Diels-Alder type adducts of chalcones and dehydroprenylphenols from the Morus root bark, such as kuwanon G (1) and sanggenon C (3), are optically active and are characterized by possessing cyclohexene ring moiety in the molecule. These compounds could be classified in four types as follows: a) chalcones and dehydro-prenylflavones (or flavanones), b) chalcones and dehydroprenyl 2-arylbenzofurans, c) chalcones and dehydroprenyloxyresveratrols, and d) chalcones and dehydroprenyl-chalcones. On the other hand, relative configuration of substituents on the cyclohexene ring has been confirmed by NMR spectra, and there are two kinds of relative configurations (all-trans, cis-trans, Fig. 1). We attemped to determinate the absolute configuration of three asymmetric centers in the cyclohexene ring using CD exciton chirality methods. Mulberrofurans C (5, cis-trans) and J (6, all-trans), both showed positive spilitting Cotton effect at about 270-320nm in the CD spectra (Fig. 6 and 7). This result can be explained owing to exciton interaction between 2-arylbenzo-furan moiety and 2,4-dihydroxybenzoyl moiety. Additionally, CD spectra of 5a and 6a, which derived from 5 and 6 with LiAlH_4, showed contrary CD curve each other at 270-350nm (Fig. 8). Taking into the account these results, absolute configuration of 5 (cis-trans) was established 3"R, 4"R, 5"S, while 6 (all-trans) was 3"S, 4"R, 5"S (Fig. 13 and 14). Furthermore, compound 10 (all-trans), which derived from kuwanon L (9) by alkali degradation, was led to cis-glycol (11) and then led to mono-p-bromo-benzoate (11a) (Chart 1). In the CD spectrum of 11a, Cotton effect owing to exciton interaction between p-bromobenzoyl moiety and 2-hydroxy-4-methoxybenzoyl moiety suggested that the absolute configuration of compound 10 is 3"S, 4"R, 5"S (Fig. 16). This result is not inconsistent with the result of mulberrofuran J (6).
Liverworts are rich sources of both terpenoids and aromatic compounds. In the course of our systematic investigation of the chemical constituents of the liverworts, the minor components of Marchantia polymorpha (Zenigoke in Japanese) (3.5kg), collected in Duisburg (W. Germany), have been studied and several macrocyclic bis(bibenzyls) were isolated as a methyl ether or an acetate and the structures determined by extensive high resolution NMR spectroscopic methods. ^1H and ^<13>C NMR data were fully assigned by using 2D (C/H correlation and long-range C/H correlation) and LSPD techniques. Biological activities of these compounds are listed in the Tables 3 and 4.
Recently we have found a novel antifungal substance possessing growth self-inhibiting activity from cherry brown rot fungus M. fructicola, which was named chloromonilicin (1). 1 was isolated from the culture filtrate of this fungus, and its novel structure with β-chloro-α,β,γ,δ-unsaturated seven-membered lactone ring was determined on the basis of chemical and spectroscopic evidence. In addition, a new xanthone metabolite obtained was identified as 4-chloropinselin (7), suggesting 7 to be a probable precursor of 1. These bromine congeners, bromo-monilicin (8) and 4-bromopinselin (9), were prepared for biological activity and biosynthetic studies by inoculation of M. fructicola into a KBr-containing medium. Antimicrobial activity of 8 was about one-fourth of that of 1. Biosynthetic incorporation of 9 into 8 was accomplished. To the best of our knowledge, this is the first instance of Baeyer-Villiger type oxidation of phenolic metabolites. In addition to 8 and 9, a new benzophenone derivative, which was named moniliphenone, was isolated and identified as 12. Deuterated 12's were prepared by treatment of 12 with methanol-d_4 in the presence of catalytic amount of sulfuric acid and was shown to be straightforwardly incorporated into 7 and 1. Furthermore, an anthraquinone pigment, chrysophanol, was isolated as a probable precursor of 12. These observations show that the biosynthesis of 1 in M. fructicola proceeded in the sequence chrysophanol (13)→12→7→1.
Alternaria solani, causal organism of early blight disease on potato produces several metabolites whose structures have been clarified. Recently presence of two other host specific toxin in the culture filtrate of the fungus has been reported. From the filtrate four new phytotoxic compounds, solanapyrones A(1), B(2), C(3) and zinnolide(4) have been isolated. These structures have been elucidated on the basis of spectroscopic data and chemical reaction. The structure of 2 was confirmed by correlation with 1. Further, zinnolide(4) was converted to a known phthalide 9 to prove the structure. Absolute configuration of solanapyrone A(1) was determined by the application of CD exiton chirality method to the dibenzoate derivative 13. In order to confirm the structure and to develop an effective synthetic method, synthesis of solanapyrone A(1) has been completed as follows. The pyrone moiety, the thioacetal 16, was prepared from dehydroacetic acid through 5 steps, and the diene moiety, the aldehyde 19, was derived from sorbic acid. Aldol condensation of 16 with 19 and dehydration of the product afforded the triene 20, which was subjected to intramolecular Diels-Alder reaction heating at 170-190℃ to yield a mixture of two products. The mixture was treated with thallium nitrate to give (±)-solanapyrone A(1), and the diastereoisomer 21 in a ratio of 2:3. The spectroscopic data of (±)-1 are completely identical with those of natural sample.
