Kazuo Nagasawa

Entropy‐Controlled Catalytic Asymmetric 1,4‐Type Friedel–Crafts Reaction of Phenols Using Conformationally Flexible Guanidine/Bisthiourea Organocatalyst

One of the most important aspects of protein function is the motion that occurs in response to substrate binding. In the dynamics of enzyme catalysis, multiple weak hydrogenbonding interactions in the polypeptide that are controlled by interrelated enthalpy and entropy changes play a significant role in governing the conformational changes that take place. In contrast, the development of asymmetric organocatalysts has rarely focused on hydrogen-bond donors that have conformationally flexible scaffolds as a likely consequence of difficulties in controlling the conformation of acyclic skeletons. However, recently our research group has successfully demonstrated the utility of conformationally flexible guanidine/bisthiourea organocatalysts 1 for organocatalytic carbon–carbon bond-forming reactions. Herein, we describe studies that have led to the development of new acyclic C3-linked guanidine/bisthiourea organocatalysts 2. Analysis of these processes shows that the catalytic effect resides in a trade off between enthalpies and entropies of activation and reveals the existence of dramatic concentration effects. This investigation has uncovered a unique catalytic stereodiscrimination process controlled only by differences in the activation entropies. The primary aim of this study was to extend our newly developed organocatalytic system to asymmetric 1,4-additions reactions of nitroolefins. A plausible interaction mode for the catalytic reactions of nitroolefins with nucleophilic anions is shown in Scheme 1. In the reactive complex involving an acyclic guanidine/bisthiourea organocatalyst, the thiourea moiety can interact with the nitro group in the acceptor and ionic interactions with the guanidinium cation can orient a nucleophilic anion. We envisaged that a long chiral spacer between the two centers in the catalyst would be required for the promotion of the 1,4-addition reactions that take advantage of these synergistic proximity effects. In the current study, we initially selected catalytic asymmetric Friedel–Crafts (FC) reactions 16] of phenol derivatives. Although a variety of electron-rich aromatic compounds such as indoles, pyrroles, and furans have been successfully utilized as nucleophiles in 1,4-addition processes, 16] asymmetric reactions of phenol derivatives have been rarely studied. The difficulty in employing phenol derivatives in these processes could be a result of two intrinsic factors that are related to the fact that phenoxide anions generated in situ 1) often promote ligand exchange with metal catalysts, and 2) can participate in reactions that take place with low levels of chemoand regioselectivity. In 2007, Chen and co-workers developed the first 1,4-type of FC reaction of naphthols with nitroolefins that utilize cinchona-based thiourea catalysts. These processes give ortho-selective FC products with 85– 95% ee. However, the undesired dimeric furans that are formed in these reactions cannot be easily separated from the target chiral phenols. Following this early study, most catalytic reactions of phenols were designed to prepare pyrans c] and chromanes through C-alkylation/O-cyclization cascade processes. Thus, to broaden the utility of this process in the preparation of chiral phenols, alternative approaches have been explored to repress the inherent cascade pathway. To evaluate the catalytic activities of newly designed C3tethered guanidine/bisthiourea catalysts, initial studies were conducted using sesamol (3a) and nitroalkene 4 a (1.0 equiv) as substrates. As the results displayed in Table 1 show, 2 effectively promotes nucleophilic addition at the C6 position of 3a to selectively afford the corresponding Scheme 1. The structures of 1 and 2, and working model for 1,4additions with nitroolefins.

Solution structure of an intramolecular (3 + 1) human telomeric g-quadruplex bound to a telomestatin derivative.

Guanine-rich human telomeric DNA can adopt secondary structures known as G-quadruplexes, which can be targeted by small molecules to achieve anticancer effects. So far, the structural information on complexes between human telomeric DNA and ligands is limited to the parallel G-quadruplex conformation, despite the high structural polymorphism of human telomeric G-quadruplexes. No structure has been yet resolved for the complex with telomestatin, one of the most promising G-quadruplex-targeting anticancer drug candidates. Here we present the first high-resolution structure of the complex between an intramolecular (3 + 1) human telomeric G-quadruplex and a telomestatin derivative, the macrocyclic hexaoxazole L2H2-6M(2)OTD. This compound is observed to interact with the G-quadruplex through π-stacking and electrostatic interactions. This structural information provides a platform for the design of topology-specific G-quadruplex-targeting compounds and is valuable for the development of new potent anticancer drugs.

