Isolation and Structure Elucidation of New Cytotoxic Macrolides Halosmysins B and C from the Fungus Halosphaeriaceae sp. Associated with a Marine Alga.

Two new cytotoxic metabolites, halosmysins B and C, have been isolated from the fungus Halosphaeriaceae sp. OUPS-135D-4 separated from the marine alga Sargassum thunbergii. These chemical structures have been elucidated by 1D and 2D NMR, and HRFABMS spectral analyses. The new compounds had the same 14-membered macrodiolide skeleton as halosmysin A, which was isolated from this fungal strain previously. As the unique structural feature, a diketopiperazine derivative and a sugar are conjugated to the 14-membered ring of halosmysins B and C, respectively. The absolute stereostructures of them were elucidated by the chemical derivatization such as a hydrolysis, the comparison with the known compounds (6R,11R,12R,14R)-colletodiol and halosmysin A, and a HPLC analysis of sugar. In addition, their cytotoxicities were assessed using murine P388 leukemia, human HL-60 leukemia, and murine L1210 leukemia cell lines. Halosmysin B was shown to be potent against all of them, with IC50 values ranging from 8.2 ± 1.8 to 20.5 ± 3.6 µM, though these values were slightly higher than those of halosmysin A.

6-Arylcoumarin as a Scaffold of Photofunctional Molecules with OFF-ON-OFF Type Regulation.

Coumarin has been utilized as a core structure of photofunctional molecules, such as fluorescent sensors and photoremovable protecting groups. Here, we show that the 6-arylcoumarin moiety can provide OFF-ON-OFF type regulatory functionality for such compounds. To illustrate its utility, we synthesized a coumarin derivative bearing two phenolic hydroxy groups at 7-position and on 6-aryl group as a fluorescent sensor showing an OFF-ON-OFF change in fluorescence intensity in response to an increase in pH from a strongly acidic condition. Further, we show that the efficiency of photoreaction of other derivatives with the same hydroxyl groups is switched from "OFF" at pH 3 and 6 to "ON" at pH 9 and then to OFF at pH 12, enabling their application as switchable photoremovable protective groups. These features arise from sequential deprotonation of two hydroxyl groups: the monoanionic form is responsible for the photoinduced fluorescence and photoreaction.

Halosmysin A, a Novel 14-Membered Macrodiolide Isolated from the Marine-Algae-Derived Fungus Halosphaeriaceae sp.

Halosmysin A, a new 14-membered macrodiolide with an unprecedented skeleton, was isolated from the fungus Halosphaeriaceae sp. OUPS-135D-4, which, in turn, was obtained from the marine algae Sargassum thunbergii. The chemical structure of the macrodiolide was elucidated using 1D and 2D NMR, as well as high resolution fast atom bombardment mass (HRFABMS) spectral analysis. The absolute stereochemistry was determined via chemical derivatization and comparison with a known compound, (6R,11R,12R,14R)-colletodiol. Additionally, halosmysin A was shown to be very potent against murine P388 leukemia, human HL-60 leukemia, and murine L1210 leukemia cell lines, with IC50 values ranging from 2.2 ± 3.1 to 11.7 ± 2.8 µM.

Recent Advances in Chemical Tools for the Regulation and Study of Protein Lysine Methyltransferases.

Protein lysine methyltransferases (PKMTs) are epigenetic regulators that modulate gene transcription and physiological functions by catalyzing the post-translational methylation of specific lysine residues of substrate proteins, such as histones. They are considered to be candidate drugs for the treatment of various diseases, including acute myeloid leukemia, and in the past decade, potent and selective inhibitors of individual PKMTs have been developed. Some are currently under clinical trial. In this review, we will focus on some breakthrough PKMT inhibitors, and discuss chemistry-based methods available for elucidation of the physiological functions of PKMTs and methylated proteins.

Synthesis and Conformational Analysis of Alternately N-Alkylated Aromatic Amide Oligomers.