Alternaria alternata strawberry pathotype cause Alternaria black spot desease of strawberry (Morioka-16). This pathogen produces three host-specific toxins, AF-toxin I, II, and III (1, 2, and 3). These toxins expressed different biological activities as shown in Table 1. These chemical structures were determined by chemical derivation and spectroscopic analyses. Each toxin were a mixture of three stereoisomers. Major component was (2E,4E,6Z)-stereochemistry (1a, 2a, and 3a) in each case. The structure of 2b was very similar to those of AK-toxin I and II (6 and 7). (2E,4Z,6E)-stereoisomers (1b, 2b, and 3b) were minor component in our AF-toxin series. The presence of secondary hydroxy group (OH) seems to be very impotant for the toxicity to japanese pear (Nijisseiki).
Emericella striata 80-NE-22 is a fungus (Ascomycetes) isolated from cumin collected in Nepal. From its mycelial extract, novel macrocyclic epipolythiodioxopiperazines, emestrin (1) and emestrin B (2), were isolated along with shamixanthone, emericellin, sterigmatocystin, and paxilline. The structures of 1 and 2 were determined on the basis of the spectroscopic investi-gation of their derivatives and X-ray crystallography of emestrin methanol solvate. Emestrin (1) has strong antifungal activity, especially against Microsporum spp. and Trichophyton spp. Three compounds relating to 1, dethiosecoemestrin (3), aurantioemestrin (4) and violaceic acid (5), were isolated from the culture filtrate, and their structures were elucidated by the spectroscopic investigation. Isolation of dethiosecoemestrin (3) and aurantioemestrin (4) along with emestrin (1) and emestrin B (2) from the same fungus suggested the biodegradation of epidithiodioxopiperazines into trioxopiperazines. Emestrin (1) was also isolated from several species belonging to the genus Emericella; E. foveolata, E. quadilineata, E. acristata, and E. parvathecia.
Boxazomycins A, B and C are antibiotics isolated from Pseudonocardia species. Their structures have been elucidated as 1, 2 and 3, respectively, by use of modern NMR technics including INADEQUATE, NH-COSY, NH-COLOC and CP/MAS. In the last spectrum, signals of carbons which are bonded to a nitrogen atom are splitted (or broadened), and this technic has been found to be very useful in the present work. In the INADEQUATE spectrum of 4, connectivity peak between C-4' and 5' was not observed, because the chemical shifts of both the carbons are very close. This problem was overcome by converting boxazomycin A(1) into diphosphate(7), the ^<13>C-NMR spectrum of which revealed the long-range coupling(Jcp) from 5'-O-P to C-4'. This finding suggests that C-4' is connected with C-5'. Assignments of all the nitrogen atoms were done by analysis of NH-COSY and NH-COLOC spectra. The structure of boxazomycins is unique in that a benzoxazole moiety is combined with a pyrimidine ring.
Carbazomycins A (II) and B (I), the first antibiotics having a carbazole nucleus, were produced by Streptoverticillium ehimense and inhibit mainly the growth of phytopathogenic fungi. Their structures were determined by spectral and chemical means including X-ray analysis. The present paper describes the biosynthetic studies on the major component, carbazomycin B (I), performed by feeding experiments with ^<14>C- and ^<13>C-labeled precursors followed by ^<13>C-NMR measurements. The results summarized in Fig. 7 indicate that tryptophan is incorporated into the ten carbons of the carbazole nucleus of I except for C-1 and C-2, the methoxyl group is introduced from methionine, C-1 and C-10 of I are derived from acetate, and C-2 and C-11 of I are derived from C-2 and C-3 of pyruvate or alanine which are also incorporated into C-1 and C-10 of I via acetate. During the biosynthetic studies of I, we found six minor components, carbazomycins C (III), D (IV), E (V), F (VI), G (VII) and H (VIII). Their structures have been elucidated by ^1H- and ^<13>C-NMR spectroscopy and chemical correlation, and the structures of III and VII have been confirmed by X-ray analysis. Carbazomycins G (VII) and H (VIII) have a unique structure with partly oxidized carbazole nucleus.
Allosamidin(1), a novel insect chitinase inhibitor, was isolated from the mycelium of Streptomyces sp. Allosamidin showed strong inhibitory activity against the chitinases of the silkworm, Bombyx mori, in vitro, and also prevented its larval ecdysis in vivo. The structure of allosamidin was elucidated as 1 by acid hydrolysis experiments and analysis of 2D-NMR spectra. 1 is a basic pseudotrisaccharide consisting of N-acetyl-D-allosamine and a novel aminocyclitol derivative(3), termed allosamizoline. Methylallosamidin(2) was isolated from the mycelium of the unidentified actinomycetes and characterized as 2 by spectral comparison with 1.
During the cource of our screening program for new antibiotics, herbimycin C (1), trienomycins A (6), B (7) and C (8). awamycin (9) and hitachimycin (stubomycin) (10) were isolated and characterized. Each antibiotic except for 10 possesses macrocyclic lactam structure containing benzenoid or naphthalenoid moiety as a chromophore. Structures 1 and 6-9 were elucidated mainly through the comparative NMR analysis with the known ansamycin antibiotics and the structure of 10 was established by the chemical degradations. Further, "Celmer's model" which was applied to the stereochemistry and biogenecis of macrolide antibiotics was applied to those of ansamycin antibiotics such as macbecin I (14). naphthomycin A (15). rifamycin B (16) and streptovaricin C (17) in which the absolute configurartions have been established. Consequently, the absolute configurations of herbimycin A (2) and 9 except for C-6 and C-7 were proposed as shown in Fig. 2. through the model.
