Ingenol, isolated from the genus Euphorbia, has been of great interest as a synthetic target because of its unusual structure involving an "inside-outside" bridged BC ring coupled with a broad spectrum of biological activities. We herein describe a new entry to ingenane skeleton synthesis via a tandem cyclization-rearrangement strategy. Cross-aldol condensation of acetaldehyde with bromoketone 7, which was easily prepared from commercially available cyclohexanol 5, resulted in selective formation of (E)-enone 8. In the presence of LiBr, 8 was treated with lithium enolate of tert-butyl acetate to give hydroxy ester 9 as a single isomer. Stereoselective construction of the trans-decaline framework was achieved by intramolecular alkylation reaction of 9 promoted by tert-butylmagnesium chloride and LDA. Ester 10 was converted into dichloroolefin 12 via reduction, Swern oxidation, and Horner-Emmons reaction. Successive treatment of 12 with excess butyllithium and paraformaldehyde afforded a propargyl alcohol, which was acetylated and treated with Co-2(CO)8 to give cyclization-rearrangement precursor 1. Under the influence of methyl(2,6-dimethyl-4-nitrophenoxy)aluminum trifluoroacatate, cobalt complex 1 underwent intramolecular electrophilic addition of the propargyl cation moiety to the ethylidene carbon followed by the pinacol-type rearrangement to yield cobalt complex 4 having an ingenane skeleton. Birch reduction of 4 followed by dibromocyclopropanation and methylation afforded 17 containing a complete CD ring system of ingenol.
According to Wenkert's hypothesis of diterpene biogenesis, atisirene (1a), kaurene (2a), trachylobane (3a), and hibaene (4) are arised from a nonclassical carbonium ion 6 as common intermediate. Several reports have appeared which describe the interconversion of diterpenes based on this hypothesis via Wagner-Meervein rearrangement. Herein we describe a novel and efficient methodology for converting kaurene skeleton into atisirene skeleton via radical 1,2-vinyl group migration reaction as a key step. (A) Construction of kaurene skeleton We had already reported the synthesis of racemic alcohol 14 by means of palladium(II)-catalyzed cycloalkenylation reaction as a key step. Four carbon homologation of 14 gave tetraene 18, which was then transformed into pentacyclic compuond 19 via stereocontroled intramolecular Diels-Alder reaction. In order to establish the stereochemistry of 19, we next investigated the conversion of 19 into kaurenoic acid (2b) as follows. Carbometoxylation of 19, followed by 1,4-reduction, gave ester 20. After stereoselective introduction of methyl group into 20, ethylene acetal moiety was hydrolyzed to give methyl 12-oxo-kaurenoate (21). Surprisingly, Wolff-Kishner reduction of 21, accompanying hydrolysis of methyl ester, gave (±)-kaurenoic acid (2b) together with (±)-trachyloban-19-oic acid (3b) in 3: 1 ratio. (B) Construction of atisirene skeleton Methyl 12-oxo-kaurenoate (21) was converted into thioimidazolide 22, which was then treated with "Bu_3SnH in the presence of AIBN in refluxing toluene. Under these reaction conditions, 3-exo type cyclization of homoallylic radical, followed by fragmentation, resulted in an overall 1,2-vinyl group migration to give methyl atisirenoate as a sole product. Subsequent hydrolysis gave (±)-atisirenoic acid (1b). (C) Enantioselective synthesis of the key intermediate 26 Chiral lactam 23 was alkylated with ICH_2CH_2OMOM in the presence of LDA to give 24. Partial reduction of 24, followed by hydrolysis and successive aldol condensation, furnished (4S)-26, transformable into three diterpenes described above.
Claenone (1) is a dolabellane type of marine diterpenoid, isolated from the Okinawan soft coral Clavularia sp., showing potent antiproliferative activity. We describe herein a total synthesis of claenone (1). Syntheses of dolabellatrienone and dolabellatrienol are also described. The sequential Michael reaction of kinetic enolate of cyclopentenone derivative 2 with optically active α,β-unsaturated ester 3 afforded bicyclo[2.2.1]heptane derivative 4a and 4b (4a: 4b=5.3: 1). Ketone 4a was converted to β-hydroxyketone 8 in several steps. Retro-aldol reaction of 8 was successfully carried out by treatment of NaH in the presence of 15-crown-5 to afford diketone 9, which was then converted to tetrasubstituted cyclopentane derivative 13 (Scheme 1). Reaction of the lithio derivative of sulfone 13 with allylic bromide 16, prepared from hydroxyacetone, gave 17. Stepwise elongation of the upper side chain in 17 leading to allylic alcohol 23 was effected through 20 and 21. Asymmetric epoxidation of allylic alcohol 23, followed by mesylation gave epoxymesylate 24. The cyclization of epoxymesylate 24 was carried out by treatment with KHMDS to afford 25. Sulfone 25 was converted to allylic alcohol 27 in the following steps: 1) hydrolysis of ketal, 2) isomerization of olefine, 3) methylation of ketone, and 4) removal of the phenylsulfonyl group. Finally, allylic alcohol 27 was treated with PDC to complete the synthesis of claenone (1) (Scheme 3). Dolabellatrienone was synthesized from the synthetic intermediate 23, through dolabellatrienol (Scheme 4).
4-Arylhexahydroindole derivatives 5 were synthesized from 2-arylcyclohexenyl allylamine derivatives 4, which have a large protecting group on nitrogen, using zirconium-promoted cyclization. Reaction of 4e with Cp_2ZrBu_2 followed by treatment with MeMgBr and then O_2 gave 2a in 63% yield by a one-pot reaction, since the approach of O_2 to zirconium was prevented by the aryl group. The total syntheses of (±)-mesembrane and (±)-mesembrine were achieved starting from 2a. To synthesize these natural products in a chiral form, the starting allylamine derivative 24 (80% yield, 86%ee, recrystallized from MeOH, 99%ee with 79% recovery) was prepared from allyl carbonate 22a and N-tosylallylamine 23 using palladium-catalyzed asymmetric amination in the presence of (S)-BINAPO as a chiral ligand. (-)-Mesembrane and (-)-mesembrine were synthesized from this allylamine 24.
Heterobimetallic complexes function as both a Brφnsted base and a Lewis acid, making possible a variety of new asymmetric catalyses. Among them, ALB [AlLibis(binaphthoxide)] complex is an effective catalyst for asymmetric Michael reactions, tandem Michael-aldol reactions, and hydrophosphonylations of aldehydes. Herein we describe the applications of these reactions to practical catalytic asymmetric syntheses of several bioactive compounds. 1) Practical catalytic asymmetric synthesis of strychnos alkaloids The key Michael reaction promoted efficiently by ALB (0.3mol%) and KO/Bu (0.27mol%) in the presence of activated MS4A gave 2 in 98% yield and 99%ee. The Michael adduct 2 was converted to the tricyclic compound 9 using a Fisher indol synthesis and an amide coupling as key steps. Finally a first catalytic asymmetric total synthesis of 20-deethyl tubifolidine has been achieved through a key cyclization of 9 using DDQ. We hope to reveal our catalytic asymmetric total synthesis of (-)-tubifolidine, being currently under investigation, at the meeting. 2) Practical catalytic asymmetric synthesis of a prostaglandin derivative The three-component coupling product 10 was synthesized using a key tandem Michael-aldol reaction promoted efficiently by ALB (10mol%) in 86% yield and 91%ee, before the conversion to the enone 11. After recrystallization, optically pure 11 was transformed to the enone 14 through a hydrosilylation, a stereoselective reduction, and an aldol condensation via titanium-enolate, as key steps. Finally 11-deoxy-PGF_<1α> has been efficiently synthesized through a key asymmetric epoxidation of 14 using TBHP catalyzed by La-BINOL complex.
Protein kinase C (PKC) is a key enzyme family involved in cellular signal transduction and tumor promotion. It consists of a catalytic domain for protein phosphorylation and a regulatory domain which binds the endogenous messenger diacyl glycerol or exogenous agents such as the phorbol ester-type tumor promoters. The phorbol ester-binding site in PKC is a cysteine-rich domain (CRD) at the N-terminal regulatory region. The CRD consisting of ca. 50 amino acids has six conserved cysteines and two histidines in the pattern HX_<12>CX_2CX_<13>CX_2CX_4HX_2CX_7C where X is a variable amino acid residue. With the discovery of at least eleven PKC isozymes, increasing importance is placed on isozyme specific analysis of function. Isozyme-selective activators or inhibitors represent powerful tools to analyze the role of PKC and become new medicinal leads for diseases related to PKC activation like the vascular complications by hyperglycemia. However, the rational development of such compounds has been hampered by limited information on the structure of the phorbol ester-PKC-phosphatidylserine aggregate. Since pure PKC isozymes are not easily obtainable from natural sources, the CRD's of PKC consisting of ca. 50 amino acids (γ-CRD1, γ-CRD2, η-CRD1, andη-CRD2) have been prepared by solid phase synthesis to establish the models of PKCγ and PKCη for development of new medicinal leads. These peptides were efficiently folded upon zinc treatment to produce PKC regulatory domain surrogates that bind [^3H]phorbol 12,13-dibutyrate (PDBu) with high affinity comparable to native PKC itself, suggesting that these peptides serve as effective models for native PKC. Tumor promoter analogues are most promising as lead compounds for isozyme selective activation or inhibition of PKC since they specifically recognize PKC. Using the PKC surrogate peptides, we have identified new ligands (4a, 4b) with high CRD selectivity based on our recently reported strategy for the design of conformationally restricted indolactam derivatives. Furthermore, we have found that cadmium ion would become an effective tool for controlling isozyme selective regulation of PKC. As exemplified in the PKC surrogate peptides, recent solid phase peptide synthesis as well as DNA recombination technique has brought a new drug design strategy.
