NPC 2010 Vol. 5 No. 2 253 - 258 Natural Product Communications Aristolactams, 1-(2-C-Methyl-β-D-ribofuranosyl)-uracil and Other Bioactive Constituents of Toussaintia orientalis Josiah O. Odaloa, Cosam C. Josepha, Mayunga H.H. Nkunyaa*, Isabel Sattlerb, Corinna Langeb, Gollmick Friedrichb, Hans-Martin Dahseb and Ute Möllmanb a Department of Chemistry, University of Dar es Salaam, P.O. Box 35061, Dar es Salaam, Tanzania b Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute, Beutenbergstrasse 11a, 07745 Jena, Germany nkunya@chem.udsm.ac.tz, mnkunya@tcu.go.tz Received: September 1st, 2009; Accepted: November 23rd, 2009 The new aristolactam alkaloid toussalactam {2-hydroxy-1,6-dimethoxy-5H-dibenzo[cdf]indol-4-one} and the known ones, namely aristolactam AII, aristolactam BII, piperolactam C and aristolactam FII; 1-(2-C-methyl-β-D-ribofuranosyl)-uracil, 3,4,5-trimethoxyphenyl-β-D-glucopyranoside, and three catechinoids were isolated from the cytotoxic Toussaintia orientalis Verdc stem and root bark extracts, and their structures established based on analysis of spectroscopic data. The aristolactams exhibited antimicrobial and antiinflammatory activity, aristolactam FII showing almost the same level of activity as the standard anti-inflammatory agent Indomethacin. The compounds also exhibited either mild or no antiproliferative and cytotoxic activities, except aristolactam FII that showed the same level of cytotoxicity as the standard drug Camptothecin. 1-(2C-Methyl-β-D-ribofuranosyl)-uracil, which is being reported for the first time as a natural product, was inactive in the antibacterial, antifungal, antiinflammatory, antiproliferative and cytotoxicity assays. Keywords: Toussaintia orientalis, Annonaceae, Aristolactams, 1-(2-C-methyl-β-D-ribofuranosyl)-uracil, Antimicrobials, Antiproliferative, Cytotoxicity, Antiinflammatory. In East Africa several Annonaceae species are used as herbal medicines [1,2]. This has inspired us to investigate the nearly 90 Annonaceae species occurring in Tanzania, some of which having been taxonomically described only recently [3,4]. Others, such as Toussaintia oriantalis Verdc., are reported to occur only in Tanzania where their ecological habitats are systematically being destroyed through human activities, thus threatening them with imminent extinction. Therefore, our investigations are focused on determining the bioactive or other chemical constituents of such endangered plant species. This paper reports the isolation, structural determination, and anti-bacterial, antifungal, antiproliferative, cytotoxic and antiinflammatory activities of the constituents of T. oriantalis, which include the hitherto unreported aristolactam alkaloid toussalactam (1), as well as the known analogues aristolactam AII, aristolactam BII, piperolactam C and aristolactam FII (2–5) [6-8], the nucleoside 1-(2-C-methyl-β-Dribofuranosyl)-uracil (6), previously reported only as a R'' O R' 2 O NH 1 R 5a 10 4 5 5 NH 6 10a 6 R''' 6a N 2 O HO 5' 8 R 1: 2: 3: 4: 5: OMe OMe OMe OMe OMe R' R'' R''' OH OH OMe OMe OH H OMe H H H H OMe H OMe H O Me 4' 1' 2' OH OH 6 synthetic product [9], 3,4,5-trimethoxyphenyl-β-Dglucopyranoside [10], and three catechinoids. Structure 1 for toussalactam was established based on analysis of spectroscopic data [11,12], the C-6 substitution being indicated by the absence of a 1 H NMR signal at ca. δ 7.2 that is diagnostic of H-6 in aristolactams [13]. Furthermore, the 13C NMR data indicated the two methoxy groups were bis orthosubstituted at C-1 and C-6 {δ OCH3) = ca. 60 ppm [14]}, 254 Natural Product Communications Vol. 5 (2) 2010 Odalo et al. O H HN O H H N H H O CH3 HO H H OH OH Figure 1: Important H/C HMBC correlations for toussalactam (1). the