Sporaviridins (SVD) are basic glycoside antibiotics produced by Streptosporangium viridogriseum. They exhibit strong inhibitory activity against Gram-positive bacteria, acid-fast bacteria and trichophyton. We have already reported on the structural determination of the three constituent pentasaccharides (viridopentaose A, B and C) of N-acetylsporaviridins (N-Ac-SVD). Recently, we succeeded in the isolation of major components and the structure elcidation of N-Ac-SVD. N-Ac-SVD are composed of six components (N-Ac-SVD-A_1, A_2, B_1, B_2, C_1 and C_2) whose molecular weights are all about 2200. Treatment of each component of N-Ac-SVD with DBU gave two pseudoaglycones (N-Ac-pAG-U and L) and one of three viridopentaoses. These pseudoaglycones are an epimeric pair with respect to C-13 and have still two sugar moieties (D-glucose and N-acetyl-L-vancosaminide). A variety of degradative reactions and spectroscopic analyses of N-Ac-pAG-U and L showed that the aglycone moieties are 34-membered polyol macrocyclic lactone with a hemiketal system and three double bonds including a conjugated diene. Finally, the total structures of N-Ac-SVD were determined as shown in Fig. 1.
Rhizoxin (1a) is a novel 16 membered anti-fungal and anti-tumor macrolide isolated from Rhizopus chinensis, the pathogen of the rice seedling blight. The fungus produced also the homologues, 1b-3b, 4 and 5. Biosynthesis of rhizoxin was studied and incorporation of 1 mole serine, 12 moles acetate and 6 methionin methyls was established by feeding experiment using ^<13>C labeled precursors. Rhizoxin inhibited the mitosis the tumor cells in a manner similar to that of vincristine and showed similar chemoterapeutic effect to those of vicristine. The mode of action of rhizoxin was investigated. In vitro polymerization of microtubule proteins purified from prcine brains was completely inhibited at a 10×10^<-6>M concentration of rhizoxin. Activity of rhizoxin against tubulin polymerization was compared with those other antitubulin drugs such as colchcine, vinblastine and ansamitocin P-3. The homologues of rhizoxin, 1b-3b, also inhibited grouth of rice seedling roots and of P. oryzae similarly. Anti-tubulin activities of these compounds were also the same extent as rhizoxin.
In the course of our screening program for biologically active substances, we found that Streptomyces tsukubaensis No. 9993 produced a potent immunosuppressant, tentatively designated FR 900506. The structure of FR 900506, C_<44>H_<69>NO_<12> mp127-9°, has been elucidated as shown 1 to be 23-membered-ring lactone having allylic and, cyclohexyl propenyl groups on the ring by means of chemical degradation and spectral evidences. The absolute stereostructure of 1 has been determined by X-ray diffraction study and by isolating (s)-pipecolinic acid(2) from 6N HCl hydrolysis products of 1.
The structure of a new polyether antibiotic 6270 (C_<44>H_<76>O_<14>, mp. 115-118℃, [α]^20_D-11.5° (c 1.0, MeOH)), which was produced by Nocardiopsis sp. 6270, was determined as shown in Fig. 6 by the use of new NMR techniques such as heteronuclear multiple bond correlation spectroscopy (HMBC) and 2D homo Hartman Hahn (2D HOHAHA). HMBC proved to be a very powerful method for structural elucidation of complicated molecules, especially polyketide metabolites consisting of propionate units.
For many years, it has been known that the molting of crustaceans is induced by the molting hormone (MH) released from the molt gland, Y-organ; furthermore, the molting is controlled by the molt-inhibiting hormone (MIH) produced in the X-organ of the eyestalks (ES). The chemical study on MIH from eyestalks of crabs has led to the isolation and characterization of 3-hydroxy-L-kynurenine (3) as one of the compounds exhibiting molt-inhibiting activity in the in vitro bioassay system. Xanthurenic, acid (4), a metabolite of 3-hydroxy-L-kynurenine, showed strong MIH activity in the bioassay. Thus, we estimated that xanthurenic acid formed during the incubation acts directly as the inhibitor of ecdysone biosynthesis.
During our studies on bioactive metabolites from marine organisms, we have examined pharmacological effects of extracts of various tunicates. A series of β-carboline compounds with specific activities has been isolated from tunicates of Eudistoma species. They are eudistomin A〜Q isolated from the Caribbean tunicate Eudistoma olivaceum and eudistomidin-A isolated from the Okinawan tunicate Eudistoma glaucus. These structures were assigned on the basis of the spectral data including ^1H and ^<13>C NMR, as simple β-carbolines (eudistomin D, J, N and O), 1-pyrrolinyl-β-carbolines (eudistomin G, H, I, P, Q and eudistomidin-A), 1-pyrrolyl-β-carbolines (eudistomine A and M) and tetrahydro-β-carbolines containing a novel oxathiazepine ring (eudistomin C, E, F, K and L), respectively. Antiviral activity in vitro was found for endistomin C〜E, H, I, K and L, while eudistomin A, D and J induced Ca^<2+> release from sarcoplasmic reticulum like caffeine. Bromoeudistomin D (BED), a monobrominated derivative of eudistomin D, was 400 times more potent than caffeine in the Ca^<2+>-releasing activity. Eudistomidin-A exhibited potent calmodulin antagonistic activity to be 15 times more potent than W-7, a well-known calmodulin antagonist. The structures of these β-carboline tunicate metabolites will be discussed in relation to their pharmacological activities.
