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Papers - Arai, Midori

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Natural Compounds with BMI1 Promoter Inhibitory Activity from Mammea siamensis and Andrographis paniculata

Fujii Kazuki, Hara Yasumasa, Arai Midori A., Sadhu Samir K., Ahmed Firoj, Ishibashi Masami

Chemical and Pharmaceutical Bulletin (The Pharmaceutical Society of Japan) 70 ( 12 ) 885 - 891 2022.12

ISSN 00092363

A new coumarin derivative (1) and 30 known compounds were isolated from Mammea siamensis and Andrographis paniculata, guided by B cell-specific Moloney murine leukemia virus insertion region 1 (BMI1) promoter inhibitory activity. Among the isolated compounds, 15 compounds showed BMI1 promoter inhibitory activity, and five compounds were found to be cytotoxic. 14-Deoxy-11,12-dehydroandrographolide (18) was highly cytotoxic to DU145 cells with an IC50 value of 25.4 µM. Western blotting analysis of compound 18 in DU145 cells suggested that compound 18 suppresses BMI1 expression.

病原微生物の浸潤進化に学ぶ休眠遺伝子活性化と創薬

荒井緑

上原記念生命科学財団研究報告集 ((公財)上原記念生命科学財団) 36 1 - 5 2022.12

Dihydromaniwamycin E, a Heat-Shock Metabolite from Thermotolerant Streptomyces sp. JA74, Exhibiting Antiviral Activity against Influenza and SARS-CoV-2 Viruses

S Saito, K Funayama, W Kato, M Okuda, M Kawamoto, T Matsubara, ...

Journal of Natural Products 85 (11), 2583-2591 (American Chemical Society (ACS)) 85 ( 11 ) 2583 - 2591 2022.11

ISSN 01633864

Dihydromaniwamycin E (1), a new maniwamycin derivative featuring an azoxy moiety, has been isolated from the culture extract of thermotolerant Streptomyces sp. JA74 along with the known analogue maniwamycin E (2). Compound 1 is produced only by cultivation of strain JA74 at 45 °C, and this type of compound has been previously designated a "heat shock metabolite (HSM)" by our research group. Compound 2 is detected as a production-enhanced metabolite at high temperature. Structures of 1 and 2 are elucidated by NMR and MS spectroscopic analyses. The absolute structure of 1 is determined after the total synthesis of four stereoisomers. Though the absolute structure of 2 has been proposed to be the same as the structure of maniwamycin D, the NMR and the optical rotation value of 2 are in agreement with those of maniwamycin E. Therefore, this study proposes a structural revision of maniwamycins D and E. Compounds 1 and 2 show inhibitory activity against the influenza (H1N1) virus infection of MDCK cells, demonstrating IC50values of 25.7 and 63.2 μM, respectively. Notably, 1 and 2 display antiviral activity against SARS-CoV-2, the causative agent of COVID-19, when used to infect 293TA and VeroE6T cells, with 1 and 2 showing IC50values (for infection of 293TA cells) of 19.7 and 9.7 μM, respectively. The two compounds do not exhibit cytotoxicity in these cell lines at those IC50concentrations.

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Noaoxazole, a new heat shock metabolite produced by thermotolerant Streptomyces sp. HR41

S Saito, S Suzuki, MA Arai

The Journal of Antibiotics 75 (9), 509-513 (Springer Science and Business Media LLC) 75 ( 9 ) 509 - 513 2022.09

ISSN 00218820

The thermotolerant strain Streptomyces sp. HR41 was found to produce compound 1 only in a 45 °C culture, and not at the standard temperature. We previously designated this type of compound as a “heat shock metabolite” (HSM). NMR and MS analytical techniques were used to determine that the chemical structure of 1 comprised a methylated-oxazole ring and a linear chain moiety modified with a terminal amide group. Thus, 1 was shown to be a new curromycin analog, which we have designated noaoxazole (1). Compound 1 weakly activated Notch signal reporter activity without exhibiting cytotoxicity against assay cells at the same concentration.

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Corrrection to “Two Bioactive Compounds, Uniformides A and B, Isolated from a Culture of Nocardia uniformis IFM0856T in the Presence of Animal Cells”

Yasumasa Hara, Keiichiro Watanabe, Akiko Takaya, Itsuki Ebihara, Teruhisa Manome, Midori A. Arai, Takashi Yaguchi, Masami Ishibashi

Organic Letters (American Chemical Society (ACS)) 24 ( 31 ) 5867 - 5867 2022.08

ISSN 1523-7060

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Identification of 1β, 2α-epoxytagitinin C as a Notch inhibitor, oxidative stress mechanism and its anti-leukemia activity

Y Makita, S Saito, A Tsuchiya, M Ishibashi, MA Arai

Journal of natural medicines 76, 234-243 (Journal of Natural Medicines) 76 ( 1 ) 234 - 243 2022.01

ISSN 13403443

Notch signaling plays crucial roles in cell differentiation and proliferation, but aberrant activation of this signaling results in tumorigenesis and cancer progression. Notch signaling is thus a promising drug target for oncotherapy, and the development of Notch signaling inhibitors is eagerly awaited. Notch inhibitory activity-guided fractionation of a Spilanthes acmella extract led to the identification of five sesquiterpene lactones: tagitinin A (1), 1β,2α-epoxytagitinin C (2), tagitinin C (3), orizabin (4), and 2α-hydroxytirotundin (5). 1β,2α-Epoxytagitinin C (2) exhibited Notch signaling inhibition, with an IC50 of 25.6 μM, and was further evaluated for its activity against HPB-ALL, a Notch-activated leukemia cell line. Compound 2 showed potent cytotoxicity against HPB-ALL (IC50 1.7 μM) and arrested the cell cycle at the G2/M phase, but did not induce apoptotic cell death. Notch inhibitory mechanism analysis suggested that compound 2 transcriptionally suppresses Notch1 mRNA. In addition, we found that oxidative stress induction is critical for Notch signaling inhibition and the cytotoxicity of compound 2. This is the first mechanism of small molecule Notch inhibition. Our results demonstrate that 1β,2α-epoxytagitinin C (2) is a potential anti-leukemia agent and further investigation of this compound is warranted. Graphical abstract: [Figure not available: see fulltext.].

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Two Bioactive Compounds, Uniformides A and B, Isolated from a Culture of Nocardia uniformis IFM0856 (T) in the Presence of Animal Cells (vol 24, pg 4998, 2022)

Y Hara, K Watanabe, A Takaya, I Ebihara, T Manome, MA Arai, T Yaguchi, ...

ORGANIC LETTERS 24 (31), 5867-5867 2022

Corrrection to “Two Bioactive Compounds, Uniformides A and B, Isolated from a Culture of Nocardia uniformis IFM0856T in the Presence of Animal Cells”

Y Hara, K Watanabe, A Takaya, I Ebihara, T Manome, MA Arai, T Yaguchi, ...

Organic Letters 24 (31), 5867-5867 2022

Erratum: Corrrection Two Bioactive Compounds, Uniformides A and B, Isolated from a Culture of Nocardia uniformis IFM0856Tin the Presence of Animal Cells (Organic Letters (2022 …

Y Hara, K Watanabe, A Takaya, I Ebihara, T Manome, MA Arai, T Yaguchi, ...

Organic Letters 2022

Evaluation of Naturally Occurring HIF-1 Inhibitors for Pulmonary Arterial Hypertension.

Midori A Arai, Kota Sakuraba, Yoshinori Makita, Yasumasa Hara, Masami Ishibashi

Chembiochem : a European journal of chemical biology (ChemBioChem) 22 ( 18 ) 2799 - 2804 2021.09

ISSN 14394227

Pulmonary arterial hypertension (PAH) is a rare and severe progressive disorder characterized by high pulmonary artery pressure. Chronic hypoxia causes a metabolic disorder and the Warburg effect in pulmonary arterial smooth muscle cells (PASMCs). Pyruvate dehydrogenase kinase 1 (PDK1) is a key enzyme in Warburg effect increased by hypoxia-inducible factor (HIF-1). We constructed a cell-based luciferase assay system for HIF-1 inhibitors. Using this system, six HIF-1 inhibitors were identified. Among these inhibitors, the effect of tagitinin C (1) on PASMC was investigated. Tagitinin C (1) clearly decreased the amount of HIF-1β and the HIF-1 target PDK1. This result indicates that HIF-1 inhibitors effectively decrease PDK1 activity, which is a cause of the metabolic disorder and Warburg effect observed in PASMCs. Identifying naturally occurring HIF-1 inhibitors could provide novel insights into the development of PAH medications.

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Target Protein-Oriented Isolations for Chemical Biology based on Natural Products

Arai Midori A., Ishibashi Masami

Journal of Synthetic Organic Chemistry, Japan (The Society of Synthetic Organic Chemistry, Japan) 79 ( 7 ) 684 - 693 2021.07

ISSN 00379980

Natural products have contributed to development of medicine for a long time. The structures and bioactivities of natural products often exceed the human knowledge and imagination. We have developed target protein-oriented natural products isolation methods to quick find bioactive compounds from natural resources (compounds mixture). This review summarizes our recent results including protein beads methods and protein plate method for neural stem cells differentiation activators and new cancer drug candidates. Isolated compounds accelerated neural stem cells differentiation and/or showed cytotoxicity against cancer cells. Syntheses of isolated compounds were also described. As protein binding ability tightly related bioactivity, protein based natural products isolation would be a powerful way to find new candidate of medicines.

Target Protein-Oriented Isolations for Bioactive Natural Products

MA Arai

Chemical and Pharmaceutical Bulletin 69 (6), 503-515 (The Pharmaceutical Society of Japan) 69 ( 6 ) 503 - 515 2021.06

Accepted, ISSN 00092363

Natural products are very attractive for development of medicine. Their structure and bioactivities are often beyond human knowledge and imagination. We have developed isolation methods for target protein-oriented natural products so as quickly to discover bioactive compounds from natural resources. This review summarizes our recent results including protein beads methods for neural stem cells differentiation activators and new cancer drug candidates. Syntheses of isolated compounds are described. We also developed protein plate method for identification of protein–protein interaction inhibitors. Because protein binding ability is tightly related to bioactivity, protein-based natural products isolation is a powerful means to find new candidate medicines.

Practical Stereoselective Synthesis of C3‐Spirooxindole‐ and C2‐Spiropseudoindoxyl‐Pyrrolidines via Organocatalyzed Pictet‐Spengler Reaction/Oxidative Rearrangement Sequence

Masaru Kondo, Naoki Matsuyama, Tin Z. Aye, Irshad Mattan, Tomoyuki Sato, Yoshinori Makita, Masami Ishibashi, Midori A. Arai, Shinobu Takizawa, Hiroaki Sasai

Advanced Synthesis & Catalysis (Wiley) 363 ( 10 ) 2648 - 2663 2021.05

ISSN 16154150

A stereoselective synthetic route to chiral C3-spirooxindole- and C2-spiropseudoindoxyl-pyrrolidines was accomplished by an enantioselective organocatalyzed Pictet-Spengler reaction of tryptamines and isotryptamines followed by a diastereoselective oxidative rearrangement using eco-friendly oxidants (i. e., NaOCl ⋅ 5H2O and Oxone®). This sequential reaction enables rapid access to chiral C3-spirooxindole- and C2-spiropseudoindoxyl-pyrrolidines in a one-pot process. A Wnt signaling inhibitory assay of the prepared enantioenriched spiro compounds demonstrated that they exhibited moderate activities. (Figure presented.).

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Isolation of nocobactin NAs as Notch signal inhibitors from Nocardia farcinica, a possibility of invasive evolution

MA Arai, I Ebihara, Y Makita, Y Hara, T Yaguchi, M Ishibashi

The Journal of Antibiotics 74 (4), 255-259 (Journal of Antibiotics) 74 ( 4 ) 255 - 259 2021.04

ISSN 00218820

Notch signaling inhibitors with the potential of immune suppressor production by pathogenic bacteria for easy host infection were searched from extracts of Nocardia sp. Nocobactin NA-a (compound 1) and nocobactin NA-b (compound 2), which have been suggested as pathogenesis factors, were isolated from N. farcinica IFM 11523 isolated from the sputum of a Japanese patient with chronic bronchitis. Compounds 1 and 2 showed Notch inhibitory activities with IC50 values of 12.4 and 17.6 μM, respectively. Compound 1 and 2 decreased of Notch1 protein, Notch intracellular domain, and hairy and enhancer of split 1, which is a Notch signaling target protein. In addition, compounds 1 and 2 showed cytotoxicity against mouse macrophage-like cell line RAW264.7 with IC50 values of 18.9 and 21.1 μM, respectively. These results suggested that the Notch inhibitors production by pathogenic bacteria may increase pathogen infectivity.

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Acacienone, a terpenoid-like natural product having an unprecedented C20 framework isolated from Acacia mangium leaves

Y Hara, Y Totsugi, H Ichikawa, S Harada, K Fujii, F Ahmed, SK Sadhu, ...

Journal of natural medicines 75, 99-104 (Journal of Natural Medicines) 75 ( 1 ) 99 - 104 2021.01

ISSN 13403443

A novel C20 natural product, acacienone (1), was isolated from the leaves of Acacia mangium collected in Bangladesh. The structure of compound 1 was elucidated by spectral studies and X-ray crystallographic analysis. Acacienone (1) possesses a terpenoid-related tetracyclic framework containing 20 carbons with biogenetically unusual structural features: (i) vicinal C1-branches at the C-3 and C-4 positions in the A ring, and (ii) a cyclopentenone D ring in an androsterone-like assembly, lacking a methyl group at the C-13 position.

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Isolation of Peptidolipin NA Derivatives From the Culture of Nocardia arthritidis IFM10035T in the Presence of Mouse Macrophage Cells

Y Hara, D Tanimura, T Manome, MA Arai, T Yaguchi, M Ishibashi

Heterocycles 104 (1), 185-190 (Heterocycles) 104 ( 1 ) 185 - 190 2021

ISSN 03855414

We isolated two peptidolipin NA derivatives, L-Val (6) peptidolipin NA and peptidolipin NA, named as compounds 1 and 2, respectively. These compounds were produced in the co-culture extracts of Nocardia arthritidis IFM10035T and the mouse macrophage cell line J774.1, but not in the single culture of N. arthritidis. Among the two compounds along with nocarjamide (3) isolated from the co-culture of N. tenerifensis and J774.1, compounds 1 and 3 showed higher cytotoxicity against J774.1, with IC50 values of 116 μM and 25 μM, respectively. In addition, compound 1 showed a TRAIL-resistance-overcoming activity in the human gastric adenocarcinoma AGS cells.

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Evaluation of Naturally Occurring HIF‐1 Inhibitors for Pulmonary Arterial Hypertension

MA Arai, K Sakuraba, Y Makita, Y Hara, M Ishibashi

ChemBioChem 22 (18), 2799-2804 2021

Practical Stereoselective Synthesis of C3‐Spirooxindole‐ and C2‐Spiropseudoindoxyl‐Pyrrolidines via Organocatalyzed Pictet‐Spengler Reaction/Oxidative …

M Kondo, N Matsuyama, TZ Aye, I Mattan, T Sato, Y Makita, M Ishibashi, ...

Advanced Synthesis & Catalysis 363 (10), 2648-2663 2021

Target protein-oriented isolation of Hes1 dimer inhibitors using protein based methods.

Midori A Arai, Kaori Morita, Haruka Kawano, Yuna Makita, Manami Hashimoto, Akiko Suganami, Yutaka Tamura, Samir K Sadhu, Firoj Ahmed, Masami Ishibashi

Scientific reports (Scientific Reports) 10 ( 1 ) 1381 - 1381 2020.12

Accepted

Natural products isolation using protein based methods is an attractive for obtaining bioactive compounds. To discover neural stem cell (NSC) differentiation activators, we isolated eight inhibitors of Hes1 dimer formation from Psidium guajava using the Hes1-Hes1 interaction fluorescent plate assay and one inhibitor from Terminalia chebula using the Hes1-immobilized beads method. Of the isolated compounds, gallic acid (8) and 4-O-(4"-O-galloyl-α-L-rhamnopyranosyl)ellagic acid (11) showed potent Hes1 dimer formation inhibitory activity, with IC50 values of 10.3 and 2.53 μM, respectively. Compound 11 accelerated the differentiation activity of C17.2 NSC cells dose dependently, increasing the number of neurons with a 125% increase (5 μM) compared to the control.

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Total synthesis of lindbladione, a Hes1 dimerization inhibitor and neural stem cell activator isolated from Lindbladia tubulina.

