Natural products from selaginella

Chapter Introduction to Natural Products Natural Products There are two kinds of organic comfpounds in Nature, primary metabolites and secondary metabolites Primary metabolites are compounds such as proteins, carbohydrates, fats and nucleic acids which play an essential role for the existence of the organisms which produce them Inference with one of these functions of the primary metabolism might cause the death of the producing organism They have a universal distribution in plants, microorganism and mammal and their structures are found to be the essentially same among different species, apart from minor variation Secondary metabolites, also called “natural products”, are compounds of natural origin that only occur in a limited number of species They are not essential for the survival of the producing organism Natural products are usually small molecules and many of them are used by man as drugs, coloring agents, food additives, insecticides, etc (1) Natural products attract researchers from various fields due to their great structural and chemical diversities and biological activities Two examples of natural products will help to clarify the definition Morphine (1), a drug widely used and abused by man, can only be found in two species of poppy, Papaver somniferum and Papaver setigerum Penicillins (2-3) which have great value as antibiotics are produced only by a few species of fungi (1) Both morphine and penicilins are not essential for the life processes of the species producing them but may play some unknown biological activities (1) HO H N O H H O NMe N S Me Me O HO (1) CO2H (2) Penicillin G H N O O H N O S Me Me CO2H (3) Penicillin V The above definitions distinguishing primary and secondary metabolites leave a grey area at the boundary For example, fatty acids and sugars are described as primary metabolites in most cases, but some representatives are extremely rare and found only in a very limited number of species and thus should be considered as secondary metabolites Many steroids are widely distributed in various species and have a vital role in the organisms, and therefore should be considered as primary metabolites While others are of restricted occurrence, should therefore be regarded as secondary metabolites Though natural products have complicated and diverse structures, only a limited number of building blocks are employed in the biosynthesis of natural products The most important are acetyl coenzyme A (acetyl-CoA) (4) (2), shikimic acid (5) (3, 4) and mevalonic acid (6) (5) Acetyl-CoA is the precursor for many fatty acids, aromatic polyketides, macrolides and polyethers, while shikimic acid is used in the production of aromatic compounds, such as coumarins, flavonoids, cinnamic acids, etc Mevalonic acid, derived from acetyl-CoA, is the precursor for many terpenoids and steroids Terpenoids and steroids can also be synthesized by a non-mevalonate pathway, e.g deoxyxylulose phosphate (5) Classfication of compounds not by species or structures but by biosynthetic pathway is a more rational approach O Me H N S OH H N O Me Me O O OH O P P OH O O O HO O P O O HO HO OH N NH2 N HO Me CO2H OH (5) N N (4) CO2H O OH (6) Natural Products as drug It is the biological activities of natural products that stimulate the study of natural products In 19th century more than twenty biological active natural products were isolated, including morphine (1), codeine (7), quinine (8), caffeine (9), nicotine (10), camphor (11), strychnine (12), and cocaine (13) Until now natural products remain the best sources of new drug discovery due to their structural diversity and complexities From 1981 to 2005 42% new drugs are natural products, natural products derivatives or natural products pharmacophore (6) RO OMe Me O H NMe H N HO O N O Me N N Me N N OH (1) R=H (7) R=Me (8) (9) O OMe N N Me Me Me N