Constituents of two Japanese species of Ophiorrhiza (Rubiaceae) were studied. Ophiorrhiza kuroiwai Mak. was found to contain four known alkaloids, i.e. harman (I), camptothecin (II), 9-methoxycamptothecin (III), 10-methoxycamptothecin (IV), and three new alkaloids, i.e. lyalosidic acid (V), ophiorine A (VI) and ophiorine B(VII). From O. japonica Bl. three known alkaloids, i.e. harman (I), lyaloside (VIII), and 6-hydroxyharman (XVI), and six new compounds, i.e. lyalosidic acid (V), ophiorine A (VI), ophiorine B (VII), 10-hydroxylyalosidic acid (XVII), ophiorine A-methyl ester (XIV), and ophiorine B-methyl ester (XV), were isolated. Synthesis of lyaloside (VIII) using L-tryptophan (IX) and secologanin (X) was made. Interconversion between ophiorines A, B and lyaloside (VIII) was also carried out. Action of emulsin on lyalosidic acid (V) or lyaloside (VIII) was found to give rise to harman (I) as the result of a facile fragmentation of the intermediary hydrolysis product. Similarly, 6-hydroxyharman (XVI) was given from 10-hydroxylyalosidic acid (XVII). These results suggest a possibility that harmans widely distributing in Rubiaceous plants accompanying monoterpenoid indole alkaloids may be secondarily formed from β-carboline type monoterpenoid indole alkaloids through this or similar mechanisms.
Soles of the genus Pardachirus possess ichthyotoxic defense secretion. Three homologous peptides, pardaxin P-1 (1), pardaxin P-2 (2), and pardaxin P-3 (3), have been isolated from the secretion of P. pavoninus, where the ichthyotoxic and shark repellent steroid glycosides, pavoninins, had already been isolated. The amino acid sequence of each pardaxin P, a linear peptide comprising 33 conventional amino acid residues, was obtained as shown in Fig. 3 by means of an automated Edman degradation, and was corroborated by solid-phase synthesis thereof. The peptides are ichthyotoxic, hemolytic, and markedly surfactant. They are also indicated to be shark repellent. The surfactant property is clearly indicated by their amphiphilic sequences and also by their probable secondary structures. These peptides show physical, pharmacological, and structural similarities to a bee venom, melittin, and are expected to constitute a good pharmacological tool, when used in combination with melittin, to probe mechanisms of action involving permeabilization of phospholipid bilayer membranes.
In search of new bioactive substances from marine organisms, we have investigated the chemical constituents of two Okinawan marine sponges of Theonella sp. (Theonellidae). 1) Aminosesquiterpenes From the acetone extract of a marine sponge of Theonella sp. (Theo-84-HM-1), which was collected in the coral reef of Hatomajima, Okinawa Prefecture, four new aminosesquiterpenes: aminobisabolene (1), aminobisabolenol (2), isoaminobisabolenol-a (3), and isoaminobisabolenol-b (4), were isolated. Based on the chemical and physicochemical evidence, including the X-ray analysis of the bromoacetate (1a) of 1, their absolute stereostructures have been determined. 2) Theonellapeptolides From the EtOAc soluble portion of the acetone extract of a marine sponge of Theonella sp. (Theo-83-ZM-1), which was collected in the coral reef of Zamamijima, Okinawa Prefecture, five new peptolides: theonellapeptolides Ia (10), Ib (11), Ic (12), Id (13), and Ie (14), were isolated. These peptolides inhibit development of the fertilized eggs of the sea urchin Hemicentrotus pulcherrimus. The structure of the major peptolide, theonellapeptolide Id (13), has been determined on the basis of chemical and physicochemical evidence. Theonellapeptolide Id (13) is a characteristic tridecapeptolide which comprises N-methyl and D amino acids in high ratio.
Trichosporins are a peptide mixture isolated from culture filtrate of Trichoderma polysporum, which is antagonistic to Leutinus edodes. The pure component, trichosporin (TS)-B-V, has been obtained by preparative HPLC from Trichosporin mixture. This novel peptide has been shown to consit of one acetylated N-terminal residue, 19 amino acids and a phenylalaninol, C-terminal amino alcohol. Moreover, since this peptide contains a high proportion of α-aminoisobutyric acid (Aib) and has a moleculor weight near 2000, it belongs to the class of peptaibophols. Sequence determination (Fig. 8) was based on positive ion FAB mass spectrometry. High-field NMR data with two-demensional NMR assignment techniques were very useful in this study. Furthermore, sequences of the congeners, TS-B-I-a, -III-a, and -III-d were elucidated as shown in Fig. 8.
The structure of FR900359, a novel cyclic depsipeptide from Ardisia crenata sims that shows inhibition of platelet aggregation and activity to decrease the blood pressure, has been determined as 3-acetamide-22-benzyl-10-[1-(3-hydroxy-4-methyl-2-propionamidopentanoyloxy)-2-methylpropyl]-4-isopropyl-7-(1-methoxyethyl)-19-methylene-8,13,14,16,20-pentamethyl-1,5-dioxa-8,11,14,17,20-pentaazacyclodocosane-2,6,9,12,15,18,21-heptone by ^1H- and ^<13>C-NMR and Mass spectrometry. FR900359 consisted of ten units: alanine, N-methylalanine, β-hydroxyleucine, 3-phenyllactic acid, acetic acid, propionic acid and novel amino acids N-methyldehydroalanine and N,O-dimethylthreonine (1:1:3:1:1:1:1:1, by molar ratio). Of particular significance of this cyclic depsipeptide are the novel amino acid constituents N-methyldehydroalanine and N,O-dimethylthreonine. As far as we know, the former has been known only in a toxin from the blue-green alga, Microcystis aeruginosa, and the latter has not yet been found in nature till now.