Prostaglandins (PGs) such as Δ^<12>-PGJ_2 and Δ^7-PGA_1 methyl ester that possess a cross-conjugated dienone unit exhibit unique anti-tumor and anti-viral activities independent of intracellular cAMP levels. These compounds are transported reversibly into cultured cells and accumulate in nuclei via covalent interaction, eliciting growth inhibition. PGA_1 methyl ester, a simple cyclopentenone analog, is less potent. The unique cellular behavior of the dienone PGs correlates well with their chemical properties. The PGs react specifically with thiol nucleophiles such as glutathione. Michael addition of thiols to Δ^7-PGA_1 methyl ester, an alkylidenecyclopentenone derivative, occurs facilely at the endocyclic C(11) position rather than at the C(7) position. This process is reversible and, in solution phase, the adducts are in equilibrium with the considerable amounts of free PG and thiols. However, the reaction of this PG with Sepharose-bound thiols, regarded to be plausible mimics of protein thiols, is irreversible and the resulting adducts are dissociated only by alkali treatment. On the other hand, PGA_1 methyl ester reacts with soluble or polymer-anchored thiols at lower rates thanΔ^7-PGA_1 methyl ester but the resulting thiol adducts are substantially more stable than those of the dienone PGs. This tendency of the PGA_1 methyl ester causes its equilibrium to shift to the adduct formation. The reversibility of the Michael reaction of PGs with thiols is consistent with their intracellular behavior and biological activities. Since glutathione adducts of PGs have no anti-proliferative activities for cancer cells, the intracellular free PGs are presumed to interact with target molecules to cause cell growth inhibition. The involvement of the ATP-dependent glutathione S-conjugate export pump (GS-X pump) in the efflux of PGs is clarified. Actually, the PG undergoes ATP-dependent uptake by inside-out vesicles of plasma membrane prepared from GS-X pump overexpressed HL-60/R-CP cells, proving the existence of the active extrusive transport mechanism of PGs from the cells. The newly designed GS-X pump inhibitor effects the increase of antiproliferative activity of Δ^7-PGA_1 methyl ester against the same tumor cells by blocking the GS-X pump, indicating a possibility of new cancer chemotherapy. In nuclei, Δ^7-PGA_1 methyl ester induces p21, a CDK inhibitor, independent of p53, arresting the cell cycle at G1 phase.
Phosphatidylinositol 3,4,5-trisphosphate (PIP_3: 1) is a polyphosphoinositide produced from phosphatidylinositol 4,5-bisphosphate (PI4, 5P_2) by phosphatidylinositol-3 kinase (PI-3 kinase) in response to a wide range of growth factor stimulation. It is still unclear what a important role of PIP_3 is. To investigate how PIP_3 interact with proteins in the intracellular signal transduction system, we have developed two affinity gels with novel PIP_3 analogs. IP_3-APBgel is a affinity gel bearing 1-O-acyl type ligand (IP_3-APB: 6) and is expected to associate with proteins which specifically recognize head group of PIP_3. Using this IP_3-APBgel, we have isolated a novel protein with a molecular mass of 43kD from bovine brain extract and was termed PIP_3 binding protein. The protein bound PIP_3 with a higher affinity than it did inositol 1,3,4,5-tetrakisphosphate (IP_4), PI4, 5P_2, phosphatidylinositol 3,4-bisphosphate (PI3, 4P_2) and phosphatidylinositol 3-phosphate (PI3P), suggesting that the binding to PIP_3 was specific. It contained one zinc finger motif and two pleckstrin homology (PH) domains and the entire sequence was 83% similar to centaurin α, another PIP_3 binding protein. The binding activity was weaker in the mutants with a point mutation in the conserved sequences in each PH domains and the introduction of both mutations caused loss of the binding activity. These suggest PIP_3BP binds PIP_3 throuth two PH domains present in the molecule. To detect PIP_3 binding proteins in a more exact manner, we designed PIP_3 analog containing a glycerol group as well as fatty acid groups (PIP_3-APB: 12). One of the peptides identified from the lysates of calf thymus had a molecular mass of 70kD and was identified to be the Tec protein-tyrosine kinase. This interaction between the PIP_3 analog beads and Tec could be efficiently inhibited by free PIP_3. Significantly higher concentrations of PI4, 5P_2, PI4P and IP_4 were required to compete this interaction. Studies with the various deletion mutants of Tec protein revealed that the binding site of PIP_3 was localized within the PH domain of Tec. Now we have demonstrated that our affinity gels are very useful biochemical tools to detect PIP_3 associated proteins. Moreover, we isolated a novel PIP_3 binding protein, Tee and Aktγ (which was also isolated by PIP_3-APBgel). These proteins have PH domains which turned out to be essential to interact with PIP_3. This PIP_3-PH interaction is likely to have a significance in the PI-3 kinase signal transdution pathway.
N-Acetyl neuraminic acid (Neu5Ac (3)) is incorporated at the nonreducing terminal position of glycoconjugates which are found in cell membranes and in nervous systems of various living organisms. These sialyl conjugates play an essential role in biological molecular recognition processes, such as cell adhesion and differentiation phenomena. For the elucidation of these biological properties and functions, the supply of natural and nonnatural sialyl conjugates with a definite structures is indispensable. The ulosonic acids such as Neu5Ac (3), KDO (1), KDN (2) have the α-ketoacid moieties which exist in a 6-membered cyclic hemiacetal form. To establish a general synthetic method for monosaccharides containing the α-ketoacid structures, we examined the synthesis of Neu5Ac (3) utilizing alkylation of 2-alkoxy-2-cyanoacetate 20, an acylanion equivalent of alkyl glyoxylate, with bromide 19 derived from the commercially available D-glycero-D-gulo-heptose-1,4-lactone (11). We also examined the syntheses of KDO (1) and KDN (2) based on alkylation of 2-alkoxy-2-cyanoacetate 7 with the sugar-derived iodide 6 and triflate 15. One of the most difficult problem in the synthesis of sialyl conjugate is the stereoselective glycosidation of sialic acid with α-glycosidic linkage. First, we examined a new method for α-glycoside formation of sialyl conjugate utilizing the concept of "long-range participation". When 2-methylthioethyl ester was introduced into the carboxylic acid in the sialic acid as neighboring group and DME was used as solvent, highly α-selective glycosylation was achieved in moderate yields. To improve the yields, we examined alkylation of 2-alkoxy-2-phenylthioacetate 34 with allyl bromide 36 followed by an intamolecular glycosylation. Phenylthio-group serves as not only a stabilizer of an enolate in the alkylation but also a leaving group in the glycosylation. This method are underway in our laboratory.
Carzinophilin (1) is an antitumor antibiotic isolated from Streptomyces sahachiroi by Hata et al. in 1954. While the structure of 1 has been revised several times over 30 years, Armstrong et al. reported in 1991 that the ^1H- and ^<13>C-NMR spectra of 1 were superimposable on these of azinomycin B. The latter compound is an antitumor antibiotic bearing a characteristic 1-azabicyclo[3.1.0]hexan-2-ylideneglycine system. It is also disclosed that 1 is one of the strand cross-linking reagents for DNA. These unique history, novel structure, and potent antitumor activity make 1 as well as its related compounds the attractive targets for total synthesis. In the course of our synthetic studies on the model compounds 10 and 23 of 1, we have achieved in developing several methods useful for the synthesis of1: (1) synthesis of 2-methylidene-1-azabicyclo[3.1.0]hexanes 10 and 23 by the aziridine formation under basic conditions; (2) stereoselective construction of the C12, C13-dioxygenated (carzinophilin numbering) pyrrolidine 15 from 2,3,5-tri-O-benzylarabinofuranose (12) by employing the Nicotra's protocol; (3) elaboration of pyrrolidin-2-ylideneglycine 20 by the coupling reaction of thioimidate 15 with 4-phenylazlactone followed by activation of the azlactone ring by introducing an Alloc group into the N9 position (carzinophilin numbering) and subsequent ring opening with amine 19; and (4) construction of β-hydroxyenamide system by deprotection of protected 2-N-acylamino-3-ketoaldehyde. Based on the results accumulated in these model studies, the synthesis of 13-desacetyl-12,13-O-dibenzyl-4-O-methykarzinophilin (2) was achieved. However, subsequent deprotection of the two benzyl groups in 2 to obtain 1 met with failure. In this synthesis, the stoichiometric asymmetric dihydroxyation of allylic alcohol 26 employing (DHQ)_2PHAL, the Sharpless ligand, effected stereoselective introduction of the C18 and C19 oxygen functionalities in 2. The synthesis of 2 constitutes the first example which successfully constructed the full carbon framework involved in 1. It was also revealed that the 1-azabicyclo[3.1.0]hexan-2-ylideneglycine system corresponding to the central core skeleton of 1 is quite reactive, and that 2 and 10 readily alkylate two molecules of thiophenol at the C10 and C21 positions. These results seem to mimic the reactivity of 1 which behaves as an alkylating reagent for DNA. Some carzinophilin congeners synthesized in these studies were found to exhibit potent in vitro cytotoxicity [for example, IC_<50> (P388)=0.0032μg/mL (9) and 0.0021μg/mL (10)] which is almost equal to that of adriamycin. In antitumor activity assay (in vivo), 9 clearly reduced the volume of Sarcoma 180 similarly to mitomycin C while a decrease of body weight was observed for the tested mice.
