range against L1210, Molt4/C8, CEM and MCF7 cells (i. e. IC50: 7 to 23 lM). It was less cytostatic against CACO-2 cell cultures. The corresponding uracil derivative 13b was cytostatic at 26-38 lM against L1210, Molt4/C8, CEM and MCF7 cells. Alike 13a, 13b was also markedly less cytostatic against CACO-2 cells (Table 1). Previously, the corresponding hexenopyranosyl derivatives have been evaluated on their cytostatic activity against CACO-2 cells and found signifiwww.archpharm.com Arch. Pharm. Chem. Life Sci. 2009, 342, 353 – 360 Exomethylene and Keto D-Lyxopyranonucleoside Analogues 355 Reactions and conditions: (i) Silylated base, SnCl4, dry CH3CN; (ii) NH3 / MeOH; (iii) CH3COCH3, 2,2-DMP, p-TsOH; (iv) PDC, Ac2O, dry CH2Cl2 / DMF (5 : 3); (v) Ph3PCH3Br, NaH, t-amyl alcohol in THF, 0–258C; (vi) HCOOH / CH2Cl2 (1 : 1); (vii) dry Tol / DMF (4: 1), I2-imidazole-Ph3P, 15 min; (viii) (CH3CO)2O, pyridine; (ix) dry Tol / DMF (4 : 1), CHI3-imidazole-Ph3P, 1208C, 90 min; (x) PDC, (CH3CO)2O, dry CH2Cl2. Scheme 1. Synthesis of compounds 8a and 8b. Table 1. Cytostatic activity of test compounds. IC501 (lM) Compound 7a 7b 8a 8b 13a 13b 1 L1210 Molt4/C8 CEM MCF7 CACO-2 >500 >500 147 € 0 210 € 15 7.0 € 0.4 26 € 4.0 >500 >500 213 € 15 210 € 15 11 € 5.0 38 € 3.0 >500 >500 220 € 55 256 € 64 11 € 5.0 36 € 9.0 220 € 20 209 € 8.0 176 € 11 172 € 25 23 € 7.0 35 € 9.0 500 488 € 16 320 € 27 230 € 9 77 € 7.0 134 € 13 50% Inhibitory concentration. cantly more inhibitory (IC50 in the lower micromolar range [26] than the pentenopyranosyluracil derivatives. However, the CACO-2 cell assay was performed during 10 days of drug exposure [26] whereas in the current study, only a 3-day exposure period was used for determining the IC50 value. This can explain the different cytostatic potential of the compounds. The IC50 values found for the other tumor murine and human cell lines were comparable for both types of compounds. i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Conclusion In conclusion, the synthesis of unsaturated exomethylene lyxopyranonucleoside analogues bearing thymine and uracil was undertaken. The target nucleosides 8a, b and their key intermediates saturated exomethylene 7a, b and keto unsaturated 13a, b were tested for their inhibitory effects on the proliferation of murine leukemia cells (L1210), human T-lymphocyte cells (Molt4/C8, www.archpharm.com 356 N. Tzioumaki et al. Arch. Pharm. Chem. Life Sci. 2009, 342, 353 – 360 CEM), human mammary breast carcinoma (MCF7), and human colon carcinoma cells (CACO-2). The unsaturated keto analogues 13a, b were endowed with pronounced cytostatic activity, indicating that the presence of the keto group in the sugar moiety is indispensable. Although the exomethylene derivatives 7a, b showed marginal, if any, significant inhibitory activity, the 29,39-unsaturation somewhat enhanced their activity, suggesting that the carbon-carbon double bond has not much influence on the biological activity compared to the keto moiety. Conversely to the previously reported keto and exomethylene mannopyranosyl nucleoside analogues [26], the newly synthesized molecules showed similar inhibitory activity, establishing the non requisiteness of the presence of the primary hydroxyl group. Whereas, the mode of action of these analogues is still unclear, the unsaturated keto system might function as an acceptor in a Michael-addition mechanism. It would therefore be interesting to further explore the structureactivity relationship of the compounds on their cytostatic potential. from sodium / benzophenone before use and pyridine stored over pellets of potassium hydroxide. We are grateful to Mrs. Lizette van Berckelaer, Leentje Persoons, Frieda De Meyer, Vickie Broeckx, and Leen Ingels for dedicated technical help. The financial support was provided by the ”Geconcerteerde Onderzoeksacties” (GOA no. 05/19) of the K. U. Leuven. 