In the course of our search for bioactive metabolites from Japanese marine invertebrates, we encountered two sponges, Acanthella klethra and Haliclona sp., which showed marked activity in antifungal and starfish egg assays. Bioassay-guided isolation afforded four new cytotoxic compounds; two new diterpene isonitriles named kalihinene (1) and isokalihinol B (2) along with the known compound kalihinol A (3) from A. klethra and two macrocyclic alkaloids, haliclamines A (4) and B (5) from Haliclona sp. Structures of isocyanoditerpenes were deduced mainly by interpretation of 500MHz NMR spectra including COSY, NOE and decoupling experiments as well as by comparison of spectral data with those of kalihinols. Kalihinene was found to contain a unique cis-dehydro-decalin system. Kalihinene and isokalihinol B were not only antifungal but also cytotoxic against P388 leukemia cells with IC_<50> 1.2 and 0.8μg/mL, respectively. Structure elucidation of haliclamines was done by extensive 500MHz NMR analyses including COSY, HMBC, NOESY and decoupling experiments in different solvents as well as by HREIMS analyses. Finally, the structures were confirmed by dehydrogenation with Pd-C. Haliclamines A and B inhibited not only cell division of fertilized sea urchin eggs at 5 and 10μg/mL, respectively, but also growth of L1210 and P388 leuekemia cells at IC_<50> 1.5 and 0.9, and 0.75 and 0.39μg/mL, respectively.

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During our studies on bioactive metabolites from Okinawan marine organisms, we have isolated iejimalides A(1) and B(2), two novel 24-membered macrolides with powerful antileukemic activity, from the Okinawan tunicate Eudistoma cf. rigida. The structures were elucidated from spectral data including 2D NMR. Compounds 1 and 2 also inhibited Na^+, K^+-ATPase. Eudistomidins B(3), C(4) and D(5), novel β-carbolines with potent cytotoxicity, have been isolated from the Okinawan tunicate Eudistoma glaucus. The stereostructure of 3 was elucidated from the NMR and CD data, while that of 4 was determined through synthesis of 6-O-methyl-10(R)-eudistomidin C. Compound 3 inhibited Na^+,K^+-ATPase but activated actomyosin ATPase, while compound 4 showed calmodulin-antagonistic activity. Eudistomidin D(5) induced Ca^<2+> release from sarcoplasmic reticulum (SR). 9-Methyl-7-bromoeudistomin D(6) synthesized based on structure-activity relationship between caffeine and eudistomin D was ca. 1000 times more potent than caffeine in SR Ca^<2+>-releasing assay. Therefore, compound 6 is used as a valuable tool for elucidating molecular mechanism of Ca^<2+> release from SR.

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Marine organisms are excellent sources for biologically active compounds. In our screening for biologically active metabolites from marine sponges, the crude extracts (CHCl_3-MeOH, 9:1) of the Caribbean sponge Ptilocaulis spiculifer and the Red Sea sponge Hemimycale sp. were found to show a remarkable activity. These extracts were separated by chromatography to afford the same antitumor, antiviral, and antifungal active compound, ptilomycalin A (1). The structure of 1 was elucidated by spectroscopic analyses and chemical reactions of its derivatives (2 and 4). Ptilomycalin A (1) has a new carbon skeleton possessing a polycyclic guanidine moiety, which is quite different from those of known guanidino compounds such as tetrodotoxins and saxitoxins. It should be also noted that spermidine is connected with the polycyclic skeleton through an ω-hydroxylated fatty acid.

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Ciguatoxin (CTX) is the principal toxin of ciguatera, which is a fish poisoning of a dinoflagellate origin. The toxin (0.35mg) was isolated from the moray eel Gymnothorax javanicus and its less polar congener (0.74mg) from the causative organism, Gambierdiscus toxicus. We successfully elucidated their structures, 1 and 2 only by spectroscopic methods. High resolution FABMS suggested the formula of CTX to be C_<60>H_<86>O_<19>. The structure elucidation was carried out using the ^1H-^1H 2D-NMR methods. Although most proton connectivity was elucidated by ^1H-^1H COSY, the skeletal chain was interrupted at the following four positions; 1) protons (H23-H30) on a middle-membered ring that presumably undergoes a slow conformational change; 2) a methyl-bearing methine at the heavily overlapping region of ^1H NMR spectra (H-39); 3) a quaternary carbon bearing a methyl and a oxygen (C33); 4) a spiro-ketal carbon (C52). The first part was assigned by 2D-NMR measurements at ?25℃, which revealed the presence of a 9-membered ether-ring. The second part was clarified by simulating the decoupling difference spectrum using the coupling constants obtained from MM2 calculations. The third and last parts were solved by detecting NOE's at ?25℃ on Me-56/H37, Me-60/H-53 or H-48/H-55 and by DQF COSY, which showed a long range coupling between H-32 and Me-56. ^1H NMR assignments of 1 and 2 are shown in Table I. The structure, 1, partly resembles those of brevetoxins or yessotoxin. However, toxicity (0.35ug/kg, mice, ip.) of CTX is several hundreds times more potent than those of the other two toxins. The difference in activities is an interesting subject for future studies on structure-activity relationship among the compounds of this class.

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Trichoderma spp. are active as mycoparasites and, therefore, can serve as potential biocontrol agents. We found in dual culture studies on agar medium that Trichoderma harzianum killed most wood decay fungi examined. New antibiotic peptides, trichokindin (TK)-I〜X, which belong to a class of peptaibols like alamethicin and suzukacillin, were isolated from the spores of T. harzianum. The primary structure of TK-X(10) was clearly established by FABMS and FABMS/MS. The N-terminal of (10) is protected by an acetyl group and the C-terminal is linked with an isoleucinol (Iol). The peptide contains a high proportion of abnormal amino acids, α-aminoisobutyric acid (Aib) and isovaline (Iva). The peptides, trichokindin-IV〜IX (4〜9), have all about 1800 of molecular weight and have a similar structure, in which an aminoalcohol is Iol or leicinol (Lol), to TK-X. The other peptides, trichokindin-I〜III (1〜3), whose molecular weights are about 1200, are also protected by an acetyl group. Their total structure are not yet elucidated.

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The solitary digger wasp Philanthus triangulum preys on honeybees with paralyzing venom, and then feeds the victims to their larvae. T. Piek and his colleagues have shown that the venom contains low molecular weight, polyamine-like components, philanthotoxins (PTX), which exhibit a variety of pharmacological properties on insect skeletal muscle. We isolated a potent glutamate receptor antagonist in this wasp venom, and determined the structure as being philanthotoxin (PhTX)〜433 (1) by spectral analyses and synthesis. L-Glutamate is a putative excitatory neurotransmitter at many synapses in vertebrates and invertebrates. Glutamate receptors are presumably involved in higher neural functions such as memory and learning, and in neurological disorders, e. g., epilepsy and Huntington's disease. With the aim of the isolation of the glutamate receptor, we next explored the structure-activity relationships of PhTX analogs, which resulted in higher active analogs such as 10 and 11. We then synthesized the radioactive analogs labeled with ^<125>I. Furthermore, syntheses of the ligands for affinity chromatography and the photoaffinity labeling reagents were investigated.