Antitumor antibiotic kedarcidin consists of an apoprotein and chromophore 1. We describe herein a synthetic study directed towards 1 and revised structure of 1. α-Chloroazatyrosyl naphthoamide fragment 2 was synthesized from naphthalene carboxylic acid 14 and chloroazatyrosine derivative 15. NMR spectra of synthetic α-chloroazatyrosyl naphthoamide 2 was not identical with the reported data. β-Chloroazatyrosyl naphthoamide 17 was then prepared. Its spectral data were identical with those of the fragment of 1. Optically active 17 was synthesized through the optical resolution of β-chloroazatyrosine derivative 16 whose absolute configuration was determined by modified Mosher's method. The absolute configuration of the fragment degraded from 1 was elucidated to be R by comparison of the optical rotations and CD. Thus, the structure of kedarcidin chromophore has been revised as 18 based on the above syntheses and NOEs. Optically pure enediyne 22 with the revised absolute configuration was synthesized. Furthermore, an efficient method for synthesizing an aryl ether was established. CsF-mediated coupling of 26 with epoxide 27 was achieved under the mild conditions.
Clinical early events in inflammation, the allergic response, and tumor metastasis involve interactions between leukocytes and endothelial cells. A variety of cytokinins and related chemical mediators control both leukocyte adhesion and subsequent intercellular invasion via the regulation and expression of cellular adhesion molecules, inhibition of cell-cell adhesion thus holds promise for the treatment of diverse pathologies. In the course of a screening program aimed at cell adhesion inhibitors obtained from microoragnisms, macrosplelides A (1) to D (4) were discovered from the fermentation broth of Microsphaeropsis sp. FO-5050. Macrosphelides are the first 16-membered-ring antibiotics embodying three lactone linkages (e.g., Macrotriolides). The macrosphelide A (1) strongly inhibit the adhesion of human-leukemia (HL-60) cells to human-umbilical-vein endothelical cells (HUVEC) in dose-dependent fasion (IC_<50>=3.5μM). Preliminary studies suggest that 1 prevents cell-cell adhesion by inhibiting the binding of sialyl Lewis^x to E-selectin. 1 also proved to be orally active against B-16 tumor metastasis to the lung in mice. We detemined the complete relative and absolute stereochemistries of 1, via X-ray crystallography and the Kakisawa-Kashman modification of Mosher's NMR method. The stereostructure of 2 was determined by chemical correlation with 1. Moreover, we accomplished the first total synthesis of 1. As our point of departure we selected the asymmetric dihydroxylation of (E,E)-hexa-2,4-dienoic acid t-butyl ester (5), which affords the (4S,5S)-diol (6) (85%ee). Selective monosilylation of 6 (TBDMSCl, DMAP, CH_2Cl_2) provided the desired ether (7). Mitsunobu inversion (PPH_3, DEAD, HCOOH; NH_3/MeOH) at C(4) of 7 furnished 8. Protection of 8 as the MEM ether and hydrolysis (0.2N NaOH, MeOH/THF/H_2O) then gave 10, whereas desilylation of 9 generated the second building block (11) (Bu_4NF, THF). Condensation of carboxylic acid (10) and alcohol (11) via the Keck protocol (DCC, DMAP, CSA, CH_2Cl_2) and desilylation of the resultant ester (12) (AcOH/THF/H_2O) produced 13. The third fragment, TBDMS ether (14) of (3S)-hydroxybutyric acid and coupled with 13 (DCC, DMAP, CSA, CH_2Cl_2). Removal of the silyl and t-butyl moieties in 15 (TFA/CH_2Cl_2/thioanisole) provided seco acid (16), which smoothly underwent Yamaguchi macrolactonization (DMAP, 2,4,6-trichlorobenzoyl chloride). Finally, deprotection of 17 (TFA/CH_2Cl_2) gave synthetic 1, identical in all respects with a sample of the natural product. The first total synthesis of 1 has thus been achieved via a highly convergent, efficient strategy (11 steps, 10.6% overall yield).
Vicenistatin (1) is an antitumor antibiotic, isolated from Streptomyces sp. HC-34, having a novel structure including a 20-membered macrocyclic lactam and aminosugar (vicenisamine: 2). Among other characteristic features is its significant activity especially against human leukemia and human colon carcinoma. Synthetic studies have now been launched to analyze essential chemical features in expressing biological activities and to further develop novel compounds useful in cancer chemotherapy. The absolute stereochemistry of the aglycon part was first determined by synthetic approach. Relevant MTPA ester derivatives of two fragments (aminoalcohol and triol), which include the chiral center C18 and the C6-C7 portion, respectively, were degradatively derived from natural vicenistatin, and compared with the respective synthetic specimen prepared from the known compounds by stereochemically defined chemistry. The absolute configuration was determined to be 6S, 7S, 18S. Total synthesis of vicenistatin was the next issue. We envisioned that the final macrocyclization should be either by macrolactamization (path A) or an intramolecular Wittig-Horner reacton (path B). The crucial precursors were respectively synthesized convergently from two major components. One fragment was synthesized from cyclopropyl methyl ketone and the other amino fragment from (S)-citronellol. Two key reactions were involved, that is, the Suzuki's coupling allowed to connect these two components and the subsequent Evans' asymmetric aldol reaction to construct the remaining two vicinal chiral centers. We were successful in the final cyclization (path A) to construct the whole skeleton of the aglycon. The aminosugar part (vicenisamine) is essential in expressing biological activities as well, and its synthesis and derivatization is naturally important. Our route starting from a known chiral epoxyalcohol obtained by Sharpless' asymmetric epoxidation involved two key reactions: intramolecular epoxide opening promoted by Lewis acid and Brown's asymmetric allylation.
Aplyronine A (1), a novel macrolide isolated from the sea hare Aplysia kurodai, shows potent antitumor activity and interacts with actin, the protein in cytoskeleton. The enantioselective synthesis of 1 was achieved by a convergent approach. Starting from imides 4 and ent-4, the C5-C20 segment 8 and the C21-C34 segment 9 were synthesized by using the Evans aldol reaction and the Sharpless epoxidation reaction as key steps. Julia olefination between the segments 8 and 9 followed by four-carbon homologation reaction and Yamaguchi lactonization led to the 24-membered lactone 10, which was converted into aplyronine A (1) by construction of the terminal N-formyl enamine structure and subsequent introduction of the two amino acids. The use of the [(3,4-dimethoxybenzyl)oxy]methyl group as a protecting group for the hydroxyl at C29 was crucial for this synthesis. The artificial analogs of aplyronine A (1) were synthesized, and the structure-activity (cytotoxicity and actin depolymerizing activity) relationships were examined. The side chain portion of 1 was found to play an essential role in both biological activities, and the trimethylserine moiety and two hydroxyl groups in 1 turned out to be important in the potent cytotoxicity.
Polycavernoside A (1) has been isolated as one of the sources of human intoxication from the red alga Polycavernosa tsudai by Yasumoto et al. in 1993. They have proposed the planar structure of 1, which is a novel macrolide disaccharide, on the basis of ^1H and ^<13>C NMR spectra. However, although the respective relative configurations of the tetrahydrofuran ring, the tetrahydropyran ring, and the sugar moieties have been clarified, the absolute configuration of the whole molecule has not been established yet. We have started the total synthesis of this compound, because of its unusual molecular structure, the significant toxic activity, and the lack of a satisfactory natural source. We describe herein the construction of the southern part (9), macrolactone part (25), and triene model compound (34) as well as the determination of the relative configration of 1.
The wide occurrence of medium-sized ring ethers in a variety of Laurencia species and in the organisms which feed on this alga has become a feature of marine natural products. Much attention has recently been focused on efficient approaches toward these systems. We disclose the efficient method by intramolecular opening of hydroxy epoxides promoted by the (Bu_3Sn)_2O/Lewis acid system, and their application to the synthetic studies on (+)-isolaurepinnacin(2) and (+)-laurencin(4). Regio- and stereoselective cyclization of the hydroxy epoxides would be the most straightforward access to the stereoselective construction of medium-sized ring ethers. The reaction was designed based on the concept that both activation of a hydroxyl group by formation of a tin ether and an epoxy group by coordination of Lewis acid would promote the cyclization reaction. Expected S_N2 process and exo-mode cyclization would stereospecifically afford either α,ω-cis- or α,ω-trans cyclic ethers depending on the stereochemistry of the hydroxyl group and the epoxy group. At first, model compounds 7a, 7b were investigated to explore the general cyclization reaction. After preparation of a tin ether by treatment with (Bu_3Sn)_2O in toluene, several Lewis acids were examined. In the presence of Zn(OTf)_2, Sn(OTf)_2, Yb(OTf)_3, La(OTf)_3, or Eu(OTf)_3, the cyclization reaction proceeded to afford the corresponding 7-membered cyclic ether 8a, 8b as a sole product in high yield regardless of the geometry of the epoxy group. The application to the synthesis of 8-membered cyclic ether 10a, 10b gave unsatisfactory result. The successful result was obtained by using of Eu(fod)_3 as Lewis acid. The extension of this methodology to the stereospecific construction of α,ω-cis- and trans-disubstituted derivatives were also investigated and gave good result. Synthetic study of (+)-isolaurepinnacin(2), a representative marine natural products containing the 7-membered cyclic ether, was examined by using the above methodology as a key step. The key intermediate 19 was prepared from the acetylene fragment 16 and the epoxy fragment 17. Cyclization of 19 promoted by (Bu_3Sn)_2O/Eu(fod)_3 proceeded smoothly to afford the cyclic ether skeleton 22 as a sole product. To the epoxide 23 obtained from 22, a trans-enyne structure was stereoselectively introduced via the Corey's method. After halogenations of the hydroxyl groups at C_6 and C_<13> with inversion of configuration, total synthesis of (+)-2 has been achieved. Synthetic study of (+)-laurencin(4), a representative 8-membered cyclic ether, was next examined. Retrosynthetic analysis showed the analogous key intermediate 30. Cyclization of 30 by using the above methodology was successful to give 31 stereoselectively in high yield. Introduction of a trans-enyne structure to 33 by the similar procedure to the synthesis of (+)-2 and modifications of the hydroxyl groups at C_6 and C_<12> furnished (+)-4.