Alternately N-alkylated aromatic amides such as 1-3 bearing various side chains were designed and synthesized as novel helical foldamers. The CD spectra of oligomers with chiral side chains showed a positive Cotton effect, which indicates that these oligomers take helical conformations in solution. The CD intensity gradually increased with increasing chain length, and pentamer 3d showed remarkably strong CD signals in chloroform. The absorption maxima of the UV spectra were increasingly red shifted with increasing chain length, in contrast to the case of poly( p- N-alkylbenzamide)s. Structure optimization of the oligomers based on the crystal structure of 1a as the monomer unit supported the formation of helical structure with a large cavity and also suggested intramolecular hydrogen bond formation between secondary amides. The results of calculation were consistent with the observed spectroscopic features.

Conformational Properties of Aromatic Oligoamides Bearing Pyrrole Rings.

N-Alkylbenzanilides generally exist in cis conformation both in the crystalline state and in various solvents, and this cis conformational preference can be utilized to construct dynamic helical oligoamides. Here, we synthesized the pyrrole-containing amides 2-5 and their oligomers 6-8 and examined their conformations in the crystalline state and in solution. All the N-methylated amides showed cis conformational preference in solution, but the ratio of the cis isomer was decreased when the amide bond was attached at the 4-position of the pyrrole ring, probably because the destabilization of the trans conformer due to electronic repulsion between the pyrrole π electrons and the amide carbonyl lone-pair electrons is reduced due to the small torsion angle between the 5-membered N-pyrrole and the amide bond. In the crystalline state, N-methylated amides showed cis structure, except for compound 5, and cis conformational preference was observed for the pyrrole amides. The CD spectra of oligoamides 15-18 bearing chiral N-substituents were consistent with the presence of dynamic and well-defined chiral foldamers, which were structurally distinct from N-alkylated poly( p-benzamide)s 1.

Sucupiranins A-L, Furanocassane Diterpenoids from the Seeds of Bowdichia virgilioides.

Twelve new furanocassane diterpenoids, sucupiranins A-L (1-12), and three known compounds (13-15) were isolated from the seeds of Bowdichia virgilioides. The structures of the compounds were elucidated via 1H and 13C NMR analysis, including 2D NMR (1H-1H COSY, HSQC, HMBC, and NOESY); HRMS data; and X-ray crystallographic analysis. The absolute configurations were defined using their electronic circular dichroism (ECD) spectra by applying the exciton chirality method to the bis-p-bromobenzoate of compound 13. Sucupiranin J (10) inhibited lipopolysaccharide-induced nitric oxide production (IC50 30.6 µM), whereas sucupiranins J (10), K (11), and 13 exhibited weak antimalarial activity against Plasmodium falciparum K1 with IC50 values of 32.2, 23.5, and 22.9 µM and selectivity indices of 4.3, 1.9, and >12.0 (MRC-5/K1), respectively.

Steric structure-activity relationship of cyproheptadine derivatives as inhibitors of histone methyltransferase Set7/9.

Set7/9 is a histone lysine methyltransferase, but it is also thought to be involved in a wide variety of pathophysiological functions. We previously identified cyproheptadine, which has a characteristic butterfly-like molecular conformation with bent tricyclic dibenzosuberene and chair-form N-methylpiperidine moieties, as a Set7/9 inhibitor. In this work, we synthesized several derivatives in order to examine the steric structure-inhibitory activity relationship. We found that even a small change of molecular shape due to reduction or replacement of the 10,11-olefinic bond of the tricyclic ring generally resulted in a drastic decrease of the inhibitory activity. Our results should be useful not only for development of more potent and selective inhibitors, but also for the construction of novel inhibitor scaffolds.

Development of 6-arylcoumarins as nonsteroidal progesterone antagonists. Structure-activity relationships and fluorescence properties.