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Several cell organelles assembled from microtubules (MTs) and other proteins perform an important role in cell biology. MTs are found in a great variety of situations in cells, and may occur singly, loosely ordered into bundles, or in highly ordered arrays of a define pattern. These variations are presumably related to the different functions of MTs. In order to understand such functions, several naturally occurring compounds have been used as molecular prober.1,2) T-1 and curvularin have been found to be effective mitotic inhibitors which induced barrel-shaped spindles in sea urchin eggs due to disorder of the microtubule organizing centers (MTOCs) in centrosomes.3-7) MTs are also involved in the pronuclear fusion (PF) of sea urchin eggs. Early literature contains few reports of this process, and a better understanding of the detailed role and nature of MTs awaits the discovery of new types of specific inhibitors toward PF. We made the first systematic search for microbial products that might have PF inhibitory activity using a novel bioassay system. A Streptomyces sp. KO2388 was discovered, which produced a novel γ-pyrone, named streptopyrone(Structure: 2), with pronounced inhibitory activity toward PF. A fungus, Curvularia harveyi, was found to produce a new mitotic arrester(Structure: 3), which was named herveynone and its structure was elucidated as shown in Fig.. This compound effectively inhibited spindle formation and arrested the first cell division of sea urchin eggs. The mode of operation of these compounds are in progress.

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Lampteroflavin (1), a riboflavin α-D-riboside was isolated in extraordinary small amount from the luminous mushroom, Lampteromyces japonicus (Fig 1), which was available only two weeks in a year. Extraction method was improved to utilize only alive gills under aeration instead of using the whole body (Fig 3), and the method was established as Scheme 1. It's structure has been elucidated by chromatographic and spectroscopic analyses(1). It's fluorescence spectrum was identical to the bioluminescence spectrum of the mushroom, having maximum at 524nm (Fig 2). We concluded that 1 was responsible to the bioluminescence mechanism as the light emitter, since 1 was only the fluorescent constituent in fresh gills. Previous report that illudin S (lampterol) or ergosta-4,6,8(14),22-tetraen-3-one(2) could be the emitter is thus unlikely judging from the weak fluorescent intensity and the different maximum wavelength from that of mush-room bioluminescence. Lampteroflavin (Table 1) was hydrolyzed with dil. mineral acid to give riboflavin and D-ribose. Riboflavin was identified by HPLC, ^1H NMR, UV, Fluorescence and FAB mass spectrometry. D-ribose was acetylated and then confirmed by ^1H NMR, CD and tandem mass spectrometry. Riboflavin and D-ribose was connected together with α-glycosidic linkage which was determined by ^<13>C NMR of the anomeric carbon (δ=103.2ppm)(3), NOSEY spectrum (H-1" being close to H-3" and H-5') and ^1H NMR pattern of anomeric proton. The total structure of lampteroflavin was confirmed through its chemical synthesis.

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Peridinin (1) and pyrrhoxanthin (3) are unique C_<37>-skeletal nor-carotenoids owing to the presence of a 4-ylidenebutenolide system carrying an allene or an acetylene function in the main polyene chain. Both pigments were found from the planktonic algae, dinoflagellates causing "red tide". Main pigment peridinin is known as an auxiliary light harvesting pigment for photosynthesis. Toward a total synthesis of these carotenoids, a novel synthetic method of carotenoidal ylidenebutenolides was developed via a reaction of the conjugated formyl ester (8) with the various allylic sulfones in the presence of LDA at ?78℃ (route C). In this reaction, addition, cyclization, and elimination took place successively in one pot to give expected products in moderate yields. By using this method, the first synthesis of peridinin (1) and pyrrhoxanthin (3) was achieved by the reaction of the C_<15>-epoxy formyl ester (35) with the conjugated C_<22>-allenic sulfone (23) or C_<22>-acetylenic sulfone (41). The formyl ester (35) was synthesized through 12 steps starting from (30) as shown in Scheme 6 which included the palladium catalyzed olefination of the enol triflate, the preparation of the allylic sulfone catalyzed by palladium (0) complex, and functionalization of the allylic sulfone. On the other hand, the sulfones (23) and (41) were prepared in 3 steps from the known allenic (21) and (22) or acetylenic (38) and (39) apo-carotenals, respectively. The condensation reaction between the formyl ester (35) and the sulfone (23) or (41) produced peridinin (1) or pyrrhoxanthin (3) accompanying its 11'E-isomer (36) or (43). Spectral properties of the synthetic (1) and (3) were good agreement with those of the respective specimens.

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Fucosterol 24,28-epoxide (1) is a key intermediate in the conversion of sitosterol to cholesterol in phytophagous insects. e have investigated the stereochemical fate of the diastereotopic C-26 and C-27 methyl groups of the epoxide during an enzymatic conversion into desmosterol (2). Incubation of the chirally labeled fucosterol (24R,28R)-epoxides, having ^<13>C at pro-S- (6) or pro-R- (7) methyl group with a cell free enzyme system obtained from guts of the silkworm, Bombyx mori, and ^<13>C-NMR analysis of the product have revealed that the conversion is stereospecific with the C-25 prochiral center and the pro-S and pro-R methyl groups of (1) turn into (Z)- and (E)-methyl of (2). The similar transformation can be achieved in a chemical manner; treatment of fucosterol epoxide 3-benzoate with BF_3 etherate affords three products, desmosterol benzoate, a ketone and an aldehyde. The mechanism of this chemical reaction was also investigated from the stereochemical point. The results suggested that: 1) the formation of desmosterol benzoate involves a carbonium ion intermediate, 2) formation of the ketone (involves migration of hydrogen) takes place with the retention of the stereochemistry at the migration terminus (C-24), and 3) formation of the aldehyde (involves migration of methyl group) proceeds with the inversion of stereochemistry at the migration terminus. Another example, which follows the aforementioned stereo-chemical course, of trisubstituted epoxide-carbonyl rearrangement is also provided.

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A number of studies on opiates that contain a C-6 carbonyl function have suggessted that they can more readily be converted to enols or enolic derivatives than conventional ketones. For example, it has been reported that dihydromorphinone and related ketones are relatively resistant to addition by Grignard reagents and can form tertiary alcohols only with organolithium compounds. Further, crystallographic studies have shown ring C to be in a flattened chair conformation. In this symposium we report chemical studies that provide evidence for the greater stability of opiate products that contain a 6,7-double bond. Treatment of naltrexone, dihydromorphinone, or related opiates that contain a 6-keto group with acetic anhydride or t-butyldimethylsilyl chloride under mild conditions afforded enol derivetives having a 6,7-double bond. Naltrexone also was found to undergo the Robinson annelation reaction with methylvinyl ketone with facility and in high yield to give the β-hydroxyketone. We applied the abnormal reaction of naltrexone to the syntheses of opioid receptor antagonists, nor-BNI 13(pyrrole derivative) and NTI(indole derivative). We also report the intramolecular rearrangement of the acetyl group in 14-acetylnaltrexone.