Novel Non-Steroidal/Non-Anilide Type Androgen Antagonists with an Isoxazolone Moiety

3-Substituted (Z)-4-(4-N,N-dialkylaminophenylmethylene)-5(4H)-isoxazolones and related compounds were designed and prepared as candidates for structurally novel androgen antagonists. Several compounds showed potent anti-androgenic activity as assessed by nuclear androgen receptor binding assay and growth inhibition assay using androgen-dependent Shionogi carcinoma cells SC-3. They were approximately 10--220 times more potent than flutamide in these assay systems. They also showed anti-androgenic activity toward prostate tumor cell line LNCaP, which has an aberrant nuclear androgen receptor.

A nonclassical vitamin D receptor pathway suppresses renal fibrosis

The TGF-β superfamily comprises pleiotropic cytokines that regulate SMAD and non-SMAD signaling. TGF-β-SMAD signal transduction is known to be involved in tissue fibrosis, including renal fibrosis. Here, we found that 1,25-dihydroxyvitamin D3-bound [1,25(OH)2D3-bound] vitamin D receptor (VDR) specifically inhibits TGF-β-SMAD signal transduction through direct interaction with SMAD3. In mouse models of tissue fibrosis, 1,25(OH)2D3 treatment prevented renal fibrosis through the suppression of TGF-β-SMAD signal transduction. Based on the structure of the VDR-ligand complex, we generated 2 synthetic ligands. These ligands selectively inhibited TGF-β-SMAD signal transduction without activating VDR-mediated transcription and significantly attenuated renal fibrosis in mice. These results indicate that 1,25(OH)2D3-dependent suppression of TGF-β-SMAD signal transduction is independent of VDR-mediated transcriptional activity. In addition, these ligands did not cause hypercalcemia resulting from stimulation of the transcriptional activity of the VDR. Thus, our study provides a new strategy for generating chemical compounds that specifically inhibit TGF-β-SMAD signal transduction. Since TGF-β-SMAD signal transduction is reportedly involved in several disorders, our results will aid in the development of new drugs that do not cause detectable adverse effects, such as hypercalcemia.

A novel glucosylation reaction on anthocyanins catalyzed by acyl-glucose-dependent glucosyltransferase in the petals of carnation and delphinium.

This work describes a glucosylation reaction at the 5/7 positions of anthocyanins in the petals of carnations and delphiniums. Unusually, this reaction is catalyzed by acyl-glucose–dependent glucosyltransferases that belong to glycoside hydrolase family 1. This modification mechanism may play an important role in generating variation in anthocyanins. Glucosylation of anthocyanin in carnations (Dianthus caryophyllus) and delphiniums (Delphinium grandiflorum) involves novel sugar donors, aromatic acyl-glucoses, in a reaction catalyzed by the enzymes acyl-glucose–dependent anthocyanin 5(7)-O-glucosyltransferase (AA5GT and AA7GT). The AA5GT enzyme was purified from carnation petals, and cDNAs encoding carnation Dc AA5GT and the delphinium homolog Dg AA7GT were isolated. Recombinant Dc AA5GT and Dg AA7GT proteins showed AA5GT and AA7GT activities in vitro. Although expression of Dc AA5GT in developing carnation petals was highest at early stages, AA5GT activity and anthocyanin accumulation continued to increase during later stages. Neither Dc AA5GT expression nor AA5GT activity was observed in the petals of mutant carnations; these petals accumulated anthocyanin lacking the glucosyl moiety at the 5 position. Transient expression of Dc AA5GT in petal cells of mutant carnations is expected to result in the transfer of a glucose moiety to the 5 position of anthocyanin. The amino acid sequences of Dc AA5GT and Dg AA7GT showed high similarity to glycoside hydrolase family 1 proteins, which typically act as β-glycosidases. A phylogenetic analysis of the amino acid sequences suggested that other plant species are likely to have similar acyl-glucose–dependent glucosyltransferases.

Thalidomide and its analogues as cyclooxygenase inhibitors

Thalidomide showed cyclooxygenase (COX)-1/2 inhibitory activity with a potency comparable to that of aspirin. Structural development studies of thalidomide resulted in potent COX-1/2 inhibitors, and COX-1-selective and COX-2-selective inhibitors.

Anti-androgens with full antagonistic activity toward human prostate tumor LNCaP cells with mutated androgen receptor.