1-(a-D-Lyxopyranosyl)thymine 3a The authors have declared no conflict of interest. Synthesis of 1-(2,3,4-trideoxy-4-methylene-a-pent-2enopyranosyl)thymine 8a 1-(2,3,4-Tri-O-acetyl-a-D-lyxopyranosyl)thymine 2a A mixture of thymine (2.87 g, 22.72 mmol), hexamethyldisilazane (HMDS) (5.94 mL, 28.17 mmol), and saccharin (191.45 mg, 1.05 mmol) in dry CH3CN (85 mL) was refluxed for 30 min under nitrogen. To this were added 1,2,3,4-tetra-O-acetyl-D-lyxopyranose 1 (5.16 g, 16.23 mmol) and SnCl4 (2.7 mL, 22.72 mmol). The reaction mixture was refluxed for 1 h, neutralized with saturated sodium bicarbonate, and then extracted with CH2Cl2. The organic extract was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by flash chromatography (EtOAc / hexane, 7 : 3) to give compound 2a (4.49 g, 72%, Rf = 0.38) as a clear viscous oil. [a]D22 = 16.6 (c = 0.130, CHCl3). UV (CHCl3) kmax: 260 nm (e = 6926). 1H-NMR (CDCl3) d: 8.79 (br s, 1H, NH), 7.15 (s, 1H, H-6), 6.03 (d, J19,29 = 9.9 Hz, 1H, H19), 5.49 (t, J29,39 = J39,49 = 3.2 Hz, 1H, H-39), 5.26 (dd, 1H, H-29), 4.89 (m, 1H, H-49), 4.09 (m, 2H, H-5a9, H-5b9), 2.22, 2.21, 1.99 (3s, 9H, 3OAc), 1.95 (s, 3H, 5-CH3). ESI-MS (m/z): 385.04 [M + H+]. Anal. Calcd. for C16H20N2O9: C, 50.00; H, 5.25; N, 7.29. Found: C, 49.75; H, 5.32; N, 7.51. Compound 2a (4.49 g, 11.68 mmol) was treated with ammonia / MeOH (saturated at 08C, 0.48 L). The solution was stirred overnight at room temperature and, then, was concentrated under reduced pressure to give compound 3a (2.95 g, 98%, Rf = 0.40 in EtOAc / MeOH, 8 : 2) as a white foam. [a]D22 = – 10.0 (c = 0.500, MeOH). UV (MeOH) kmax: 261 nm (e = 6302). ESI-MS (m/z): 259.38 [M + H+]. Anal. Calcd. for C10H14N2O6: C, 46.51; H, 5.46; N, 10.85. Found: C, 46.96; H, 5.32; N, 10.41. 1-(2,3-O-Isopropylidene-a-D-lyxopyranosyl)thymine 4a Experimental Chemistry Melting points were recorded in a Mel-Temp apparatus (Kleinfeld Labortechnik GmbH, Gehrden, Germany) and are uncorrected. Thin-layer chromatography (TLC) was performed on Merck precoated 60F254 plates (Merck, Germany). Reactions were monitored by TLC on silica gel, with detection by UV light (254 nm) or by charring with sulphuric acid. Flash chromatography was performed using silica gel (240 – 400 mesh, Merck). 1HNMR spectra were recorded at room temperature with a Bruker 400 MHz spectrometer (Bruker Bioscience, USA) using chloroform-d (CDCl3) and methanol-d4 (CD3OD). Chemical shifts are reported in parts per million (d) downfield from tetramethylsilane (TMS) as internal standard. Mass spectra were obtained with a Micromass Platform LC (ESI-MS; Micromass, Manchester, UK). Optical rotations were measured using Autopol I polarimeter (Rudolph Research, Hackettstown, NJ. USA). All reactions were carried out in dry solvents. CH2Cl2 was distilled from phosphorous pentoxide and stored over 4E molecular sieves. Acetonitrile, toluene (Tol), and N,N-dimethyl formamide (DMF) were distilled from calcium hydride and stored over 3E molecular sieves. THF was freshly distilled under nitrogen i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim To a stirred suspension of 3a (2.95 g, 11.45 mmol) in anhydrous acetone (215 mL) and 2,2-dmp (11 mL) was added p-toluenesulfonic acid (p-TsOH) monohydrate (0.44 g, 2.29 mmol). After 3 h the resulting solution was neutralized with triethylamine so that pH did not exceed 7. The solution was concentrated and the residue was purified by flash chromatography (EtOAc) to give 4a (2.77 g, 81%, Rf = 0.34) as a white foam. [a]D22 = 22.0 (c = 0.505, CHCl3). UV (CHCl3) kmax: 260 nm (e = 1625). 