Lipase-mediated asymmetrization of the meso-diol (4) afforded the optically pure monoacetate (5) in good yield. To prepare potential chiral 2,5-cyclohexadienone synthons, 5 was first oxidized to the enone (8) in an excellent yield. On the other hand, 5 was transformed into the mono-pivalate (7) which in turn was oxidized to the enone (9) in good overall yield. Both 8 and 9 are virtually enantiomeric and serve as potential synthons of chiral 4-oxygenated 2,5-cyclohexadienones. Fortuitously, we found that the enone [(-)-2], a potential chiral synthon of 2,5-cyclohexadienone (1), was generated in one step in an excellent yield when the acetate (5) was refluxed with ammonium formate in the presence of a catalytic amount of PdCl_2(PPh_3)_2. The same reaction also occurred with the pivalate (7) to furnish the enantiomeric enone [(+)-2]. The reaction was found to take place by unprecedented suprafacial 1,4-hydrogen transfer mechanism which was proven by deuterium labeling experiment. To demonstrate potential of the optically pure enones thus obtained as potential chiral 2,5-cyclohexadienone synthons, synthesis of some natural products and the related compounds was investigated. As we have anticipated the reactions occurred in completely diastereoselective manners to both enone double bond and the carbonyl group owing to the biased frameworks. Furthermore, the regeneration of the masked double bond could also be carried out without difficulty by thermal retro-Diels-Alder cleavage. Reflecting these stereochemical characteristics of the substrates, the following four products, 2-aminoheptanedioic acid (16), conduritol C (27), eutipoxide B (39), and carvone (46) as well as 5-substituted-2-cyclohexenones (17) including 5-trialkylsilyl- and 5-trialkylstannyl-2-cyclohexenones have been synthesized in both enantiomeric forms.
The terpenoids 1-3, which posess both intriguing structural features and biological activities (except for 1), contain asymmetric quaternary carbon centers whose efficient and enantio divergent construction is the important issue for the asymmetric total synthesis. We now wish to report the first total synthesis of the marine furanoterpene 1 and the formal total syntheses of (-)-dihydroactinidiolide 2, which exhibits pheromone and phytotoxic activities, and (-)-anastrephin 3, an insect sex attracting pheromone, employing the intramolecular cycloaddition reaction as the key steps. 1. Total Synthesis and Determination of the Absolute Configuration of Furanoterpene 1 The chiral aldehyde 6, which was prepared in an optically active form by using the methodology for assembling the asymmetric quaternary carbon centers developed by Yamamoto, was converted into 1 employing the fused furan construction based on the intramolecular [3+2] cycloaddition of nitrile oxide developed by us. The absolute configuration of the quaternary asymmetric carbon in 1 was determined as S. Alternatively, the intermediate 9 was synthesized more conveniently by using the PLE mediated asymmetric hydrolysis of the functionalyzed prochiral diester 18. 2. Formal Total Syntheses of (-)-Dihydroactinidiolide 2 and (-)-Anastrephin 3 The protected diol 7, an intermediate for the synthesis of 1, was converted into the amide 36a, which was treated with triflic anhydride and collidine to give diastereoselectively (5:1) the bicyclic cyclobutanone 39a via the intramolecular [2+2] cycloaddition of the in situ generated keteniminium salt 37a in 68% yield. The major diastereomer was converted into the lactone 41, an easily convertible immediate precursor to 2. For the synthesis of anastrephin 3, the olefinic lactone 48, a key intermediate for the total synthesis of Tadano, was synthesized similarly via the [2+2] cycloaddition of the nor-keteniminium salt generated from 45. However, the efficiency of 1,2-asymmetric induction during the cycloaddition is disappointingly low (2:1).
The Ireland-Claisen rearrangement of allyl ester enolates has frequently been applied to the stereocontrolled C-C bond formation because of its high diastereo-selectivity. However, being "self-immolative", the reaction is not suited for the asymmetric induction. In order to develop a new methodology in asymmetric synthesis, we investigated the Claisen rearrangement of amide enolates and found that 1) the enolate derived from N-(2E)-butenyl-N-butylpropanamide rearranged with excellent internal asymmetric induction (syn:anti=199:1) (eq. 2), that 2) the reaction of amide enolates containing chiral alkyl groups (R^*) on the nitrogen proceeded with high selectivity in relative asymmetric induction in addition to the syn selectivity described above, and that 3) the rearrangement can be extended to the acetamides with a heteroatom at the α-position (Table 2). In order to test its applicability, (-)-verrucarinolactone and D-allo-isoleucine were synthesized (eq. 5 and 6). In a continuing effort to promote this potentially useful reaction, we attempted a synthesis of (-)-isoiridomyrmecin ((-)-1), a bioactive principle of Actinidia polygama with unique activity toward felids and an attractive target of many synthetic efforts because of the presence of the 4 contiguous chiral centers in a monoterpenoid carbon skeleton. Starting from dl-alcohol 6 derived from 2-methylcyclopentanone, (-)-1 was synthesized with excellent stereoselectivity in 6 steps utilizing the aza-Claisen rearrangement as a key step (eq. 7 and 8). Another notable feature is the introduction of N,N,N',N'-tetramethylazodicarboxamide (TMAD)-Bu_3P, a new Mitsunobu reagent system, applicable to the preparation of allylic secondary amines in general(eq. 7).
Although the palladium-promoted cycloalkenylation reaction of an olefinic silyl enol ether is a powerful strategy for construction of polycyclic system, little is known about successful application of the above reaction to biologically active natural product syntheses. Herein we report the total syntheses of (±)-hirsutene (4) and (±)-stemodin (18) using diastereoselective palladium-promoted cycloalkenylation reaction. (I) Total Synthesis of (±)-Hirsutene (4) A sequence leading from trans-6-methyl-3-cyclohexenecarboxylic acid (6) to the cis, anti, cis linear triquinane hirsutene (4) is described in which the key step utilizes an acid catalyzed intramolecular conjugate addition (8→9a) and a Pd^<2+>-promoted highly stereocontrolled cyclization (10→12). (II) Total Synthesis of (±)-Stemodin (18) Intramolecular Diels-Alder reaction of the triene (24) and Pd^<2+>-promoted cyclization reaction of the olefinic silyl enol ether of 29 have been utilized as the key steps for a conceptually new, highly diastereocontrolled formal total synthesis of (±)-stemodin (18).
The allenic carotenoid fucoxanthin 1 is known as an auxiliary light harvesting pigment for photosynthesis in the sea. We have accomplished the first total synthesis of optically active 1. In order to construct the fucoxanthin skeletal compound 2, two kinds of Wittig salts 3 and 5 were synthesized from the common intermediate 9a, which was prepared from the optically active hydroxy ketone 6 in good yield. Stereoselective DIBAL reduction of the anti-epoxide 11b derived from 9a in two steps and subsequent MnO_2 oxidation gave the allenic aldehyde 13, which was converted to the allenic Wittig salt 5. Rearrangement of the α-acetylenic alcohol 9a to the α,β-unsaturated ketone 14 catalyzed by oxometallic derivatives and subsequent iodine-catalyzed isomerization of the 6,7-exo-double bond into the 5,6-endo position provided the intermediate 15, leading to the 8-oxo-Wittig salt 3. The 8-oxo-Wittig salt 3 was condensed with the dialdehyde 4 to yield an isomeric mixture of 8-oxo-apocarotenals 16 and 17. Palladium-catalyzed isomerization of the mixture gave the all-E-isomer 16 as a sole product. Condensation between the TES ether 18 of 16 and the allenic Wittig salt 5 followed by reacetylation of 3'-hydroxyl group and deprotection of silyl group gave the skeletal compound 2. Epoxidation of the 5,6-double bond in 2 provided fucoxanthin 1. Spectral properties of the synthetic 1 were identical with those of the natural specimen.