third substituent being a C-2 OH group. The COSY, HMQC and HMBC interactions (Figure 1) indicated the inter-atomic connectivity of the aristolactam skeleton and the substitution pattern of the phenanthrenoid carbacyclic system, thus confirming structure 1. Aristolactams form a small group of modified aporphinoids that exhibit antibacterial, antimalarial, cytotoxic, and platelet aggregation inhibition activities. These compounds are distributed in the families Annonaceae, Aristolochiaceae, Menispermaceae, Monimimaceae and Piperaceae [7,15]. Structure 6 for 1-(2-C-methyl-β-D-ribofuranosyl)-uracil was based on analysis of the 1H and 13C NMR spectra, as well as HMBC interactions (Figure 2), indicating the presence of a C-2 methylated pentafuranose sugar linked to the uracil moiety through the anomeric carbon. In the 1H NMR spectrum the J3,4 value of 9.2 Hz was indicative of the β-configuration at the anomeric carbon [16-19], as further corroborated from the UV and CD spectra [21-23]. The CD and 1H NMR spectral data, as well as the strong H-6/H-3α NOE correlation, also indicated both the anti and C-3α endo conformation for the sugar unit in solution [17], and an anti orientation for the base. NOE further indicated a cis configuration for 2β-C-Me/H-3α. Compound 6 is reported for the first time as a plant natural product, which is also unprecedented among nucleosides since so far they have been obtained only from marine sponges [23,24]. Nucleoside analogues have been used to treat viral infections [26-28] and are potential leads to new antiviral agents [28-30]. The light petroleum, CH2Cl2 and MeOH stem and root bark extracts showed activity in the brine shrimp test (LC50 72.4, 22.3 and 19.2, and 57.6, 1.3 and 17.2 µg/mL respectively), the stem bark extracts being the most active. This is the source of the aristolactams that showed the highest antibacterial, antiinflammatory, antiproliferative and cytotoxic activities. In the antibacterial assay the aristolactams 1 – 5 exhibited growth inhibition effects against M. vaccae, but only Figure 2: Important H/C HMBC correlations for 1-(2-C-methyl-β-Dribofuranosyl)-uracil (6). mild activity against all other microbial strains, with 1 and 5 more active against the fungi and M. vaccae than against the other organisms tested (Table 1). Generally, the active compounds showed better efficacy against bacterial than fungal strains, the fungus P. notatum (P1) being more susceptible as compared with the other fungi, S. salmonicolor and C. albicans. Most of the compounds exhibited moderate activity against P. notatum (P1), compound 1 being the most active against this fungal strain (Table 1). Against all the test organisms, compound 6 and 3,4,5-trimethoxyphenyl-βD-glucopyranoside showed no activity. The results in Table 1 indicate that increased oxygenation of the aristolactam skeleton generally enhanced antibacterial activity, especially at C-6, which also increased antifungal activity. Since inhibition of rat liver cytosol NAD (P) linked 3α-hydroxysteroid dehydrogenase is correlated to anti-inflammatory activity in humans, that test was applied to compounds 3–6, 3,4,5-trimethoxy-phenyl-β-D-glucopyranoside and epicatechin-4β,8-epicatechin (Table 2). Aristolactams 3–5 were moderately active, epicatechin-4β,8epicatechin was only mildly active, while both 6 and were 3,4,5-trimethoxyphenyl-β-D-glucopyranoside inactive. Compared with the standard anticancer drugs Taxol®, Colchicine and Camptothecin, the aristolactams also exhibited some antiproliferative activity, compound 4 being the most potent against L-929, whereas 5 was the most active against the K-562 cell lines (Table 3), having the same level of cytotoxicity as Camptothecin. Compound 