Chemistry of southeast asian plants 1

CHEMISTRY OF SOUTHEAST ASIAN PLANTS LE CONG THUAN (B. Sc. (Hons.), Vietnam National University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2005 Acknowledgements I would like to thank the following people for their help and support, without whom this project would not have been possible: I am indebted to my supervisor, Associate Professor Leslie John Harrison, for providing me with the chance to carry out research in his group and for the valuable trainings that I had received during my journey to discover the richness of nature. His patiently guidance and constant encouragement has guided me to the very end of this project. I express my sincere gratitude to Professor Shinro Tachibana of the National University Hospital and Dr. Chidambaram of the National Cancer Center for their guidance in biological assays. I am also grateful to the Director and staff of the WHO Immunology Center and the Paediatric Laboratory for the permission of using their facilities for cell culture and bioassay screening. I also wish to thank my fellow researchers A. S. Md. Sofian, Wong Chek Ming, Wang Yanmei, Teo Ee Ling, Wu Ji-en, Ge Xiaowei, and Zhang Guodong, for the time that we shared in the laboratory and for their support throughout my project. Special thanks also go to Miss Shanon Sng Poh Tee, Miss Wong Siew Ying, Mdm Han Yanhui, and Miss Peggy Ler of the NMR Laboratory, and last but not least, Mdm Wong Lai Kwai and Mdm Chen Lijun of the NUSChem/Finnigan-MAT Mass Spectrometry Laboratory for their hard work and precious assistance. I would like to thank the National University of Singapore for the financial support and for the award of the postgraduate scholarship. Finally, I am indebted to my family, especially my father and my mother, as everything would not have been possible without their unconditioned love and support. i Table of Contents Chapter - General introduction……………………………………………… Chapter - The Celastraceae………………………………………………… 30 2.1. Introduction……………………………………………………………… 30 2.2. Constituents of Salacia chinensis L…………………………………… . 48 2.3. Experimental…………………………………………………………… 68 Chapter - The Hepaticae…………………………………………………… . 79 3.1. Introduction………………………………………………………… 79 3.2. Chemistry of Pallavicinia……………………………………………… 93 3.3. Constituents of Singaporean Pallavicinia cf. lyellii…………………… 95 3.4. Experimental……………………………………………………… . 131 3.5. Constituents of Vietnamese Pallavicinia lyellii………………………… 137 3.6. Experimental……………………………………………………………. 165 Chapter - Pain and nociceptin receptor……………………………………. 170 4.1. Introduction…………………………………………………………… 170 4.2. Results and discussion………………………………………………… 190 4.3. Experimental……………………………………………………………. 193 References……………………………………………………………………… 195 Appendix……………………………………………………………………… 214 ii Summary From the stem bark of Salacia chinensis L. collected in Vietnam, eight triterpenoids have been isolated and identified, including the new natural product 3,11-dioxoolean-12-en28-oic acid. The known compounds are oleanolic acid, betulin 3-caffeate, krukovine A, morolic acid, 28,30-dihydroxylup-20(29)-en-3-one, 3β-hydroxy-11-oxoolean-12-en-28oic acid, and 3β,28-dihydroxy-12-oleanen-11-one. Phytochemical investigation of the Singaporean liverwort Pallavicinia cf. lyellii led to the isolation of five novel diterpenoids, together with two known diterpenoids, (-)-3,14clerodadien-13-ol and (-)-sacculatal. The five novel diterpenoids were named pallavicinins B-F. In addition, three bis(bibenzyls) were also obtained, two of which are novel. The known compound is perrottetin E, and the two novel bis(bibenzyls) are 8hydroxymarchantin C and 7-oxoriccardin D. The novel compounds isolated from the Vietnamese Pallavicinia lyellii are two highly modified labdane diterpenoids (pallavicinins G-H), two dimeric diterpenoids (pallavicidine A-B), and a clerodane, 4β-hydroxyclerodane. Four known diterpenoids, pleuroziol, levierol, ent-kaur-16-en-19-ol, and (-)-manool were also isolated and identified. Screening for potential nociceptin receptor (NOP) ligand revealed that the crude extract of Vietnamese P. lyellii has higher binding affinity to the NOP than the endogenous ligand OFQ/N. Further investigation of the Vietnamese P. lyelli is required to isolate and identify this potential ligand for NOP, as well as establish its pharmacological profile. iii List of Tables Table 2-1. The Celastraceae s.l.