In the course of our search for bioactive metabolites from a Japanese marine invertebrates, we found that lipophilic extract of a sponge Discodermia calyx showed a strong activity both in the starfish egg assay and in the cytotoxicity test, which led us to isolate active components, named calyculins A-D (1-4). Calyculins inhibit cell division of fertilized starfish and sea urchin eggs at very low concentrations (MIC: 0.01, 0.01, 0.005, and 0.005μg/ml, respectively; H. pulcherrimus). Calyculin A is also highly cytotoxic to tumor cells (IC_<50>: 1.75×10^<-3>μg/ml; L1210). The structure of the major component, calyculin A(1) was determined mainly by 2-D NMR and X-ray crystallographic analyses, while those of the minor components 2-4 were elucidated by comparing their spectral data with those of 1. Calyculins are extraordinary spiro-ketals containing an unusual array of functional groups such as amide, oxazole, phosphate, nitrile moieties as well as tetraen system bearing the vicinal dimethyl groups. Roles and biosynthesis of this new class of natural products are interesting problems.
Bryostatins 12 (12, 3.7mg) and 13 (13, 0.7mg) were isolated from〜1000kg of the marine bryozoan Bugula neritina (Linnaeus) found in the Eastern Pacific Ocean (California). The new bryostatins led to strong cell growth inhibitory (PS cell line ED_<50> 0.014 and 0.0054μg/mL) and antineoplastic activity (for bryostatin 12, a 47-68% increase in life extension at 30-50μg/kg) against the murine P388 lymphocytic leukemia (PS system). In addition to the previously known constituents, bryostatin 1(1), 2(2) and 3(3), of Bugula neritina from this ocean area the presence of bryostatins 8 (8, 13.2mg) and 9 (9, 16.4mg) were also established. A detailed series of ^1H-^1H-COSY, 2D-J-resolved, and ^1H-13-C-2D-shift correlated NMR experiments combined with ^<13>C-NMR and SP-SIMS studies were employed to elucidate the structures of bryostatins 12 (12) and 13 (13).
The lipophylic extract of the cultured terrestrial bluegreen alga Scytonema pseudohofmanni Bharadwaja contains five novel cytotoxic macrolides, scytophycins A (1), B (2), C (3), D (4), and E (5). The structures of 1-5 have been determined mainly from spectroscopic data. The relative stereochemistries of the scytophycins are based on an X-ray crystallographic analysis of a transformation product of scytophycin C (8) and comparison of the ^1H and ^<13>C NMR spectra of 1-5. The absolute stereochemistries of the scytophycins have been deduced from a circular dichroism study of the dibenzoate ester (11) obtained in five steps from scytophycin B and comparison of the CD curves of 1-5. These novel secondary metabolites appear to be biogenetically related to the marine natural products swinholide A (12), ulapualide A (13), and kabiramide C (14).
Diarrhetic Shellfish Poisoning, which is caused by ingestion of bivalves fed on toxic dinoflagellates, has become one of the largest natural food poisonings from marine source. The poisoning has broken out at various places in Japan as well as in Europe and a number of the total patients has exceeded ten thousand so far. The authers have already identified two toxins responsible for the poisoning, DTX1 (II) from Japanese mussels and okadaic acid (I) from Europian mussels. In the present work, a main toxic constituent of Japanese scallops has been elucidated to be 7-O-acylDTXl. Its acyl moiety consists of various unsaturated fatty acids (C18:4, C20:5, C22:5 etc.) and the highly unsaturated fatty acyloxy group seems to play a substantial roll in bioactivity of the toxin. Causative organism of the poisonings in Europe has identified as a dinoflagellate Dinophysis acuminata because okadaic acid was detected in the extract of the alga by a fluorometric HPLC analysis. A novel diol ester of okadaic acid (PLT1a, VIII) was isolated from a cultured dinoflagellate Prorocentrum lima. Five novel diol compounds (X-XIV) were also obtained from a hydrolysate of the ester fraction.
Large number of pharmacologically active natural products have been isolated from the marine organisms. Since 1981, we have carrying out the investigation of pharmacologically active compounds from marine organisms forcusing on sponges which collected in southern Pacific water such as Australia, Okinawa and Palau. We isolated many pharmacologically active compounds from the marine invertebrates. In this paper we have tested aldose reductase inhibitor assay to about 100 species of Palauan sponges. Aldose reductase inhibitor is one of the most important therapeutical use for galactsemic cataract. Extracts of the sea sponges Dysidea sp., Ircinia ramosa and Dactylospongia metachromia showed aldose reductase inhibitory activities at the concentration of 400μg/ml. Active principles were polybromo phenol(1), palinurin(2) and sesterterpene lactone(4) respectively. We isolated the aldose reductase inhibitory active new sesterterpenoidal acid(5) from the other Dysidea sp. which structure was elucidated by spectroscopic method.
In our continuing studies on the marine red algae of the genus Laurencia, we newly investigated the constituents of Laurencia obtusa collected at Teuri Island, Hokkaido, Japan. This species is a prolifie source of halogenated metabolites and several halogenated sesquiterpenoids, diterpenoids and C_<15>-nonterpenoids have been isolated. A marked variation in the major chemical constituents from this species collected at different locations has been observed. The crude extracts of Japanese species exhibited the strong cytotoxic properties (ED_<50> of 0.18μg/ml) against P388 cells and ten new compounds 1, 2, 4, 6, 8, 11, 12, 13, 14, 15 have been isolated together with known compounds, humulene and thyrsiferol. The structural determination of the new compounds except for 6 was performed by chemical and spectroscopic methods and the structure of the remaining compound 6 was elucidated by X-ray crystallographic method. Bromo cyclic ethers consisting of squalene carbon skeleton 8, 11, 12, 13, 14, 15 were found to show a remarkably cytotoxic properties (ED_<50>; 0.3〜100ng/ml) against P388 in vitro cell line.