1H-NMR (CDCl3) d: 7.35 (s, 1H, H-6), 5.49 (d, J19,29 = 8.6 Hz, 1H, H-19), 4.30 (m, 1H, H-39), 4.23 (dd, J29,39 = 4.9 Hz, 1H, H-29), 3.93 – 3.89 (m, 3H, H-49, H-5a9, H-5b9), 1.84 (s, 3H, 5-CH3), 1.51, 1.31 (2s, 6H, 2CH3). ESI-MS (m/z): 299.35 [M + H+]. Anal. Calcd. for C13H18N2O6: C, 52.34; H, 6.08; N, 9.39. Found: C, 52.65; H, 5.84; N, 9.63. 1-(2,3-O-Isopropylidene-a-L-erythro-pentopyranosyl-4ulose)thymine 5a A mixture of 4a (1.97 g, 6.60 mmol), PDC (2.98 g, 7.93 mmol), and Ac2O (1.87 mL, 19.80 mmol) was stirred in 80 mL of dry CH2Cl2 / DMF (5 : 3) for 1 h under nitrogen at room temperature and was concentrated in vacuo. Purification by flash chromatography (EtOAc / hexane, 8 : 2) yielded pure 5a (1.64 g, 84%, Rf = 0.36 in EtOAc / hexane, 9 : 1) as a white foam. [a]D22 = 8.0 (c = 0.533, CHCl3). UV (CHCl3) kmax: 260 nm (e = 3879). 1H-NMR (CDCl3) d: 8.43 (br s, 1H, NH), 7.11 (s, 1H, H-6), 5.51 (d, J19,29 = 5.2 Hz, 1H, Hwww.archpharm.com Arch. Pharm. Chem. Life Sci. 2009, 342, 353 – 360 19), 4.81 (dd, J29,39 = 7.2 Hz, 1H, H-29), 4.70 (d, 1H, H-39), 4.36, 4.27 (q, AB-system, J = 17.9 Hz, 2H, H-59), 1.97 (s, 3H, 5-CH3), 1.55, 1.41 (2s, 6H, 2CH3). ESI-MS (m/z): 297.32 [M + H+]. Anal. Calcd. for C13H16N2O6: C, 52.70; H, 5.44; N, 9.46. Found: C, 52.92; H, 5.78; N, 9.61. 1-(4-Deoxy-2,3-O-isopropylidene-4-methylene-a-Lerythro-pentopyranosyl)thymine 6a To a stirred suspension of Ph3PCH3Br (6.53 g, 18.28 mmol) and tamyl alcohol (2.18 mL, 19.94 mmol) in dry THF (56 mL) was added NaH (0.76 g, 60% in oil, 31.90 mmol) at 08C under nitrogen and the reaction mixture was stirred for 2 h at ambient temperature. To this yellow phosphorous ylide was added a solution of 5a (1.64 g, 5.54 mmol) in dry THF (9 mL) dropwise, at 08C under nitrogen. After the mixture was stirred for 30 min at ambient temperature, the reaction mixture was quenched with saturated sodium bicarbonate and extracted with EtOAc. The organic layer was washed with water, dried with sodium sulfate, and evaporated. The residue was purified by flash chromatography (EtOAc / hexane, 5 : 5) to give 6a (0.99 g, 61%, Rf = 0.3), as a white foam. [a]D22 = – 6.0 (c = 0.433, CHCl3). UV (CHCl3) kmax: 265 nm (e = 6874). 1H-NMR (CDCl3) d: 8.55 (br s, 1H, NH), 7.10 (s, 1H, H-6), 5.74 (d, J19,29 = 5.0 Hz, 1H, H-19), 5.43, 5.36 (br s, 2H, methylene), 4.77 (d, J29,39 = 8.9 Hz, 1H, H-39), 4.48, 4.32 (q, AB-system, J = 13.2 Hz, 2H, H-59), 4.18 – 4.15 (m, 1H, H-29), 1.93 (s, 3H, 5CH3), 1.64, 1.43 (2s, 6H, 2CH3). ESI-MS (m/z): 295.26 [M + H+]. Anal. Calcd. for C14H18N2O5: C, 57.13; H, 6.16; N, 9.52. Found: C, 57.40; H, 6.42; N, 9.43. 1-(4-Deoxy-4-methylene-a-L-erythropentopyranosyl)thymine 7a Compound 6a (0.99 g, 3.38 mmol) was dissolved in a mixture of CH2Cl2 (12 mL) and HCOOH (12 mL, 90%). The solution was stirred for 3 h at room temperature and then concentrated under reduced pressure. The residue was purified by flash chromatography (EtOAc / hexane, 9 : 1) to give 7a (0.72 g, 84%, Rf = 0.14) as a white foam. [a]D22 = – 14.0 (c = 0.605, CHCl3). UV (CHCl3) kmax: 260 nm (e = 6538). 1H-NMR (CDCl3) d: 10.36 (br s, 1H, NH), 7.19 (s, 1H, H-6), 6.03 (d, J19,29 = 9.3 Hz, 1H, H-19), 5.22, 5.12 (br s, 2H, methylene), 4.61 (d, J29,39 = 1.9 Hz, 1H, H-39), 4.57, 4.14 (q, AB-system, J = 12.6 Hz, 2H, H-59), 3.69 (d, 1H, H-29), 1.85 (s, 3H, 5-CH3). ESI-MS (m/z): 255.28 [M + H+]. Anal. Calcd. for C11H14N2O5: C; 51.97, H; 5.55, N; 11.02. Found: C, 52.23; H, 5.76; N, 10.88. 