6 and 3,4,5-trimethoxyphenyl-β-Dglucopyranoside were inactive in the antiproliferative and cytotoxicity assays. Experimental General experimental procedures: CC: Silica gel 60 (0.063-0.200 mm, Merck) and Sephadex® LH-20 (Pharmacia); TLC: Kieselgel 60 F 254 precoated on Bioactive constituents of Toussaintia orientalis Natural Product Communications Vol. 5 (2) 2010 255 Table 1: Antibacterial and antifungal activity (zones of inhibition in mm) of the isolated compounds. Compounds/microorganisms Toussalactam (1) Aristolactam AII (2) Aristolactam BII (3) Piperolactam C (4) Aristolactam FII (5) 1-(2-C-Methyl-β-D-ribofuranosyl)-uracil (6) 3,4,5-Trimethoxyphenyl-β-D-glucopyranoside Ciprofloxacin (5 µg/ml) Amphotericin (10 µg/ml) B1 14 0 0 12 12 0 0 28 -- B3 13 0 0 12 15p 0 0 18 -- B4 0 0 0 0 0 0 0 23/32p -- B7 --0 0 -0 0 --- B9 0 0 0 0 0 0 0 22/27 -- M4 23/29p 14 16p 13p 20p 0 0 22p -- H4 0 0 --13p 0 0 -14 H8 13 0 0 0 11p 0 0 -20 P1 17 15 0 0 13 0 0 -18 p = Partial inhibition; 0 = inactive; -- = Test not carried out on these microorganisms because of small quantity of the compounds. Bacterial species tested: B1 = Bacillus subtilis ATTC 6633 (IMET) NA; B3 = Staphylococcus aureus (IMET 10760) SG511; B4 = Escherichia coli SG 458; B7 = Pseudomonas aeruginosa SG 137 (IMET 10480); B9 = Pseudomonas aeruginosa K 799/61 and M4 = Mycobacterium vaccae IMET 10670. Fungal species used: H4 = Sporobolomyces salmonicolor SBUG 549; H8 = Candida albicans BMSY 212 and P1 = Penicilium notatum. Table 2: Antiinflammatory activity of the isolated compounds. Compounds % 3α-Hydroxysteroid dehydrogenase inhibition Aristolactam BII (3) Piperolactam C (4) Aristolactam FII (5) 1-(2-C-Methyl-β-D-ribofuranosyl)-uracil (6) 3,4,5-Trimethoxyphenyl-β-D-glucopyranoside Epicatechin-4β,8-epicatechin Indomethacin (standard) 30 µg/mL 87 65 47 0 0 11 93 3 µg/mL 31 19 30 0 0 0 27 IC50 (µg/mL) 0.3 µg/mL 11 16 24 0 0 0 10 4.6 14.5 --0 0 0 4.6 Table 3: Antiproliferative and cytotoxic activity of the isolated compounds. Compounds Aristolactam BII (3) Piperolactam C (4) Aristolactam FII (5) 1-(2-C-Methyl-β-D-ribofuranosyl)-uracil (6) 3,4,5-Trimethoxyphenyl-β-D-glucopyranoside Taxol® Colchicine Camptothecin plastic plates (Merck, 0.20 mm); visualizing: UV/VIS and Dragendorff’s or anisaldehyde spray [31]; m.p. Electrothermal 9100 (uncorrected); UV spectra: Hitachi 200-20 spectrophotometer; IR spectra: JASCO FT/IR4100 spectrometer; 1H NMR (300 and 500 MHz) and 13 C NMR (75 MHz) in DMSO-D6: Bruker DRX-300, internal standard TMS (1H NMR) and solvent signal for 13 C NMR; ESIMS: 70 eV on an HP 5990/5988A spectrometer; HRESIMS: JEOL JMS-HX 110 mass spectrometer. Plant materials: The stem and root barks were collected in October 2004 from Zaraninge forest reserve at the edge of Saadani National Park, Bagamoyo District in Tanzania. The plant species was identified at the Herbarium of the Department of Botany, University of Dar es Salaam, where a voucher specimen No. FMM 3330 is preserved. Extraction and isolation: The air-dried and powdered stem and root barks (1 Kg each) were subjected to sequential extraction in light petroleum, CH2Cl2 and Antiproliferative activity (µg/mL) L-929 (GI50) K-562 (GI50) 50 50 13.2 21.8 19.1 5.7 50 50 >50 >50 0.1 0.01 0.9 0.02 0.02 0.002 Cytotoxicity (µg/mL) HeLa (CC50) 9.6 36.3 0.2 50 >50 0.01 0.006 0.2 MeOH at room temp, each 2 x 72 h. Vacuum liquid chromatography (VLC) of the concd. cytotoxic (brine shrimp test) CH2Cl2 stem bark extract (6.9 g) on silica gel and then CC (silica gel: light petroleum/EtOAc gradient) yielded 1 (4.2 mg), 4 (48.1 mg), 5 (26.6 mg), 3 (6.5 mg), and 2 (5.3 mg) in that sequence. VLC (silica gel: light petroleum/EtOAc gradient) of the MeOH extracts, then CC (silica gel: light petroleum/EtOAc gradient), then Sephadex® LH-20, MeOH/CH3Cl, 1:1 v/v) yielded (-)-epicatechin, a mixture of (-)-epicatechin and (+)-catechin, epicatechin-4β,8-epicatechin, 6 and 3,4,5-trimethoxyphenyl-β-D-glucopyranoside. Toussalactam (2-hydroxy-1,6-dimethoxy-5Hdibenzo[cdf]indol-4-one, 1) Brownish yellow powder (yield 4.2 mg). MP: 255-258°C. Anisaldehyde spray – yellow. UV, λmax (log ε) 202 (4.42), 236 (4.54), 278 (4.45), 289 (4.43) and 391 (3.82) nm. 256 Natural Product Communications Vol. 5 (2) 2010 IR (film) νmax: 3224, 2926, 1684, 1647, 1609, 1558, 1541, 1507, 1456, 1437, 1362, 1330, 1248, 1200, 1173, 1110, 1037, 982, 960, 926, 869, 844, 802, 766, 744 and 720 cm-1. 1 H NMR: δ 4.01 (3H, s, 1-OMe), 4.05 (3H, s, 6-OMe), 7.60 (1H, ddd, J = 7.8, 7.2, 1.7 Hz, H-6), 7.61 (1H, s, H-3), 7.65 (1H, ddd, J = 7.8, 7.2, 1.7 Hz, H-8), 8.17 (1H, dd, J = 7.5, 1.8 Hz, 9), 9.16 (1H, dd, J = 7.5, 2.2 Hz, H-10) and 10.95 (1H, br s, N-H). 13 C NMR: δ 168.1 (C=O), 151.5 (C-2), 149.0 (C-1), 133.8 (C-6), 130.2 (C-6a), 127.3 (C-10a), 127.2 (C-8), 126.9 (C-10), 126.0 (C-5a), 125.8 (C-9), 122.3 (C-7), 120.9 (C-3a), 120.6 (C-5), 117.7 (C-10b), 113.4 (C-3), 60.8 (6-OMe) and 59.3 (1-OMe). HRESIMS, m/z 296.0967 ([MH]+, calculated for C17H14NO4 = 296.0893), 295 ([M]+, 15), 294 (100), 280 (9), 279 (71) and 264 (18). 1-(2-C-Methyl-β-D-ribofuranosyl)-uracil (6) White crystals (yield, 561 mg). MP: 117-118oC (MeOH/EtOAc). [α]D20: +67.81 (c 0.20, MeOH). Anisaldehyde spray – pink. UV, λMeOHmax (log ε): 213 (3.14) and 263 (3.88). IR (film) νmax: 3595, 3097, 1697, 1662, 1631, 1473, 1418, 1398, 1379, 1274, 1184, 1123, 1094, 1079, 1051, 1033, 949, 880 and 732 cm-1. HRESIMS, m/z 258.22804 (calc. for C10H14N2O6, 258.08519), m/z (% rel. int.) 257 (23), 214 (100) and 166 (40). 1 H NMR: δ 1.15 (3H, s, 2'-Me), 3.77 (1H, dd, J = 12.5, 2.6 Hz, H-5'a), 3.83 (1H, d, J = 9.2 Hz, H-3'), 3.91 (1H, td, J = 9.2, 2.3, H-4'), 3.97 (1H, dd, J = 12.5, 2.6 Hz, H-5'b), 5.67 (1H, d, J = 8.1 Hz, H-5), 5.95 (1H, s, H-1') and 8.13 (1H, d, J = 8.1, H-6). 13 C NMR: δ 166.0 (C-4), 152.4 (C-2), 142.5 (C-6), 102.3 (C-5), 93.1 (C-1'), 83.9 (C-4'), 80.0 (C-2'), 73.4 (C-3') 60.5 (C-5) and 20.2 (2'-Me). Brine shrimp test: This was carried out according to a standard procedure [32], using brine shrimp (Artemia salina Leach) larvae as the indicator organisms, which were hatched in artificial seawater prepared from sea salt (3.8 g) in distilled water (1000 mL), and then filtered. The mature nauplii were collected after 48 h of hatching. Each sample was tested at concentrations of 240, 120, 80, 40, 24 and 8 μg/mL in DMSO, in triplicate vials, each containing 10 brine shrimp larvae. An additional vial with only the solvent, DMSO, and 10 shrimp larvae was used as the control. The number of surviving larvae after 24 h exposure was established and the LC50 values (the concentration required to kill 50% of the larvae) were determined using Probit analysis [33]. Odalo et al. Agar diffusion assay for antimicrobial activity (antibacterial and antifungal activity): The agar diffusion method was used for the determination of antibacterial and antifungal activity of the isolated compounds against the microorganisms listed in Table 5, obtained from the Hans Knolls Institute for Natural Product Research and Infectious Biology (HKI) in Jena, Germany. Ca. 9 mL of Müller-Hinton agar for bacteria and Sabouraud Dextrose agar for fungi (Oxoid, UK) were poured into Petri dishes (9 cm diameter) and inoculated with the respective test organisms. Wells (4 cm) were punched out of the solid agar using pipette tips, and 1 mL of 50 µg/mL solutions of the test compounds and control antibiotics (Ciprofloxacin, 5 µg/mL and Amphotericin, 10 µg/mL) were placed in each