Kansuinine A (1) is a jatrophane diterpene isolated from a root of Euphorbia kansui Liou, known as Chinese herb “kansui” ¹⁾. It has a highly oxidized oxygen-bridged twelve-memberd ring. In addition, there exist seven contiguous stereogenic centers in which four carbon centers are located on five-membered ring. The biological activity of 1, strong promotion of a production of nerve growth factor (NGF) in a L-M cell, attract much attention as a lead compound of pharmaceutical agent for neurodegenerative diseases such as Alzheimer’s disease²⁾. In particular, while 1 displayed the strongest activity in known NGF-inducers, the increase in cytotoxicity was not shown in a range of higher-dose conditions. Due to the intriguing bioactivity and structure of 1, we undertook a total synthesis of 1.

Our retrosynthetic analysis was shown in Scheme 1. Twelve-membered ring of 1 would be constructed by NHK macrocyclization. The precursor 2 would be obtained by the assembly of two fragments 3 and 4 by an aldol reaction and subsequent hemiketalization. The first issue toward total synthesis of 1 is efficient construction of five-membered ring that possesses “all cis” substituents on four carbon centers.

First, we attempted regio- and stereoselective ring opening reactions of epoxide 6a. While desired product was not obtained under various conditions, an acid promoted rearrangement of vinyl group was found to give ketone 12. This result led to change of substrate into 6b, and the desired ketone 14 was obtained by treatment of TMSOTf/HMDS. Additional two-step transformations afforded fragment 3’. Next, fragment 4’ was synthesized from known aldehyde 17 by six-step operations that involved Cr-mediated allylation and stereoselective addition of vinyl group to aldehyde. Finally, assembly of two fragments 3’ and 4’ was achieved by the aldol reaction to give 23. This coupling reaction was comprised of a cascade reaction of aldol reaction of dianion 21 with 4’, migration of TES group, and hemiketalization of intermediate 22. Transformation of 23 to 2 is currently underway, and we plan to obtain 1 by NHK macrocyclization of 2.

Microorganisms have a tremendous capacity to produce structurally diverse metabolites with disparate activities. Therefore we have constructed a microbial metabolites fraction library. The library has been prepared based on basic chromatographic techniques by HPLC and MPLC. To discover a novel metabolite efficiently and quickly from the library, we have developed a unique database named an NPPlot (Natural Products Plot) based on the physicochemical property of a metabolite obtained by PDA-LC/MS analysis of each fraction in the library. The NPPlot is a distribution map of metabolites for a strain and each metabolite is appeared as a dot in 2-dimentional area by retention time for X-axis and m/z value for Y-axis.

On the screening for structurally unique metabolites by the NPPlot from the fraction library, four dots showing unusual alignment were found in a fraction library generated from Streptomyces sp. RK85-270. Among four dots, compounds 1 and 2 were isolated and their structures were elucidated based on spectroscopic methods including extensive NMR techniques and MS/MS analysis to confirm the amino acid sequences. They belong to a cyclic octadepsipeptide, which form the ester between the hydroxyl group of threonine and the carboxyl group of proline, containing two leucines, two prolines, valine, phenylalanine, threonine, and N-methyl-tyrosine. Threonine residue was n-propionylated or acetylated at N-terminal.

Their antiproliferative, antibacterial, and antimalarial activities were evaluated in vitro. Both of them did not show any significant effects on proliferation and bacterial growth at the concentration of 30 mM, however showed antimalarial activity with the same IC₅₀ values of 1.5 mM.

Thiopeptides are produced mainly by actinomycetes and typically contain highly modified sulfur-containing peptides, which have a characteristic macrocycle knotted with pyridine or piperidine, a six-membered nitrogen-containing ring. Although more than 100 thiopeptides have been discovered, the number of validated gene clusters involved in their biosynthesis is lagging. We used genome mining to identify a silent thiopeptide biosynthetic gene cluster responsible for biosynthesis of lactazoles from Streptomyces lactacystinaeus OM-6519. To date, the ring size of macrocyclic thiopeptide is limited to 26, 29, or 35 atoms, while lactazoles are structurally novel thiopeptides with a 32-membered macrocycle. The 2-oxazolyl-6-thiazolylpyridine core with the 3-position connected to tryptophan through an amino linkage also provides a unique structure in thiopeptides. Lactazoles did not show any antimicrobial activities, however, we found that inhibitory activities for the bone morphogenetic protein (BMP) signal cascade in vivo, which could add to a new aspect of therapeutic treatment against to thiopeptide family antibiotics.

We demonstrate that lactazoles originate from the simplest cluster, containing only six unidirectional genes (lazAto lazF). It is the smallest cluster among the known thiopeptide biosynthetic gene clusters. The structure gene, lazA contains the precursor peptide sequence, and it is classified into a phylogenetically distinct clade. We show that lazC is involved in the macrocyclization process, leading to central pyridine moiety formation by gene disruption. The lazC disruptant accumulated a linear compound with fully matured structure.

Substitution of the endogenous promoter with that of godA, a gene involved in goadsporin biosynthesis results in an approximately 30-fold increase in lactazole A production. A using the godA promoter to regulate the lactazole biosynthetic machinery, production of two analogs, S11C and W2S, was achieved.

Thus, this compact biosynthetic machinery has high potency to lend large diversity to the thiopeptide core structures. Our approach facilitates the production of a more diverse set of thiopeptide structures, increasing the semisynthetic repertoire for use in drug development.

1. Onaka H, Nakaho M, Hayashi K, Igarashi Y, & Furumai T (2005) Cloning and characterization of the goadsporin biosynthetic gene cluster from Streptomycessp. TP-A0584. Microbiology 151(Pt 12):3923-3933.

2. Hayashi S, Ozaki T, Asamizu S, Ikeda H, Omura S, Oku N, Igarashi Y, Tomoda H, & Onaka H (2014) Genome mining reveals a minimum gene set for the biosynthesis of 32-membered macrocyclic thiopeptides lactazoles. Chem Biol 21(5):679-688.

Iron is an essential element for organisms as it is requisite in various biological processes including primary metabolism. However, most iron under aerobic conditions exists as insoluble Fe(III) at neutral pH and is hard to be taken up by organisms. To overcome this problem, microbes produce and secrete low molecular weight metabolites called siderophores which can capture and solubilize Fe(III). During the course of our screening for siderophores from microbial extracts, we discovered a novel siderophore named chlorocatechelin A (1) and a related metabolite chlorocatechelin B (2).

The molecular formula of 1 was established as C₂₆H₃₀Cl₂N₆O₁₁ on the basis of HR-ESIMS and NMR spectral data. The partial structure of 1 was elucidated by 2D NMR analysis to be composed of two units of 4-chloro-2,3-dihydroxybenzoic acid (CDB), one unit of arginine and one unit of N^δ-hydroxy-N^δ-formyl ornithine (hfOrn). The connectivity of these residues was elucidated by 2D NMR, MS/MS and deuterium shift analysis. The absolute stereochemistries of amino acid residues were determined to be D-configurations by advanced Marfey’s method. The structure of 2was determined in the same manner as 1.

To unambiguously determine the structure of 1, we conducted total synthesis of 1. We planned to synthesize 1 by conjugating the left segment 6 and the right segment 10. The left segment 6 was synthesized from o-vanillin (3) and D-arginine in 12 steps. The right segment 10 was synthesized from 1-benzyl D-glutamate (7) in 6 steps. Deprotection of the Boc group from 10 and subsequent condensation with 6 commenced 11. We tried to deprotect all of Bn groups from 11 under a hydrogen atmosphere, resulting in producing dechlorinated compounds due to low reactivity of Bn ester. Therefore, Bn ester was first cleaved in an alkaline condition and then the other Bn groups were deprotected with hydrogen to give 1. NMR spectra and other physicochemical properties of synthesized 1 were identical with those of natural 1.

Iron binding properties of 1 and 2 were determined by spectrophotometric titration and cyclic voltammetry experiments. The molecular ratios of Fe(III)-1 complex and Fe(III)-2complex were 1:1 and 1:3, respectively. The redox potentials of Fe(III)-1 and Fe(III)-2 were -578 mV and >-450 mV, respectively, indicating that Fe(III)-1 complex was more stable than Fe(III)-2 complex.

Production of 1 and 2 were rarely observed in iron rich conditions, suggesting these two compounds were produced as siderophores. On the other hand, we observed degradation of acylguanidine moiety of 1 to produce the lower-affinity siderophore 2 in acidic conditions. Producing labile compounds is just a waste of energy unless the decomposition plays any productive roles. For clarifying biological rationalities of 1 and 2, their biological effects to the producer and other bacteria are under investigation.

Palytoxin (PLTx, C₁₂₉H₂₂₃N₃O₅₄) and its congeners occur widely in marine environments, threatening public health. Structural diversity of the toxins is known but the limited availability of samples coupled with the large and complex nature of the molecules has posed an seirious obstacle to chemical studies. We challenged the complex structures using liquid chromatography coupled to time-of-flight mass spectrometer (LC-TOF). Emphasis was laid on elucidation of the mechanism of fragmentation and assignment of all fragment ions with structures. PLTx and palytoxin carboxylic acid (PLTx-COOH) were first chosen to understand the effect of different terminal moieties. Measurements were made using both positive and negative modes.