Progesterone is involved in multiple physiological processes, including female reproduction, via binding to the progesterone receptor (PR). We have developed 6-arylcoumarins such as 5 and 6 as non-steroidal PR antagonists with receptor-binding-dependent fluorescence. In this study, we investigated the structure-activity relationships and fluorescence properties of coumarin derivatives bearing a heterocyclic aromatic moiety. Among these derivatives, 7c (IC50: 34nM) and 10b (IC50: 24nM) showed more potent PR-antagonistic activity than lead compounds 5 (IC50: 500nM) and 6 (IC50: 65nM) in alkaline phosphatase (AP) assay. Compound 9b showed solvent-dependent fluorescence intensity, exhibiting strong fluorescence in the presence of PR LBD only in buffer solution. On the other hand, 10b showed a solvent-dependent shift of the fluorescence maximum wavelength in the presence of PR LBD. These results indicate that 6-arylcoumarin will be a useful scaffold for PR antagonists and fluorescent probes targeting PR.

Design and synthesis of tetraol derivatives of 1,12-dicarba-closo-dodecaborane as non-secosteroidal vitamin D analogs.

Vitamin D receptor (VDR), a nuclear receptor for 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3, 1), is a promising target for multiple clinical applications. We recently developed non-secosteroidal VDR ligands based on a carbon-containing boron cluster, 1,12-dicarba-closo-dodecaborane (p-carborane), and examined the binding of one of them to VDR by means of crystallographic analysis. Here, we utilized that X-ray structure to design novel p-carborane-based tetraol-type vitamin D analogs, and we examined the biological activities of the synthesized compounds. Structure-activity relationship study revealed that introduction of an ω-hydroxyalkoxy functionality enhanced the biological activity, and the configuration of the substituent significantly influenced the potency. Among the synthesized compounds, 4-hydroxybutoxy derivative 9a exhibited the most potent activity, which was equal to that of the secosteroidal vitamin D analog, 19-nor-1α,25-dihydroxyvitamin D3 (2).

Structural development of p-carborane-based potent non-secosteroidal vitamin D analogs.

Non-secosteroidal vitamin D receptor (VDR) ligands are promising candidates for many clinical applications. We recently developed novel non-secosteroidal VDR agonists based on p-carborane (an icosahedral carbon-containing boron cluster) as a hydrophobic core structure. Here, we report the design, synthesis and biological evaluation of carborane-based vitamin D analogs bearing various substituents at the diol moiety. Among the synthesized compounds, methylene derivative 31 exhibited the most potent vitamin D activity, which was comparable to that of the natural hormone, 1α,25(OH)2D3. This compound is one of the most potent non-secosteroidal VDR agonists reported to date, and is a promising lead for development of novel drug candidates.

Synthesis and structure-activity relationship of p-carborane-based non-secosteroidal vitamin D analogs.

1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3: 1] is a specific modulator of nuclear vitamin D receptor (VDR), and novel vitamin D analogs are therapeutic candidates for multiple clinical applications. We recently developed non-secosteroidal VDR agonists bearing a p-carborane cage (a carbon-containing boron cluster) as a hydrophobic core structure. These carborane derivatives are structurally quite different from classical secosteroidal vitamin D analogs. Here, we report systematic synthesis and activity evaluation of carborane-based non-secosteroidal vitamin D analogs. The structure-activity relationships of carborane derivatives are different from those of secosteroidal vitamin D derivatives, and in particular, the length and the substituent position of the dihydroxylated side chain are rather flexible in carborane derivatives. The structure-activity relationships presented here should be helpful in development of non-secosteroidal vitamin D analogs for clinical applications.

Structures of histone methyltransferase SET7/9 in complexes with adenosylmethionine derivatives.