………………………………………………… 31 Table 2-2. The Celastraceae in Vietnam ………………………………………… 31 Table 2-3. Distribution of triterpenoid quinonemethides in Salacia species ……. 40 Table 2-4….……………………………………………………………………… 53 Table 2-5….……………………………………………………………………… 64 Table 2-6….……………………………………………………………………… 66 Table 3-1………………………………………………………………………… 100 Table 3-2………………………………………………………………………… 104 Table 3-3………………………………………………………………………… 109 Table 3-4………………………………………………………………………… 112 Table 3-5………………………………………………………………………… 115 Table 3-6………………………………………………………………………… 126 Table 3-7………………………………………………………………………… 130 Table 3-8………………………………………………………………………… 141 Table 3-9………………………………………………………………………… 147 Table 3-10……………………………………………………………………… 150 Table 3-11……………………………………………………………………… 155 Table 3-12……………………………………………………………………… 158 Table 4-1. Chemical mediators and their effect on C-fibres.…………………… 173 Table 4-2. Metabolism fate of OFQ/N in different tissues ……………………. 188 Table 4-3………………………………………………………………………… 190 Table 4-4………………………………………………………………………… 190 iv List of Schemes Scheme 1-1. Biosynthetic relationship of terpenoids Scheme 1-2. Non-mevalonate pathway to isoprenoids . 11 Scheme 1-3. Biosynthetic relationship of cyclic monoterpenoids 13 Scheme 1-4. Biosynthesis of gibberellin A3……………………………………. 16 Scheme 1-5. Formation of β-amyrin . 17 Scheme 1-6. Biosynthesis of cholesterol from squalene oxide 18 Scheme 1-7. Biosynthetic relationship of flavonoids . 21 Scheme 1-8……………………………………………………………………… 23 Scheme 1-9. Biosynthesis of cocaine in Erythroxylon coca……………………. 24 Scheme 1-10. Biosynthesis of retronecine……………………………………… 25 Scheme 1-11. Biosynthesis of nicotine…………………………………………. 26 Scheme 1-12. Biosynthesis of mescaline……………………………………… 27 Scheme 2-1. Biosynthetic relationship between quinonemethides and 7-oxoquinonemethides……………………………………………. 39 Scheme 2-2. Proposed biosynthetic pathway of celastrol and pristimerin……… 43 Scheme 2-3.……………………………………………………………………… 67 Scheme 4-1. Proposed biosynthetic pathway of morphine in Papaver somniferum……………………………………………… 179 Scheme 4-2. Total synthesis of morphine………………………………………. 181 v List of spectra in appendix H- and 13C-NMR of 3,11-dioxoolean-12-en-28-oic acid (146) 215-216 H- and 13C-NMR of pallavicinin B (249) . 217-218 H- and 13C-NMR of pallavicinin C (250) . 219-220 H- and 13C-NMR of pallavicinin D (251) . 221-222 H- and 13C-NMR of pallavicinin E (252) . 223-224 H- and 13C-NMR of pallavicinin F (253) . 225-226 H- and 13C-NMR of 8-hydroxymarchantin C (267) . 227-228 H- and 13C-NMR of 7-oxoriccardin D (268) . 229-230 H- and 13C-NMR of 4β-hydroxyclerodane (269) . 231-232 H- and 13C-NMR of pallavicidine A (270) . 233-234 H- and 13C-NMR of pallavicidine B (271) 235-236 H- and 13C-NMR of pallavicinin G (273) . 237-238 H- and 13C-NMR of pallavicinin H (274) . 239-240 1D- and 2D-NMR of fraction G4-19-19 241-244 vi Chapter General introduction Humans have extracted natural products from plants and animals for a long time, and used them in crude form to treat diseases, as poisons for warfares and hunting, as stimulants, etc. Papaver somniferum (poppy juice) and Glycyrrhiza glabra (licorice) are some examples which are still in use today, for the same purpose. The natural compounds shown in Fig. 1-1 have been used in crude form for centuries before they were isolated in pure form in the 19th century. Natural products have attracted and motivated chemists of many generations, due to the fact that mother nature seems to be an unlimited source of structurally and pharmacologically interesting compounds. N H OH H N N Me H H H O O Me H (1) Ephedrine (used to treat asthma and hay fever) (2) Strychnine (potent convulsant) HO O O Me N H MeN NMe H O HO (3) Morphine (analgesic, narcotic) N Me N (4) Caffeine (mild stimulant to the nervous system) Fig. 1-1 Primary and secondary metabolites Natural products, as the term implies, are those biological molecules which originate from living organisms such as plants, animals, and insects. The study of natural products is the investigation of their structure, formation, use, and purpose in the organism. There are two major classes of metabolites, primary and secondary metabolites. Compounds that make up the fundamental and essential process of life, e.g. nucleic acids, carbohydrates, etc. are primary metabolites. Secondary metabolites are small molecules, e.g. terpenoids, alkaloids, phenols, pigments, etc., which are not vital for the survival or well-being of the organism, usually confined to a particular group of closely related