Misc

Basic helix-loop-helix (bHLH) repressor- and activator-type transcription factors play essential roles in proliferation and differentiation of neural stem cells (NSCs). Hes1 and Hes5, bHLH repressor-type factors, lead NSCs to remaining undifferentiation. The transcription of Hes1 and Hes5 are regulated by Notch signaling pathway. Therefore, inhibition of Notch signaling pathway would accelerate NSCs differentiation into neurons and glial cells. To find inhibitors of Notch signaling pathway from natural resources, we constructed a cell-based reporter assay of NICD-mediated transcription.By using this assay system, we isolated twelve xanthones (1-12) including a new compound (12) from Garcinia speciosa (bark). Structure of 12was elucidated by spectroscopic data analyses. Among the isolated compounds, 2strongly inhibited the transcriptional activity of NICD. Western blot analysis of 2showed decrease in Hes1 and Hes5 protein expression. NICD was also decreased, but NotchDE was increased. These results suggested that 2inhibits Notch signaling pathway by the inhibition of g-secretase. Five cardenolide glycosides (13-17) were isolated from Nerium indicum (leaves). 13inhibited the transcriptional activity of NICD with IC50 values of 0.12 mM. Western blot analysis of 13showed decrease in Hes1 and Hes5 protein expression. Uscharin (18) and calotropin (19) were also found as Notch signaling inhibitors. Next, we examined the effect of 13, 18 and 19on NSCs differentiation. These compounds accelerated differentiation of mouse NSCs (MEB5) into neurons and glial cells.

Basic helix-loop-helix (bHLH) repressor- and activator-type transcription factors play essential roles in proliferation and differentiation of neural stem cells (NSCs). Hes1 and Hes5, bHLH repressor-type factors, lead NSCs to remaining undifferentiation. The transcription of Hes1 and Hes5 are regulated by Notch signaling pathway. Therefore, inhibition of Notch signaling pathway would accelerate NSCs differentiation into neurons and glial cells. To find inhibitors of Notch signaling pathway from natural resources, we constructed a cell-based reporter assay of NICD-mediated transcription.By using this assay system, we isolated twelve xanthones (1-12) including a new compound (12) from Garcinia speciosa (bark). Structure of 12was elucidated by spectroscopic data analyses. Among the isolated compounds, 2strongly inhibited the transcriptional activity of NICD. Western blot analysis of 2showed decrease in Hes1 and Hes5 protein expression. NICD was also decreased, but NotchDE was increased. These results suggested that 2inhibits Notch signaling pathway by the inhibition of g-secretase. Five cardenolide glycosides (13-17) were isolated from Nerium indicum (leaves). 13inhibited the transcriptional activity of NICD with IC50 values of 0.12 mM. Western blot analysis of 13showed decrease in Hes1 and Hes5 protein expression. Uscharin (18) and calotropin (19) were also found as Notch signaling inhibitors. Next, we examined the effect of 13, 18 and 19on NSCs differentiation. These compounds accelerated differentiation of mouse NSCs (MEB5) into neurons and glial cells.

Neural stem cells (NSCs) can differentiate to neural cells such as neurons and astrocytes, therefore regenerative medicine based on natural products are urgent to neural diseases. We recently focused on the basic helix-loop-helix (bHLH) transcriptional factors, which control proliferation and differentiation of NSCs. To quickly find the ligands of bHLH factors from natural products extract, we developed the Hes I immobilized sepharose beads-HPLC method. Flavonoid glycosides (1-3) have been isolated rapidly by this method. Compounds 1 and 3 had the inhibitory activity against the Hes 1 dimer formations, and accelerated the neuron differentiation of mouse neural stem cells (MEB5). As a "natural products + diversity-oriented synthesis (DOS)" derived library, the heterocyclic-substituted chromones or flavonoids library was constructed by our efficient synthesis method and solid phase synthesis. By constructed cell-based assay, several compounds were found as moderate Hes 1 promoter inhibitors. Compound 4 actually inhibited Hes 1 mRNA expression in NSCs. Interestingly, 4 led the NSCs to astrocyte selective differentiation. We also searched upregulators of Ngn2 expression, which is bHLH differentiation activator. The cell-based assay for Ngn2 promoter activity was constructed. By Ngn2 promoter activity guided separation, several activators were isolated from Butea superba. Of them, three compounds (5-7) showed neuron selective differentiation of NSCs.

[GRAPHICS]A chiral bis(isoxazoline) ligand (SPRIX) containing a rigid spiro skeleton was designed and synthesized via a double intramolecular nitrile oxide cycloaddition, The optically pure SPRIX greatly accelerated the Cu(acac)(2)-catalyzed asymmetric reaction of diisopropylzinc with cyclohexenone to give the Michael adduct with 49% ee. This is the first example of the use of an isoxazoline ligand for a transition metal catalyzed reaction. The X-ray crystallographic analysis of the hexacoordinated copper complex of SPRIX confirmed the ligand character.

The first chiral isoxazoline ligands bearing spiro skeleton are developed. The spiro bis(isoxazoline) ligands (SPRIXs) are readily synthesized from diethyl malonate via double intramolecular nitrile oxide cycloaddition as a key step. The complex of (M, S, S)-R-SPRIX and Pd(OCOCF3)(2) promoted the catalytic asymmetric Wacker-type cyclization of alkenyl alcohols to give optically active cyclic ethers for the first time. Furthermore, starting from dialkenyl alcohol, the enantioselective tandem cyclization via oxy-palladation was achieved with up to 95% ee. Asymmetric ligands other than SPRIXs promote neither the Wacker-type reaction nor the tandem reaction.

Combination of Pd(OCOCF3)(2) and (M,S,S)-iPr-SPRIX ligand promoted the intramolecular cyclization of 2-alkynoates very effectively to form alpha-methylene-gamma-butyrolactones in good yields with up to 92% ee. (C) 2003 Elsevier Science Ltd. All rights reserved.