SET7/9 is a protein lysine methyltransferase that methylates histone H3 and nonhistone proteins such as p53, TAF10 and oestrogen receptor α. In previous work, novel inhibitors of SET7/9 that are amine analogues of the coenzyme S-(5'-adenosyl)-L-methionine (AdoMet) have been developed. Here, crystal structures of SET7/9 are reported in complexes with two AdoMet analogues, designated DAAM-3 and AAM-1, in which an n-hexylaminoethyl group or an n-hexyl group is attached to the N atom that replaces the S atom of AdoMet, respectively. In both structures, the inhibitors bind to the coenzyme-binding site and their additional alkyl chain binds in the lysine-access channel. The N atom in the azaalkyl chain of DAAM-3 is located at almost the same position as the N-methyl C atom of the methylated lysine side chain in the substrate-peptide complex structures and stabilizes complex formation by hydrogen bonding to the substrate-binding site residues of SET7/9. On the other hand, the alkyl chain of AAM-1, which is a weaker inhibitor than DAAM-3, binds in the lysine-access channel only through hydrophobic and van der Waals interactions. Unexpectedly, the substrate-binding site of SET7/9 complexed with AAM-1 specifically interacts with the artificial N-terminal sequence of an adjacent symmetry-related molecule, presumably stabilizing the alkyl chain of AAM-1.

Development of functional molecules for elucidation of the physiological roles of several nuclear receptors and their endogenous ligands.

Nuclear receptors and their endogenous ligands are involved in key biological functions, including development, homeostasis and metabolism, and functional molecules that can modulate receptor activities are of interest for basic research to elucidate the signaling pathways, as well as having potential therapeutic applications. Here, we summarize our recent work on the development of nuclear receptor ligands, focusing on thyroid hormone receptor (TR) antagonists, fluorescent ligands for progesterone receptors (PR), and inhibitors of histone methyltransferase (HMT). We have developed a series of potent TR antagonists bearing a thiazolidinedione group as a bioisoster of a polar amino acid group. Utilizing our library of fluorescent coumarin derivatives, we obtained a PR antagonist that exhibits fluorescence enhancement upon binding to PR. We also developed a series of HMT inhibitors based on the structure of adenosylmethionine (AdoMet), a cofactor in the methylation reaction, by introducing various alkylamino groups onto the nitrogen atom in place of the sulfur atom of AdoMet. Our compounds should be useful in functional analysis of these nuclear receptors and investigations of their signaling pathways.

Secoergostane- and ergostane-type steroids from Pleurotus cornucopiae var. citrinopileatus.

In this study, we described the isolation of an 8,14-secoergostane-type, a 9,11-secoergostane-type, and three ergostane-type steroids from the fruiting bodies of Pleurotus cornucopiae var. citrinopileatus. The structure of (22Z)-3ß,5α,11-trihydroxy-9,11-secoergosta-7,22-diene-6,9-dione, previously reported, have been revised to (22E). Their structures were established using NMR, UV, IR, and mass spectroscopic analyses. Three of the isolated compounds were found to exhibit inhibitory activity on the production of nitric oxide in lipopolysaccharide-stimulated RAW264.7 macrophages with IC50 values of 21.3, 17.6, and 23.1 µM, respectively.

Boron cluster-based development of potent nonsecosteroidal vitamin D receptor ligands: direct observation of hydrophobic interaction between protein surface and carborane.

We report here the design and synthesis of a novel vitamin D receptor (VDR) agonist whose hydrophobic core structure is p-carborane (1,12-dicarba-closo-dodecaborane, an icosahedral carbon-containing boron cluster having remarkable thermal and chemical stability and a characteristically hydrophobic B-H surface). This carborane-based VDR ligand exhibited moderate vitamin D activity, comparable to that of the natural hormone, despite its simple and flexible structure. X-ray structure analysis provided direct evidence that the carborane cage binds to the hydrophobic surface of the ligand-binding pocket of the receptor, promoting transition to the active conformation. These results indicate that the spherical B-H surface of carborane can function efficiently as a hydrophobic anchor in binding to the receptor surface, thereby allowing induced fitting of the three essential hydroxyl groups on the alkyl chains to the appropriate positions for interaction with the VDR binding site, despite the entropic disadvantage of the flexible structure. We suggest that carborane structure is a promising option in the design of novel drug candidates.

ASKA technology-based pull-down method reveals a suppressive effect of ASK1 on the inflammatory NOD-RIPK2 pathway in brown adipocytes.