Midori A Arai, Yuna Makita, Yumi Yamaguchi, Haruka Kawano, Akiko Suganami, Yutaka Tamura, Masami Ishibashi

Scientific reports (Scientific Reports) 10 ( 1 ) 21433 - 21433 2020.12

Lindbladione (1) is a neural stem cell differentiation activator isolated from Lindbladia tubulina by our group. Hes1 dimerization inhibitory activity of lindbladione (1) was discovered using our original fluorescent Hes1 dimer microplate assay. We also found that lindbladione (1) accelerates the differentiation of neural stem cells. We conducted the first total synthesis of lindbladione (1) via Heck reaction of 1-hexene-3-one 7 with iodinated naphthoquinone 12, which was provided by Friedel-Crafts acylation followed by Claisen condensation, in the presence of Pd (II) acetate. Careful deprotection of the benzyl groups of 13 successively provided lindbladione (1). Synthesized lindbladione (1) exhibited potent Hes1 dimer inhibition (IC50 of 2.7 μM) in our previously developed fluorescent Hes1 dimer microplate assay. Synthesized lindbladione (1) also accelerated the differentiation of C17.2 mouse neural stem cells into neurons dose dependently, increasing the number of neurons by 59% (2.5 μM) and 112% (10 μM) compared to the control. These activities are comparable to those of naturally occurring lindbladione (1) isolated from L. tublina.

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Isolation and evaluation of cardenolides from Lansium domesticum as Notch inhibitors.

Anna Tsuchiya, Yoshinori Makita, Takashi Koyano, Thaworn Kowithayakorn, Masami Ishibashi, Midori A Arai

Journal of natural medicines (Journal of Natural Medicines) 74 ( 4 ) 758 - 766 2020.09

Accepted, ISSN 13403443

Since Notch signaling plays important roles in cell proliferation and differentiation, aberrant activation of this signaling contributes to cancer progression. In neural stem cells, Notch signaling inhibits differentiation by activating HES1 expression. Therefore, Notch signaling inhibitors may be candidates for new anticancer drugs or have applications in neural regenerative medicine. In this study, six naturally occurring Notch inhibitors were isolated from the methanol (MeOH) extract of Lansium domesticum using our novel cell-based assay. Hongherin (2), a cardiac glycoside, demonstrated potent Notch inhibitory activity with an IC50 of 0.62 μM and was found to be cytotoxic in HPB-ALL human T cell acute lymphoblastic leukemia cells. Hongherin (2) also induced the differentiation of C17.2 neural stem cells to neurons, causing a 65% increase in differentiation compared to the control. Mechanistically, hongherin (2) reduced the amount of Notch1 (full length) and mastermind-like protein (MAML). This indicates that hongherin (2) inhibits Notch signaling through a dual mechanism involving the reduction of both Notch1 and MAML protein levels.

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Cadinane sesquiterpenoids isolated from Santalum album using a screening program for Wnt signal inhibitory activity

Tomoyuki Sato, Midori A. Arai, Yixizhuoma, Yasumasa Hara, Takashi Koyano, Thaworn Kowithayakorn, Masami Ishibashi

Journal of Natural Medicines (Journal of Natural Medicines) 74 ( 2 ) 476 - 481 2020.03

Accepted, ISSN 13403443

© 2019, The Japanese Society of Pharmacognosy. Upon screening compounds having Wnt signal inhibitory activity through evaluating TCF/β-catenin transcriptional (TOP) activity, eight cadinane sesquiterpenoids, including three new compounds (1–3), were isolated from wood extracts of Santalum album (Santalaceae). Structures of compounds 1–3 were elucidated by spectral data to have a cadinane skeleton with an aromatic ring. Of the eight compounds isolated, compound 4, identified as mansonone I, was found to be active against TOP, having an IC50 of 1.2 μM.

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Isolation of inohanalactone, a γ-butyrolactone, from Nocardia Inohanensis IFM0092T

Natsumi Kobayashi, Yasumasa Hara, Midori A. Arai, Shoko Hara, Tohru Gonoi, Takashi Yaguchi, Masami Ishibashi

Heterocycles (Heterocycles) 101 ( 1 ) 312 - 317 2020

Accepted, ISSN 03855414

© 2020 The Japan Institute of Heterocyclic Chemistry. Because research on the genus Nocardia has not progressed as compared to the extensively studied genus Streptomyces, Nocardia is considered as a useful undeveloped resource for exploring natural products. In this study, a new γ-butyrolactone derivative, named inohanalactone (1), was isolated from Nocardia inohanensis IFM0092T. Inohanalactone possesses an aliphatic side chain containing 8 carbons, a vicinal diol, and a cis double bond. Inohanalactone was produced by N. inohanensis IFM0092T in the modified Czapek-Dox medium, whereas it was not produced in other media such as Nutrient broth, Waksman, and Yeast-Malt-Glucose media.

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Identification of BMI1 promoter inhibitors from Streptomyces sp. IFM-11958

Yusuke Yokoyama, Midori A. Arai, Yasumasa Hara, Masami Ishibashi

Bioorganic and Medicinal Chemistry (Bioorganic and Medicinal Chemistry) 27 ( 13 ) 2998 - 3003 2019.07

Accepted, ISSN 09680896

© 2019 Elsevier Ltd B-cell-specific Moloney murine leukemia virus region 1 (BMI1) is a central component of polycomb repressive complex 1 (PRC1), which maintains epigenetic repression of genes expression via chromatin condensation. BMI1 overexpression downregulates the expression of tumor suppressor genes, such as p16Ink4a and PTEN. BMI1 expression is upregulated in cancer stem cells (CSCs). Therefore, inhibitors of BMI1 expression have potential as therapeutic agents for cancer. This study aimed to identify BMI1 promoter inhibitors from actinomycetes. Using a recently constructed BMI1 promoter assay, we isolated three known compounds, elaiophylin (1), 2-methylelaiophylin (2), and nocardamin (3), from Streptomyces sp. IFM-11958 that inhibited BMI1 promoter activity with IC50 values of 30 nM, 447 nM, 22 µM, respectively. Elaiophylin (1) was the most potent. Western blot and PCR analyses revealed that elaiophylin (1) inhibited BMI1 expression at the mRNA level in human prostate cancer cells (DU145). Elaiophylin (1) also inhibited the sphere-forming activity of human hepatocellular carcinoma cells (Huh7), indicating that elaiophylin (1) suppresses the self-renewal capacity of CSCs. Elaiophylin (1) is the first BMI1 promoter inhibitor isolated from actinomycete metabolites.

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Nonactic Acid Derivatives Isolated from Streptomyces werraensis IFM12104 in a Screening Program for BMI1 Promoter Inhibitory Activity

Yusuke Yokoyama, Midori A. Arai, Yasumasa Hara, Masami Ishibashi

Natural Product Communications (Natural Product Communications) 14 ( 7 ) 2019

Accepted, ISSN 1934578X

© The Author(s) 2019 Upon screening for compounds with B cell-specific Moloney murine leukemia virus region 1 (BMI1) promoter inhibitory activity, 3 nonactic acid derivatives (1-3) were isolated from Streptomyces werraensis IFM12104. Although spectral data of compound 1 were identical with those reported in literature for 4α-(3, 5-dihydroxy-heptyl)-3α-methyl-2-oxetanone (4), compound 1 was revealed to be homononactic acid by X-ray analysis. Compounds 2 and 3 were identified as bishomononactic acid and nonactic acid using spectral data. The 3 compounds 1 to 3 inhibited BMI1 promoter activities with IC50 values of 240, 160, and 180 μM, respectively.

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The Notch inhibitor cowanin accelerates nicastrin degradation

Midori A. Arai, Ryuta Akamine, Anna Tsuchiya, Tatsuro Yoneyama, Takashi Koyano, Thaworn Kowithayakorn, Masami Ishibashi

Scientific Reports (Nature Publishing Group) 8 ( 1 ) 2018.12

Accepted, ISSN 2045-2322

Aberrant activation of Notch signaling contributes to the pathogenesis of several different types of cancer, and Notch pathway inhibitors may have significant therapeutic potential. Using a unique cell-based assay system, we isolated twelve compounds, including one new natural product from Garcinia speciosa, that inhibit the Notch signaling pathway. HES1 and HES5 are target genes of the Notch cascade, and compound 2, referred to as cowanin, decreased the protein levels of HES1 and HES5 in assay cells. Furthermore, cowanin (2) showed potent cytotoxicity against human leukemic HPB-ALL cells. The Notch signaling inhibitory activity of cowanin (2) is linked to the increased degradation of nicastrin, which is one of the components of the γ-secretase complex. To the best of our knowledge, this is the first example of a compound with Notch pathway inhibitory activity mediated by nicastrin degradation.

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Coculture of a Pathogenic Actinomycete and Animal Cells to Produce Nocarjamide, a Cyclic Nonapeptide with Wnt Signal-Activating Effect

Yasumasa Hara, Midori A. Arai, Kazufumi Toume, Hyuma Masu, Tomoyuki Sato, Katsuko Komatsu, Takashi Yaguchi, Masami Ishibashi

Organic Letters (Organic Letters) 20 ( 18 ) 5831 - 5834 2018.09

Accepted, ISSN 15237060

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GLI1 Inhibitors Identified by Target Protein Oriented Natural Products Isolation (TPO-NAPI) with Hedgehog Inhibition.

Midori A Arai, Fumie Ochi, Yoshinori Makita, Tetsuhiro Chiba, Kyohei Higashi, Akiko Suganami, Yutaka Tamura, Toshihiko Toida, Atsushi Iwama, Samir K Sadhu, Firoj Ahmed, Masami Ishibashi

ACS chemical biology (ACS Chemical Biology) 13 ( 9 ) 2551 - 2559 2018.09

Accepted, ISSN 15548929

This report describes the development of a target-protein-oriented natural-products-isolation (TPO-NAPI) method for Hedgehog inhibitors and the direct GLI1 inhibitor, 5'- O-methyl-3-hydroxyflemingin A (3), which inhibited hedgehog (Hh) signal transduction and diminished characteristics of cancer stem cells. Eight natural products (including three newly described products) that directly bind to GLI1 were rapidly obtained via the TPO-NAPI method developed using GLI1 protein-immobilized beads. 5'- O-Methyl-3-hydroxyflemingin A (3) inhibited Hh signaling (IC50 7.3 μM), leading to decreasing production of the Hh target proteins BCL2, PTCH1, and BMI1. 5'- O-Methyl-3-hydroxyflemingin A (3) was cytotoxic to Hh-related cancer cells. CD experiments revealed that 5'- O-methyl-3-hydroxyflemingin A (3) directly bound GLI1 ( Kd = 7.7 μM). Moreover, 5'- O-methyl-3-hydroxyflemingin A (3) diminished cancer stem cell characters of Huh7 such as sphere formation and production of the cancer stem cell marker EpCAM. These results suggest that Hh inhibitors can efficiently suppress the activity of cancer stem cells.

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Total syntheses of schizandriside, saracoside and (±)-isolariciresinol with antioxidant activities

Mana Sampei, Midori A. Arai, Masami Ishibashi

Journal of Natural Medicines (Springer Tokyo) 72 ( 3 ) 651 - 654 2018.06

Accepted, ISSN 13403443

Lignans are widely distributed in plants and exhibit significant pharmacological effects, including anti-tumor and antioxidative activities. Here, we describe the total synthesis of schizandriside (1), a compound we previously isolated from Saraca asoca by monitoring antioxidative activity using the 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay. Starting from a tandem Michael-aldol reaction, the lignan skeleton was synthesized in 6 steps, including a cyclization step. To determine the stereochemistry of 1, we synthesized the natural product (±)-isolariciresinol (18) from alcohol 17. Comparison of the spectral data showed good agreement. Glycosylation was investigated using four different glycosyl donors. Only the Koenigs–Knorr condition using silver trifluoromethanesulfonate with 1,1,3,3-tetramethylurea provided the glycosylated product. Deprotection and purification using reverse-phase high-performance liquid chromatography gave schizandriside (1) and its diastereomer saracoside (2). Synthesized 1, 2 and 18 showed antioxidant activity with IC50 = 34.4, 28.8, 53.0 μM, respectively.

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The Notch Inhibitors Isolated from Nerium indicum

Midori A. Arai, Ryuta Akamine, Narumi Hayashi, Takashi Koyano, Thaworn Kowithayakorn, Masami Ishibashi

Journal of Natural Products (American Chemical Society) 81 ( 5 ) 1235 - 1240 2018.05

Accepted, ISSN 01633864

Notch signaling plays a crucial role in differentiation and cell maintenance, but once aberrantly activated, it contributes to cancer progression. Notch inhibitors were isolated from plant extracts and tested using an originally constructed cell-based assay system. We isolated eight compounds from Nerium indicum that showed inhibition of the Notch signaling pathway. HES1 and HES5 are target genes of the Notch signaling pathway, and oleandrin (1) decreased the protein levels of HES1 and HES5 in assay cells. Oleandrin (1) showed potent cytotoxicity against HPB-ALL cells and decreased HES1 and the Notch intracellular domain in these cells. The main mechanism of action of 1 appears to be inhibition of Notch signaling by acceleration of Notch intracellular domain degradation.

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New Flavanol Dimers from the Bark of Celtis tetrandra and Their TRAIL Resistance-Overcoming Activity

Seephonkai Prapairat, Ishikawa Ryusho, Arai Midori A, Kowithayakorn Thaworn, Koyano Takashi, Ishibashi Masami

NATURAL PRODUCT COMMUNICATIONS (Natural Product Communications) 13 ( 4 ) 427 - 430 2018.04

Accepted, ISSN 1934578X

In the courses of our search for bioactive natural products on TRAIL resistance-overcoming activity, the active EtOAc extract from the bark of Celtis tetrandra was chemically investigated. The fractionation of the extract resulted in the isolation of flavanols; (+)-afzelechin (1), (-)-epiafzelechin (2), (-)-catechin (3), and two new flavanol dimers; (-)-epiafzelechin-(4α8)-(-)-catechin (4) and (-)-epiafzelechin-(4α 8)-(-)-epicatechin (5). All isolated compounds 1-5 were tested for their TRAIL resistance-overcoming activity against AGS cells at the concentrations from 5-20 µM. It is likely that the flavanol dimers 4 and 5 exhibited weak activity in the range of the concentration of 10-20 µM as compared with the standard.

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Dehydropropylpantothenamide isolated by a co-culture of Nocardia tenerifensis IFM 10554T in the presence of animal cells

Yasumasa Hara, Midori A. Arai, Kanae Sakai, Naoki Ishikawa, Tohru Gonoi, Takashi Yaguchi, Masami Ishibashi

Journal of Natural Medicines (Journal of Natural Medicines) 72 ( 1 ) 280 - 289 2018.01

Accepted, ISSN 13403443

© 2017, The Japanese Society of Pharmacognosy and Springer Japan KK, part of Springer Nature. A new amide, named dehydropropylpantothenamide (1), was obtained by a co-culture of Nocardia tenerifensis IFM 10554T in the presence of the mouse macrophage-like cell line J774.1 in modified Czapek-Dox (mCD) medium. Compound 1 was synthesized from d-pantothenic acid calcium salt in 6 steps. The absolute configuration of natural compound 1 was determined by comparisons of the optical rotation and CD spectra of synthetic 1. In the present study, a new method for producing secondary metabolites was demonstrated using a “co-culture” in which the genus Nocardia was cultured in the presence of an animal cell line.

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Isolation of Resistomycin from a Terrestrial Actinomycete with TRAIL Resistance-overcoming Activity

Liu Xuefeidan, Arai Midori A, Toume Kazufumi, Ishibashi Masami

NATURAL PRODUCT COMMUNICATIONS (Natural Product Communications) 13 ( 1 ) 65 - 66 2018.01

Accepted, ISSN 1934578X

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) potently induces apoptosis in most cancer cells, but not in normal cells, and, thus, has attracted intense interest as a promising antitumor agent. Activity-guided fractionation of the culture broth of the actinomycete CKK1172 strain led to the isolation of the antibiotic resistomycin (1) using human gastric adenocarcinoma AGS cells. Compound 1 exhibited TRAIL resistance-overcoming activity at 2 μM with 38% more inhibition than a treatment with 1 alone (without TRAIL) in TRAIL-resistant AGS cells.

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Isolation of nabscessin C from nocardia abscessus IFM 10029T and A study on biosynthetic pathway for nabscessins

Shoko Hara, Yasumasa Hara, Midori A. Arai, Yoko Kusuya, Hiroki Takahashi, Takashi Yaguchi, Masami Ishibashi

Chemical and Pharmaceutical Bulletin (Chemical and Pharmaceutical Bulletin) 66 ( 10 ) 976 - 982 2018

Accepted, ISSN 00092363

© 2018 The Pharmaceutical Society of Japan A new aminocyclitol derivative, designated nabscessin C (1), was isolated from Nocardia abscessus IFM 10029T. Nabcessin C is an isomer of nabscessins A (2) and B (3) with different positioning of the acyl group. Absolute configuration of nabscessin A was determined by conversion into the 2-deoxy-scyllo-inosamine pentaacetyl derivative (4) by hydrolysis and acetylation of 2. The biosynthetic pathway of nabscessins is proposed based on gene expression analysis.