A novel antibiotic AB3217-A (1) was isolated in 1992 from the fermentation broth of Streptomyces platensis AB3217. It exhibits anti-mite, antimicrobial, and herbicidal activities. The unique structure of 1 was elucidated by spectroscopic means and its absolute configuration was established by X-ray crystallographic analysis. We now report the synthetic studies of AB3217-A. AB3217-A was divided into two portions, namely the deacetylanisomycin segment A and the β-D-xylofuranose segment B. The epoxide 3, which was prepared from dimethyl L-tartrate (2) according to the known procedure, was transformed to pyrrolidine-aldehyde 10 by cyclization of aminoepoxide in the presence of Lewis acid as a key step. This aldehyde 10 was subjected to the stereoselective anisylation followed by successive oxidation and stereoselective reduction to afford the desired deacetylanisomycin segment 14. α-Phenylthioglycoside 20, corresponding to the β-D-xylofuranose segment, was prepared from diacetone-D-glucose (15) by a conventional procedure. The coupling of deacetylanisomycin segment 14 and β-D-xylofuranose segment 20 was realized by treatment with NaN(TMS)_2 in DMF to afford the coupling product 21, which was subjected to cyclization using NBS to give the 9-membered ring product 23. We are now investigating deprotection of 23 to accomplish the total synthesis of AB3217-A.
In the last few years, numerous new indole and oxindole alkaloids were isolated from the Chinese medicinal plant, Gelsemium elegans Benth., by us and other researchers. Our interest in the relationship of various skeletons of Gelsemium alkaloids has led us to consider their biogenetic pathway and to investigate our proposal by chemical transformations. Here, we describe the following three newly developed chemistry on the Gelsemium alkaloids. I: 3,3-Disubstituted oxindole (16) could be converted to the Na-methoxy-oxindole (20) via tungstate-catalyzed oxidation of the indoline derivative (17). This method was applied to the synthesis of the Gelsemium alkaloids, (5), (27), (28) and (56). II: Based on a biogenetic speculation, a humantenine-type Na-methoxyoxindole alkaloid, humantenirine (27), was prepared from a sarpagine-type indole alkaloid, gardnerine (21), via stereoselective rearrangement to the oxindole, olefin inversion, and introduction of a methoxy group onto the Na-function. III: 20-Hydroxydihydrorankinidine (28), a biogenetically hypothetical intermediate to the new skeletal type alkaloid, gelselegine (8) (Fig. 5), was synthesized from ajmaline (33) in 22 steps. Gardnerine (21) was transformed into the gelselegine skeleton (53), which was then converted to the natural gelsedine-type alkaloid, gelsemicine (56) in a highly stereoselective manner. Starting from 21 the first and biomimetic construction of another gelselegine-type alkaloid (60) having a hydroxy group at C-19 was achieved via a biogenetically hypothetical aziridine intermediate (59).
Tumor-promoting teleocidins are known to exist in an equilibrium between two conformational states, the twist form and the sofa form, in solution. Benzolactam-Vs (BL-Vs), in which the indole ring of indolactam was replaced by a benzene ring and ring size was prescribed by methylelene, were designed in an attempt to reproduce the active conformation of teleocidins and synthsized. The conformational analysis was carried out by ^1H-NMR. The 8-membered lactam (bezolactam-V8, 4) exists only in twist form in solution. Both the 9-membered lactam (benzolactam-V9, 5) and the 10-membered lactam (benzolactam-V10, 6) exist only in sofa form in solution. It has been reported that the biological activities of IL-V are reinforced by introduction of alkyl chain. Alkylated benzolactams (BL-V8-310 (10), BL-V9-310 (11)) were synthesized for the evaluation of the biological activities. The 8-membered lactam (10) showed stronger biological activities than those of indolactam-V. On the other hand, 9-membered lactam (11) showed above 10^3-times less activities than those of 10. These results clearly indicate that the active conformation for tumor-promoting activities of teleocidins is close to the twist form.
Remarkably high regio- and stereoselective approach for the syntheses of dendrobatid alkaloids (+)-homopumiliotoxin 223G (1), (+)-allopumiliotoxin 267A (2) and (+)-allopumiliotoxin 339A (3) have been developed. As a model study for the syntheses of these alkaloids, we initially undertook intramolecular chromium(II)-mediated cyclization of the racemic N-iodoalkenyl piperidine 9, which smoothly proceeded by treatment with CrCl_2 (5 equiv) and catalytic NiCl_2 (2.5mol%) in DMF to form a 1.3:1 epimeric mixture of 2-hydroxy-3(E)-alkylidene-trans-quinolizidines 20a and 20b. When the alternative chiral N-iodoalkenyl piperidine 18 was subjected to the identical cyclization conditions, the 3(E)-alkylidene-trans-quinolizidine 22a with the axially oriented 2-hydroxy group was formed as a single isomer. The first total synthesis of (+)-homopumiliotoxin 223G (1) has been achieved from 22a via deprotection of a benzyl group and removal of the acetoxy group in a single operation. Based on these model studies, we then undertook the enantioselective total synthesis of (+)-allopumiliotoxin 267A (1). For the synthesis of (+)-allopumiliotoxin 267A (1) coupling of the two segments, (E)-vinyl iodide 33, obtained via stereospecific palladium-catalyzed hydrostannation, and the pyrrolidine derivative 38, gave the N-iodoalkenyl pyrrolidine 39, which underwent intramolecular chromium(II)-mediated cyclization after cleavage of the dimethyl acetal, exclusively providing 41 with complete retension of the required E alkenyl geometry. Subsequent cleavage of the benzyl group furnished 2. Synthesis of allopumiliotoxin 339A (3) was next investigated by employing the strategy developed for 2. The side chain segment, (E)-vinyl iodide 50, was prepared via high degree of stereo- and regioselective reactions involving Evans alkylation and palladium-catalyzed hydrostannation. Intramolecular nickel(II)/chromium(II)-mediated cyclization of the N-iodoalkenyl pyrrolidine 54, available via coupling 50 with pyrrolidine derivative 52, led to exclusive formation of 55, which was deprotected to afford 3.
Neurotrophic agents such as nerve growth factor (NGF), a well-characterized protein, are required for the survial and function of neurons. In 1991 we reported the isolation and characterization of the first non-protein neurotrophic factor, (+)-lactacystin, a novel sulfur-containing γ-lactam produced by a culture broth of Streptomyces sp. OM-6519. Lactacystin induces neuritogenesis and causes a transient increase in the intracellular cAMP level in Neuro 2a neuroblastoma cells. Corey and Reichard recently reported the first total synthesis of lactacystin. Herein we describe a concise alternative approach, designed to afford easy access to both the natural product and a variety of analogues. Key steps in the elaboration of the lactam moiety include the stereoselective hydroxymethylation of oxazoline 11 and an asymmetric allylboration which introduces the hydroxyl and methyl substituted at C(6) and C(7), respectively. As our point of departure, 2(R), 3(S)-γ-hydroxyleucine methyl ester (10) was treated with methly benzimidate to furnish the trans-disubstituted oxazoline 11. Aldol condensation with formaldehyde via the Seebach protocol then gave 12 exclusively (>98% de). Moffat oxidation of 12 afforded aldehyde 13, which was subjected without purification to allylboration with (E)-crotyldiisopinocampheylborane as described by Brown. The desired γ-methly homoallylic alcohol 15 was thus obtained. Conversion of 16 to carboxylic acid 17 entailed ozonolysis and reductive workup followed by selective oxidation. The key γ-lactam 18 could be elaborated by catalytic transfer hydrogenation of 16 and ester hydrolysis. The amino side chain was incorporated via thioesterification of 18 with BOPCl and N-acetyl-L-cystein allyl ester. Finally, deallylation of 19 gave pure (+)-lactacystin as colorless needles. In summary, the development of an economic and versatile synthetic approach to lactacystin (10 steps, 13% overall yield) should permit the preparation of useful quantities of (+)-lactacystin and its analogues, greatly facilitatin the on going pharmacological studies of neurotrophic factors.