Hemibrevetoxin B (1), isolated from cultured cells of the red tide organism Gymnodinium breve, is the smallest member of the polycyclic ether marine toxins and has about half of the skeleton of brevetoxins. Much attention has been paid to the synthesis of 1 owing to its unique structure and strong biological activities and, recently, three total syntheses of hemibrevetxin B have bee reported. We report here a new approach to the synthesis of hemibrevetoxin B based on recent oxiranyl anion methodology developed in our laboratory. The strategy is illustrated by the stereocontrolled synthesis of 2, which is elaborated by sequential coupling of three chiral sulfonyl-stabilized oxiranyl anions 4b, 5b, and 6b to the monocyclic diol 3. The key reactions of our synthesis are alkylation of oxiranyl anions, 6-endo cyclization, and one-carbon ring expansion of a 6-membered cyclic ether to a 7-membered ring by trimethylsilyldiazomethane. These reactions were used in an iterative fashion to synthesize compound 2. Since Yamamoto has already described the conversion of 2 into hemibrevetoxin B, our synthesis of 2 constitutes the formal total synthesis of the natural product.
Our concept of the total synthesis toward the marine polyether ciguatoxin is based on acetylene biscobalthexacarbonyl complex-mediated ether cyclizations. The cyclization of medium size ether rings according to Scheme 1 is recently established in our laboratory. The reaction proceeds via a biscobalthexacarbonyl complex-stabilized cationic intermediate, which subsequently undergoes endo cyclization to give syn/trans products. Medium size ether rings with 7-9 members have been synthesized along this line and thus the applicability in the ciguatoxin synthesis is given. Decomplexation of cobalt-acetylene was carried out under high pressure H_2-atmosphere in the presence of Wilkinson catalyst; more recently with tributyltin hydride or triethylsilane to provide olefin compounds. The ABC-fragment was synthesized starting with the BC-system 15, derived from α-methyl-D-glucose in several steps (Scheme 4). After a Lewis acid catalyzed glycosylation that coupled fragment 15 with the pentose derivative 16, the acetylenic intermediate 18 was obtained. Treatment with Co_2(CO)_8 lead to the precursor for a cyclization reaction that yielded the 7-membered A-ring with the adjacent side chain. In model studies focussing on the EFG-fragment the 7- and 9-membered ether rings 4, 8 and 25 were constructed using tri-O-acetyl-D-glucal as a commercially available precursor. The 6/7-ring system 4 was achieved after treating the Co-complex 2 with BF_3・OEt_2 to produce the 7-membered ring in compound 3, which was decomplexed using a Rh-catalyst (Scheme 1, 2, 3). The 6/9-ring system 8 was obtained in a similar procedure. The 6/7-ring system 25 that will lead to the EFG-fragment was obtained after acid catalyzed cyclization of Co-complex 23 and subsequent treatment with RhCl(PPh_3)_3/H_2 (100atm)(Scheme 5). The retrosynthetic study of the JKLM-fragment was also based on a Co-complex mediated cyclization to build the 7-membered K-ring. The key intermediate for this step will be accomplished with the connection of the J-ring 35 and the LM-fragment 37, derived from tri-O-acetyl-D-galactal and 5-menthyloxy-2-furanone, respectively. The various stereogenic centers were introduced by a stereoselective epoxidation followed by a regioselective epoxide opening and a heteroconjugate addition in the case of the compound 35 (Scheme 6). A stereoselective Michael addition to 5-menthyloxyfuranone provided the two methyl groups in fragment 37. Since a migration of the double bond, formed during the decomplexation process, occurred sometimes as a side reaction, a new method for the transformation of acetylene Co-complexes into olefins was developed. Treatment of the Co-complex with either nBu_3SnH or HSiEt_3 leads to cis olefins or vinyl silanes, respectively, in good yields without migration of the resulting double bond (Scheme 7).
Ciguatoxin (CTX1B, 1) and its congeners are the toxic principles of ciguatera, a widespread seafood poisoning of dinoflagellate origin, and reported to bind to the same site of voltage-sensitive sodium channels (VSSC) as brevetoxins, another class of structurally related marine toxins. The ciguatoxin molecule consists of 12 trans-fused cyclic ethers ranging from six- to nine-membered, where another five-membered oxacycle is spirally attached at one end. The most remarkable structural feature is that oxonene ring F in the central region of the molecule undergoes a rather slow conformational change in solution. Thus, ring F functions as a hinge in the conformational alternation, which is speculated to play an important role in its binding to VSSC. However, little is known about the detailed mechanism of this conformational change. From the synthetic point of view, construction of the oxonene ring system is a formidable and challenging synthetic objective because severe difficulties such as unfavorable entropy factors and transannular interactions have to be overcome. We now report the first achievement in synthesis fused oxonene ring systems 2 and 3 and their conformational analysis on the basis of the dynamic NMR studies. The present synthesis of 6-9-6 tricyclic system 2 involves as the key steps (i) Lewis acid-mediated intramolecular condensation of γ-alkoxyallylsilane-acetal to yield O-linked oxacycles and (ii) SmI_2-mediated intramolecular Reformatsky-type reaction to construct an oxonane ring. As seen for ciguatoxin, ^1H and ^<13>C NMR signals on the oxonene ring in 2 extremely broadened at room temperature. As the temperature decreased, these signals sharpened and separated into two sets of peaks, which corresponded to the two alternating conformers with a ratio of about 1: 1 in pyridine-d_5 solution. The structures of these conformers were assigned by ^3J_<H,H> data at -20℃ and appeared to closely mimic the ring F of ciguatoxin except the ratio of the two conformers. The free energy of activation for this conformational change was estimated to be approximately 14kcal/mol from the coalescence temperature (28℃). The present strategy for the construction of a fused oxonene ring was also successfully applied to the synthesis of the 6-6-9-7-6-6 hexacyclic system 3. Compound 3 also exists in an equilibrium of two conformational states of the oxonene ring as was the case with 2. In order to clarify the relationships between conformational flexibility and biological activity, further investigation toward the synthesis of decacyclic ciguatoxin model compound 29 is currently underway.
Chiral BINAP-Rh(I)-catalyzed asymmetric desymmetrization (asymmetrization) of a series of the tricyclic meso-1,4-enediols and their bis-ethers having bicyclo[2.2.1]heptane and bicyclo[2.2.2]octane background has been examined for the construction of versatile chiral building blocks served as chiral cyclohexenone and chiral cyclohexadienone. The isomerization reaction occurred facilely to give the keto-alcohols or the keto-ethers in excellent yields with high optical purities when the bis-ethers were used as substrates though the products from the alcohols did not have high optical purities. Interestingly, the optical specificities observed between the alcohols and the ethers were inversed in the presence of the same chiral BINAP-Rh(I) catalyst. The reaction was confirmed to proceed via a suprafacial 1,3-hydrogen migration by deuterium labeling experiment. The reaction was also extended to the isomerization of a monocyclic seven membered meso-1,4-enediol bis-silyl ether which afforded the optically active hydroxy-ketone in an excellent yield with moderate optical purity (〜70%ee). The chiral monocyclic product thus obtained was utilized for the first enantiocontrolled synthesis of the tropane alkaloid (-)-physoperuvine The resulting optically enriched tricyclic products having bicyclo[2.2.1]heptenene background were used as a versatile chiral building block serving as chiral cyclohexenoids and chiral cyclohexadienoids. Namely, owing to their rigid biased framework, they allowed diastereoselective modification from convex face and, moreover, they generate the double bond by thermal retro-Diels-Alder reaction making themselves as versatile chiral building blocks. Their potential as chiral building blocks were demonstrated by diastereo- and enantiocontrolled construction of a variety of natural products and their key intermediates ranging from a simple monoterpenoid to a complex steroid molecule, such as 4-hydroxy-2,6,6- and 4-hydroxy-3,5,5-trimethyl-2-cyclohexenones, malingolide, isoretronecanol, tracheranthamidine, shikimic acid, tricholomenyn A, calcitriol A ring, etc.