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Synthesis and Evaluation of Fuligocandin B Derivatives with Activity for Overcoming TRAIL Resistance

Arai Midori A, Masuda Ayaka, Suganami Akiko, Tamura Yutaka, Ishibashi Masami

CHEMICAL & PHARMACEUTICAL BULLETIN (Chemical and Pharmaceutical Bulletin) 66 ( 8 ) 810 - 817 2018

Accepted, ISSN 00092363

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling pathway induces apoptosis in cancer cells but not in normal cells. Therefore, this pathway has attracted attention regarding possible clinical treatment of cancer. However, many cancer cells demonstrate TRAIL resistance. To overcome this problem, small molecules that sensitize cancer cells to TRAIL are desired. Heterocyclic derivatives of the natural product, fuligocandin B (2), with activity for overcoming TRAIL resistance were synthesized, and their activity was evaluated. Of the synthetic molecules, the quinoline derivative (10g) showed potent activity against TRAIL-resistant gastric adenocarcinoma cells. After a docking study of the target protein valosin-containing protein, 7'-amino fuligocandin B (10m) was designed and synthesized. Compound 10m also showed good activity for overcoming TRAIL resistance. 10m produced a 49.7% difference in viability with TRAIL at 30 µM compared to without TRAIL. This activity was better than that of fuligocandin B (2).

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Boesenberols I-K, new isopimarane diterpenes from Boesenbergia pandurata with TRAIL-resistance overcoming activity

Utpal K. Karmakar, Midori A. Arai, Takashi Koyano, Thaworn Kowithayakorn, Masami Ishibashi

TETRAHEDRON LETTERS (PERGAMON-ELSEVIER SCIENCE LTD) 58 ( 40 ) 3838 - 3841 2017.10

Accepted, ISSN 0040-4039

TRAIL is a promising anticancer agent due to its unique ability to kill tumor cells selectively without harming non-malignant cells. However, some cancer cells are reported to be TRAIL-resistant. Searching potent TRAIL agonist to overcome resistance is an important strategy for its successful use as an anticancer agent. As a part of our continuous research on natural inhibitors, activity-guided fractionation of the MeOH extract of Boesenbergia pandurata rhizomes afforded three (1-3) new isopimarane diterpenes, boesenberols I-K, together with two (4-5) known compounds. The structures of these new compounds were elucidated using HRESIMS, IR, 1D and 2D NMR spectroscopy. All compounds showed TRAIL-resistance overcoming activity. (C) 2017 Elsevier Ltd. All rights reserved.

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Notch Inhibitors from Calotropis gigantea That Induce Neuronal Differentiation of Neural Stem Cells

Yoneyama, Tatsuro, Arai, Midori A., Akamine, Ryuta, Koryudzu, Kazune, Tsuchiya, Anna, Sadhu, Samir K., Ahmed, Firoj, Itoh, Motoyuki, Okamoto, Ryuichi, Ishibashi, Masami

JOURNAL OF NATURAL PRODUCTS (AMER CHEMICAL SOC) 80 ( 9 ) 2453 - 2461 2017.09

Accepted, ISSN 0163-3864

Neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease occur due to loss of the structure and function of neurons. For the potential treatment of neurodegenerative diseases, accelerators of neuronal differentiation of neural stem cells (NSCs) have been focused on and a cell-based assay system for measuring Notch signaling pathway activity was constructed. Using this assay system, eight compounds isolated from Calotropis gigantea were identified as inhibitors of the Notch signaling, pathway. Hes1 and Hes5 are target genes of the Notch signaling pathway, and compound 1, called uscharin, decreased the protein levels of Hes1 and Hes5 in assay cells and MEB5 cells (mouse NSCs). Furthermore, uscharin (1) enhanced the differentiation of MEB5 cells into neurons. The mechanism of uscharin (1) for the Notch signaling inhibitory activity would be acceleration of the degradation of the Notch intracellular domain (NICD) in the MEB5 cells.

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Efficient Synthesis of Heterocyclic Flavonoids with Hedgehog Signal Inhibitory Activity

Midori A. Arai, Takao Utsumi, Natsuki Yanase, Teruhisa Fujimatsu, Masami Ishibashi

CHEMICAL & PHARMACEUTICAL BULLETIN (PHARMACEUTICAL SOC JAPAN) 65 ( 8 ) 784 - 795 2017.08

Accepted, ISSN 0009-2363

The hedgehog (Hh) signaling pathway performs important roles in embryonic development and cellular proliferation and differentiation. However, in many cancer cells Hh signaling is aberrantly activated, which has provided a strong impetus for the development of Hh pathway inhibitors. To address this, we synthesized a series of heterocyclic flavonoids and evaluated their Hh signaling inhibitory activity on cancer cell lines using our cell-based assay system. Of the synthetic flavonoids, compounds 4a and g showed good inhibitory activity (IC50 was 16.8 and 21.8 mu m, respectively), and were cytotoxic toward human pancreatic (PANC1) and prostate (DU145) cancer cells in which Hh signaling was activated. Compounds 4a and g had moderate selectivity against PANC1 cells. Western blotting analyses revealed that PTCH and GLI1 expression was reduced after treatment with these compounds. Overall, these synthetic flavonoids represent promising new additions to our expanding panel of Hh pathway inhibitors, and with further development these molecules may ultimately be considered for clinical use.

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Germacranolides from Enhydra fluctuans with TRAIL-resistance Abrogating Activity

Firoj Ahmed, Utpal K. Karmakar, Midori A. Arai, Naoki Ishikawa, Samir K. Sadhu, Masami Ishibashi

NATURAL PRODUCT COMMUNICATIONS (NATURAL PRODUCTS INC) 12 ( 8 ) 1169 - 1170 2017.08

Accepted, ISSN 1934-578X

TRAIL selectively kills cancer cells without harming most normal cells. However, TRAIL resistance is a major problem for its therapeutic use. Developing a strategy to overcome resistance is crucial for the successful use of TRAIL as an antitumor agent. In our screening program of natural products that can abrogate TRAIL resistance, four germacranolides (1-4) were isolated from the aerial parts of Enhydra fluctuans. All compounds exhibited potent TRAIL resistance overcoming activity against TRAIL-resistant human gastric adenocarcinoma (AGS) cells.

Identification of BMI1 Promoter Inhibitors from Beaumontia murtonii and Eugenia operculata

Yui Kaneta, Midori A. Arai, Naoki Ishikawa, Kazufumi Toume, Takashi Koyano, Thaworn Kowithayakorn, Tetsuhiro Chiba, Atsushi Iwama, Masami Ishibashi

JOURNAL OF NATURAL PRODUCTS (AMER CHEMICAL SOC) 80 ( 6 ) 1853 - 1859 2017.06

Accepted, ISSN 0163-3864

B-Cell-specific Moloney murine leukemia virus insertion region 1 (BMI1) is a core component of the polycomb repressive complex 1 (PRC1). Abnormal expression of BMI1 is associated with a number of human malignances and cancer stem cells (CSCs), which cause chemotherapy resistance. Therefore, small molecules that inhibit BMI1 expression are potential candidates for cancer therapy. In this study, a cell-based reporter gene assay was developed that allowed BMI1 promoter activity to be measured in 293T human embryonic kidney cells based on luciferase expression levels. Using this screening assay, the methanol-soluble extracts of Beaumontia murtonii and Eugenia operculata were selected as leads. Bioassay-guided fractionation of the extracts led to the isolation of three known cardenolides (13) and one new compound (4) from B. murtonii and two known triterpenoids (5 and 6) and one new compound (7) from E. operculata. These seven compounds inhibited BMI1 promoter activity (IC50 range 0.09323.0 mu M), and the most active compound, wallichoside (1), was further evaluated. Western blot analysis revealed that wallichoside (1) decreases BMI1 protein levels in HCT116 human colon carcinoma cells, and flow cytometry analysis showed that it significantly reduced levels of the CSC biomarker epithelial cell adhesion molecule. Wallichoside (1) also inhibited sphere formation of Huh7 human hepatocellular carcinoma cells, indicating that it diminished the self-renewal capability of CSCs.

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Total synthesis of agalloside, isolated from Aquilaria agallocha, by the 5-O-glycosylation of flavan

Midori A. Arai, Yumi Yamaguchi, Masami Ishibashi

ORGANIC & BIOMOLECULAR CHEMISTRY (ROYAL SOC CHEMISTRY) 15 ( 23 ) 5025 - 5032 2017.06

Accepted, ISSN 1477-0520

Agalloside (1) is a neural stem cell differentiation activator isolated from Aquilaria agallocha by our group using Hes1 immobilized beads. We conducted the first total synthesis of agalloside (1) via the 5-O-glycosylation of flavan 25 using glycosyl fluoride 20 in the presence of BF3 center dot Et2O. Subsequent oxidation with DDQ to flavanone 2 and deprotection successively provided agalloside (1). This synthetic strategy holds promise for use in the synthesis of 5-O-glycosylated flavonoids. The synthesized agalloside (1) accelerated neural stem cell differentiation, which is a result comparable to that for the naturally occurring compound 1.

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Hes1-Binding Compounds Isolated by Target Protein Oriented Natural Products Isolation (TPO-NAPI)

Midori A. Arai, Mitsuha Tanaka, Kana Tanouchi, Naoki Ishikawa, Firoj Ahmed, Samir K. Sadhu, Masami Ishibashi

JOURNAL OF NATURAL PRODUCTS (AMER CHEMICAL SOC) 80 ( 2 ) 538 - 543 2017.02

Accepted, ISSN 0163-3864

Hairy and enhancer of split 1 (Hes1) is a transcription factor that acts in neural stem cells to inhibit differentiation. We recently developed target protein oriented natural products isolation (TPO-NAPI) using Hes1-immobilized beads to identify activators of neural stem cells. Isomicromonolactam (1), staurosporin (2), and linarin (3) were isolated as Hes1-binding compounds using the TPO-NAPI method. Of these, compound 1 enhanced neural stem cell differentiation. Using truncated Hes1 proteins, the binding region of Hes1 for 1 was estimated to be in the C-terminal half that includes a TLE/Grg binding site. The differentiation-promoting activity of inohanamine (4) is also reported.

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ISOLATION AND EVALUATION OF HEDGEHOG INHIBITORS FROM CHRISTMAS GRASS (THEMEDA ARGUENS)

Yoneyama, Tatsuro, Arai, Midori A., Koyano, Takashi, Kowithayakorn, Thaworn, Ishibashi, Masami

HETEROCYCLES (PERGAMON-ELSEVIER SCIENCE LTD) 95 ( 1 ) 210 - 222 2017.01

Accepted, ISSN 0385-5414

The hedgehog (Hh) signaling pathway is an important regulator of embryonic development, tissue patterning, cellular proliferation and differentiation. However, aberrant activation of the pathway is associated with tumorigenesis. In this study, the Hh signal inhibitory activity of plant extracts was measured with a cell-based assay system that targets GLI1-mediated transcription. Activity guided analysis of MeOH extracts of christmas grass (Themeda arguens) led to the isolation of four inhibitory compounds: aciculatin (1), 7-de-O-methylaciculatin (2), 8-C-beta-D-boivinopyranosylapigenin (3) and aciculatinone (4). Compound 1 strongly inhibited Hh/GLI1-mediated transcriptional activity with an IC50 value of 1.8. mu M, and disrupted the formation of the GLI1-DNA complex by EMSA. 1 and 3 were cytotoxic for human prostate (DU-145) and breast (MCF-7) cancer cells. These compounds may serve as new tools for the molecular dissection of Hh pathway activation.

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Notch Inhibitors from Calotropis gigantea That Induce Neuronal Differentiation of Neural Stem Cells

Tatsuro Yoneyama, Midori A. Arai, Ryuta Akamine, Kazune Koryudzu, Anna Tsuchiya, Samir K. Sadhu, Firoj Ahmed, Motoyuki Itoh, Ryuichi Okamoto, Masami Ishibashi

Journal of Natural Products (American Chemical Society) 80 ( 9 ) 2453 - 2461 2017

Accepted, ISSN 1520-6025

Neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease occur due to loss of the structure and function of neurons. For the potential treatment of neurodegenerative diseases, accelerators of neuronal differentiation of neural stem cells (NSCs) have been focused on and a cell-based assay system for measuring Notch signaling pathway activity was constructed. Using this assay system, eight compounds isolated from Calotropis gigantea were identified as inhibitors of the Notch signaling pathway. Hes1 and Hes5 are target genes of the Notch signaling pathway, and compound 1, called uscharin, decreased the protein levels of Hes1 and Hes5 in assay cells and MEB5 cells (mouse NSCs). Furthermore, uscharin (1) enhanced the differentiation of MEB5 cells into neurons. The mechanism of uscharin (1) for the Notch signaling inhibitory activity would be acceleration of the degradation of the Notch intracellular domain (NICD) in the MEB5 cells.

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Valosin-containing Protein is a Target of 5 '-l FuligocandinB and Enhances TRAIL Resistance in Cancer Cells

Midori A. Arai, Shota Taguchi, Kazuhiro Komatsuzaki, Kento Uchiyama, Ayaka Masuda, Mana Sampei, Mamoru Satoh, Sayaka Kado, Masami Ishibashi

CHEMISTRYOPEN (WILEY-V C H VERLAG GMBH) 5 ( 6 ) 574 - 579 2016.12

Accepted, ISSN 2191-1363

Fuligocandin B (2) is a novel natural product that can overcome TRAIL resistance. We synthesized enatiomerically pure fuligocandin B (2) and its derivative 5'-l fuligocandin B (4), and found that the latter had an improved biological activity against the human gastric cancer cell line, AGS. We attached a biotin linker and photoactivatable aryl diazirine group to 5'-l fuligocandin B (4), and employed a pull-down assay to identify valosin-containing protein (VCP/p97), an AAA ATPase, as a 5'-l fuligocandin B (4) target protein. Knock-down of VCP by siRNA enhanced sensitivity to TRAIL in AGS cells. In addition, 4 enhanced CHOP and DR5 protein expression, and overall intracellular levels of ubiquitinated protein. These data suggest that endoplasmic reticulum stress caused through VCP inhibition by 4 increases CHOP-mediated DR5 up-regulation, which enhances TRAIL-induced cell death in AGS cells. To the best of our knowledge, this is the first example to show a relationship between VCP and TRAIL-resistance-overcoming activity in cancer cells.

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Boesenberols, Pimarane Diterpenes with TRAIL-Resistance-Overcoming Activity from Boesenbergia pandurata

Utpal K. Karmakar, Naoki Ishikawa, Midori A. Arai, Firoj Ahmed, Takashi Koyano, Thaworn Kowithayakorn, Masami Ishibashi

JOURNAL OF NATURAL PRODUCTS (AMER CHEMICAL SOC) 79 ( 8 ) 2075 - 2082 2016.08

Accepted, ISSN 0163-3864

TRAIL is a potent and selective inducer of apoptosis in most cancer cells while sparing normal cells, which makes it an attractive target for the development of new cancer therapies. In a screening program on natural resources with the ability to abrogate TRAIL resistance, the bioassay guided fractionation of Boesenbergia pandurata rhizomes resulted in the isolation of 17 pimarane diterpenes and a monoterpene. Among these, compounds 1-8, named boesenberols A-H, are new pimarane diterpenes. All compounds exhibited TRAIL-resistance-overcoming activity in TRAIL-resistant AGS cells. Subtoxic doses of the major compound 9 sensitized AGS cells to TRAIL-induced apoptosis by up-regulating apoptosis-inducing proteins, such as DR4, DRS, p53, Fas, CHOP, Bak, and cleaved caspases-3,-8, and -9, and down-regulating the levels of cell survival proteins, such as Bcl-2, c-FLIP, and GSK-3 beta, in TRAIL-resistant AGS cells. Furthermore, compound 9 did not decrease the viability of noncancerous (HEK293) cells at concentrations up to 30 mu M.

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Cerasoidine, a Bis-aporphine Alkaloid Isolated from Polyalthia cerasoides during Screening for Wnt Signal Inhibitors

Takumi Shono, Naoki Ishikawa, Kazufumi Toume, Midori A. Arai, Hyuma Masu, Takashi Koyano, Thaworn Kowithayakorn, Masami Ishibashi

JOURNAL OF NATURAL PRODUCTS (AMER CHEMICAL SOC) 79 ( 8 ) 2083 - 2088 2016.08

Accepted, ISSN 0163-3864

A new bis-aporphine alkaloid, cerasoidine (1), was isolated from the root extract of Polyalthia cerasoides together with the known bis-aporphine bidebiline E (2) during screening for compounds with Wnt signal inhibitory activities. The structure of cerasoidine (1) was established by X-ray analysis and shown by chiral HPLC analyses and electronic circular dichroism to be a 57:43 mixture of R(-)- and S(+)-atropisomers. Bidebiline E (2) exhibited inhibition of transcriptional activity of TCF/beta-catenin with an IC50 value of 20.2 mu M and was also found to inhibit Wnt signaling by decreasing nuclear beta-catenin.

