The Annonaceous acetogenins, that are endemic to certain plants of the Annonaceae, are of much interest especially due to their unique structural features and their significant cytotoxic, antitumor, pesticidal, antiinfective and antifeedant activities. More than 200 compounds belonging to this family have been isolated to date, and most of them posses one or more tetrahydrofuran rings, together with a terminal α, β-unsaturated γ-lactone unit on a C-35 or C-37 long carbon chain. In addition to this major group of acetogenins, there are several congeners bearing an epoxide group in place of the tetrahydrofuran ring. Herein, we report total synthesis of three mono-tetrahydrofranyl acetogenins and two epoxide containing acetogenins. A synthetic study on a nonadjacent bis-tetrahydrofuran acetogenin, 4-deoxygigantecin is now underway. 1. Synthesis of solamin (2) and reticulatacin (3) A total synthesis of the natural products, solamin (2) and reticulatacin (3) is described. The monotetrahydrofuran moiety 9 of 1 and 2 was constructed by a fifteen-step reaction sequence, starting from lauryl bromide (4) and 2-propyn-1-ol (5). A palladium-catalyzed cross coupling reaction of 9 and γ-lactone 10 or 11, and subsequent three-step sequence led to 2 and 3, respectively. 2. Synthesis of (10R)- and (10S)-corossoline (12) A convergent stereoselective total synthesis of (10R)- and (10S)-corossoline (12) has been performed via a multi-step process. Comparison of the mp, [α]D, IR, and NMR data of both synthetic materials with those reported for natural corossoline did not allow for the strict determination of the configuration at the C-10 hydroxy group of 12. However, a slight chemical shift difference at the C-10 methine proton was observed in the ^1H-NMR spectra of the corresponding tris(S)-MTPA esters of synthetic (10R)- and (10S)-12, indicating that if the tris(S)-MTPA ester of natural one is available, the stereochemistry at the C-10 hydroxyl group of corossoline will be established. 3. Synthetic studies on 4-deoxygigantecin (22) Synthetic studies on 4-deoxygigantecin (22) is described. The bistetrahydrofuran moiety 27 of 22 was constructed by an eleven-step reaction sequence, starting from 23 and 24. A palladium-catalyzed cross coupling reaction of 27 and γ-lactone 28 afforded 29. Studies directing toward a total synthesis of 22 is now underway. 4. Synthetic studies on epoxyacetogenins. (i) Synthesis of (20R, 21S, 37S)- and (20S, 21R, 37S)-epoxyrollin A (30) A synthesis of two possible diastereoisomers (20R, 21S, 37S)- and (20S, 21R, 37S)-30 corresponding to the epoxy lactone that has been proposed for epoxyrollin A, a stuructural representive of biosynthetic precursors of tetrahydrofuran annonaceous acetogenins, is described. The ^<13>C-NMR and tandem mass spectral data of the synthetic samples were not in accordance with those recorded for natural epoxyrollin A. Consequently, the structure of epoxyrollin A needs to be revised. (ii) Synthesis of (15S, 16R, 19S, 20R, 36S)-diepomuricanin (31) An eleven-step reaction sequence starting from enantiomerically pure (-)-muricatacin (6) afforded the key intermediate 38, which was then converted to (15S, 16R, 19S, 20R, 36S)-diepomuricanin (31) via introduction of an acetylene unit and a coupling reaction with iodo lactone synthon 41.

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In continuation of our studies on microbes that produce insecticidal compounds, we obtained a fungal isolate of Penicillium sp. NTC-47 from soil. When grown on okara (insoluble residue of whole soybean), this strain showed the toxic effect to brine shrimps (Artemia salina). Bioassay-guided purification Iead to the isolation of two active compounds 1 and 2, and one related compound 3. The structure of 2 was established spectroscopically, and finally by X- ray crystarography to be 3-methoxy-4, 5-dihydroxy-4- (4'-methoxyphenyl)-isoquinolinone. 3 was deduced to be 5-deoxy-2. 1 had the same isoquinolinone carbon skeleton with an additional C_<10> side chain, which contained a trans- double bond, three tertiary methyls, an oxymethylene, a-CH_2-CH2-group and two quaternary carbons. The structure of 1 was elucidated spectroscopically and it was shown that 1 was a mixture of two epimers at C_<11>. The LD_<50> values of 1 and 2 against brine shrimps were 0.9 and 20μg/ml, respectively, indicating that the side chain of 1 enhances the toxicity. On the other hand, 3 showed no activity. These facts strongly indicated that the phenolic hydroxyl is necessary to exhibit the toxicity.

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Cyanobacteria produce toxic peptides such as microcystins and nodularins, which are known as the lethal hepatotoxins. Many research groups have isolated other types of peptides than these hepatotoxins from cyanobacteria. We have also continued to focus on these peptide compounds produced by toxic and non-toxic cyanobactera and the biosynthetic relationship between such peptides and toxic peptide compounds. In the present study we would like to report that two types of cyclic and linear peptides, nodulapeptins A (1), B (2) and spumigins A〜C (3〜6), were isolated together with nodularins from a toxic cayanobacterium, Nodularia spumigena AVl and a novel peptide compound, spumigin HKVV (7), was isolated from a non-toxic cyanobacterium, N. spumigena HKVV. Their structures were determinated by 2D-NMR techniques, MS/MS experiments and the advanced Marfey's method. The structures of nodulapeptins are similar to those of anabaenoeptins which were isolated from the toxic cyanobacteria, Anabaena sp. that co-produces microcystins. Recently, Murakami et al. reported the isolation and the structural determination of aeruginosins from Microcystis aeruginosa and the proposed structures for spumigins A〜C have a resemblance to those for these compounds. On the other hand, spumigin HKVV has a quite novel structure comprised of 2-azabicyclo[3.3.1]non-3-carboxylic acid, Ile, O-methlated glyceric acid sulfate, glucose and a cyclic guanidine moiety and its partial structure seems to be similar to those of spumigins A〜C. The production of nodulapeptins and anabaenopeptins are suggested to be characteristic for the toxic cyanobacteria, because they were not isolated from the non-toxic cyanobacterium.

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Camptothecin (1) is well known for the remarkable inhibitory activities against tumor cells and topoisomerase I. Synthetic and biological studies about camptothecin and related compounds have been extensively carried out. In the course of chemical studies of camptothecins, we have already found that Ophiorrhiza pumila Champ. (Rubiaceae) produces not only camptothecin (1) but also a variety of related alkaloids (2-5). We herein report the synthetic studies of these alkaloids based on a biogenetic hypothesis as well as the chemical investigation of the secondary metabolites in cell cultures of this plant. I. Synthetic Studies of Camptothecin Related Compounds Vincoside lactam (8) was converted to 3(R)-deoxypumiloside tetraacetate (13) through a reductive deoxygenation at C-7 position in 3(R)-pumiloside tetraacetate (10). Synthetic 13 was identified with the tetraacetate of natural deoxypumiloside. From this result, H-3 stereochemistry of natural deoxypumiloside, which has been previously reported as α-form, was revised to be β orientation. Strictosamide (7) could also be converted to 3(S)-deoxypumiloside tetraacetate (14). By exhaustive investigation of the chemical constituents in O. pumila, it was found that O. pumila produces both of 3(R)-deoxypumiloside (16) and 3(S)-deoxypumiloside (17). II. Chemical Investigation of the Metabolites in Cell Cultures of Ophiorrhiza pumila From callus cultures induced from leaf segments of O. pumila, eight anthraquinones including two new ones and cholesterol were isolated. No trace of camptothecin related alkaloids was detected. New anthraquinones were identified as 1-hydroxy-2-hydroxymethyl-3-methoxyanthraquinone (22) and 2-butoxymethyl-1,3-dihydroxyanthraquinone (25) by spectroscopic analyses. Regenerated plant, which was formed from callus cultures through differentiation, produced camptothecin (1), pumiloside (4), 3(R)-deoxypumiloside (16), strictosamide (7) and loganin. New camptothecin alkaloid (26) was also isolated from acetylated n-BuOH fraction and identified as 9-O-β-D-glucosylcamptothecin pentaacetate (26). Hairy roots of O. pumila were obtained by the inoculation of Agrobacterium rhizogenes to regenerated plants. From hairy roots, pumiloside (4), 3(R)- and 3(S)-deoxypumiloside (16, 17), strictsamide (7), and two anthraquinones (24, 27) were isolated.

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Recently, a number of naturally occurring cyclic peptides with unique structures and biological activities have been isolated. However, despite their importance, surprisingly few studies of higher plants occurring cyclic peptides exist in the literature. In our investigation of bioactive cyclic peptides from higher plants, new cyclic peptide, called segetalins A-H (1-8), were isolated from the seeds of Vaccaria segetalis (Caryophyllaceae), which are used to activate blood flow and promote milk secretion, and also to treat amenorrhea and breast infections in China. The structures of segetalins A-H were elucidated by 2D NMR, ESI-MS/MS, and chemical degradations. Conformational analysis of segetalins A, B, G, and H, which showed estrogen-like activity, was made by using X-ray, high field NMR and computational methods. To propose the 3D requirements to exhibit the estrogen-like activity, superposition using DISCO was undertaken and each cyclic peptide was suggested to possess a partial conformation similar to that of segetalin A in the region of Trp-Ala-Gly-Val. The sequence and coformation may play an important role to show the activity. To elucidate the relation between their conformation and estrogen-like activity, backbone transformation with Lawesson's reagent was examined. Thionation of segetalins A and B with Lawesson's reagent in dioxane gave each two dithionated products; dithiosegetalins Al (9) and A2 (10), and dithiosegetalins B1 (11) and B2 (12). The only dithiosegetalin A2 showed estrogen-like activity. Conformational preference of dithiosegetalins analyzing by NMR and computational methods suggested that dithiosegetalins A2, dithiosegetalins B1 and B2 took different solution conformations from their parent compounds.