Me Me H O N O N O O O (10) (11) (12) (13) Natural products play a key important role in anti-cancer drugs Taxol® and epothilones are molecules found to kill tumor cells with the mechanism of stabilization of microtubules Taxol ® (14), isolated from the bark of the Pacific Yew tree, Taxus brevifolia Nutt (Taxaceae) (7), is the first compound known to act as a promoter of microtubule assembly (8), and it is used to cure ovarian cancer, breast cancer, non-small-cell lung cancer, small-cell lung cancer, squamous cancers of the head and neck and various other cancers It is the best-selling anticancer drug in history, with commercial sales of well over $1 billion in 1998 (9) Initially the only way to obtain Taxol® was by removing the bark of mature yew trees This damage was of course fatal to the tree! The supply problem was solved by the discovery that the European yew tree Taxus baccata was a renewable source of Taxol® -type metabolite By harvesting and extracting the needles, 10-deacetylbaccatin (15) can be isolated and converted to Taxol® in high yield (Scheme 1) Me O O NH O O Me Me Me O HO O O OH HO O OH Me H Me O Me O HO Me Me Me HO O OH Me Me Me HO O O H O O H HO O O O (15) Me O C6H5 N NaH, THF, O O C6H5 OSi(Pr/)3 Me HF/pyridine O Me O O (14) HO O OH Me O NH O O Me O OH O OH Me Me Me HO O O H O Me O O Scheme Partial synthesis of Taxol® Epothilones A (16) and B (17), were isolated from the culture extract of the cellulose-degrading mycobacterium Sorangium cellulosum (Myxococcales) strain So ce90 (10) They kill tumor cells through a mechanism similar to that of Taxol®, with almost identical IC50 values to those of Taxol® It is effective against a number of Taxol® -resistant tumor cell lines (11, 12), showing that it may be a more efficient anti-cancer drug than Taxol® Its supply problem was solved by microorganism fermentation Me O HO Me Me Me Me N O Me O OH O (16) S Me HO O Me Me Me Me S N Me O Me O OH O (17) The general shortage of organs for transplants means that any drug that increases the chance of organ survival is greatly needed FK506 (18), a 23-membered macrolide lactone, isolated from Streptomyces tsukubaensis no 9993 (13), and cyclosporine (19), produced by fungi of the genus Tolypocladium in submerged cultures (14), are immunosuppressive drugs conventionally used in the treatment of organ transplant patients They are effective in ensuring the short-term survival of the transplant, but ultimately the organ is still rejected in the long term, so the patients have to take such drugs for the rest of their lives A progress in the discovery of immunosuppressive drugs was made when Rapamycin (20) was isolated in 1975 from the bacterial strain Streptomyces hygroscopicus found in a soil sample on Easter Island (15) It also appears to cause fewer side effects than the standard anti-rejection treatments due to its novel mode of action (16) HO H MeO Me O Me H N O Me OH O O O OH O Me Me OMe OMe (18) Me Me Me Me H N Me HO H H Me C Me C N O O N N C C C N O H N Me C O H O Me Me O H H C Me O H H O O H O N N C N C C N C Me H Me H Me H O Me Me Me Me Me Me Me Me Me Me N H (19) Me O Me H OH OMe O N H O O Me O OH O Me H OMe Me Me OMe OH Me (20) Chemical Ecology Some natural products are produced for easily appreciated ecological reasons, e.g as toxic materials to defend against predators or compete with other species, as volatile attractants toward other organisms especially animals, and as coloring agents to attract or warn off other species For example, sessile or slow-moving animals, such as sponges, nudibranch mollusks, corals and amphibians are famous for their ability to produce a wide range of chemicals that are usually very toxic, to serve as chemical defenses against predators The generally low overall algal abundance on tropical reefs has been attributed to the intense grazing activities of herbivorous fishes and sea