well. The Petri dishes were then incubated at 30ºC and 35ºC for the test bacterial and fungal strains, respectively, for 20 h and the average diameter of the inhibition zone surrounding the wells was measured. Microplate dilution assay for minimum inhibitory concentrations (MIC) determination: The minimum inhibitory concentrations (MIC) of the isolated compounds with good antimicrobial activity in the agar diffusion tests were determined using a serial microplate dilution assay against each test bacterial species. This was determined by 2-fold serial dilution of the compounds beyond the level where no inhibition of growth of the bacterial strains Bacillus subtilis ATTC 6633 (IMET) NA (B1), Staphylococcus aureus (IMET 10760) SG511 (B3), Mycobacterium smegmatis SG987 (M2), M. aurum SB66 (M3), M. vaccae IMET 10670 (M4) and M. fortuitum M. Fort. (M5) was observed. The compounds were reconstituted to 100 µg/mL in DMSO and 100 µL aliquots of the fractions were serially diluted by 50% with water in 96-well microplates. Muller-Hinton (MH) broth culture (1%) was inoculated with the test bacteria and then incubated at 37ºC overnight, and 100 µL aliquots of the resulting culture were added to each well. Ciprofloxacin (100 µg/mL) was used as the reference antibiotic and two wells were used as sterility and growth controls, respectively, with the sterility control containing only Oxoid MH broth, while the negative growth control contained both MH broth as well as the test organism. The microplates were sealed and incubated at 37ºC at 100% relative humidity for 18 h. As an indicator of bacterial growth, 40 µL aliquots of a 0.2 mg/mL solution of p-iodonitrotetrazolium violet (INT) dissolved in water were added to the microplate wells. Antiinflammatory activity [3α-hydroxysteroid dehydrogenase (3α-HSD) assay]: The source of 3αhydroxysteroid dehydrogenase used in the bioassay was liver from adult male Sprague-Dawley rats (150-200 g). The liver was excised and homogenized in 3 volumes of Bioactive constituents of Toussaintia orientalis 50 mM Tris-HCl of pH 8.6 containing 250 mM sucrose, 1 mM dithiothreitol and 1 mM EDTA. Homogenates were centrifuged (100,000 x g for 30 min) and the resulting supernatant (cytosol containing 3αhydroxysteroid dehydrogenase) was used for enzyme assays without further processing, after being prepared according to the method described by Penning [34], and having attained a final specific activity of 3.58 µM of 5β-dihydrocortisone reduced/min/mg of protein). The reduction of 5β-dihydrocortisone was monitored by measuring the changes in the absorbance of the pyridine nucleotide at 340 nm. Each assay (1.0 mL) contained potassium phosphate buffer (pH 6.0, 0.840 mL of 1M), NADPH (20 µL of 9 M), 5β-dihydrocortisone (10 µL of 5 mM), and acetonitrile (30 µL). The reactions were initiated by the addition of enzyme (30-50 µg of either cytosolic protein or 0.6 µg of purified enzyme), and optical density change was followed over a period of 5 min. Control incubation experiments by addition of the cytosol in which either 5β-dihydrocortisone or NADPH was absent indicated that the presence of both substances was required before the cytosol would promote a change in absorbance at 340 nm. The % inhibition of the isolated compounds was generated at 30, 3 and 0.3 µg/mL concentration. Increasing amounts of the isolated compound were added to the standard assay system, and the concentration of the compound required to reduce the rate of 5β-dihydrocortisone Natural Product Communications Vol. 5 (2) 2010 257 reductions by 50% (IC50) was computed from the resulting natural logarithm dose-response curves. Antiproliferative and cytotoxicity assay: This assay was carried out by the