species, or to a single species, or even to a single strain growing under certain conditions. The term “natural products” is usually reserved for these secondary metabolites. Many of these compounds have certain function in the organisms that produce them: they can act as repellents, attractants, allelopathic reagents, phytoallexins, sex pheromones, etc. Tens of thousands of natural products have been characterized, and there are certainly many more thousands of compounds still waiting to be discovered. However, as scientists discover novel natural products, the difference between primary and secondary metabolites becomes less clearly defined: there are amino acids that not play any vital role at all, while many sterols must be considered as primary metabolites, since they play an essential role in many organisms. The biosynthesis of natural products involves three key building blocks: amino acids, shikimate, and acetate. Amino acids are biosynthetic precursors of alkaloids and peptide antibiotics (e.g. penicillins). Shikimate is the key starting material of many aromatic compounds, such as aromatic amino acids, cinnamic acids, and certain polyphenols. Acetate, in its active form (thioester with coenzyme A), is the precursor of many classes of compounds including polyacetylenes, polyphenols, prostaglandins, and macrocyclic antibiotics. A derivative of acetyl thiocoenzyme A, mevalonate, is the branch point leading to the terpenoids, steroids, and carotenoids. Role of enzymes in the biosynthesis of natural products The metabolites are synthesized (and degraded) in living organisms via series of chemical reactions (metabolic pathways). These reactions are theoretically reversible and are well-known in any laboratory. However, these reactions are performed in nature at a much higher rate, efficiency, and selectivity. The reason is that each reaction is mediated by a specific biological catalyst called an enzyme. Most enzymes catalyse the known types of reaction: oxidation, reduction, elimination, hydroxylation, hydrolysis, etc. Enzymes can increase the rates of these reactions by as much as 109 to 1012 fold, due to certain advantages: - Specificity: Each enzyme only catalyses one particular type of reaction, and only accepts substrates that feature a stereospecific structure. Furthermore, the enzyme-substrate complex may be formed in such a way that the substrate is forced into transition-state configuration. - The aprotic environment of the active site necessitate the transfer of H+, thereby enhance reaction rate, since most enzymic reactions are acid-base catalysis. - The enzyme-substrate complex is stabilized by non-covalent interactions, therefore lowering the number of interactions, as well as energy needed for the product releasing step. Using HMQC and HMBC data (Table 2-2), the structures of rings A to C were built up in the similar manner as for 146, and the position of the ketone group is confirmed at C-3 by HMBC correlations (Fig. 2-5). 25 O 24 23 Fig. 2-5 The 1,1-disubstituted alkene protons H2-29 showed HMBC correlation with one oxygenated carbon [δC 65.1 (C-30)], confirming the presence of a 3- hydroxyisopropenyl side chain. Finally, protons of the second hydroxymethylene group showed HMBC correlations with C-16, C-17 and C-22, confirmed that this oxygenated carbon is attached to C-17. The important HMBC correlations of rings D and E are shown in Fig. 2-6. 29 HOH2C 30 19 22 17 13 16 CH2OH 15 Fig. 2-6 This triterpenoid was originally isolated from Maytenus canariensis (which also belongs to the Celastraceae family) as a minor compound, together with another lupane 63 triterpenoid.105 This is the second report of the isolation of (152) from nature, also as a very minor constituent. Table 2-5. 1H-NMR (300 M Hz), 13C-NMR (125 M Hz) and HMBC spectral data of compound 152 in CDCl3. HMBC No. δC δH a 1.89 (m) 39.6 a 1.38 (m) 2.46 (m) a 1, 3, 10 34.1 218.0 47.3 a 1.32 (m) 54.9 1.45 (m) a 19.7 a 1.46 (m) 33.6 40.9 1.38 (m) a 49.7 10 36.9 11 1.43 (m) a 21.5 a 1.26 (m) 12 1.41 (m) a 26.8 1.11 (m) a 13 1.66 (m) a 18 37.4 14 42.8 15 1.72 (m) a 13 27.0 1.11 (m) a 16 1.95 (m) a 14, 18 29.2 17 47.8 18 1.52 (m) a 13, 17, 19 49.4 a 19 2.30 (m) 18, 20 43.5 20 154.5 21 2.12 (m) a 31.8 22 1.90 (m) a 33.8 a 1.12 (m) 23 1.07 (s) 3, 4, 5, 24 26.7 24 1.02 (s) 3, 4, 5, 23 21.0 25 0.92 (s) 1, 5, 9, 10 16.0 26 1.06 (s) 7, 8, 9, 14 15.8 27 0.99 (s) 8, 13, 14, 15 14.7 28a 3.79 (dd, J = 1.7, 11.2 Hz) 16, 17, 