1-(2,3,4-Trideoxy-4-methylene-a-pent-2enopyranosyl)thymine 8a Ph3P (2.22 g, 8.49 mmol), iodine (1.08 g, 4.25 mmol), and imidazole (0.29 g, 4.25 mmol) were added to the suspension of 7a (0.72 g, 2.83 mmol) in 78 mL of dry Tol / DMF (4 : 1). The reaction mixture was heated (808C, oil bath) under nitrogen for 15 min and concentrated in vacuo. The residue was purified by flash chromatography (EtOAc / hexane, 9 : 1) and yielded 8a (0.34 g, 55%), as a white foam. [a]D22 = – 8.0 (c = 0.500, CHCl3). UV (CHCl3) kmax: 260 nm (e = 6389). 1H-NMR (CDCl3) d: 8.44 (br s, 1H, NH), 7.06 (s, 1H, H-6), 6.58 (dd, J19,39 = 1.5 Hz, J29,39 = 10.0 Hz, 1H, H-39), 6.41 (br s, 1H, H-19), 5.62 (d, 1H, H-29), 5.04, 4.99 (br s, 2H, methylene), 4.39, 4.33 (q, AB-system, J = 13.9 Hz, 2H, H-59), 1.85 (s, 3H, 5-CH3). ESI-MS (m/z): 221.34 [M + H+]. Anal. Calcd. for C11H12N2O3: C, 59.99; H, 5.49; N, 12.72. Found: C, 59.82; H, 5.68; N, 12.93. i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Exomethylene and Keto D-Lyxopyranonucleoside Analogues 357 1-(4-O-Acetyl-2,3-O-isopropylidene-a-Dlyxopyranosyl)thymine 9a Compound 4a (0.80 g, 2.68 mmol) was dissolved in a mixture of pyridine (2.3 mL) and Ac2O (1.2 mL, 12.72 mmol). The reaction was carried out at room temperature for 2 h, then quenched with MeOH at 08C and concentrated. The residue was purified by flash chromatography (EtOAc / hexane, 8 : 2) to give 9a (0.87 g, 95%, Rf = 0.54) as a white solid, m.p.: 158 – 1608C. [a]D22 = 12.0 (c = 0.583, CHCl3). UV (CHCl3) kmax: 265 nm (e = 7065). 1H-NMR (CDCl3) d: 8.89 (br s, 1H, NH), 7.13 (s, 1H, H-6), 5.65 (d, J19,29 = 8.6 Hz, 1H, H19), 5.17 (d, J39,49 = 1.3 Hz, 1H, H-49), 4.34 (m, 1H, H-39), 4.23 (m, 1H, H-29), 4.04 – 3.99 (m, 2H, H-5a9, H-5b9), 2.18 (s, 3H, OAc), 1.95 (s, 3H, 5-CH3), 1.62, 1.39 (2s, 6H, 2CH3). ESI-MS (m/z): 341.13 [M + H+]. Anal. Calcd. for C15H20N2O7: C, 52.94; H, 5.92; N, 8.23. Found: C, 53.30; H, 5.38; N, 8.52. 1-(4-O-Acetyl-a-D-lyxopyranosyl)thymine 10a Compound 9a (0.87 g, 2.55 mmol) was dissolved in a mixture of CH2Cl2 (8.6 mL) and HCOOH (8.6 mL, 90%). The solution was stirred overnight at room temperature and then was concentrated under reduced pressure. The residue was purified by flash chromatography (EtOAc) to give 10a (0.67 g, 88%, Rf = 0.28) as a white solid, m.p.: 229 – 2318C. [a]D22 = – 20.0 (c = 0.780, MeOH). UV (MeOH) kmax: 260 nm (e = 5073). 1H-NMR (CD3OD) d: 7.45 (d, J5b9,6 = 1.1 Hz, 1H, H-6), 5.84 (d, J19,29 = 9.7 Hz, 1H, H-19), 4.85 (d, J29,39 = 3.1 Hz, 1H, H-39), 4.12 (m, 2H, H-49, H-5a9), 3.95 (dd, 1H, H-29), 3.87 (dd, J49,5b9 = 1.1 Hz, J5a9,5b9 = 13.0 Hz, 1H, H-5b9), 2.14 (s, 3H, OAc), 1.91 (s, 3H, 5-CH3). ESI-MS (m/z): 301.12 [M + H+]. Anal. Calcd. for C12H16N2O7: C, 48.00; H, 5.37; N, 9.33. Found: C, 47.72; H, 5.78; N, 9.79. 1-(4-O-Acetyl-2,3-dideoxy-a-D-glycero-pent-2enopyranosyl)thymine 11a Imidazole (0.32 g, 4.70 mmol), Ph3P (2.46 g, 9.41 mmol), and iodoform (1.85 g, 4.70 mmol) were added to the suspension of 10a (0.67 g, 2.24 mmol) in 19 mL of dry Tol / DMF (4 : 1). The reaction mixture was heated (1008C, oil bath) under nitrogen for 2 h, concentrated in vacuo and the residue diluted with EtOAc, washed with saturated sodium bicarbonate, sodium thiosulfate and water. The organic extract was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by flash chromatography (EtOAc / hexane, 6 : 4) and yielded 11a (0.33 g, 56%, Rf = 0.45 in EtOAc) as a colorless oil. [a]D22 = 46.0 (c = 0.330, CHCl3). UV (CHCl3) kmax: 260 nm (e = 4622). 