The first total synthesis of novel antitumor antibiotic (+)-leinamycin (1) is reported. The unique structural features of leinamycin include the 1-oxo-1, 2-dithiolan-3-one moiety fused in a spiro fashion to an 18-membered lactam with an extensively conjugated thiazole ring. No other natural products with such an unusual dithiolanone moiety have been reported to date. The 18-membered lactam 19 was assembled efficiently from Seebach's dioxolanone 2 via sequential chemo- and stereoselective reactions including iterative application of our versatile thiol ester reductions. The delicate dithiolanone moiety of 1 was constructed stereoselectively by critical intramolecular delivery of a sulfur atom and the ensuing Beckmann fragmentation.
TypeIV collagenases are metalloproteinases which degrade typeIV collagen, one of the major components of basement membranes. These enzymes are thought to play an important role in tumor cell invasion. Many studies to date indicate a correlation between the amounts of these enzymes secreted and metastatic potential of tumor cells. During the course of screening for inhibitors of these enzymes, matlystatin A (1), B (2a), D (3), E (4) and F (5) were isolated from Actinomadura atramentaria. The structures of both compounds were deduced on the basis of spectroscopic analysis including several types of 2D NMR and FAB-MS/MS. In connection with our interests in the biological activities of matlystatins and our desire to determine their absolute configurations unambiguously, we planned the syntheses of matlystatins. The total synthesis of matlystatin B was accomplished by coupling 4 protected subunits: 7,9,13 and O-benzylhydroxylamine. Matlystatin A was synthesized using the carboxylic acid 15 and the amine 19 as key intermediates. Since spectral properties of 1 and 2a were identical with those of the corresponding natural products, the absolute configuration of 1 and 2a were unambiguously determined. In the next stage, the relationships between stereochemistry and inhibitory activity were studied for ten stereoisomers of matlystatin B. The structure activity relationships revealed that C2' stereochemistly plays an important role in the inhibition of typeIV collagenases.
We recently developed the stereospecific methylation of γ,δ-epoxy acrylates by trimethyl-aluminum by which both anti compounds and syn compounds can be efficiently and highly stereoselectively produced starting from (E)-γ,δ-epoxy acrylates and (Z)-epoxy acrylates, respectively (Scheme 1). We report herein a highly stereoselective construction of the eight contiguous chiral centers of ansa-chains of rifamycins and synthetic studies toward total synthesis of streptovaricin U based on the above methodology. Starting from (R)-3-benzyloxy-2-methyl-1-propanol (1), the unsaturated ester 13 having seven contiguous chiral centers of rifamycins has been highly diastereoselectively synthesized by using three times of the methylation reaction with trimethylaluminum (4→5, 9→10, 12→13) (Scheme 2). The remaining two chiral centers were introduced by the reduction of the epoxy alcohol 15 with NaBH_3CN in the presence of BF_3・Et_2O. It is noteworthy that all the stereochemistries of four secondary hydroxyl groups in the ansa-chain were stereocontrolled virtually completely by using not the asymmetric epoxidation but usual MCPBA epoxidation. The aromatic core of streptovaricin U has been regioselectively synthesized by the Diels-Alder reaction of diene 1 and dibromoquinone 2 in excellent yield (Scheme 3). On the other hand, the nine contiguous chiral centers of streptovaricin U have been constructed by using four times of the successive methylation reaction with trimethylaluminum (4→5, 7→8, 10→11, 13→14) (Scheme 4). The present work demonstrates the synthetic potential and vailidity of the methodology for the synthesis of polypropionate antibiotics.
A new cyclic depsipeptide aureobasidin A (1), isolated as a major component from the culture medium of the black yeast Aureobasidium pullulans R106, exhibits a strong antifungal activity against pathogenic fungi such as Candida albicans, Cryptococcus neoformans, and some species of Aspergillus with a low toxicity. For purpose of investigation of a structure-activity relationship of the aureobasidin family antibiotic, we attempted a total synthesis of aureobasidin A, aiming an establishment of a synthetic technique of the cyclic depsipeptide containing N-methyl amino acids, known as a difficult component in condensation reaction because of its steric bulkiness. The linkage between alle^1 and Pro^9 was chosen as a site of the final cyclization to avoid the coupling at an N-methyl amino acid as an amino component, which otherwise may diminish a yield of the cyclization reaction due to its steric characters. A linear nonapeptide(1-9) was synthesized according to the Boc strategy. Thus, a coupling between Leu^3-HOMeVal^4-Hmp^5 (Segment B) and MeVal^6-Phe^7-MePhe^8-Pro^9 (Segment C) was first attempted, followed by condensation of the coupling product (3-9) with alle^1-MeVal^2 (Segment A) by the fragment condensation. The linear nonapeptide 24 was cyclized with PyBroP in CH_2Cl_2 under high-dilution condition (10^<-3>M) to afford the cyclic monomer predominantly. The synthetic cyclic peptide thus obtained is completely identical with the natural antibiotic in all respects (TLC, HPLC, ^1H-NMR, and antifungal activities). Thus, we could achieve the first total synthesis of the unique depsipeptide, aureobasidin A.
There are many reports of the syntheses of natural products using sugars as a starting material. We examined the syntheses of some marine natural products from D-glucose. (1) Total synthesis of bengamide E (1) Bengamide E (1), one of the members of bengamide family, is a novel sponged-derived natural product and has a unique structure. Our synthetic plan is to utilized the entire carbon skeleton of D-glucose for the preparation of the side chain. Inversion of the stereochemistry of the hydroxy groups at C-3 and C-4 was accomplished according to the literature procedure. The formation of the terminal E-olefin by Julia's protocol provided 12, was condensed with cyclo-L-lysine to give 13. Deprotection afforded bengamide E (1). (2) Synthetic studies of ciguatoxin (16) Ciguatoxin (16) is the toxic principle of ciguatera, which is responsive for the most widespread food poisoning of non bacterial origin. In 1989 Yasumoto et al established the whole structure including relative stereochemistry of ciguatoxin (16) and its congener, gambiertoxin 4B (17). In order to open the way to immunoassay of these toxins and to obtain more reliable information on the absolute configuration of these toxins, we planned the preparation of the ABC ring flamework of these toxins.
Tautomycin (1) was first isolated in 1987 by Isono and co-workers and the full stereostructure was established recently. This compound is a potent inhibitor of protein phosphatases type 1 and type 2A. Here we describe the synthetic studies of 1. Synthesis of the C1-C10 segment. We applied the newly developed methodology for the synthesis of this segment. This methodology involves the selective spiroketal formation using thermodynamic equilibration and its regio- and stereoselective reduction. These reaction proceeds in high selectivity, and the reduction product 15A was leaded to the aldehyde 8 in 8 steps. Synthesis of the C11-C18 segment. Starting from methyl (S)-3-hydroxy-2-methylpropionate, the aldehyde 26 was prepared without any loss of the optical activity. This aldehyde was then treated with tributylcrotylstannane in the presence of BF_3・OEt_2 to afford 27 with moderate selectivity. The adduct 27 was further converted to the sulfone 7 in 4 steps. Synthesis of the C21-C26 segment. Optically active epoxyalcohol 29, which was obtained in 3 steps from isobutyraldehyde, was subjected to titanium(IV)-mediated ring opening reaction to yield 30 in good selectivity. Introduction of the C22 chiral centre to the olefin 32 was achieved by the diastereoselective osmylation. The desired epoxide 3 was synthesized successively with the inversion at the C22 stereocentre. Synthesis of the dialkylmaleic anhydride segment. The chirality at C3' position was introduced successfully by Sharpless's asymmetric dihydroxylation. The diol 36 was converted to diethyl dialkylmaleate 38 via the ketoester 37. Synthesis of the degradation product corresponding to C1-C18. The lithium salt of 7 was coupled with 8, and the alcohol portion was successively oxidized to give the ketosulfone 39 in good overall yield. After the phenylsulfonyl group was removed, the spiroketal moiety was selectively constructed by the deprotection sequence of the hydroxyl groups The silyl eher 40 was further leaded to 6, which was identical with the degradation product of 1 in all respects. On the other hand, the epoxyalcohol 41 was synthesized through asymmetric crotylboration and epoxydation. We now undertake the coupling of the remaining segments and complete the total synthesis of 1.