22 60.3 28b 3.32 (br d, J = 10.8 Hz) 29a 4.95 (d, J = 1.1 Hz) 19, 20, 28 107.3 29b 4.90 (br s) 30 4.12 (br s) 19, 20, 29 65.1 a ( ): Approximate position of overlapping multiplets assigned using HMQC correlations 64 3β-Hydroxy-11-oxoolean-12-en-28-oic acid Compound 153, 3β-hydroxy-11-oxoolean-12-en-28-oic acid, was obtained as a white powder, mp. 230-231 oC (lit.106 232-235 oC), [α]D +62.5 (c 0.4, CHCl3) (lit.106 +56.3). It was determined to have the molecular formula C30H46O4 by HR-EIMS (m/z M+ 470.3407). O COOH HO (153) The 1H-NMR spectrum contained signals due to one olefinic proton [δH 5.63, s, H-12), a hydroxymethine proton [δH 3.22 (dd, J = 5.8, 10.4 Hz, H-3)], seven tertiary methyl groups [δH 0.78, 0.92, 0.93, 0.94, 0.99, 1.10, and 1.36 (each 3H, s, H-24, 26, 29, 30, 23, 25 and 27)]. The 13C-NMR spectrum has thirty signals, there was one (conjugated) keto group [δC 200.4 (C-11)], one carboxyl group [δC 182.4 (C-28)], a trisubstituted double bond [δC 128.1 (C-12) and 168.3 (C-13)] and one oxygenated carbon [δC 78.8 (C-3)]. These spectral data exhibited great similarity to those of compound 146, except that the keto group at C-3 has been replaced by an α-hydroxy group, lead to structure 153. The initial assignment was based on that of compound 146, and the 2D-NMR data (HMQC and HMBC) of this compound confirmed that its structure was 153. 65 Hikino et al. had reported that the soil microbe Cunninghamella blakesleeana can convert oleanolic acid to 153.107 Later, it has been isolated from callus tissue culture of Paeonia species, along with another three triterpenoids.106 Table 2-6. 13C-NMR (125 M Hz) spectral data of 153 & 154 C 153 154 39.1 39.1 27.3 27.3 78.8 78.8 39.1 39.1 55.0 55.0 17.4 17.4 32.9 32.9 45.0 45.0 61.8 61.8 10 37.3 37.3 11 200.4 200.3 12 128.1 127.9 13 168.3 168.6 14 43.5 43.5 15 27.8 27.8 16 22.7 23.0 17 46.0 46.2 18 41.4 41.6 19 44.1 44.3 20 30.7 30.7 21 33.6 33.7 22 31.6 31.6 23 28.1 28.1 24 15.5 15.6 25 16.2 16.2 26 19.2 18.9 27 23.6 23.6 28 182.4 177.5 29 32.8 32.9 30 23.4 23.4 31 51.9 Esterification of 153 with diazomethane gave the methyl ester 154, a colorless powder, mp. 198-200 oC (lit.72 196-198 oC), [α]D +72.8 (c 0.2, CHCl3). HR-EIMS supported the molecular formula C31H48O4 (m/z M+ 484.3548). The 1H and 13 C-NMR assignments 66 were consistent with the structure (See Table 2-6). Oxidation of the ester 154 using PCC in dichloromethane gave a diketone which is identical with the methyl ester 147. The conversion is shown in Scheme 2-3. CH2N2 / Et2O O O COOH HO COOMe HO (154) (153) PCC / CH2Cl2 O O COOH COOMe CH2N2 / Et2O O O (146) (147) Scheme 2-3. 67 -Dihydroxy-12-oleanen-11-one (= 11-oxoerythrodiol) Compound 155, 3β,28-dihydroxy-12-oleanen-11-one, was obtained as a white powder, mp. 146-148 oC (lit.108 145-147 oC), [α]D +53.6 (c 0.32, CHCl3) (lit.108 +60.9). It was determined to have the molecular formula C30H48O3 by HR-EIMS (m/z M+ 456.3605). O CH2OH HO (155) The 1H-NMR spectrum contained signals due to one olefinic proton [δH 5.57 (s, H12)], a hydroxymethylene group [δH 3.47 (d, J = 10.8 Hz, H-28a) and 3.21 (d, J = 10.8 Hz, H-28b)], seven tertiary methyl groups [δH 0.80, 0.89, 0.92, 1.00, 1.11, 1.13 and 1.39 (each 3H, s, H-24, 29, 30, 23, 26, 25 and 27)]. Amongst the thirty signals in the C-NMR spectrum were those of one keto group [δC 200.0 (C-11)], a trisubstituted 13 double bond [δC 128.3 (C-12) and 169.3 (C-13)], one oxygenated methine [δC 78.8 (C3)], and one oxygenated methylene [δC 69.7 (C-28)]. These spectral data are similar to those of compound 153, the most important difference being the replacement of the carboxyl group by a primary hydroxyl group in the 13 structure of 155 was confirmed by comparison of its C-NMR spectrum of 155. The 13 C-NMR data with that of the literature.108 68 This compound was first isolated from the methanolic extract of the wood of Viburnum awabuki, along with three other oleanane-type triterpenoids.108 Subsequently, it was isolated from the twigs of Ilex macropoda,109 and from Cephalaria transylvanica, in glycoside form.110 69 2.3. Experimental Chromatography Liquid chromatography was carried out using Kieselgel 60 (230-400 mesh, Merck), Lichroprep RP-18 (40-63 µm, Merck), RP-8 (40-63 µm, Merck) and Lichroprep Diol (40-63 µm, Merck). Gel permeation chromatography was performed using Sephadex LH-20 eluting with MeOH-CHCl3 (1:1). HPLC was carried out using Waters system (Waters 600 Controller, Waters 600 pump and Waters 