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Bioactive Secondary Metabolites with Unique Aromatic and Heterocyclic Structures Obtained from Terrestrial Actinomycetes Species

Mohamed S. Abdelfattah, Midori A. Arai, Masami Ishibashi

CHEMICAL & PHARMACEUTICAL BULLETIN (PHARMACEUTICAL SOC JAPAN) 64 ( 7 ) 668 - 675 2016.07

Accepted, ISSN 0009-2363

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Constituents from Entada scandens with TRAIL-resistance Overcoming Activity

Firoj Ahmed, Kazufumi Toume, Naoki Ishikawa, Midori A. Arai, Samir K. Sadhu, Masami Ishibashi

NATURAL PRODUCT COMMUNICATIONS (NATURAL PRODUCTS INC) 11 ( 7 ) 993 - 995 2016.07

Accepted, ISSN 1934-578X

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has emerged as a promising anticancer agent because of its ability to selectively kill tumor cells. But TRAIL-resistance is a major problem of its therapy. A search for compounds for abrogating TRAIL-resistance has, thus, become an important strategy for anticancer drug discovery. In search of bioactive natural products for overcoming TRAIL-resistance, we previously reported some compounds with TRAIL-resistance overcoming activity. Bioassay guided fractionation of Entada scandens led to the isolation of four compounds (1-4). Of the isolates, compounds 1 and 3 showed moderate TRAIL-resistance overcoming activity in TRAIL-resistant human gastric adenocarcinoma (AGS) cells.

Stereochemical Assignment and Biological Evaluation of BE-14106 Unveils the Importance of One Acetate Unit for the Antifungal Activity of Polyene Macrolactams

Kohei Fujita, Ryosuke Sugiyama, Shinichi Nishimura, Naoki Ishikawa, Midori A. Arai, Masami Ishibashi, Hideaki Kakeya

JOURNAL OF NATURAL PRODUCTS (AMER CHEMICAL SOC) 79 ( 7 ) 1877 - 1880 2016.07

Accepted, ISSN 0163-3864

Heronamides are a class of potent antifungal metabolites produced by marine-derived actinomycetes. The number of hydroxy groups and the stereochemistry of the two hydroxylated methine carbons are important for the activity of heronamide C, whereas the effect of the hydrocarbon chains is not known. In this study, the stereochemistry and the biological activity of BE-14106, another member of the heronamide class of antibiotics, isolated from an actinomycete Actinoalloteichus cyanogriseus IFM 11549 was investigated. Spectroscopic analysis coupled with photo- and O-2-induced conversion revealed that BE-14106 and the heronamides had the same stereochemistry. BE-14106 showed potent growth inhibition against fission yeast cells with an MIC value of 0.50 mu M (0.21 mu g/mL), being 4 times less potent than heronamide C, which revealed the importance of the structure of the hydrocarbon tail for the activity.

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Calotropis giganteaからのノッチシグナル伝達経路阻害を標的とした神経幹細胞分化促進剤の探索

米山達朗, 荒井緑, 岡本隆一, Sadhu Samir K., Ahmed Firoj, 石橋正己

日本薬学会年会要旨集 ((公社)日本薬学会) 136年会 ( 2 ) 59 - 59 2016.03

ISSN 0918-9823

Bioassay-Guided Isolation of Compounds from Datura stramonium with TRAIL-Resistance Overcoming Activity

Utpal K. Karmakar, Kazufumi Toume, Naoki Ishikawa, Midori A. Arai, Samir K. Sadhu, Firoj Ahmed, Masami Ishibashi

NATURAL PRODUCT COMMUNICATIONS (NATURAL PRODUCTS INC) 11 ( 2 ) 185 - 187 2016.02

Accepted, ISSN 1934-578X

TRAIL is a potent inducer of apoptosis in most cancer cells, but not in normal cells, and therefore has deserved intense interest as a promising agent for cancer therapy. In the search for bioactive natural products for overcoming TRAIL-resistance, we previously reported a number of active compounds. In our screening program on natural resources targeting overcoming TRAIL-resistance, activity-guided fractionation of the MeOH extract of Datura stramonium leaves led to the isolation of three alkaloids - scopolamine (1), trigonelline (2), and tyramine (3). Compounds 1, 2, and 3 exhibited TRAIL-resistance overcoming activity at 50, 150, and 100 mu M, respectively in TRAIL-resistant AGS cells.

Hes1 inhibitor isolated by target protein oriented natural products isolation (TPO-NAPI) of differentiation activators of neural stem cells

Midori A. Arai, Naoki Ishikawa, Mitsuha Tanaka, Kenji Uemura, Noriko Sugimitsu, Akiko Suganami, Yutaka Tamura, Takashi Koyano, Thaworn Kowithayakorn, Masami Ishibashi

CHEMICAL SCIENCE (ROYAL SOC CHEMISTRY) 7 ( 2 ) 1514 - 1520 2016

Accepted, ISSN 2041-6520

The Hes1 dimer inhibitor, agalloside (2), which can accelerate the differentiation of neural stem cells is described. Six natural products, including one new natural product, which bind to Hes1 were rapidly isolated by a developed "target protein oriented natural products isolation" (TPO-NAPI) method using Hes1-immobilized beads. Of the six compounds, 2 inhibited Hes1 dimer formation at both the protein-and cellular level. Neural stem cells treated with 2 differentiated to neurons with longer neurites than cells treated with varproic acid or retinoic acid. Moreover, 2 exhibited specificity for neurons. This promotion of differentiation was supported by an increase in the mRNA expression of the proneural genes, Mash1 and Ngn2, which were inhibited by Hes1.

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Synthesis of rocaglamide derivatives and evaluation of their Wnt signal inhibitory activities

Midori A. Arai, Yuuki Kofuji, Yuuki Tanaka, Natsuki Yanase, Kazuki Yamaku, Rolly G. Fuentes, Utpal Kumar Karmakar, Masami Ishibashi

ORGANIC & BIOMOLECULAR CHEMISTRY (ROYAL SOC CHEMISTRY) 14 ( 11 ) 3061 - 3068 2016

Accepted, ISSN 1477-0520

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Hedgehog/GLI-mediated transcriptional activity inhibitors from Crinum asiaticum

Midori A. Arai, Ryuta Akamine, Samir K. Sadhu, Firoj Ahmed, Masami Ishibashi

JOURNAL OF NATURAL MEDICINES (SPRINGER JAPAN KK) 69 ( 4 ) 538 - 542 2015.10

Accepted, ISSN 1340-3443

The inhibition of the hedgehog (Hh) signaling pathway has emerged as an attractive anti-cancer strategy. As part of our continuing search for natural inhibitors of the Hh/GLI1 signaling pathway, we isolated three alkaloids (1-3) from Crinum asiaticum. Compounds 1 and 3 showed potent Hh/GLI1-mediated transcriptional inhibitory activity and exhibited cytotoxicity against human pancreatic (PANC1) and prostate (DU145) cancer cells. Our data revealed that compounds 1 and 3 clearly inhibited the Hh signaling pathway by down-regulating the expression of GLI-related proteins (PTCH and BCL2) in DU145 cells.

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Phenolic compounds from the bark of Oroxylum indicum activate the Ngn2 promoter

Rolly G. Fuentes, Midori A. Arai, Samir K. Sadhu, Firoj Ahmed, Masami Ishibashi

JOURNAL OF NATURAL MEDICINES (SPRINGER JAPAN KK) 69 ( 4 ) 589 - 594 2015.10

Accepted, ISSN 1340-3443

A reporter gene assay that detects neurogenin 2 (Ngn2) promoter activity was utilized to identify compounds that induce neuronal differentiation. Ngn2 is a basic helix-loop-helix transcription factor that activates transcription of pro-neural genes. Using this assay system and an activity-guided approach, seven phenolic compounds were isolated from the methanol extract of Oroxylum indicum: 1 oroxylin A, 2 chrysin, 3 hispidulin, 4 baicalein, 5 apigenin, 6 baicalin, and 7 isoverbascoside. Compounds 1 and 2 induced an estimated 2.7-fold increase in Ngn2 promoter activity, whereas 3 increased the activity by 2.5-fold. Furthermore, 1 and 2 enhanced neuronal differentiation of C17.2 cells, which are multipotent stem cells.
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Coronaridine, an iboga type alkaloid from Tabernaemontana divaricata, inhibits the Wnt signaling pathway by decreasing beta-catenin mRNA expression

Kensuke Ohishi, Kazufumi Toume, Midori A. Arai, Samir K. Sadhu, Firoj Ahmed, Masami Ishibashi

BIOORGANIC & MEDICINAL CHEMISTRY LETTERS (PERGAMON-ELSEVIER SCIENCE LTD) 25 ( 18 ) 3937 - 3940 2015.09

Accepted, ISSN 0960-894X

Four alkaloids, voacangine (1), isovoacangine (2), coronaridine (3), and coronaridine hydroxyindolenine (4), were isolated from the MeOH extract of Tabernaemontana divaricata aerial parts by activity-guided fractionation for Wnt signal inhibitory activity. Compounds 1-4 exhibited TCF/beta-catenin inhibitory activities with IC50 values of 11.5, 6.0, 5.8, and 7.3 mu M, respectively. Of these, coronaridine (3) decreased beta-catenin levels in SW480 colon cancer cells, while this decrease in beta-catenin was not suppressed by a co-treatment with 3 and MG132, a proteasome inhibitor. These results suggested that the decrease observed in beta-catenin levels by coronaridine (3) did not depend on a proteasomal degradation process. On the other hand, the treatment of SW480 cells with coronaridine (3) caused a decrease in beta-catenin mRNA levels. Thus, coronaridine (3) may inhibit the Wnt signaling pathway by decreasing the mRNA expression of beta-catenin. (C) 2015 Elsevier Ltd. All rights reserved.

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Hedgehog inhibitors from Withania somnifera

Yoneyama, Tatsuro, Arai, Midori A., Sadhu, Samir K., Ahmed, Firoj, Ishibashi, Masami

BIOORGANIC & MEDICINAL CHEMISTRY LETTERS (PERGAMON-ELSEVIER SCIENCE LTD) 25 ( 17 ) 3541 - 3544 2015.09

Accepted, ISSN 0960-894X

The hedgehog (Hh) signaling pathway performs an important role in embryonic development and in cellular proliferation and differentiation. However, aberrant activation of the Hh signaling pathway is associated with tumorigenesis. Hh signal inhibition was evaluated using a cell-based assay system that targets GLI1-mediated transcription. Activity-guided isolation of the Withania somnifera MeOH extract led to the isolation of six compounds: withaferin A (1) and its derivatives (2-6). Compounds 1 and 2 showed strong inhibition of Hh/GLI1-mediated transcriptional activity with IC50 values of 0.5 and 0.6 mu M, respectively. Compounds 1, 2, 3, and 6 were cytotoxic toward human pancreatic (PANC-1), prostate (DU145) and breast (MCF7) cancer cells. Furthermore, 1 also inhibited GLI1-DNA complex formation in EMSA. (C) 2015 Elsevier Ltd. All rights reserved.

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Calotropis giganteaからのノッチシグナル伝達経路阻害剤の探索

米山達朗, 荒井緑, 岡本隆一, Sadhu Samir K., Ahmed Firoj, 石橋正己

日本生薬学会年会講演要旨集 ((一社)日本生薬学会) 62回 172 - 172 2015.08

ISSN 0919-1992

Hedgehog inhibitors from Artocarpus communis and Hyptis suaveolens

Midori A. Arai, Kyoko Uchida, Samir K. Sadhu, Firoj Ahmed, Takashi Koyano, Thaworn Kowithayakorn, Masami Ishibashi

BIOORGANIC & MEDICINAL CHEMISTRY (PERGAMON-ELSEVIER SCIENCE LTD) 23 ( 15 ) 4150 - 4154 2015.08

Accepted, ISSN 0968-0896

The hedgehog (Hh) signaling pathway plays crucial roles in cell maintenance and proliferation during embryonic development. Naturally occurring Hh inhibitors were isolated from Artocarpus communis and Hyptis suaveolens using our previously constructed cell-based assay system. Bioactivity guided fractionation led to the isolation of 15 compounds, including seven new compounds (4, 5, 6, 7, and 9-11). The isolated compounds showed cytotoxicity against a cancer cell line (PANC1) in which Hh signaling was abnormally activated. Several compounds (12-14; GLI1 transcriptional inhibition IC50 = 7.6, 4.7, and 4.0 mu M, respectively) inhibited Hh related protein (BCL2) expression. Moreover, compounds 1, 12, and 13 disrupted GLI1 and DNA complex formation. (C) 2015 Elsevier Ltd. All rights reserved.

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Sesquiterpenes with TRAIL-resistance overcoming activity from Xanthium strumarium

Utpal K. Karmakar, Naoki Ishikawa, Kazufumi Toume, Midori A. Arai, Samir K. Sadhu, Firoj Ahmed, Masami Ishibashi

BIOORGANIC & MEDICINAL CHEMISTRY (PERGAMON-ELSEVIER SCIENCE LTD) 23 ( 15 ) 4746 - 4754 2015.08

Accepted, ISSN 0968-0896

The ability of TRAIL to selectively induce apoptosis in cancer cells while sparing normal cells makes it an attractive target for the development of new cancer therapy. In search of bioactive natural products for overcoming TRAIL-resistance from natural resources, we previously reported a number of active compounds. In our screening program on natural resources targeting overcoming TRAIL-resistance, activity-guided fractionations of the extract of Xanthium strumarium led to the isolation of five sesquiterpene compounds (1-5). 11 alpha, 13-dihydroxanthinin (2) and 11 alpha, 13-dihydroxanthuminol (3) were first isolated from natural resources and xanthinosin (1), desacetylxanthanol (4), and lasidiol p-methoxybenzoate (5) were known compounds. All compounds (1-5) showed potent TRAIL-resistance overcoming activity at 8, 20, 20, 16, and 16 mu M, respectively, in TRAIL-resistant AGS cells. Compounds 1 and 5 enhanced the levels of apoptosis inducing proteins DR4, DR5, p53, CHOP, Bax, cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9 and also decreased the levels of cell survival protein Bcl-2 in TRAIL-resistant AGS cells in a dose-dependent manner. Compound 1 also enhanced the levels of DR4 and DR5 proteins in a time-dependent manner. Thus, compounds 1 and 5 were found to induce both extrinsic and intrinsic apoptotic cell death. Compound 1 also exhibit TRAIL-resistance overcoming activity in DLD1, DU145, HeLa, and MCF7 cells but did not decrease viability in non-cancer HEK293 cells up to 8 mu M. (C) 2015 Elsevier Ltd. All rights reserved.

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Boehmenan, a lignan from Hibiscus ficulneus, showed Wnt signal inhibitory activity

Takumi Shono, Naoki Ishikawa, Kazufumi Toume, Midori A. Arai, Firoj Ahmed, Samir K. Sadhu, Masami Ishibashi

BIOORGANIC & MEDICINAL CHEMISTRY LETTERS (PERGAMON-ELSEVIER SCIENCE LTD) 25 ( 14 ) 2735 - 2738 2015.07

Accepted, ISSN 0960-894X

The Wnt signal pathway modulates numerous biological processes, and its aberrant activation is related to various diseases. Therefore, inhibition of the Wnt signal may provide an effective (or efficient) strategy for these diseases. Cell-based luciferase assay targeting the Wnt signal (TOP assay) revealed that Hibiscus ficulneus extract inhibited the Wnt signal. The activity-guided isolation of the MeOH extract of H. ficulneus stems yielded four known (1-4) lignans along with myriceric acid (5). Compounds 1-4 potently inhibited the Wnt signal with TOPflash IC50 values of 1.0, 4.5, 6.3, and 1.9 mu M, respectively. Compound 1 exhibited cytotoxicity against both Wnt-dependent (HCT116) and Wnt-independent (RKO) cells. Western blot analysis showed that 1 decreased the expression of full, cytosolic and nuclear b-catenin along with c-myc in STF/293 cells. Our results suggested that 1 may have inhibited the Wnt signal by decreasing beta-catenin levels. (C) 2015 Elsevier Ltd. All rights reserved.

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9-Hydroxycanthin-6-one, a beta-Carboline Alkaloid from Eurycoma longifolia, Is the First Wnt Signal Inhibitor through Activation of Glycogen Synthase Kinase 3 beta without Depending on Casein Kinase 1 alpha

Kensuke Ohishi, Kazufumi Toume, Midori A. Arai, Takashi Koyano, Thaworn Kowithayakorn, Takamasa Mizoguchi, Motoyuki Itoh, Masami Ishibashi

JOURNAL OF NATURAL PRODUCTS (AMER CHEMICAL SOC) 78 ( 5 ) 1139 - 1146 2015.05

Accepted, ISSN 0163-3864

Wnt signaling regulates various processes such as cell proliferation, differentiation, and embryo development. However, numerous diseases have been attributed to the aberrant transduction of Wnt signaling. We screened a plant extract library targeting TCF/beta-catenin transcriptional modulating activity with a cell-based luciferase assay. Activity-guided fractionation of the MeOH extract of the E. longifolia root led to the isolation of 9-hydroxycanthin-6-one (1). Compound 1 exhibited TCF/beta-catenin inhibitory activity. Compound 1 decreased the expression of Wnt signal target genes, mitf and zic2a, in zebrafish embryos. Treatment of SW480 cells with 1 decreased beta-catenin and increased phosphorylated beta-catenin (Ser 33, 37, Tyr 41) protein levels. The degradation of beta-catenin by 1 was suppressed by GSK3 beta-siRNA, while compound 1 decreased beta-catenin even in the presence of CK1 alpha-siRNA. These results suggest that 1 inhibits Wnt signaling through the activation of GSK3 beta independent of CK1 alpha.