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We have recently investigated the alkaloidal constituents of Gelsemium elegans, a toxic medicinal plant from Thailand and proposed the biogenetic route of these alkaloids having novel polycyclic systems. In order to support the biogenetic speculation and to develop the efficient synthetic method of these alkaloids including the minor constituents, we designed the biomimetic partial synthesis of some gelsemium alkaloids from easily obtainable compounds. Koumidine (4) is a plausible biogenetic precursor of the new alkaloid, 19-(Z)-taberpsychine (5) and its structure was revised. Both to provide support for the spectroscopic analysis and to determine the absolute configuration of these compounds, we have synthesized them from ajmaline (16). Koumidine (4) was also prepared from gardnerine (22) by the demethoxylation from indole nucleus and the inverting the configuration of the ethylidene side chain with palladium catalyst. Along the biogenetic speculation, a principal gelsemium alkaloid, koumine (6) was synthesized from 18-hydroxygardnutine (28) by the demethoxylation from indole ring and palladium-catalyzed cyclization between C_7 and C_<20> of biogenetic intermediate (27). A synthetic intermediate (45), which was corresponding to the biogenetic intermediate (12) for gelsedine (13), was prepared from ajmaline (16) in 21 steps via the deformylation (C_<21>) and the construction of five-membered D-ring having an α-ethyl group on C_<20>. Talcarpine (49), one of the macroline-type pleiocarpa alkaloid, was synthesized from (16) in 10 steps and the configuration at C_<19> was determined to be (R) by the analysis of 2D-NMR spectra.

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We have previously reported the determination of the absolute stereochemistry of the halenaquinol family, marine natural products1-4 isolated from tropical sea sponges, by the theoretical calculation of CD spectra, and also reported the total synthesis of (+)-halenaquinol (1) and (+)-halenaquinone (5).6 In this paper, we report the total synthesis of (+)-xestoquinone (4) and xestoquinol (3), and also describe the determination of their absolute configurations. Optically pure Wieland-Miescher ketone (8aR)-(-)-(15) was converted to enone (+)-(28) via the reactions of eleven steps. The Diels-Alder reaction of (+)-(28) with 3,6-dimethoxybenzocyclobutene (29) yielded adduct (30) of a tetracyclic skeleton, which was converted to (+)-xestoquinone (4) by the reactions of five steps, and then finally to xestoquinol (3). All of the spectroscopic data of the synthetic sample of xestoquinone (4), including the CD data, were identical with those of the natural xestoquinone. Therefore, the absolute configuration of xestoquinone compounds were determined to be 12bS. The CD spectrum of naphthalene-diene derivative (35) were in a good agreement with the theoretically calculated CD curve of the model compound (36). Therefore, the absolute configuration of xestoquinone compounds was also confirmed in a theoretical way. The efficient preparation procedure of optically pure Wieland-Miescher ketone is described.

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Various 14-fluorinated anthracyclines (3a, 3b, 4, 24a, 26a, 24b, and 26b) were synthesized and subjected to in vitro cytotoxicity and in vivo antitumor activity assay against P388 murine leukemia. At first, 14-fluoro-4-demethoxydaunorubicin (3a), 14-fluorodaunorubicin (3b), and 14,14-difluoro-4-demethoxydaunorubicin (4) were synthesized by employing glycosylation of the corresponding aglycones (9a, 9b, and 18) with 3-N-trifluoroacetyl-1,4-bis(O-p-nitrobenzoyl)daunosamine (10) in the presence of TMSOTf followed by deprotection. Syntheses of the 14-fluoroanthracyclinones (9a and 9b) and 14,14-difluoroanthracyclinone (18) were achieved by featuring the substitution reaction of the 14-bromo-7-deoxyanthracyclinone (6a and 6b) with a combination of Bu_4N・F and TsOH and the Reformatsky reaction of the siloxyaldehyde (14) with ethyl bromodifluoroacetate as key steps. These compounds (3a, 3b, and 4) showed prominent cytotoxicity in P388 in vitro test. Among 3a, 3b, and 4, excellent in vivo antitumor activity against P388 murine leukemia was observed for 3a and 3b. Thus, 14-fluorodaunorubicin congeners (24a, 26a, 24b, and 26b), in which the L-daunosamine residue was replaced with 2-deoxy-D-ribose and 2-deoxy-L-fucose, were synthesized by using the same glycosylation reaction of aglycones (9a and 9b) with 1,3,4-tri-O-acetyl-2-deoxy-D-ribopyranose (21) and 1,3,4-tri-O-acety1-2-deoxy-L-fucopyranose (22). Although fairly weak cytotoxicity against P388 murine leukemia was only observed for 24a and 24b, 26a and 26b exhibited prominent cytotoxicity. The 14-fluorodaunorubicins (26a and 26b) were found to show highly effective T/C values in P388 in vivo assay.

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A semi-synthetic anthracycline antibiotic, pirarubicin (1), namely 4'-O-((R)-tetrahydropyranyl)adriamycin prepared by H. Umezawa et al. as a less cardiotoxic analogue of doxorubicin (2), is widely used for treatments of leukemia and other tumors. In the search for clinically useful and orally absorbable analogues, we found that N-salicylidene derivatives of 1 were therapeutically superior to 2 in murine models. Some of them showed the possibility of being orally absorbable agents in vivo tests. Preparation and biological evaluation of N-salicylidene derivatives of 1 are studied. Daunorubicin (3) was treated with benzoylhydrazone of acrolein dimer (7) to give 8 having a unique structure, and with o-phthaladehyde to give isoindolinone 9. Both of them reduced the antitumor effect in vivo. Reaction of 1 with salicylaldehyde gave 3'-N-salicylidenepirarubicin (10), which showed marked prolongation of the survival period in experimental tumor-bearing mice by the oral administration. Among N-salicylidene analogues (15〜22) substituted with electron-donating group(s) on the aryl ring, 15 exhibited good therapeutic effects. N-Salicylidene analogues (23〜35) having electron-withdrawing group(s) were also prepared. Compound 28 showed a significant antitumor activity by oral and intraperitoneal administrations. Derivatives of forphenicine (36) which was a structural homologue of salicylaldehyde were coupled with 1 to give interesting compounds 39〜41. Compound 39 had the broadest effective dose range. Furthermore, 40 showed remarkable antitumor effect by oral administration against murine tumor. The parameters on gastrointestinal absorption among compounds synthesized are also discussed.

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The enantioselective synthesis of cis- and trans-6-amino-carbapenems which are highly resistant to renal dehydropeptidase I (DHP-I) is described. 1. Synthesis of trans-6-aminocarbapenems For the search of biologically more suitable N-substituents at C-6 in carbapenems, the versatile intermediate 7 was prepared from 6-APA(Scheme 1). Although 7 was tried to convert into the carbapenems(13a-13g) with acetyl or hydrogen on the nitrogen atom at C-6, the corresponding derivatives could not be obtained because of their physico-chemical instability. The total synthesis of the desired carbapenems(14h-14k) having high stability to DHP-I, was accomplished by N-methylation. In addition, the S-side chain derivatives(22a-22i) of 14i were efficiently prepared using the [2+2] cycloaddition reaction of ketene(15')-imine(16)(Scheme 2). 2. Synthesis of cis-6-aminocarbapenems In order to find out more potent 6-aminocarbapenems, cis-aminocarbapenems were synthesized using carbene insertion to the sulfide 23 as a key step. This reaction was regio- and stereoselective, and gave cis 24a as a sole product(Scheme 4). Compound 24a-2 was decarboxylated by hydrogenolysis and treated with Raney nickel to give the N-methylated and desulfurized derivative 26