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Farnesyl diphosphate (FPP)synthase [2.5.1.10] catalyzes the condensation of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP) and with geranyl diphosphate (GPP) to produce E,E-FPP. The specificities of the wild-type and mutant FPP synthase for artificial substrates have been studied using the substrate analogs 1-7 and 9-12, which have substituents with various chain length at 4-position of DMAPP. Previous study with the analogs showed that the substrates specificities of the bacterial enzyme was very similar to those of pig liver and chicken liver enzymes. Cysteine residues in several enzymes play important roles in their catalytic function. FPP synthase from B. stearothermophilus has two cysteine residues at positions of 73 and 289. The mutant enzymes, C73S, C2895 and C73S-C289S, in which cysteine residues were replaced with serine, showed that they all have similar substrate specificities to that of the wild-type enzyme. This result also indicates that these cysteine residues in this enzyme is not important for catalytic function as reported before. The highly conserved Gln residue at position 221 in an upstream part of the putative substrate binding site was replaced with Glu. The substrate specificity of this mutant enzyme has been also studied with some DMAPP analogs. These analogs 3 and 4 were as reactive as GPP for the wild-type enzyme. However, the mutagenesis resulted in heavy decrease of activities of the analogs tested to one twentieth of activities for the wild-type enzyme. In contrast, the K_m value of the analog 3 for the mutant enzyme Q221E was comparable to that for the wild-type enzyme. G1n 221 is involved not only in the binding of allylic substrates but also in the catalysis by FPP synthase.

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During our screening for protein farnesyltransferase (PFTase) inhibitors of microbial origin, we have found new compounds, andrastins A〜D (1〜4), from the cultured broth of Penicillium sp. FO-3929. The compounds 1〜4 were purified from EtOAc extracts by silica gel chromatography, ODS chromatography and HPLC. The molecular formulae of 1〜4 were elucidated by HR-FAB-MS to be C_<28>H_<38>O_7, C_<28>H_<40>O_7 C_<28>H_<40>O_6, and C_<26>H_<36>O_5, respectively. The structures of 1〜4 were elucidated by various NMR experiments. As 1〜4 had a ketoenol tautomer that made difficult to elucidate the structure, 2D-INADEQUATE experiments and ^<13>C-^<13>C decoupling experiments using ^<13>C-labeled 1 were carried out. The structure of 2〜4 were elucidated by NMR experiments comparing that of 1. The absolute configuration of a p-bromobenzoyl derivative of 1 was elucidated by X-ray crystallography. The X-ray crystallography was revealed that 1 has an ent-5α,14β-androstan skeleton. The configuration of 1 coincided with its NOE experiments. The biosynthetic studies of andrastins also employed ^<13>C-labeled 1. The results of ^<13>C-NMR spectra of 1 labeled with [2-^<13>C] acetate and [1,2-^<13>C] acetate suggested that andrastins were meroterpenoide that were synthesized from a farnesyl pyrophosphate and a tetraketide. The IC_<50> values of 1〜4 against protein farnesyltransferase from human THP-1 cells were 24.9, 47.1, 13.3, and 25.7μM, respectively.

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Gymnopilus spectabilis has been known as a hallucinogenic mushroom from which toxic polyisoprenepolyols such as gymnopilin have been isolated. Since similar mushroom G. aeruginosus has not been known its toxicity, we have investigated its components to isolate two types of polyisoprenepolyols, homologues of gymnopilin and gymnoprenol. The gymnopilin homologue contained an extra isoprene unit compared with known gymnopilin isolated from G. spectabilis. Because of that gymnopilin showed the neuroexitatory activity, G. aeruginosus should be regarded as a poisonous mushroom. For determination of the stereochemistry of newly isolated congeners, we have demonstrated a new procedure for analyzing the configuration at C-3' of hydroxymethylglutaric acid (HMGA) part. The absolute configuration of the ω-tert-hydroxyl was determined as S which implied the configuration of other tert-hydroxyls in 1,5-polyol system in gymnopilin. Namely, we have isolated gymnopilin (3:m=2, n=4) from G. spectabilis and transformed it to γ-Lactone (5). Model lactones having similar asymmetric center at C-5 were synthesized and Cotton effects in CD spectra were compared. γ-Lactone (5) derived from gymnopilin showed positive Cotton effect as shown with S-lactone (25). The above fact proved the S-configuration at ω-position of gymnopilin (3).

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Ornithogalum saundersiae (Liliaceae) is a perennial plant which is native to Natal, Swaziland and the eastern Transvaal. We have found that the MeOH extract of Ornithogalum saundersiae bulbs exhibited extremely potent cytotoxic activity toward human promyelocytic leukemia HL-60 cells with an LC_<50> value of 0.031μg/ml. Bioassay-guided fractionation of the MeOH extract led to the isolation of a series of cholestane glycosides (1-7) with potent cytotoxic activities toward HL-60 cells and human Tlymphocytic leukemia MOLT-4 cells. The structures of 1-7 were determined by two-dimensional NMR techniques, ^1H-^1H COSY, phase-sensitive NOESY, HMQC and HMBC spectra, and acid- and alkaline catalyzed hydrolysis. Cytotoxic activities of 1 and 3 - 7, which possess an acetyl group and an aromatic acyl group linked to the saccharide moiety, on HL-60 and MOLT-4 cells were much more potent than those of the clinically applied anticancer agents, etoposide, adriamycin (ADM) and methotrexate. Further detail examination of 1, which was obtained as a main constituent, discovered that 1 exhibited potent cytotoxic activities against various malignant tumor cells such as mouse mastrocarcinoma, human pulmonary adenocarcinoma and human pulmonary large cell carcinoma including ADM-resistant mouse leukemia (P388) and camptothecin (CPT)-resistant P388; the activities are about 10-100 times more potent than those of the clinically applied anticancer agents, miiomycin C, AMD, cisplatin, CPT and taxol (TAX). Compound 1 has little toxicity to normal human pulmonary cells. In in vivo evaluation made up to the present, 1 was remarkably effective to mouse P388 (increased life span: 59%) by one time administration of 0.01mg/kg. Accordingly, 1 could be expected to be a new anticancer agent from a higher plant next to CPT and TAX.

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From the latex of Excoecaria agallocha, skin irritant and tumor promoting substances, Excoecaria factors, have been obtained and characterized as daphnane-type diterpene esters. The resinous wood including latex, the so-called "Okinawa-jinko", has also been used as a substitute for the insence of agalwood (Jinko). We have investigated the constituents of wood of E. agallocha, and isolated nine new diterpenes (compounds 4-9 and 11-13) together with five known compounds (1-3, 10 and 14) from ether extract. The structures of these compounds were elucidated on the basis of their NMR spectra, NOE experiments and comparisons of references spectral data. Compounds 4-9, 11-12 and 13 were characterized as labdane, beyerane and kaurane-type diterpenes, respectively. As a screening study for anti-tumor-promoters, isolated compounds were examined for their inhibitory effects on the Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Of these compounds, 14 exhibited significant inhibitory effects on EBV-EA activation.

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Since the discovery of anticancer activity of taxol (1a), isolated from Taxus brevifolia in 1971, great works have been reported for the isolation of taxane diterpenoides from various species of yews and synthetic studies of taxol analogs. In Japan there are native ordinary yews; Taxus cuspidata Sieb. et Zucc. and dwarf varieties of Yew; T. cuspidata var. nana Rehd. (Kyaraboku) T. cuspidata f. luteobaccata Hayashi (Kimino-onnko) with yellow fruits. In searching of biological source for taxol analogs we examined the contents of taxol, its analogs and new taxane diterpenoids in barks, needles and fruits of Japanese dawarf varieties of T. cuspidata.. Each extract obtaned by metanol extraction of each part; heart wood, bark, needle and fruit of yew, was devided into week acidic, strong acidic, neutral and basic fractions. We found the high contents of taxol (1a) in the barks and needles of the dwarf varieties compared those of T. brevifolia. Furthermore, As new taxane diterpenoids were isolated taxa-4(20), 11-taxadiene-2 α,5 α,10β,14β-tetraol 2 α,5α,10β-triacetate-14β-(S)-2'-methylbutyrate (6), mp 108.5℃,[α]_D+42°(CHCl_3) from the heart wood of ordinary and dwarf variteies of yew (T. cuspidata), taxinine-4(20)-epoxide (11), mp 263〜265℃, [α]_D+164°(CHCl_3) from the neutral fractions of needles, 2'-hydroxy-taxine II (14) as an oil, [α]_D+38.9°(CHCl_3) with known alkaloids; taxine II (12) and 2-desacetoxyaustrospicatine (13) from the basic fractions of seeds and needles. Taxinine-4(20)- epoxide (11) was identical with the product obtaned by the epoxidation of taxinine (7) with m-chloroperbenzoic acid. The contents of taxol (1a) in the barks and the needles are different from the prefectural stock of Japanese yew to show the high content in the female stock of the dwarf variety found in Miyagi prefecture, but did not show in the male stock. Also, the seed of a dwarf variety of yew "Daisen Kyaraboku" in Totsutori prefecture show the relatively high content of taxol (1a).

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The fruit of Crescentia cujete L. (Bignoniaceae) is used as a folk medicine (local name, DAO TIEN) in Vietnam as for expectorant, antitussive, laxative and stomachic. Previous studies on the constituents of the leaves of this plant revealed the isolation of naphthoquinones and iridoid glucosides. The fruits of C. cujete afforded 15 new compounds, three iridoid glucosides (6,8,9), five iridoids (7,11-14), two 3-hydroxyoctanol glycosides (16,17), three 2,4-pentanediol glycosides (18-20) and two 4-hydoroxy-2-pentanone glycosides (21,22), along with known compounds, ajugol (1), 6-O-p-hydroxybenzoylajugol (2), aucubin (3), 6-O-p-hydroxybenzoyl-6-epi-aucubin (4), 1-dehydroxy-3, 4-dihydroaucubigenin (5),(10), acanthoside D (15), benzoic acid glucosyl ester (23) and 5-hydroxymethylfurfural (24). Structural elucidation of these compounds were carried out by means of chemical and spectral methods, especially using NMR spectroscopy including ^1H-^1H COSY, HSQC, HMBC and NOE technics.