urchins, considering the fishes have been estimated to bite the bottom in excess of 150,000 times/m2/day (17) Halimeda species are most abundant in biomass among the macroalgae exposed to herbivores in many reef systems They are avoided by generalist fishes, which suggest that Halimeda is chemically defended Halimedatrial (21), identified as the major secondary metabolite in six Halimeda species, is toxic toward reef fishes, significantly reduces feeding in herbivorous fishes, and has cytotoxic and antimicrobial activities This compound was suggested to represent a chemical defense adaptation for the successful survival of Halimeda species (18) Me Me H Me CHO H CHO CHO (21) In some cases, the origin of fish-deterrent natural products in marine invertebrates can be traced through the food-chain Most opisthobranch mollusks lack a hard external shell common to other marine snails Macrocyclic oxazole alkaloids, such as tetrahydrohalichondramide (22), tetrahydrohalichondramide (23), kabiramide B (24) and kabiramide C (25) were identified as fish-deterrent compounds present in the Spanish dancer nudibranch Hexabranchus sanguineus, a large brightly colored shell-less sea slug common to Indo-Pacific coral reefs The nudibranch passes the defensive compounds to its egg ribbons which are similarly conspicuous and physically defenseless Sponges of the genus Halichondria were found to be the true sources of the defensive metabolites of Hexabranchus sanguineus Compounds 22, 24, 25 and halichondramide (26) have been isolated from the genus Halichondria If some members of a population of Hexabranchus lack defensive chemicals due to the absence of suitable sponges in their diet, a form of automimicry will arise Automimicry means they will show the same distinctive color pattern and behavior as Hexabranchus species which possess defensive chemicals This will deter potential predators (19-22) Me OHC N Me Me OMe O OMe O Me O N O O N OH Me N O O OMe (22) Me OHC N Me Me OMe O OMe O Me O O N R1 O OR2 (23) R1=H, R2=H (24) R1=OMe, R2=CONH2 (25) R1=OH, R2=CONH2 N Me N O OH OMe 10 polar compounds then the ethyl acetate elution was applied on Sephadex LH-20 (50% MeOH in CH2Cl2) and 50 fractions were obtained B B B B Fractions 1-3 F1-F3 were combined, further separated on HPLC (Diol column, 50% ethyl acetate in hexane, UV/Vis detector, λ=254nm, flow rate=1.5ml/min) and pure compound 71 and 72 were obtained Fractions 20-25 F20-25 were combined and purified by HPLC (C18 column, 60% methanol in water, B B UV/Vis detector, λ=254nm, flow rate=1ml/min) and pure compound 55 was obtained F40-50 F40-F50 were combined and analyzed to be pure compound 49 by 1H NMR P P S involvens Extraction and Isolation The whole plant of S involvens (51g) was collected at Cameron Highland Malaysia, and identified by Professor Benito C Tan The shade-dried, powdered plant materials were extracted by methanol, the extraction defatted by hexane, then chromatographed on silica gel with ethyl acetate-hexane mixture of increasing polarity (50% up to 100%) 70 fractions were collected Fractions F41-69 were combined, further separated on silica gel column chromatography using 2% methanol in CH2Cl2 Sub-fractions F13-18 were B B B B 85 combined and purified on HPLC (Diol column, UV/Vis detector, λ=254nm, 50% ethyl acetate in hexane, flow rate=1ml/min) and pure compound 66 was obtained S intermedia var dolichocentrus Extraction and Isolation Whole plant of S intermedia var dolichocentrus K M Wong was collected at Robinson Fall, Cameron Highland Malaysia and identified by Professor Benito C Tan Shade-dried, powdered plant materials were extracted by methanol, extraction defatted by hexane, then chromatographed on silica gel using MeOH: CH2Cl2 mixture of increasing polarity (1% B B B B up to 10%) Fraction F5 was applied on Sephadex LH-20 (MeOH: CH2Cl2=50:50) and sub-fractions B B B B F21-33 