Positive ions were classified into three types depending on the charge site: C115-NH₂, amido nitrogen in the terminal unit, or partial structures produced by simultaneous two cleavages. Partial structures containing carboxyl group, a hindrance to positive ions, were often lost in fragments. Fission of a C-C bond triggered consecutive dehydration to produce conjugated polyenes. The initial monoene was absent but triene or higher conjugation gave intense ions. Bond breaks in and around the hemiketal ring gave rise to abundant ions. In the negative mode, the cleavage sites were more readily assigned. The fragment ions produced from the PLTx and PLTx-COOH molecules were assigned in full detail for the first time. Based on the achievement made, structural elucidation was carried out on new palytoxin congeners named ovatoxins produced by Ostreopsiscf. ovata (AZ) strain isolated from the Bay of Napoli. A new terminal unit of ovatoxin-b was found to be 5-amino-1,3-dihdroxypentane and a 115-deamino-oxo (aldehyde) structure was newly found in two ovatoxins, ovatoxin-a and ovatoxin-b.

The natural phytotoxin coronatine, which is composed of two individual parts, coronafacic acid and coronamic acid, exhibits various promising biological activities similar to jasmonoyl L-isoleucine, which is one of the phytohormones, through interacting COI1-JAZ complex receptor. Interestingly, coronatine induces stomatal opening in which jasmonoyl L-isoleucine is not involved as an endogenous regulator. Moreover, a diastereomer of coronatine had stomatal opening activity but did not interact with COI1-JAZ complex protein. In addition, the mutated plant lacking the function of COI1 protein, retained stomatal opening activity by coronatine. From these data, we hypothesized coronatine would have a target protein other than jasmonate receptor in the guard cells. To identify the target protein, we synthesized alkyne-tagged coronatine for Raman imaging in living guard cells.

SREBPs are transcriptional factors that control lipid and cholesterol metabolism. Activation of SREBPs in response to a decrease in the cellular sterols, the endogenous inhibitors of SREBP activation, results in acceleration of the synthesis of fatty acids, triglycerides, and cholesterol. Aberrant SREBP activity has been linked to metabolic disease states, such as obesity, fatty liver, hyperlipidemia, and atherosclerosis. Thus, inhibition of SREBP activation is a potential therapeutic approach to treating metabolic disorders.

Our group discovered a synthetic small molecule, named fatostatin (1), that inhibits the activation of SREBPs, thereby blocking the lipid biosynthesis and the accumulation of fat in obese mice.^2,3 Furthermore, we identified FGH10019 (2) as a more potent inhibitor of the SREBP activation than fatostatin by an SAR study.⁴ Our analysis suggested that fatostatin inhibits the ER-Golgi translocation of SREBPs through binding to their escort protein, the SREBP cleavage-activating protein (SCAP), at a distinct site from the sterol-biding domain. This result indicates that a possibility of the existence of unknown endogenous inhibitors that act like fatostatin.

In this study, we conducted a screening of a library consists of 280 lipid derivatives for a search of novel endogenous molecules that regulate the SREBP activity. As a result, we discovered a couple of endogenous lipid derivatives that inhibit the activation of SREBP. Further analysis suggested that the inhibitory mechanism of them is different from that of sterols and fatostatin. These results indicate that the endogenous lipid derivatives we found might be new players in the SREBP regulation.

Aminoglycoside antibiotics are clinically important class of antimicrobial agents such as streptomycin, kanamycin, gentamicin, and neomycin. Until now, we have shown that the conserved homologous enzymes that are encoded in the neomycin biosynthetic gene cluster, construct common structures of this class of antibiotics such as 2-deoxystreptamine, paromamine, neamine and ribostamycin. Thus, it is presumed that unique biosynthetic genes among the gene clusters would be responsible to the modification of common structures of aminoglycosides to expand the structural diversity. We have then investigated characteristic functions of such unique enzymes from several aminoglycoside biosynthetic machineries. In this presentation, two unique enzymatic transformations via radical reaction intermediates in the neomycin biosynthesis and kanamycin will be discussed.

Regarding the neomycin biosynthesis, a putative radical SAM enzyme NeoN found to be responsible to the epimerization at C5”’ of neomycin C to give neomycin B in the presence of SAM.

Concerning to the kanamycin biosynthesis, a non-heme iron a-ketoglutarate-dependent oxygenase KanJ found to transform kanamycin B to 2’-oxokanamycin, which was then reduced to be kanamycin A by an NADPH-dependent reductace KanK. KanJ catalyzes the hydroxylation at C2’ of kanamycin B with radical mediated reaction mechanism to afford a presumable hemiaminal intermediate that is spontaneously hydrolyzed to be 2’-oxokanamycin. KanJ and KanK are uniquely encoded enzymes in the kanamycin biosynthetic gene cluster among the related aminoglycoside antibiotic biosynthetic genes.

Obesity is a problem of great urgency all over the world. According to the investigation in 2013, 1/3 of the total population on the earth is obese or overweight. In addition, it is one of the highest risk factors for life style-related diseases including diabetes, heart diseases, and cerebral vascular disorders. Thus, the demand for effective treatments for obesity has been increasing. In view of such circumstance, we have been studied on the isolation, structure determination, and elucidation of mechanism of action of natural products that modulate lipid metabolism to propose them as promising drug leads to treat obesity and diabetes. In this presentation, we will illustrate our recent achievements about two natural products we identified, ternatin and yoshinones.

Ternatin is an unusual amino acids-containing cyclic heptapeptide derived from mushroom, Coriolus versicolor. The natural product has been shown to inhibit the fat accumulation. However, the mechanism of action of ternatin remains unclear. In this study, we tried to identify the intracellular ternatin-binding protein to solve the mechanism of action. To this end, we synthesized two ternatin derivatives, positive and negative controls, conjugated them to sepharose resin, and prepared ternatin affinity column. A whole cell extract was loaded onto the affinity column, extensively washed, then binding proteins were eluted and subjected to SDS-PAGE. We observed a protein band which was detected only in the positive control column. The amino acid sequencing of the protein is now on going.

An obese is a mixed-up result both the number and mass of adipocytes. Thus, it is important to inhibit one of these, or both, for the prevention of obese. In this project, we looked for the natural products that block adipose cell differentiation. Our efforts successfully isolated novel natural products, yoshinones A, B1, and B2 we called. Yoshinones are similar structure each other, essentially, share g-pyrone moiety. An activity evaluation test reviled that yoshinone A inhibits fat cell differentiation with EC₅₀ value of 420 nM. On the other hand, the compound did not show remarkable cytotoxicity, suggesting that yoshinone A might be a potent candidate to treat obesity.

Biosynthetic studies on the sesquarterpenes, a family of C₃₅ terpenes, in Bacillus subtilis have identified a new type of terpene cyclase, tetraprenyl-b-curcumene synthase (TS), which lacks sequence homology to any known terpene synthases. We recently revealed that Bacillus megaterium tetraprenyl-b-curcumene cyclase (TC) is a bifunctional triterpene/sesquarterpene cyclase that converts head-to-tail C₃₅ 1 and tail-to-tail C₃₀ 3 into pentacyclic 2 and bicyclic 4, respectively, in vivo (Scheme 1). In this presentation, we report novel and unnatural terpenes found from sesquarterpene cyclase (TS and TC) study.

1) We discovered head-to-tail types of novel acyclic sesterterpene 5 and triterpene 6 in B. clausii, and revealed that compounds 5 and 6 are biosynthesized by TS homolog (Bcl-TS) from GFPP and HexPP, respectively (Scheme 2). The present study revealed that TS homologs are a new family of terpene synthases that form not only sesquarterpene but also sesterterpene and triterpene. Genome mining of this new terpene synthase family will lead to discoveries of novel terpene synthases and terpenoids in the future.

2) The enzymatic cyclization of head-to-tail acyclic triterpene 6isolated from Bacillus clausii using B. subtilis TC resulted in the formation of two unnatural pentacyclic triterpenes 7and 8 (Scheme 3). It was revealed that B. subtilis TC, which forms tetracyclic terpenoid scaffold from tetraprenyl-β-curcumene in vivo, could be used to construct the 6/6/6/6/6-fused pentacyclic scaffold in vitro, suggesting that the active site cavity of TC has sufficient space to accommodate this unnatural pentacyclic scaffold.

3) We revealed that TC has an unprecedented catalytic function in cyclizing squalene from both termini and is the first onoceroid synthase (Scheme 4). Also, we report the first onoceroids from bacterial origin. Our discoveries suggested that symmetric and asymmetric onoceroids could be biosynthesized by a single enzyme via an intermediate cyclized at one terminus of squalene. Furthermore, the new function of TC enabled the synthesis of (+)-ambrein 11, a major constituent of ambergris that is difficult to obtain naturally, via a mutated squalene-hopene cyclase–catalyzed reaction from easily available squalene (Scheme 5).

The microenvironment in tumor, characterized by insufficient supply of oxygen and nutrient because of the immature vascularization, is now recognized to be key factor for altering the metabolic and proliferative pathway of the tumor cells. And the tumor cells, which have adapted to the nutrient-starved environment, are known to aggravate pathology of cancer by promoting tumor growth, angiogenesis, metastasis and response to chemotherapy and irradiation. Therefore, compounds that selectively inhibit growth of tumor cells under nutrient-deprived condition are expected to be promising anti-cancer drug leads with novel mode of action.

In the course of our study on the bioactive substancesfrom marine organisms, we constructed a bioassay system to search for selective growth inhibitors against human pancreatic cancer PANC-1 cells only under glucose-deprived condition. And, we isolated four novel compounds, biakamides A-D (1-4), from Indonesian marine sponge 05A01 as active substances. Through detailed analyses of HRMS and 1D- or 2D-NMR spectra, biakamides A-D (1-4) were found to be polyketides with unprecedented modification such as a thiazole ring, two N-methylamides and a chloromethylene moiety. We are also studying the total synthesis of 1, in order to elucidate the absolute stereostructure of 1,3-dimethylalkyl moiety and to analyze structure-activity relationship.