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Prenylated Flavonoids and Resveratrol Derivatives Isolated from Artocarpus communis with the Ability to Overcome TRAIL Resistance (vol 78, pg 103, 2015)

Toume, Kazufumi, Habu, Tadashi, Arai, Midori A., Koyano, Takashi, Kowithayakorn, Thaworn, Ishibashi, Masami

JOURNAL OF NATURAL PRODUCTS (AMER CHEMICAL SOC) 78 ( 4 ) 968 - 968 2015.04

Accepted, ISSN 0163-3864

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Scopadulciol, Isolated from Scoparia dulcis, Induces beta-Catenin Degradation and Overcomes Tumor Necrosis Factor-Related Apoptosis Ligand Resistance in AGS Human Gastric Adenocarcinoma Cells

Rolly G. Fuentes, Kazufumi Toume, Midori A. Arai, Samir K. Sadhu, Firoj Ahmed, Masami Ishibashi

JOURNAL OF NATURAL PRODUCTS (AMER CHEMICAL SOC) 78 ( 4 ) 864 - 872 2015.04

Accepted, ISSN 0163-3864

Scopadulciol (1), a scopadulan-type diterpenoid, was isolated from Scoparia dulcis along with three other compounds (2-4) by an activity-guided approach using the TCF reporter (TOP) luciferase-based assay system. A fluorometric microculture cytotoxicity assay (FMCA) revealed that compound 1 was cytotoxic to AGS human gastric adenocarcinoma cells. The treatment of AGS cells with 1 decreased beta-catenin levels and also inhibited its nuclear localization. The pretreatment of AGS cells with a proteasome inhibitor, either MG132 or epoxomicin, protected against the degradation of beta-catenin induced by 1. The 1-induced degradation of beta-catenin was also abrogated in the presence of pifithrin-alpha, an inhibitor of p53 transcriptional activity. Compound 1 inhibited TOP activity in AGS cells and downregulated the protein levels of cyclin D1, c-myc, and survivin. Compound 1 also sensitized AGS cells to tumor necrosis factor-related apoptosis ligand (TRAIL)-induced apoptosis by increasing the levels of the death receptors, DR4 and DR5, and decreasing the level of the antiapoptotic protein Bcl-2. Collectively, our results demonstrated that 1 induced the p53- and proteasome-dependent degradation of beta-catenin, which resulted in the inhibition of TCF/beta-catenin transcription in AGS cells. Furthermore, 1 enhanced apoptosis in TRAIL-resistant AGS when combined with TRAIL.

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Calotropis giganteaからのノッチシグナル伝達経路阻害剤の探索

米山達朗, 荒井緑, 小柳津和音, 岡本隆一, Sadhu Samir K., Ahmed Firoj, 石橋正己

日本薬学会年会要旨集 ((公社)日本薬学会) 135年会 ( 2 ) 226 - 226 2015.03

ISSN 0918-9823

Limonoids with Wnt signal inhibitory activity isolated from the fruits of Azadirachta excelsa

Rolly G. Fuentes, Kazufumi Toume, Midori A. Arai, Samir K. Sadhu, Firoj Ahmed, Masami Ishibashi

PHYTOCHEMISTRY LETTERS (ELSEVIER SCIENCE BV) 11 280 - 285 2015.03

Accepted, ISSN 1874-3900

The Wnt signal regulates various biological processes, and its aberrant activation is associated with the development of diseases. Thus, inhibiting the Wnt signal provides a promising strategy to treat these diseases. Our cell-based luciferase assay system, which targets the Wnt signal (TOP assay), revealed that Azadirachta excelsa inhibited the Wnt signal. The activity-guided isolation of the MeOH fruit extract of A. excelsa provided one new (1) and seven known (2-8) limonoids. Their structures were elucidated based on their spectroscopic data, and their NMR data were compared with those in the literature. Compounds 3-6 potently inhibited the Wnt signal with IC50 values of 127 nM, 300 nM, 252 nM, and 121 nM, respectively. Compound 4 exhibited selective cytotoxicity against AGS and HCT116. Western blot analysis showed that 4 did not affect the level or localization of beta-catenin, but downregulated the level of c-myc. Our results suggested that 4 may have inhibited the Wnt signal by affecting the components downstream of beta-catenin. (C) 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

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ヘッジホッグシグナル阻害活性を有するヘテロ環含有フラボノイドの合成と活性評価

荒井緑, 内海尊雄, 米山達朗, 野尻奈那, 石橋正己

日本薬学会年会要旨集 ((公社)日本薬学会) 135年会 ( 2 ) 188 - 188 2015.03

ISSN 0918-9823

Inubosins A, B, and C Are Acridine Alkaloids Isolated from a Culture of Streptomyces sp IFM 11440 with Ngn2 Promoter Activity

Midori A. Arai, Kazune Koryudzu, Masami Ishibashi

JOURNAL OF NATURAL PRODUCTS (AMER CHEMICAL SOC) 78 ( 2 ) 311 - 314 2015.02

Accepted, ISSN 0163-3864

Three new acridine alkaloids, inubosins A (1), B (2), and C (3), were isolated from an extract of a culture of Streptomyces sp. IFM 11440 using bioassay-guided fractionation. Neurogenin2 (Ngn2) is an activator-type basic helix-loop-helix transcription factor that promotes neural stem cell differentiation. Using cell-based Ngn2 promoter activity-guided screening, Streptomyces sp. IFM 11440 was found to induce Ngn2 promoter activity. The structures of 1-3 were established using spectroscopic methods, including 1D- and 2D-NMR measurements. Inubosin B (2) showed potent Ngn2 promoter activity. Moreover, inubosin B (2) increased mRNA expression of genes related to neural stem cell differentiation.

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ISOLATION OF ALKAMIDES WITH DEATH RECEPTOR-ENHANCING ACTIVITIES FROM PIPER CHABA

Hogue Tahmina, Kazufumi Toume, Midori A. Arai, Samir K. Sadhu, Firoj Ahmed, Masami Ishibashi

HETEROCYCLES (PERGAMON-ELSEVIER SCIENCE LTD) 90 ( 2 ) 1317 - 1322 2015.01

Accepted, ISSN 0385-5414

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Prenylated Flavonoids and Resveratrol Derivatives Isolated from Artocarpus communis with the Ability to Overcome TRAIL Resistance

Toume, Kazufumi, Habu, Tadashi, Arai, Midori A., Koyano, Takashi, Kowithayakorn, Thaworn, Ishibashi, Masami

JOURNAL OF NATURAL PRODUCTS (AMER CHEMICAL SOC) 78 ( 1 ) 103 - 110 2015.01

Accepted, ISSN 0163-3864

In a screening program on natural products that can abrogate tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) resistance, four new prenylated flavonoid and resveratrol derivatives (1-4) were isolated from Artocarpus communis, together with eight known prenylflavonoids (5-12). The structures of 1-4 were elucidated spectroscopically. Pannokin E (1) (2 mu M) and artonin E (5) (3 mu M) potently exhibited the ability to overcome TRAIL resistance. Artonin E (5) induced caspase-dependent apoptosis in combination with TRAIL, increased caspase 3/7 activity, and enhanced the protein levels of p53 and DR5. Moreover, this substance decreased cell viability in combination with TRAIL and enhanced the protein levels of DR5, and these effects were mediated by increases in the production of ROS (reactive oxygen species). Thus, artonin E (5) was found to induce extrinsic apoptotic cell death by the ROS- and p53-mediated up-regulation of DR5 expression in AGS cells.

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Natural compounds with Wnt signal modulating activity

Rolly G. Fuentes, Midori A. Arai, Masami Ishibashi

NATURAL PRODUCT REPORTS (ROYAL SOC CHEMISTRY) 32 ( 12 ) 1622 - 1628 2015

Accepted, ISSN 0265-0568

The Wnt signalling pathway is essential in many biological processes. The Wnt signal is associated with several diseases, particularly cancer and neurodegenerative diseases. Recently, high-throughput screening systems have been developed to rapidly identify compounds, including natural compounds, that target the Wnt signal. Some studies on natural modulators of the Wnt signal have also suggested their possible target. This review highlights some important natural compounds reported to regulate Wnt activity and describes their possible mechanism of action.

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Naturally occurring FANCF-Hes1 complex inhibitors from Wrightia religiosa

Midori A. Arai, Kenji Uemura, Nozomi Hamahiga, Naoki Ishikawa, Takashi Koyano, Thaworn Kowithayakorn, Tagrid Kaddar, Madeleine Carreau, Masami Ishibashi

MEDCHEMCOMM (ROYAL SOC CHEMISTRY) 6 ( 3 ) 455 - 460 2015

Accepted, ISSN 2040-2503

The isolation and evaluation of inhibitors of Fanconi F protein (FANCF) - haity and enhancer of split 1 (Hes1) complex are described. A high-throughput screening assay for small-molecule inhibitors of the FANCF-Hes1 complex was realized. Successful complex formation between fluorophore (Cy3)-labeled human FANCF and immobilized rat or human HES1 on a microplate was established. Screening of our plant extract library using this system resulted in the isolation of eight natural products, including two new flavonoid glycosides (3 and 4), from Wrightia religiosa. Of these compounds, 3, 5, and 7 showed potent inhibition of the FANCF-Hes1 complex formation. Compound 7 disrupted the FANCF-Hes1 complex more efficiently than the Hes1 dimer.

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Prenylflavonoids isolated from Artocarpus champeden with TRAIL-resistance overcoming activity (vol 96, pg 299, 2013)

Tomohiro Minakawa, Kazufumi Toume, Midori A. Arai, Takashi Koyano, Thaworn Kowithayakorn, Masami Ishibashi

PHYTOCHEMISTRY (PERGAMON-ELSEVIER SCIENCE LTD) 106 207 - 207 2014.10

Accepted, ISSN 0031-9422

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Ricinine: A pyridone alkaloid from Ricinus communis that activates the Wnt signaling pathway through casein kinase 1 alpha

Kensuke Ohishi, Kazufumi Toume, Midori A. Arai, Samir K. Sadhu, Firoj Ahmed, Takamasa Mizoguchi, Motoyuki Itoh, Masami Ishibashi

BIOORGANIC & MEDICINAL CHEMISTRY (PERGAMON-ELSEVIER SCIENCE LTD) 22 ( 17 ) 4597 - 4601 2014.09

Accepted, ISSN 0968-0896

Wnt signaling plays important roles in proliferation, differentiation, development of cells, and various diseases. Activity-guided fractionation of the MeOH extract of the Ricinus communis stem led to the isolation of four compounds (1-4). The TCF/beta-catenin transcription activities of 1 and 3 were 2.2 and 2.5 fold higher at 20 and 30 mu M, respectively. Cells treated with ricinine (1) had higher beta-catenin and lower of p-beta-catenin (ser 33, 37, 45, Thr 41) protein levels, whereas glycogen synthase kinase 3 beta (GSK3 beta) and casein kinase 1 alpha (CK1 alpha) protein levels remained unchanged. Cells treated with pyrvinium, an activator of CK1 alpha, had lower beta-catenin levels. However, the combined treatment of pyrvinium and 1 led to higher beta-catenin levels than those in cells treated with pyrvinium alone, which suggested that 1 inhibited CK1 alpha activity. Furthermore, 1 increased beta-catenin protein levels in zebrafish embryos. These results indicated that 1 activated the Wnt signaling pathway by inhibiting CK1 alpha. (C) 2014 Elsevier Ltd. All rights reserved.

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Themeda arguens等からのヘッジホッグ伝達経路阻害剤の探索

米山達朗, 荒井緑, 小谷野喬, Kowithayakorn Thaworn, Sadhu Samir K., Ahmed Firoj, 石橋正己

日本生薬学会年会講演要旨集 ((一社)日本生薬学会) 61回 113 - 113 2014.08

ISSN 0919-1992

Chromomycins A(2) and A(3) from Marine Actinomycetes with TRAIL Resistance-Overcoming and Wnt Signal Inhibitory Activities

Toume, Kazufumi, Tsukahara, Kentaro, Ito, Hanako, Arai, Midori A., Ishibashi, Masami

MARINE DRUGS (MDPI AG) 12 ( 6 ) 3466 - 3476 2014.06

Accepted, ISSN 1660-3397

A biological screening study of an actinomycetes strain assembly was conducted using a cell-based cytotoxicity assay. The CKK1019 strain was isolated from a sea sand sample. Cytotoxicity-guided fractionation of the CKK1019 strain culture broth, which exhibited cytotoxicity, led to the isolation of chromomycins A(2) (1) and A(3) (2). 1 and 2 showed potent cytotoxicity against the human gastric adenocarcinoma (AGS) cell line (IC50 1; 1.7 and 2; 22.1 nM), as well as strong inhibitory effects against TCF/beta-catenin transcription (IC50 1; 1.8 and 2; 15.9 nM). 2 showed the ability to overcome tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) resistance. To the best of our knowledge, the effects of chromomycins A(2) (1) and A(3) (2) on TRAIL resistance-overcoming activity, and on the Wnt signaling pathway, have not been reported previously. Thus, 1 and 2 warrant potential drug lead studies in relation to TRAIL-resistant and Wnt signal-related diseases and offer potentially useful chemical probes for investigating TRAIL resistance and the Wnt signaling pathway.

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Calotropin: A Cardenolide from Calotropis gigantea that Inhibits Wnt Signaling by Increasing Casein Kinase 1 alpha in Colon Cancer Cells

Hyun Young Park, Kazufumi Toume, Midori A. Arai, Samir K. Sadhu, Firoj Ahmed, Masami Ishibashi

CHEMBIOCHEM (WILEY-V C H VERLAG GMBH) 15 ( 6 ) 872 - 878 2014.04

Accepted, ISSN 1439-4227

Wnt signaling plays key roles in embryonic development and various human diseases. Activity-guided testing to isolate Wnt signaling inhibitors from the methanol extract of Calotropis gigantea (Asclepiadaceae) exudutes identified six Wnt inhibitory cardenolides (1-6), of which 1, 3, 5, and 6 exhibited potent TCF/-catenin inhibitory activities (IC50 0.7-3.6 nM). Calotropin (1) inhibited Wnt signaling by decreasing both nuclear and cytosolic -catenin in a dose-dependent manner, and promoted degradation of -catenin by increasing the phosphorylation of -catenin at Ser45 through casein kinase 1 (CK1). Moreover, 1 significantly increased CK1 protein and mRNA levels. The results suggest that 1 inhibits the Wnt signaling pathway by increasing CK1 protein levels. To the best of our knowledge, calotropin is the first small molecule to increase CK1 levels.

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Themeda arguens等からのヘッジホッグ伝達経路阻害剤の探索

米山達朗, 荒井緑, 小谷野喬, Kowithayakorn Thaworn, Sadhu Samir K., Ahmed Firoj, 石橋正己

日本薬学会年会要旨集 ((公社)日本薬学会) 134年会 ( 2 ) 71 - 71 2014.03

ISSN 0918-9823

Eudesmane-Type Sesquiterpenoid and Guaianolides from Kandelia candel in a Screening Program for Compounds to Overcome TRAIL Resistance (vol 75, pg 1431, 2012)

Tomohiro Minakawa, Kazufumi Toume, Midori A. Arai, Samir K. Sadhu, Firoj Ahmed, Masami Ishibashi

JOURNAL OF NATURAL PRODUCTS (AMER CHEMICAL SOC) 77 ( 2 ) 437 - 437 2014.02

Accepted, ISSN 0163-3864

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beta-Sitosterol and flavonoids isolated from Bauhinia malabarica found during screening for Wnt signaling inhibitory activity

Hyun Young Park, Kazufumi Toume, Midori A. Arai, Takashi Koyano, Thaworn Kowithayakorn, Masami Ishibashi

JOURNAL OF NATURAL MEDICINES (SPRINGER JAPAN KK) 68 ( 1 ) 242 - 245 2014.01

Accepted, ISSN 1340-3443

Screening with a cell-based luciferase assay was conducted to identify bioactive natural products which inhibit Wnt signaling activity-guided separation of an MeOH extract of Bauhinia malabarica (Caesalpiniaceae) leaves yielded five compounds, which were identified as beta-sitosterol (1), quercetin (2), 6,8-C-dimethyl kaempferol-3-O-rhamnopyranoside (3), hyperin (4), and 6,8-C-dimethyl kaempferol-3-methyl ether (5). The tested compounds 1, 3, and 5 exhibited Wnt signaling inhibitory activity, with IC50 values of 0.77, 0.74, and 16.6 mu M, respectively.