996 PDA detector) or Shimadzu LC-6A liquid chromatograph pump with Waters R401 RI detector. Phenomenex HPLC columns were used: LUNA 5µ C18 (250 x 4.6 mm), Lichrosorb 10 DIOL (250 x 4.6 mm), Partisil 10 Silica (250 x 4.6 mm) and Partisil 10 ODS-2 (250 x 4.6 mm). TLC was carried out on Si gel precoated glass plates (Merck, Kieselgel 60F254, 250 µm), C18 plates (Whatman, KC18F, 200 µm), C8 plates (Merck, HPTLC-Fertigplatten RP-8 F254), Diol Si gel precoated plates (Merck, HPTLC-Fertigplatten DIOL F254S, 200 µm), Cyano Si gel precoated plates (Merck, HPTLC-Fertigplatten CN F254S, 200 µm). Spectroscopy NMR spectra were measured using Bruker DPX-300 [300M Hz (1H) and 75M Hz (13C)], AMX-500 [500M Hz (1H) and 125M Hz (13C)] and DRX-500 [500M Hz (1H)] instruments for CDCl3 solution unless otherwise specified. Multiplicities were determined using DEPT experiments. EIMS and HR-EIMS were obtained on MICROMASS VG7035 and FINNIGAN MAT95XL-T mass spectrometers, respectively. 70 Optical rotations were recorded on a JASCO DIP-1000 automatic polarimeter using CHCl3 as solvent. Melting points were taken on Büchi B-540 or Olympus BX-41 apparatus and uncorrected values were reported. Plant material The stem bark of Salacia chinensis L. (Celastraceae) were collected May 1999 in Ba Ria province, Vietnam, and was identified by Professor Le Cong Kiet, Department of Botany, Vietnam National University, Ho Chi Minh city. A voucher sample (ref. no. LCK 853) is deposited at the herbarium of the Department of Botany, Vietnam National University. Extraction and isolation The air-dried and powdered stem bark (380 g) of Salacia chinensis was subjected to exhaustive extraction in a Soxhlet apparatus. Three solvents - hexane, ethyl acetate and methanol - were used in succession for extraction. The ethyl acetate extract (13 g) was subjected to flash chromatography eluted with an EtOAc-hexane step gradient to yield 11 fractions (A to K). Fractions A to D H-NMR spectroscopy showed that fractions A to D contained triglycerides and were not studied further. Fraction E (445 mg) after chromatography on Sephadex LH-20, followed by Diol (35% EtOAc-hexane) and RP-18 column chromatography (85% MeOH-H2O) afforded fractions. The second fraction (17 mg) gave oleanolic acid (148, 2.2 mg) and the 71 third fraction (15 mg) gave morolic acid (151, 1.5 mg) after chromatography on RP-18 silica (70% MeCN-H2O). Fraction F (630 mg) was subjected to gel permeation chromatography on Sephadex LH-20, afforded fractions (F1 to F6). From fraction F4 (67 mg), 3,11-dioxoolean-12en-28-oic acid (146, 22 mg) was isolated after column chromatography (RP-18, 7090% MeCN-H2O). Fraction F2 (44 mg), after chromatography on silica gel (20% CH2Cl2-CHCl3 to pure CHCl3) yielded compound 146 (9 mg) and krukovine A (150, 1.3 mg) after HPLC purification (LUNA C18 column, 65% MeCN-H2O). Fraction G (700 mg) after chromatography on Sephadex LH-20, gave fractions (G1 to G3). Fraction G2 (350 mg) was fractionated on silica gel (12% EtOAc-hexane to pure EtOAc), gave 160 fractions. Fraction 81 was subjected to HPLC (DIOL column, 15% acetone-hexane), lead to the isolation of an unidentified triterpenoid (G2-81a-1). Combined fractions 147-158 (32 mg) were further purified by HPLC (DIOL column, 25% acetone-hexane) and gave 3β-hydroxy-11-oxoolean-12-en-28-oic acid (153, 5.5 mg). Combined fractions 99-105 (20 mg) afforded (155, 3.2 mg), after HPLC purification (DIOL column, 20% acetone-hexane). Fraction H (700 mg) after chromatography on Sephadex LH-20, gave fractions (H1 to H4). Fraction H3 (254 mg), after column chromatography on silica gel (gradient from 10% EtOAc-hexane to pure EtOAc), followed by Diol column (gradient from 20% EtOAc-hexane to pure EtOAc) and finally by silica gel flash column (20% EtOAc-hexane), gave 28,30-dihydroxylup-20(29)-en-3-one (152, mg). Fraction H4 (94 mg), gave betulin 3-caffeate (149, 2.5 mg), after column chromatography on silica 72 gel (gradient from 10% EtOAc-hexane to pure EtOAc), RP-18 silica gel (55-80% MeCN-H2O and 60% MeCN-H2O to pure MeCN). 3,11-Dioxoolean-12-en-28-oic acid (146) C30H44O4, white powder; [α]D +77.0 (c 0.32); 1H-NMR and 13C-NMR: see Table 2-1; HR-EIMS m/z [M+] 468.3216 (C30H44O4 requires m/z 468.3235). EI-MS (rel. int.): 468 (13), 422 (52), 407 (50), 257 (100), 217 (68), 161 (45), 95 (70), 41 (92). 