Reversible phosphorylation of proteins plays an important role in intracellular signal transduction. Recently we started a project for the development of highly specific inhibitors of the biologically significant protein tyrosine phosphatases and dual-specificity phosphatases. Here we report an asymmetric synthesis of RK-682 (6) which has been reported as a potent inhibitor of a dual-specificity phosphatase VHR, and the absolute stereochemistry of RK-682 was determined to be (R) (Scheme 1). This efficient and versatile synthetic method enabled to generate a tetronic acid library containing various RK-682 analogs. Inhibition of VHR by these novel compounds was first evaluated. The structure-activity relationship suggests that hydrophobic side chain at C3 position is quite important (Table 1). To find a potential therapeutics, we next focused on phosphatases involved in the regulation of cell proliferation. Although RK-682 showed strong inhibition of VHR, its inhibitions to several other phosphatases tested were weak. Screening of the library identified several compounds which inhibit biologically important phosphatases. For example, compound 10 showed inhibition of dual-specificity phosphatases Cdc25A and Cdc25B, which are key enzymes of cell cycle progression. Compound 17 inhibited CD45, a receptor-type tyrosine phosphatase which is involved in T cell and B cell activation. It was also found that compounds 18 and 19 inhibit PTH-stimulated IL-6 production of MC3T3-E1 cells.
Seed germination of parasitic weeds of the genera Striga and Orobanche, which cause considerable damages to important gramineous crops such as corn, sorghum, and rice, is known to be stimulated by chemicals released from their host plants. The attractive idea to control such harmful weeds by using effective germination stimulants, inducing suicidal germination in the absence of their host plants, prompted various syntheses of strigol (2), the first discovered naturally occurring stimulant originally isolated from cotton (the non-host plant) root exudates, and its analogues. Sorgolactone was isolated from Sorghum bicolor, a genuine host plant, as a highly potent germination stimulant for Striga and Orobanche. Although spectroscopic details were not sufficient due to the very small amount of the stimulant obtained, the structure 1 was proposed based on ^1H-NMR, MS, and CD comparison with those of (+)-strigol. To ascertain the proposed structure of sorgolactone and also to elucidate structure-activity relationship, we studied syntheses of racemic and optically active sorgolactone and evaluated their biological activities. Synthesis of racemic sorgolactone The synthesis of the racemates of the structure proposed and its three stereoisomers was achieved by confirming the stereostructure of the intermediate (±)-12, obtained from known diketo acid 8 in three steps followed by MPLC separation from the diastereomer mixture, and the final product (±)-1 by X-ray analysis. Bioassay employing Orobanche minor seeds revealed that all of stereoisomers stimulate the germination and the order of activity was (±)-strigol=(±)-15≧(±)-18>(±)-1>(±)-17. Synthesis of optically active sorgolactone Radical cyclization of the phenylselenyl derivative of alkynyl ester 23, obtained from (S)-citronellal (19) in eight steps, afforded exo-cyclic alkene 25 in good yield. The alkene was readily transformed into a diastereomeric mixture of tricyclic lactones, which was separated into oily 11 and crystalline 12. The procedure employed for the synthesis of the racemates led to 1 from 11. Recrystallization of the product improved enantiomeric purity to 100%e.e. (determined by chiral HPLC). Bioassay of 1 and its stereoisomers is now in progress.
Campesterol (3) and dihydrobrassicasterol (4) are biosynthesized from a steroidal 24-ene precursor (desmosterol 1) via 24-methylenecholesterol (2) and 24-methyldesmosterol (5). The stereochemical courses of these transformations have been studied. by feeding regioselectively ^<13>C-labeled steroidal substrates to tissue cultures of higher plants. The formation of 24-methylenecholesterol from desmosterol involves a trans-methylation from S-adenosylmethionine followed by hydrogen migration from C-24 to C-25. The stereochemistry of the hydrogen migration was first investigated. For this purpose, ^<13>C chemical shifts of the diastereotopic C-26 and C-27 methyl groups of 24-methylene-cholesterol were unambiguously assigned by synthesizing the isopropyl pro-R-Me and pro-S-Me ^<13>C-labeled compounds. Feeding of [26-^<13>C]desmosterol to cultured cells of Catharanthus roseus followed by ^<13>C-NMR analysis of the biosynthesized 24-methylenecholesterol established that Re-face hydrogen migration takes place at the C-25 position. Next, chemically synthesized [26,27-^<13>C_2]-24-methyldesmosterol and [26,27-^<13>C_2]-24-methyl-desmosterol were fed to cultures cells of C. roseus and Oryza sativa. The former labeled sterol was shown to be transformed to campesterol and dihydrobrasicasterol, and the latter into sitosterol, demonstrating their intermediary role in the formation of 24-methyl- and 24-ethylcholesterols in higher plants. The stereochemical course of this reduction was explored by feeding regiospecifically ^<13>C labeled compounds, [E-Me-^<13>C]-(5a) and [Z-Me-^<13>C]-(5b) 24-methyldesmosterols. ^<13>C-NMR analysis of the resulting 24-methylcholesterol fraction indicated that the (E)-methyl group turned to be pro-S-Me group of campesterol and pro-R -Me group of dihydrobrassicasterol, thus demonstrating that an anti-mode of hydrogen addition occurs in both cases. This observation implies that the step of the double bond isomerization from 24-methylenecholesterol to 24-methyldesmosterol takes place in such a manner that pro-S-Me group of 2 turns (E)-Me of 5. In conclusion, the present studies established the steric course in the fromation of 24-methyl-cholesterols and allowed to correlate the C-26 and C-27 of the intermediate sterols.
Reaction catalyzed by lanosterol synthase is one of the most complex biosynthetic reactions found in nature, and its mechanism has been the subject of much research over three decades. Lanosterol synthase has attracted considerable interest from the biological and pharmaceutical viewpoints since it is situated in the center of sterol biosynthesis of eukaryotes. Membrane associated rat liver lanosterol synthase has been solubilized with Triton X-100 and for the first time purified to homogeneous protein in three steps (hydroxylapatide, isoelectric focussing and Mono Q). The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis with a molecular weight of 75kD. Eleven independent amino acid sequences (Peptide-1 to -11) of the endogenous polypeptide were obtained by sequencing the internal peptides after Lys-C or lysylendopeptidase digestion of the purified enzyme. With all the available amino acid sequence information, we tried to clone cDNA of this enzyme by PCRs. After several trials, one with a pair of sense primer based on Peptide-8 and anti-sense primer based on Peptide-9 gave an amplified ca. 200-bp DNA fragment. This fragment was subcloned into pT7Blue(R) and sequenced. Based on the sequence of this fragment, the 5'- and 3'-ends of nucleotide sequences were elucidated by RACE strategy. The full nucleotide sequence revealed the presence of 2,199-bp ORF that encodes a 733 amino acid polypeptide with a molecular mass of 83,301Da and all of the sequences of eleven internal peptides are included. The PCR-amplified ORF has been inserted into pYES2, an expression vector in yeast, under the control of galactose-inducible promoter. Significant lanosterol synthase activity has been found in the homogenate of the transformed yeast, confirming its identity as lanosterol synthase cDNA. The deduced amino acid sequence of the rat lanosterol synthase exhibits 41.1%, 38.3% and 45.3% identity to those of the oxidosqualene cyclases of yeast, Candida albicans and Arabidopsis thaliana, respectively. Taking advantage of this homology, human liver lanosterol synthase gene was cloned by PCRs. The obtained clone was successfully expressed in yeast as active enzyme.
There has been remarkable advances in the studies of oxidosqualene and squalene cyclases in the past ten years. First, complete purifications of the enzymes have been attained from several species such as vertebrates, plant, yeast and bacterium, irrespective of unstable and membrane-bound nature. Second, application of cDNA cloning technique to the cyclases have succeeded in determining the alignments of amino acids from various biological sources including human. Third, the substrate analogues including the suicide inhibitors have made a great contribution to the polycyclization mechanism. In this symposium, we report a definitive evidence that the polycyclization proceeds via the discrete carbocationic intermediate formed during the cyclization, but not via a concerted manner as proposed before; substitution of methyls with ethyl groups at 10- and 15-positions 6 halted the enzymic reaction at the monocyclic stage 7 and 8. Substrate analogue 9 with ethyl group at 15-position afforded 10 (normal cyclization) and 11 (tricyclic 6/6/5), the latter being formed under the control of Markovnikov rule. This is in contrast to the protosterol cation, which is formed under anti-Markovnikov control. Thus, oxidosqualene would be converted via the 5-membered intermediate 12 under the control of Markovnikov rule, which then undergoes a ring expansion to form 6-membered C-ring. Mechanisms of the formation of tetrahymanol and hopene from squalene itself are discussed, especially on the terminal cyclization (E-ring formation). Substrate analogs 14, 15, 18, 22 have demonstrated the cyclization mechanisms as follows: for tetrahymanol cyclase, the terminal cyclization proceeds by the process of stereoelectronic control, suggesting little participation of the enzyme, while hopene cyclase strongly binds to the terminal methyl groups to form 5-membered E-ring. From our studies, methyl groups of the substrate seem to bind the cyclase enzymes and the substrates were then subjected to the folding of chair-boat or chair-chair inside the enzymes leading to production of the desired triterpene skeletons. Over-expression of hopene cyclase was achieved successfully and the point mutations of amino acids in QW motif proved that the functions of D and W are crucial for the enzyme activity.