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Deprotonation of a cyclopropenone ketal (5) with BuLi in THF generates a lithio cyclopropene 6 which is stable at low temperatures in the presence of HMPA or TMEDA. Transmetalation with ZnCl_2 gives the corresponding zinc species 7. These metalated cyclopropenone ketals react with a variety of electrophiles to give mono-substituted cyclopropenone ketals 3 in high yield, which upon mild acidic hydrolysis give a variety of 2-substituted cyclopropenones 4 in high yield. Repetition of the alkylation/hydrolysis sequence gives 2,3-disubstituted cyclopropenones as well. The reaction of lithio cyclopropene 6 with carbonyl compounds followed by acidic hydrolysis gives α-hydroxy cyclopropenones in high yield, providing an efficient access to the novel cyclopropenone antibiotic penitricin (1). The method also allows preparation of a number of penitricin congeners. Examination of the antibiotic and cytotoxic activities of these compounds indicated that the hydroxyl group adjacent to the cyclopropenone ring is indispensable for the antibiotic activities. In order to probe the effects of the hydroxyl group, molecular structures and electronic properties of penitricin and its protonated structures have been studied with molecular orbital calculations using ab initio (HF/3-21G) and semiempirical methods (AM1).

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Aplysiatoxin (1) isolated from the digestive gland of a sea hare, Stylocheilus longicauda, is an active tumor promoter and is structurally characterized by spiroacetal and hemiacetal structures and stereochemically interesting four consecutive asymmetric centers. Our effort toward the synthesis of 1 is to be presented. 1 was dissected into five fragments, B, C, D, E, and F. Fragments, B, C, and E, were obtained in optically pure forms as 8, 13, and 24, respectively, by using asymmetric epoxidation as a key step. The synthesis of fragment D (18) started with isopropyl (2S,3R,4E)-6-benzyloxy-2-hydroxy-3-methyl-4-hexenoate (15), which was prepared by using the newly developed titanium-mediated [2,3]Wittig rearrangement. 15 was converted into the epoxide 17 stereoelectively by using iodolactonization, and 17 was further converted into the epoxy alcohol 18 in a conventional way. Coupling of C and D was achieved by treatment of 18 with the Grignard reagent 13, giving 26 regioselectively. After the removal of the unnecessary primary hydroxyl group, compound 26 was transformed into the terminal epoxide 28 with the inversion of C_9-configuration (Aplysiatoxin numbering). Thus, the stereochemistry of the part of four consecutive asymmetric centers was established. Coupling of 28 with E was effected by the treatment of 28 with the lithiated 24, giving the C_3-C_<21> fragment 29. The next stage of the synthesis, the introduction of B on the C_9 hydroxyl group was accomplished by using Yamaguchi procedure. Further transformation of 30 to 1 is now in progress.

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A one-pot three carbon ring expansion reaction involving intramolecular aldol condensation and subsequent retro-aldol cleavage is induced by treatment of β-keto esters with potassium tert-butoxide in dimethyl sulfoxide to afford functionalized 8-, 9-, 10-, and 15-membered rings, respectively. The stereochemistry of intermediate 7 was clarified to be cis-fused carbocyclic ring system with the methyl ketone in the cis-position by chemical conversion. A novel mechanistic consideration for this successful three carbon ring expansion was made by taking the dual function of electron withdrawing group (EWG) in I. This facile ring expansion was also applied to the synthesis of (-)-muscone. Treatment of compound 31 with t-BuOK led to unusual rearrangement product 33 without the formation of two carbon ring expansion product. We suggest the mechanism for this new reaction as depicted in Scheme 12. It involves a reverse intramolecular [3.3]sigmatropic rearrangement and subsequent [3.3]sigmatropic rearrangement.

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Recently, we have developed a new and efficient method for the carbon-carbon bond forming reaction by using copper(I)-catalyzed Grignard reagents with triflates, which bear a β-oxygen fuctionality. Since those starting triflates are readily accessible from simple chiral building blocks, the procedure provides a very convenient approach to synthesize a variety of natural products in optically active forms. As an application of this methodology, we have accomplished the enantioselective syntheses of (+)-exo-brevicomin (7), one of the aggregation pheromones of bark beetles, and (5R,6S)-(-)-6-acetoxy-5-hexadecanolide (16), a major oviposition attractant pheromone of the mosquito Culex pipiens fatigans, via tosyl-triflate 6 as a common key intermediate. In both examples the use of 6 has enabled us to realize the sequential C-C bond formation in one-pot operation and hence the overall yields were considerably improved. The similar strategy could be applied to the synthesis of naturally occurring hydroxy-γ-lactone 12 (termed L-factor). As an alternative pathway, the stepwise methods towards these target molecules were also examined.

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Dactylyne (1) and its isomer, isodactylyne (2), isolated from the sea hare Aplysia dactylomela, are characterized structurally by a tetrahydropyran ring with all α-oriented, four substituents, (2E)-3-bromo-2-pentenyl and pentenynyl groups at C-2 and C-6, and bromine and chlorine atoms at C-3 and C-5, and prossess central nervous system depressent activity, as evidenced by the potentiation of pentobarbital hypnosis. In view of the structural features and the biological activity, these are attractive synthetic targets. In the course of the synthetic studies on these natural products, we found that dialkylamine hydrohalides are much more effective as sources of halide nucleophiles for titanium-mediated ring openings of 2,3- and 3,4-epoxy-1-ols at the 3- and 4-positions, respectively. Interestingly, diethylaluminium chloride was found to be also effective for the regioselective opening of trans-2,3-epoxy-1-01 (6) at the 3-position. We describe herein the details of these results in Figures 1 and 2. Next, we describe application of our procedures to the synthetic approaches towards optically active 1 and 2 in Schemes 2 and 3. Starting with 3-butyn-1-01, we derived the optically pure epoxy alcohol (16), which was intramolecularly cyclized on acid treatment to give smoothly the synthetic key intermediate (22).

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Chemical synthesis of myo-inositol phosphates which are involved as metabolites in the intracellular signal transduction system have been accomplished during last three years by several groups. We have continuously made efforts to explore practical syntheses of them. The direct reaction of myo-inositol itself with an adequate electrophile may be a powerful tool for this purpose. Among electrophiles employed here, 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane(TIPSCl) was found to be especially pomising. Thus, the reaction of myo-inositol with 2.5 molar equivalent of TIPSCl in pyridine gave almost exclusively 1,6:3,4-di-TIPS derivative 20 in 66% yield. This product 20 was effectively utilized for the synthesis of myo-inositol 1,3,4,6-tetrakisphosphate 24, which was discovered recently. The reaction with benzoyl chloride was also useful. When the reaction was conducted at 90℃, 1,3,4,5-tetra-O-benzoyl-myo-inositol 25 was isolated as a main product by flashchromatography. 25 was quite easily converted to myo-inositol 1,3,4,5-tetrakisphosphate 31.