Calyculins (1) isolated from the marine sponge Discodermia calyx have strong cytotoxic activity and calyculin A has been revealed to be an inhbitor of protein phosphatases 1 and 2A. The relative stereostructures of calyculins have been determined by X-ray diffraction studies and their absolute configurations have been depicted as the structures 1 by CD spectral studies of the C_<33>-C_<37> unit. We have provided conclusive evidence supporting the reported absolute configurations of calyculins by the synthesis of the C_<33>-C_<37> unit. The intriguing biological activities of calyculins coupled with their structural curiosities have led us to investigate the total synthesis of calyculins. Retrosynthetic analysis has revealed that 1 would be constructed from four fragments A, B, C, and D, shown in Scheme 1. Fragment A could be prepared by use of the Stille coupling of the vinyl iodide unit 4 with the nitrile unit 3 followed by converting to the vinyl iodide function according to the Barton's procedure. Fragment C have been prepared by the oxazoline formation of the N-acylserine (21, 27) and the subsequent oxidation of the oxazoline. Synthesis of fragment D has been accomplished from (S)-serine using osmium tetroxide mediated dihydroxylation of the Z-olefin. The common northern part 41 of calyculins A, B, E, and F has been synthesized by coupling of the 38 with the 39, followed by N, N-dimethylation.
Neocarzinostatin chromophore (NCS-Chr., 1) and dynemicin A (2) are antitumor antibiotics that have unique structures and fascinating mode of action. These molecules inspire new strategy for the construction of the highly strained enediyne structures and novel design of the compounds to probe and mimic their chemical and biological actions. Here, we wish to report the syntheses of the NCS analogue 6 and the dynemicin analogue 27. In our synthetic plan, the key reaction for the synthesis of NCS analogue 6 is the transannular [2,3]-Wittig rearrangement of 12-membered cyclic ether 7. which allows the construction of highly strained 9-membered diyne 20. The stereoselectivity and reactivity of the Wittig reaction are predicted by MM2 transition structure model. Palladium catalyzed reaction of vinyl epoxide 11 is applied to the stereoselective introduction of the trans hydroxy groups on 5-membered ring which increase the solubility in water. The NCS analogue 6 with a phthalic acid triggering device is sufficiently stable at ambient temperature to allow it handling and shows strong DNA cleaving activity. Our strategy for the synthesis of the dynemicin analogue 27 consists of three key reactions; 1) intramolecular Diels-Alder reaction of 31 to construct the stereochemistries among C-2, C-4 and C-7, 2) cyclization of bromohydrin 30, 3) transannular [2,3]-Wittig reaction of the ether 29 to obtain the 10-membered enediyne skeleton. The stereoselectivity and reactivity of these three reactions are predicted based on molecular mechanics calculation and/or MM2 transition structure model.
Addition reaction of ester enolates possessing an α-heteroatom to a chiral imine 1 possessing (4S,5S)-4,5-dimethoxymethyl-2-methyl-1,3-dioxane ring as a chiral auxiliary is studied, in which varying the metal enolate species and the ester parts resulted in the stereodivergent synthesis of each of the possible stereoisomers. As to the preparation of α-amino derivatives, the triisopropoxytitanium enolate derived from t-butyl N,N-dibenzylglycinate reacted with the imine 1 to give directly cyclized (3R,4S)-β-lactam 2 as a sole product. Complete reversal of the diastereoselectivity was observed in the case of the corresponding chlorozinc enolete, giving (3S,4R)-β-lactam 2 exclusively. Solvent effects were also studied, and the addition of HMPA increased the product yields, but the diastereofacial selectivity was decreased in the case of the chlorozic enolate. α-Sulfenylacetates added to the imine 1 in a stereoselective fashion to give β-lactams, where the effects of the substitutents at sulfur reflected the diasteroselectivity, and a relatively small substituent, methylthio group, met with a reversal of the diastereoselectivity: the triisopropoxytitanium enolate of t-butyl methylthioacetate reacted with the imine 1 to give the directly cyclized (3R,4R)-β-lactam 3 exclusively, whereas the corresponding chlorozinc enolate afforded, after cyclization of the initially formed β-amino ester, (3R,4S)-β-lactam 3 predominantly. α-Alkoxyacetic acids also participated in the stereodivergent addition to the chiral imine 1. Although the α-methoxy derivative did not show reversal of the diastereoselectivity, α-pheoxy and t-butoxy derivatives effected the stereodivergent synthesis of the 3-alkoxy-β-lactams, in which the triisopropoxytitanium enolate gave (3R,4R)-β-lactam 4 exclusively, and the chorozinc enolate predominantly afforded (3R,4S)-β-lactam 4 after cyclization of the initially formed β-amino ester. Furthermore, the triisopropoxytitanium enolates prepared from t-butyl chloro- or fluoroacetate underwent addition reaction with the imine 1 to give (3R,4R)-β-lactam 5 (X=Cl or F) exclusively, whereas the formation of aziridine 6 was observed with the lithium or chlorozinc enolate. Factors for the reversal of the diastereoselectivity were also discussed using MM2 calculations and possible transition state models.
Carbenes or carbenoids react with olefins to provide a powerful synthetic tool for the formation of new carbon-carbon bonds in organic synthesis. It has been well recognized that an intramolecular cyclization of a γ,δ-unsaturated diazoketone in the presence of an appropriate catalyst afforded the corresponding cyclopropane derivative, whereas the similar reaction with β,γ-unsaturated diazoketone led to the formation of the α,β-unsaturated enone system. We applied this procedure to the chiral γ,δ-unsaturated diazoketone, readily accessible from (-)-carvone and discovered an interesting and useful method of forming a new carbon-carbon bond. Decomposition of the diazoketone 6 leading to the cyclohexenone derivative 7, was carried out using various kinds of catalysts such as BF_3・OEt_2, Cu(OTf)_2, Cu(acac)_2, trifluoroacetic acid and Rh_2(OAc)_4 and found that rhodium acetate was the best catalyst for carbon-carbon bond formation. It should also be noted that the stereochemistry of the newly generated stereogenic center was entirely controlled in this reaction. The procedure developed above was successfully applied to the synthesis of 4-epi-isovalerenenol, (-)-neonepetalactone, and (-)-clavukerin A.
The spined citrus bug, Biprorulus bibax Breddin, has recentry become an important pest of citrus, paticularly lemons and mandarins, in southern Australia. Oliver et al. isolated and identified a new hemiacetal 1 as the major component of the male-produced pheromone of B. bibax. Although they could not establish its absolute configuration, they found the natural 1 to exhibit a single peak when analyzed by GC on a column with a chiral stationary phase, while their synthetic (±)-1 showed two peaks. We became interested in determining the absolute configuration of the naturally occurring enantiomer of B. bibax pheromone by synthetic means. Another objective of ours was to develop an efficient synthesis of (±)-1 so as to be used in biological studies in Australia. At first, the racemic and optically active 1 were synthesized from the Diels-Alder adduct 2 via meso-diol 8. But the efficiency of the first route was unsatisfactory. We therefore devised a more efficient synthesis of 1 based on Ireland's ester enolate Claisen rearrangement. The enantiomers and racemate of 1 were synthesized by employing Claisen rearrangement of 11 as the key reaction to provide the corresponding lactone 9 via 10. Optically active 9 was prepared by employing either chemical asymmetric reduction [20→(S)-15b] or lipase-mediated asymmetric hydrolysis [17→(5R,6S)-18a] and acetylation [8→(5S,6R)-18a] as the key transformations. The absolute configuration of the naturally occurring 1 was established as 3R,4S on the basis of the chemical asymmetric synthesis of (3S,4R)-9 by starting from (S)-15b of known stereochemistry.