1 H-NMR (CDCl3) d: 8.20 (br s, 1H, NH), 7.15 (s, 1H, H-6), 6.35 (m, 2H, H-19, H-39), 5.92 (dd, J19,29 = 1.3 Hz, J29,39 = 10.0 Hz, 1H, H-29), 5.11 (m, 1H, H-49), 4.09 – 3.99 (m, J = 13.0 Hz, 2H, H-59), 2.15 (s, 3H, OAc), 1.94 (s, 3H, 5-CH3). ESI-MS (m/z): 267.29 [M + H+]. Anal. Calcd. for C12H14N2O5: C, 54.13; H, 5.30; N, 10.52. Found: C, 54.58; H, 4.72; N, 10.68. 1-(2,3-Dideoxy-a-D-glycero-pent-2-enopyranosyl)thymine 12a Compound 11a (0.33 g, 1.24 mmol) was treated with ammonia / MeOH (saturated at 08C, 17 mL). The solution was stirred overnight at room temperature and then concentrated under reduced pressure. Purification by flash chromatography (EtOAc) yielded 12a (0.19 g, 68%, Rf = 0.16) as a white solid, m.p.: 1861888C. [a]D22 = 4.0 (c = 0.230, CHCl3). UV (CHCl3) kmax: 260 nm (e = 8584). 1H-NMR (CDCl3) d: 8.56 (br s, 1H, NH), 7.21 (s, 1H, H-6), 6.40 www.archpharm.com 358 N. Tzioumaki et al. Arch. Pharm. Chem. Life Sci. 2009, 342, 353 – 360 (m, 1H, H-39), 6.29 (br s, 1H, H-19), 5.79 (dd, J19,29 = 1.2 Hz, J29,39 = 10.1 Hz, 1H, H-29), 4.15 – 4.13 (m, 1H, H-49), 4.00 (m, 2H, H-5a9, H5b9), 1.92 (s, 3H, 5-CH3). ESI-MS (m/z): 225.25 [M + H+]. Anal. Calcd. for C10H12N2O4: C, 53.57; H, 5.39; N, 12.49. Found: C, 53.42; H, 5.47; N, 12.58. in EtOAc / MeOH, 8 : 2) as a white foam, and it was used without further purification. [a]D22 = – 12.0 (c = 0.500, MeOH). UV (MeOH) kmax: 258 nm (e = 6869). ESI-MS (m/z): 245.26 [M + H+]. Anal. Calcd. for C9H12N2O6: C, 44.27; H, 4.95; N, 11.47. Found: C, 44.56; H, 5.12; N, 11.78. 1-(2,3-Dideoxy-a-pent-2-enopyranosyl-4-ulose)thymine 13a 1-(2,3-O-Isopropylidene-a-D-lyxopyranosyl)uracil 4b A mixture of 12a (0.19 g, 0.84 mmol), PDC (0.38 g, 1.01 mmol), and Ac2O (0.24 mL, 2.52 mmol) was stirred in dry CH2Cl2 (14 mL) for 2 h under nitrogen at room temperature. Purification by flash chromatography (EtOAc / hexane, 8 : 2) yielded pure 13a (0.16 g, 87%, Rf = 0.54 in EtOAc) as a white solid, m.p.: 169 – 1718C. [a]D22 = – 6.0 (c 0.305, CHCl3). UV (CHCl3) kmax: 258 nm (e = 6466). 1H-NMR (CDCl3) d: 10.12 (br s, 1H, NH), 7.36 (s, 1H, H-6), 7.15-7.12 (m, 1H, H-39), 6.59 (br s, 1H, H-19), 6.34 – 6.31 (m, 1H, H29), 4.37, 4.22 (q, AB-system, J = 16.3 Hz, 2H, H-59), 1.98 (s, 3H, 5CH3). ESI-MS (m/z): 223.24 [M + H+]. Anal. Calcd. for C10H10N2O4: C, 54.05; H, 4.54; N, 12.61. Found: C, 54.22; H, 4.38; N, 12.71. 1-(2,3,4-Trideoxy-4-methylene-a-pent-2enopyranosyl)thymine 8a To a stirred suspension of Ph3PCH3Br (0.86 g, 2.41 mmol) and tamyl alcohol (0.29 mL, 2.63 mmol) in dry THF (6.7 mL) was added NaH (0.10 g, 60% in oil, 4.19 mmol) at 08C under nitrogen and the reaction mixture was stirred for 2 h at ambient temperature. To this yellow phosphorous ylide was added a solution of 13a (0.16 g, 0.73 mmol) in dry THF (1.1 mL) dropwise, at 08C. After the mixture was stirred for 30 min at ambient temperature, the reaction mixture was quenched with saturated sodium bicarbonate and extracted with EtOAc. The organic layer was washed with water, dried with sodium sulfate, and evaporated. The residue was purified by flash chromatography (EtOAc / hexane, 9 : 1) to give 8a (0.98 g, 61%, Rf = 0.6) as a white foam. Synthesis of 1-(2,3,4-trideoxy-4-methylene-a-pent-2enopyranosyl)uracil 8b 1-(2,3,4-Tri-O-acetyl-a-D-lyxopyranosyl)uracil 2b A mixture of uracil (2.46 g, 21.99 mmol), HMDS (5.75 mL, 27.26 mmol), and saccharin (185.29 mg, 1.01 mmol) in dry CH3CN (68 mL) was refluxed for 30 min under nitrogen. To this was added 1,2,3,4-tetra-O-acetyl-D-lyxopyranose 1 (5.0 g, 15.71 mmol) and SnCl4 (2.6 mL, 21.99 mmol). The reaction mixture was refluxed for 2 h, neutralized with saturated sodium bicarbonate, and then extracted with CH2Cl2. The organic extract was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by flash chromatography (EtOAc / hexane, 7 : 3) to give compound 2b (3.79 g, 65%, Rf = 0.3) as a white foam. [a]D22 = – 32.0 (c = 0.500, CHCl3). UV (CHCl3) kmax: 258 nm (e = 9344). 