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As a part of our synthetic studies on complex carbohydrate, we wish to describe here the convenient and practical synthesis of D-erythro-C_<18> -sphingosine(2), an essential constituent of sphingoglycolilids, from the derivative of L-Serine. First, we studied the Horner-Wittig reaction of L-serylmethylphosphonate and found N-t-butoxycarbonyl-N, O-isopropylidene-L-serylmethylphosphonate(7) was suitable for this purpose. Phosphonate 7 was reacted with tetradecanal in the presence of caesium carbonate in isopropyl alcohol to afford the corresponding (E)-olefin 8 in 86% yield selectively. Next, we tried the stereoselective reduction of carbonyl group in (E)-olefin 8 and found the reaction using NaBH_4-CeCl_3 in tetrahydrofuran-methanol at room temperature gave the desired D-erythro-C_<18>-sphingosine derivative 9 in 90% yield with high stereoselectivity (erythro:threo=5:1). Because the synthesis of 2 from 9 have been reported, the formal synthesis of 2 was completed. Further, it is expected that compoud 9 can be converted easily to the glycosyl acceptor for the synthesis of sphingoglycolipids.

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N-Acetylneuraminic acid (5-acetamido-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonic acid: NeuAc) is a sugar component of oligosaccharides in glycoproteines and ganglyosides which play important roles in biological functions such as recognition and differentiation of cells. We previously succeeded to synthesize 3-deoxy-D-manno-2-octulosonic acid (KDO) via the condensation of a triflate (4) with methyl glyoxylate dithioacetal (3). In the present study, the NeuAc derivative 5 was successfully synthesized by an application of the the same methodology via the coupling reaction of 3 with epoxide 6 which possesses all asymmetric carbon atoms of NeuAc. We first prepared the mannosamine derivative 8 either from D-glucose by substitution of the hydroxyl group at C_2 with an amino group with inversion or from N-acetyl-D-glucosamine by epimerization at C_2. Epoxide 6 was then obtained by stereoselective oxidation of alkene 7 which was derived from the mannosamine derivative 8 by Wittig reaction. The condensation of the epoxide 6 with the dithioacetal 3 was proceeded with an internal exchange of the ester to give a lactone 5 with the desired NeuAc skeleton. We are now investigating the deprotection of 5 into NeuAc 1.

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An efficient enantiospecific route to the A-ring synthons, 11, 12, and 13 of active metabolites of vitamin D_3 from (R)-(-)-carvone (4) has been developed. The method involves the following major transformations, (i) highly diastereoselective Cr(II)-mediated reaction of the allylic iodide 19 with the aldehyde 20 giving the common precursor 21, (ii) dehydration of 21 under Mitsunobu reaction conditions leading to stereo- and regioselective construction of the requisite diene moiety of 11, (iii) transformation of 21 into the aldehyde 12 through oxidative cleavage of the glycol. A novel enantioselective synthesis of the hidrindanol 29, a C/D-ring synthon for the preparation of vitamin D_3 metabolites, has been achieved starting from cyclopropyl methyl ketone 34. The route relys on stereoselective construction of the hidrindan skeleton by use of newly developed chiral epoxy alcohol-initiated polyolefin cyclization (37→40). The cyclization product 40 was converted into 29 through (i) highly stereoselective introduction of the C-17 side chain by taking advantage of the established methodology involving semihydrogenation, ene reaction, and hydrogenation, (ii) degradative transformation of the C-8 side chain into the hydroxyl group.

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Immobilized plant cell cultures such as Nicotiana tabacum and Gardenia jasminoides Ellis entrapped with calcium alginate beads have been prepared and the biotransformation of synthetically important foreign substrates was examined by consecutively reusing these two immobilized cells. Immobilized cells of N. tabacum were reusable several times and enantioselectively reduced the five keto esters 1-5 to the corresponding hydroxy esters 1a-5a in reasonable chemical yields and relatively high optical yields. The consecutive reuse of the immobilized cells enhanced the rates of each biotransformation of 1-5. The time-course experiments of the present biotransformations clearly revealed that the transformation rates of 3-oxobutanoates 3 and 4, both of which bear longer n-alkyl chains in their alkoxycarbonyl groups, were faster than those of homologs 1 and 2 possessing shorter alkyl chains. The biotransformation of an aromatic ketone, acetophenone (6), with immobilized N. tabacum cells gave a chiral alcohol (S)-(-)-6a, whereas a similar one with immobilized G. jasminoides cells yielded (R)-(+)-6a.

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Six new cembranoids sarcophytol P (3), R (4), S (5), K (8), F (11), and T (13) were isolated from the soft coral Sarcophyton glaucum. Sarcophytol P (3) was shown to be the 20-hydroxy derivative of the major component sarcophytol A (1), and afforded the cyclization product 6 in CHCl_3 at room temperature, in a same way as in 1. Sarcophytols R (4) and S (5) were correlated to 1, by conversion of its 7R,8R and 7S,8S epoxide derivatives. Sarcophytol K(8) was a 13, 14-dihydroxycembranoid having a 1E,3Z-diene moiety. The absolute configuration of 8 and its 1Z,3E- and 1Z,3Z-isomers sarcophytol B (2) and J (10) were determined by circular dichroism study of their bis-p-dimethylaminobenzoate derivatives. Sarcophytols F (11) and T (13) were 1E- and 1E,3Z-isomers of 1. Compound 11 showed characteristic broadening of the ^1H-NMR chemical shifts, due to the restricted conformational interconversion. Using the three cembranoids sarcophytols F (11, 1E,3E), N (15, 1Z,3Z) and T (13,1E,3Z), spontaneous autoxidation-cyclization, in CHCl_3,was examined, in order to compare the stereochemical course of the reaction with that of 1 (1Z,3E), which affords trans-fused bicyclo[9.3.0]tetradecene systems. The 1E,3Z-isomer 13 gave the same cyclization product 18, as in the reaction of 1, even though it is isomeric at C-1,3. The 1Z,3Z isomer 15 gave 19, in consequence of the reversed geometry at C-3 of 15. The 1E,3E-isomer (11) gave the bicyclic product 22, having an antipodal fusion as compared with 19. This was confirmed by PCC oxidation of 19 and 22, giving enantiomeric ketones 23 and 24 respectively. The 1(14)-epoxide 26 was shown to be the immediate precursor of 22, and acounted for the inversion of the geometry at C-1 of the cyclization product. Compound 26 is isomeric with the epoxide 17 derived from 1. The epoxide 17 is the postulated precursor in the conversion of 13 to 18. It is noteworthy that the mode of antipodal fusion of the cyclopentane rings, in 18 and 22, was controlled by the chirality of the epoxy rings. The C-14 hydroxyl group participates in the transannular cyclization, but was found not to be the requisite functional group for the reaction. Similar treatment of cembrene C (30), the parent hydrocarbon of 1, also reacted in CHCl_3 giving the bicyclic product 32.