Further detailed examination of the extract of the same marine sponge led us to isolate two active substances named fasciospyrinadinone (11) and fasciospyrinadinol (12). Structure analysis revealed that these compounds are novel pyridine-containing terpenoids.

Among the isolated compounds, biakamide C (3) showed the most potent growth inhibitory activity against PANC-1 cells under glucose-deprived condition (IC₅₀: 0.6 μM) with more than 50-fold selectivity over that under normal condition.

New remote asymmetric induction reactions by using the E,E-vinylketene N,O-acetal possessing a chiral auxiliary have been developed and applied to the syntheses of natural products. The vinylogous Mukaiyama aldol reaction of the E,E-vinylketene N,O-acetal with an aldehyde in the presence of excess amount of TiCl₄ (4 equiv) gave a synadduct predominantly, while conditions including one equivalent of TiCl₄afforded an anti adduct in a stereoselective manner. The reaction of the E,E-vinylketene N,O-acetal with an acetal produced a syn adduct in high stereoselectivity. All stereoisomers of 2,4,6-trimethyloctanoic acid derivative have been synthesized in a few steps by using the remote asymmetric induction reaction and the sequential regio- and stereoselective reductions. The short step synthesis of septoriamycin A, an anti-leishmanial compound, has been achieved by using this methodology. Additionally, the remote asymmetric induction-type acylation reaction of the E,E-vinylketene N,O-acetal has been developed and applied to the formal synthesis of khafrefungin, a polypropionate antibiotic.

The remote asymmetric induction reaction has also been applied to the synthesis of oxygenated propionates. The anti adduct was submitted to the sequential conversion including reduction to allylic alcohol, stereoselective epoxidation, and semi-pinacol rearrangement to give an aldehyde possessing four stereogenic centers.

Exploration of novel chemical space has been crucial to discover novel drug leads for both emerging and traditional therapeutic targets. Natural products remain to be one of the most attractive sources for drug discovery because of not only their structural complexity and diversity but also their chemical space apart from that of synthetic libraries. It is thus great challenge to develop efficient methods to expand the natural product chemical space.

Natural product biosynthesis begins with formation of basic skeleton by using fundamental building blocks come from primary metabolism, which undergoes multi-step modifications and rearrangements to form a variety of end products. The structural motif of multi-potent intermediates vastly branching to various pathways directly leads to the chemical diversity. 3,5-Dimethylorsellinic acid, which is biosynthesized by fungal non-reducing polyketide synthase (NR-PKS), is converted into a huge number of different flameworks, resulting in the production of vast array of meroterpenoids. While, utilization of multi-potent intermediates is strictly limited under metabolism in their original resources. Therefore, artificial methods, such as their heterologous expression and chemical transformation, of multi-potent intermediates make it possible to create structurally diverse pseudo-natural products and enable us to access to novel chemical space.

We previously isolated structurally diverse chaetophenols from Chaetomiumfungi as novel natural products by using chemical epigenetic approach, and also revealed that simple aromatic compound PM-1, biosynthesized by an NR-PKS gene, pksCH-2, is a common intermediate of chaetophenols. Thus, we focused on PM-1 as a multi-potent intermediate in chaetophenol biosynthesis, and generated various novel pseudo-natural products by over expression of pksCH-2 and PM-3 encoding gene, Ts0021, in Aspergillus oryzae and artificial chemical transformation of PM-2. We therefore, demonstrated that multi-potent intermediate in natural biosynthetic pathway has great potential to produce novel chemical entities and increase the chemical diversity.

The genus Kopsia has been used as a folk medicine and is a rich source of biologically active alkaloids. These alkaloids have been reported to have unique polycyclic skeletons. Lundurines, which were first isolated from Kopsia tenuis by Kam and co-workers in 1995, are some of the most unique alkaloids in this category.¹ These compounds have an intriguing hexacyclic framework that includes an unprecedented cyclopropane-fused indoline skeleton.

In addition to their interesting structures, lundurines have also been shown to have intriguing biological activities. Lundurines B (1) and D (2) exhibit appreciable toxicity toward B16 melanoma cells, and also reverse multidrug-resistance in vincristine-resistant KB cells.^2b These findings suggested that lundurines are promising leads for new anti-tumor drugs. However, no total synthesis of lundurines, or even a synthetic approach to their skeleton, has been reported to date.

Here we report a first total synthesis of (±)-lundurine A (2) and B (1). ^4,7

Isoschizogamine (1), isolated from Schizozygia caffaeoides by Renner in 1963,¹ is a polycyclic alkaloid containing an aminal functionality. The initially assigned structure of 1, an epimer of schizogamine, was revised by extensive structural analysis to bean unprecedented hexacyclic structure possessing the [5.6.6.6]diazafenestrane skeleton including the aminal functionality and a quaternary carbon center. While this intriguing structure prompted synthetic chemists to launch synthetic studies on isoschizogamine, only one racemic and one enantioselective total synthesis of 1 have been reported to date. In this paper, we report a stereocontrolledtotal synthesis of 1 utilizing a late-stage C-H functionalization of the tetracyclic lactam and unprecedented cascade cyclization as key processes.

Our synthesis commenced with enantioselective construction of the quaternary carbon center by asymmetric 1,2-reduction of enone 10 according to the protocol developed by Ikariya and Johnson-Claisen rearrangement of allylic alcohol 12. After extensive optimization, we found that the Johnson-Claisen rearrangement of 12 was successfully carried out with Me(OEt)₃, MS4A, in Hunig’s base as solvent under microwave irradiation to provide the cyclohexene 13 in a satisfactory yield without loss of optical purity. Then,construction of the tetracyclic quinolone skeleton was conducted by an acid-promoted cascade cyclization of 16 with CSA in DMSO at 150 °C to afford tetracyclic quinolone product 19 in good yield as a sole isomer. Compound 22, a substrate for the C-H functionalization, was prepared through ketone 19 by diastereoselective introduction of the amino ethyl side chain. After extensive investigations, we succeeded in the late stage C-H functionalization adjacent to the quaternary carbon center using a combination of CrO₃ and n-Bu₄NIO₄. Then, the crucial formation of aminal was executed by activation of hemiaminal moiety using Bi(OTf)₃ to provid the desired aminal 5 in high yield. After formation of the dehydropiperidine ring and requisite functional group manipulations, we completed a total synthesis of (–)-isoschizogamine (1).

Psiguadial B (1), which was isolated from an evergreen shrub of Myrtaceae in 2010, exhibits antitumor activity against human hepatoma cells. The natural product possesses a unique hybrid skeleton containing a tricyclic terpenoid moiety and two aromatic rings. We herein report the total synthesis of 1 using a new method for constructing the polycyclic skeleton of 1 through the cascade cyclization reaction of dicobalt acetylene complex 6.

Alkyne 15 having an allylsilane moiety and two leaving groups was prepared from d-hexanolactone in 9 steps transformations. After converting to cobalt complex 6, the double cyclization reaction was accomplished by using dichloroaluminum 2,4-dichlorophonoxide to afford cyclic cobalt complex 4 in quantitative yield. Treatment of 4 with cerium(IV) ammonium nitrate (CAN) gave maleic anhydride 16 which was reacted with 3,5-dimethoxyphenol in the presence of AgBF₄. The resulting ether 17 underwent the cyclization reaction upon heating with NBS and AIBN followed by treatment with silica gel, giving rise to benzopyran derivative 19 as a single diastereomer. Maleic anhydride 19 was converted to diester 23, and the product was subjected to the reduction with samarium(II) iodide followed by isomerization under basic conditions to give diester 25. The cyclobutane ring was constructed by the intramolecular cyclization of iodo nitrile 28 that was obtained from 25 through the regioselective substitution reaction of cyclic sulfonate 26 with KCN. After converting the cyano group of nitrile 29 into a methyl group, the formyl groups of the aromatic ring were introduced through the Suzuki-Miyaura coupling of dibromide 31 with vinylboronic anhydride pyridine complex followed by oxidative cleavage of the vinyl groups. Finally, the total synthesis of 1 was accomplished through cleavage of the methyl ethers of 33 (28 steps in total, 0.25% overall yield).

The A-type procyanidins, such as procyanidin A₂ (1) and cinnamtannin B₁ (2), are unique among the oligomeric catechins by a characteristic dioxabicyclo[3.3.1]nonane platform formed by a double interflavan linkage. Potential bioactivities of these compounds are starting to be revealed, such as insulin-enhancing activity of 2 associated with diabetes remedy,^[3] although detailed study has been hampered by the scarcity of natural products.

In addressing the chemical synthesis, the key challenge is the viability to construct the double interflavan linkages, where worth considering is the putative biogenesis of such double interflavan linkages.

We report herein the stereoselective syntheses of procyanidin A₂ (1) and cinnamtannin B₂ (2) based on the annulation strategy. Ethylenedioxy-bridged flavan units were prepared by DDQ oxidation of protected flavan units, which allowed dual activation at C2 and C4 position of the flavan unit, enabling annulation with nucleophilic units to construct double linked skeleton in regio- and stereoselective manner.

(+)-Ryanodine (1) has potent modulatory activity toward an intracellular calcium release channel known as the ryanodine receptor. Compound 1 possesses five hydroxy groups, a hemiacetal, a pyrrole-carboxylate ester, and eleven consecutive stereocenters including eight tetrasubstituted carbons within the complex pentacyclic ABCDE-ring system. The densely functionalized structure of 1 poses a formidable synthetic challenge. Herein, we report the first asymmetric total synthesis of 1.