1H-NMR (CDCl3) d: 8.86 (br s, 1H, NH), 7.30 (d, J5,6 = 8.2 Hz, 1H, H-6), 5.96 (d, J19,29 = 9.9 Hz, 1H, H-19), 5.75 (d, 1H, H-5), 5.41 (t, J29,39 = J39,49 = 3.2 Hz, 1H, H-39), 5.18 (dd, 1H, H-29), 4.82 (d, 1H, H-49), 4.05, 3.98 (q, AB-system, J = 13.3 Hz, 2H, H59), 2.15, 2.13, 1.92 (3s, 9H, 3OAc). ESI-MS (m/z): 371.36 [M + H+]. Anal. Calcd. for C15H18N2O9: C, 48.65; H, 4.90; N, 7.56. Found: C, 49.14; H, 5.24; N, 7.41. 1-(a-D-Lyxopyranosyl)uracil 3b Uracil derivative 3b was synthesized from 2b by the similar procedure as described for 3a. 3b was obtained (2.47 g, 99%, Rf = 0.34 i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Uracil derivative 4b was synthesized from 3b by the similar procedure as described for 4a. It was purified by flash chromatography (EtOAc) (2.24 g, 78%, Rf = 0.57 in EtOAc / MeOH, 9 : 1) and 4b was obtained as a white foam. [a]D22 = 6.0 (c = 0.485, MeOH). UV (MeOH) kmax: 258 nm (e = 4457). 1H-NMR (CD3OD) d: 10.05 (br s, 1H, NH), 7.63 (d, J5,6 = 8.1 Hz, 1H, H-6), 5.61 (d, 1H, H-5), 4.52 (d, J19,29 = 6.4 Hz, 1H, H-19), 4.34 – 4.29 (m, 2H, H-29, H-39), 3.90 – 3.82 (m, 3H, H-49, H-5a9, H-5b9), 1.44, 1.26 (2s, 6H, 2CH3). ESI-MS (m/z): 285.31 [M + H+]. Anal. Calcd. for C12H16N2O6: C, 50.70; H, 5.67; N, 9.85. Found: C, 50.85; H, 5.80; N, 9.98. 1-(2,3-O-Isopropylidene-a-L-erythro-pentopyranosyl-4ulose)uracil 5b Uracil analogue 5b was synthesized from 4b by the similar procedure as described for 5a. It was purified by flash chromatography (EtOAc / hexane, 8 : 2) and yielded pure 5b (0.85 g, 69%, Rf = 0.45 in EtOAc / hexane, 9 : 1) as a white foam. [a]D22 = 4.0 (c = 0.500, CHCl3). UV (CHCl3) kmax: 258 nm (e = 2444). 1H-NMR (CDCl3) d: 8.02 (br s, 1H, NH), 7.31 (d, J5,6 = 8.0 Hz, 1H, H-6), 5.85 (d, 1H, H5), 5.52 (d, J19,29 = 5.3 Hz, 1H, H-19), 4.77 (m, 1H, H-29), 4.69 (d, J29,39 = 7.2 Hz, 1H, H-39), 4.37, 4.30 (q, AB-system, J = 17.9 Hz, 2H, H-59), 1.56 (2s, 6H, 2CH3). ESI-MS (m/z): 283.22 [M + H+]. Anal. Calcd. for C12H14N2O6: C, 51.06; H, 5.00; N, 9.93. Found: C, 51.38; H, 5.11; N, 9.68. 1-(4-Deoxy-2,3-O-isopropylidene-4-methylene-a-Lerythro-pentopyranosyl)uracil 6b Uracil analogue 6b was synthesized from 5b by the similar procedure as described for 6a. It was purified by flash chromatography (EtOAc / hexane, 7 : 3) to give 6b (0.46 g, 54%, Rf = 0.54 in EtOAc / hexane, 9 : 1) as a white foam. [a]D22 = – 10.0 (c = 0.500, CHCl3). UV (CHCl3) kmax: 258 nm (e = 8024). 1H-NMR (CDCl3) d: 8.26 (br s, 1H, NH), 7.26 (d, J5,6 = 8.4 Hz, 1H, H-6), 5.77 (d, J29,39 = 7.3 Hz, 1H, H-39), 5.73 (d, 1H, H-5), 5.53, 5.43 (br s, 2H, methylene), 4.76 (br s, 1H, H-19), 4.47, 4.32 (q, AB-system, J = 12.7 Hz, 2H, H-59), 4.11 (m, 1H, H-29), 1.63, 1.48 (2s, 6H, 2CH3). ESI-MS (m/z): 281.29 [M + H+]. Anal. Calcd. for C13H16N2O5: C, 55.71; H, 5.75; N, 9.99. Found: C, 56.12; H, 5.93; N, 9.85. 1-(4-Deoxy-4-methylene-a-L-erythropentopyranosyl)uracil 7b Uracil analogue 7b was synthesized from 6b by the similar procedure as described for 7a. It was purified by flash chromatography (EtOAc / hexane, 9 : 1) to give 7b (0.32 g, 82%, Rf = 0.13) as a white foam. [a]D22 = -12.0 (c = 0.480, MeOH). UV (MeOH) kmax: 258 nm (e = 5825). 