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Soft corals belonging to the genera Pachyclavularia and Briareum on the Great Barrier Reef, Australia are now known to be extremely close taxonomically. Based on classical taxonomic characters, it is difficult to distinguish between colonies of the two genera, and yet Pachyclavularia belongs to the order Stolonifera, while Briareum is a Gorgonacean coral. Chemical studies of the two groups suggest that Pachyclavularia species produce cembranoid diterpenes, while Briareum species generally yield briarane diterpenes. Chemical investigation of a soft coral tentatively ascribed to the genus Pachyclavularia afforded the known diterpene, acetoxycladiellin (3), and three new briarane diterpenes (4), (5) and (6). The major briarane (4) was found to be ichthyotoxic to the mosquitofish Gambusia affinis at 2-5 ppm. The basic skeleton and the common substitution pattern of the cyclohexane ring in the briaranes (4), (5) and (6) was deduced from ^1H and ^<13>C-n.m.r. spectral comparisons with the known metabolite (2) (Tables 1 and 2.). High field n.m.r. experiments, including COSY-45 and short and long range ^<13>C-^1H shift correlated 2-D n.m.r. spectroscopy, permitted the broad structural features to be deduced for (4) and (5). N.O.e. difference spectroscopy permitted stereochemical detail to be added to these structures. Compound (6) was isolated in only trace amounts, and was not stable. Only ^1H-n.m.r. data was available for this compound, and the structure should be regarded as tentative. The ^1H-n.m.r. spectrum of (4) at room temparature in CDCl_3 was extremely broad due to slow exchange between several conformations. Molecular mechanics calculations on compound (4) revealed three major conformations for (4), and their likely proportions in an equilibrium mixture as 4a, 52%; 4b, 39%; 4c, 9%. At 345K in C_6D_6, the ^1H n.m.r. spectrum sharpened, but the coupling constants measured under these conditions were averaged conformational values. They did not relate to the J values calculated for the various minimised conformations. The isolation of briarane diterpenes from a soft coral attributed to the genus Pachyclavularia castes some doubt on its taxonomy.

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From fresh rhizomes of the fern, Polypodiodes niponica (NAKAI) CHING (=Polypodium niponicum NAKAI, Polypodiaceae), the new migrated dammarane hydrocarbon, aonena-3,21-diene (1) was isolated and its structure was established by spectral and chemical methods. Compound 1 is the first example of a final migrated product belonging to the dammarane triterpenoid with a S configuration at C-18 in the side chain. In addition, two oily migrated malabaricane hydrocarbons, podioda-7,17,21-triene (2) and podioda-8,17,21-triene (3) were isolated from the same fern materials. These are also the first example of a migrated malabaricane triterpenoid. From the fresh roots of the composite plant, Picris hieracioides LINNE subsp. japonica (THUNB.) KRYLOV. (Compositae), gammacer-16-en-3β-yl acetate (4), having a new basic triterpenoid skeleton, was isolated along with its corresponding 3α- and 3β-ols (5,6) and their structures were established by physico-chemical and single X-ray crystallographic methods. Two new compounds, pichierenyl acetate (7) and isopichierenyl acetate (8), were also isolated and their structures were confirmed by an acid induced rearrangement experiment of the compound 4.

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In the course of our studies on dried bulbs of Fritillaria genus (Chinese medicine "Bei-mu"), novel steroidal alkaloids having new skeletons were isolated from Fritillaria ussuriensis Maxim. along with known alkaoids. Ussuriedine (5) and ussuriedinone (6) have a seven ring skeleton formed by C-C bond between C-18 and C-27 of 5a-cevanine skeletone. Ussurienine (3) and ussurienone (4), which were isolated from the HCl-MeOH hydrolyzed alkaloid fraction, were shown to be artifacts derived from 5 and 6, respectively. Additional new alkaloid, pingbeinone (7) has a novel skeleton lacking C-18 carbon of 5α-cevanine skeleton. This is the first example of C_<26> steroidal alkaloid from Fritillaria genus. It is notable that F. ussuriensis contains new alkaloids which were assumed to be drived from the modification at C-18 (and C-27 for 5 and 6) of 5α-cevanine skeleton. As the HPLC analysis of 19 kinds of Fritillaria species cultivated in China showed that 3 was detected only in F. ussuriensis, it is important to clarify the biosynthetic pathway of these alkaloids and the relation between F. ussuriensis and other Fritillaria species.

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Alstonia scholaris R.Br. is grown widely in South-East Asian and South Asian districts. The studies on the constituents have been carried out on the plants originated from Thailand, India, or Pakistan, so far. In order to establish the feature of the component alkaloids in the plant grown in Philippines, the isolation and identification of the alkaloids were attempted on the leaves of A. scholaris in Philippines as well as those from Indonesia, Thailand and Taiwan. 1. Isolation of the new alkaloids. Alschomine and isoalschomine, N_b-C_5 secoalkaloid, were isolated from the leaves from Taiwan and Thailand, along with the known alkaloids, e.g. picrinine, picrarinal and nareline, and the structures were elucidated by X-ray analysis of alschomine. The structures of AsP-5a and 5b, (major alkaloids from the Philippine leaves), were established to be 6,7-seco-alstonamine and N-demethyl-6,7-seco-alstonamine, respectively. AsP-5a seems to be the same compound as 6,7-seco-angustilobine B, recently isolated from A. angustiloba. Along with AsP-5a and 5b, 19-hydroxy-tubotaiwine was isolated from the Philippine leaves. 2. Characteristic feature of the component alkaloids in each district. The picrinine-type alkaloids (Group I) are popular in the leaves from Thailand and Taiwan but not in the leaves from Philippines. The alstonamine-type and seco-alstonamine-type alkaloids are dominant in those from Philippines. From Indonesian leaves, vallesamine and 6,7-seco-alstonamine in Group IV were obtained as major alkaloids, and pseudo-akuammigine, picraline, and N_a-methylburnamine in Group I were isolated instead of picrinine and picralinal in those from Taiwan.

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Chiriquitoxin (CHTX, 1), isolated from the Costa Rican frog Atelops chiriquiensis, has drawn the interest of physiologists because of its effects on both Na and K currents of exitable cell membranes. However, the structure has remained undetermined. We isolated 6mg of CHTX from 100g of the frog skin and deduced the molecular formula C_<13>H_<20>N_<4>O_<10> from HRFABMS data. The positive reaction of CHTX to ninhydrine reagent indicated the presence of NH_2 in the molecule. The FT-IR and NMR spectra data together with H_5IO_6 oxdation experiments suggested that CHTX was a tetrodotoxin (TTX, 2) analog, in which 11-CH_2OH was replaced by CH(OH)CH(NH_2)COOH or CH(NH_2)CH(OH)COOH. In a D_2O solution containing 1%CF_3COOD and 4%CD_3COOD, CHTX formed a 13,6-lactone (4) of which CH(NH2) was exchanged with deuterium. Thus the substituent group at C-6 was presumed to be CH(OH)CH(NH_2)COOH. The stereochemistries of C-6 (S), C-11 (R), and C-12 (S) were suggested by NOE measurements and difference spectra.

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In the course of our search for antibacterial oligostilbenes in Carex spp. (Cyperaceae), a novel tetrastilbene named kobophenol A (5), having a unique 2,3,4,5-tetraaryltetrahydrofuran unit, has been isolated from subterranean parts of C. kobomugi Ohwi. Methanol extracts of fresh roots and rhizoma of C. kobomugi were fractionated by a series of solvent partitions followed by column and thin layer chromatography to afford a major active principle, kobophenol A (5), as a pale yellow oil along with previously known oligostilbenes, miyabenol C (3) and ε-viniferin (4). All isolates showed antibacterial activity against Staphylococcus aureus. Kobophenol A (5) has a molecular formula of C_<56>H_<44>O_<13>, which corresponds to a tetrastilbene. Permethylation of 5 yielded a decamethyl ether (6). The plane structure of 5 was elucidated principally with the aid of 2D-NMR (HH-, CH- and long range CH-COSY) as well as the UV and 1D-NMR results. The EI-MS fragmentation pattern of 6 well supports this structure. The relative stereochemistry of 5 has been assigned from results of 2D PSNOESY and 1D NOE difference spectrum experiments as rel-(7aR,8aR,7bS,8bS,7cS,8cS,7dS,8dR).