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The absolute connfiguration of kiwiionoside (1), coexists with actinidioionoside (9) in Actinidiaceae, was determined to be 3S, 5R, 6R, and 9R by single crystal X-ray analysis. The absolute configuration of actinidioionoside (9) was determined by use of an aglucone of 1 by means of C-13 NMR glucosidation shift rules proposed by Tori et al.. That of roseoside, from Raphanus satives, was elucidated to be 6S and 9R by the results of X-ray diffraction method. It was clarified that the glucosidation shift rules was applied to ionone type glucosides regardless of measuring solvents by the comparison of the data of C-13 NMR between the enzymatic degradation product, (6S, 9R)- vomifoliol (2a), and the glucosides (2, 3 and 4). Furthermore, the absolute structure of (6R, 9S) and (6S, 9S)-roseosides (3 and 4) was revealed on the basis of the above fact, respectively. The absolute configuration of (6R, 9S)-3-oxo- a -ionol glucoside was revised to be (6S, 9S)-glucoside (12), by using the glucosidation shift rules. According to the avobe rules, we could lay down the proper parameters of C-13 NMR glucosidation shifts (allyl alcohol and sterically hindered case) in the field of ionone type glucosides.

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Capsanthin esters were separated from paprika (Capsicum annuum) oleoresin, and their structures were determined without saponification.3) The major diesterified and monoesterified capsanthins were identified as 3,3'-lauroylmyristoylcapsanthin (LM-CP) and 3'-myristoylcapsanthin (M-CP), respectively. HPLC, UV and 2D-densitometoric analyses indicated that 3'-O-esterified capsanthins exist in a much larger quantity than the 3-O-esterifed ones. The time-course accumulation of capsanthins was then observed in several cultivar of C. annuum fruits. Esterified capsanthins appeared almost simultaneously with the appearance of capsanthin itself and the amount of the 3'-O-esterified one was larger than that of the 3-O-esterified one during all stage of ripening (Fig. 3). These results suggested that esterification at the cyclohexene ring is the rate-determining step in the biosynthesis of esterified capsanthin and that the 3'-hydroxyl group on the cyclopentane ring is esterified much faster than the 3-hydroxyl group on the cyclohexene ring (Fig. 2). Several reports have suggested that esterified capsanthins were more stable than capsanthin itself.2) To verify this fact, we monitored the degradation of capsanthin, LM-CP and M-CP in paprika powder and the extract during storage under illumination. The amount of capsanthin was reduced time-dependently; however, the amounts of the esterified ones increased in the early stage but, then decreased as fast as capsanthin (Fig. 4). The LC-MS results suggested that the increase in the early stage was ascribed to the isomerization of the naturally occurring Z-isomers. We next studied the stability of individual capsanthin and LM-CP in solutions. In the same solvent, the degradation curves for both compounds were quite similar. On the basis of these data, it was concluded that the stability of capsanthin and the esterified capsanthins do not significantly differ from each other.

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The toad poison bufadienolides having novel steroidal A/B cis and C/D cis structure with α-pyrone ring at C17-position exhibit a variety of biological activities. To carry out the systematic studies on the biological activities of the bufadienolides, we investigated their cytotoxcities against cell line of human liver cancer PLC/PRF/5 and other cancer cells which are HepG2, HeLa-S3, KB, PC-3 cell including some resistance cells. Our research was extended to studies on relationship the bufadienolides structure and antineoplastic activity. Natural bufadienolide exhibited a potent cytotoxicity against PLC cell than their isomers and related compounds. For cytotoxycities against PLC cell, 16 compounds showed comparatively potent active (IC50 10^<-4>〜10^<-3>μg/ml)(Table 1). Hellebrigenin having 19-CHO group was most active (IC50 1.6×10^<-4>μg/ml). Next, bufalin-3-acetate, gamabufotalin, bufalin, scillarenin, bufotalin, telocinobufagin et. al showed strong cytotoxicities. The 14 β-hydroxy derivatives showed the higher activites than 14β, 15β-epoxy, 14α, 15α-epoxy and α-pyrone ring opening compounds. Therefore, we classified the bufadienolides, cardenolides and their derivatives into the five groups, A〜E, and discussed relationship between structure and activity on each group. The most important factors were found to be the α-pyrone ring, 14β-OH or 14β, 15β-epoxy, 19-CHO, 11α-OH and 16β-OAc groups (Fig. 2 and 3). We also found that the D-ring structure and 3-substituent structure contributed much to the activites for PLC cell. On the base of activities, we attempted to approch to structure/activity correlation by computable calculation. We first calculated to find possible pharmacophores given 16 compounds having potent cytotoxicity against PLC and resistance PLC cell using DISCO(Distance Comparisions). A pharmacophore moldel was established, as indicated in Fig. 4. Then, we determained the structure/activity relationship 80 compounds by CoMFA (Comparative Molecular Field Analysis). Finally, we discussed about the antineoplastic activities of bufadienolides such as cell line of the HepG2, HeLa-S3, KB, PC-3 and P388 cells, including some resistance cell in the comparion with PLC/PRF/5 activities. As the interesting antineoplastic compounds, scillarenin (5), bufalin-3-O-[N-(tert-butoxycarbonyl)hydraidolsuccinate (8), bufalin-3-suberate (10) and its CH3 ester (15), Δ^2/Δ^3-bufalin (81), and Δ^<14>-bufalin (45) were found (Table 2). Also, in the difference of activities of bufadienolides between the original cancer cells and the Resistance cancer cells, the drastic change was not fouund.

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The conidia of a plant anthracnose fungus pathogenic to strawberry, Colletotrichum fragariae, germinate in water depending on the population, where the germination is inhibited under crowded conditions. The inhibition is considered to be caused by some germination-inhibiting substances exuded from conidia. Such germination self-inhibitors were searched for in the extract of the fungal culture and several active compounds were isolated. They were (E)- and (Z)-3-ethylidene-1,3-dihydroindol-2-one(1 and 2), (2R)-(3-indolyl) propionic acid(3), (2E,4E)-2,4-hexadienal(4) and two new compounds named colletofragarones Al(5) and A2(6). The structures of colletofragarones were determined spectroscopically to be tricyclic compounds with the molecular formulae of C22H26O6, respectively, which characteristically have a 10-membered ring lactone and a conjugated triene-containing side chain. The biosynthesis study using ^<13>C-labeled compounds revealed that all of the carbon atoms in colletofragarone were of acetate origin. HPLC analysis indicated that the compounds 1, 2, 5 and 6 were actually exuded from the conidia in water under crowded conditions, suggesting that these compounds control the conidial germination of this fungi.

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On the last Symposium in 1995, we reported the isolation and structure elucidation of several immunosuppressive components, new α-pyrones named multiforisins A〜F from Gelasinospora multiforis and new 2-furanone derivatives tentatively named GK-2〜-5 (Chart1) from G.kobi except for GK-4. We report now the structure of the main immunosuppressive component of G.kobi, GK-4, and five immunosuppressive components tentatively named DG-1〜-5, which have newly been isolated from Diplogelasinospora grovesii, Chemical and spectroscopic investigation including application of the new Mosher's method indicated that the structure of GK-4 with absolute configuration was expressed to be 1, which was supposed to be a new macrocyclic sesterterpenetriol(see Chart 5). The structure of the main immunosuppressive principle of D.grovesii, DG-2, was elucidated to be a new a-pyrone derivative 9, mainly on the basis of its spectroscopic data. Other immunosuppressive principles of the fungus, DG-1, -3, -4, and -5 were also deduced to be a dimeric dilactone formed from two molecules of 3-hydroxybutyric acid (8), 10-epi-isomer of colletodiol(10), colletodiol(11), and isosclerone (12), respectively (see Chart 6,7,8).Among them,11 and 12 had been formerly isolated from Colletotrichum capsici and Sclerotinia sclerotiorum, respectively. Immunosuppressive activities evaluated with their IC_<50> values against mitogen-induced proliferations of mouse spleen lymphocytes of all of these fungal immunosuppressive components GK-2〜-5, DG-1〜-5 and their structure-immunosuppressive activity relationships were also discussed. We proposed to name GK-4 kobiin, GK-2, -3, and -5 kobifuranones A, B,and C, and DG-2 grovesiin, respectively.

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In our ongoing search for biologically active metabolites from cyanobacteria, we have found that Oscillatoria agardhii (NIES-204, 205) are a rich source of novel protease inhibitors. Here we mainly report the isolation and structure elucidation of agardhipeptins A (1) and B (2), and aeruginosin 205-A (3), though we succeeded in the isolation of several types of protease inhibitors from O. agardhii. O. agardhii (NIES-204, 205) were obtained from the NIES-collection (Microbial Culture Collection, the National Institute for Environmental Studies, Japan) and cultured in 10L glass bottles containing CB medium. The 80% MeOH extracts of lyophilized cyanobacteria were subjected to solvent partitions, ODS column chromatography followed by ODS HPLC to yield 1-3. Molecular formulae of 1 and 2 were determined as C_<43>H_<51>N_<11O>_7 and C_<57>H_<69>N_<110>_8, respectively, by HRFAB-MS and NMR data. Amino acid analyses of the hydrolyzates of 1 and 2 gave Pro, His, Leu, Trp, and Gly for 1, and Ala, Val, Leu, Pro, and Trp for 2. Interpretation of HMBC and NOESY spectra revealed the structure of 1 and 2. Absolute stereochemistry of amino acid residues in 1 and 2 was determined to be L-form by HPLC analysis of the acid hydrolyzates derivatized with Marfey's reagent. Agardhipeptin A (1) inhibited plasmin with an IC_<50> of 65μg/mL but agardhipeptin B (2) had no plasmin inhibitory activity. Molecular formula of 3 was decided to be C_<34>H_<53>N_<6O>_12ClS from FAB-MS and NMR data. A detailed analysis of the 2D NMR data, including ^1H-^1H COSY, HMQC, HMBC, NOESY and HOHAHA revealed the presence of Pla sulfate (phenyllactic acid 2-O-sulfate), Hleu (3-hydroxyleucine), Ccoi (2-carboxy-6-chlorooctahydroindole), Agma (agmatine), and xylopyranose. The structure of 3 was elucidated by the interpretation of HMBC and NOESY correlations. Absolute stereochemistry of Pla sulfate and xylopyranose was determined to be L- and D-form by HPLC analysis of menthyl ester of acid hydrolyzate and GC analysis of trifluoroacetyl isopropyl derivative, respectively. The absolute configuration of Ccoi and Hleu remains to be defined. Aeruginosin 205-A (3) inhibited trypsin and thrombin with the IC_<50> of 0.07 and 1.5μg/ml, respectively.