Molecular Natural Product Research group of HKI in Jena, Germany, as described in the literature [35], using the cell lines K-562 (human chronic myeloid leukemia) and L-929 (mouse fibroblast) for antiproliferative effects (GI50, concentration which inhibited cell growth by 50%), and against HeLa cells for cytotoxicity (GC50 = concentration at which cells were destroyed by 50%; used partially in referring to the lysis of cells). Taxol®, Colchicine and Camptothecin were used as the standard antiproliferative and cytotoxic drugs. Acknowledgements - JOO thanks the Germany Academic Exchange Services (DAAD) and NAPRECA for funding these studies through the DAAD/NAPRECA Fellowship Scheme, which also supported the collaborative research engagement with the Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll-Institute in Jena, Germany. Part of the research was funded through Sida/SAREC support to the Faculty of Science, University of Dar es Salaam. We thank Mr F. Mbago of the Herbarium, Department of Botany at the University of Dar es Salaam for the location and identification of the investigated plant species. References [1] Verdcourt B. (1971) Flora of Tropical East Africa - Annonaceae, Crown Agents, London, p. 1,9. [2] Kokwaro JO. (1993) Medicinal Plants of East Africa, 2nd edition, Kenya Literature Bureau, Nairobi. [3] Verdcourt B. (1996) Sanrafaelia, a new genus of Annonaceae from Tanzania. Garcia de Orta, Series Botanica, 13, 43-44. [4] Couvreur TLP, Van Der Ham RWJM, Mbele YM, Mbago FM, Johnson DM. (2009) Molecular and morphological characterization of a new monotypic genus of Annonaceae, Mwasumbia, from Tanzania. Systematic Botany, 34, 266-276. [5] Deroin T, Luke Q. (2005) A new Toussaintia (Annonaceae) from Tanzania. Journal of East African Natural History, 94, 165-174. [6] Desai SJ, Prabhu BR, Mulchandani NB. (1988) Aristolactams and 4,5-dioxoaporphines from Piper longum. Phytochemistry, 27, 1511-1515. [7] Chia YC, Chang FR, Teng CM, Wu YC. (2000) Aristolactams and dioxoaporphines from Fissistigma balansae and Fissistigma oldhamii. Journal of Natural Products, 63, 1160-1163. [8] Sun NJ, Antoun M, Chang CJ, Cassady JM. (1987) New cytotoxic aristolactams from Pararistolochia flos-avis. Journal of Natural Products, 50, 843-846. [9] Beigelman LN, Ermolinsky BS, Gurskaya GV, Tsapkina EN, Karpeisky MY, Mikhailov SN. (1987) New synthesis of 2′-Cmethylnucleosides starting from D-glucose and D-ribose. Carbohydrate Research, 166, 219-232. [10] Shimomura H, Sashida Y, Oohara M, Tenma H. (1988) Phenolic glucosides from Parabenzoin praecox. Phytochemistry, 27, 644-646. [11] Olsen CE, Tyagi OD, Boll PM, Hussaini FA, Parmar VS, Sharma NK, Teneja P, Jain SC, (1993) An aristolactam from Piper acutisleginum and revision of the structures of piperolactam B and D. Phytochemistry, 33, 518-520. [12] Chen YC, Chen JJ, Chang YL, Teng CM, Lin WY, Wu CC, Chen IS. (2004) A new aristolactam alkaloid and anti-platelet aggregation constituents from Piper taiwanense. Planta Medica, 70, 174-177. [13] Likhitwitayawuid K, Wiasathien L, Jongboonprasert V, Krungkrai J, Aimi N, Takayama H, Kitajima M. (1997) Antimalarial alkaloids from Goniothalamus tenuifolius. Pharmacology Letters, 7, 99-102. 258 Natural Product Communications Vol. 5 (2) 2010 Odalo et al. [14] Makriyannis A, Knittel JJ. (1979) The conformational analysis of aromatic methoxy groups from carbon-13 chemical shifts and spin-lattice relaxation times. Tetrahedron Letters, 20, 2753-2756. [15] Daneluttea AP, Costantina MB, Delgadob GE, Braz-Filho R, Kato MJ. (2005) Divergence of secondary metabolism in cell suspension cultures and differentiated plants of Piper cernuum and P. crassinervium. Journal of Brazilian Chemical Society, 16, 1425-1430. [16] Davies DB. (1978) Conformations of nucleosides and nucleotides. Progress of Nuclear Magnetic Resonance Spectroscopy, 12, 135-225. [17] Stevens JD, Fletcher HG. (1968) The proton magnetic resonance spectra of pentofuranose derivatives. Journal of Organic Chemistry, 33, 1799-1805. [18] Kam BL, Barascut JL, Imbach JL. (1979) A general method of synthesis and isolation, and an N.M.R.