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Mallotus japonicus MUELL. ARG. (Euphorbiaceae) is a deciduous tree that sprouts red-colored buds and is widely distributed in Japan. The leaves of the tree are used as a treatment for boils. We have examined cytotoxic constituents in pericarps of the tree. In a investigation of the CHCl_3-soluble fraction, which was found to show significant cytotoxic activity in the KB cell culture system, eight new phloroglucinol derivatives named mallotolerin (5), isomallotolerin (6), mallotochromene (7), butyrylmallotochromene (8), isobutyrylmallotochromene (9), mallotophenone (10), mallotochromanol (11) and isomallotochromanol (12) were isolated together with five known compounds, 1-4 and 13. Their structures have been characterized by their respective chemical and spectral data. The unambiguous assignments of ^<13>C-NMR spectra of compounds 7-10 and 12 were performed by means of 2D NMR of long rang ^1H-^<13>C shift correlation. These compounds were assessed for growth inhibiting activity against KB, human larinx (Hep-2) and lung (PC-13) carcinoma cells as well as mouse B-16 melanoma, leukemias P-388 and L-5178Y cells. Most of these derivatives were prove to be significantly cytotoxic in culture. One of them 3-(3,3-dimethylally1)-5-(3-acety1-2,4-dihydroxy-5-methy1-6-methoxybenzy1)-phroracetophenone (1) showed an excellent cytotoxicity against all target tumor cells and marked prolongation of life-span in mice bearing L-5178Y. Analysis of the DNA histogram obtained by flow cytometry showed that 1 inhibited the cell cycle progression.

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Based on the chromatographic survey which revealed wide distribution of dehydroellagitannins, geraniin (1) and mallotusinic acid (2), in Euphorbia, Mallotus, Aleurites, Sapium and Phyllanthus species of Euphorbiaceous plants, and also on the isolation of euphorbins A (3) and B (4), dimeric hydrolysable tannins, having the geraniin structure as a part of their molecules, from Euphorbia hirta, a further search of oligomers of novel structural units was carried out. Three new dimers, euphorbins C (5), D (6) and E (21) were isolated from the same plant and their structures were elucidated. Euphorbins C and D are dimers of new class which possess, in addition to the geraniin structure in their molecules as in 3 and 4, a unique new tetrameric galloyl (euphorbinoyl) group which is biogenetically regarded as the product of C-O oxidative couplings between two galloyl groups in a monomer and hexahydroxydiphenoyl group in the another monomer. Euphorbin E is the first example of the dimeric hydrolyzable tannins having a dehydroeuphorbinoyl group as a linking unit between two monomers. Euphorbin A (3) and an analogous new dimer, euphorbin F were also isolated from E. tirucallii, the latter of which was found to have structure (26). E. prostrata was found to contain a different type of hydrolyzable tannin oligomers, rugosins D (27), E (28) (dimers) and G (29) (trimer).

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Simple anthocyanins are unstable in dilute aqueous solutions around pH 4-7, but some highly acylated anthocyanins were found to be stable in such solutions. Structures of several highly acylated anthocyanins such as gentiodelphin from Gentiana makinoi, platyconin from Platycodon grandiflorum, cinerarin from Cenecio cruentus, heavenly blue anthocyanin (HBA) from Ipomoea tricolor, zebrinin from Zebrina pendula, lobelinins from Lobelia erinus, ternatins from Clitoria ternatea, etc. have been elucidated mainly by means of ^1H NMR including the NOE relief method. Stability of the anthocyanins has been assumed to come from intramolecular sandwichtype stacking of anthocyanidin nucleus and two aromatic acyl moieties. Recently, we have succeeded in obtaining ^1H NMR spectra of the anhydrobase form of some of the complex anthocyanins in a neutral solution, which enabled us to support the stacking.

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KS-506f(1), p(2), a(3), x(4), g(5) have been isolated from culture broth of Mortuerella vinacea as a potent inhibitors of calmodulin-dependent phosphodiesterase. Structures of these novel sulfer-containing depsides are described. KS-506f and p were identified to be 1 and 2. 3, 4, 5 were found to be the dimeric compounds composed by component I and II through S-S linkage, which are converted each other by disproportionation reaction. The structure of 3 was determined to be dimeric thiocarboxylic acid of component I by COLOC experiment, degradation to afford 1 by methanolysis, and chemical conversion to methylthioester (8) and cyclic diester (9). Confirmation of the structures of 4 and 5 were accomplished using COLOC data of 3-oxobutylthio ether of component II (10) to be dithioester and disulfide, respectively. Methanolysis of KS-506s gave KS-506h(6) and m(7). 6 was methyl ester of component I. The COLOC spectrum of 7 suggested that 7 is the 3-oxobutylthio ester of component I. 7 was produced on treatment 4 with Ph_3P and methyl vinyl ketone, which also assisted its structure.

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Screening of new insecticidal and acaricidal antibiotics was carried out with reference to anti-brine shrimp activity from actinomycete strains. As a result, one actinomycete isolates identified as Streptomyces sioyaensis SA-1758 was found to produce a new substance, altemicidin. Altemicidin was purified by activated charcoal, Diaion CHP-20P and Sephadex LH-20 column chromatographies. The molecular formula was determined as C_<13>H_<20>N_4O_7S by elemental analysis, mass spectra and ^<13>C NMR spectrum. The structure was determined to be (1R,2S,3aR,7aS)-4-carbamoy1-2-hydroxy-6-methyl-1-(sulfamoylacetamido)-2,3,3a,6,7,7a-hexahydro-6-azaindene-1-carboxylic acid by the spectroscopic analysis and X-ray crystallographic analysis of its derivatives. Altemicidin belongs to the monoterpene alkaloid. Altemicidin showed not only acaricidal activity but also antitumor activity. And the compound showed no antimicrobial activity.

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Two new cyclic octapeptides, named lyciumins A (1) and B (2), have been isolated from Lycii Radicis Cortex, the root barks of Lycium chinense Mill. (Solanaceae), which has been used for an antifebrile and a tonic as traditional crude drug. Their structures have been elucidated by chemical (analysis of amino acids produced by hydrolysis using mineral acid, chymotrypsin and proline specific endopeptidase) and spectroscopic (assignments of ^1H-NMR spectra by ^1H-^1H COSY and determination of sequences of amino acids by ROESY) means. The structure of 1 has been characterized as (glycyl^4Cα, tryptophan^8indole N^1)-cyclo-pyroglutaminyl-prolyl-tyrosyl-glycyl-valyl-glycyl-seryl-tryptophan. The structure of 2 has likewise been deduced to be a peptide replaced by Trp in 2 instead of Tyr in 1. These plant origin peptides are unique examples and they are novel in terms of cyclic one with linkage between the indole N^1 of Trp^8 and the Ca of Gly^4. Furthermore, they have been shown to possess inhibiting actions for renin and ACE activities, i.e. inhibitions of renin activities for 1, 19.4% (40μg/ml); for 2, 32% (40μg/ml), inhibitions of ACE activities for 1, 90.9% (100μg/R.M.); for 2, 79.0% (100μg/R.M.).