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As a part of our search for functional molecules in edible fungi, we report here the characterization and structural determination of complex lipids (1-6) including new type of glycosyl phosphosphingolipids in Hypsizigus marmoreus (Bunashimeji, a mushroom) and Pleurotus citrinopileatus (Nireouma, a mushroom) by B/E constant linked scan fast atom bombardment (FAB) mass spectrometry. The structures of complex lipids were determined to be O-(1,2-di-O-linoleoylglycero-3-phosphoryl)ethanolamine (1), (4E,8E)-N-2-hydroxyhexadecanoyl-1-O-β-glucopyranosyl-9-methyl-C18-sphinga-4,8-dienine (2), phosphodihexose N-(2-hydroxyoctadecanoyl)-4-hydroxy-C18-sphinganine (3), phosphodihexose N-(2-hydroxyhexadecanoyl)-4-hydroxy-C18-sphinganine (4), phosphodihexose N-(2-hydroxytetracosanoyl)-4-hydroxy-C18-sphinganine (5), and phosphodihexose N-(2-hydroxydocosanoyl)-4-hydroxy-C18-sphinganine (6). In particular, location of the double bonds in the long-chain base of 1 and in the fatty acid of 2 were clearly determined by the B/E constant linked scan method. The structure of cerebroside having a long-chain base of 9-methyl-C18-sphinga-4,8-dienine could be determined in general by the presence of characteristic fragment ions of [C19-sphingadienine + H- H_2O]^+ at m/z 276 and [C19-sphingadienine + H]^+ at m/z 294, and the fatty acid carbon number could be calculated from the characteristic fragment ion of [ceramide -180]^+ ([MH - GlcOH - 180]^+) in positive ion mode FAB mass spectrometry. In the structural determination of 3-6, the ions of m/z 421 and 720 in the negative ion mode analyses are assigned to be characteristic of phosphodihexose and phytosphingosine containing phosphodihexose, respectively. This is a useful methodology for the structural determination of other unstable natural products such as lipids.

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For the determination of 3D molecular structure and absolute configuration (R/S) of natural organic compounds, an X-ray crystallography method is the most powerful technique. However, the method can not be used if a compound does not crystallize. A general technique for determination of the stereochemistry is the use of chemical reaction to decompose into small fragments for relating with the known compounds. If the both methods can not work, the NMR Distance Geometry Method, its appearance some ten years ago, that is an epoch-making method of analyzing the 3D molecular structure of protein in solution, can be used as an important method. The NMR Distance Geometry Method may be broadly classified into two optimization methods. The first is the method of taking three dimensional position relationships of each atom constituting the molecule as xyz variables of the Descartes coordinate system, is generally used. The second is the method of taking only the angle of rotation (dihedral angle) around the chemical bond in a dihedral angle coordinate system, in which the bond length of the bond between atoms in the molecule and bond angle are fixed, as the variable. The latter is the DADAS method (Distance Analysis in Dihedral Angle Space). This is used in the MolSkop as the DADAS90 program for calculations of molecular structure, using the constraint data of distance between atoms in a molecule obtained from the NMR data, the dihedral angle constraint data from the J spin-spin coupling constants. The DADAS90 can determine a stable molecular structure from the aspects of stereochemistry, thanks to the retention of chirality (R/S) even during the calculations for the optimum structure. The new technique, stereochemistry structure determination method used by computer, with the MolSkop system and a Chiral program that newly developed, applied to determine the stereochemistry of three cyclic natural organic compounds, cyclothiazomycin, sekothrixide and promothiocin. This technique can be also used for analysis of stereo structures of natural organic compounds, R/S determination, conformation analysis, and for steric positioning analysis of composite molecules.

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Camphorsultam (1) is very useful for both the enantioresolution of carboxylic acids and the determination of their absolute configurations by X-ray structure analysis. The camphorsultam amide method has the following advantages; i) a diastereomeric mixture of camphorsultam amides is easily separable by HPLC on silica gel; ii) camphorsultam amides generally crystallize as large prismatic crystals suitable for X-ray diffraction; iii) the absolute configurations of the amides obtained can be determined by the X-ray Bijvoet method using the anomalous dispersion effect of a sulfur atom and also by the internal reference method using the camphor part with known absolute configuration; iv) both the optically active carboxylic acid and camphorsultam can be easily recovered. This camphorsultam method can be extended to the cases of alcohols or amines if proper linking moieties connecting a camphorsultam group with alcohols or amines are designed, as shown in 6. We designed chiral phthalic acid amide (7) and it was found that the chiral phthalic acid amide method was effective for the enantioresolution and the determination of the absolute configurations of alcohols and amines. By this method, the obtained esters or amides were separated into enantiomers by HPLC on silica gel, and the crystalline esters or amides were subjected to X-ray structure analysis to determine their absolute configurations. In the chiral phthalic acid amide method, however, we have had difficulties like a long retention time in the chromatographic separation, because a diastereomeric mixture of some esters or amides is less soluble in the organic solvent. In order to solve this problem, the chiral dichlorophthalic acid amide method was developed. Chiral dichlorophthalic acid amide (11) enabled us to have a shorter retention time and a better separation in HPLC. These camphorsultam methods are thus very useful for the enantioresolution of carboxylic acids, alcohols and amines, and also for the determination of their absolute configurations.

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New phytotoxic metabolites, named pyriculone and zingibertriol, were isolated and their structures were elucidated by spectroscopic as well as chemical reactions. Pyriculone was successfully prepared by DDQ oxidation of pyriculol and found to be more toxic to the rice plant than pyriculol. Its production by the rice blast disease fungus, Pyricularia oryzae Cavara (Magnaporhte grisea (Hebert) Barr), had tendency to observed by the fresh field isolate, while some strain being stocked in laboratory could not be observed for the production of pyriculone. Zingibertriol is a first sesquiterpenene found among the blast disease fungi. The productivity of which was found to be restricted among the blast fungus, P. zingiberi, from mioga. The aromatics (phenylpropenes and indene) as well as aliphatics (limonene and β-ionone) were found to be metabolized by the blast disease fungi to dihydroxylated as well as monohydroxylated metabolites. The carbonyls (indanone and β-ionone) were also found to be reduced by the fungi. The stereospecificity of the conversion were different among the substrates.

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Liverworts (Hepaticae) are known to be rich sources of both terpenoids and aromatic compounds with biological activities. The Hepaticae occasionally produce their own peculiar phenolic bis(bibenzyl) derivatives. In the course of our systematic investigation of the chemical constituents of liverworts belonging to the Blasiaceae, we isolated four novel cyclic bis(bibenzyl) dimers, pusilatins A-D (1-4) and two depsides (16, 17) together with nine phenolic compounds (5, 6, 9-15) from Blasia pusilla L. and a novel optically active cyclic bibenzyl-dihydrophenanthrene derivative.(+)-cavicularin (18) from Cavicularia densa Steph. Their structures were characterized by a combination of spectroscopy, X-ray crtstallogarphic analysis and chemical evidences. Further investigation of the phenolic constituents of the thallic liverworts, we isolated a new cyclic bis(bibenzyl) dimer, pusilatin E (8) from Riccardia multifida (L.) S. Gray subsp. decrescens (Steph.) Furuki which was easily derived from riccardin A (7) by coupling reaction using Mn(OAc)_3 ・2H_2O. Previously, we isolated pusilatin D from B. pusilla and proposed the structure (4b) linked by ether C12-O-C1''' bond on the basis of comparison of the ^<13>C NMR data of its acetate with those of the related acethyl compounds. However, the structure (4b) was revised to 4a possessing C10'-O-C11" linkage on the basis of the HMBC analysis. (+)-Cavicularin (18) might be formed by intramolecular phenolic oxidative coupling between 3' and 10' position of riccardin C. On the other hand, riccardin C dimers, pusilatins A-D (1-4) might be biosynthesized by intermolecular coupling between two molecules of riccardin C. These bibenzyl derivatives are significant chemical markers of the Blasiaceae. Pusilatins B (2) and C (3) showed DNA polymerase β inhibitory activity.

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In the course of our study of the phenolic compornents of Thymelaeaceous plant, four new C-7/C-2", C-8/C-3" biflavonoids, daphnodorin E-H (1-4) and a spirobiflavonoid, daphnodorin I (5), were isolated from the roots of Daphne odora together with several know compounds. The structure of 1 including absolute configuration were established 25, 2"R, 3"R from spectral means. The compound 2 was decided to be a stereoisomer of 1 by comparison of spectral data with 1. The plane structures of 3 and 4 were deduced to be 3-hydroxy derivates of 1 and 2, respectively, from spectral data. The absolute configuration of 3 and 4 were determined by comparison of their CD spectra with those of 1 and 2 and by modified Moshe's method. The structure of 5 including absolute configuration was determined by chemical transformation of 5 into daphnodorin B (19) and by comparison of spectral data with genkwanol A (18).

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The family Guttiferae includes about 50 genera and nearly 1000 species. Because many species in this pantropical family have been used as traditional medicine, Guttiferaeous plants are important natural resources to search for biologically active principles. The family is well-known to be an abundant container of prenylated xanthones, biflavonoids, coumarins and benzophenones. Recently, various bioactivities of xanthones and coumarins have been reported. From this stand point of view, we investigated the chemical constituents in Guttiferaeous plants along with their bioactivity. Chemical investigation Chemical constituents in Garcinia (5 species), Calophyllum (2), Mammea (1), Cratoxylum (1) and Harungana (1) species resulted in isolation eighty-five xanthones, five benzophenones, four anthraquinones, three anthranones, nine flavonoids and two coumarin derivatives, in addition to three benzophenone-xanthone dimers with a new skeleton. Among them, forty-nine isolates were new compounds. Anti-bacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) After the first screening for antibacterial activity of extracts prepared from Guttiferaeous plants anginst S. aureus NIHJ 209_p, we found the potent activity in the benzene extract of pericarps of Garcinia mangostana. The extract was bioassay-guidedly fractionated and purified to achieve some compounds with anti-MRSA activity. An active isolate, xanthone, α-mangostin (70), showed minimum inhibitory concentration (MIC) to be ranged from 1.57 to 12.5 μg/ml. Other xanthones isolated and characterized were determined their anti-MRSA activity. Rubraxanthone (78), which has a similar substitutional pattern as 70, from G. dioica, exhibited the highest activity against staphylococcal strains (MIC: 0.313-1.25μg/ml) among them and the activity was stronger than that of vancomycin (3.13-6.25μg/ml). On the other hand, benzophenone derivatives, xanthochymol (61) and garcinol (65) from the pericarps of G. subelliptica and G. purpurea, also showed the strong anti-MRSA activity (MIC: 3.13-12.5μg/ml). The concentration is nealy equal to that of vancomycin.