1H-NMR (CDCl3) d: 7.36 (d, J5,6 = 8.2 Hz, 1H, H-6), 5.94 (d, J19,29 = 9.3 Hz, 1H, H-19), 5.76 (d, 1H, H-5), 5.23, 5.15 (br s, 2H, methylene), 4.58 (d, J29,39 = 2.9 Hz, 1H, H-39), 4.54, 4.17 (q, ABsystem, J = 12.6 Hz, 2H, H-59), 4.60 – 4.54 (m, 2H, H-5a9, H-5b9), 3.62 (dd, 1H, H-29). ESI-MS (m/z): 241.25 [M + H+]. Anal. Calcd. for C10H12N2O5: C, 50.00; H, 5.04; N, 11.66. Found: C, 50.41; H, 5.22; N, 11.43. www.archpharm.com Arch. Pharm. Chem. Life Sci. 2009, 342, 353 – 360 1-(2,3,4-Trideoxy-4-methylene-a-pent-2enopyranosyl)uracil 8b Uracil analogue 8b was synthesized from 7b by the similar procedure as described for 8a. It was purified by flash chromatography (EtOAc / hexane, 8 : 2) (0.14 g, 52%) and 8b was obtained as a white foam. [a]D22 = 8.0 (c = 0.425, CHCl3). UV (CHCl3) kmax: 258 nm (e = 6249). 1H-NMR (CDCl3) d: 8.24 (br s, 1H, NH), 7.35 (d, J5,6 = 8.1 Hz, 1H, H-6), 6.66 (d, J29,39 = 10.0 Hz, 1H, H-39), 6.47 (br s, 1H, H19), 5.72-5.68 (m, 2H, H-29, H-5), 5.11, 5.07 (br s, 2H, methylene), 4.45, 4.38 (q, AB-system, J = 14.0 Hz, 2H, H-59). ESI-MS (m/z): 207.29 [M + H+]. Anal. Calcd. for C10H10N2O3: C, 58.25; H, 4.89; N, 13.59. Found: C, 58.42; H, 4.63; N, 13.51. 1-(4-O-Acetyl-2,3-O-isopropylidene-a-Dlyxopyranosyl)uracil 9b Uracil derivative 9b was synthesized from 4b by the similar procedure as described for 9a. It was purified by flash chromatography (EtOAc / hexane, 5 : 5) (0.99 g, 86%, Rf = 0.52 in EtOAc / hexane, 8 : 2) and 9b was obtained as a white solid, m.p.: 185 – 1868C. [a]D22 = 36.0 (c = 0.505, CHCl3). UV (CHCl3) kmax: 258 nm (e = 8015). 1 H-NMR (CDCl3) d: 8.29 (br s, 1H, NH), 7.32 (d, J5,6 = 8.01 Hz, 1H, H6), 5.80 (d, 1H, H-5), 5.64 (d, J19,29 = 8.6 Hz, 1H, H-19), 5.16 (br s, 1H, H-49), 4.33 (br s, 1H, H-39), 4.17 (m, 1H, H-29), 4.06, 4.01 (q, AB-system, J = 13.2 Hz, 2H, H-59), 2.16 (s, 3H, OAc), 1.62, 1.39 (2s, 6H, 2CH3). ESI-MS (m/z): 327.33 [M + H+]. Anal. Calcd. for C14H18N2O7: C, 51.53; H, 5.56; N, 8.59. Found: C, 51.79; H, 5.38; N, 8.82. 1-(4-O-Acetyl-a-D-lyxopyranosyl)uracil 10b Uracil derivative 10b was synthesized from 9b by the similar procedure as described for 10a. It was purified by flash chromatography (EtOAc) (0.77 g, 89%, Rf = 0.17) and 10b was obtained as a white solid, m.p.: 196 – 1978C. [a]D22 = – 18.0 (c = 0.500, MeOH). UV (MeOH) kmax: 258 nm (e = 9495). 1H-NMR (CD3OD) d: 10.60 (br s, 1H, NH), 8.07 (d, J5,6 = 8.1 Hz, 1H, H-6), 5.86 (d, J19,29 = 9.1 Hz, 1H, H19), 5.77 (d, 1H, H-5), 4.97 (br s, 1H, H-39), 4.25 (br s, 1H, H-49), 4.14, 3.94 (q, AB-system, J = 13.0 Hz, 2H, H-59), 3.83 (m, 1H, H-29), 1.90 (s, 3H, OAc). ESI-MS (m/z): 287.71 [M + H+]. Anal. Calcd. for C11H14N2O7: C, 46.16; H, 4.93; N, 9.79. Found: C, 46.32; H, 4.75; N, 9.83. 1-(4-O-Acetyl-2,3-dideoxy-a-D-glycero-pent-2enopyranosyl)uracil 11b Uracil derivative 11b was synthesized from 10b by the similar procedure as described for 11a. It was purified by flash chromatography (EtOAc) (0.40 g, 59%, Rf = 0.42) and 11b was obtained as a white solid, m.p.: 150 – 1518C. [a]D22 = 48.0 (c = 0.500, CHCl3). UV (CHCl3) kmax: 258 nm (e = 10696). 1H-NMR (CDCl3) d: 8.72 (br s, 1H, NH), 7.36 (d, J5,6 = 8.1 Hz, 1H, H-6), 6.36 (m, 2H, H-19, H-39), 5.90 (d, J29,39 = 10.1 Hz, 1H, H-29), 5.78 (d, 1H, H-5), 5.11 (m, 1H, H-49) 4.063.99 (m, 2H, H-5a9, H-5b9), 2.13 (s, 3H, OAc). ESI-MS (m/z): 253.29 [M + H+]. Anal. Calcd. for C11H12N2O5: C, 52.38; H, 4.80, N, 11.11. Found: C, 52.51; H, 5.02; N, 11.48. 1-(2,3-Dideoxy-a-D-glycero-pent-2-enopyranosyl)uracil 12b Compound 12b was synthesized from 11b by the similar procedure as described for 12a. It was purified by flash chromatography (EtOAc) (0.23 g, 70%, Rf = 0.12) and 12b was obtained as a white foam. [a]D22 = 14.0 (c = 0.454, MeOH). UV (MeOH) kmax: 260 nm (e = 2117). 