A dipolar trimethylenemethane (TMM) generated thermally from a 1,1-dialkoxy-2-methylenecyclopropane is an useful three-carbon 1,3-dipole for the [3+2] cycloaddition reaction. It reacts with an electron deficient olefin or an oxime to afford a five-membered carbo- and heterocycle. We report here the syntheses of natural and unnatural biological active compounds based on the [3+2] cycloaddition of the TMM. 1. Synthesis of (±)-allokainic acid Kainoid, as exemplified by kainic acid, is a potent agonist of the glutamate receptor. We synthesized allokainic acid by using the [3+2] cycloaddition of the TMM derived from 1 and methyl glyoxylate O-benziloxime. 2. Synthesis and biological activity of fullerene carboxylic acid Although the chemical behavior of fullerenes has suggested possible biological activity of fullerenes, there has been no direct evidence of such activity. We synthesized a water miscible fullerene, and found that it shows distinct biological activity under light irradiation. The water miscible fullerene carboxylic acid 16 was prepared by the [3+2] cycloaddition of a dipolar TMM with C_<60> followed by esterification of the resultant fullerene alcohol 14 with succinic anhydride. The carboxylic acid 16 showed distinct biological activity, e.g. cytotoxicity, enzyme inhibitory activity, and DNA cleaving activity under irradiation of low-energy visible light.
Aplyronine A (1) is a potent antitumor macrolide isolated as a minute component from the sea hare Aplysia kurodai. The gross structure of 1 was determined by the spectral analysis and chemical degradation. We report herein the absolute stereostructure of 1 and the result of the synthetic study. Chemical degradation of 1 afforded five fragments 2-6. Enantioselective syntheses of the fragments 4a and 5a were performed to disclose the absolute stereochemistry of seven chiral centers (C7-C10, C13, C17, and C19). HPLC analyses of the fragments 2 and 3 using chiral columns disclosed that N,N,O-trimethylserine and N,N-dimethylalanine moieties of 1 were scalemic mixtures (S:R=52:48 for 2; 72:28 for 3). On the basis of the result of the previous and present studies, the absolute stereostructure of aplyronine A (1) was elucidated unambiguously. The synthetic study of 1 was carried out using the Evans aldol reaction and the Sharpless epoxidation reaction as key steps. Starting from N-propionylurethane 11, the C5-C20 segment 13 and the C21-C34 segment 16 were synthesized. The Julia coupling reaction of 13 with 16 followed by four-carbons homologation and lactonization provided 24-membered lactone 17.
Halichondrins are marine natural products isolated from Halichondria okadai Kadota in low yield. Halichondrin B (1) is a representative member and exhibits remarkably powerful antitumor activity in vitro and in vivo. Additionally, 1 is a considerably complex molecule containing 32 asymmetric carbons, substituted tetrahydrofuran and pyran and spiroketal rings, and a large membered lactone. During course of some synthetic studies of 1, Kishi's group succeeded in the first total synthesis of 1 in 1992. For the purpose of establishing a stereoselective and efficient synthetic pathway of 1, we begun to synthesize 1 in 1989. We report here a stereocontrolled synthesis of four fragments, 2,3,5 and 6. As our synthetic program was illustrated in scheme 1, halichondrin B (1) was expected to be synthesized from four convenient fragments, C_1-C_<15> (2), C_<16>-C_<26> (3), C_<27>-C_<36> (5) and C_<37>-C_<54> (6). Tricyclic compound (18a) corresponding to C_1-C_<13> part was stereoselectively synthesized from D-glucose. The C_<16>-C_<26> fragment (3) was also stereoselectively prepared from D-malic acid. Two fragments (5,6) were stereoselectively synthesized from dimethyl L-tartrate as a common starting material. Coupling between the lithium enolate of 3, generated by treatment with LDA, and the aldehyde (5) proceeded smoothly and stereoselectively to afford the β-hydroxy ester (41) as a sole product. Construction of E ring was achieved by treatment with BF_3・OEt_2 in the presence of Et_3SiH in CH_2Cl_2. Other coupling programs toward the total synthesis of 1 is now in progress.
Sialic acid(NeuAc) is a ubiquitous component of glycoconjugates such as glycoprotein and glycosphingolipids. However, stereoselective and efficient introduction of NeuAc has been considered as a difficult task. In order to solve this problem, we developed the strategy depicted in Scheme 1 which takes advantage of the neighbouring group participation by the C-3 substituent (Y). As expected, NeuAc donor 4 gave the α-glycoside in a completely stereoselective manner. This methodology was then successfully applied to the synthesis of disialoganglioside GD3. (Scheme 2) Due to complete stereoselectivity and linkage specificity of glycosyltransferase(GT)-catalyzed reactions, the combined chemical and enzymatic approach is highly promising in oligosaccharide synthesis. Among various GTs, sialyltransferases(STs) are considered to be of particular importance because of the well-recognized difficulty in chemical synthesis of NeuAc glycosides. In order to make ST-assisted strategy practical and widely applicable, the substrate specificities of 2,3-STs were investigated and it was found that some modifications at C-2 position of GalNAc or GlcNAc are acceptable. (Scheme 3) The product derived from 2-O-acyl-lactose derivative 10e was further converted into ganglioside GM3. (Scheme 4) In addition, we developed the novel multienzyme system shown in Scheme 5, which utilizes trans-sialidase in combination with ST. This system was demonstrated to be applicable to the synthesis of NeuAc containing oligosaccharide which is not obtainable by ST alone.
In the field of carbohydrate chemistry, differentiation of the potentially competing functions is rather formidable. For example, the extension of an oligosaccharide at a single glucose entity requires the identification of one of five hydroxyls to function as glycosyl acceptor. Chemists carry out multi-step protection-deprotection processes by the combination of many protective groups, such as esters, ethers, carbonates, ketals etc., to resolve this problem. Generation of a catalytic antibody which recognizes and catalyzes the hydrolysis of certain ester bond as programmed by the hapten might simplify oligosacchride synthesis. We designed and synthesized hapten-KLH conjugate 5 to generate monoclonal antibodies that catalyze selective hydrolysis of the 4-positioned ester of glucose derivatives. The immunization elicited fourteen IgG which bound to the hapten portion. Two IgG were found to catalyze the hydrolysis reaction of the substrate 17 at the directed 4-position selectively with significant rate enhancement compared to the uncatalyzed background reaction. One antibody (17E11) was further characterized in detail.
Plant cell cultures have been known to transform enantioselectively synthetic important foreign substrates as well as secondary metabolities. We have investigated the biotransformation of organic xenobiotics such as β-keto esters 1-5 and aromatic ketones 6-9 by immobilized plant cell cultures, in order to learn the stereoselectivity and the mechanistic pathway in terms of synthetic chemistry. Immobilized cells of Nicotiana tabacum enantioselectively reduced some β-keto esters 1-5 to the corresponding (S)-hydroxy esters 1a-5a in relatively high optical yields. Immobilized cells of Gardenia jasminoides reduced aromatic ketones 6 and 7 to their corresponding (R)-alcohols 6a and 7a, whereas the cells of Daucus carota and N. tabacum converted the same ketones to the (S)-alcohols 6a and 7a, respectively. Immobilized cells of D. carota and G. jasminoides could be found to be effective biocatalysts for synthesizing both the enantiomers of 1-phenyletnanol (6a) with a high enantiomeric purity of >99% ee.