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Sanggenon A (2) isolated from Chinese crude drug "Sang-Bai-Pi" has a unique flavanone derivative having an isoprenyl group at the C-3 position and a hydroxyl group at the C-2 position in the molecule. However, 2D ^<13>C-^1H long-range shift correlation spectra (COLOC and HMBC) revealed the structure of sanggenon A to be not the formula (2) but to be the formula (2^1), in which the isoprenyl group and the hydroxyl group locate at the C-2 and C-3 positions, respectively. Sanggenon A trimethyl ether (2e) having a diketone structure derived by exhaustive methylation with dimethyl sulfate gave a phenazine derivative by treatment with o-phenylenediamine under acidic condition. This result indicated the trimethyl ether has a α-diketone partial structure. Furthermore, an analogous experiment of sanggenon A with o-phenylene-diamine affoded a phenazine derivative, indicating that a hemiketal group locates at the C-3 position in the molecule. Thus the formation of the phenazine derivative gave a conclusively evidence for revision of the structure of sanggenon A from the formula (2) to the formula (2^1). Diels-Alder type adducts having a sanggenon A type of skeleton have been also isolated from "Sang-Bai-Pi". Taking the revised structure of sanggenon A into the account, the structures of these Diels-Alder type adducts should be revised to new ones. Indeed, sanggenon C octamethyl ether (4'a) gave a phenazine derivative as in the case of sanggenon A trimethyl ether (2'e). The structures of sanggenon A type of compounds from Chinese crude drug "Sang-Bai-Pi" have thus been revised.

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In our previous studies, we elucidated the structure of the unstable yellow precursor of carthamin (5). The elucidated structure of 5 suggested that it was biogenetically derived from safflower yellow B (3) in the florets of safflower. The transformation of 3 with cultured safflower cells was investigated to prove this hypothesis. Safflower Yellow B was transformed to red carthamin (1) and safflomin-A (2) in the safflower cells cultured on the Murashige-Skoog medium which lacks NH_4NO_3 and KH_2PO_4. The transfomation of 3 by the crude enzyme extracted from the above safflower cells, gave 2 and 6 in good yields. The compound 6 was unstable and easily converted to precursor (5) by the addition of glyoxylic acid. Actually, a very small amount of glyoxilic acid was detected in the safflower cell. Further, the treatment of 5 with the cell-free homogenate of culltured safflower cells gave 1 quantitatively. From these results, it was confirmed that 1 was biogenetically derived from 3 in the safflower cell by way of compound 6 and 5. Further, it was also confirmed that the treatment of 3 in the concentrated phosphate buffer solution, gave 2 and 6 almost quantitatively. It may be found the convenient method to produce 1 from 3 in the application of this transformation.

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As marine microorganisms live in different environment and are seemed to have different metabolic systems from those of terrestrial ones, they are likely to produce new chemical substances. In the course of our screening for novel compounds by using an HPLC/MS with a photo diode array detector, we found novel alkaloids, Ulbactins Al (3), A2 (4), B (5) and enantiomer of Aerugine (1) which were produced by Vibrio sp. B-93, as well as Ulbactins C1 (7) and C2 (8) which were produced by Alteromonas sp. D-12. Vibrio sp. B-93 and Alteromonas sp. D-12 were also found to produce known compounds, Yersiniabactin (2) and Aerugine (6), respectively. The structure of 1-8 were elucidated on the basis of MS, UV, IR, 1^H- and ^<13>C-NMR spectroscopic studies. 1 and 6 were identified to he (-)-(S)-Aerugine and (+)-(R)-Aerugine, respectively, based on the comparison of those specific rotations with synthetic ones. 2 was found to be one of the diastereomers of Yersiniabactin (isomer II). All of the UV spectra of 3-5, 7 and 8 showed the similar pattern, which attributed to 2-(o-hydroxyphenyl)-2-thiazoline moiety. 3 and 4 were suggested to have additional one thiazolidin ring with hydroxymethyl by 1^H-1^H COSY and HSQC, allowing us to establish the structures shown in Fig. 1. 5 was found to have 2-(2-(o-hydroxyphenyl)-2-thiazoline-4-yl)thiazolidine, which was the same partial structure as 3 and 4. An extra C_5H_8O_2 unit was considered to build bicyclo-ring system with thiazolidin through amide bond, which was supported by IR and 2D-NMR. 7 and 8 had two thiazolidin rings, 4''' position of which was attached by 2-amino-1,3-propanediol residue through amide bond. 3, 4, 7 and 8 tended to racemize in CDCl_3", solutions.

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The toxic strain of freshwater cyanobacterium(blue-green algae) Oscillatoria agardhii forms waterblooms in lakes and drinking water reservoirs, and produces heptacyclic peptide hepatotoxins named microcystins. During investigations of cyclic peptide toxins of the toxic strain of O. agardhii, we found a novel chymotrypsin-inhibiting cyclic decapeptide containing (3a-cis)-1,2,3,3a,8,8a-hexahydro-3a-(3-methyl-2-butenyl)-pyrrolo[2,3-b]indol-2-carboxylic acid (oscillatoric acid, Osc), and a novel cyclic peptide (oscillamide H). We now describe the isolation and structure elucidation of oscillatorin(Fig. 1.) and oscillamide H(Fig. 4.). The extract from freeze-dried cells(99.1g) of O. agardhii(NIES-610) with chloroform-methanol-water(1:3:1, v/v) was dried at reduced pressure, and was suspended with 5% acetic acid aqueous solution. The suspension was filtered. The filtrate inhibited chymotrypsin potently. The inhibitor was fractionated by solid-phase extraction using ODS cartridge(Sep-Pak ODS). The inhibitor fraction was further purified by reverse-phase HPLC(Purosphare ODS, 20mm I.D.×25cm, flow rate, 6.0ml/min) with methanol(75%) containing 0.05M phosphate (pH 3.0). The yield of the inhibitorin, oscillatorin, was 8.5mg. The inhibitor is a colorless amorphous solid: λmax(H_2O) 283nm(logε 3.4). The [M+H]^+ ion of the positive HRFABMS using glycerol as a matrix was observed at m/z 1240.6456. From the results, the molecular formula of oscillatorin was established to be C_<60>H_<85>O_<14>N_<15>(Calcd for M+H: 1240.6478, Δ-2.2mmu). The extensive NMR analyses of 1^H-1^H COSY and HMBC spectra revealed an abnormal amino acid residue which was (3a-cis)-1,2,3,3a,8,8a-hexahydro-3a-(3-methyl-2-butenyl)-pyrrolo[2,3-b]indol-2-carboxylic acid (oscillatoric acid, Osc)(Fig. 2.). The sequence of oscillatorin was mostly deduced by HMBC correlations from N-H to C=O (Fig. 3.). Oscillamide H was isolated from another strain of O. agardhii(NIES-595) by the same procedure. Oscillamide H is a colorless amorphous solid: λmax(H_2O) 278nm. The [M+H]^+ ion of the positive HRFABMS using glycerol as a matrix was observed at m/z 930.4891. From the results, the molecular formula of oscillamide H was established to be C_<60>H_<85>O_<14>N_<15>(Calcd for M+H: 930.4976, Δ-8.5mmu). By the extensive NMR analyses, the structure of oscillamide H(Fig. 4. and Fig. 5.) was established.

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Tumor cells that survive initial chemotherapy often increase their resistance to not only the original drug but also to other unrelated drugs. The phenomenon, termed multi-drug resistance (MDR), results in the ultimate failure of chemotherapy. Dendroamide A (1), one of three new bistratamide-type cyclic hexapeptides containing thiazole (Tzl) and methyloxazole (mOzl) residues, from the terrestrial blue-green alga Stigonema dendroideum Fremy, exhibits multi-drug-resistance reversing activity. The gross structure of the three compounds, dendroamide A-C (1-3), were determined by NMR and mass spectral analysis. Their absolute stereochemistries were determined by Marfey and chiral GC/MS analysis of derivatives formed from acid hydrolysis of the intact and ozonized peptides. As indicated in Figure 1, dendroamide A was more potent than verapamil in its ability to increase the accumulation of [^3H]vinblastine in P-glycoprotein-overexpressing breast carcinoma (MCF-7/ADR) cells, reaching 〜750% of control at 5μM and maximal effect at 20μM. And as indicated in Figure 2A, MCF-7/ADR cells were killed much more effectively by combinations of daunomycin or actinomycin D and dendroamide A than with identical doses of the drugs alone. Dendroamide A concentrations of 0.6μM or greater increased the sensitivity of MCF-7/ADR cells to vinblastine (Figure 2B) to that of parental MCF-7 cells, indicating that this compound is able to completely overcome P-glycoprotein-mediated MDR in this model system. The abilities of dendroamide A are characteristic of MDR-reversing agents which act as antagonists for drug transport by P-glyciprotein.

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Marine dinoflagellates of the genus Amphidinium, which are symbiotically associated with the marine flatworms Amphiscolops sp., have been revealed to produce chemically interesting and biologically significant metabolites, such as amphidinolides. To search for further unique compounds from dinoflagellates Amphidinium sp., we studied another strain of Amphidinium sp. (Y-52), which was isolated from host flatworm Pseudaphanostoma luteocoloris, and isolated a new polyhydroxyl compound, luteophanol A (1), consisting of C_<57> linear aliphatic chain possessing one sulfate ester and two tetrahydropyran rings. The host invertebrate P. luteocoloris was collected at Zanpa, Okinawa, and the symbiotic dinoflagellate Amphidinium sp. (Y-52) was mass cultured unialgally at 25℃ for 2 weeks in sea water medium enriched with 1% ES supplement. The cultured alga was harvested by centrifugation and extracted with MeOH-toluene (3:1). The BuOH-soluble fraction was purified with gel filteration and reversed-phase MPLC affoded luteophanol A (1, 0.002 %, wet weight). The molecular formula of 1, C_<60>H_<102>O_<25>S, was established by ESI-MS and HRFAB-MS. The planar structure of 1 was deduced by detailed analysis of the ^1H and ^<13>C NMR spectra aided with 2D NMR techniques including DQF COSY, HSQC, HSQC-HOHAHA, PFG HMBC experiments. Luteophanol A (1) showed no antifungal activity, but exhibited weak antimicrobial activity against Gram-positive bacteria.