Anti-androgens were designed based on the principle of inhibiting the folding of helix 12 of the nuclear androgen receptor. The prepared anti-androgens exhibited full antagonistic activity toward human prostate tumor LNCaP cells with T877A point-mutated nuclear androgen receptor, as far as examined, towards which other known anti-androgens, including hydroxyflutamide, are inactive or act as androgen agonists.

Rif1 binds to G quadruplexes and suppresses replication over long distances

Rif1 regulates replication timing and repair of double-strand DNA breaks. Using a chromatin immunoprecipitation–sequencing method, we identified 35 high-affinity Rif1-binding sites in fission yeast chromosomes. Binding sites tended to be located near dormant origins and to contain at least two copies of a conserved motif, CNWWGTGGGGG. Base substitution within these motifs resulted in complete loss of Rif1 binding and in activation of late-firing or dormant origins located up to 50 kb away. We show that Rif1-binding sites adopt G quadruplex–like structures in vitro, in a manner dependent on the conserved sequence and on other G tracts, and that purified Rif1 preferentially binds to this structure. These results suggest that Rif1 recognizes and binds G quadruplex–like structures at selected intergenic regions, thus generating local chromatin structures that may exert long-range suppressive effects on origin firing.

Disabling c-Myc in Childhood Medulloblastoma and Atypical Teratoid/Rhabdoid Tumor Cells by the Potent G-Quadruplex Interactive Agent S2T1-6OTD

We investigated here the effects of S2T1-6OTD, a novel telomestatin derivative that is synthesized to target G-quadruplex–forming DNA sequences, on a representative panel of human medulloblastoma (MB) and atypical teratoid/rhabdoid (AT/RT) childhood brain cancer cell lines. S2T1-6OTD proved to be a potent c-Myc inhibitor through its high-affinity physical interaction with the G-quadruplex structure in the c-Myc promoter. Treatment with S2T1-6OTD reduced the mRNA and protein expressions of c-Myc and hTERT, which is transcriptionally regulated by c-Myc, and decreased the activities of both genes. In remarkable contrast to control cells, short-term (72-hour) treatment with S2T1-6OTD resulted in a dose- and time-dependent antiproliferative effect in all MB and AT/RT brain tumor cell lines tested (IC50, 0.25–0.39 μmol/L). Under conditions where inhibition of both proliferation and c-Myc activity was observed, S2T1-6OTD treatment decreased the protein expression of the cell cycle activator cyclin-dependent kinase 2 and induced cell cycle arrest. Long-term treatment (5 weeks) with nontoxic concentrations of S2T1-6OTD resulted in a time-dependent (mainly c-Myc–dependent) telomere shortening. This was accompanied by cell growth arrest starting on day 28 followed by cell senescence and induction of apoptosis on day 35 in all of the five cell lines investigated. On in vivo animal testing, S2T1-6OTD may well represent a novel therapeutic strategy for childhood brain tumors. Mol Cancer Ther; 9(1); 167–79

Ligand-Induced Conformational Changes with Cation Ejection upon Binding to Human Telomeric DNA G-Quadruplexes

The rational design of ligands targeting human telomeric DNA G-quadruplexes is a complex problem due to the structural polymorphism that these sequences can adopt in physiological conditions. Moreover, the ability of ligands to switch conformational equilibria between different G-quadruplex structures is often overlooked in docking approaches. Here, we demonstrate that three of the most potent G-quadruplex ligands (360A, Phen-DC3, and pyridostatin) induce conformational changes of telomeric DNA G-quadruplexes to an antiparallel structure (as determined by circular dichroism) containing only one specifically coordinated K(+) (as determined by electrospray mass spectrometry) and, hence, presumably only two consecutive G-quartets. Control ligands TrisQ, known to bind preferentially to hybrid than to antiparallel structures, and L2H2-6M(2)OTD, known not to disrupt the hybrid-1 structure, did not show such K(+) removal. Instead, binding of the cyclic oxazole L2H2-6M(2)OTD was accompanied by the uptake of one additional K(+). Also contrasting with telomeric G-quadruplexes, the parallel-stranded Pu24-myc G-quadruplex, to which Phen-DC3 is known to bind by end-stacking, did not undergo cation removal upon ligand binding. Our study therefore evidences that very affine ligands can induce conformational switching of the human telomeric G-quadruplexes to an antiparallel structure and that this conformational change is accompanied by removal of one interquartet cation.