One of the most important defensive systems in plants against pathogen attack is the production of antimicrobial compounds called phytoalexins. We have demonstrated that accumulation of the flavonoid phytoalexin, sakuranetin is mediated by endogenous production of JA in stressed rice leaves, and also shown that sakuranetin production is strongly elicited by exogenously applied JA. We suggested that the further metabolism of JA into its amino acid conjugates would be needed to furnish the signal transduction pathway and showed the importance of amino acid conjugates of JA in phytoalexin production. On the mechanism(s) by which exogenously applied JA elicits phytoalexin production in rice leaves, we find that cytokinin (a plant hormone) counteracts the JA-inducible sakuranetin production, and ascorbic acid (free radical generator) accelerates the JA-inducible sakuranetin production. We speculate that active oxygen species (AOS) might be connected with the JA-inducible sakuranetin production from above results and observed that exogenously applied AOS elicit the sakuranetin production. We also find that methionine (an amino acid) also elicits phytoalexin production in rice. Studying of elicitation mechanism(s) by methionine in relation to JA is in progress in our laboratory.
Three novel trichothecene sesquiterpenes, trichothecinols A (1), B (2) and C (3) were isolated from the fungus Trichothecium roseum (TMI-32358) together with some known analogues such as trichothecin (4). They showed inhibitory effect on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and thus were classified as anti-tumor promoters in vitro. We focused on the potent activity of 1 and further examined its inhibitory effect on the tumor promotion in mouse skin in vivo. Surpprisingly, while 1 reduced both the rate of papilloma-bearing mice and the number of papillomas per mouse in the presence of TPA, 1 itself enhanced the tumor promotion in the absence of TPA and induced the formation of papillomas. In addition, malignant conversion of papillomas to carcinomas was caused after 20 weeks without 1. On the other hand, 1 did not enhance the tumor promotion in vitro in the absence of TPA at all. Accordingly, 1 was indicated to be a new class of tumor promoter that can be distinguished from TPA and teleocidin. In order to clarify the peculiar action of 1 in the tumor promotion, a structure-activity relationship was investigated with the natural products and compounds derived from 1 and 4. In anti-tumor promoting effect in vitro, it was found that the presence of the isocrotonyl ester and conjugated ketone was most important for increasing the activity. The presence of the 3-hydroxyl group was also effective when the ketone carbonyl group was reduced. Interestingly 4 did not enhance the tumor promotion neither in vitro nor in vivo at all although the structure of 4 differs from 1 only in the lack of 3-hydroxyl group and its antitumor promoting activity in vitro was comparable to that of 1. Therefore, it was strongly suggested that the 3-hydroxyl group plays a crutial role in the tumor promotion.
Astaxanthin is known as a red carotenoid distributed in salmons, shrimps, and crabs. We reported the first isolation of an astaxanthin-producing bacterium Agrobacterium aurantiacum, and the elucidation of astaxanthin biosynthetic pathway at levels of the genes and enzymes through the cloning of the biosynthetic genes and the expression in E. coli. We describe here the isolation and the structure determination of new carotenoid glucosides isolated from the polar fractions of Me_2CO extracts of A. aurantiacum, and production of astaxanthin (di) glucoside by E. coli transformants through approach of metabolic engineering. When A. aurantiacum was cultured in the medium containing glucose, two new carotenoid glucosides, (3S, 3'S)-astaxanthin β-D-glucoside (1) and (3S, 3'R)-adonixanthin 3-β-D-glucoside (2), were isolated. These structures were determined by spectral means. Two transformed strains, E. coli JM109 (pACCAR25, pAK916) and E. coli JM109 (pACCAR16, pAK96K), were constructed. The both strains have the seven carotenoid biosynthetic genes, crtE, crtB, crtI, crtY, crtZ, crtW, and crtX, which were isolated from the bacterium Erwinia uredovora or A. aurantiacum. From the former strainas, compound 1 was isolated as a major component. From the individual strains, new carotenoid glucosides (3S, 3'S)-astaxanthin β-D-diglucoside (3) and (3S, 3'R)-adonixanthin 3'-β-D-glucoside (4) were isolated. These structures were also determined by spectral means. The biological activities of hydrophilic carotenoids produced by natural or transformed strains will be investigated.
1. Structures of Julibrosides in Albiziae Cortex and Their Cytotoxicity. The dried stem bark of Albizia julibrissin DURAZZ, Albiziae Cortex, is used as tonic in China and Japan. Since it had been found that the saponin fraction from this crude drug showed a cytotoxic activity against KB cell (IC_<50> 12.8μg/ml) by the general screening, we had investigated the genuine saponins in this crude drugs to reveal the following results: a. Three new triterpenoidal saponins named Julibroside I-III were isolated and their structures were determined based on spectral and chemical evidence. After alkaline hydrolysis, the prosapogenins were also prepared and tested for cytotoxicity. b. The structure-activity relationships for KB cell cytotoxicity of Julibrosides and prosapogenins suggested that both an α-L-arabinofuranosyl-(1→4)-[β-D-glucopyranosyl-(1→3)]-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl (a mimosatetraosyl) ester unit and a monoterpene-quinovopyranosyl moiety are crucial substituents for cytotoxicity. The hydroxy group at C-16 of aglycone may play an important role in mediating cytotoxicity and the N-acetylglucosamine moiety at C-3 seems to enhance activity because Julibroside III showed the strongest cytotoxicity. 2. Synthesis of Neosaponins by Chemical Trans-Glycosylation. From the above evidence (1.b.), the mimosatetraosyl moiety is essential to show cytotoxicity. While, during the course of studies on the leguminase plants, we found that the β-fabatriosyl [α-L-rhamnopyranosyl (1→2)-β-D-galactopyranosyl-(1→2)-β-D-glucuronopyranosyl] triterpene glycosides (oleanane glucuronides) are effective for experimental liver injuries. Since the oligosaccharide parts of natural glycosides seemed to play an important role for cytotoxic and hepatoprotective activities, we had planed to synthesis neosaponins by chemical transglycosylation and examined biological activities. a. The ester glycosidic bond (mimosatetraosyl moiety) could be selectively cleaved with lithium iodide in the presence of lutidine and allyl alcohol to give an allylglycoside. The allyl tetrasaccharide was converted to a ceramide like double-tailed glycolipid, following ozonolysis, reductive amination, and N-acylation. Futhermore the tetrasaccharide is converted to an α-trichloroimidate and glycosylated to diosgenin and glycyrrhitinic acid, respectively. Following deprotectin, a diosgenin and a glycyrrhitinic acid tetraglycosides were obtained. b. Glycyrrhizine hydrolase (GHase) selectively cleaves an ether linked end glucuronic acid at C-3 position of triterpene. The β-fabatriosyl moiety obtained by GHase from Soyasaponin I was glycosylated to diosgenin by the method as described above. c. The obtained neosaponins (1〜5) were tested for cytotoxicity against PC-6 and P388 cell lines. Although they were less active than CDDP and dioscin, compounds 2, 3, 4 showed some cytotoxicity. In spite of having the same tetrasaccharide(1〜4), the potency was different due to difference of aglycone part. The compounds 3, 4, 5 were tested for hepatoprotective activity using in vitro immunological liver injury model. Since the compound 5 showed an activity, the β-fabatriosyl moiety is appeared to be important for the hepatoprotective effect.
In the course of our search for biologically active substances from marine organisms, we isolated a potent cytotoxic polyketide named callystatin A (1) from the marine sponge Callyspongia truncata collected at Goto Islands in Nagasaki Prefecture. An acetone extract of the titled fresh sponge (1.0kg) showing cytotoxic activity was subjected to bioassay-guided separation to furnish 1 (1.0mg, 1.0×10^<-4>% from the fresh sponge) as an active substance. Callystatin A (1) exhibited extremely potent cytotoxicity against KB cells at IC_<50> 10pg/ml. The plane structure of 1 having α,β-unsaturated δ-lactone, two conjugated diene, and β-hydroxyketone portions was elucidated by detailed NMR analysis. The absolute configuration of C-19 linked to a hydroxyl group was determined to be R by modified Mosher method. The stereochemistry around β-hydroxyketone portion was established as 16R, 18S, and 20S by comparison of the NMR data of 1 with those of (-)-ebelactone, 12-epi-(-)-ebelactone, and some related polyketides. Additionally, the unidentified 5R and 10R configurations were revealed by comparative CD analysis of 1 and the four model compounds (2, 3, 16, 17). The model compounds were synthesized through high E-selective Wittig olefination between an aldehyde 9, whose acetal moiety was easily converted to α,β-unsaturated δ-lactone, and allylic tributylphosphorus ylides 14, 20 as a key step. In connection with callystatin A (1), several related antitumor antibiotics were isolated from actinomyces and their plane structures are clarified. To our knowledge, this is the first example of elucidation of the absolute stereostructure among the related cytotoxic polyketides.