were combined and chromatographed on silica gel using MeOH: CH2Cl2: acetic B B B B acid mixture of increasing polarity, 30 fractions were collected Sub-fractions F21-25 were combined and confirmed to be compound 76 by 1H NMR, F43-51 were combined P P and found to be compound 46 F70-75 were combined and found to be compound 49 Methylation of amentoflavone 4'-methyl ether (46) Amentoflavone 4'-methyl ether (46) (4 mg), mixed with K2CO3 (150mg) and CH3I (1ml), B B B B B B were dissolved in 5ml methanol The mixture was refluxed for 4-5 hours and checked with TLC When the reaction was complete, the reaction mixture was dried in vacuo at room temperature and extracted with water-CH2Cl2 The CH2Cl2 layer was dried in vacuo B B B B B B B B and confirmed to be amentoflavone hexmethyl ether (77) using 1H NMR spectroscopy P P 86 S frondosa Warb Extraction and Isolation Whole plant of S frondosa Warb (80g) was collected at Beckok Malaysia and identified by Professor Benito C Tan The shade-dried, powdered plant materials were extracted by methanol then defatted by hexane The extraction was eluted on a short silica gel column by ethyl acetate to remove strongly polar compounds The ethyl acetate elution was applied on Sephadex LH-20 (50% MeOH in CH2Cl2) and 14 fractions were obtained B B B B F1-F5 F1-F5 were combined and further purified on a small Sephadex LH-20 column (50% MeOH in CH2Cl2), the sub-fraction F5 was purified by HPLC (Diol column, 50% ethyl B B B B acetate in hexane, UV/Vis detector, λ=254nm, flow rate=1.5ml/min), pure amentoflavone 7''-methyl ether (50) was obtained Sub-fraction F6 was purified on HPLC (Diol column, 50% ethyl acetate in hexane, UV/Vis detector, λ=254nm, flow rate=1.5ml/min) and pure 2'',3''-dihydrodelicaflavone (71) was obtained F15-F21 F15-F21 were combined and eluted on silica gel column using 2% methanol in dichloromethane, pure 2,3-dihydro-3',3'''-biapigenin (78) was obtained S willdenowii (Desv.) Baker Extraction and Isolation The whole plant of S willdenowii (Desv.) Baker (88g) was collected at the rainforest of Bekok Malaysia and identified by Professor Benito C Tan The shade-dried, powdered plant materials were extracted by methanol then defatted by hexane The extraction was 87 eluted on silica gel flash column by ethyl acetate to remove strongly polar compounds The ethyl acetate elution was applied on Sephadex LH-20 (50% MeOH in CH2Cl2) and B B B B 30 fractions were obtained F3 F3 was purified by HPLC (Diol column, 55% ethyl acetate in hexane, UV/Vis detector, λ=254nm, flow rate=1ml/min) and pure amentoflavone 4',7''-dimethyl ether (79) was obtained F13 F13 was further separated by HPLC (Diol column, 50% ethyl acetate in hexane, UV/Vis detector, λ=254nm, flow rate=1.5ml/min) and two compounds have been obtained, 2'',3''-dihydroisocryptomerin (61), and 2'',3''-dihydro-4'-O-4'''-biapigenin 7''-methyl ether (80) F25-30 F25-30 was combined and analyzed to be amentoflavone (49) by 1H NMR P P S roxburghii (Hk & Gr.) Spring Extraction and Isolation Whole plant of S roxburghii (Hk & Gr.) Spring (67g) was collected at Gunung Belumut Malaysia and identified by Professor Benito C Tan The shade-dried, powdered plant materials were extracted by methanol, extraction combined (6.1g) and defatted by hexane The extract was eluted on a short silica gel column by ethyl acetate to remove strongly 88 polar compounds The ethyl acetate elution (1.3g) was applied on Sephadex LH-20 (50% methanol in dichloromethane) and 26 fractions were obtained F13: F13 (30mg) was analyzed to be pure amentoflavone 4'-methyl ether (46) by 1H NMR P P F18-24: Fractions F18-24 were combined (200mg) and chromatographed on silica gel column using 5% methanol in dichloromethane, and 30 fractions were collected Sub-fraction F7 was purified on HPLC (Diol column, 50% ethyl acetate in hexane, UV/Vis detector, λ=254nm, flow rate=1ml/min) and pure 