On the constituents of Asclepiadaceous plants, a couple of Cynanchum sp. plants were investigated. We wish to report here the structures of the new compounds, atratogenin A(1) from C. atratum, cynajapogenin A(2) from C. japonicum, neocynapanogenin A(13) of the aglycone of neocynapanoside A(14) from C. paniculatum, and boerharigenin A(5) and B(6) from C. boerharifolium. Both atratogenin A(1) and cynajapogenin A(2) contain a 2,3 disubstituted furan ring which may be derived from the C-15 aldehyde and C-20 oxo group formed by oxidative cleavage at C-14,15 bond of hypothetical precursor such like 14β-hydroxy pregnenolone (4) which is isolated from C. paniculatum. The latter compoud(2) lacked C-18 methyl group. Neocynapanogenin A(13), an aglycone of 14, has an exo α,β unsaturated five membered lactone ring in addition to the nine membered lactone like glaucogenins type compound. Both 5 and 6 have the same substitution system in the ring D and defferent in that the former has benzoyloxygenated C-19 methyl. A plausible biogenetical pathway for pregnane type aglycones of the Cynanchum plants is given in the chart 1.
Mechanisum of the diazomethane degradation for the sugar-aglycone linkage of the gypsogenin 3-O-glycoside(1) was studied and the useful degradative reaction was applied to the structure determination of quillayasaponin. Since three kinds of functional groups of 1, the 4α-CHO group in the aglycone, COOH and 4-OH group in the glucuronic acid were presumed to contribute to the degradative reaction, the necessarity of the each functional group was examined using a model compound 7, and only the 4α-CHO group was showed to be essential. By taking the above evidence and the usual reaction products of aldehyde with diazomethane into account, the reaction must be proceeded through such a oxide intermediate as shown in the scheme 6. QS-III(25), a major acylated genuine bisdesmoside of the so-called quillayasaponin, afforded a 28-O-glycosidal triterpenoid(29) still possessing acyl moiety by this diazometane degradation. On the basis of chemical and spectral evidence, especially, comparison of the ^<13>C NMR spectra of 29 and its desacyl compound(30)(Table 1), the site of linkage of acyl moiety of 29 was determined. The structure of 25, therefore, was complex acylated oligosaccharide as shown in scheme 8.
We had obtained five steroidal glycosides including solamargine and solasonine from the fresh immature berries of Solanum nigrum L. Now, we have isolated six components SN-a-f (1-6) from the berries immersed in cold MeOH during two years without crush of the plant bodies. They have been shown to be the steroidal alkaloids oxidized selectively at C-12 or/and C-27, and are recognized to be derived from solasonine and solamargine probably by reaction of the included enzymes. While, two novel glycosides, TA-a (11) and TA-b (12), have been obtained from the fresh berries of Tubocapsicum anomalum Makino and their structures have been characterized as new C_<28> steroidal lactone glucosides possessing a novel side chain with the C-21 methyl group fused to the lactone ring such as acnistin type.
In the course of searching for the physiologically active substances in the plant pittosporum tobira Ait (Japanese name "Tobera") we have isolated six new sesquiterpene glycosides designated as pittosporanosides A_1, A_2,A_3,A_4,A_5, and A_6 as repellent active substances against the blue mussel Mytilus edulis from the fresh leaves. These structures were determined based on chemical and spectral evidence and X-ray crystal analysis. Pittosporanosides are novel glycosides consisting of viridiflorol as aglycone and fucose, arabinose or unique sugar substituted with angeloyl or senecioyl groups as sugar moieties, as shown with (1), (2), (3), (9), (10), and (11) respectively.
The efforts to study natural products for highly oxygenated sesquiterpenelactones, have resulted as obtained three novel guaianolides, Ezoartemin(II), Ezomontanin(IV), Dihydroezomontanin(V), and a novel eudesmanolide, Ezoalantonin(VII) from Artemisia feddei and A. momtana (Ezoyomogi in Japanese) both of the Compositae fam. The conformational structure of (II) has been decided by the hydrogenation of (I) which had confirmed by X-ray analysis. The configuration of (VII) has been cleared as 1,α ax, 2,α eq, and 3,α-ax, 4,α eq-diepoxyalantolide by X-ray analysis. Both of (II) and (VII) have two epoxy groups and each group is located to the same side of the same ring (A-ring). The conformational skeltones of (IV) and (V) are the same as (I), moreover, each compound has a peroxide type (-O-O-) oxygen bridge between C-1 and C-4 positions.
A large amount of 20-hydroxyecdysone was orally administered to larvae of the tobacco budworm, Heliothis virescens, in order to investigate its detoxification mechanisms. Four major relatively nonpolar metabolites were isolated from their frass. These compounds were identified as the 22-linoleate, 22-palmitate, 22-oleate and 22-stearate of 20-hydroxyecdysone using various forms of spectroscopy, including NMR. This is the first report of this type of metabolites from an insect.
When the (E,E)-farnesol was enzymatically synthesized from an equimolar mixture of 3,3-dimethylallyl pyrophosphate (DMAPP) and (E)- or (Z)-[4-^2H]isopentenyl pyrophosphate (IPP) in the presence of iodoacetamide (IAA) with the cell-free extract of Pisum sativum, occurrence of the unusual addition of allylic residue to the re-re face of the carbon-carbon double bond of IPP was found. This unusual addition was indicated by the chirality of C-4 and C-8 of the deuterated farnesol obtained. The elimination of the pro-2R hydrogen of IPP occurred during the enzymatically formation of farnesol from an equimolar mixture of DMAPP and (R)- or (S)-[2-^2H]IPP by incubation with the same enzyme solution as above in the presence of IAA. These findings indicated occurrence of the anti elimination of the pro-2R hydrogen atom. The addition of the allylic residue to the re-re face of the carbon-carbon double bond of IPP as well as the anti elimination of the pro-2R hydrogen atom are the first unusual examples in E-prenyl chain elongation in the biosynthesis of (E,E)-farnesol and squalene.