The calyculins [calyculin A (1)], unique polyketides bearing a variety of functionalities isolated from the Japanese marine sponge Discodermia calyx, exhibit antitumor, smooth muscle contractile, and tumor promotion activities, which are most likely to be attributable to inhibition of protein phosphatases 1 and 2A. Eight known calyculins A(1)-H, which differ in the presence or absence of methyl group on C32 and in the geometry of C2-C3 and C6-C7 double bonds, display similar inhibitory activity against protein phosphatases 2A. Further examination of D. calyx extracts led to isolation of five more calyculin derivatives, calyculin J(2), calyculinamide A(3), calyculinamide F(4), des-N-methyl-calyculin A(5), and dephosphonocalyculin A(6). Structures of 2-6 were assigned on the basis of spectral data and chemical transformation. Calyculin J(2) had a bromine atom and an additional tetrahydrofuran ring. The structure including the absolute stereochemistry has been established by chemical transformation of calyculin A. Calyculinamide A(3) was found to have a terminal amide group instead of the nitrite in 1 by analysis of spectral data. This was further substantiated by hydrolysis of 1. Dephosphonocalyculin A (6) behaved a more polar compound than other calyculins in both normal phase and reversed phase chromatographies. The FAB mass spectrum exhibited an (M+H)^+ ion at m/z929, 80 units smaller than that of calyculin A, whereas the gross structure of 6, which was identical with that of 1 except for the phosphate ester on the C17 oxygen function, was easily assigned on the basis of the COSY, HMQC, and HMBC spectra. The structure including the absolute stereochemistry has been confirmed by chemical transformation of 6 and 1 to a common compound. Compounds 3, 4, 6 showed inhibitory activity against protein phosphatase 2A similar to that of calyculin A. However, IC_<50> values of 2 and 5 were 100 and 150 times higher than that of calyculin A.
Marine microorganisms are potentially prolific sources of highly bioactive secondary metabolites that might represent useful leads in the development of new pharmaceutical agents. As part of our ongoing search for new antitumour metabolites produced by microorganisms from marine organisms, gymnastatins A (1)-H (8) have been isolated from a strain of Gymnasella dankaliensis originally separated from the marine animal Halichondria japonica. Furthermore, pericosines A (11), B (12), macrosphelides E (13) and F (14) have been isolated from a strain of Periconia byssoides separated from the marine animal Aplysia kurodai. The stereostructures of these metabolites have been elucidated by spectroscopic analyses and some chemical transformations. Among these compounds, 1-3, 6, 7, 11 and 12 exhibited significant cytotoxicity against cultured P388 cells.
In tropical areas, sporadic but highly fatal seafood poisonings are known to occur after eating sharks, turtles, and sardines. We identified the causative toxins and pointed out that they were distinctly different from ciguatoxin both chemically and etiologically. [Shark Poisoning] From 30g of the shark liver that was implicated in massive human intoxication, two toxins were isolated and named carchatoxin-A and -B, respectively. Compared with ciguatoxin, carchatoxins were less polar by HPLC, killed mice in shorter periods, and caused only mild diarrhea. They inhibited depolarization of Na channels, as does ciguatoxin. The very tiny amounts of the toxins prevented us from elucidating their structures by NMR spectroscopy. Nevertheless, effort is being continued to obtain further structural information. [Turtle Poisoning] About 90g of the left-over meal was made available for experiments. From the low polarity, weak basicity, and inflammatory nature of the turtle toxin, we predicted that the toxin could be lyngbyatoxin-A or its analogs. Subsequent analysis by HPLC and LC/MS confirmed the presence of lyngbyatoxin-A and thus suggested the toxin source to be blue-green algae tangling on Zostera. [Clupeotoxism] Clupeotoxin isolated from a head of the sardine, Herklotsichtys quadrimaculatus, implicated in human fatality was identified to be palytoxin, based on its chromatographic, cytotoxic, and hemolytic properties, and neutralization assay results obtained by using an anti-palytoxin antibody. This is the first time to determine the cause of clupeotoxism. The dinoflagellate Ostreopsis siamensis was suggested to be the primary source of the toxin.
Identification of significant populating conformers in extremely flexible molecules is a matter of long standing interest. We have developed a useful and very reliable method for conformational analysis of a flexible compound using the information obtained from the chemical shifts. It is based on the chemical shift simulation method which utilized the calculation of the change in proton chemical shifts produced by nearby aromatic ring. In this paper we report a successful application of our method to the conformational analysis of an oligosaccharide in aqueous solution using molecular dynamics calculations. The tetrasaccharide sialyl Lewis^X derivatives have known to be a ligand for endothelial leukocyte adhesion molecule-1 (ELAM-1, E-selectin). We selected the sialyl Lewis^X derivatives (1-3) for the conformational analysis. Our method consists of four stages: 1) the generation of the probable structures, 2) estimation of the induced proton shifts in these structures caused by the aromatic ring, 3) the selection of the most likely conformers by comparison of the observed and calculated induced shift, and 4) molecular dynamics simulation starting from these candidate structures to obtain a conformer which satisfying the observed induced shifts. According to our strategy, we obtained a converged structure by the molecular dynamics simulation with Amber^* force field. It gave a satisfying agreement of the induced shifts of 25 C-H protons of the five saccharide units with those of the observed. (R^2=0.90)
Bupleuri Radix, which is one of the most important component in Chinese traditional medicine, has been used as an antiinflammatory, antipyretic, and antihepatotoxic agent. The botanical origin of this natural medicine was prescribed as the roots of Bupleurum falcatum ("Mishimasaiko" in Japanese) in Japanese Pharmacopea XIII. In recent years, due to the poor supply of Japanese Bupleuri Radix, various Chinese Bupleuri Radix have been imported and commonly used in the traditional preparations. In the course of our studies in search of bioactive principles from Chinese natural medicine, we have found that the glycosidic fraction from a Chinese Bupleuri Radix, the roots of B. scorzonerfolium, showed potent protective effect on liver injury induced by D-galactosamine and lipopolysaccharide. From the active glycosidic fraction, sixteen new triterpene saponins called bupleurosides I (1)-XVI (16) were isolated together with sixteen known saponins. The structures of bupleurosides were elucidated on the basis of physicochemical evidence using 2D-NMR technique (H-H, C-H COSY, NOESY, HMBC) and chemical findings, which included the synthesis of the adonitol-glucoside part of bupleuroside XVI (16). The hepatoprotective effect of bupleurosides and known related saponins was examined by monitoring the inhibition of cytotoxicity induced by D-galactosamine in primary cultured rat hepatocytes. Among them, bupleurosides III (3), VI (6), IX (9), and XIII (13) and saikosaponin b_3 (28) were found to exhibit the inhibitory activity. In addition, by examination of the structure requirement in saponin for the hepatocytoprotective effect, the 11-oxygenated functional group, the 16β- and 28-hydroxyl groups, and the 3-O-diglycoside structure were found to be essential to exerting the activity. By utilizing anodic oxidation as the key reaction, bupleuroside-type saponins were readily synthesized from naturally abundant olean-12-ene oligoglycosides without previous protection of the hydroxyl groups in starting oligoglycosides.
The petal of blue morning glory, Ipomoea tricolor cv. heavenly blue, changes its color from purplish red to light blue during blooming. The pigment, heavenly blue anthocyanin, was distributed in the vacuoles of upper and lower epidermis and not in parenchyma. We measured pHv of living petal cell directly using proton selective-microelectrode. The pHv of purplish red buds was 6.6 and that of blue open flower was 7.7. This study provides the clear evidence that the color change is caused by the increase of pHv during blooming. The protoplasts were prepared from the pigment cells and then the vacuolar membrane were provided. By immunoblot technique H^+-ATPase and H^+-PPase retained both in the buds and the open flower petals. PPi- and ATP-dependent H^+-transport activities in vacuolar membrane vesicles from the buds and the open flower petals were almost in the same level, although no activities in those from the faded flower petals. HBA produced three isomers by irradiation of UV-B light in the acidic methanol solution, of which the outer two caffeic acids were isomerized to Z-configuration. The inner one was not because it might be strongly stacking to the anthocyanidin nucleus. Anthocyanin is generally stable in strong acidic condition. However, under UV-irradiation, HBA in the acidic medium was not stable, but in neutral condition relatively stable. It is concerning that the intramolecular stacking of HBA between caffeic acid and the chromophore is stronger in the neutral condition than in acidic medium. HBA showed very strong fluorescence in neutral aq. solution, while in acidic very weak. The strong fluorescence may play a role of UV-light quenching and protecting system.
Leaf-movements of plant mostly fall into two categories: rhythmic slow leaf-movement in nyctinastic plants, and more rapid seismonastic movements, that occur in a limited number of the nyctinastic plants. The rapid leaf-movement of Mimosa pudica L. is most well-known of all the plant movements. It has been already verified in the early beginnig of 20th century that this movement is caused by some chemical substances. But, identification them has been unsuccessful, and thought to be impossible. However, we have succeeded in the isolation of chemical factor for the leaf-movement of mimosa plant. That is the mixture of three chemical substances, two of the three were identified as potassium malate and magnesium transaconitate. The mixture of these chemical substances was effective at 10^<-8>-10^<-9>M. And all these three components were necessary for causing leaf-movement. Nyctinastic movement in the circadian rhythm observed in plants is known to be controlled by an internal clock. We have isolated several bioactive substances that cause the leaf-closing movement in these plants, e.g. potassium chelidonate (2) from Cassia mimosoides L., trigonelline (3) from Aeshenomene indica L., and phyllanthurinolactone (4) from Phyllanthus urinaria. Each leaf-movement factor was effective at the concentration of 10^<-6>-10^<-7>M, and only for the original plant, not for other nyctnastic plants. This result strongly suggests the existence of different leaf-movement factor in every nyctinastic plants. Recently, we have isolated two inversely effective leaf-movement factors, potassium D-idarate (5) as leaf-closing factor and potassium lespedezate (7) as leaf-opening factor from one nyctinastic plant, Lespedeza cuneata G. Don., and the MeOH-extract of the plant had inverse bioactivity between daytime and night. Thus, the nyctinastic leaf-movement is controlled by the balance in the concentration between inversely effective bioactive substances.