Our studies on the resistance mechanisms and chemical modifications of aminoglycoside antibiotics led to the synthesis of arbekacin (ABK), which was refractory to most aminoglycoside-modifying enzymes in resistant bacteria. In 1990, ABK was launched into Japan as a chemotherapeutic agent for the treatment of infection caused by methicillin-resistant Staphylococcus aureus (MRSA). By 1992 only a few MRSA strains moderately resistant to ABK (6.25-12.5μg/ml) were clinically isolated. ABK was modified by reaction with excess of an enzyme preparation extracted from an ABK-resistant strain and three inactivated products, mainly ABK 2"-phosphate along with small amounts of 6'-N-acetyl-ABK and the doubly modified ABK were obtained. Based on these results, replacement of the 2"-OH by NH_2 in dibekacin (DKB) or in ABK was designed to obtain potent active derivatives against MRSA. Conversion of the 2"-OH by DMSO-DCC oxidation followed by reductive amination with NH_4OAc-NaBH_3CN gave 2"-amino-2"-deoxy-DKB (D1) and -ABK (A1). Their 5-deoxy, 5-epifluoro and 5-epiamino derivatives were also synthesized. All 2"-amino-ABK derivatives showed excellent activities against MRSA and Gram-negative bacteria, as expected. Among them, A1 having low toxicity was selected as a candidate for anti-MRSA agent.
A general method based on difference circular dichroic spectroscopy for assigning multiple chiral centers in acyclic polyols is presented. The validity of this method was demonstrated using twelve 1,2,4-triols, eight 1,2,4,6-tetrols, and four 1,2,4,6,8,10-hexols as model componds with established absolute configuration. Model Polyols were transformed into two acylated derivatives; perbenzoates (II) and 1-0-pivaloyl-perbenzoates (III). Subtraction of the CD spectrum of III from that of II gave a difference CD (DIF CD) spectrum, where benzoate/benzoate interactions between the secondary chiral centers canceled out. The Cotton effect of the DIF CD spectrum reflects only the exciton interaction of the terminal 1,2-dibenzoate system. A positive sign of the DIF CD is correlated with (2S)-configuration and a negative one with (2S)-configuration. The intensity of the Cotton effect (A-Value) is diagnostic of the relative configuration of 2,4-positions. In the case of 2,4-syn isomers, the absolute value of A is higher than 10, whereas that of 2,4-anti isomers shows below 10. Moreover, in the CD study of 1-0-pivaloyl-2,4,6-tri-benzoates the following general rule is concluded: either a distinctive negative or positive couplet is observed if the tribenzoate contains odd number of 1,3-anti moieties or a very weak couplet if an even number of 1,3-anti or 1,3-syn moieties are present. Combination of this rule and the DIF CD method allowed us to determine the absolue configuration of 1,2,4,6-tetrols, even if the relative stereochemistry is unkown.
In order to overcome the problems of a widely used NMR technique, HMBC, we have developed a new technique named decoupled-HMBC (D-HMBC) which enables to observe ^1H-^<13>C long-range coupling separated by four or five bonds with small coupling constants (J<2Hz). Its application for structural studies of a new peptide, SF2741B, and a macrolide antibiotic, monazomycin are described. In addition, two new NMR techniques, HMBC-COSY and HMBC-HOHAHA, which are combinations of HMBC and COSY, or HMBC and HOHAHA pulse sequences, are proposed. These methods enable to reveal complicated and/or overlapped proton spin systems, which are often difficult to analyze by conventional techniques including HMBC. Their applications to structural studies of complicated natural products showed several advantage of these techniques over the HMBC method.
As basic studies to clarify the role of glycosphigolipids and to produce new medicines from marine natural products, we have been conducting isolation, structure elucidation and synthesis of biologically active starfish glycosphigolipids. In this time, we wish to report the usefulness of FAB MS/MS (Tandem MS) spectrometry to structure elucidation of starfish glycosphingolipids. The sphingosine-type glucocerebrosides S-1-3〜5, which were isolated from the starfish Stellaster equestris, were characterized as described in Chart 1 on the basis of chemical and spectral evidences. Especially, the position of conjugated double bond in the side chain was determined from the characteristic fragments observed in the negative ion FAB MS/MS spectrum (Fig. 1) of diol derived from the native glucocerebroside. The ganglioside molecular species LG-2, which was obtained from the starfish Astropecten latespinosus, was completely methylated followed by separated with reversed phase HPLC to give a methylated major component LG-2M-5 in a pure state. The CAD spectrum originated from ceramide ion (m/z 680) in the positive ion FAB MS/MS spectrum of LG-2M-5 reveals characteristic peaks indicating the composition of ceramide moiety (Fig.2). Thus the structure of the major component in LG-2 was elucidated as shown in Chart 2. The above mentioned fragmentation due to the ceramide ion was verified from the measurements of positive ion FAB MS/MS spectra of cerebroside permethylates prepared from four known starfish cerebrosides.
Trichoderma spp. are active as mycoparasites. Therefore, they can serve as potential biocontrol agents. Many peptaibols have been isolated from Trichoderma spp. Their structures are characterized as follows; 1) both N- and C-terminal amino acids are protected, 2) a high proportion of abnormal amino acids, α-aminoisobutyric acid (Aib) and isovaline (Iva) are incorporated, and 3) a labile Aib-Pro peptide bond is present. New peptaibols, trichocellins (TC)-A and B have been isolated from the conidia of T. viride. The primary structures were clearly established by Ion Spray-MS, MS/MS and NMR. TCs are composed of 20 amino acid residues. The N-terminals are protected by an acetyl group and the C-terminal amino acids are linked with a phenylalaninol. Then, we examined the effect of TCs on catecholamine secretion from cultured bovine adrenal chromaffin cells. TCs induced catecholamine secretion. In comparison with TCs-A, TC-B-II showed very low activity. This result suggests that TC-B-II has poor channel formation activity. Based on the CD and NMR studies, there was no significant difference between the secondary structures of TC-A-II and B-II. It was deduced that the ion channel formation by TC-B-II is inhibited by the ionic amino acid residue, Glu.
Antibiotic C-1027, a novel antitumor chromoprotein isolated from the broth filtrate of Streptomyces globisporus C-1027, shows extremely potent cytotoxicity against KB carcinoma cells (IC_<50> 0.1ng/ml) in vitro and antitumor activity toward tumor-bearing mice in vivo. These activities are correlated with the ability of the antibiotic to cause DNA double-strand scission. The antibiotic consists of an apoprotein and a labile chromophore (C-1027-Chr) that is responsible for the biological activity of C-1027. The chromophore is readily separated from its apo-protein by extraction, but the exceeding instability in the protein-free state hampered the structure elucidation. The similar situation has been also observed in the other chromoprotein antibiotics of this family such as neocarzinostatin (NCS), macromomycin, auromomycin (AUR), actinoxanthin and kedarcidin. Among them, NCS is the only one whose chromophore structure has been elucidated, and very recently, the structural novelty of kedarcidin chromophore has been disclosed by the Bristol-Myers Squibb group. We have characterized an inactive but more stable reaction product (2) of C-1027-Chr, which was prepared by treatment of C-1027-Chr in ethanol. It possesses a macrocyclic structure together with oxazolinate and aminosugar moieties as side chains. Its benzodihydropentalene core structure suggested to us the presence of an enediyne in the native C-1027-Chr. We disclose herein the novel structure of C-1027-Chr (1) and the cycloaromatization mechanism leading to product (2), which would explain its extreme potency in terms of cytotoxicity and ability to cause DNA double-strand scission.
In the screening of microbial metabolites active against tumors, we found that Streptomyces chartreusis D329 produced chartreusin(1). Though 1 itself was not utilized clinically, elsamicin A(2) (one of natural products) showed high therapeutic activities against murine tumor and was expected to be useful based on its antitumor activities. Therefore, we tried to mutate the Streptomyces with N-methyl-N'-nitro-N-nitrosoguanidine in order to produce the novel antibiotics related to 1. From analyses of about 3500 strains, we found a mutant strain (D329-185 strain, FERM BP-3269) which produced demethylchartreusin (3) and D329C compound (4). As a result of further analysis of the other products in the fermentation broth of the mutant strain, novel chrymutasin A (5), B (6) and C (7) were isolated. The structure of 4 was determined from spectral studies in comparison with 1. The structures of 5, 6 and 7 were determined from spectral studies, incorporation studies of ^<13>C-labeled-acetates and the synthesis of derivative. Compound 5 showed the strongest cytotoxicities among 4, 5, 6 and 7, and hence antitumor activities of 5 in vivo were assayed. Compound 5 showed that the T/C value was 173% against Meth A murine fibrosarcoma (ip-ip).