Purine alkaloids (caffeine and other methylxanthines) are important nitrogen compounds in non-alcoholic beverages prepared from caffeine-containing plants, i.e., cocoa, coffee, cola, guarana, mate, tea and so on. We have demonstrated that caffeine is synthesized in tea and coffee plants through the steps: purine nucleotides in nucleotide pool (AMP and/or GMP→IMP→XMP)→xanthosine→7-methylxanthosine→7-methylxanthine→theobromine→caffeine. Evidence was obtained that purine biosyntheses de novo and by salvage pathways are separately compartmentalized in tea shoot tips. Administration of [8-^<14>C]hypoxanthine or [8-^<14>C]guanine showed a preferential incorporation of ^<14>C into the GMP of RNA, while there was more incorporation of [1-^<14>C]glycine or [^<14>C]methylamine into AMP of RNA than GMP of RNA. Further, after administration of a 'pulse' of [8-^<14>C]adenine, almost all of the [8-^<14>C]adenine supplied disappeared by 30h, and ^<14>C-labeled caffeine and purine nucleotide (AMP and GMP) synthesis increased throughout the experimental period, whereas the ^<14>C of xanthosine, 7-methylxanthosine, 7-methylxanthine and theobromine increased during the first 10h incubation period, followed by a steady decrease. Extracts prepared from tea leaves or fruits and callus cultures of coffee with Polyclar AT contained three methyltransferase activities catalysing the transfer of methyl groups from S-adenosylmethionine to xanthosine, producing 7-methylxanthosine, to 7-methylxanthine, producing theobromine, and to theobromine, producing caffeine, respectively. Similar extracts from coffee fruits or callus cultures exhibited 7-methyl-N^9-nucleoside hydrolase activity, which is responsible for the hydrolysis of 7-methylxanthosine to 7-methylxanthine and D-ribose. The metabolism (biodegradation), physiological significance and ecological function of purine alkaloids in tea and coffee plants are also discussed.
A genaral synthetic method of D-hexoses and D-pentoses with chirally deuterated hydroxymethyl groups was presented. The method involves two key steps of a stereospecific photobromination of 1,6-anhydro-D-hexopyranose or 1,5-anhydro-D-pentofuranose derivatives and a deuteride reduction of C6-exo (or C5-exo) brominated sugars with n-Bu_3SnD or LiEt_3BD. These deuterated sugars are strongly expected to be useful not only for the biosynthetic or enzymatic studies on carbohydrates but also for their NMR or Mass analyses. Here, we applied our deuterium labeling method to the study on the stereochemical patlways of galactose oxidase reaction and conformational analyses of mono-, di- and trisaccharides in solutions. The galactose oxidase (Dactylium dendroides) reactions of 100% e.e. (6S)- and (6R)-(6-^2H_1)-Me-β-D-galactopyranosides revealed that there were two mechanisms for the dehydrogenation process: an efficient pro-S hydrogen atom specific and far less efficient nonspecific or pro-R specific oxidations. ^1H-NMR studies on the mono, (1→6)-disaccharides and branched trisaccharides with chirally deuterated hydroxymethyl groups clarified conformational preferences about the C5-C6 bond of these sugars in aqueous solution. The conformations were discussed in terms of the configurations at C1, C2 and C4 positions and also of glycosidic linkages.
During the course of our studies to clarify the photobleaching process of rhodopsin (1) by chemical methods, two kinds of rhodopsin analogues [(4) and (18)] were synthesised. The former (4) was prepared from 11-cis-locked cyclopentatrienylideneretinal (5) which has a high degree of coplanarity, particularly in the C_9-C_<14> triene part. The latter (18) was derived from the 9-cis bicyclic retinal analogue (15) which is the first 6s-cis-fixed example in the retinal analogues. A circular dichroism (CD) spectrum of (4), having a non-twisted structure around a 12s-trans bond, showed a negligible α-band. A comparison of the CD data for rhodopsin and cycloheptatrienylidenerhodopsin (12) with our results shows that the presence of the α-band in the induced CD of rhodopsin is due to the twisted 12s-bond in the chromophore. The conformationally 6s-cis-fixed 9-cis-rhodopsin analogue (18) exhibited an absorption maximum at 539nm, the longest wavelength observed so far for visual pigment analogues with the chromophore possessing the same number of conjugated double bonds as that of 9-cis-rhodopsin. In addition, (18) showed an interesting CD spectrum [α-band: 527nm(+16), β-band: 328nm(-8.2)]. A negative sign for the β-CD-band has not been reported so far for artificially prepared visual pigments. Photochemical behaviours of this pigment (18) at low temperatures suggest that a structural change in the neighbourhood of the β-ionone ring seems to be necessary for the conversion of bathorhodopsin to lumirhodopsin.
In the field of the synthesis of polyoxoantibiotics or the related polyfunctional natural products, the use of chiral synthon is quite advantageous. We now report the synthesis of optically active 3-hydroxy-2-methyl propionate derivative I, based on the asymmetric reduction including immobilized microorganisms with prepolymer and kinetical resolution with industrialized enzyme (lipase). 1. Determination of absolute structure of (-)-oudemansin B (1) (-)-oudemansin B was synthesized from the two different chiral synthons, hydroxy lactone 5a and (2R,3S)-epoxy ester 20b obtained by microbial asymmetric chiral induction method. Natural product was found to possess the 9S, 10S absolute configuration. 2. Asymmetric reduction with immobilized microbial cells by prepolymer method Asymmetric reduction of 2-methyl 3-oxopropionate derivative II with immobilized microbial cells was carried out. It was found that by proper selection of prepolymer in the stage of immobilization, chemical and optical yields were improved significantly compared with the use of native microorganisms. 3. Highly enantioselective hydrolyses of 3-hydroxy or acetoxy 2-methyl esters with industrialized lipase Highly enantioselective hydrolyses of 3-hydroxy or acetoxy 2-methyl esters with lipase Candida cylindracea MY-50, lipase "Amano A" and lipase "Amano A-6" isolated from Aspergillus niger, are described.