The "Advanced Marfey's method" has been developed to non-empirically determine the absolute configuration of constituent amino acids in a peptide. This method consists of Marfey's method as chromatographic technique for the separation of amino acids into each enantiomer, the detection of an amino acid by mass spectrometry and a procedure for obtaining the corresponding enantioner from ether the L- or D-amino acid. We introduced the "D,L-FDLA derivatization" for a procedure for obtaining the corresponding enantioner instead of the conventional chemical racemization. The derivatization with an equal mixture of D- and L-FDLA was able to give the desired enantiomer from its L- or D-amino acid on the HPLC chromatogram. The procedure was successfully applied to the determination of the absolute configuration of Ahp, one of constituent amino acids in aeruginopeptin 228-A (1) by a cyanobacterium. It was also suggested that the "Advanced Marfey's method" including "D,L-FDLA derivatization" is also applicable to the determination of absolute configuration of primary amino compounds except for amino acids on the basis of the proposed separation mechanism of this method. Therefore, we carried out the fundamental approaches of this method to primary amino compounds. As the results, we confirmed its applicability to several primary amino compounds, and the method was successfully applied to the characterization of not only constituent amino acids but also an amino compound in microginin (3) produced by a cyanobacterium.
Solitary wasps paralyze insects or spiders with stinging venom and feed the paralyzed victims to their larvae. Accordingly, the venom should contain a variety of constituents acting on nervous systems. However, only a few constituents of the venom have been chemically characterized, despite the fact that thousands of solitary wasp species are inhabiting on the earth. We report here the isolation, structure and biological effects of novel peptides in the solitary wasp venom. Eumenine mastoparan-AF (1) was isolated from the venom of the Eumenine wasp Anterhynchium flavomarginatum micado, which was responsible for the block of neurotransmission in lobster leg. The congeners 2-4 were also obtained, but they turned out to be the hydrolysate by the protease in the venom. The structure of 1 was similar to that of mastoparan (5), a mast cell degranulating peptide in the venom of a hornet wasp, and the peptide 1 was indeed as active as 5 in stimulation of histamine release from rat peritoneal mast cells. The peptide 3 was active in this assay as well, whereas the peptides 2 and 4 were inactive. As-A (6) and As-B (7) were the major peptides in the venom of the spider wasp Anoplius samariensis, of which the pentapeptide 6 blocked the neurotransmission in lobster leg. Bm-17 (8) was obtained from the venom of another spider wasp Batozonellus maculifrons, but totally inactive in the lobster leg muscle preparation.
A novel inhibitor of aflatoxin production of Aspergillus parasiticus was isolated from the mycelial extracts of Streptomyces sp. MRI142 and termed aflastatin A (1). The molecular formula of 1 was determined as C_<62>H_<115>NO_<24>, and its structure was elucidated by NMR and chemical degradation experiments. By analyzing a variety of 2D-NMR spectra of 1, a partial polyhydroxylated structure (A) was identified. Based on the structure of A, 1 was oxidized with NaIO_4, and three fragment molecules, 2, 3 and 4, were obtained. Since all carbon atoms contained in 1 were involved in the partial and fragment structures, its carbon chain was constructed with them. Finally, the stereochemistry of a tetrahydropyran ring was determined by J values and NOEs among the ring protons. Thus, the total structure of aflastatin A was characterized as 1, which is a unique tetramic acid derivative having a highly oxygenated long alkyl chain. To determine the absolute configuration of 4, 2,4-dimethyl-1,6-hexanediol dibenzoate (6) was prepared from 4. By comparison of its CD spectrum with those of opticaly active (2R, 4S)- and (2S, 4S)-6, which were derivatized from cycloheximide, the configurations of natural 6 were determined to (2S, 4R). On the other hand, N-methylalanine from 4 was determined to have a D configuration. The incorporation experiments using ^<13>C-labeled acetate and propionate suggested that most of the C_2 and C_3 units involved in the alkyl chain moiety of 1 were biosynthesized from acetic and propionic acid. Unexpectedly, no incorporation was observed in one of the C_2 units and two of the C_4 units in the alkyl chain. Aflastatin A inhibited aflatoxin production in A. parasiticus at the concentration of 0.5μg/mL completely, but didn't inhibit its growth at the same concentration.
Ceramide, the product of sphingomyelin hydrolysis, has been reported to play an important role in signal transduction. In response to several stimuli including TNFα and IL-1β, the ceramide level increases in cells in advance of stimuli-mediated physiological and pathological processes. Thus, inhibition of sphingomyelinase(SMase) may lead to regulation of ceramide levels and to therapy for inflammation and autoimmune diseases. With this aim in mind, fermentation broths of microorganisms were screened for SMase inhibitors and scyphostatin(1) was discovered in a mycelial extract of Dasyscyphus mollissimus. The structural study of 1 was carried out by spectroscopic investigations and some chemical transformations. Then, it was established that 1 was composed of a fatty acid and an amino alchohol substituted with a highly oxygeneted cyclohexenone moiety. The structural similarity between 1 and ceramide represented by the N-acyl-amino alcohol moiety suggests that 1 may exhibit inhibitory activity as a substrate or product analogue of the enzymatic reaction. As far as we know, there are no previous reports about a neutral SMase inhibitor with significant activity, and 1 may be a useful probe for studying neutral SMase-mediated signal transduction.
In order to clarify the precise sugar-recognition mechanism of pradimicin, we analyzed the supramolecular formation (Supramolecule I, II and III) using the derivative BMY-28864 (B), Ca^<2+> (C) and methyl-α-D-mannoside (M)(Fig. 1). Major obstacle to the NMR analysis of the supramolecule formation was its insolubility in D_2O. We found the NMR condition to analyze the chelate formation of B with C in D_2O containing Py-d_5 (Fig. 2). BMY-28864 can be precipitated by addition of excess amounts of M and C in the aqueous solution. After washing with water and lyophilization, the precipitate was dissolved in DMSO-d_6. ^1H-NMR spectrum of B and M in this mixture showed the ratio of one to one. In the aqueous solution, the positive first Cotton effect in the CD spectra increased as the ratio of C to B increases. The maximum CD intensity was obtained from mixtures that the ratio of C to B exceeded 0.34. In view of the above result, we concluded that the ratio of B: M: C in the supramolecule is 3: 1: 3. After a number of unsuccessful experiments, we finally succeeded in the formation of single crystals of the supramolecule III. The crystallographic study of the supramolecule III was carried out by using synchrotron radiation in the Photon Factory of National Laboratory for High Energy Physics in Japan. Although high resolution data could not be obtained, the crystals belong to the trigonal system, and the cell constant is a=b=21.6, c=46.5Å. These NMR, CD, and X-ray data afforded a model that two calcium ions are located at the center of the molecule on a crystallographic 3-fold axis. B is self-associated with each other as BB in a right-handed screw manner. The three self-associated Bs are placed around the 3-fold axis(Fig. 4). The computer-aided modeling and the docking study revealed the manner of intermolecule association between B and M (Fig. 4). The proposed supramolecule model affords a mechanism of action of pradimicin which is consistent with the biological data up to present.
Infectious diseases due to antibiotic-resistant bacteria pose a threat to public health around the world. Especially, nosocomial infections caused by methicillin-resistant Staphylococcus aureus (MRSA) in hospitals have become a serious clinical problem. Therefore, an antibacterial agent is clinically desirable. In the course of our screening system for anti-MRSA antibiotics, we found that a novel cyclic depsipeptide antibiotic, WAP-8294A_2(1) produced by a Gram-negative bacterium Lysobacter sp. showed higher in vivo activity against MRSA. We report the structure and activity of this antibiotic. This antibiotic is composed of Gly, L-Leu, L-Glu, D-Asn, D-Trp, D-threo-β-hydroxyasparagine, N-methyl-D-Phe, N-methyl-L-Val, and two residues of L-Ser, D-Orn, and D-3-hydroxy-7-methyl-octanoic acid. The structure of 1 was determined mainly by 2D NMR experiments including HMBC and ROESY techniques. 1 was as active as vancomycin against MRSA clinical isolates (MIC: 0.78μg/mL) and interestingly, this activity was highly enhanced by the addition of 10% human serum (MIC: <0.1μg/mL). No activity of 1 was observed against Gram-negative bacteria, yeasts and fungi. In vivo efficacies of 1 and vancomycin were assessed in the experimental systemic MRSA infection of mice. The mean ED_<50> values of 1 and vancomycin against nine MRSA strains were 0.38mg/kg and 5.3mg/kg, respectively, indicating that 1 is 14 times more active than vancomycin.
In order to overcome the problems of HMBC associated with the setting of delay time for observing small long range couplings and short T_2 with broad methylene proton signals, we have developed a new technique, 3D-HMBC. In the pulse sequence of this method, delay time (Δ), which has to be fixed to an appropriate value (typically 60msec) in the conventional 2D-HMBC, is replaced by a variable parameter (t_1). This modification enables to cover wide delay time range needed to observe from small to large long range couplings and thus gives better HMBC spectra than obtained by the conventional method. In addition, we have developed a new NMR pulse sequence (3D-J-HMBC) useful for observing indirectly long range ^<13>C-^<13>C couplings separated by two bonds by modification of the 3D-HMBC. Its application for biosynthetic studies of a fungal metabolite, agonomic acid is explained.