Amphidinolides are a series of cytotoxic macrolides isolated from the laboratory-cultured dinoflagellates Amphidinium sp., which are symbionts of Okinawan marine flatworms Amphiscolops sp. Further investigation on search for other cytotoxic components of this dinoflagellate has been continuously progressed by us to isolate two novel 15- and 19-membered macrolides, amphidinolides J (1) and K (2), possessing cytotoxic activity against L1210 and KB cells in vitro (IC_<50> values, 1:2.7 and 3.9μg/mL, respectively; 2:1.65 and 2.9μg/mL, respectively). The planar structure of amphidinolide J (1) was deduced by detailed analysis of the ^1H and ^<13>C NMR data aided with 2D NMR spectra and synthesis of three ozonolysis products firmly established the absolute stereochemistry of six chiral centers of 1 to be 3R,9R,10R,13R,14R, and 15R. Amphidinolide K (2) was isolated as a minor constituent and its gross structure was elucidated on the basis of extensive 2D NMR investigations (^1H-^1H COSY, HSQC, HMBC, HOHAHA, and NOESY) using limited quantity of the sample. The dinoflagellate Amphidinium sp. is quite unique because it produces a number of cytotoxic macrolides possessing a variety of carbon-skeletons; they, however, have similar structural characteristics (e.g., odd-numbered macrocyclic lactone ring and the presence of exo-methylene units), appearing to be biogenetically related to one another.
New marine steroids, aragusterol A (1), B (2) and C (3), were isolated from the Okinawan sponge of the genus, Xestospongia. Aragusterol A and C strongly inhibited the cell proliferation of tumor cells in vitro, and also showed potent in vivo antitumor activity toward L1210 in mice. The planar structure of aragusterol A (1) was elucidated based on spectroscopic analysis and chemical reaction (Scheme I). PCC oxidation of 1 gave 4,5 and 6. Zn-AcOH reduction of 6 gave 7 which was shown to be identical with 7 obtained from hecogenin acetate (8), elucidating the planar structure of the nucleus as well as the stereochemistry of the nucleus except for C-12. The structure of the side chain was determined by formation of 12 on treating 10 with H_5IO_6 followed by NaBH_4 and then p-nitrobenzoyl chloride. The stereochemistry at C-12 was determined based on the coupling constant of H-12 and observation of NOE between H-12 and H-14 in 1. The 22R configuration was elucidated by applying modified Mosher's method, and the stereochemistry at C-20 was determined based on observation of NOE between H-21 and H-22 in 11. The stereochemistry at C-24, 25 and 26 was established by the synthesis of 12 and 23 as shown in Scheme II and III. The structure of aragusterol B was shown to be 2 except for the stereochemistry at C-20 on the basis of spectroscopic analysis and chemical reaction (Scheme IV). The S configuration at C-20 was determined by X-ray crystallographic analysis. The structure of aragusterol C (3) was also established by X-ray crystallographic analysis.
The macrolide bryostatin 1-13 isolated from sea mat Bugula neritina(Bryozoa) exhibit highly potent antineoplastic activity. Bryostatin 1 has been isolated only from the specimen of the Pacific coast (Monterey, America). On the other hand, bryostatin 4 and 5 have been isolated as the main compounds of each Gulf of Florida, Gulf of California and Gulf of Sagami specimens. Investigation has been started to be finding the macrolide bryostatins of the Japanese coast Bugula neritina by use of a new bioassay screening that inhibit cell division of sea urchin egg. The resulting bryostatin 10 as main bioactive compound has been isolated from Gulf of Aomori (ASAMUSHI) and Gulf of Ohzuchi (OHZUCHI) specimens of Bugula neritina. Specially, the yield of bryostatin 10 in ASAMUSHI specimen was 10^<-5>% highly (Scheme 1). The structure of bryostatin 10 has been established to be structure 1 (Fig. 1) by new detailed 1D and 2D NMR (^1H and ^<13>C) techniques such as DEPT, ^1H-^1H COSY, ^1H-^<13>C COSY, HMQC (Fig. 2), HMBC, COLOC and NOESY. By these experiments the perfect chemical shifts of ^1H and ^<13>C of bryostatin 10 structure (1) were obtained (Table 1). Also, the ester group at C-7 has been proved to be the pivalate by mainly HMBC experiment (Previously, the ester was assigned to the isovalerate.). Interesting behavior of ^1H and ^<13>C chemical shifts of bryostatin 10 in exchange of the experimental temperature from R.T. to 50℃ was observed at 3-OH, 2-H, 5-H, 7-H, 30-H, 34-H, etc. Bryostatin 10 exhibits against cell division of sea urchin egg (IC_<50> 1.6×10^<-1>μg/ml) and also against people liver cell PLC/PRF/5 (IC_<50> 7.3×10^<-1>μg/ml). On the other hand, antineoplastic activity (in vitro) against the P-388 lymphocytic leukemia with bryostatin 10 displayed substantial cell growth inhibitory (IC_<50> 1.85×10^<-3>μg/ml), which was similar to the previous result (at America).
In our screening program on biologically active fungal metabolites, two new azaphilones having monoamine oxidase inhibitory (MAOI) activity, TL-1 (1) and -2 (2), were isolated from an Ascomycete, Talaromyces luteus In our successive work to search related compounds to 1 and 2 from Talaromyces fungi, three new azaphilones named TL-3 (3),-4 (4) and -5 (5) from T. luteus, and four new ones named TH-1 (8),-2 (9),-3 (10) and -4 (11) from T. helicus have been isolated. Physico-chemical and spectral data of 3 and 4 suggested that 3 is similar to 1 and is constructed with the partial structures 3A and 3B, and 4 is an isomer of 3. The ^1H- and ^<13>C-NMR data of 3, 4 and their tetrahydro derivative (6) showed that structures of TL-3 and -4 were deduced to be 3 and 4 which is the 11Z isomer of 3, respectively. Structure of TL-5 was estimated to be 5 in comparison of the NMR data of 5 with those of chaetoviridin A (7). Physico-chemical and spectral data of 8-11 suggested that 8-11 are isomeric each other and 8 is similar in part to 7-epi-sclerotiorin (12). The NMR data of 8 indicated that 8 is constructed with the partial structures 8A and 8B_1 or 8B_2. The fact that 8 gives 13 on alkaline degradation and 14 on methylation suggested the structure 8 for TH-1 which contains 8A and 8B_1. Comparison of the NMR data of 9-11 with those of 8 indicated that structures of TH-2, -3 and -4 are deduced to be 9, 10 and 11, respectively. From the comparison of MAOI activity of these azaphilones, it is suggested that the OH at position 8 and the conjugated unsaturated system from position 3 to 11 are indispensable to exhibit the MAOI activity. The OH at position 8 seemed to be also important to exhibit suppressive activity against lymphocyte proliferation.
In the course of our screening program for cytoprotective substances of microbial origin, we have isolated two new compounds named rumbrin and thiazohalostatin. Rumbrin was produced by a fungi identified as Auxarthon umbrinum. The molecular formula was determined as C_<20>H_<20>NO_3Cl by high resolution FAB-MS and the structure was elucidated by a variety of two-dimentional NMR tecniques as shown in Fig. 2. Furthermore, the structure was confirmed by 2D INADEQUATE experiment using [1,2-^<13>C_2] acetate labeled rumbrin. Thiazohalostatin was produced by a actinomycete identified as Actinomadura sp., The molecular formula was determined as C_<20>H_<25>N_2O_4SCl_3 by high resolution FAB-MS and elemental analyses. The structure was elucidated by the NMR spectral analyses on thiazohalostatin and its derivatives (trimethylthiazohalostatin and tribromo analog of thiazohalostatin) as shown in Fig. 5. Rumbrin and thiazohalostatin showed cytoprotective and antioxidant activities as shown in Table 5.