To take advantage of the intrinsic symmetric substructure of 1, we designed C₂-symmetric tricycle 5 as the key intermediate. First, dearomatizing Diels-Alder reaction of 3 and 4, followed by asymmetric methanolysis, led to optically active bicyclo[2.2.2]octene (+)-12. The pairwise functionalizations of C₂-symmetric compounds and construction of the AB-ring system via the transannular aldol reaction afforded the key intermediate 5.

The carbon skeleton of 1 was constructed from 5. After Co (II)-catalyzed oxidative desymmetrization of C₂-symmetric tricycle 5, the bridgehead radical allylation of 22 installed the C11-tetrasubstituted stereocenter of 6. The regio- and stereoselective introduction of the two carbon chains at the C6 and C2-position and construction of the C-ring by ring-closing metathesis completed the synthesis of 8, which has the entire carbon skeleton of 1.

The asymmetric total synthesis of (+)-ryanodine (1) has been accomplished from 8. Removal of C6-TMS group and the acetonide, followed by the stereoselective reduction of C3-ketone, afforded pentaol 31. The regioselective protection of the four tertiary hydroxy groups of 31yielded bisboronate 32. Condensation of 32 with protected glycine Dgave rise to glycine ester 33. Treatment of 33 with vinamidinium salt E efficiently constructed the pyrrole-carboxylate ester at the C3-position, generating 35. Finally, deprotection and hydrogenation of the isopropenyl group delivered (+)-ryanodine (1).

Batrachotoxin (1) is a steroidal neurotoxin originally isolated from the skin of Columbian poison dart frogs. This molecule acts as a potent agonist of voltage-gated sodium channels (Na_vs). Because Na_vs are responsible for the initiation and propagation of action potentials in excitable cells and are related to severe diseases like epilepsy, arrhythmia and chronic pain, its unique property as a modulator of Na_vs has been intensely investigated by electrophysiology. However, the molecular determinant of the interaction of Na_vs and batrachotoxin has not been elucidated yet because of the scarce natural availability. Thus we initiated a synthetic studies aiming for establishing an efficient synthetic route to provide enough sample for Na_v investigations.

Our synthesis commenced with preparation of enone 10 and alkenyl bromide 14 from known, optically active molecules (Scheme 2). 1,2-Addition of the lithiated 14 to enone 10 afforded adduct 15 and it was converted to radical cyclization precursor 17 (Scheme 3). The desired tandem radical cyclization was triggered by tributyltin radical at high temperature and the C-13 quaternary center was established successfully. Removal of the silyl tether, IBX oxidation of the secondary alcohol and oxidative cleavage of the TMS-alkene furnished ketoaldehyde 20. The following construction of ring E was achieved by reductive amination, acylation and treatment with sodium ethoxide in the presence of ethanol (Scheme 4). After protection of the ketone of lactam 22, oxidative cleavage of the stannylated alkene was thoroughly investigated but was unsuccessful (Scheme 5). It was revealed that the stanylated alkene successfully converted to an enal by copper chloride and molecular oxygen. Thus Curtius rearrangement was utilized and the following hydrolysis afforded ketone 28. The subsequent Stille coupling successfully furnished the methyl ketone side chain. Stereoselective reduction of three carbonyl groups of enone 29 is currently underway.

Caprazamycin A (1) was isolated from Streptomyces sp. MK730-62F2 in 2003 by Igarashi and co-workers, which exhibits anti-mycobacterial activity against multi-drug resistant tuberculosis¹ (Figure 1). Its structure features a uridine, an aminoribose, a diazepane ring system and a fatty acid side chain. Although the total synthesis of caprazol,which has no fatty acid side chain, has been reported by Matsuda³ and Shibasaki⁴, respectively, no total synthesis ofcaprazamycins has been accomplished to date. In this report, we will report a synthesis of caprazol core 8 and a careful introduction of the labile fatty acid side chain into the diazepane core.

The highlights of this work are (1) an introduction of the fatty acid side chain into 1,4-diazepane model 23in which it was found that stepwise introduction of fatty acid side chain 22and carboxylic acid 20 was robust for accessing the unstable structure; (2) a diastereoselective aldol reaction using thiourea organocatalyst 15 for synthesis of syn-b-hydroxy-a-amino acid derivative 13; (3) an efficient construction of caprazol core 8 by the Mitsunobu reaction.

We are now working on the first total synthesis of caprazamycin A from the caprazol core 8 by introduction of the labile fatty acid side chain.

We attempted the catalytic asymmetric total synthesis of caprazamycin B (1), a lipo-nucleoside antibiotic with anti-tuberculosis activity, in which four key stereoselective transformations were employed. The stereochemistries of the anti-b-hydroxy-a-amino acid moiety embedded in the diazepanone system and the syn-b-hydroxy-a-amino acid moiety at the juncture of uridine- and diazepanone parts were controlled using enantioselective anti-selective nitroaldol reaction catalyzed by Nd/Na-chiral amide ligand (dr = 12:1, 95% ee), and diastereoselective isocyanoacetate aldol reaction (dr = 88:12), respectively.

For the synthesis of the side chain moiety, desymmetrization of 3-methylglutaric anhydride with the (S)-Ni₂-(Schiff base) complex as the catalyst furnished the chiral monobenzylester (88% ee), and thioamide-aldol reaction with the catalyst comprising mesitylcopper, (R,R)-Ph-BPE, and 2,2,5,7,8-pentamethylchromanol installed the b-hydroxythioamide in good enantioselectivity (93% ee).

Further transformations completed the catalytic asymmetric total synthesis of (+)-caprazol (2), a core structure of caprazamycin B and itself a natural product.

Azadirachtin (1), which was isolated from the neem tree Azadirachta indica A. Juss (Meliaceae), exhibits potent antifeedant and growth inhibitory activities. Due to its complicated structure, only one total synthesis of 1 has been reported by the Ley group to date. Herein, we report the formal asymmetric synthesis of 1 by utilizing the tandem radical cyclizaiton approach developed in our laboratory.

The Diels-Alder reaction – decarboxylation – Claisen rearrangement strategy was used to construct the basic structure of the left-hand segment (22R24). The product 24 was subjected to an oxidative ring-opening reaction and a subsequent aldol reaction to give 28 with the correct stereochemistry at C4-position. After a coupling of the left-hand segment 31 with the right-hand segment 39prepared from the known compound 32, an S_N2’ reaction with methyl copper reagent afforded the allene 41. The key tandem radical cyclization reaction of 41 was realized by treatment with Bu₃SnH and AIBN in DMF to give 42 in a moderate yield. The compound 42could be converted into the Ley’s intermediate 45 in 9 steps, resulting in the formal asymmetric synthesis of 1. Our synthesis which requires a longest linear sequence of 30 steps to the intermediate 45 is much more efficient than the Ley’s synthesis (53 steps).

Although 2-aminoimidazoles are found in many bioactive compounds and pharmaceuticals, their synthetic methods are very limited. We have developed a new method to access to the substituted 2-aminoimidazoles, as inspired by an arginine post-translational modification by lipid metabolites. In addition to the arginine derivatives, variously substituted phenyl guanidines participated in the smooth reaction with the unsaturated aldehydes, providing the substituted 2-aminoimidazoles, through a sequence of imine formation and intramolecular conjugation addition. Based on the established method, a one-pot library synthesis of 2-aminoimidazoles was also developed using the variously substituted aniline derivatives as the starting materials.

We then applied the method to natural product, ageladine A, which was isolated from a marine sponge, Agelas Nakamurai, and shows significant antiangiogenic activity. Our synthesis focuses on generating the natural product and its derivatives from the simple guanidines through the sequential multi-step reactions in one-pot operation, namely, i) formation of 2-aminoimidazole ring, ii) enamine formation derived from ammonia, iii) imine formation with substituted aldehydes, iv) azaelectrocyclization, v) autooxidation, and vi) cleavage of aryl protection groups. Based on this strategy, ageladine A and its pyridine derivative with strongest antiangiogenic activity developed so far, were synthesized in one-pot process, 10-20% overall yields.

We further prepared more than 20 derivatives of ageladine A; some of them showed prominent biological activity, which is totally different from ageladine A. We thus achieved the bioactivity remodelling of natural product, based on the one-pot total synthesis of natural product.

Tirandamycins A-D (1-4) and their congeners (5and 6) were isolated from a culture broth of Streptomyces sp. These compounds possess characteristic structures consisting of the various oxidation patterns of dioxabicyclo[3.3.1]nonane skeleton with four contiguous stereogenic centers and a tetramic acid moiety. They exhibit a variety of biological activities including antibacterial, antiviral, and antitumor activities. In particular, tirandamycin B (2) has attracted much attention as a lead scaffold to discover and develop antifilarial drugs. Their unique structures and intriguing biological activities prompt us to address the synthesis of tirandamycins.

We expected that our cinchona alkaloid catalyzed Morita-Baylis-Hillman (MBH) reactions can be feasible for the construction of their polypropionate sutructures. Thus, the four contiguous stereogenic centers can be constructed from adehyde 13by the combination of　β-ICD or α-ICPN-catalyzed MBH reaction and MgBr₂ or Rh catalyst mediated diastereoselective hydrogenation.