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CONSTITUENTS FROM THE RHIZOMES OF CURCUMA COMOSA AND THEIR WNT SIGNAL INHIBITORY ACTIVITIES

Rolly G. Fuentes, Kazufumi Toume, Midori A. Arai, Takashi Koyano, Thaworn Kowithayakorn, Masami Ishibashi

HETEROCYCLES (PERGAMON-ELSEVIER SCIENCE LTD) 88 ( 2 ) 1501 - 1509 2014.01

Accepted, ISSN 0385-5414

Activity-guided fractionation of the Curcuma comosa rhizome MeOH extract that inhibited Wnt signal activity led to the characterization of two new diarylheptanoids (1 and 2) together with six known diarylheptanoids (3-8) and two known sesquiterpenoids (9 and 10). Their structures were identified by 1D and 2D NMR, HRESIMS, optical rotation, and chemical evidences. Compounds 1 and 8 exhibited dose-dependent inhibitory activity against beta-catenin/TCF transcriptional activity.

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Physalin H from Solanum nigrum as an Hh signaling inhibitor blocks GLI1-DNA-complex formation

Midori A. Arai, Kyoko Uchida, Samir K. Sadhu, Firoj Ahmed, Masami Ishibashi

BEILSTEIN JOURNAL OF ORGANIC CHEMISTRY (BEILSTEIN-INSTITUT) 10 134 - 140 2014.01

Accepted, ISSN 1860-5397

Hedgehog (Hh) signaling plays an important role in embryonic development, cell maintenance and cell proliferation. Moreover, Hh signaling contributes to the growth of cancer cells. Physalins are highly oxidized natural products with a complex structure. Physalins (1-7) were isolated from Solanum nigrum (Solanaceae) collected in Bangladesh by using our cell-based assay. The isolated physalins included the previously reported Hh inhibitors 5 and 6. Compounds 1 and 4 showed strong inhibition of GLI1 transcriptional activity, and exhibited cytotoxicity against cancer cell lines with an aberrant activation of Hh signaling. Compound 1 inhibited the production of the Hh-related proteins patched (PTCH) and BCL2. Analysis of the structures of different physalins showed that the left part of the physalins was important for Hh inhibitory activity. Interestingly, physalin H (1) disrupted GLI1 binding to its DNA binding domain, while the weak inhibitor physalin G (2) did not show inhibition of GLI1-DNA complex formation.

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Calotropin, a cardenolide from Calotropis gigantea, with Wnt signal inhibition activity through increase of casein kinase 1 alpha

Park, H. Y., Toume, K., Arai, M. A., Sadhu, S. K., Ahmed, F., Ishibashi, M.

Planta Medica 80 ( 10 ) 818 - 818 2014

Accepted

New aromatic heterocycles with TRAIL resistance overcoming activity isolated from actinomycetes collected in Chiba area

Abdelfattah, M. S., Toume, K., Arai, M. A., Ishibashi, M.

Planta Medica 80 ( 10 ) 2014

Accepted

Prenylflavonoids isolated from Artocarpus champeden with TRAIL-resistance overcoming activity

Tomohiro Minakawa, Kazufumi Toume, Midori A. Arai, Takashi Koyano, Thaworn Kowithayakorn, Masami Ishibashi

PHYTOCHEMISTRY (PERGAMON-ELSEVIER SCIENCE LTD) 96 299 - 304 2013.12

Accepted, ISSN 0031-9422

In a screening program for bioactive natural products which can overcome Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-resistance, three prenylflavonoids, named pannokin A-C, were isolated from a MeOH extract of Artocarpus champeden (Moraceae) roots, together with three known prenylflavonoids. The structures of pannokin A-C were elucidated by spectroscopic analysis. These of the prenylflavonoids in combination with TRAIL, showed cytotoxic activity in sensitizing TRAIL-resistant human gastric adenocarcinoma (AGS) cells. Of these compounds, heterophyllin increased caspase 3/7 activity when combined with TRAIL in AGS cells, and enhanced the expression of DR4 and DR5 mRNA. Moreover, heterophyllin up-regulated mRNA expression of CCAAT/enhancer-binding protein-homologous protein (CHOP) which was reported to be an important regulator of DR5 expression. Thus, heterophyllin was presumed to cause a CHOP-dependent up-regulation of DR5 expression resulting in apoptosis in AGS cells. (C) 2013 Elsevier Ltd. All rights reserved.

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Xylogranin B: A Potent Wnt Signal Inhibitory Limonoid from Xylocarpus granatum

Toume, Kazufumi, Kamiya, Kentaro, Arai, Midori A., Mori, Naomi, Sadhu, Samir K., Ahmed, Firoj, Ishibashi, Masami

ORGANIC LETTERS (AMER CHEMICAL SOC) 15 ( 23 ) 6106 - 6109 2013.12

Accepted, ISSN 1523-7060

Xylogranin B (2) was isolated from Xylocarpus granatum (Meliaceae) leaves, by use of a cell-based luciferase screening system targeting a Wnt signaling pathway. Compound 2 inhibited TCF/beta-catenin transcriptional activity (IC50 48.9 nM) and exhibited strong cytotoxicity against colon cancer cell lines. Compound 2 significantly decreased beta-catenin protein levels in nuclei but not in the cytosol. These results indicated that a decrease in beta-catenin levels in nuclei by 2 resulted in the Wnt signal inhibitory effects of 2.

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Hh signaling inhibitors from Vitex negundo; Naturally occurring inhibitors of the GLI1-DNA complex

Midori A. Arai, Teruhisa Fujimatsu, Kyoko Uchida, Samir K. Sadhu, Firoj Ahmed, Masami Ishibashi

Molecular BioSystems 9 ( 5 ) 1012 - 1018 2013.05

Accepted, ISSN 1742-206X

The hedgehog (Hh) signaling pathway has crucial roles in embryonic development, cell maintenance and proliferation, and is also known to contribute to cancer cell growth. New naturally occurring Hh inhibitors (1, 7 and 9) were isolated from Vitex negundo using our previously constructed cell-based assay. Bioactivity guided isolation provided 9 natural compounds including a new diterpene, nishindanol (9). Compounds 7 and 9 showed cytotoxicity against cancer cell lines in which Hh signaling was aberrantly activated. Vitetrifolin D (7
GLI1 transcriptional inhibition IC50 = 20.2 μM) showed inhibition of Hh related protein (PTCH and BCL2) production. Interestingly, the constructed electrophoresis mobility shift assay revealed that vitetrifolin D (7) disrupted GLI1 binding on its DNA binding domain. epi-Sclareol (8
inactive), possessing a similar structure to 7, did not show inhibition of GLI1-DNA complex formation. This is the first example of naturally occurring inhibitors of GLI1-DNA complex formation. © 2013 The Royal Society of Chemistry.

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Merging Chemistry and Biology in Emerging Countries

Takayoshi Suzuki, Midori A. Arai, Mitsue Nakashima, Motonari Uesugi

CHEMISTRY & BIOLOGY (CELL PRESS) 20 ( 4 ) 461 - 465 2013.04

Accepted, ISSN 1074-5521

Chemical biology is not a new discipline, as research at the interface of chemistry and biology has been growing for decades. However, the field still needs regional and global support in emerging countries. The meeting organized by Asian Chemical Biology Initiative in Bangkok, Thailand in January 2013, brought together regional communities of young and established researchers to nurture chemical biology research programs and highlight some of the newer developments. Here, we report on the meeting and some of the key research topics discussed.

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Catalytic Asymmetric Synthesis of Mixed 3,3 '-Bisindoles and Their Evaluation as Wnt Signaling Inhibitors

Takayoshi Arai, Yushi Yamamoto, Atsuko Awata, Kentaro Kamiya, Masami Ishibashi, Midori A. Arai

ANGEWANDTE CHEMIE-INTERNATIONAL EDITION (WILEY-V C H VERLAG GMBH) 52 ( 9 ) 2486 - 2490 2013

Accepted, ISSN 1433-7851

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Naturally occurring Ngn2 promoter activators from Butea superba

Midori A. Arai, Kazune Koryudzu, Takashi Koyano, Thaworn Kowithayakorn, Masami Ishibashi

MOLECULAR BIOSYSTEMS (ROYAL SOC CHEMISTRY) 9 ( 10 ) 2489 - 2497 2013

Accepted, ISSN 1742-206X

Neurogenin2 (Ngn2), an activator-type bHLH transcriptional factor, promotes differentiation of neural stem cells into neurons by transcription of pro-neural genes. To find neural stem cell accelerators from the extract library of natural resources, we used a two-step screening including a Ngn2 promoter reporter gene screening and differentiation assay screening of neural stem cells. A reporter gene assay that can detect Ngn2 promoter activity by luciferase expression was constructed using C3H10T1/2 cells. Using this primary cell-based screening, Butea superba was found to include Ngn2 promoter activators from our tropical plant extract libraries. Bioassay-guided fractionation of this plant extract led to the isolation of 18 natural products, including pterocarpans and isoflavonoids. Dehydromaackiain (1), formononetin (6), (-)-variabilin (13), (-)-medicarpin (14), rothindin (17) and ononin (18) showed 1.8-2.8 times higher Ngn2 promoter activity at 5 mu M compared with control. Of active natural compounds, 3'-methoxydaidzein (3) showed promotion of neurite outgrowth of C17.2 in a secondary screen. 3'-Methoxydaidzein (3) increased mRNA expression of pro-neural transcriptional factors (Ngn2, Ngn1, NeuroD2), a mature neuron-specific enzyme GAD1 and a pro-neural neurotrophic growth factor neurotrophin 3 (NT3) in C17.2 neural stem cells.

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GRISEOVIRIDIN AND CYCLIC HYDROXAMATES FOUND IN A SCREENING PROGRAM FOR WNT SIGNAL INHIBITOR

Tamai, Yuuya, Toume, Kazufumi, Arai, Midori A., Ishibashi, Masami

HETEROCYCLES (PERGAMON-ELSEVIER SCIENCE LTD) 86 ( 2 ) 1517 - 1524 2012.12

Accepted, ISSN 0385-5414

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Cycloartane Triterpenes and Ingol Diterpenes Isolated from Euphorbia neriifolia in a Screening Program for Death-Receptor Expression-Enhancing Activity

Toume, Kazufumi, Nakazawa, Takafumi, Hoque, Tahmina, Ohtsuki, Takashi, Arai, Midori A., Koyano, Takashi, Kowithayakorn, Thaworn, Ishibashi, Masami

PLANTA MEDICA (GEORG THIEME VERLAG KG) 78 ( 12 ) 1370 - 1377 2012.08

Accepted, ISSN 0032-0943

In our screening program for natural products that increase death-receptor 5 expression, seven new cycloartane triterpenes, euphonerins A-G (1-7), and 3-O-acetyl-8-O-tigloylingol (8), a new ingol diterpene, were isolated from the MeOH extract of Euphorbia neriifolia leaves, together with 3,12-di-O-acetyl-8-O-tigloylingol (9), (24R)-cycloartane-3 beta,24,25-triol (10), and three known flavonols (11-13). The structures of 1-8 were elucidated by spectroscopic analysis. Among these compounds, 1-11 showed death-receptor 5 expression enhancing activity.

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Eudesmane-Type Sesquiterpenoid and Guaianolides from Kandelia candel in a Screening Program for Compounds to Overcome TRAIL Resistance

Tomohiro Minakawa, Kazufumi Toume, Midori A. Arai, Samir K. Sadhu, Firoj Ahmed, Masami Ishibashi

JOURNAL OF NATURAL PRODUCTS (AMER CHEMICAL SOC) 75 ( 8 ) 1431 - 1435 2012.08

Accepted, ISSN 0163-3864

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BIOACTIVE NATURAL PRODUCTS FROM MYXOMYCETES HAVING EFFECTS ON SIGNALING PATHWAYS

Masami Ishibashi, Midori A. Arai

HETEROCYCLES (PERGAMON-ELSEVIER SCIENCE LTD) 85 ( 6 ) 1299 - 1332 2012.06

Accepted, ISSN 0385-5414

The myxomycetes (true slime molds) are an unusual group of primitive organisms that may be assigned to one of the lowest classes of eukaryotes. We studied the chemical constituents of a laboratory culture of plasmodia as well as field-collected fruit bodies of myxomycetes, and isolated various compounds with unique chemical structures and bioactivities. We recently also performed screening studies of natural products targeting signaling pathways related to basic studies of diseases and life sciences. Among myxomycetes constituents, we found several bioactive compounds that have effects on signaling pathways, including a cycloanthranilylproline with TRAIL resistance-overcoming activity, peptide lactone derivatives with Wnt signal inhibitory activity, a naphthoquinone pigment with Hes1 dimerization inhibition activity, and bisindole alkaloids with hedgehog signal inhibitory activity. Here we describe our recent results of their isolation, structure elucidation, synthesis, and findings in bioactivity studies.

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Katorazone, a new yellow pigment with a 2-azaquinone-phenylhydrazone structure produced by Streptomyces sp IFM 11299

Mohamed S. Abdelfattah, Kazufumi Toume, Midori A. Arai, Hyuma Masu, Masami Ishibashi

TETRAHEDRON LETTERS (PERGAMON-ELSEVIER SCIENCE LTD) 53 ( 26 ) 3346 - 3348 2012.06

Accepted, ISSN 0040-4039

An unusual alkaloid with a 2-azaquinone-phenylhydrazone structure, katorazone (1), and other metabolites were isolated from the ethyl acetate extract of Streptomyces sp. IFM 11299. The chemical structure of katorazone (1) was elucidated by 1D and 2D NMR analyses together with HR-ESI mass spectrometry. Katorazone (1) showed a synergistic effect in combination with TRAIL and decreased the viability of human gastric adenocarcinoma (AGS) cells. (C) 2012 Elsevier Ltd. All rights reserved.

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Phorbol Esters with Wnt Signal-Augmenting Effects Isolated from Excoecaria indica

Yamaguchi, Tatsuhiro, Toume, Kazufumi, Arai, Midori A., Ahmed, Firoj, Sadhu, Samir K., Ishibashi, Masami

NATURAL PRODUCT COMMUNICATIONS (SAGE PUBLICATIONS INC) 7 ( 4 ) 475 - 477 2012.04

Accepted, ISSN 1934-578X

A New Resin Glycoside from Ipomoea maxima

Kazune Koryudzu, Midori A. Arai, Firoj Ahmed, Samir K. Sadhu, Masami Ishibashi

NATURAL PRODUCT COMMUNICATIONS (NATURAL PRODUCTS INC) 7 ( 2 ) 219 - 220 2012.02

Accepted, ISSN 1934-578X

NONACTIN AND RELATED COMPOUNDS FOUND IN A SCREENING PROGRAM FOR WNT SIGNAL INHIBITORY ACTIVITY

Yuuya Tamai, Kazufumi Toume, Midori A. Arai, Akinori Hayashida, Hikaru Kato, Yoshikazu Shizuri, Sachiko Tsukamoto, Masami Ishibashi

HETEROCYCLES (PERGAMON-ELSEVIER SCIENCE LTD) 84 ( 2 ) 1245 - 1250 2012.01

Accepted, ISSN 0385-5414

A screening study using a luciferase assay to identify natural products with inhibitory activity against Wnt signaling was carried out. Fractionation of the culture extract of an actinomycete, strain CKK179, led to the identification of nonactin (1), monactin (2), and dinactin (3) as active compounds. We examined the effects of dimeric dinactin (4), isolated from another actinomycete, Streptomyces sp. (YM09-028), on Wnt signaling activity and found that 1 - 4 inhibited TCF/beta-catenin transcriptional activity with IC50 values of 0.6 - 7.4 nM.