As a part of our synthetic studies on biologically active compounds consisting of five membered ring, we have succeeded in the synthesis of prostaglandins and the related compounds, such as carbacyclin and 11-deoxy PGE_1 by means of enzymic procedure. 1. Synthesis of carbacyclin using microbial reduction. The kinetical resolution of (±)-1 was screened by means of the microbial reduction using forty species of yeast. Among these, Kloeckera saturnus and Pichia farinosa afforded the optically active (-)-1 in 17% (96%ee) and 27% (93%ee) yields, respectively. The chemical conversion of (-)-1 into the key intermediate (9) for carbacyclin was achieved by ring-contraction with Tl(NO_3)_3. 2. Synthesis of 11-deoxy PGE_1 using enzymic hydrolysis. Enzymic hydrolysis of the meso-diacetate (16) was studied. Among the tested enzymes, Rhizopus delemar lipase afforded the optically active monoacetate ((-)-20) in 84% yield (>99%ee). The monoacetate ((-)-20) was effectively converted into the synthetic intermediate (33) for 11-deoxy PGE_1.
Stegobinone 1 is the sex pheromone produced by the female drugstore beetle (Stegobium paniceum), which is known as a pest for stored foods and crops. Its structure 1 was assigned by Prof. Kuwahara et al. in 1978. Very recentry, Dr. H. Kodama of Japan Tobacco Inc. found another pheromone component named stegobiol 26. We now report the synthesis of these two pheromones. The starting materials, we employed, were of microbial origin. (16,17,27) The key-step of our synthesis was the intramolecular acylation reaction follwed by cyclization and deprotection. Our synthetic (2S,3R,1'R)-stegobinone 1 and (2S,3R,1'S,2'S)-stegobiol 26 showed same spectral properties (IR, 500 ^1H NMR, MS, CD) as those of the natural 1 and 26 excellently.
Both enantiomeric alcohol derivatives 1S and 1R were prepared from easily available L-(-)-ethyl lactate through (2S)-1-tosyl-propan-2-ol and its positional isomer (2S)-2-tosyl-propan-l-ol, respectively, in excellent yield. Muscarine and aplasmomycin C_<12>-C_<17> segment were obtained starting from 1S and 1R, respectively, as shown in scheme 3 and 4. (S)- and (R)-Sulcatol were also simply synthesized by the reaction of 1S and 1R with an allylic sulfide 16 (scheme 5). Another synthetic application of the pair of enantiomers was presented for the synthesis of (+)- and (-)-nonactic acid.
A series of biologically active 6-substituted-5,6-dihydro-5-hydroxy(or acyloxy)-2H-pyran-2-ones, (-)-osmundalactone(1) from Osmunda japonica(Akaboshi zenmai), (+)-phomalactone(2) from Nigrospore sp. and Phoma sp., (+)-acetylphomalactone(4) and (+)-asperlin(3) from Aspergillus sp. and their isomers were synthesized in optically active forms. 2,3-O-cyclohexylidene-D-glyceraldehyde(6) was converted to (S)-2-benzoyloxypropanal and (E),(S)-2-benzoyloxy-3-pentenal. The reaction between latter aldehyde and propargyl alcohol derivative gave, after several steps, the aforementioned dihydropyranones and their isomers. The signs of optical rotations of the dihydropyranones were largely dependent on the absolute configurations of C-4, that is, 5-(S)-dihydropyranones showed dextrorotatory, while 5-(R)-dihydropyranones were levorotatory. Biological activities of the newly synthesized compounds will be presented.
Synthesis of angularly tricyclopentanoid sesquiterpenes, was investigated via tricyclo[220.127.116.11^<1,5>]dodecane derivatives using intramolecular Diels-Alder reaction or intramolecular double Michael reaction as a key step. (1) Highly Stereoselective Total Synthesis of (±)-3-Oxosilphinene via Intramolecular Diels-Alder Reaction-(E,E)-3-(8-Phenylthio-octa-5,7-dien-2-yl)-2-methyl-2-cyclopenten-1-one (19) was prepared from the bromocyclopentenone (16) in three steps. Cycloaddition of 19 gave only one stereoisomer of the tricyclo[18.104.22.168^<1,5>]dodecene (20) having all correct four contiguous asymmetric centers. The cyclo-adduct (20) was converted into the tricyclo[22.214.171.124^<4,8>]undecane (25) via Wolff rearrangement. According to usual procedures, the ester (25) was then transformed into (±)-3-oxosilphinene (1). (2) Synthetic Study of Pentalenene and Pentalenic Acid via Intramolecular Double Michael Reaction-Barbier reaction of 4,4-dimethyl-2-cyclopenten-1-one (34) followed by oxidation with pyridinium chlorochromate gave the enone (36), which was converted into the bis-enone (33) in four steps. Intramolecular double Michael reaction of 33, carried out by heating with trimethylsilyl chloride, triethylamine, and zinc chloride, gave tricyclo[126.96.36.199^<1,5>]dodecanediones (40), which were subjected to Wolff rearrangement to afford the tricyclo[188.8.131.52^<4,8>]undecanes (41) possessing all carbon skeleton of natural products (4 and 5).