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As a part of our study on larval settlement (attachment and metamorphosis) of sessile marine invertebrates, we are searching the settlement inducing and inhibiting substances from various marine organisms. Here we present our new findings on the metamorphosis inducing compounds on ascidian Halocynthia roretzi larvae and the antifoulants against barnacle Balanus amphitrite larvae. Metamorphisis of ascidian larvae has been thought to be induced by some chemical cues. a) We found the conditioned medium with the ascidian H. roretzi larvae induced the larval metamorphosis of the conspecies. The metamorphosis-inducer has been isolated, and the strucutre was identified as lumichrome (1). b) From various marine organisms, we have isolated almost 40 compounds including 24 new ones as the metamorphosis-inducers on the ascidian. For example, from a marine sponge Callyspongia sp. (to be), ten acyclic polyacetylene derivatives were obtained (e.g. 2 and 3). A marine sponge Stelletta sp. afforded a norsesequiterpenamide with two guanidium moieties, stellettadine A (4), the absolute stereochemistry of which was determined by degradation into 2-methylglutaric acid. From the marine sponge Anthosigmella aff. raromicrosclera, two pipecolate derivatives (5 and 6) were obtained. On MeOH extracts of more than 300 marine invertebrates, we have performed antifouling screening against barnacle larvae (B. amphitrite). Following the screening result, we attempted the isolation and structure determination of antifoulants to afford more than 60 compounds including almost 30 new ones. a) From the marine sponge Acanthella cavernosa, 26 antifoulants were obtained. Kalihinane-type compounds with isocyano, formamino, and/or isothiocyano group(s) showed prominent antifouling activities, some of which were stronger than that of cupric sulfate. Steroids with epidioxy group (e.g. 7) delayed the timing of larval settlement. b) Nudibranchs of the family Phillidiidae afforded 11 sesquiterpenoid antifoulants (e.g. 8). c) From the marine sponge Pseudoceratina purpurea, seven antifoulants (9 - 15) including a unique cyanoformamide derivative (14) were obtained. d) The marine sponge Agelas mauritiana afforded oroidin and its unique dimer (16). e) From a marine sponge Axinyssa sp., three new sesquiterpenoids with dichloromethyleneamino group (17-19) were obtained. f) A soft coral Dendronephthya sp. afforded four new D-seco-steroids (e.g. 20). These research results will become important on understanding the more concise mechanism of larval settlement and for development of commercial antifouling agents.

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Two new marine prostanoids, preclavulone lactone I (1) [C_<20>H_<28>O_3, [α]_D-168.0°(CHCl_3)] and II (2) [C_<20>H_<28>O_3, [α]_D-110.0°(CHCl_3)], were isolated from the Okinawan soft coral, Clavularia viridis. Their plane structures were elucidated based on spectroscopic analysis involving two-dimensional NMR spectra (^1H-1^H COSY, ^<13>C-^1H COSY, HMBC). The stereostructure of 1 was determined from the results of the total synthesis. The synthesis was performed starting from (S)-(-)-malic acid via a key intermediate 8, which possesses a trans configuration of the iodomethyl group and ω side chain. (4S, 8R, 12S)-1 was synthesized by the reaction of 8 with acetylenic compound 14 having an S-configration followed by modification of the functional groups. (4R, 8R, 12S)-1 was also synthesized similarly. The spectral data of the synthetic (4S, 8R, 12S)-1 were identical with those of natural preclavulone lactone I (1), but the sign of the optical rotation of the synthetic compound was shown to be different from that of natural 1, demonstrating the absolute stereochemistry of (4R, 8S, 12R) for 1. Preclavulone lactone I (1) is regarded as an important intermediate in the biosynthesis from preclavulone A (5) to clavulones.

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A new amino acid dysiherbaine (1), was isolated from a Micronesian sponge Dysidea herbacea. The structure was determined spectroscopically by using FABMS, ESIMS, FABMS/CID/MS, and one- and two-dimensional NMR experiments of 1 and its dimethyl derivative 2 to be a novel di-amino di-carboxylic acid, which consisted of a cis fused-hexahydrofuro[3,2-b] pyran ring substituted with a 3-[2-amino propanoic acid] side chain. The relative configuration of the bicyclic portion of 1 was determined by 3^J_<H.H>analysis and deference NOE experiments, and that of acyclic side chain was assigned by additional ^<2.3>J_<C.H>analysis, measured by conventional proton NMR and newer hetero half-filtered TOCSY (HETLOC) and phase sensitive HMBC experiments. A systemic administration of 1 induced typical neurotoxic symptoms in mice which were reminiscent of neuroexcitatory amino acids, such as domoic acid. Dysiherbaine inhibited bindings of [^3H]kainic acid (KA) and [^3H]1-amino-3-hydroxy-5-methyl-4-isoxazol-propionic acid (AMPA), but not not [^3H]CGS-19755, an NMDA receptor agonist, to the rat brain synaptic membranes, suggesting that 1 is a specific agonist of KA/AMPA class glutamate receptors in the central nervous system.

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During our studies on bioactive substances from Okinawan marine organisms, we recently examined extracts of the sponge Hippospongia sp., and isolated a novel acetylenic compound, taurospongin A (1). The monomethyl ester derivative (2), C_<41>H_<73>O_9NS, derived from 1, was revealed to be an acetylenic compound consisting of a taurine and two (trihydroxyl and unsaturated) fatty acids on the basis of analysis of the spectral data including several types of 2D NMR spectra (^1H-^1H gradient COSY, HMQC, decoupling HMBC, and gradient HMBC). Amino acid analysis of hydrolysis products of 1 showed the presence of taurine. Two fragments, trihydroxylamide (3) and unsaturated fatty acid methyl ester (4), were obtained by methanolysis of 2 to elucidate the lengths of the methylene chains contained in 3 and 4, respectively. Relative stereochemistry of 1,3-diol moiety of 3 was shown as syn from the ^<13>C NMR data of the acetonide (5) prepared from 3 by applying the Rychnovsky rule. Studies for determining the absolute stereochemistry of 1 are currently in progress by preparation of two possible diasteromers of 5. Taurospongin A (1) showed inhibitory activity against c-erbB-2 Kinase (IC_<50> 28μg/mL), and exibited no cytotoxicity.

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Yessotoxin (1, YTX) was isolated as one of the causative toxins of diarrhetic shellfish poisoning (DSP). Its planar structure was elucidated by using NMR spectroscopies and by negative FAB MS/MS. Structurally, YTX is closely related to brevetoxin B and ciguatoxin 4B in having a ladder-shape polycyclic ether skeleton and an unsaturated side chain. In view of the increasing interest in the molecular mechanism of the action of polycyclic ether toxins on sodium channels or cell membranes, we elucidated the stereostructure of YTX to compare with that of brevetoxin B and searched for its analogs. YTX and its analogs were isolated from the digestive glands of DSP-infested scallops, Patinopecten yessoensis, collected in 1993 in Mutsu Bay, Japan, following the previously reported procedures, modifying only the final step of purification; a Develosil ODS-7 column (Nomura Chemicals) with MeOH:MeCN:H_2O (4:2:7) for 1, and MeOH: MeCN:H2O (4:2:9) for 2 and 3 by Yasumoto and co-workers. The relative stereochemistry of 1, 2, and 3 was determined by NOE experiments. We have been developing new chiral anisotropic reagents, such as 2NMA, 9ATMA, 2ATMA, and PGME, which make it possible to determine the absolute configurations of organic compounds by NMR. Among these reagents, 2NMA [methoxy-(2-naphthyl)acetic acid] is the most convenient for secondary alcohols in that (i) the anisotropy of its naphthalene ring is much greater than that of the benzene ring of MTPA (conventional modified Mosher's method), giving greater Δδ(δR- δS) values, and (ii) the enantiomerically pure reagent is easily prepared in substantial amounts. We reported here determination of the absolute configuration of YTX by means of 2NMA. Attempted esterification at 32-OH of YTX with 2NMA was unsuccessful because of its axial orientation. Therefore, we focused on introducing 2NMA at 4-position, which is blocked by a sulfate group. Solvolysis of YTX was facilitated by heating a solution of YTX in pyridine-dioxane (1:1, 120℃, 3h) to give bisdesulfated YTX (5: DS-YTX). When DS-YTX was treated with an excess of acetic anhydride in pyridine at 0℃ for 45 min, a mixture of recovered DS-YTX, monoacetate (6), and diacetate, separable by HPLC, was obtained. Monoacetate 6 (600μg) having free OH at C-4 was divided in two portions, and each portion was treated with (R) and (S)-2NMA, EDC, DMAP, and NEt_3 in chloroform for 43.5 h at room temperature. Each product was purified by HPLC to give pure (R) (7) and (S)-2NMA esters (8), respectively. By analyzing the COSY and HOHAHA spectra of 7 and 8, much higher Δδvalues [δR(7)-δS(8)] of the protons with much greater magnitude than those anticipated for MTPA were obtained (Fig. 4). From these results, the absolute configuration of the hydroxy group at C-4 was determined to be S, and, since the relative stereochemistry of the other asymmetric centers has been established, the present finding led to the absolute configuration of YTX as shown in 1.