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Mechanism of the anti-proliferative action of 25-hydroxy-19-nor-vitamin D-3 in human prostate cells

Eiji Munetsuna, Sachie Nakabayashi, Rie Kawanami, Kaori Yasuda, Miho Ohta, Midori A. Arai, Atsushi Kittaka, Tai C. Chen, Masaki Kamakura, Shinichi Ikushiro, Toshiyuki Sakaki

JOURNAL OF MOLECULAR ENDOCRINOLOGY (BIOSCIENTIFICA LTD) 47 ( 2 ) 209 - 218 2011.10

Accepted, ISSN 0952-5041

According to the prevailing paradigm, 1 alpha-hydroxylation of 25-hydroxyvitamin D-3 (25(OH)D-3) and its analogs is a prerequisite step for their biological effects. We previously reported that 25-hydroxy-19-nor-vitamin D-3 (25(OH)-19-nor-D-3) had anti-proliferative activity in a cell line, PZ-HPV-7, which was derived from human non-cancerous prostate tissue, and suggested that 25(OH)-19-nor-D-3 acted after 1 alpha-hydroxylation by vitamin D 1 alpha-hydroxylase (CYP27B1). However, metabolic studies of 25(OH)-19-nor-D-3 using recombinant CYP27B1 revealed that 25(OH)-19-nor-D-3 was rarely subjected to 1 alpha-hydroxylation. Therefore, in this report, we attempted to clarify the mechanism of 25(OH)-19-nor-D-3 action in intact cells using PZ-HPV-7 prostate cells. After incubating the cells with 25(OH)-19-nor-D-3, eight metabolites of 24-hydroxylase (CYP24A1) were detected, whereas no products of CYP27B1 including 1 alpha,25-dihydroxy-19-nor-vitamin D-3 (1 alpha,25(OH)(2)-19-nor-D-3) were found. Furthermore, the time-dependent nuclear translocation of vitamin D receptor (VDR) and the subsequent transactivation of cyp24A1 gene in the presence of 25(OH)-19-nor-D-3 were almost identical as those induced by 1 alpha,25(OH)(2)-19-nor-D-3. These results strongly suggest that 25(OH)-19-nor-D-3 directly binds to VDR as a ligand and transports VDR into the nucleus to induce transcription of cyp24A1 gene. In addition, knock down of cyp27B1 gene did not affect the anti-proliferative activity of 25(OH)-19-nor-D-3, whereas knock down of VDR attenuated the inhibitory effect. Thus, our results clearly demonstrate that the anti-proliferative activity of 25(OH)-19-nor-D-3 is VDR dependent but 1 alpha-hydroxylation independent, suggesting that 25(OH)D-3 analogs such as 25(OH)-19-nor-D-3 could be attractive candidates for anticancer therapy. Journal of Molecular Endocrinology (2011) 47, 209-218

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Acoschimperoside P, 2 '-acetate: a Hedgehog signaling inhibitory constituent from Vallaris glabra

Yusnita Rifai, Midori A. Arai, Takashi Koyano, Thaworn Kowithayakorn, Masami Ishibashi

JOURNAL OF NATURAL MEDICINES (SPRINGER TOKYO) 65 ( 3-4 ) 629 - 632 2011.07

Accepted, ISSN 1340-3443

Complete (1)H and (13)C NMR assignments of acoschimperoside P, 2'-acetate (1) and a new cardiac glycoside (2), isolated from the leaves of Vallaris glabra, are described. Compound 1 was active in the assay for Hedgehog signaling inhibition. In further experiments, this compound showed a strong cytotoxicity against human pancreatic (PANC1) and human prostate (DU145) cancer cells. The expression of GLI-related proteins (PTCH and BCL-2) in a dose-dependent manner was also inhibited by 1.

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Approaches to Neural Stem Cells and Cancer Cells Based on Natural Products

Midori A. Arai

CHEMICAL & PHARMACEUTICAL BULLETIN (PHARMACEUTICAL SOC JAPAN) 59 ( 4 ) 417 - 426 2011.04

Accepted, ISSN 0009-2363

We have developed "protein- and cell-based screen methods" for the isolation of new natural products. By using VDR (vitamin D receptor) immabilized magnetic beads, two new natural products were isolated rapidly. To find inhibitors of Hes1 protein, which is one of the important transcriptional factors in neural stem cells, a Hes1 dimer plate assay was developed, and then first Hes1 dimer inhibitors were found from our natural products library. As a "cell-based screen method," a reporter gene assay for screening Hh (hedgehog) signaling inhibitors was constructed. New Hh signaling inhibitors were isolated from our natural extracts library. We evaluated their Hh inhibitory activity in Hh related protein synthesis and cytotoxicity against cancers, in which Hh signaling is aberrantly activated. In addition to the naturally made compounds library (extracts library), "small molecules based on natural products" were synthesized. The total synthesis of Melleumin A, B, which were isolated by our group, was achieved. From their synthetic derivatives, inhibitors of the Wnt signal, which has been reported to cause colon cancers, were discovered. Moreover, flavanone and chromone were selected as natural product scaffolds. Many flavonoids and chromones with diverse heterocyclic units were constructed using our efficient synthetic method.

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Cycloartane Triterpenes Isolated from Combretum quadrangulare in a Screening Program for Death-Receptor Expression Enhancing Activity

Toume, Kazufumi, Nakazawa, Takafumi, Ohtsuki, Takashi, Arai, Midori A., Koyano, Takashi, Kowithayakorn, Thaworn, Ishibashi, Masami

JOURNAL OF NATURAL PRODUCTS (AMER CHEMICAL SOC) 74 ( 2 ) 249 - 255 2011.02

Accepted, ISSN 0163-3864

In our screening program for natural products that increase DR5 (death-receptor 5) expression, nine new cycloartane triterpenes, combretanones A-G (1-7), combretic acid A (8), and combretic acid B (9), were isolated from a MeOH extract of Combretum quadrangulare leaves. The known oleanane triterpenes (10, 11) and six known flavonols (12-17) were also isolated. The structures of 1-9 were elucidated by spectroscopic studies. Compounds 7, 9, 12, 16, and 17 enhanced DRS expression, and 16 showed TRAIL-resistance abrogating activity.

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Tubiferic Acid, a New 9,10-Secocycloartane Triterpenoid Acid Isolated from the Myxomycete Tubulifera arachnoidea

Yukihito Ippongi, Takashi Ohtsuki, Kazufumi Toume, Midori A. Arai, Yukinori Yamamoto, Masami Ishibashi

CHEMICAL & PHARMACEUTICAL BULLETIN (PHARMACEUTICAL SOC JAPAN) 59 ( 2 ) 279 - 281 2011.02

Accepted, ISSN 0009-2363

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2-Methoxy-1,4-naphthoquinone isolated from Impatiens balsamina in a screening program for activity to inhibit Wnt signaling

Naomi Mori, Kazufumi Toume, Midori A. Arai, Takashi Koyano, Thaworn Kowithayakorn, Masami Ishibashi

JOURNAL OF NATURAL MEDICINES (SPRINGER TOKYO) 65 ( 1 ) 234 - 236 2011.01

Accepted, ISSN 1340-3443

A screening study using a luciferase assay to identify natural products which inhibit Wnt signaling was carried out. The bioassay-guided fractionation of aerial parts of a plant, Impatiens balsamina, led to the isolation of 2-methoxy-1,4-naphthoquinone (1) as an active compound. Compound 1 inhibited the TCF/beta-catenin (TOP) transcriptional activity (IC(50) 2.9 A mu M), while it decreased the transcriptional activity of FOP (mutated TCF-binding site)-transfected cells at > 5 A mu M.

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New Hedgehog/GLI signaling inhibitors from Excoecaria agallocha

Yusnita Rifai, Midori A. Arai, Samir K. Sadhu, Firoj Ahmed, Masami Ishibashi

BIOORGANIC & MEDICINAL CHEMISTRY LETTERS (PERGAMON-ELSEVIER SCIENCE LTD) 21 ( 2 ) 718 - 722 2011.01

Accepted, ISSN 0960-894X

The inhibition of the Hedgehog (Hh) signaling pathway has emerged as an anti-cancer strategy. Three flavonoid glycosides including 2 new compounds (1-2) were isolated from Excoecaria agallocha as Hedgehog/GLI1-mediated transcriptional inhibitors and exhibited cytotoxicity against human pancreatic (PANC1) and prostate (DU145) cancer cells. Our data revealed that compound 1 clearly inhibited the expression of GLI-related proteins (PTCH and BCL-2) and blocked the translocation of GLI1 transcription factors into the nucleus in PANC1. Deleting the Smoothened (Smo) function in PANC1 treated with 1 led to downregulation of the mRNA expression of Ptch. This study describes the first Hh signaling inhibitor which blocks GLI1 movement into the nucleus without interfering with Smo. (C) 2010 Elsevier Ltd. All rights reserved.

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New hedgehog/GLI-signaling inhibitors from Adenium obesum

Midori A. Arai, Chikashi Tateno, Takashi Koyano, Thaworn Kowithayakorn, Seiichiro Kawabe, Masami Ishibashi

ORGANIC & BIOMOLECULAR CHEMISTRY (ROYAL SOC CHEMISTRY) 9 ( 4 ) 1133 - 1139 2011

Accepted, ISSN 1477-0520

The aberrant hedgehog (Hh)/GLI signaling pathway causes the formation and progression of a variety of tumors. We recently constructed a cell-based screening system to search for Hh/GLI signaling inhibitors from natural resources. Using our screening system, Adenium obesum was found to include Hh/GLI signaling inhibitors from our tropical plant extract libraries. Bioassay-guided fractionation of this plant extract led to the isolation of 17 cardiac glycosides (1-17), including 3 new compounds (4, 9, 16). These compounds showed strong inhibitory activities, especially the IC(50) of 17 is 0.11 mu M. The inhibition of GLI-related protein expression with 3, 9, 11, 15 and 17 was observed in human pancreatic cancer cells (PANC1), which express Hh/GLI components aberrantly. The expressions of GLI-related proteins PTCH and BCL2 were clearly inhibited. These compounds also showed selective cytotoxicity against two cancer cell lines, with less effect against normal cells (C3H10T1/2). RT-PCT examinations showed that Ptch mRNA expression by 3, 11, 15 and 17 was inhibited.

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COMPOUND ANALYSIS VIA GRAPH KERNELS INCORPORATING CHIRALITY

J. B. Brown, Takashi Urata, Takeyuki Tamura, Midori A. Arai, Takeo Kawabata, Tatsuya Akutsu

JOURNAL OF BIOINFORMATICS AND COMPUTATIONAL BIOLOGY (IMPERIAL COLLEGE PRESS) 8 63 - 81 2010.12

Accepted, ISSN 0219-7200

High accuracy is paramount when predicting biochemical characteristics using Quantitative Structural-Property Relationships (QSPRs). Although existing graph-theoretic kernel methods combined with machine learning techniques are efficient for QSPR model construction, they cannot distinguish topologically identical chiral compounds which often exhibit different biological characteristics. In this paper, we propose a new method that extends the recently developed tree pattern graph kernel to accommodate stereoisomers. We show that Support Vector Regression (SVR) with a chiral graph kernel is useful for target property prediction by demonstrating its application to a set of human vitamin D receptor ligands currently under consideration for their potential anti-cancer effects.

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STUDIES ON THE SYNTHESIS AND METABOLISM OF 14-epi-2 alpha-(3-HYDROXYPROPYL)-19-NORVITAMIN D-3 AND ITS 2 beta-ISOMER

Atsushi Kittaka, Hideki Hara, Kaori Yasuda, Masashi Takano, Midori A. Arai, Daisuke Sawada, Hiroshi Saito, Kazuya Takenouchi, Tai C. Chen, Toshiyuki Sakaki

HETEROCYCLES (PERGAMON-ELSEVIER SCIENCE LTD) 82 ( 1 ) 649 - 661 2010.12

Accepted, ISSN 0385-5414

Two derivatives of 14-epi-1 alpha,25-Dihydroxy-19-norvitamin D-3, 14-epi-2 alpha-(3-hydroxypropyl)-1 alpha,25-dihydroxy-19-norvitamin D-3 (14-epi-MART-10) and its 2-epimeric analog (14-epi-MART-11), were synthesized using Julia coupling reaction to connect between the C5 position (steroidal numbering) of an A-ring precursor ketone derived from (-)-quinic acid and the C6 position of a protected 14-epi-CD-ring benzothiazole sulfone. The coupling and deprotection reactions generated a mixture of 14-epi-MART-10 and 14-epi-MART-11 in a moderate yield. The C2-isomers were then separated as their pivalate forms. The C2-stereochemistry of 2 alpha- and 2 beta-isomers was determined by H-1 NMR studies including NOE experiments. The pivaloyl group was removed under basic conditions to obtain the target molecules of 14-epi-MART-10 and 14-epi-MART-11. The metabolism of these two new analogs was further studied in a reconstituted cell-free human CYP24A1 system to elucidate the potential mechanism of their super agonistic action on vitamin D receptor. Our results indicate that epimerization at C14 makes the analogs less susceptible to CYP24A1 degradation and therefore more bio-available, leading to enhanced biological activities.

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Constituents of Pongamia pinnata Isolated in a Screening for Activity to Overcome Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Resistance

Tomohiro Minakawa, Kazufumi Toume, Firoj Ahmed, Samir Kumar Sadhu, Takashi Ohtsuki, Midori A. Arai, Masami Ishibasha

CHEMICAL & PHARMACEUTICAL BULLETIN (PHARMACEUTICAL SOC JAPAN) 58 ( 11 ) 1549 - 1551 2010.11

Accepted, ISSN 0009-2363

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A Bisindole Alkaloid with Hedgehog Signal Inhibitory Activity from the Myxomycete Perichaena chrysosperma

Akinori Shintani, Kazufumi Toume, Yusnita Rifai, Midori A. Arai, Masami Ishibashi

JOURNAL OF NATURAL PRODUCTS (AMER CHEMICAL SOC) 73 ( 10 ) 1711 - 1713 2010.10

Accepted, ISSN 0163-3864

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Practical Total Synthesis of Fuligocandins A and B

Midori A. Arai, Junya Seto, Firoj Ahmed, Kento Uchiyama, Masami Ishibashi

SYNLETT (GEORG THIEME VERLAG KG) ( 16 ) 2498 - 2502 2010.10

Accepted, ISSN 0936-5214

Fuligocandins A and B, cycloanthranilylproline derivatives isolated from the myxomycete Fuligo candida, were synthesized efficiently by a Meyer-Schuster rearrangement, which gave Z-isomer with high selectivity. The absolute stereochemistry at C-11a of the natural products was determined to be S by comparison of the optical rotation of the natural products with synthetic counterparts derived from L-proline. This report is the first total synthesis of optically active fuligocandin B.

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Synthesis of vitamin D analogs as ligands for mutant human vitamin D receptor (Arg274Leu)

Shinobu Honzawa, Naoyuki Takahashi, Atsushi Yamashita, Takayuki Sugiura, Midori A. Arai, Shigeaki Kato, Masaaki Kurihara, Atsushi Kittaka

ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY (AMER CHEMICAL SOC) 240 2010.08

Accepted, ISSN 0065-7727

Terpenoids and a Flavonoid Glycoside from Acacia pennata Leaves as Hedgehog/GLI-Mediated Transcriptional Inhibitors

Yusnita Rifai, Midori A. Arai, Takashi Koyano, Thaworn Kowithayakorn, Masami Ishibashi

JOURNAL OF NATURAL PRODUCTS (AMER CHEMICAL SOC) 73 ( 5 ) 995 - 997 2010.05

Accepted, ISSN 0163-3864

Overexpression of glioma-associated oncogene 1 (GLI1), which has been characterized as a terminal effector and a target gene of the Hedgehog (Hh) signaling pathway, is associated with the development of cancer. A cellular screen was applied utilizing of a GLI-dependent luciferase reporter in human keratinocyte cells (HaCaT) and identified two terpenoids (1 and 2) and a flavonoid glycoside (5) from Acacia pennata as Hh/GLI inhibitors. Compounds 1, 2, and 5 exhibited selective cytotoxicity against human pancreatic (PANC1) and prostate (DU145) cancer cells with no toxic effect on normal cells. This result was consistent with a dose-dependent reduction of the protein levels of antiapoptotic BCL-2 and the tumor suppressor patched 1 protein (PTCH). Additionally, treatment of 1 downregulated mRNA expression of Ptch in PANC1, suggesting that the compound has an inhibitory effect on the transcription of Hh/GLI.

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Constituents of Amoora cucullata with TRAIL resistance-overcoming activity

Firoj Ahmed, Kazufumi Toume, Samir K. Sadhu, Takashi Ohtsuki, Midori A. Arai, Masami Ishibashi

ORGANIC & BIOMOLECULAR CHEMISTRY (ROYAL SOC CHEMISTRY) 8 ( 16 ) 3696 - 3703 2010

Accepted, ISSN 1477-0520

In search of bioactive natural products for overcoming TRAIL resistance from natural resources, we previously reported a number of active compounds. Bioassay-guided fractionation of mangrove, Amoora cucullata, collected from Sundarbans Mangrove Forest, Bangladesh, led to the isolation of four new compounds (1-4), along with seven known compounds (5-11). Of the isolates, compounds 1, 5, 8, and 9 showed TRAIL resistance-overcoming activity, among which 8 showed the most potent activity and enhanced TRAIL-induced apoptosis in TRAIL-resistant human gastric adenocarcinoma (AGS) cells through the activation of caspase-3/7, enhancing the expression of DR4 and DR5 mRNA in AGS cells. Cell death caused by the combined treatment of 8 and TRAIL was inhibited by human recombinant DR5/Fc and DR4/Fc chimera proteins, indicating that 8 sensitizes TRAIL-resistant AGS cells to TRAIL through the induction of DR4 and DR5.