In this symposium, we will report a first asymmetric total synthesis of (-)-tirandamycin B (2). The synthesis involves a highly enantio- and diastereoselective formation of 11 from 13, formation of bicyclic ester 9 from 10 via Achamatowictz reaction and construction of 2 via a HWE reaction of aldehyde 8 with phosphonate 7. In addition, we report the total syntheses of tirandamycin A (1) and D (4) via deoxygenation of 9, and the formal syntheses of streptolydigin (5) and streptolydiginone (6) and total synthesis of streptolic acid via intramolecular S_N2’ reaction of halo alcohol 22 derived from 9.

Since 1970, cancer has been holding the first place of the mortality rate in Japan. Causes of cancer are considered to be related to aging, lifestyle and other environmental conditions. From now on, cancer is a severe health problem because cancer mortality rate would be rapidly increased among an aging Japanese society.

In our previous study, Sesquiterpene Lactones (SLs) isolated from yacon leaf such as enhydrin, uvedalin and sonchifolin exhibited an anticancer promoter activity coupled with TPA-induced deformation of Raji cells. Furthermore, the SLs showed cytotoxicity against HeLa cell, which was caused by inducted of apoptosis as a consequence of caspase-3/7 activation. In this study, dimeric SLs, enhydrofolin (5) and uvedafolin (6), from yacon leaf were newly found in the extract and also exhibited cytotoxicity in HeLa, HL60 and B16 cell lines.

Dimeric SLs (IC₅₀ 5; 1.69 and 6; 1.93 μM, respectively) are found to be the most effective substances among 8 SLs and typical cancer drug, etoposide (IC₅₀ 3.2 μM) on HeLa cells. Common functional group of these SLs for the activity-structure relationship was 1) the a-methylene-g-lactone, 2) the ester linkage of Dimeric SL between two sets of SL monomers, and 3) the acetyl group at the C-9 for the great cytotoxicity.

Wnt signaling pathway has a key roles in various cellular process such as cell proliferation and tissue maintenance. Aberrant activation of Wnt signal can initiate the development of several diseases including cancer, Alzheimer’s disease, and diabetes. Using cell-based luciferase screening assay system for a Wnt signaling pathway (TCF/β-catenin transcription), activity-guided exploration of our natural resources extracts library were conducted. Detailed investigation on mode of action of isolated compounds were also carried out.

Six cardenolides (1-6) were isolated from the methanol extract of Calotropis gigantea (Asclepiadaceae) exudate, and compounds 1, 3, 5, and 6 strongly inhibited TCF/β-catenin transcription activity (IC₅₀0.7-3.8 nM). Compounds 1, and 3-6showed potent cytotoxicity (IC₅₀ 1.8-7.0 nM) against Wnt dependent colon cancer cell (SW480, DLD1, HCT116), without cytotoxicity against RKO (Wnt independent colon cancer) cells. Calotropin (1) inhibited Wnt signaling by reducing both nuclear and cytosolic β-catenin levels in SW480 cells. Further investigation using inhibitors revealed that 1 potentiate the degradation of β-catenin by increasing the phosphorylation of β-catenin at Ser45 mediated by casein kinase 1α (CK1α). Moreover, 1 significantly enhanced CK1α protein and mRNA levels.

Three limonoides (7-9) were isolated from methanol extract of Xylocarpus granatum (Meliaceae) leaves, including two new limonoids, named as xylogranin A (7) and B (8). Compounds 7 and 8 inhibited TCF/β-catenin transcription activity. Treatment of SW480 cells with 8 led to a significant decrease of β-catenin in nuclei but not in the cytosol, and c-myc and PPARδ, target proteins of Wnt signaling pathway. These results indicated that a decrease of β-catenin in nuclei by 8contributed the Wnt inhibition of 8.

The life cycle of Dictyostelium discoideum consists of growth and developmental phases. In the growth phase, amoebae of this organism multiply by binary fission, and they initiate development when the food source is exhausted. In the developmental stage, they aggregate and form multicellular structures called slugs composed of prespore and prestalk cells. At fruiting body construction, these cells differentiate into spores and stalk cells, respectively. Previously, we described two prespore cell-inducing factors in conditioned medium (CM) of D. discoideum; one was a glycoprotein named prespore cell-inducing factor (y factor, or PSI-1), and the other, a dialyzable factor(s). Here we report on the identification of a unique alkylbenzoquinone, dictyoquinone (1), as the dialyzable factor that plays significant roles in the multicellular development of D. discoideum and its related species.

CM (~12 L) prepared from D. discoideum was separated into three fractions using Amberlite XAD-2 resin. A fraction eluated with MeOH–H₂O (9:1) exhibited the highest pre-spore inducing activity. Activity guided fractionation with Sephadex LH-20, HPLC equipped with ODS and C8 columns was carried out to give the factor (>400 μg). The structure of the compound was determined to be 2-hydroxy-5-methyl-6-pentylbenzoquinone by spectral analysis and chemical synthesis, and by their prespore-cell inducing activity. The synthetic compound has prespore-cell-promoting activity similar to the natural one, with half-maximal induction at a concentration as low as 40 pM. It was also found that the factor induces aggregation in an aggregation-deficient mutant of a related species, Polysphodilium violaceum. Both these activities are sensitive to positional isomerism with 6-methyl-5-pentyl isomer showing no detectable activity. Fremy salt treatment of MPBD (3), which was also obtained from D. discoideum, gave dictyoquinone (1). MPBD (3) is probably a biogenetical precursor of dictyoquinone (1).

Jawsamycin (1), produced by Streptoverticillium fervens HP-891, is a potent antifungal agent for various phytopathogenic fungi. The structure consists of a highly unusual polycyclopropanated fatty acid and 5’-amino-5’-deoxy-5,6-dihydrouridine. Notably, all cyclopropane moieties in 1 have the same absolute stereochemistry. To elucidate biosynthetic machinery of characteristic polycycropropane ring system, a series of administration studies and functional analysis of the biosynthetic enzymes found in the jawsamycin biosynthetic gene cluster have been conducted.

Feeding experiments with D-[U-¹³C₆]glucose, [1,3-¹³C₂]glycerol, L-[Me-¹³C]- methionine showed that carbon chain of polycyclopropanated fatty acid is constructed by acetate unit and the additional C1 unit of cyclopropane moiety was derived from S-adenosyl methionine. Incorporation of cyclopropanated diketide precursor 3 indicated that cyclopropanation occurs at the stage of a,b-unsaturated thioester bound to PKS. Bioinformatic analysis of draft genome sequence enabled us to identify jawsamycin biosynthetic gene cluster (jaw) consisting of nine open reading frames. A series of gene inactivations and in vitro analysis of key enzymes in the biosynthetic pathway established function of those enzymes. On the basis of these results, we propose a novel mechanism for the construction of the unique polycyclopropanated backbone involving a radical S-adenosyl methionine (SAM) cyclopropanase collaborating with an iterative PKS. Our reconstitution system sets the stage for studying the catalytic mechanism of this intriguing contiguous cyclopropanation.

Teleocidin B is an indole alkaloid isolated from Streptomyces sp. Due to its unique chemical structure and ability to activate protein kinase C, it has attracted interest in the areas of organic chemistry and cell biology. While the structure-function relationships of teleocidin B have been investigated by using natural and synthetic derivatives, the reaction mechanism for the formation of its indole-fused six-membered ring remained unclear. In this presentation, we report the identification of teleocidin B biosynthetic genes and the functional and structural characterization of tailoring enzymes.

We found a gene cluster including tleA (non-ribosomal peptide synthetase), tleB (P-450), and tleC (prenyltransferase) and a discrete gene tleD (methyltransferase) in S. blastmyceticus genome⁸. To determine their roles in teleocidin B biosynthesis, we introduce these genes in Streptomyces lividans. The strain produces teleocidin B and its derivatives, thus indicating that the tle genes are responsible for teleocidin B biosynthesis. In vitro analyses of tailoring enzymes revealed that TleC catalyzes a rare reverse-prenylation of geranyl group to indolactam V to produce lyngbyatoxin A, and TleD produces teleocidin B by methylation and cyclization in one-pot reaction. To the best our knowledge, this is the first report of C-methyltransferase catalyzing methylation to facilitate terpenoid cyclization.

Plant hormones are a group of structurally diverse small compounds that orchestrate the cellular processes governing proper plant growth and environmental adaptation. In order to grasp the details of the hormonal action, we have to study their not only inherent activities but also cross talk between plant hormones. To study the cross talk of plant hormones, plant chemical activators, such as probenazol and uniconazol, have made great contribution to understand hormonal actions together with actual use in agriculture. However, there is toughy barrier to use plant chemical activators due to the limitation in available source of the activators. For example, to our best knowledge, there is no plant chemical activator to stimulate plant to accumulate ABA. In this report, we presented theobroxide as a chemical activator for stimulating plant to accumulate ABA via BG1, and the resulting ABA derived from ABA-GE showed antagonistic effect against GA and SA. We also found that root-applied theobroxide was transported to upper part of plant. In many cases, these antagonistic effects were suggested by exogenously applied ABA, whose concentration was far from biologically relevant concentration, or using mutant or transgenic plants. However, we firstly accomplished to reveal these biological phenomena using theobroxide as a plant chemical activator. Our results demonstrate that ABA level can be chemically controlled and ABA-GE is available source affording ABA for plant to exhibit ABA dependent biological phenomena.

Bacteria-infected cells are known to produce higher level of nitric oxide and reactive oxygen species. Although these reactive chemicals can contribute directly to kill pathogens, in this study, we focus on antibacterial autophagy regulation via downstream mediator.