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Ciguatoxin (1) is the principal toxin of the seafood poisoning, which is termed ciguatera and is prevalent in tropical and subtropical areas. The toxin structure including the relative stereochemistry was determined using NMR methods by our group. However, configurations at C2 and C5 remained undetermined due to the extrmely low amount of 1. In order to determine the absolute configuration at C2, we protected 1,2-diol of CTX and then cleaved the 3,4-double bond by osmium oxidation followed by NaBH_4 reduction. The resultant alcohol was esterified with a chiral fluorometric reagent (S)-(+)-2-tert-butyl-2-methyl-1,3-benzodioxo-le-4-carboxylic acid and compared with synthetic references on two HPLC systems. Starting with only 5μg of 1 the absolute configuration at C2 was successfully determined to be S. To determine the absolute configuration at C5, 100μg of CTX4A was reacted with ρ-bromobenzoyl chloride to yield tris-bromobenzoyl derivative. The CD spectrum of CTX4A-tris-ρ-bromobenzoate was comparable with that of ρ-bromobenzoate derived from a synthetic (5R) AB fragment. This result indicated that the absolute configuration at C5 is R. Consequently, determining (2S), (5R) configuration allowed us to assign whole stereostructure of 1. A new ciguatoxin analog, CTX2A1 (2), isolated from moray eels was deduced to have the molecular formula C_<57>H_<84>O_<18> from FABMS. The structure of 2 was elucidated to be CTX3C-2,3-diol by detailed analysis of NMR spectra. This is the first CTX analog to be oxidised in the A ring and is an additional example to show diverse metabolic modification of CTXs to occur in fish.

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In the course of our screening program for new antitumor antibiotics, the fermentation broth filtrate of Streptomyces sp. AJ9493 was found to produce active substance. The active compound, N1999A2, was purified by Diaion HP-20 column chromatography, ethyl acetate extraction, silica gel column chromatography, ODS column chromatography and preparative ODS HPLC. The methanolysis compound of N1999A2 was deduced to 1-naphthoate derivative by X ray crystal analysis and NMR assignments. The structure of N1999A2 was established by using variety of NMR technique, especially HMBC spectrum on several J values. N1999A2 was new enediyne antibiotic, similar to Neocarzinostatin chromophore (NCS-Chr). The major difference between N1999A2 and NCS-Chr was lack of the amino sugar in N1999A2, which was considered as the reason why N1999A2 was stable in spite of non-protein chromophore. N1999A2 was strongly inhibit the growth of tumor cell lines and bacteria. The IC_<50> of cytotoxicity to HCT-15 cell was 0.008nmol/ml.

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Pamamycins, a novel family of macrodiolides with MW's ranging from 593 to 663, are produced by Streptomyces alboniger IFO 12738. Pamamycin-607 (MW 607) has aerial mycelium-inducing activity in an aerial mycelium-less mutant of S. alboniger. We examined the structure-activity relationship of pamamycins in view of alkyl substit-uents, dimethylamino group and macrodiolide ring. Nine new pamamycin homologues with MWs 593, 621 and 635 were isolated using a combination of ODS and NH_2 HPLCs, and their structures determined by GC-MS. The aerial mycelium-inducing activity mostly was lost when R_3 or R_4 was changed from CH_3 to C_2H_5. Replacement of the other alkyl substituents, R_1, R_2 and R_5, caused a drop to 30-40% of the original activities. In consideration with CPK model, pamamycin-607 has the conformation that both R_3 and R_4 extend from one side of the molecule. Next, we prepared a desdimethylamino derivative, a ring-opened compound and two constituent hydroxy acids by chemically degrading pamamycin-607, and examined their aerial mycelium-inducing activity. Desdimethylamino derivative lost activity, indi-cating that dimethylamino group was indispensable for activity. The ring-opened com-pound showed about 1/2 the activity of pamamycin-607, whereas the larger hydroxy acid had only 1/30 of the activity and the smaller acid had no activity. Thus, the larger hydroxy acid part was the principal component responsible for activity and the smaller hydroxy acid part may help the activity of the larger hydroxy acid part. We found that Ca^<2+> regulates the aerial mycelium formation of many actinomycetes including S. alboniger and that Ca^<2+> signal modulators such as Ca^<2+> channel blockers and calmodulin inhibitors inhibit aerial mycelium formation of S. alboniger. Based on these observations, we examined the effects of pamamycin-607 on intracellular concen-tration of Ca^<2+>. As a result, pamamycin-607 induced a transient increase in the intracellular concentration of Ca^<2+> dose-dependently.

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Chloropeptin, a novel peptide antibiotic isolated from a culture broth of S treptomyces sp. WK-3419, is a potent inhibitor of the binding between gp120 envelope glycoprotein of HIV-1 and the CD4 receptor of HIV-1 on the surface of human T cells (IC_<50> 32nM by the ELIS A method). Chloropeptin also inhibits the cytopathic effect assayed in HIV-1-infected Molt-4 cells (IC_<50> 1.6μM) and the syncitium formation in Molt-4 as well as HIV-1-infected Molt-4 cells (IC_<50> 0.7μM). We now report the absolute configuration of chloropeptin, elucidated via a combination of NMR, degradative and molecular modeling techniques. The molecular formula was determined by HR-FAB-MS to be C_<61>H_<45>N_7O_<15>Cl_6. The ^1H- and ^<13>C-NMR spectra indicated the presence of six aromatic amino acids and an oxoaromatic acid. The complete connectivity of chloropeptin was subsequently established utilizing COSY, HMQC and HMBC spectroscopy. Acid hydrolysis furnished (D)-4-hydroxyphenylglycine and (D)-3,5-dichloro-4-hydroxyphenylglycine; their absolute configurations were determined by optical rotation and chiral TLC. The stereochemistry at C_α of residues, tyrosine, 3,5-dihydroxyphenylglycine and tryptophan, were determined using a combination of high-temperature molecular dynamics (MD) calculations, NOE data, and J values. Among a lot of conformers obtained from the MD calculations, one satisfying all of distance and dihedral constraints could be found in case that the absolute stereochemistry at C_α of residue, tyrosine, 3,5-dihydroxyphenylglycine and tryptophan, was assigned as L, D, and D configuration, respectively.

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The aromatic polyketide, actinorhodin, belongs to a class of benzoiso-chromanequinone (BIQ) antibiotics whose other examples include nanaomycins, frenolicins, and dihydrogranaticin. Streochemical control in pyran ring formation involved in BIQ biosynthesis was investigated in the actinorhodin producer, Streptomyces ceolicolor. The relevant genetic region (5.7kbp), the actVI of the act cluster (actinorhodin biosynthetic genes), was sequenced. Six open reading frames (ORFs) were identified: ORFB, ORFA, ORF1, ORF2, ORF3, ORF4. The deduced ORFs' products show some significant similarities with other known proteins: ORF1/b-hydroxybutyrylCoA dehydrogenase (Clostridium acetobacterium), ORF2 (4)/deoxyerythronolideB synthase (DEBS) enoyl reductase domain (Saccharopolyspora erythrea). These fmdings together with gene disruption studies on each ORF allow a scheme to be proposed (Scheme1) for the middle steps of the actinorhodin biosynthesis, leading to the BIQ chromophore. It was suggested that the actVI-ORF1 and ORF2/4 products reduce stereospecifically at C-3 and C-15, respectively. Functional assignment of actVI ORFs was carried out using a series of engineered derivatives of Streptomyces expression plasmid, pRM5 which carries the Polyketide Synthase (PKS) genes and the others involved in the early biosynthetic steps. The act cluster deficient strain, CH999 was transformed with the engineered plasmids followed by the chemical analysis of the metabolites. The transformants carrying pIJ5660 (pRM5 + actVI-ORF1) produced the yellow pigment 4, suggesting that actVI-ORF1 indeed functions as a reductase which determines the C-3 chiral centre. Present address: Faculty of Pharmaceutical Sciences, The University of Tokyo

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In the course of our studies on aroma formation mechanism in oolong tea (Camellia sinensis var. sinensis cvs. Shuixian and Maoxie), we have shown in molecular basis that most of alcoholic tea aroma are generated by endogenous enzymatic hydrolysis of glycosidic aroma precursors during tea manufac-turing. We have isolated and identified main alcoholic aroma precursors of geraniol, linalool, 2-phenyl-ethanol, benzyl alcohol, linalool oxides I and II (trans- and cis-linalool 3,6-oxides) and methyl salicylate as β-primeverosides (6-O-β-D-xylopyranosyl-β-D-glucopyranosides), guided by an enzymatic hydroly-sis (with the acetone powder prepared from cv. Yabukita) followed by GC and GC-MS analyses. Aroma precursors of linalool oxides III and IV (trans- and cis-linalool 3,7-oxides) and (Z)-3-hexenol were exceptionally isolated as 6-O-β-D-apiofuranosyl-β-D-glucopyranosides and a β-D-glucopyrano-side, respectively, from the oolong tea leaves. In cv. Yabukita for green tea benzyl alcohol was found to be stored as ca. 1:1 mixture of β-primeveroside and β-D-glucopyranoside. As a preliminary experiment we purified a new glycosidase β-primeverosidase) from fresh leaves of cv. Yabukita by ammonium sulfate precipitation followed by chromatographies with CM-Toyopearl and Mono S-HR. This glycosidase was found to be one of the main glycosidases involved in the alcoholic aroma formation in tea leaves. This is the second example of β-primeverosidases. Purification of the β-primeverosidase from fresh oolong tea leaves (cv. Shuixian) was also carried out in the same manner. The molecular weight was shown to be 61kDa and 60.3kDa by SDS-PAGE and TOFMS, respectively. Its pI and optimum temperature and pH are 9.5, 45℃ and 4,respectively. The enzyme is stable below 45℃, and between pH 3 to 5. These enzymic characteristics are quite similar to those of the β-primeverosidase from cv. Yabukita. The β-primeverosidase was found to hydrolyze a β-apiofuranoside into each constituent dissacharide and aglycon without further hydrolysis as effectively as in the cse of a β-primeveroside. Amounts of alcoholic aroma precursor and glycosidase activity in each part of the tea shoot (cvs. Yabukita and Izumi) were indirectly measured by means of a crude enzyme assay. The aroma precursors were abundant in young leaves and decreased as the leaf aged. Glycosidase activity also decreased as leaves aged, but was high in stems.

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We have recently demonstrated that several minor components which amount to only 2-3 wt % of the lipoteichoic acid (LTA) fraction from Enterococcus hirae ATCC 9790 possess cytokine-inducing activity, whereas the major component of over 90 wt % has no activity. In the present paper, we report an improved separation procedure and structural analysis of both biologically active and inactive components. In order to obtain sufficient active components for their structural study, their rapid and efficient separation was achieved by using batch- and stepwise elution from Octyl Sepharose followed by anion exchange membrane chromatography. The chemical structure of the inactive major component was first elucidated. The products from 47% hydrofluoric acid (HF)-hydrolysis were isolated and characterized by means of NMR and MS. The phosphodiester linkage between the hydrolysis products were determined by NMR spectra of alkali-treated inactive component without HF degradation. The structure was deduced as shown in 2, which coincides with the structure proposed by Fischer for LTA. This so-called "LTA structure" thus proved to be not responsible for the cytokine-inducing activity. The immuno-stimulating bioactive components from LTA contained rhamnose or mannose in addition to the common constituents found in the above bio-inactive component and were resistant to HF degradation. The chemical structure of these active components is now being examined in details.