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The Indian Ocean sea hare Dolabella auricularia (Japanese name: Tatsunamigai) has been found to be an exceptionally products. In 1981, from 100Kg of animals, the nine new cell growth inhibitory substances, which were designated Dolastatin 1-9 (each ca. 1mg) were isolated. In them, only structure of Dolastatin 3 was determined to be 2 by the results of the detail NMR spectra and finally total synthesis. Subsequently, from the new Indian Ocean specimens (1600Kg) of Tatsunamigai, we isolated the most important antineoplastic substance Dolastatin 10(3), in addition to the isolation of Dolastatin 11(4),and Dolastatin 12(5), and recent isolation of Dolastatin 13(6) and Dehydrodolastatin 13(7).6_ Structure of Dolastatin 10(3), which was an unique linear pentapeptide, was recently confirmed by the total synthesis. Structures of Dolastatin 11(4), Dolastatin 12(5), Dolastatin 13(6) and Dehydrodolastatin 13(7), which were the cyclic depsipeptide, were illustrated. Evidence of structures was based on the detail 400MHz NMR 2D experiences (e.g. 'H-'H cosy, 'H-'H relayed cosy, 'H-^<13>C cosy, NOE, HMBC etc.) and the HRMS data (e.g. SP-SIMS, FAS, EI, GC-MS etc.). In this paper, the recnt results. were discussed.

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Several hepatotoxic peptides have been isolated from cyanobacteria. Microcystis aeruginosa and M. viridis produce at least nine microcystins as shown in Fig. 1. We previously established an effective isolation and analysis method for these peptides. During separation of these toxins using our method we found that the purified microcystins include consistently 15-20% of minor components (Fig. 2). In this paper we describe isolation and structure determination of the minor components that microcystins LR and RR accompany. The naturally occurring cell samples were treated according to our isolation method. The three minor components, the "small peak" from the RR fraction, and the "small" and "unknown" peaks from the LR fraction could be successfully isolated as shown in Fig. 3. The physicochemical and toxic properties of the five components thus obtained are briefly summarized in Table 1. Both small peaks of microcystins LR and RR possess the same molecular weights and amino acid compositions as the Large peaks. However, while the large peaks of microcystins LR and RR showed the same toxicity as the reported values, no toxicity was found for the small peaks of both toxins up to 1.2mg/kg. Additionally, differences were recognized in the UV and NMR spectra between the large and small peaks. Therefore, the ^1H- and ^<13>C-NMR spectra were carefully examined using COSY, HMQC and HMBC techniques (Table 2). Finally, application of ROESY could make clear that the small peaks are geometrical isomers at C-7 of Adda moiety of the parent microcystins (Figs. 4 and 5).

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In the course of our research project on plant growth regulators among microbial metabolites, we isolated two macrolide plant growth inhibitors, kaimonolide A (KA) C_<40>H_<68>O_<12> and kaimonolide B (KB) C_<35>H_<58>O_<10>, from Streptomyces No. 4155 isolated from a soil sample collected near Mt. Kaimon in Kagoshima Prefecture. Mainly by ^1H-, ^<13>C-NMR, ^1H-^1H, ^1H-^<13>C, ^1H-^<13>C long range COSY NMR and HOHAHA, the structure of the dehydrate of KA was elucidated and consequently KA was determined to be a 22-membered macrolide. Similarly the structure of KB, an 18-membered macrolide, was determined by means of the analysis of nmr spectra. In a bioassay using lettuce seedlings (Lactuca sativa L., cv. Great Lakes 366), KA inhibited root elongation by about 50% even at 0.2 ppm, and gave rise to necrosis at 0.5 ppm. On the other hand KB inhibited root elongation by about 50% at 10 ppm.

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The ethyl acetate extract of the sea hare Aplysia kurodai collected in Mie Prefecture was subjected to chromatographic separation, resulting in the isolation of a novel fatty acid metabolite (1) and the congeners [2, 3 (3a+3b)]. The major metabolite (1) was found to possess phospholipase A_2 activating activity. The planar structure of 1 was deduced by means of 2D NMR spectroscopy (^1H-^1H and ^<13>C-^1H COSY) and chemical derivatization. The octahydro derivative (6) of the metabolite (1), after acetylation, was cleaved by osmylation followed by periodate oxidation to afford two aldehydes (7, 8): the structure of 1 was unambiguously established by the detailed spectral analysis of 7 and 8. On the basis of the spectral data, the congener 2 was found to be the tetrahydro derivative of 1, while the congener 3 was shown to be a 1:1 mixture of 17, 18-dihydro-1 (3a) and 17',18'-dihydro-1 (3b).

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ACT-toxin AI and AIII are host-specific toxins produced by Alter-naria alternata tangerine pathotype causing citrus brown spot disease in USA. They were isolated from both culture filtrates and spore germinating fluid of the toxin producer and the chemical structures were elucidated to be 6 and 7, which contain the conjugated carboxylic acid moieties found in AK-toxins (4 and 5) and AF-toxins (1, 2 and 3) produced by A. alternata Japanese pear pathotype and strawberry pathotype, respectively. We suppose that ACT-toxin AI is an original form produced by enzyme system of the fungus and ACT-toxin AII is an artifact originated by photochemi-c al isomerization of ACT-toxin AI. They exibited potent toxicitiesn of only to the host-plant (citrus) but also to the other plant (Japanese pear). Conjugated acid derivatives, 13b and 13c, were also isolated from culture filtrates of ACT-toxins producer and identified witht he authentic samples isolated from both culture filtrates of AK-toxins producer and AF-toxin producer. Since the decatrienoic acid 13 had been determined to be a biological precursor of AK-toxins, biosynthetic pathway of AK-toxins, AF-toxins and ACT-toxins must be very similar as shown in the scheme.

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We have synthesized ^<13>C-labeled precursors such as glycine, methionine and glutamic acid for the biosynthesis of porphyrinoid. We also established the regioselective synthesis of ^<13>C-labeled ALA (δ-aminolevulinic acid) in the same method from ^<13>C-labeled sodium acetate, and utilize these in the study of biosynthesis of chlorophyll and bacteriochlorophyll. L-[1-^<13>C]glutamic acid-incorporated chlorophyll showed ^<13>C-enriched peaks at sp^2 region. This result shows ring carbons are derived from glutamic acid by Beale route in E. gracilis. Incorporation of [2-^<13>C]glycine to E. gracilis gave chlorophyll labeled at 10b methyl ester (52.4 ppm). In the same way, L-[^<13>CH_3] methionine was incorporated and showed enriched peak at 52.4 ppm. It indicates glycine was metabolized into methionine and was incorporated into one side chain of chlorophyll in E. gracilis. [2-^<13>C]glycine-incorporated bacteriochlorophyll showed ^<13>C-enriched peaks at sp^2 region. Also the feeding experiment of [5-^<13>C]ALA into bacteriochlorophyll showed the same result. It indicates ALA was derived from glycine by Shemin route in R. spheroides. Furthermore feeding experiment of ^<13>C-labeled ALA in 50% D_2O medium and that of ^<13>C, ^2H-doubly labeled ALA into bacteriochlorophyll showed α,β isotope-shifted peaks at ring carbons. It showes ring protons at C5a, C3, C4, C8 position of bacteriochlorophyll are derived from water in R. spheroides.

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