A downstream mediator of nitric oxide signaling, 8-nitro-cGMP, has been recently identified in various mammalian cell lines. This nitrated version of cGMP modifies Cys residues of proteins (protein S-guanylation) to modulate their functions. In this study, we found that endogenous 8-nitro-cGMP promotes autophagic exclusion of invading group A Streptococci (GAS) from murine macrophages. Interestingly, cytosolic GAS were partly modified by the S-guanylation at their surface, and the S-guanylation level was significantly higher with the autophagosome-encapsulated GAS than the cytosolic counterpart. This finding suggests a possible role of S-guanylation as a tag to recruit bacteria for autophagic degradation. Further analysis revealed that S-guanylation-positive bacteria were selectively modified by Lys63-linked polyubiquitin chains, which is a known molecular determinant for selective transport to autophagosomes via autophagy receptor binding.

Collectively, the results of present study suggest that 8-nitro-cGMP is an endogenous autophagy enhancer that defines targets for sequestration in autophagosomes by recruiting ubiquitin chains.

Reference: C. Ito et al., Molecular Cell, 52, 794-804 (2013).

Seven new manzamine alkaloids, acantholactam (3), pre-neo-kauluamine (5), and acanthomanzamines A-E (7-11) were isolated from three specimens of marine sponges Acanthostrongylophora ingens, which were collected in North Sulawesi, Indonesia. Acantholactam (3) contains a g-lactam ring N-substituted with a (Z)-2-hexenoic acid moiety and is proposed to be biosynthetically derived from manzamine A (1) by oxidative cleavage of the eight-membered ring. Pre-neo-kauluamine (5) was converted to the dimer neo-kauluamine (6) during storage in the freezer for two months, suggesting non-enzymatic dimerization. Acanthomanzamines A and B (7and 8) are the first examples, containing a tetrahydroisoquinoline instead of a b-carboline in manzamine-related alkaloids. Acanthomanzamine C (9) contains a hexahydrocyclopenta[b]pyrrol-4(2H)-onering that may be converted from an eight-membered ring in manzamine A. Acanthomanzamines D and E (10 and 11) have an additional oxazolidine and 2-methyloxazolidine rings, respectively, which fuse to the manzamine skeleton. The biological activities, such as cytotoxic activity against HeLa cells, inhibitory activity against the proteasome, and inhibitory activity against the accumulation of cholesterol esters in macrophages, were tested with the isolated compounds. These three activities were markedly lower in 3 than in other compounds. This result clearly indicated that the presence of the eight-membered ring in the manzamines was essential for them to show their biological activities.

Nature has given us a large number of bioactive compounds available as therapeutic agents or biological tools. In spite of their great potential, not a few natural products are lacking in detailed studies on their biological activities. It remains challenging how natural products exhibit their unique activities. Our research group has been searching for novel bioactive molecules from marine organisms. Additionally, we have recently attempted to understand the biological activities of natural products. In this presentation, our group especially focuses on discovery and biological mechanism of “biselyngbyasides”, novel 18-membered ring macrolides isolated from marine cyanobacteria.

Biselyngbyasides were discovered through bioassay-guided fractionation of cyanobacterial extracts. The methanol extract of Lyngbya sp. was partitioned between ethyl acetate and water. The ethyl acetate layer was concentrated and further partitioned between n-hexane and 90% aqueous methanol. The 90% aqueous methanol layer, which showed remarkable growth-inhibitory activity against HeLa cells, was subjected to fractionation with ODS silica gel column chromatography and reversed-phase HPLC to give biselyngbyasides (2-6). Cell viability assay and DNA ladder analysis showed that biselyngbyasides induced apoptosis against HeLa cells and HL60 cells. Through the screening with human cancer cell lines, we noticed that the screening result of biselyngbyaside was somewhat similar to that of thapsigargin, a known inhibitor of sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA). This screening result provided motivation to evaluate the effects of biselyngbyasides on cytoplasmic Ca²⁺. Fura-2 method revealed that biselyngbyasides increased cytoplasmic Ca²⁺concentration against HeLa cells. Using RT-PCR analysis, we detected mRNA expression of endoplasmic reticulum (ER)-stress markers.

Recently, we found that biselyngbyasides target the calcium pump SERCA. biselyngbyaside (1) and biselyngbyolide B (2) showed remarkable inhibitory activity against SERCA1a and 2a. This presentation provides an understanding of biological mechanism of the marine macrolides inspired by screening profile.

Physalins (1), which have a steroidal skeleton, were reported to show various unique biological activities including their inhibitory activity on the transcriptional activation of NF-κB. In order to elucidate biological function of right-side unique structure of physalins, we had first established the strategy towards cage-shaped DEFGH-ring moiety via the domino-type transformation and completed the synthesis of Bn-DEFGH (3). Biological evaluation of natural products and synthetic compounds revealed that 3 showed moderate inhibitory activity on NF-κB activation and shared the same mode of action as 1. These results indicated that DEFGH-ring motif along with a hydrophobic functionality has a potential to serve as a non-steroidal NF-κB inhibitor. Therefore, we envisioned the construction of the cage-shaped molecules library and set compound 5, 6and 7 along with 1 as target molecules to create more potent inhibitors. What we challenge in this synthetic research is the creation of these designed molecules on the way to the synthetic studies towards 1. This time, we successfully established most of their synthetic methodologies listed below.

Synthetic strategies for these molecules are summarized in Scheme 1. The characteristic DEFGH-ring system 8 would be formed from its precursor 9. Thus, three key transformations to construct C8–C14 bond, AB-rings and C12–C13 bond to convert 2 to 9 (such as 13, 16, 17 and 18) were investigated.

Bromopyrrole alkaloids are known to be one of the most common metabolites contained in marine sponges. These alkaloids have attracted widespread interest due to their fascinating chemical structures with high N to C ratio (~1:2) and intriguing biological activities. In our search for new metabolites from marine organisms, we have reported several bromopyrrole alkaloids from the extract of an Okinawan marine sponge Agelas sp. (SS-162). Further investigation of the extract has resulted in the isolation of five new bromopyrrole alkaloids, agelamadins A-E (1-5). The structures of 1-5 were elucidated on the basis of detailed spectroscopic analysis and TDDFT ECD calculation.

Agelamadins A (1) and B (2) are structurally unique dimeric bromopyrrole alkaloids having an agelastatin-like 5/5/6/5 tetracyclic moiety and an oroidin-like liner moiety in common. Agelamadins A (1) and B (2) were assigned as racemate by chiral HPLC analysis. Agelamadins C-E (3-5) are bromopyrrole alkaloids with a bicyclic skeleton including a fused morpholine and benzene moieties. The absolute configuration of the a-carbon of 3 was assigned as S by application of PGME method. To the best of our knowledge, agelamadins C-E (3-5) are the first example for heterodimers comprising a bromopyrrole alkaloid and a tryptophan derivative.

Agelamadins A (1) and B (2) exhibited antimicrobial activity against Bacillus subtilis (MIC, 16 mg/mL, each), Micrococcus luteus (MIC, 4.0 and 8.0 mg/mL, respectively), and Cryptococcus neoformans (IC₅₀, 8.0 and 4.0 mg/mL, respectively).

A total synthesis of halichondrin A, the phantom member in the halichondrin class of natural products, is reported. The highlights of synthesis include: (1) synthesis of C1−C19 building block 8 via a catalytic asymmetric Cr-mediated coupling of iodoacetylene 4 and aldehyde 6 in presence of bromoolefin moiety followed by face-selective epoxidation of vinylogous ester 7; (2) synthesis of the right-half of halichondrin A 13 via an asymmetric Ni/Cr-mediated coupling of vinyl bromide 8with aldehyde 11, followed by base-induced furan formation, and Shiina macrolactonization; (3) coupling of C1–C38 building block 15 and C39–C54 building block 16 by Ni/Cr reagent; (4) synthesis of halichondrin A with use of a newly discovered, highly selective TMSOTf-mediated equilibration of C38-epi-halichondrin A (22) to halichondrin A (1). Two pieces of evidence are presented unambiguously to establish the structure of halichondrin A thus synthesized: one is the synthesis of norhalichondrin A (2) and/or homohalichondrin A (3) from C1–C38 building block 15, the common intermediate used for the synthesis of halichondrin A, and C39–C53 building block 18 and/or C39–C55 building block 20. The other is the study of the proton chemical shift difference between synthetic halichondrin A (1) and known members of this class of natural products.

Mycalolide B (1) is a trisoxazole macrolide isolated from the marine sponge Mycale sp.,¹ which shows various biological activities, such as cytotoxicity, antifungal activity, and potent actin-depolymerizing activity.² A number of trisoxazole macrolides that are structurally related to mycalolide B have been isolated, and recently, total syntheses of mycalolide A³ and ulapualide A⁴ have been accomplished.

Based on the finding that olefin metathesis is a useful method for connecting the C19–C20 double bonds in mycalolide analogs,^6b we expected that structure of 1 could be efficiently constructed from a C1–C19 trisoxazole segment 4 and a C20–C35 side-chain segment 5. First, 5 was synthesized by using the Julia–Kocienski olefination between sulfone 6 and aldehyde 7 as a key step. To construct macrolactone 3 from compounds 4 and 5, we examined two approaches. Due to the higher stereoselectivity on the metathesis reaction, the cross metathesis / macrolactonization approach was preferred to the esterification / RCM approach. After the 11 steps including the installation of the C35 N-methyl enamide and the C30 ester moieties, we accomplished the total synthesis of (–)-mycalolide B (1).