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Detailed analysis of the ^1H and ^<13>C NMR spectra of three fructose-oligosaccharides (1-3) isolated from Asparagus officinalis L. (Liliaceae) was carried out. All compounds consist of one unit of glucose and three units of fructose. The ^1H NMR signals of the compounds are severely overlapped in the region of δ_H 3.45-3.85ppm and the circumstances are the same in the ^<13>C NMR. It was quite difficult to make assignments of the signals using conventional NMR methods. Hence, overcoming these signal-overlapping problems required some special NMR techniques to be applied as follows. Assignments of each signal of C-5 and C-6 of the fructose residues which could not be separated in 2D spectra were achieved by 1D INAPT (Insensitive Nuclei Assigned by Polarization Transfer) method. Since δ_C of C-6 of the fructoses are close each other and H-6 of the fructoses are also present in the crowded region, those protons could not be assigned by HMQC or HIMBC spectrum because of limited resolution in the carbon axis of these methods. However, the limited δ_C range (less than 4ppm) of C-6 methylenes of the compounds enabled high resolution in the carbon axis when a methylene-selected HSQC method, a modified version of E-HSQC, was applied. Finally, quaternary C-2 of fructoses gave no correlations with H-3 and H-4 of the residues by HMBC or INAPT. In this case, heteronuclear ^<13>C {^1H} NOE experiments in which each of the H-3 was selectively excited or saturated were effective for their assignments.

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We have already demonstrated the aromatic C-H/olefin coupling reaction with the aid of Pd (II) complex. These include the preparation of 4-bromodehydrotryptophan 2 as a building block for the synthesis of indole alkaloids. Here we would like to report an asymmetric synthesis of chanoclavine-I(3), an ergot alkaloid, which utilizes 4-bromodehydrotryptophan 2 via a Pd (II)-mediated C-H/olefin coupling reaction. Our design is outlined in scheme 2. Treatment of 4-bromoindole 1 with a stoichiometric amount of Pd (II) complex was expected to give intermediate 1A (via route A) without affecting the C-Br bond. Insertion of olefin to 1A followed by syn-β-H elimination was then anticipated to generate 4-bromodehydrotryptophan 2, a compound which was expected to be amenable to further functionalization. As shown in scheme 3, treatment of 4-bromoindole 1 and enamide 4 with Pd(OAc)_2 (1 equiv), NaHCO_3, and chloranil as an additive in 1,2-dichloroethane provided 4-bromodehydrotryptophan 2 in 87% yield. Having completed the synthesis of 4-bromodehydrotryptophan 2, we applied it to the syntesis of (-)-chanoclavine-I (3). Retrosynthetic analysis of (-)-chanoclavine-I (3) suggested that 4-bromodehydrotryptophan 2 might be a good precursor (scheme 5). The key step in the conversion of 4-bromodehydrotryptophan 2 into (-)-chanoclavine-I (3) involves the intramolecular cyclization of 12 (12→11). As shown in Scheme 6, attempts to cyclize 12 to 11 under radical conditions led to disappointing results. On the other hand, Heck cyclization of 12 gave the cyclized product 14. Ultimately, compound 18 was reached (see scheme 7). Not unexpectedly, a variety of attempts to achieve the deprotection of N-Boc group in 18 to complete the synthesis of (-)-chanoclavine-I (3) failed. An alternative route (scheme 8) to (-)-chanoclavine-I (3) required preparation of 22, a key intermediate in Oppolzer's synthesis of (±)-chanoclavine-I. As shown in scheme 8, 14 was converted to the key intermediate 22. This intermediate was further converted to (-)-chanoclavine-I (3) according to Oppolzer's route. As both synthetic sample and natural product have negative optical rotations, the absolute configuration of (-)-chanoclavine-I (3) has been assigned to be the one shown in scheme 8. We wish to add that no racemization occurred in our synthetic route.

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Erythrina Alkaloids are remarkable for their strong curare-like action appeared on oral administration. Total syntheses of those which have an aromatic group at ring D (aromatic Erythrina alkaloids) have been accomplished, but synthesis of so-called non-aromatic Erythrina alkaloids, all of which have an unsaturated lactone ring at ring D instead of an aromatic group, is still elusive. This study is based on a strategy involving the following three steps: 1) construction of the erythrinan skeleton having a furan ring through Diels-Alder reaction of furodioxopyrroline (2) with an activated butadienes, 2) conversion to oxirane carboxylic acid (11) via oxidative fission of the furan ring, 3) construction of the unsaturated δ-lactone by one carbon homologation followed by cyclization. The conversion of the γ-lactone (26) into β-erythroidine (1) is currently under progress. The preliminary experiments with model compounds will also be presented.

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We have studied the generation of cation radicals from some stannyl compounds by the oxidation with metallic oxidants and their cleavage to carbocations or carbon radicals. For example, the oxidation of α-stannyl carbamates generates their cation radicals, which are cleaved into carbocations of carbamates by the elimination of the stannyl radical. In addition, α-stannyl alkanoates are oxidized to generate the corresponding α-radicals of alkanoates and stannyl cation. Cations or radical intermediates generated in this way are utilized in the carbon-carbon bond forming reactions; they react with various olefinic compounds, such as silyl enol ethers (Eq. 1, 2). (-)-Stemonamide (1), isolated by Lin et al. from roots and rhizomes of Stemona (Stemonaceae), has been used as an anticough agent and an insecticide in traditional medicines of China and Japan. In this symposium, we report a total synthesis of (±)-stemonamide by applying the above-mentioned oxidative coupling reactions of the stannyl compounds with silyl enol ethers for the construction of the carbon framework. All the carbon units of stemonamide 1, except the methyl group, were arranged by applying our oxidative coupling of three components, 5, 6, and 11 in 4 steps (Eq. 3). The transformation of the intermediate 15 to the tricyclic compound (2) was performed under the conditions described in Eq. 4〜6. The introduction of the methyl unit proceeded stereoselectively from the convex side of 2. Thus, synthesis of (±)-stemonamide (1) was accomplished in a total of 12 steps.

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Huperzine A (1) is an alkaloid isolated from Huperzia serrata (Thunb.) Trev. The gross structure of 1 was determined by Liu et al. in 1986 (Figure 1). This natural product exhibits a potent inhibitory activity against acetylcholinesterase (AChE). Consequently, it is expected that 1 serves as a therapeutic agent for the treatment of Alzheimer's desease. The total synthesis of racemic 1 was achieved by Qian et al. in 1989 and by Kozikowski et al. in 1989 and in 1993, independently. The asymmetric synthesis of (-)- 1 was also reported by Kozikowski et al. in 1991. We wish to present here the synthesis and in vitro AChE inhibitory activity of optically active (-)-huperzine A (1) and its fluorinated analogues. The asymmetric synthesis of (-)-1 was achieved by employing two novel methods. Thus, one is Michael addition of 2 with acrolein in the presence of chiral base (Scheme 1). The good optical yield (64% ee) of the adduct was obtained by employing (-)-cinchonidine (5) as a chiral base. The other one is asymmetric allylation of 2 employing a π-allylpalladium complex (Scheme 2). The use of ferrocenyl derivative 7 as a chiral ligand gave the best enantioselectivity (49% ee). Optically pure (+)-4 obtained by recrystallization was converted to (-)-1 by Kozikowski's procedure (Scheme 3). Next, we designed and synthesized some fluorinated huperzine A analogues (Figure 2) so far not reported. The trifluoromethyl-substituted compounds 11-13 were efficiently synthesized by a method featuring addition of trifluoromethyltrimethylsilane promoted by fluorine anion and Corey-Winter's olefin formation reaction as key steps (Scheme 4-6). Additionally, the synthesis of the mono-fluorinated compound 26 was accomplished by a method which employs fluorination of a hydroxy group (Scheme 7). With completion of the synthesis of (-)-1, 11-13, 26, in vitro AChE inhibitory activity was next assessed (Table 1). All these fluorinated analogues were found to still retain AChE inhibitory activity. Taking into account their racemic forms, analogues 11 and 26 were estimated to be 40- and 20-fold less potent than 1, respectively. These studies on the structure-activity relationships should hold promise for designing novel analogues of 1 which may exhibit characteristic profiles.

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Acronycine (1) is an alkaloid isolated from the bark of the Australian scrub ash Baurella simplicifolia (ENDL.) Hartley (syn. Acronychia baueri SCOTT) (Rutaceae). When 1 was treated with hot methanolic HCl, two dimers (3,4), a trimer(6), two tetramers (7, 9) and two pentamers (8, 10) of noracronycine (2) were obtained.Two of these seven compounds (4, 9) possessed a rearranged partial structure. The dimeric compound (3) was obtained selectively in 41% yield when 2 was treated with conc. H_2SO_4-MeOH (1:1) at r.t. On the other hand, when norisoacronycine (5) was treated with hot methanolic HCl, a dimeric compound (17) was obtained. The structure of this compound was elucidated by comparison of the ^1H and ^<13>C NMR spectra of this compound with those of 3 and 4. By treatment of dihydronoracronycine (11) with conc. H_2SO_4 at r.t., dihydronorisoacronycine (12) and dihydronoralloacronycine (14) were obtained together with 1,3-dihydroxy-10-methylacridone (13) and 15. It was reported that noracronycine (2) was obtained in quantitative yield when the HCl salt of acronycine was heated at 140℃. On the other hand, when it was heated at 250℃, various acridone derivatives were obtained and one of the main products was dihydronorisoacronycine (12). In this reaction, four novel acridone derivatives 20-23 were obtained. Cytotoxic activities of 16 acridone derivatives obtained through these experiments were evaluated and it was found that 1,3-dihydroxy-10-methylacridone (13) and 1,3-dihydroxy-2,10-dimethylacridone (20) showed activity against VLB resistant KB cells (KB-VI).

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