3,11-Dioxoolean-12-en-28-oic acid, Me ester (147) C31H46O4, yellowish oil; [α]D + 15.1 (c 0.09); 13 C-NMR: 39.8 (C-1), 34.2 (C-2), 217.2 (C-3), 47.7 (C-4), 55.4 (C-5), 18.7 (C-6), 32.3 (C-7), 43.6 (C-8), 61.1 (C-9), 36.9 (C-10), 199.6 (C-11), 127.8 (C-12), 169.1 (C-13), 44.9 (C-14), 27.8 (C-15), 22.9 (C-16), 46.2 (C-17), 41.7 (C-18), 44.3 (C19), 30.7 (C-20), 33.7 (C-21), 31.6 (C-22), 26.5 (C-23), 21.4 (C-24), 15.5 (C-25), 18.8 (C-26), 23.4 (C-27), 177.5 (C-28), 32.8 (C-29), 23.5 (C-30), 51.9 (Me); HR-EIMS m/z [M+] 482.3397 (C31H46O4 requires m/z 482.3391). EI-MS (rel. int.): 482 [M+] (87), 454 (47), 407 (28), 343 (13), 317 (73), 276 (100), 257 (82), 217 (100), 189 (91), 161 (29), 95 (43), 55 (37), 41 (26). Oleanolic acid (148) C30H48O3, white powder, [α]D +67.0 (c 0.22); 1H-NMR: 5.28 (1H, t, J = 3.3 Hz, H-12), 3.22 (1H, dd, J = 4.5, 10.1 Hz, H-3); 13C-NMR: 38.4 (C-1), 27.2 (C-2), 79.0 (C-3), 38.8 (C-4), 55.3 (C-5), 18.3 (C-6), 32.7 (C-7), 39.3 (C-8), 47.7 (C-9), 37.1 (C-10), 23.0 (C-11), 122.7 (C-12), 143.6 (C-13), 41.7 (C-14), 27.7 (C-15), 23.4 (C-16), 46.5 (C-17), 41.1 (C-18), 45.9 (C-19), 30.7 (C-20), 33.8 (C-21), 32.4 (C22), 28.1 (C-23), 15.5 (C-24), 15.3 (C-25), 17.1 (C-26), 25.9 (C-27), 182.1 (C-28), 33.1 (C-29), 23.6 (C-30); HR-EIMS m/z [M+] 456.3553 (C30H48O3 requires m/z 73 456.3598); EI-MS m/z (rel. int.): 456 (2), 438 (25), 410 (40), 327 (6), 300 (23), 252 (100), 216 (94), 165 (89), 103 (76), 55 (95). Betulin 3-caffeate (149) C39H56O5, yellowish amorphous powder, [α]D +32.4 (c 0.25); H-NMR: 4.68 and 4.59 (1H each, d, J = 2.1 Hz, H2-29), 3.81 and 3.34 (1H each, d, Jgem = 10.8 Hz, H2-28), 2.38 (1H, ddd, J = 5.5, 10.5, 10.5 Hz; H-19), 1.03 (3H, s, H3- 26), 0.99 (3H, s, H3-27), 0.91 (3H, s, H3-24), 0.88 (3H, s, H3-23), and 0.87 (3H, s, H325); 13 C-NMR: 167.3 (C-9'), 150.4 (C-20), 146.0 (C-4'), 144.1 (C-7'), 143.7 (C-3'), 127.9 (C-1'), 122.4 (C-6'), 116.6 (C-8'), 115.5 (C-2'), 114.3 (C-5'), 109.7 (C-29), 81.0 (C-3), 60.6 (C-28), 55.4 (C-5), 50.3 (C-9), 48.8 (C-17), 47.8 (C-18), 47.8 (C-19), 42.7 (C-14), 41.0 (C-8), 38.4 (C-1), 38.1 (C-4), 37.3 (C-13), 37.1 (C-10), 34.2 (C-7), 34.0 (C-22), 29.7 (C-21), 29.2 (C-16), 28.0 (C-23), 27.0 (C-15), 25.2 (C-12), 23.8 (C-2), 20.9 (C-11), 19.1 (C-30), 18.2 (C-6), 16.7 (C-24), 16.2 (C-25), 16.0 (C-26), 14.8 (C27); HR-EIMS m/z [M+] 604.4135 (C39H56O5 requires m/z 604.4123); EI-MS m/z (rel. int.): 591 (1), 497 (2), 433 (3), 428 (3), 316 (4), 253 (8), 197 (12), 149 (41), 83 (100), 57 (54). Krukovine A (150) C30H46O3, colorless powder; [α]D +45.0 (c 0.13); 1H-NMR: 5.61 (1H, s, H-12), 0.90 (3H, s, H3-27), 0.92 (3H, s, H3-29), 1.07 (3H, s, H3-24), 1.10 (3H, s, H3-23), 1.15 (3H, s, H3-26), 1.26 (3H, s, H3-25), and 1.40 (3H, s, H3-30); 13 C-NMR: 39.8 (C-1), 34.2 (C-2), 217.1 (C-3), 47.8 (C-4), 55.5 (C-5), 18.8 (C-6), 32.1 (C-7), 43.6 (C-8), 61.0 (C-9), 36.7 (C-10), 199.3 (C-11), 128.2 (C-12), 169.8 (C-13), 45.3 (C-14), 25.9 (C-15), 21.6 (C-16), 37.0 (C-17), 42.7 (C-18), 45.0 (C-19), 31.1 (C-20), 30.6 (C21), 33.9 (C-22), 26.4 (C-23), 21.4 (C-24), 15.7 (C-25), 18.5 (C-26), 23.4 (C-27), 69.7 (C-28), 32.9 (C-29), 23.3 (C-30); HREIMS m/z [M+] 454.3443 (C30H46O3 requires m/z 74 454.3442); EI-MS m/z (rel. int.): 454 [M+] (58), 289 (66), 248 (94), 190 (70), 161 (73), 95 (100), 55 (89), 43 (99). Morolic acid (151) C30H48O3, white powder; [α]D +32.7 (c 0.15); 1H-NMR: 5.17 (1H, s, H-19), 3.21 (1H, dd, J = 6.0, 11.0 Hz; H-3), 0.76 (3H, s, H3-25), 0.77 (3H, s, H3-27), 0.86 (3H, s, H3-24), 0.96 (3H, s, H3-23), 0.97 (3H, s, H3-30), 0.98 (3H, s, H3-26), and 1.00 (3H, s, H3-29); 13C-NMR: 38.9 (C-1), 27.4 (C-2), 79.0 (C-3), 39.0 (C-4), 55.5 (C5), 18.2 (C-6), 34.6 (C-7), 40.7 (C-8), 51.2 (C-9), 37.2 (C-10), 20.9 (C-11), 26.0 (C12), 41.4 (C-13), 42.6 (C-14), 29.4 (C-15), 33.5 (C-16), 48.0 (C-17), 136.9 (C-18), 133.3 (C-19), 32.1 (C-20), 33.5 (C-21), 33.4 (C-22), 28.0 (C-23), 16.6 (C-24), 15.4 (C25), 16.0 (C-26), 14.9 (C-27), 179.9 (C-28), 30.3 (C-29), 29.1 (C-30); HR-EIMS m/z [M+] 456.3631 (C30H48O3 requires m/z 456.3598); EI-MS m/z (rel. int.): 456 [M+] (5), 438 (7), 394 (22), 351 (14), 248 (28), 203 (52), 163 (100), 121 (56), 95 (62), 43 (70). 