Taxol (paclitaxel, 1), isolated from the bark of Taxus brevifolia, has been widely used as an anti-cancer therapeutic reagent. It consists of a highly oxidized tetracyclic framework including an 8-membered ring, a bridgehead olefin and a unique oxetane. Although a number of excellent total syntheses have been reported, taxol (1) still remains as one of the most formidable synthetic targets.

In designing an efficient synthetic route, we employed the convergent strategy using the Shapiro coupling of A-ring moiety 13 with C-ring moiety 3, which was derived from readily available D-glucose (2) (Scheme 2). The next cyclization to form the multi-functionalized 8-membered ring (B-ring) was the most challenging step in our total synthesis. Ultimately, we developed the novel SmI₂-mediated cyclization of a substrate bearing an aldehyde and an allylic benzoate, with 6aand 6b isolated in 66% combined yield (Scheme 3). The proper choice of the stereochemistry at C-13 secondary alcohol and the leaving group was crucial in this reaction. Substrates 15a-c did not afford any desired product under the same conditions. The salient features of this reaction were i) high reactivity to construct the medium-sized ring, ii) promising stability of the allylic benzoate, which was important in the multi-step synthesis, and iii) quick differentiation of the resulting olefin and hydroxyl groups. The strained bridgehead olefin was then installed by double Chugaev reaction of bis(xanthate) 19, giving 7 in 94% yield (Scheme 4). The reaction took place in complete regioselectivity without generation of the exo olefin. From the resulting diene 7, we successfully achieved the formal synthesis of taxol (1) via the construction of the oxetane ring.

Bucidarasins were isolated in 2002 from extract of Bucida buceras. These compounds showed potent cytotoxicity against human tumor cell lines with IC₅₀ values of 0.5–1.9 μM. Moreover, their potency was retained in drug-resistant tumor cell lines.^[1] Bucidarasins are a family of clerodane diterpenoids, possessing a unique tricyclic ring system incorporating a cis-dehydrodecalin core with a fused tetrahydrofuran. The tricyclic scaffold contains up to eight stereogenic centers, six of which are contiguous and include two all-carbon quaternary stereogenic centers. To the best of our knowledge , the total synthesis of these fascinating compounds has not been reported. We have constructed the cis-dehydrodecalin core of bucidarasins via a highly stereoselective Diels–Alder reaction using a chiral building block prepared by us,^[3] and have achieved the first total synthesis of bucidarasins A and C, tricyclic clerodane diterpenoids, which also elucidated the absolute structure of their natural forms.

A seco-prezizaane type sesquiterpene jiadifenin (1) consists of highly oxygenated cage-like tetracycle with a cyclic hemi-acetal, γ-lactone, and six stereogenic centers. In addition to its unique structure, jiadifenin was found to significantly promote neurite outgrowth in the primary cultured fetal rat cortical neurons. This bioactivity indicates that jiadifenin is a potential candidate for the treatment of neurodegenerative disorders such as Alzheimer’s disease. In this symposium, we report a formal synthesis of jiadifenin using Pd-catalyzed reactions as key steps.

Our synthetic strategy focused on application of Pd-chemistry for the construction of the jiadifenin’s ABC ring system with quaternary centers. Mizoroki-Heck reaction would be used to construct the A ring with the C-9 quaternary carbon, and a tandem Tsuji-Trost cyclization/lactonization sequence would be employed to establish the BC ring system.

A bromoester 5 was prepared from commercially available diethyl 4-oxopimelate (3) in 5 steps, and Mizoroki-Heck reaction of 5 was carried out with Pd(OAc)₂/(o-tol)₃P catalytic system. Interestingly, protic solvent such as MeOH dramatically promoted the reaction to give rise to the corresponding A ring compound 6 in 99% yield. Subsequently, a key cyclic carbonate 16 requisite for Tsuji-Trost tandem reaction was converted from 6 in several steps. This tandem cyclization was investigated under a variety of conditions such as Pd catalyst, solvent, ligand, and base. As results, aprotic solvents mainly gave rise to O-alkylated product 20, whereas protic solvents provided C-alkylation 17 toward Tsuji-Trost reaction. The use of bidentate ligand BINAP was crucial for control of the facial selectivity of oxidative addition in Tsuji-Trost reaction. Addition of LiOAc was found to accelerate the lactonization step to give the desired 18. After optimization, we were pleased to find that Pd(OAc)₂/(±)-BINAP and LiOAc system in t-BuOH brought about the Tsuji-Trost tandem cyclization of 16 to afford 18 in 65% yield. Finally, Theodorakis’s intermediate 2 was derived from 18 in 7 steps, thus accomplishing a formal synthesis of jiadifenin (1).

Piperidamycins (1-3), isolated from mutant strains of a soil-isolated Streptomycesspecies¹⁾, are 18-membered cyclohexadepsipeptides consisting of rare amino acids such as three piperazic acids and one g-hydroxypiperazic acid. Piperidamycins exhibit a strong antibacterial activity against gram negative bacteria and an anaerobic gram-negative bacillus. To elucidate the absolute and relative stereochemistries and the structure-activity relationships of piperidamycins, we planned a total synthesis of piperidamycin F (3). Prior to the synthesis of 3, we initially accomplished the synthesis of natural product JBIR-39 (4).

The synthesis of oligopiperazic acids is one of the key issues. We found that Sc(OTf)₃ effectively promoted the acylation of 9 with acid chloride 10, and the synthesis of the desired tetrapeptide 7 was achieved in a moderate yield. Further elongation of N- and C-terminus leading to 24, followed by removal of the all protecting groups furnished desired 4a. However, the spectral data of 4a was not identical to that of the natural product. It was found that chemical shifts of a-methylserine and g-hydroxypiperazic acid were different from those of the natural product, thus we synthesized three diastereomers 4b-4d. Since the spectra data of 4b was in good agreement with that of the natural 4, the structure of JBIR-39 including its absolute configuration was determined as shown in Scheme 5. We next attempted the synthesis of the expected structure of piperidamycin F (3a) based on the structural information of 4, and details of the synthetic study for 3a will also be discussed.

Entamoeba histolyticamembrane lipids contain inositol phospholipids EhPIa and EhPIb, which were shown to activate Natural Killer T (NKT) cells and induce selective IFN-gbut not IL-4 production in NKT cells via CD1d restricted manner. Because of these biological activities and the unique structures of EhPIa and EhPIb having characteristic long-chain fatty acids, we synthesized these compounds to clarify the detailed biological activities.

In the present study, we developed new synthetic methods including regioselective phosphorylation reaction of myo-inositol with utilizing chiral phosphorylating reagents such as chiral binaphtol as a chiral auxiliaries and Ni catalyzed sp³-sp³ cross coupling reaction for the synthesis of the long-chain fatty acids. We also adopted a new protecting group strategy utilizing Allyl and Alloc groups as the permanent protecting groups for hydroxyl groups in the inositol. Based on these newly developed methods, we achieved the first total syntheses of C30-EhPIa-cis 1a, C30-EhPIb-cis 2a, C30-EhPIb-trans 2b and C28-EhPIb 2c.

Extensin, hydroxyproline-rich glycoprotein (HRGP) [1] which is the structural motif of plant extracellular matrix proteins, possesses unique highly glycosylated, hydrophilic and repeating Ser₁Hyp₄ pentapeptideunit which has been proposed to include post-translational hydroxylation at proline residue and subsequent oligo-L-arabinosylations (Fig. 1) at all of the resultant hydroxyprolines as well as galactosylationat serin residue. Here, we wish to report the stereoselective synthesis of one of the highly glycosylated motifs, Ser(Galp₁)-Hyp(Araf₄)-Hyp(Araf₄)-Hyp(Araf₃)-Hyp(Araf₁) (Fig. 2). The synthesis has been completed by the applications of 2-(naphthyl)methyl (NAP) ether-mediated intramolecular aglycon delivery (IAD) [2] to the stereoselective constructions of the Ser(Galp₁) and Hyp(Araf_n) (n = 1, 3, 4) fragments as well as Fmoc-solid phase peptide synthesis (SPPS) of backbone peptapeptide [6] (Fig. 3).

Application of our NAP-IAD to the construction of Hyp(L-Araf_n) (n = 1, 3, 4) as well as Ser(Galp₁) derivatives has been achieved successfully with complete stereoselectivity(Sch. 1, 2) which had been already applied to the synthesis of the substrate [5] for b-L-arabinofuranosidase [4]. Subsequently Fmoc-SPPS was carried out using Fmoc- deprotectionwith pipridine and coupling using COMU which has been used for the synthesis of CLV3 [3] with an exception with piperidinein the presence of HOBt and using DIC–HOBt for the coupling of the dipeptideII in order to avoid the diketopiperazine formation (Sch. 3). Structural analysis of 1 has been carried out by using NMR and Mass in detail which is leading the glycopeptide 1 into polyProII helix-like structure (Fig. 4).

Peptide antibiotics are biosynthesized by two distinct machineries: (i) ribosome-based synthesis, and (ii) ribosome-independent synthesis exemplified by nonribosomal peptide synthesis. Pheganomycin (PGM) consists of the nonproteinogenic amino acid (S)-2-(3,5-dihydroxy-4-methoxyphenyl)-2-guanidinoacetic acid (1) at the N-terminus and a proteinogenic core peptide derived from NVKDGPT or NVKDR. The biosynthetic gene cluster responsible for the catalysis was identified in Streptomyces cirratus, containing a gene encoding a precursor peptide, including both core peptides, and several genes plausibly encoding enzymes for 1 biosynthesis and tailoring reactions. We searched for the gene responsible for the peptide bond formation between 1 and the peptides in the cluster, and identified pgm1, which has an ATP-grasp domain. A pgm1-deletion mutant lost PGM productivity, and recombinant PGM1 catalyzed the ATP-dependent peptide bond formation. This is the first example of cooperative peptide synthesis achieved by ribosomes and peptide ligases using a peptide as a nucleophile. PGM1 accepted a variety of peptides as the nucleophile and its flexibility was comprehended by the crystal structure of PGM1 and mutagenesis analyses.