Recent studies have shown that adipose tissue is an immunological organ. While inflammation in energy-storing white adipose tissues has been the focus of intense research, the regulatory mechanisms of inflammation in heat-producing brown adipose tissues remain largely unknown. We previously identified apoptosis signal-regulating kinase 1 (ASK1) as a critical regulator of brown adipocyte maturation; the PKA-ASK1-p38 axis facilitates uncoupling protein 1 (UCP1) induction cell-autonomously. Here, we show that ASK1 suppresses an innate immune pathway and contributes to maintenance of brown adipocytes. We report a novel chemical pull-down method for endogenous kinases using analog sensitive kinase allele (ASKA) technology and identify an ASK1 interactor in brown adipocytes, receptor-interacting serine/threonine-protein kinase 2 (RIPK2). ASK1 disrupts the RIPK2 signaling complex and inhibits the NOD-RIPK2 pathway to downregulate the production of inflammatory cytokines. As a potential biological significance, an in vitro model for intercellular regulation suggests that ASK1 facilitates the expression of UCP1 through the suppression of inflammatory cytokine production. In parallel to our previous report on the PKA-ASK1-p38 axis, our work raises the possibility of an auxiliary role of ASK1 in brown adipocyte maintenance through neutralizing the thermogenesis-suppressive effect of the NOD-RIPK2 pathway.

Modulation of intramolecular heterodimer-induced fluorescence quenching of tricarbocyanine dye for the development of fluorescent sensor.

Various approaches have been used to modulate the fluorescence changes of sensors in the presence of target analytes, including intramolecular interaction between fluorophores or between fluorophore and other molecular species, like resonance energy transfer (RET). Here, we focus on fluorescence quenching by intramolecular heterodimer complex formation, which can be modulated over a shorter distance range than RET. We synthesized several conjugates of tricarbocyanine, which is a near-infrared fluorophore, with several quencher candidates via flexible short linker structure, and examined their fluorescence properties. Of our synthesized compounds, the dabcyl group proved to be the best quencher via heterodimer complex formation. The fluorescence of tricarbocyanine-dabcyl conjugates in aqueous media was almost completely quenched, and there was a dramatic fluorescence enhancement when heterodimer formation was blocked. These results suggested a design approach to develop fluorescence sensors for probing proximity relationships and structural transitions.

Development of novel bisubstrate-type inhibitors of histone methyltransferase SET7/9.

Histone modification, for example, by histone deacetylase (HDAC) and histone lysine methyltransferase (HMT), plays an important role in regulating gene expression. To obtain novel inhibitors as tools for investigating the physiological function of members of the HMT family, we designed and synthesized novel inhibitors, which are amine analogues of adenosylmethionine (AdoMet; the cofactor utilized in the methylation reaction) bearing various alkylamino groups coupled via an ethylene linker. The inhibitory activities of these compounds towards SET7/9, an HMT, were evaluated. It was found that introduction of an alkylamino group increased the inhibitory activity.

Latent enamine functionality of 5-methyl-2,3-dihydropyrazines.

Tautomerization of methyl-substituted dihydropyrazine (DHP) derivatives to their latent enamine form was investigated theoretically and empirically. Among two types of hydrogen transfer model simulated by means of density functional theory calculation, a simple intramolecular hydrogen shift mechanism for 5,6-dimethyl-2,3-dihydropyrazine (1) and 5-methyl-6-phenyl-2,3-dihydropyrazine (3) required high activation energies for tautomerism, while a water-assisted intermolecular hydrogen transfer mechanism gave smaller activation energies (about 160 kJ/mol). Examination of the deuterium exchange reaction of 3 in 50% (v/v) D(2)O/dimethyl sulfoxide-d(6) solution revealed temperature-dependent and stepwise deuterium exchange of the 5-methyl group. Reaction of compound 3 with phenyl isocyanate in acetonitrile afforded a mono adduct (7) at the 5-methyl group, and a cyclic adduct (8). These results represent evidence of tautomerism of 5-methyl-2,3-dihydropyrazines (imine forms) to the latent enamine tautomers, and suggest that DHPs may behave as enamines to a significant degree under physiological conditions.