28,30-Dihydroxylup-20(29)-en-3-one (152) C30H48O3, colorless powder; [α]D +32.0 (c 0.15); 1H-NMR: 4.95 (1H, d, J = 1.1 Hz, H-29a), 4.90 (1H, br s, H-29b), 4.12 (2H, s, H2-30), 3.79 (1H, dd, J = 1.7, 11.0 Hz, H-28a), 3.32 (br d, J = 11.0 Hz, H-28b), 2.30 (1H, m, H-19), 0.92 (3H, s, H3-25), 0.99 (3H, s, H3-27), 1.02 (3H, s, H3-24), 1.06 (3H, s, H3-26), and 1.07 (3H, s, H3-23); 13C-NMR: 39.6 (C-1), 34.1 (C-2), 218.0 (C-3), 47.3 (C-4), 54.9 (C-5), 19.7 (C-6), 33.6 (C-7), 40.9 (C-8), 49.7 (C-9), 36.9 (C-10), 21.5 (C11), 26.8 (C-12), 37.4 (C-13), 42.8 (C-14), 27.0 (C-15), 29.2 (C-16), 47.8 (C-17), 49.4 (C-18), 43.5 (C-19), 154.5 (C-20), 31.8 (C-21), 33.8 (C-22), 26.7 (C-23), 21.0 (C-24), 16.0 (C-25), 15.8 (C-26), 14.7 (C-27), 65.1 (C-28), 107.3 (C-29), 60.3 (C-30); HREIMS m/z [M+] 456.3576 (C30H48O3 requires m/z 456.3598); EI-MS m/z (rel. int.): 423 (20), 407 (22), 367 (44), 245 (18), 205 (86), 149 (63), 95 (87), 55 (100), 41 (90). 75 3β-Hydroxy-11-oxoolean-12-en-28-oic acid (153) C30H46O4, white powder; [α]D +62.5 (c 0.4); 1H-NMR: 5.63 (1H, s, H-12), 3.22 (1H, dd, J = 5.8, 10.4 Hz, H-3), 2.97 (1H, dd, J = 3.9, 13.6 Hz, H-18), 2.82 (1H, dt, J = 3.3, 13.6 Hz, H-1β), 2.31 (1H, s, H9), 2.06 (1H, dt, J = 3.2, 14.3 Hz, H-16), 0.78 (3H, s, H3-24), 0.92 (3H, s, H3-26), 0.93 (3H, s, H3-29), 0.94 (3H, s, H3-30), 0.99 (3H, s, H3-23), 1.10 (3H, s, H3-25), and 1.36 (3H, s, H3-27); 13 C-NMR: 39.1 (C-1), 27.3 (C-2), 78.8 (C-3), 39.1 (C-4), 55.0 (C-5), 17.4 (C-6), 32.9 (C-7), 45.0 (C-8), 61.8 (C-9), 37.3 (C-10), 200.4 (C-11), 128.1 (C-12), 168.3 (C-13), 43.5 (C-14), 27.8 (C-15), 22.7 (C-16), 46.0 (C-17), 41.4 (C-18), 44.1 (C19), 30.7 (C-20), 33.6 (C-21), 31.6 (C-22), 28.1 (C-23), 15.5 (C-24), 16.2 (C-25), 19.2 (C-26), 23.6 (C-27), 182.4 (C-28), 32.8 (C-29), 23.4 (C-30); HR-EIMS m/z [M+] 470.3407 (C30H46O4 requires m/z 470.3391); EI-MS m/z (rel. int.): 470 [M+] (35), 424 (44), 391 (45), 303 (46), 257 (72), 217 (64), 175 (59), 95 (70), 55 (72), 43 (100). 3β-Hydroxy-11-oxoolean-12-en-28-oic acid, Me ester (154) C31H48O4, colorless amorphous powder, [α]D +72.8 (c 0.2, CHCl3); 1H-NMR: 5.58 (1H, s, H-12), 3.58 (3H, s, H3-31), 3.16 (1H, dd, J = 5.1, 9.7 Hz, H-3), 2.95 (1H, br d, J = 10.6 Hz, H-18), 2.77 (1H, br d, J = 13.4 Hz, H-1β), 2.26 (1H, s, H-9), 1.99 (1H, dt, J = 3.0, 14.1 Hz, H-16), 1.30 (3H, s, H3-27), 1.05 (3H, s, H3-25), 0.94 (3H, s, H3-23), 0.88 (3H, s, H3-30), 0.87 (3H, s, H3-29), 0.86 (3H, s, H3-26), and 0.74 (3H, s, H3-24); 13C-NMR: 200.3, 177.5, 168.6, 127.9, 78.8, 61.8, 55.0, 51.9, 46.2, 45.0, 44.3, 43.5, 41.6, 39.1, 39.1, 37.3, 33.7, 32.9, 32.9, 31.6, 30.7, 28.1, 27.8, 27.3, 23.6, 23.4, 23.0, 18.9, 17.4, 16.2, 15.6; HREIMS m/z [M+] 484.3548 (C31H48O4 requires m/z 484.3548); EI-MS m/z (rel. int.): 484 [M+] (28), 451 (12), 317 (64), 276 (87), 271 (100), 175 (67), 135 (32), 69 (14), 41 (8). 76 3β,28-Dihydroxy-12-oleanen-11-one (155) C30H48O3, white powder; [α]D +53.6 (c 0.32); 1H-NMR: 5.57 (1H, s, H-12), 3.47 (1H, d, J = 10.8 Hz, H-28a), 3.21 (1H, d, J = 10.8 Hz, H-28b), 0.80 (3H, s, H3-24), 0.89 (3H, s, H3-29), 0.92 (3H, s, H3-30), 1.00 (3H, s, H3-23), 1.11 (3H, s, H3-26), 1.13 (3H, s, H3-25), and 1.39 (3H, s, H3-27); 13CNMR: 39.2 (C-1), 27.3 (C-2), 78.8 (C-3), 39.2 (C-4), 55.0 (C-5), 17.5 (C-6), 32.7 (C7), 45.4 (C-8), 61.8 (C-9), 37.0 (C-10), 200.0 (C-11), 128.3 (C-12), 169.3 (C-13), 43.4 (C-14), 25.9 (C-15), 21.6 (C-16), 37.1 (C-17), 42.7 (C-18), 45.0 (C-19), 31.1 (C-20), 33.9 (C-21), 30.6 (C-22), 15.6 (C-23), 28.1 (C-24), 16.4 (C-25), 18.6 (C-26), 23.4 (C27), 69.7 (C-28), 32.9 (C-29), 23.4 (C-30); HR-EIMS m/z [M+] 456.3605 (C30H48O3 requires m/z 456.3598); EI-MS m/z (rel. int.): 456 [M+] (44), 407 (18), 289 (67), 248 (72), 189 (60), 175 (72), 135 (73), 55 (69), 43 (100). Reactions All reactions were monitored using TLC. Crude products were purified by minicolumn chromatography. Procedures for common reactions carried out in this work are described below. Methylation with CH2N2 The compound is dissolved in diethyl ether, then the reaction was carried out in the Aldrich’s MMNG diazomethane-generator. The reaction mixture was left to stand in the fumehood at room temperature for an hour. Removal of the diethyl ether afforded the crude product. Oxidation using PCC / CH2Cl2 PCC was suspended in CH2Cl2, stirred for 30 minutes at room temperature. Compound was dissolved in CH2Cl2, then added to the above PCC suspension and stirred at room 77 temperature, until the solid turned to dark brown. The reaction mixture was then filtered and washed with diethyl ether (3 times). Solvent was removed from the combined filtrate under reduced pressure to afford the crude product. 78 [...]