Azoline heterocycles are often found in the backbones of peptidic natural products and are important structural motifs that play critical roles in their diverse bioactivities. Some of such azoline-containing peptides (Az-peptides) are produced via posttranslational modifications of ribosomally synthesized precursor peptides, in which cysteine, serine, and threonine residues are converted to their corresponding azolines by a cyclodehydratase. Here we have devised an in vitro biosynthesis system of Az-peptides, referred to as FIT–PatD system, by the integration of a cell-free translation system with a posttranslational cyclodehydratase PatD. In the FIT–PatD system, three biosynthesis reactions, transcription of substrate DNAs, ribosomal synthesis of precursor peptides, and subsequent posttranslational azoline installation, proceed in a coupled manner. The FIT–PatD system has facilitated mutagenesis studies on a wide array of precursor peptide sequences, unveiling the substrate recognition determinants for PatD catalysis and unprecedented in vitro substrate tolerance of PatD. This system also allows “one-pot” synthesis of a various Az-peptide derivatives expressed from synthetic DNA templates. The FIT–PatD system could be a powerful synthetic tool to produce a wider array of Az-peptides than produced in nature.

Onion (Allium cepa L.), garlic (Allium sativum L.), and Welsh onion (Allium fistulosum L.) belong to the genus of Allium in Liliaceae family. In particular, garlic is ranked at the top of the list of designer foods showing anticancer effects by the National Cancer Institute. In general, the biological activities of onion and garlic can be classified into two categories: cardiovascular disease prevention and cancer prevention.

Respective commercial onion, Welsh onion, and Chinese garlic were extracted with acetone. The acetone extracts of onion and garlic showed the potential to inhibiting the polarizationof M2 activated macrophages being capable of suppressing tumor-cell proliferation. Next, they were separated on silica gel to provide novel sulfides named onionins A₁, A₂, and A₃from both onion and Welsh onion, and garlicnins B₁, B₂, B₃, B₄, C₁, C₂, C₃, L-1-L-4, E, and F from garlic. They were devided into two types, one is novel cyclic sulfoxide type of 3,4-dimethyltetrahydrothiophene S-oxide derivatives, and another is linear acyclic sulfide type as shown in Chart 1.

The proposed production of cyclic sulfoxides is shown in Chart 1: Allyl sulfenic acid derived from (+)-S-allyl L-cysteine sulfoxide (alliin) present in garlic yields diallyl thiosulfinate (allicin), which was transformed into 1-propenyl 1-propenethiosulfinate via double-bond rearrangement and then become converted to 2,3-dimethyl- butanedithial 1-oxide via [3,3]-sigmatropic rearrangement. Next, the generated compound was ring-closed to form a thiophene derivative, which was attached with 1-propenesulfenic acid (b) or/and allyl thiosulfenic acid (a) derived from allicin to finally produce the cyclic sulfoxides.

Next, we examined the effects of onionin A₁ and garlicnin C on tumor progression and lung metastasis in mouse osteosarcoma (LM8)-bearing mice model. Our results showed that onionin A₁ and garlicnin C were effective agents for osteosarcoma in both in vitro and in vivo models, and the antitumor effects observed in vivo are likely caused by reversing the antitumor immune system. Activation of the antitumor immune system by onionin A₁ or garlicnin C might be an effective adjuvant therapy for patients with osteosarcoma and other malignant tumors.

The identification and characterization of these novel sulfides isolated from onion, Welsh onion, and garlic will contribute to the accumulation of information on new chemicals and pharmaceutical substances in the Allium sulfide group. Based on these findings, pharmacological investigations will be conducted in the future to develop natural and healthy foods and anti-cancer agents that can prevent or combat disease.

Vineomycin B₂ (1), an anthracycline antibiotic, was isolated by O̅mura et al. in 1977. Because of its important biological activity and novel molecular architecture, 1 is a prime target for both chemical and biological studies. Although six elegant syntheses of the aglycon moiety, vineomycinone B₂ methyl ester (2), have been reported so far, the total synthesis of 1 has not been accomplished to date. Herein, the development of novel chemoselective glycosylations using 2,3-unsaturated sugars and its application to the first total synthesis and the SAR studies of 1 are described.

The aglycon segment 14 and the glycon segment 17 were prepared via C-glycosylation using an unprotected sugar and powerful chemoselective O-glycosylation using 2,3-unsaturated sugar, respectively, as the key steps. Furthermore, effective and simultaneous introduction of the two glycon moieties 17 to the aglycon part 14 by concentration-controlled glycosylation led to the total synthesis of 1.

In addition, nine oligosaccharide analogs (33-41) of vineomycin B₂were designed and synthesized, and their cytotoxicities against cancer cell lines, MCF-7 and sarcoma 180, were evaluated. As a result, it was found that analogs 33, 40 and 41, which possess the aculoside moiety and/or anthraquinone moiety, showed significant cytotoxicity against these cell lines. These results suggest that 1 shows significant cytotoxicity toward cancer cell lines by two major pathways.

A toxic red tide occurred in Wellington Harbor, New Zealand in early 1998 and caused massive kills of marine life. More than 500 cases of human illness were also reported. The causative organism was identified as a new dinoflagellate Karenia brevisulcata. Cultured K. brevisulcata produces two classes of ladder-shaped polyether compounds, brevisulcenals (KBTs) and brevisulcatic acids (BSXs). The molecular weights of KBTs are over 2,000 and those of BSXs are nearly 900. KBTs have potent mouse lethality and cytotoxicity. On the other hand, BSXs have toxicity to Neuro-2a.

Five BSXs were isolated from extracts of K. brevisulcata by partitionings and by various column chromatographies. Detailed analysis of various NMR spectra led to elucidate the structures of BSXs. BSXs consist of 9-fused ether rings, g-lactones and carboxylic acid side chains and have two conformational isomers at low temperature presumably originated from medium-membered ether rings in the middle of the molecule. The structure of a major component, BSX-4 was confirmed by a high energy CID MS/MS experiment using MALDI-SpiralTOF-TOF. The g-lactone was essential for their neurotoxicity.

(+)-Vibsanin A (1) was isolated by Kawazu from leaves of Viburnum odoratissimum Ker. (sweet viburnum) in 1980 and showed piscicidal activity. The structure is characterized by an 11-membered ring skeleton containing an all-carbon asymmetric quaternary stereocenter. A variety of other related compounds have been found in plants. Although many research groups have been interested in vibsanins as a synthetic target, the total synthesis of vibsanins that possess an 11-membered ring skeleton has not yet been reported. Accordingly, we embarked on the synthesis of 1, aiming at a definite determination of its stereochemistry.

To realize a convergent synthesis of 1, the target molecule was divided into the upper and the lower segments. The upper segment 4 was synthesized as an enantioenriched form, starting from commercially available 4-pentyn-1-yl acetate (7), in 7 steps through Sharpless asymmetric epoxidation. The lower segment 5 was synthesized by Barbier-type allylation using geranyl chloride (9) and chiral aldehyde 16 as a key step for the construction of an all-carbon quaternary stereocenter. The two segments 4 and 5 were connected to afford the coupling product 24, which was converted to aldehyde 27 in 3 steps including the introduction of a 3-methylcrotonyl group. The 11-membered ring skeleton was constructed by the intramolecular Nozaki–Hiyama–Kishi reaction of 27. Finally, the total synthesis of 1 was achieved by a two-step conversion from the cyclized product 28. The synthetic 1 was identical in NMR spectra and an optical rotation to those of natural sample, thereby establishing the absolute stereochemistry of (+)-vibsanin A.

The genus Armillaria belonging to the family Physalacriaceae is a well-known edible mushroom throughout the world.On the other hand, the genus has been known as a serious plant pathogen which causes root rot in various plant species. Furthermore, it is known that penetration of Armillaria mycelia to the fungus Entoloma abortivum and Wynnea americana induces deformity of the fruiting bodies of those mushrooms. These facts mean that Armillaria produces allelopathic substance(s). However, isolation of allelopathic substance(s) from the genus Armillaria have not been reported. Therefore, we tried to isolate compounds with plant and fungus growth regulatory activity from the fungus.

The culture broth of Armillaria sp. strain 488 or 543 was filtrated and then concentrated under reduce pressure. The concentrated filtrate was successively partitioned between n-hexane and water, EtOAc and water, and then n-BuOH and water. Isolation of the active compounds from the fungus was guided by their growth-regulating activity against lettuce. The active fractions, n-hexane-soluble and EtOAc-soluble parts were fractionated by chromatography. As a result, seven novel compounds (1, 2, 16-20) and theirteen known compounds (3-15) were isolated and their structures were determined by spectroscopic data analyses and chemical reactions.

All the compounds inhibited hypocotyl and root growth of the lettuce at 1 μmol/paper with significant differences. However, several compounds promoted the plant growth at lower concentrations, significantly. In addition, 7 inhibited the mycelial growth of Coprinopsis cinerea and Flammulina velutipes, and 12 inhibited the mycelial growth of C. cinerea. Furthermore, treatment of mycelia of Hypsizygus marmoreus with 7 induced deformity of the fruiting bodies of the fungus.