In our screening program for new phospholipase C (PLC) inhibitors of microbial origin, we discovered a novel PLC inhibitor hispidospermidin (1) from the culture broth of Chaetosphaeronema hispidulum (Cda) Moesz NR7127. Hispidospermidin (1) was isolated by Amberlite IRC-50 and Diaion HP-21 column chromatography and solvent extraction. The molecular formula was determined to be C_<25>H_<47>N_3O by HREI-MS and NMR spectral data. Its structure was elucidated as a cage compound with a trimethylspermidine side chain based on various 2D NMR studies. The trans relation between 8a-H and C-10 was established from the coupling constant ^3J C10-H8a=5Hz obtained by the J C-H resolved 2D spectroscopy. The cis relations between 6-CH_3 and 10-Hβ and between 11-H and 3-CH_3 were determined by the NOE experiments on 1. The absolute configuration of 1 was elucidated by the modified Mosher's method on the (R)- and (S)-MTPA amides of a derivative (4) of 1. 1 inhibits rat brain PLC with an IC_<50> of 16μM, whereas the primary amine 4 does not at concentrations up to 7.4mM. Since spermine (IC_<50>=59μM) and spermidine (IC_<50>=1.2mM) also inhibit the PLC weakly, it is suggestive that the polyamine moiety in 1 is essential for the inhibitory activity, and the hydrophobic substituents in 1 potentiate the inhibitory activity.
Lepidimoide(1) was isolated as a novel allelopathic substance, which promoted the shoot growth of different plant species but inhibited the root growth, from mucilage of germinated cress (Lepidium sativum L.) seeds. For example, lepidimoide promoted the hypocotyl growth of etiolated Amaranthus caudatus L. at concentrations higher then 3μM and inhibited the root growth at concentrations higher than 100μM. The growth-promoting activity in hypocotyls was 20 or 30 times as much as that of gibberellic acid (GA_3). The structural study of lepidimoide, with spectral analyses and some chemical evidence, has showed that lepidimoide 1 is regarded as the uronic acid derivative bearing an α, β-unsaturated carboxylate bonded to rhamnose via α-glucoside linkage. Thus, the intriguing structure as well as its unique biological activity prompted us to determine the absolute configuration of lepidimoide(1), by total synthesis. In this meeting we wish to report a biological activity, isolation, structural determination, total synthsis and absolute configuration of lepidimoide 1 and the structure-activity relationships of lepidimoide analogues.
Aklavinone (2) is an aglycone of aclacinomycin A (1) which is an important antitumor drug. Genes for the biosynthesis of aklavinone were cloned from Streptomyces galilaeus 3AR-33, an aklavinone-producing mutant, by use of the actI and actIII polyketide synthase gene probes. Restriction mapping and Southern analysis of the DNA cloned in a λ phage vector established that the DNA represented three different regions of S. galilaeus 3AR-33 genome that contained 3.4, 2.5 and 4.1kb BamHI fragments which hybridized with actIII. Of those, only the 3.4kb fragment hybridized with actI. Complementation experiments with specifically blocked mutants confirmed that the cloned 3.4kb BamHI fragment contains the genes required for the early stage of polyketide synthesis in aklavinone biosynthesis. A 5.5kb region including 3.4kb BamHI fragment of the S. galilaeus 3AR-33 aklavinone gene cluster was sequenced. FRAME analysis revealed the presence of five possible open reading frames (ORFs). Four ORFs (aknX, aknB, aknC and aknD) run divergently from the adjacent aknA gene. The deduced gene product of aknA is homologous to known oxidoreductases. The predicted aknB and aknC gene products are homologous to β-ketoacylsynthses of fatty acid synthases. The aknD gene product is homologous to acyl carrier proteins. The aknX product does not resemble any other known proteins. These results show the aklavinone polyketide synthetase is a multienzyme complex which consists of separate polypeptides rather than the multifunctional polypeptides for the macrolide polyketide synthases.
2-Deoxystreptamine 1 is a common central aglycon in the major group of clinically important aminocyclitol antibiotics. A crucial chemistry in the biosynthesis of 1 is the formation of the precursor, 2-deoxy-scyllo-inosose 3, from D-glucose 2 via the intramolecular C-C bond formation between its C-1 and C-6. The reaction is proposed to proceed via the oxidation at C-4 of D-glucose-6-phosphate 4, followed by elimination of inorganic phosphate and subsequent reduction at C-4 and aldol-type cyclization. The mechanistic similarity is proposed between the biosynthesis of 3 and the dehydroquinate synthase in the shikimate pathway. It is thus quite intriguing from enzymological and evolutionary viewpoints to compare more closely the features of the 2-deoxy-scyllo-inosose synthase reaction with the dehydroquinate synthase reaction. However, nothing has been known at the enzyme level as to, for example, whether a single enzyme is involved or several dissociable enzymes cooperate to form 3. To focused on these questions, we first established the quantitative assay method of 3, by using GC-MS SIM analysis of its trimethylsilyl ether and HPLC analysis of its O-(4-nitrobenzyl)oxime. The in vitro formation of 3 from 4 in the presence of nicotinamide adenine dinucleotide (NAD) was successfully observed in the precipitate fraction obtained by 30〜45% saturation of ammonium sulfate from the 10,000xg supernatant of the sonicated Streptomyces fradiae IFO 13147. Next, the involvement of C-4 hydrogen of 4 in 2-deoxy-scyllo-inosose synthase was investigated with a partially purified enzyme of the above-mentioned ammonium sulfate precipitate. Mass spectrometric and ^2H NMR analysis of the enzyme reaction product from D-[4-^2H]-4 showed that deuterium is retained at the C-6 position of 3. The result suggests a single enzyme of the 2-deoxy-scyllo-inosose synthase is responsible for the intramolecular cyclization of D-glucose-6-phosphate with a catalytic turn-over of the NAD cofactor to form 2-deoxy-scyllo-inosose.
Farnesyl diphosphate (FPP) synthase catalyzes the condensation of geranyl diphosphate (GPP) with isopentenyl diphosphate (IPP). Changes in the structure of GPP during the biosynthesis of FPP were studied by ^1H, ^<13>C and ^<31>P NMR measurements. (1) Substrate specificity: The ability of FPP synthase to identify the substrates for the biosynthetic process was tested by using GPP, linalyl diphosphate (LPP), dihydrolinalyl diphosphate (DHLPP) and citronellyl diphosphate (CPP) as substrates. LPP gives 4% of GPP by non-enzymatic isomerization, and then the so-formed GPP was transformed into FPP by condensation with IPP by FPP synthase. On the other hand, DHLPP and CPP remained unchanged. Thus, only the primary, allylic double bond-bearing GPP acts as substrate for FPP synthase. (2) Conformation changes of GPP by chelation of divalent metal ions: Mn^<2+> and Mg^<2+> were used as divalent metal ions. (i) Changes in the ^<13>C and ^<31>P NMR spectra indicated a one to one chelation of divalent metal ion to the diphosphate moiety, which in turn weakens the C-OP bond of GPP and facilitates the elimination of the diphosphate. (ii) ^1H and ^<31>P NMR relaxation measurements revealed that chelation of divalent metal ion induces a folding of the carbon chain of GPP. (3) NMR detection of geranyl cation: The condensation of [1-^<13>C]GPP with IPP under control of the FPP synthase activity by 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and dithiothreitol (DTT) was monitored by ^<13>C NMR. Besides the signals for C-1 of GPP and C-5 of FPP, a signal at δ 164.5 assigned to geranyl cation was observed (the intensity of the latter was 8.8% that of C-1 of GPP). This signal disappeared when the enzyme activity was inhibited by DTNB, but reappeared when activity was recuperated by addition of DTT. Therefore, geranyl cation is formed and acts as intermediate in the biosynthesis of FPP.