2,3-dihydro-3',3'''-biapigenin (78) (10mg) was obtained Sub-fractions F10-17 were combined and further separated on HPLC (Diol column, 60% ethyl acetate in hexane, UV/Vis detector, λ=254nm, flow rate=1ml/min) and HPLC Peak-4 was purified on HPLC again (ODS column, 60% acetone in water, RI detector, λ=254nm, flow rate=1ml/min) and pure 2,3-dihydroamentoflavone (82) was obtained Sub-fractions F21-30 were combined and analyzed to be pure ametoflavone (49) by 1H NMR P P Amentoflavone 4'-methyl ether (46) Yellow solid, UV (methanol) λmax: 191.2, 200.8, 271.4, 329.2, 434.2 nm IR(KBr)νmax : B B B B 1169.8, 1243.4, 1361.1, 1608.6, 1655.2, 3423.7 cm-1 1H NMR (acetone-d6, 500MHz): P P P P B B δ 13.15 (1H, s, 5''-OH), 12.97 (1H, s, 5-OH), 8.17 (1H, dd, J=2.3, 8.8Hz, H-6'), 8.15 (1H, d, J=2.3Hz, H-2'), 7.59 (2H, d, J=8.8Hz, H-2'''/6'''), 7.38 (1H, d, J=8.8Hz, H-5'), 89 6.83 (2H, d, J=8.8Hz, H-3'''/5'''), 6.76 (1H, s, H-3), 6.66 (1H, s, H-3''), 6.53 (1H, d, J=1.9Hz, H-8), 6.44 (1H, s, H-6''), 6.25 (1H, d, J=1.9Hz, H-6), 3.85 (3H, s, (-) ESIMS m/z: 551.5 [M-H]-, (+) ESIMS m/z: 553.3 [M+H]+ , (-) HRESIMS: 4'-OMe), P P P P 551.1003 [M-H]- (C31H19O10, calcd 551.0978) P P B B B B B B Amentoflavone (49) Yellow solid, IR(KBr)νmax: 1030, 1112, 1169, 1246, 1288, 1366, 1493, 1577, 1608, 1655, B 3136 cm-1 P P B H NMR (acetone-d6, 500MHz): P P B B 13.16 (1H, s, 5''-OH), 13.00 (1H, s, 5-OH), 8.07 (1H, dd, J=2.3, 8.7Hz, H-6'), 8.00 (1H, d, J=2.3Hz, H-2'), 7.57 (2H, d, J=8.8Hz, H-2'''/6'''), 7.05 (1H, d, J=8.7Hz, H-5'), 6.77 (1H, s, H-3''), J=8.8Hz, H-3'''/5'''), 6.72 (2H, d, δ 6.68 (1H, s, H-3), 6.46 (1H, d, J=2.3Hz, H-8), 6.40 (1H, s, H-6''), 6.19 (1H, d, J=2.3Hz, H-6) Amentoflavone 7''-methyl ether (50) Yellow solid, 1H NMR (acetone-d6, 500MHz): δ 13.30 P P B B (1H, s, OH-5''), 12.99 (1H, s, OH-5), 8.07 (1H, d, J=2.2Hz, H-2'), 8.03 (1H, dd, J=2.5, 8.8Hz, H-6'), 7.64 (2H, d, J=8.8Hz, H-2'''/6'''), 7.24 (1H, d, J=8.8Hz, H-5'), 6.82 (2H, d, J=8.8Hz, H-3'''/5'''), 6.71 (1H, s, H-3), 6.68 (1H, s, H-3''), 6.60 (1H, s, H-6''), 6.51 (1H, d, J=2.2Hz, H-8), δ 6.24 (1H, d, J=2.2Hz, H-6), 3.91(3H, s, 7''-OMe) Delicaflavone (55) Yellow solid, 1H NMR (acetone-d6, 500MHz): δ 12.92 (1H, s, OH-5''), 12.36 (1H, s, P P B B OH-5), 8.01 (2H, d, J=9.3Hz, H-2'''/6'''), 7.95 (2H, d, J=9.2Hz, H-2'/6'), 7.29 (2H, d, 90 J=8.8Hz, H-3'''/5'''), 6.97 (2H, d, J=8.8Hz, H-3'/5'), 6.69 (1H, s, H-3''), 6.60 (1H, d, J=1.9Hz, H-8), 6.53 (1H, d, J=1.9Hz, H-8''), 6.31 (1H, d, J=1.9Hz, H-6), 6.26 (1H, d, J=1.9Hz, H-6'') 2'',3''-Dihydroisocryptomerin (61) Yellow solid UV (methanol) λmax: 218.2, 272.2, 288.6, 334.2nm 1H NMR (acetone-d6, B B P 500MHz): δ 12.94 (1H, s, 5-OH), 12.10 (1H, s, 5''-OH), B P B 8.00 (2H, d, J=8.8Hz, H-2'/6'), 7.44 (2H, d, J=8.4Hz, H-2'''/6'''), 7.05 (2H, d, J=9.3Hz, H-3'/5'), 6.93 (2H, d, J=8.4Hz, H-3'''/5'''), 6.68 (1H, s, H-3), 6.54 (1H, d, J=2.3Hz, H-8), 6.37 (1H, s, H-8''), 6.26 (1H, d, J=1.9Hz, H-6), 5.58 (1H, dd, J=2.8, 13.0Hz, H-2''), 3.88 (3H, s, OMe-7''), 3.25 (1H, dd, J=12.9,17.1Hz, H-3''α), 2.77 (1H, dd, J=3.0, 17.0Hz, H-3''β) H NMR (DMSO-d6, 500MHz): δ 12.87 (1H, s, 5-OH), 12.02 (1H, s, 5''-OH), 8.01 (2H, P P B B d, J=8.8Hz, H-2'/6'), 7.37 (2H, d, J=8.4Hz, H-2'''/6'''), 6.99 (2H, d, J=8.8Hz, H-3'/5'), 6.85 (1H, s, H-3), 6.82 (2H, d, J=8.8Hz, H-3'''/5'''), 6.49 (1H, d, J=1.9Hz, H-8), 6.46 (1H, s, H-8''), 6.20 (1H, d, J=1.9Hz, H-6), 5.59 (1H, dd, J=2.8, 13.0Hz, H-2''), 3.80 (3H, s, OMe-7''), 3.41 (1H, dd, J=13.0,17.1Hz, H-3''α), 2.77 (1H, dd, J=2.8, 17.6Hz, H-3''β) ESIMS: 152.9 (14.5), 254.0 (14.2), 281.0 (17.7), 391.0 (11.3), 406.1 (16.1), 434.0 (57.3), 435.1 (37.1), 554.1 (100) HREIMS: 554.1229 (C31H22O10, calcd 554.1213) B B B B Robustaflavone 4',7''-dimethyl ether (66) P H NMR (acetone-d6, 500MHz): δ 13.20 (1H, s, OH-5''), 13.02 (1H, s, OH-5), 8.08 (1H, P B B dd, J=2.3, 8.8Hz, H-6'), 7.96 (2H, d, J=8.8Hz, H-2'''/6'''), 7.9 (1H, d, J=2.3Hz, H-2'), 