... the known skeletal types The discovery of mevalonic acid (MVA) (18 ) in 19 56 3 lead to the identification of isopentenyl pyrophosphate (IPP) (19 ) in 19 59,4-5b and its isomer, dimethylallyl pyrophosphate (DMAPP) (20),5c as the activated C5 units These are the biological equivalents of isoprene Formation of the main structural types of terpenoid are shown in Scheme 1- 1.6 8 OPP OPP H isomerase ? (IPP) PPO... formation of pyrrolizidine alkaloids advances through a route that is analogous to that of quinolizidine alkaloids, which involved the condensation of two ornithine molecules The biosynthetic pathway of retronecine (53), as shown in Scheme 1- 10, was proposed based on 13 C and 15 N labelled experiments. 21 COOH H2N H CHO H2N NH2 H2N NH2 H2N NH2 NH2 NH2 + 2 [H] H N N CHO CHO CHO HO CH2OH N H N N (53) Scheme 1- 10... cyclization product of a pentaketide precursor (10 ) The biosynthetic precursor of citrinin in the filamentous fungus Monascus ruber, however, was proved to be a tetraketide instead.2 6 COMe HO O OH Me COMe Me OH O (8) Me Me Me O Me O O O O CoAS COOH O OH (9) O (10 ) Terramycin (11 ), tetracycline (12 ), and aureomycin (13 ) are probably derived from cyclization of a nonaketide The biosynthetic pathway of these effective... chair-chair-chair-boat conformation (Scheme 1- 5),6 the well established biosynthetic pathway of cholesterol (38) proceeds via the chair-boat-chair-boat conformation (Scheme 1- 6) ,18 a with the formations of ring A, and probably ring B, are concerted with the initial epoxide protonation .18 b H+ O H (36) H HO H H H H H HO HO H H (37) Scheme 1- 5 Formation of -amyrin 17 Enz-AH+ H O (36) H H H HO ring expansion... of the peyote cactus Lophophora williamsii The biosynthesis of this hallucinogenic alkaloid, as shown in Scheme 1- 12, involve decarboxylation of tyrosine, followed by oxidation to form dopamine (58), an intermediate commonly found in biosynthesis of many alkaloids of this group, including the isoquinolines and the benzylisoquinolines NH2 COOH NH2 NH2 NH2 HO OH OH MeO OH (58) OMe OMe (57) Scheme 1- 12... in the currently accepted biosynthetic pathway of gibberellin A3 (35), the most active among these plant hormones, are shown in Scheme 1- 4 .19 15 H H H H H H H O H H H HOOC OPP (34) OH H H O CHO HO COOH H O HOOC (35) Scheme 1- 4 Biosynthesis of gibberellin A3 As mentioned earlier, the precursor of triterpenoids (C30) and steroids (C27-C29) is squalene ( 21) , which is abundant in shark liver oil The first... (15 ), and thermarol (16 ) 7 OH (14 ) (15 ) (16 ) Isoprene (17 ) was suggested as the building block for terpene biosynthesis: The terpenes can be formed by condensation of successive isoprene units in a head-to-tail fashion This so-called “isoprene rule” was later replaced by Ruzicka’s “biogenetic isoprene rule”, in which the C5 unit is isoprene-like, and the basic skeletal type formed by condensation of. .. strains of Streptomyces.6 Me OH H H Cl H R H O OH (11 ) R = OH (12 ) R = H H O NMe2 OH H CONH2 OH OH H OH Me NMe2 OH CONH2 OH OH O OH O (13 ) The second major biosynthetic pathway from acetate leads to a large and structurally diverse group, collectively known as the isoprenoids The common structural characteristic of this group is that they contain an integral number of C5 units, as shown for -myrcene (14 ),... Biosynthesis of retronecine The biosynthesis of pyridine alkaloids, such as nicotine (54) from tobacco plants, is straightforward: an electrophilic aromatic substitution at C-3 of the pyridine moiety, which comes from nicotinic acid (55) or its enzyme-bound thiol ester, by the C4N unit originates from ornithine The overall pathway is shown in Scheme 1- 11, together with the isolated enzymes that mediated some of. .. Monoterpenes (C10) OPP H Sesquiterpenes (C15) OPP (GPP) OPP (IPP) (E,E)-Farnesyl PP (FPP) PPO OPP OPP H (FPP) Geranyl geranyl PP (GGPP) (FPP) Diterpenes (C20) Squalene (C30) Triterpenes (C30) Scheme 1- 1 Biosynthetic relationship of terpenoids 9 It is noteworthy that the C30 intermediate squalene ( 21) was formed by joining two units of farnesyl pyrophosphate (FPP) tail-to-tail, instead of the regular . synthesis of morphine………………………………………. 18 1 v List of spectra in appendix 1 H- and 13 C-NMR of 3 ,11 -dioxoolean -12 -en-28-oic acid (14 6) 215 - 216 1 H- and 13 C-NMR of pallavicinin B (249) 217 - 218 . 217 - 218 1 H- and 13 C-NMR of pallavicinin C (250) 219 -220 1 H- and 13 C-NMR of pallavicinin D (2 51) 2 21- 222 1 H- and 13 C-NMR of pallavicinin E (252) 223-224 1 H- and 13 C-NMR of pallavicinin. 1 H- and 13 C-NMR of pallavicidine B (2 71) 235-236 1 H- and 13 C-NMR of pallavicinin G (273) 237-238 1 H- and 13 C-NMR of pallavicinin H (274) 239-240 1D- and 2D-NMR of fraction G4 -19 -19