-spectroscopic study, of tetraO-acetyl-D-aldopentofuranoses. Carbohydrates Research, 69, 135-142. [19] Jenkins SR, Arison B, Walton E. (1968) Branched-chain sugar nucleoside. IV. 2’-Methyladenosine. Journal of Organic Chemistry, 33, 2490-2494. [20] Guo FQ, Li Ai, Huang LF, Liang YZ, Chen BM. (2006) Identification and determination of nucleosides in Cordyceps sinesis and its substitutes by high performance liquid chromatography with mass spectrometric detection. Journal of Pharmacological and Biomedical Analysis, 40, 623-630. [21] Miles DW, Inskeep WH, Robins MJ, Winkley MW, Robins RK, Eyring H. (1970) Circular dichroism of nucleoside derivatives. IX. Vicinal effects on the circular dichroism of pyrimidine nucleosides. Journal of American Chemical Society, 92, 3872-3881. [22] Ingwall JS. (1972) Circular dichroism of nucleosides. I. Anomeric pairs of the D-pentofuranosides of adenine. Journal of American Chemical Society, 94, 5487-5495. [23] Searle PA, Molinski TF. (1995) Trachycladines A and B: 2’-C-methyl-5’-deoxyribofuranosyl nucleosides from the marine sponge Trachycladus laevispirulifer. Journal of Organic Chemistry, 60, 4296-4298. [24] Ichiba T, Nakao Y, Scheuer PJ, Sata NU, Kelly-Borges M. (1995) Kumusine, a chloroadenine riboside from a sponge, Theonella sp. Tetrahedron Letters, 36, 3977-3980. [25] De Clercq E. (2005) Antiviral drug discovery and development: Where chemistry meets with biomedicine. Antiviral Research, 67, 56-75. [26] De Clercq E. (2003) Clinical potential of the acyclic nucleoside phosphonates cidofovir, adefovir, andtenofovir intreatment of DNA virus and retrovirus infections. Clinical Microbiological Review, 16, 569-596. [27] Naesens L, De Clercq E. (2001) Recent developments in herpes virus therapy. Herpes, 8, 12-16. [28] Donia M, Hamann MT. (2003) Marine natural products and their potential applications as antiinfective agents. Lancet Infectious Diseases, 3, 338-348. [29] Suckling CJ. (1991) Chemical approaches to the discovery of new drugs. Science Progress, 75, 323-359. [30] Carroll SS, Tomassini JE, Bosserman M, Getty K, Stahlhut MW, Eldrup AB, Bhat B, Hall D, Simcoe AL, LaFemina R, Rutkowski CA, Wolanski B, Yang Z, Migliaccio G, De Francesco R, Kuo LC, MacCoss M, Olsen DB. (2003) Inhibition of hepatitis C virus RNA replication by 2′-modified nucleoside analogs. Journal of Biological Chemistry, 278, 11979-11984. [31] Stahl E. (1969) Thin-layer chromatography, a laboratory handbook. Springer-Verlag, New York, p. 857. [32] Meyer BN, Ferrigini RN, Jacobsen LB, Nicholas DE, McLaughlin JL. (1982) Brine shrimp: A convenient general bioassay for active plant constituents. Planta Medica, 45, 31-34. [33] Finney DJ. (1971) Probit analysis, 3rd ed., Cambridge University Press, Cambridge, 333 pp. [34] Penning TM. (1985) Inhibition of 5ß-dihydrocotisone reduction in rat liver cytosol: A rapid spectrophotometric screen for nonsteroidal anti-inflammatory drug potency. Journal of Pharmacological Science, 74, 651-654. [35] Dahse HM, Schlegel B, Gräfe U. (2001) Differentiation between inducers of apoptosis and nonspecific cytotoxic drugs by means of cell analyzer and immunoassay using the cell lines K-562 (human chronic myeloid leukemia), and L-929 (mouse fibroblast) for antiproliferative effects and HeLa (human cervix carcinoma) for cytotoxic effects. Pharmazie, 56, 489-491.