7.27 (1H, d, J=8.8Hz, H-5'), 6.94 (2H, d, J=8.8Hz, H-3'''/5'''), 6.92 (1H, s, H-3), 91 6.74 (1H, s, H-8''), 6.72 (1H, s, H-3''), 6.55 (1H, d, J=2.3Hz, H-8), 6.25 (1H, d, J=2.3Hz, H-6), 3.90 (3H, s, OMe-7''), 3.87 (3H, s, OMe-4'), 2''S, 3''-Dihydrodelicaflavone (71) Yellow solid, UV (methanol) λmax: 202.0, 207.0, 212.0, 227.0, 270.5, 289.5, 332.0, 503.0 B B nm IR(KBr)νmax: 3468, 1647, 1606, 1506, 1461 cm-1 1H NMR (acetone-d6, 500MHz): B B P P P P B B δ 12.45 (1H, s, 5''-OH), 12.15 (1H, s, 5-OH), 7.97 (2H, d, J=8.7Hz, H-2'/6'), 7.50 (2H, d, J=8.7Hz, H-2'''/6'''), 7.15 (2H, d, J=8.7Hz, H-3'''/5'''), 6.97 (2H, d, J=9.1Hz, H-3'/5'), 6.59 (1H, d, J=2.1Hz, H-8), 6.29 (1H, d, J=2.1Hz, H-6), 5.96 (1H, d, J=2.1Hz, H-8''), 5.94 (1H, d, J=2.1Hz, H-6''), 5.51 (1H, dd, J=3.0, 13.0Hz, H-2''), 3.19 (1H, dd, J=13, 17Hz, H-3''α), 2.77 (1H, dd, J=3.0, 17.0Hz, H-3''β) EIMS: 540.1(1.8), 446.1(1.8), 375.0(2.2), 354.0(0.7), 306.0(1.5), 279.1(9.6), 227.0(3.0), 213.1(3.0), 167.0(29), 149.0(100) HREIMS m/z: 540.1047 (C30H20O10 calc as: 540.1050) B B B B B B 6′′-Methyl-2′′,3′′-dihydrodelicaflavone (72) Yellow solid, UV (methanol) λmax: 203.6, 211.2, 225.4, 270.4, 293.2, 339.2nm B IR(KBr)νmax: 1080.4, 2454.4, 1103.7, B cm-1 P P P B B 1170.8, 1224.8, 1359.8, 1507.1, 1608.7, 1643.5 H NMR (acetone-d6, 500MHz): δ 12.45 (1H, s, 5-OH), 12.42 (1H, s, 5''-OH), P B B 7.98 (2H, d, J=8.8Hz, H-2'/6'), 7.49 (2H, d, J=8.8Hz, H-2'''/6'''), 7.14 (2H, d, J=8.8Hz, H-3'''/5'''), 6.97 (2H, d, J=8.8Hz, H-3'/5'), 6.59 (1H, d, J=1.9Hz, H-8), 6.29 (1H, d, J=2.3Hz, H-6), 6.03 (1H, s, H-8''), 5.47 (1H, dd, J=2.8, 13.0Hz, H-2''), 3.16 (1H, dd, J=13.0, 17.1Hz, H-3''α), 2.77 (1H, dd, J=3.2, 17.0Hz, H-3''β), 1.97 (3H, s, Me-6'') 92 EIMS: 43.0 (100), 83.0 (23.3), 149.0 (44.5), 166.9 (16.8), 191.0 (6.6), 256.9 (4.4), 265.0 (4.4), 304.0 (5.1), 388.9 (13.1), 553.8 (6.6) HREIMS m/z: 554.1211(C31H22O10 calc as: B B B B B B 554.1213) (2S, 2′′S)-3',3'''-Binaringenin (76) Yellow solid, UV (methanol) λmax: 191.6, 210.2, 288.6 nm IR(KBr)νmax: 1087(C-O-C), B B B B 1161, 1354, 1500, 1642 (C=O), 2926, 3456(-OH, broad peak) cm-1 P P P H NMR P (acetone-d6, 500MHz): δ 12.17 (2H, s, 5-OH/5'''-OH), 7.52 (2H, d, J=2.3Hz, 2'/2'''), B B 7.43 (2H, dd, J=2.3, 8.3Hz, 6'/6'''), 7.06 (2H, d, J=8.3Hz, 5'/5'''), H-8/8''), 5.94 (2H, d, J=2.3Hz, H-6/6''), (2H, dd, J=12.9, 17.1Hz, H-3/3''α), 5.98 (2H, d, J=1.9Hz, 5.52 (2H, dd, J=2.8, 12.9Hz, H-2/2''), 3.25 2.77 (2H, dd, J=3.0, 17.0Hz, H-3/3''β) ESIMS m/z: 541.4 1127 [M-H]- (-) HRESIMS m/z: 541.1127 [M-H]- (C30H21O10 calc 541 1135) P P P P P P B B B B B B B Amentoflavone hexamethyl ether (77) IR(KBr)νmax: 1035, 1262, 1340, 1425, 1463, 1511, 1603, 1640, 2842, 2927, 3438 cm-1 1H B B P NMR (CDCl3, 500MHz): B H-2'), B δ 7.97 (1H, dd, J=2.3, 8.8Hz, H-6'), P P P 7.86 (1H, d, J=2.3Hz, 7.40 (2H, d, J=8.8Hz, H-2'''/6'''), 7.15 (1H, d, J=8.8Hz, H-5'), 6.80 (1H, s, H-3), 6.79 (2H, d, J=8.8Hz, H-3'''/5'''), 6.75 (1H, s, H-3''), 6.54 (1H, s, H-6''), 6.50 (1H, d, J=1.9Hz, H-8), 6.36 (1H, d, J=1.9Hz, H-6), 4.08 (3H, s, 7''-OMe or 5''-OMe), δ 3.95 (3H, s, 5-OMe), δ 91 (3H, s, 5''-OMe or 7''-OMe), 3.85 (3H, s, 7-OMe), δ 3.78 (3H, s, 4'-OMe), δ 3.77 (3H, s, 4'''-OMe) (+) ESI m/z: 623.3 [M+H]+ (+) HRESIMS: 623.1939 P P [M+H]+ (C36H31O10, calcd 623.1917) P P B B B B B B 93 2,3-Dihydro-3',3'''-biapigenin (78) UV (methanol) λmax: 218.0, 272.2, 288.6, 334.2nm 1H NMR (acetone-d6, 500MHz): δ B B P P B B 13.01 (1H, s, 5''-OH), 12.17 (1H, s, 5-OH), 8.00 (1H, d, J=2.3Hz, H-2'''), 7.95 (1H, dd, J=2.3, 8.3Hz, H-6'''), 7.56 (1H, d, J=2.3Hz, H-2'), 7.48 (1H, dd, J=2.3, 8.4Hz, H-6'), 7.17* (1H, d, J=8.3Hz, H-5'), 7.09* (1H, d, J=8.3Hz, H-5'''), 6.71 (1H, s, H-3''), 6.56 (1H, d, J=1.9Hz, H-8''), 6.25 (1H, d, J=1.9Hz, H-6''), 6.01 (1H, d, J=2.3Hz, H-8), 5.95 (1H, d, J=2.3Hz, H-6), 5.55 (1H, dd, J=2.8, 13.0Hz, H-2), 3.27 (1H, dd, J=13.0, 17.1Hz, H-3α), H-3''β signal was overlapped by solvent peak (Values with superscript may be interchangeable.) P H NMR (DMSO-d6, 500MHz): δ 13.00 (1H, s, 5''-OH), P B B dd, J=2.3, 8.3Hz, H-6'''), H-6'), 12.15 (1H, s, 5-OH), 7.89 (1H, 7.83 (1H, d, J=2.3Hz, H-2'''), 7.34 (1H, dd, J=2.3, 8.3Hz, 7.33 (1H, d, J=2.3Hz, H-2'), 7.02 (1H, d, J=8.3Hz, H-5'''), 6.94 (1H, d, J=8.3Hz, H-5'), 6.80 (1H, s, H-3''), 6.48 (1H, d, J=1.9Hz, H-8''), 6.18 (1H, d, J=1.9Hz, H-6''), 5.90 (1H, d, J=1.9Hz, H-8), 5.88 (1H, d, J=2.4Hz, H-6), 5.49 (1H, dd, J=3.0, 13.0Hz, H-2), 3.35 (1H, dd, J=13.0,17.0Hz, H-3α), 2.71 (1H, dd, J=3.0,17.0Hz, H-3 β) EIMS: 43.0 (95.1), 55.0 (100), 69.0 (71.5), 83.0 (48.0), 97.1 (38.2), 111.1 (24.4), 153.0 (37.4), 362.0 (19.5), 511.0 (7.3), 540.0 (34.2) Amentoflavone 4',7''-dimethyl ether (79) Yellow solid UV (methanol) λmax: 214.6, 229.0, 271.6, 335.6, 432.6nm 1H NMR B B P P (acetone-d6, 500MHz): δ 13.20 (1H, s, 5''-OH), 13.00 (1H, s, 5-OH), 8.08 (1H, dd, J=2.8, B B 8.8Hz, H-6'), 8.01 (2H, d, J=8.8Hz, H-2'''/6'''), 7.89 (1H, d, J=2.3Hz, H-2'), 7.28 (1H, d, J=8.8Hz, H-5'), 7.05 (2H, d, J=8.8Hz, H-3'''/5'''), 6.92† (1H, s, H-3), 6.74† (1H, s, 94 H-3''), 6.71† (1H, s, H-6''), 6.54 (1H, d, J=1.9Hz, H-8), δ 6.25 (1H, d, J=1.9Hz, H-6), 3.90* (3H, s, 4'-OMe), 3.87* (3H, s, 4'-OMe) (Values with the same superscript may be interchangeable) P H NMR (DMSO-d6, 500MHz): δ 13.12 (1H, s, 5''-OH), 12.92 (1H, s, 5-OH), 8.08 (1H, P B B dd, J=2.3, 8.8Hz, H-6'), 8.01 (2H, d, J=8.8Hz, H-3'''/5'''), 7.83 (1H, d, J=2.3Hz, H-2'), 7.25 (1H, d, J=8.8Hz, H-5'), 6.98 (2H, d, J=8.8Hz, H-2'''/6'''), 6.95 (1H, s, H-6''), 6.90 (1H, s, H-3), 6.86 (1H, s, H-3''), δ 6.50 (1H, d, J=2.3Hz, H-8), 6.19 (1H, d, J=2.3Hz, H-6), 3.82* (3H, s, 4'-OMe), 3.80* (3H, s, 4'-OMe) (Values with superscript may be interchangeable) EIMS: 68.9 (5.9), 152.9 (9.2), 267.5 (11.1), 369.1 (4.6), 520.0 (9.2), 535.1 (100), 566.1 (19.6) HREIMS: 566.1236 (C32H22O10, calcd 566.1213) B B B B B B B B 2'',3''-Dihydro-4'-O-4'''-biapigenin 7''-methyl ether (80) Yellow solid 1H NMR (acetone-d6, 500MHz): δ 12.95 (1H, s, 5-OH), 12.20 (1H, s, P P B B 5''-OH), 7.98 (2H, d, J=9.3Hz, H-2'/6'), 7.18 (2H, d, J=8.4Hz, H-2'''/6'''), 7.05 (2H, d, J=9.3Hz, H-3'/5'), 6.77 (2H, d, J=8.4Hz, H-3'''/5'''), 6.68 (1H, s, H-3), 6.54 (1H, d, J=1.9Hz, H-8), 6.33 (1H, s, H-8''), 6.26 (1H, d, J=2.3Hz, H-6), 5.54 (1H, dd, J=3.3, 12.0Hz, H-2''), 3.89 (3H, s, OMe-7''), 3.22 (1H, dd, J=12.0,17.1Hz, H-3''α), 2.90 (1H, dd, J=3.0, 17.6Hz, H-3''β) EIMS: 68.9(41.1), 152.9(32.3), 254.0(26.6), 434.0(60.5), 554.1(100) 2,3-Dihydroamentoflavone (82) Yellow solid, 1H NMR (acetone-d6, 500MHz): δ 13.16 (1H, s, 5''-OH), 12.17 (1H, s, P P B B 5-OH), 7.65 (2H, d, J=8.7Hz, H-2'''/6'''), 7.59 (1H, d, J=1.7Hz, H-2'), 7.46 (1H, dd, 95 J=2.1, 8.3Hz, H-6'), 7.09 (1H, d, J=8.0Hz, H-5'), 6.91 (2H, d, J=8.8Hz, H-3'''/5'''), 6.63 (1H, s, H-3''), 6.39 (1H, s, H-6''), 6.07 (1H, d, J=1.7Hz, H-8), 5.92 (1H, d, J=1.7Hz, H-6), 5.52 (1H, dd, J=2.8 13.2Hz, H-2), H-3 signals were overlapped by solvent 96 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Mann, J 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surfaces of invertebrates are remarkably clean, a chemical suppression of fouling by involvement of natural products seems highly plausible considering the enormous number of antibiotical and cytotoxic natural products isolated from invertebrates (24) Aplysina fistularis, exudes aerothionin (28) and homoaerothionin (29) into the surrounding seawater at rates of 8.9×10-3-7.7×10-4... species of Selaginella are used as traditional medicines in various countries Selaginella tamariscina (Beauv.), with a more popular Korean name “Keoun Back”, is used to treat advanced cancer patients It has shown the ability to modify gene expression and cytokine production (32) Selaginella delicatula (Desv.), as a folk medicine in China, is used to treat hepatitis and cancer (33) Some other Selaginella. .. bond Only 2'',3''-dihydroisocryptomerin (61) has been reported from S willdenowii (Desv ex Poir) Baker (51) OH 8'' MeO 8 OH O 1' O 1''' 3'' O 4' HO O 4''' 2 3 6 OH O (61) 6 Robustaflavone series: This comprises two apigenin units connected together through a 3'-6'' C-C bond Robustaflavone (62) has been reported from several species from Selaginella (34, 53, 58, 59), and its O-methyl derivatives robustaflavone... 