1H-NMR (CDCl3) d: 8.20 (br s, 1H, NH), 7.41 (d, i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Exomethylene and Keto D-Lyxopyranonucleoside Analogues 359 J5,6 = 8.1 Hz, 1H, H-6), 6.40 (m, 1H, H-39), 6.29 (d, J19,29 = 1.7 Hz, 1H, H-19), 5.79 (dd, J29,39 = 10.1 Hz, 1H, H-29), 5.75 (d, 1H, H-5), 4.15 (br s, 1H, H-49), 3.98 – 3.95 (m, 2H, H-5a9, H-5b9). ESI-MS (m/z): 211.38 [M + H+]. Anal. Calcd. for C9H10N2O4: C, 51.43; H, 4.80; N, 13.33. Found: C, 51.52; H, 4.67; N, 13.57. 1-(2,3-Dideoxy-a-pent-2-enopyranosyl-4-ulose)uracil 13b Compound 13b was synthesized from 12b by the similar procedure as described for 13a. It was purified by flash chromatography (EtOAc) (0.20 g, 89%, Rf = 0.45) and 13b was obtained as a white solid, m.p.: 158 – 1608C. [a]D22 = -10.0 (c = 0.486, CHCl3). UV (CHCl3) kmax: 258 nm (e = 3613). 1H-NMR (CDCl3) d: 9.03 (br s, 1H, NH), 7.29 (d, J5,6 = 8.1 Hz, 1H, H-6), 6.89 (dd, J19,39 = 1.5 Hz, J29,39 = 10.5 Hz, 1H, H-39), 6.67 (br s, 1H, H-19), 6.46 (dd, J19,29 = 1.8 Hz, 1H, H-29), 5.82 (d, 1H, H-5), 4.43-4.34 (m, 2H, H-5a9, H-5b9). ESI-MS (m/z): 209.21 [M + H+]. Anal. Calcd. for C9H8N2O4: C, 51.93; H, 3.87; N, 13.46. Found: C, 52.24; H, 3.68; N 13.61. 1-(2,3,4-Trideoxy-4-methylene-a-pent-2enopyranosyl)uracil 8b Uracil analogue 8b was synthesized from 13b by the similar procedure as described for 8a. It was purified by flash chromatography (EtOAc / hexane, 8 : 2) (0.11 g, 57%, Rf = 0.5 in EtOAc) and 8b was obtained as a white foam. Antiviral assays The antiviral assays, other than the anti-HIV assays, were based on inhibition of virus-induced cytopathicity in HEL (herpes simplex virus type 1 (HSV-1) (KOS), HSV-2 (G), vaccinia virus, and vesicular stomatitis virus), Vero (parainfluenza-3, reovirus-1, Sindbis, Coxsackie B4, and Punta Toro virus), or HeLa (vesicular stomatitis virus, Coxsackie virus B4, and respiratory syncytial virus) cell cultures. Confluent cell cultures in microtiter 96-well plates were inoculated with 100 CCID50 of virus (1 CCID50 being the virus dose to infect 50% of the cell cultures). After a 1 h virusadsorption period, residual virus was removed, and the cell cultures were incubated in the presence of varying concentrations (200, 40, 8, …, lM) of the test compounds. Viral cytopathicity was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. The methodology of the anti-HIV assays was as follows: human CEM (l36105 cells/cm3) cells were infected with 100 CCID50 of HIV(IIIB) or HIV-2(ROD)/mL and seeded in 200 lL wells of a microtiter plate containing appropriate dilutions of the test compounds. After 4 days of incubation at 378C, HIVinduced CEM giant cell formation was examined microscopically. Cytostatic assays Murine leukemia L1210, human lymphocyte Molt4/C8 and CEM, human breast carcinoma MCF7, and human colon carcinoma CACO-2 were seeded in 96-well microtiter plates at 50 000 – 75 000 cells/well in RPMI-1640 culture medium in the absence or presence of serial dilutions of the test compounds. After 2 days (L1210) or 3 days (other cell lines), the cell numbers were counted using a Coulter counter. The cytostatic concentration was calculated as the CC50, or the compound concentration required reducing cell proliferation by 50% relative to the number of cells in the untreated controls. CC50 values were estimated from graphic plots of the number of cells (percentage of control) as a function of the concentration of the test compounds. www.archpharm.com 360 N. Tzioumaki et al. Arch. Pharm. Chem. Life Sci. 2009, 342, 353 – 360 References [19] F. Leclercq, K. Antonakis, Carbohydr. Res. 1994, 263, 309 – 313. [1] W. Zhou, G. Gumina, Y. Chong, J. Wang, et al., J. Med. 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