3,5-Di-O-caffeoylquinic acid (68) (34), (-)-lirioresinol A (69) (34), (-)-lirioresinol B (70) (34), have been reported from Selaginella species (34) OH OH HO OH OH O O OH O MeO O O (68) OH OH MeO OMe MeO OMe H H O O O O H H MeO OMe OH (69) MeO OMe OH (70) 29 Chapter 3 Selaginella ciliaris (Retz.) Spring 3.1 Introduction Selaginella ciliaris is a creeping, spreading and short species, with stem laterals close together... C3-O-C4''' bond Delicaflavone (55) has only been reported from S delicatula (Desv.) (34) None of its O-methyl derivative has been reported HO 4' 1' O 8 6 4''' O 8'' HO O O 1''' OH OH 3'' 6'' OH O (55) 25 3 Hinokiflavone series: This comprises two apigenins linked together by a C4'-O-C6'' bond Hinokiflavone (56) has been isolated from several species of Selaginella (53, 54, 57-62), and its O-methyl derivatives... have been isolated from Selaginella species OR3 R2O 4' 8 R1O O 1''' 3'' O OH O 1' O 8'' 4''' 2 3 6 OH O (56) R1=H, R2=H, R3=H (57) R1=H, R2=Me, R3=H (58) R1=H, R2=Me, R3=Me (59) R1=Me, R2=Me, R3=H 4 2,3-dihydrohinokiflavone series: This comprises a naringenin and an apigenin linked together by a C4'-O-C6'' bond In this series only 2,3-dihydroisocryptomerin (60) has only been reported from S delicatula... N N N N H O OH Br O Br OMe (28) Br MeO Br Br Br HO O H N N OMe O OH N H N O O (29) 12 Chapter 2 Selaginellaceae and Biflavonoids The Selaginellaceae are an ancient group of fern allies composed of about 750 species widely distributed worldwide (26) They are a distinctive family containing the single genus Selaginella, only distantly related to other families such as the Lycopodiaceae and Isoetaceae... Starting from a cinnamoyl-CoA unit (43), three molecules of malonyl-CoA are coupled to give 44, which is then cyclized via a Claisen condensation reaction to give a chalcone (45) Chalcones act as precursors for various flavonoid derivatives Flavanone (34) is formed from a chalcone via a Michael-type nucleophilic attack of OH on the α,β–unsaturated ketone Many flavonoid variants can be produced from the... HO O HO HO HO HO HO OH OH O O HO O O H H O OH O (47) OH O (48) 23 3 1 2 Fig 9 CD spectra of 2S-naringin (curve 1), racemic naringin (curve 2) and 2S-naringenin (curve 3) Figure from (50) The reported biflavonoid compounds in Selaginella can be further divided according to the flavonoid-flavonoid linkage patterns, namely, the amentoflavone series, the delicaflavone series, the hinokiflavone series, the... constituent in Selaginella species (34, 51) Its O-methyl derivatives, e.g (46) amentoflavone 4'-methyl ether (52), amentoflavone 7''-methyl ether (50) 7,7''-dimethyl ether (51) (53), amentoflavone (34), amentoflavone 7,4'-dimethyl ether (52) (52, 54, 24 55), amentoflavone 7,7'',4'''-trimethyl ether (53) 7,4',7'',4'''-tetramethyl ether (54) (34, 56), amentoflavone (34, 52, 53, 56) have been reported from various ... OH (6) Natural Products as drug It is the biological activities of natural products that stimulate the study of natural products In 19th century more than twenty biological active natural products. .. Until now natural products remain the best sources of new drug discovery due to their structural diversity and complexities From 1981 to 2005 42% new drugs are natural products, natural products. .. suppression of fouling by involvement of natural products seems highly plausible considering the enormous number of antibiotical and cytotoxic natural products isolated from invertebrates (24) Aplysina