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Total synthesis and evaluation of Wnt signal inhibition of melleumin A and B, and their derivatives

Midori A. Arai, Shuwa Hanazawa, Yujiro Uchino, Xiaofan Li, Masami Ishibashi

ORGANIC & BIOMOLECULAR CHEMISTRY (ROYAL SOC CHEMISTRY) 8 ( 23 ) 5285 - 5293 2010

Accepted, ISSN 1477-0520

The total synthesis of melleumin A (1), a novel cyclic depsipeptide isolated from the myxomycete Physarum melleum, and 3-epi-melleumin A (6) was achieved. Melleumin A-like compounds were also designed and synthesized; analysis of these melleumin A-like compounds showed moderate Wnt signal inhibition. Comparison of the inhibition activity of melleumin B and its three epimers, melleumin A, 3-epi-melleumin A and three melleumin A-like compounds led to further investigation of the structural conformation of the active molecules. The scaffold of melleumin is a potential target in the search for "peptide-like" Wnt signaling inhibitors.

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Search for Bioactive Natural Products Targeting Cancer-Related Signaling Pathways

Masami Ishibashi, Midori A. Arai

JOURNAL OF SYNTHETIC ORGANIC CHEMISTRY JAPAN (SOC SYNTHETIC ORGANIC CHEM JPN) 67 ( 11 ) 1094 - 1104 2009.11

Accepted, ISSN 0037-9980

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The first Hes1 dimer inhibitors from natural products

Midori A. Arai, Ayako Masada, Toshiyuki Ohtsuka, Ryoichiro Kageyama, Masami Ishibashi

BIOORGANIC & MEDICINAL CHEMISTRY LETTERS (PERGAMON-ELSEVIER SCIENCE LTD) 19 ( 19 ) 5778 - 5781 2009.10

Accepted, ISSN 0960-894X

In the present study, we developed a high-throughput screening system for small molecule-inhibitors of the basic helix-loop-helix (bHLH) transcriptional repressor factor Hes1. Successful dimerization of Hes1 immobilized on a microplate and fluorophore (Cy3)-labelled Hes1 was confirmed. Using this system, several natural products were identified as the first Hes1 dimer inhibitors. Of these, two compounds which were isolated from myxomycetes (true slime molds) inhibited Hes1 from N box-dependent suppression of the gene expression in C3H10T1/2 cells. (C) 2009 Published by Elsevier Ltd.

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Synthesis and Biological Activities of 14-epi-MART-10 and 14-epi-MART-11: Implications for Cancer and Osteoporosis Treatment

Atsushi Kittaka, Hideki Hara, Masashi Takano, Daisuke Sawada, Midori A. Arai, Kenichiro Takagi, Takayuki Chida, Yoshifumi Harada, Hiroshi Saito, Kazuya Takenouchi, Seiichi Ishizuka, Keiko Hayashi, Shinichi Ikushiro, Toshiyuki Sakaki, Takayuki Sugiura, Tai C. Chen

ANTICANCER RESEARCH (INT INST ANTICANCER RESEARCH) 29 ( 9 ) 3563 - 3569 2009.09

Accepted, ISSN 0250-7005

The 14-epimer of MART-10, namely 14-epi-MART-10 (14-epi-2 alpha-(3-hydroxypropyl)-1 alpha,25-dihydroxy-19-norvitamin D(3)) and its 2-epimeric analog (14-epi-MART-11) were efficiently synthesized using the Julia coupling reaction to connect between the C5 and C6 positions (steroid numbering). An A-ring precursor was prepared from (-)-quinic acid as shown in the previous MART-10 synthesis. The novel 14-epi-CD-ring coupling partner with an elongated two carbon unit as a sulfone was synthesized from 14-epi-25-hydroxy Grundmann's ketone in good yield. The subsequent coupling reaction followed by a deprotection step afforded a mixture of 14-epi-MART-10 and 14-epi-MART-11 in 40% yield. To separate 14-epi-MART-10 and 14-epi-MART-11, each primary hydroxyl group was esterified with a pivaloyl group and the resulting pivalates 2 alpha and 2 beta were separated by high performance liquid chromatography. After the separation, the C2-stereochemistry of each (2 alpha or 2 beta) was determined by (1)H NMR (nuclear magnetic resonance) studies including NOE (nuclear Overhauser effect) experiments. The pivaloyl group was removed under basic conditions to obtain the target molecules of 14-epi-MART-10 and 14-epi-MART-11, respectively. The VDR (vitamin D receptor)-binding affinity, HL-60 (human promyelocytic leukemia) cell differentiation activity, antiproliferative activity in PZ-HPV-7 (immortalized normal prostate) cells and transactivation activity of the osteocalcin promoter in HOS (human osteoblast cell line) cells (serum-free conditions) were investigated. In addition, the effects on bone mineral density (BMD) and the blood and urine calcium concentrations of ovariectomized (OVX) rats were examined. 14-epi-MART-10 has much greater antiproliferative and cell differentiation activities compared to 1 alpha,25-dihydroxyvitamin D(3) (1 alpha,25(OH)(2)D(3)).

Synthesis of a 1 alpha-C-methyl analogue of 25-hydroxyvitamin D-3: interaction with a mutant vitamin D receptor Arg274Leu

Shinobu Honzawa, Naoyuki Takahashi, Atsushi Yamashita, Takayuki Sugiura, Masaaki Kurihara, Midori A. Arai, Shigeaki Kato, Atsushi Kittaka

TETRAHEDRON (PERGAMON-ELSEVIER SCIENCE LTD) 65 ( 34 ) 7135 - 7145 2009.08

Accepted, ISSN 0040-4020

Vitamin D-3 analogues have been developed for a mutant vitamin D receptor (VDR), Arg274Leu. The mutant VDR has a Mutation at Arg274, which forms an important hydrogen bond with 1 alpha-OH of 1 alpha,25-dihydroxyvitamin D3 to anchor the ligand tightly in the VDR ligand binding pocket. Stereoselective synthesis of the A-ring part of the novel vitamin D analogue, 2 alpha-(3-hydroxypropyl)-1 alpha-methyl-25-hydroxyvitamin D3 (4), from D-galactose was accomplished with the key steps of the introduction of the methyl and allyl groups to the chiral building blocks. The new analogue 4 is ca. 7.3-fold more active than the natural hormone 1 alpha,25-dihydroxyvitamin D-3 (1). (C) 2009 Elsevier Ltd. All rights reserved,

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Development of Chiral Spiro Ligands for Metal-Catalyzed Asymmetric Reactions

Gan B. Bajracharya, Midori A. Arai, Priti S. Koranne, Takeyuki Suzuki, Shinobu Takizawa, Hiroaki Sasai

BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN (CHEMICAL SOC JAPAN) 82 ( 3 ) 285 - 302 2009.03

Accepted, ISSN 0009-2673

This account focuses our works on the development of chiral spiro ligands bearing N-heterocycles as metal-coordinating units and their applications in the metal-catalyzed asymmetric reactions. The spiro bis(isoxazoline) ligands (SPRIXs), spiro bis(isoxazole) ligands, and spiro (isoxazole-isoxazoline) ligands were readily synthesized through intramolecular double nitrile oxide cycloaddition of the corresponding dioximes as a key step. An unprecedented activation of olefins was displayed by the Pd-11 complexes of these chiral spiro ligands in the enantioselective oxidative cyclizations, for example; the asymmetric tandem cyclization of dialkenyl alcohol via oxy-palladation produced a bicyclic ether in excellent enantioselectivity and enantioselective version of the aminocarbonylation proceeded for the first time. Other hitherto known chiral ligands we examined failed to promote these reactions.

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A Method for the Rapid Discovery of Naturally Occurring Products by Proteins Immobilized on Magnetic Beads and Reverse Affinity Chromatography

Midori A. Arai, Eiji Kobatake, Takashi Koyano, Thaworn Kowithayakorn, Shigeaki Kato, Masami Ishibashi

CHEMISTRY-AN ASIAN JOURNAL (WILEY-V C H VERLAG GMBH) 4 ( 12 ) 1802 - 1808 2009

Accepted, ISSN 1861-4728

A highly efficient screening method for naturally occurring products that bind to a specific target protein was demonstrated by using hVDR magnetic beads. The native ligand 1 alpha,25(OH)(2) VD(3) (1) was selectively bound by hVDR magnetic beads when present in a mixture of natural compounds. Furthermore, this method was shown to be applicable to the identification of natural products that interact with a specific protein immobilized on the beads from an extract of a natural resource. Two new natural compounds were isolated by this method. This approach will be helpful for the discovery of novel, naturally occurring products that bind to specific target proteins. This method has the further advantages that it can identify the HPLC peak corresponding to the target compound for isolation, as well as provide important UV, CD, or MS profile information.

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Hedgehog/GLI-mediated transcriptional inhibitors from Zizyphus cambodiana

Midori A. Arai, Chikashi Tateno, Takahiro Hosoya, Takashi Koyano, Thaworn Kowithayakorn, Masami Ishibashi

BIOORGANIC & MEDICINAL CHEMISTRY (PERGAMON-ELSEVIER SCIENCE LTD) 16 ( 21 ) 9420 - 9424 2008.11

Accepted, ISSN 0968-0896

The aberrant hedgehog (Hh)/GLI signaling pathway causes the formation and progression of a variety of tumors. By screening tropical plant extracts by using our screening system, Zizyphus cambodiana was found to include Hh/GLI signaling inhibitors. Bioassay-guided fractionation of this plant extract led to the isolation of three active pentacyclic triterpenes, colubrinic acid (1), betulinic acid (2) and alphitolic acid (3), as potent inhibitors. The inhibition of GLI-related protein expression with 1 or 2 was observed in HaCaT cells with exogenous GLI1, or human pancreatic cancer cells (PANC1), which express Hh/GLI components aberrantly. The expressions of GLI-related proteins PTCH and BCL2 were clearly inhibited by 1 or 2. We also examined the cytotoxicity of these active compounds against PANC1, human prostate cancer cells (DU145) and mouse embryo fibroblast cells (C3H10T1/2). The cytotoxicity against cancer cells (PANC1 and DU145) by 1 or 2 would be caused by inhibition of the expression of the anti-apoptosis protein BCL2. These pentacyclic triterpene inhibitors showed an important relationship between Hh/GLI signaling inhibition, the decrease of BCL2, and cytotoxicity against cancer cells. (C) 2008 Elsevier Ltd. All rights reserved.

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SYNTHESIS AND EVALUATION OF ISOMERS OF MELLEUMIN B FROM THE MYXOMYCETE PHYSARUM MELLEUM; AN APPROACH FOR WNT SIGNAL INHIBITOR TRUSTED IN NATURE

Midori A. Arai, Yujiro Uchino, Shuwa Hanazawa, Xiaofan Li, Naoki Kimura, Masami Ishibashi

HETEROCYCLES (PERGAMON-ELSEVIER SCIENCE LTD) 76 ( 2 ) 1425 - 1438 2008.11

Accepted, ISSN 0385-5414

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Naturally occurring small-molecule inhibitors of hedgehog/GLI-mediated transcription

Takahiro Hosoya, Midori A. Arai, Takashi Koyano, Thaworn Kowithayakorn, Masami Ishibashi

CHEMBIOCHEM (WILEY-V C H VERLAG GMBH) 9 ( 7 ) 1082 - 1092 2008.05

Accepted, ISSN 1439-4227

The aberrant hedgehog (Hh)/GLI signaling pathway causes the formation and progression of a variety of tumors. To search for Hh/GLI inhibitors, we screened for naturally occurring inhibitors of the transcriptional activator GLI1 by using a cell-based assay. We identified zerumbone (1), zerumbone epoxide (2), staurosporinone (9), 6-hydroxystourosporinone (10), orcyriaflavin C (71) and 5,6-dihydroxyarcyrioflovin A (12) as inhibitors of GLI-mediated transcription. In addition, we isolated physolins F (17) and B (18) from Physalis minima, which are also potent inhibitors. These compounds also inhibited GLI2-mediated transactivation. Semiquantitative RT-PCR and Western blotting analysis further revealed that 1, 9, 17, and 18 decreased Hh-related component expressions. We also show that inhibitors of GLI-mediated transactivation reduce the level of the antiapoptosis BcI2 expression. Finally, these identified compounds were cytotoxic to PANC1 pancreatic cancer cells, which express Hh/GLI components. These results strongly suggest that the cytotoxicity of the compounds to PANC1 cells correlates with their inhibition of GLI-mediated transcription.

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Vitamin D hormone: Structural refinement of the seco-steroidal skeleton and the nuclear receptor mediated biological activity

Atsushi Kittaka, Daisuke Sawada, Masashi Takano, Nozomi Saito, Tomoyuki Katayama, Kanako Shindo, Yuya Tsukuda, Shinobu Honzawa, Midori A. Arai, Hiroshi Saito, Ken-ichiro Takagi, Kazuya Takenouchi, Seiichi Ishizuka, Toshiyuki Sakaki, Tai C. Chen

ANTICANCER RESEARCH (INT INST ANTICANCER RESEARCH) 28 ( 3A ) 1618 - 1619 2008.05

Accepted, ISSN 0250-7005

The 2 alpha-(3-hydroxypropyl) group as an active motif in vitamin D-3 analogues as agonists of the mutant vitamin D receptor (Arg274Leu)

Honzawa Shinobu, Yamamoto Yasuhiro, Yamashita Atsushi, Sugiura Takayuki, Kurihara Masaaki, Arai Midori A, Kato Shigeaki, Kittaka Atsushi

BIOORGANIC & MEDICINAL CHEMISTRY 16 ( 6 ) 3002 - 3024 2008.03

Accepted, ISSN 0968-0896

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Determination of absolute stereochemistry, total synthesis, and evaluation of peptides from the myxomycete Physarum melleum

Shuwa Hanazawa, Midori A. Arai, Xiaofan Li, Masarni Ishibashi

BIOORGANIC & MEDICINAL CHEMISTRY LETTERS (PERGAMON-ELSEVIER SCIENCE LTD) 18 ( 1 ) 95 - 98 2008.01

Accepted, ISSN 0960-894X

The absolute stereochemistry of melleumin A (1) and B (2), novel peptide compounds isolated from the myxomycete Physarum melleum, was determined by synthesis of their segments and by a modified Mosher's method. Total synthesis of melleumin B (2) was achieved by a stereoselective method, which provided further evidence for all the absolute stereochemistries of melleumin B (2). The Wnt signal inhibitory activities of 2 and its 10R-epimer 19 were evaluated. Compound 19 showed moderate inhibition of Wnt signal transcription, which suggests that melleumin analogues might be useful as Wnt signal inhibitors. (C) 2007 Elsevier Ltd. All rights reserved.

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Hedgehog signaling inhibitors isolated from myxomycetes and a Solanaceae plant

Ishibashi, M., Hosoya, T., Tateno, C., Arai, M. A.

Planta Medica 74 ( 9 ) 909 - 909 2008

Accepted

Efficient Synthesis of Chromone and Flavonoid Derivatives with Diverse Heterocyclic Units

Midori A. Arai, Mina Sato, Keisuke Sawada, Takahiro Hosoya, Masami Ishibashi

CHEMISTRY-AN ASIAN JOURNAL (WILEY-V C H VERLAG GMBH) 3 ( 12 ) 2056 - 2064 2008

Accepted, ISSN 1861-4728

Chromones and flavonoids are important bioactive compounds. We envisioned that new heterocyclic-substituted chromones or flavonoids might act as new bioactive compounds. To obtain diverse molecules, we developed an efficient one-pot synthesis by Michael aldol reaction of chromone and flavonoid derivatives bearing heterocyclic units. The 2,3-heterocyclic-substituted chromones were obtained in one step. Moreover, the use of substituted benzaldehydes and subsequent addition of heterocyclic aldehydes gave 3-pyridyl-substituted flavones. We also examined these one-pot reactions in the solid phase. To introduce an additional point of diversity into the molecules, Suzuki-Miyaura coupling was performed. Furthermore, we identified the cytotoxicity of the synthesized compounds against cancer cells (PANC1 and HeLa cells). Several compounds were cytotoxic to these cancer cells.

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Total synthesis of melleumins A and B and their analogs: Determination of stereochemistry and Wnt signal inhibitory activity

Ishibashi, M., Hanazawa, S., Uchino, Y., Li, X., Arai, M. A.

Planta Medica 74 ( 9 ) 2008

Accepted

Kinetic studies of 25-hydroxy-19-nor-vitamin D-3 and 1 alpha,25-dihydroxy-19-nor-vitamin D-3 hydroxylation by CYP27B1 and CYP24A1

Naoko Urushino, Sachie Nakabayashi, Midori A. Arai, Atsushi Kittaka, Tai C. Chen, Keiko Yamamoto, Keiko Hayashi, Shigeaki Kato, Miho Ohta, Masaki Kamakura, Shinichi Ikushiro, Toshiyuki Sakaki

DRUG METABOLISM AND DISPOSITION (AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS) 35 ( 9 ) 1482 - 1488 2007.09

Accepted, ISSN 0090-9556

Our previous study demonstrated that 25-hydroxy-19-nor-vitamin D-3[25(OH)-19-nor-D-3] inhibited the proliferation of immortalized noncancerous PZ-HPV-7 prostate cells similar to 1 alpha,25-dihydroxyvitamin D-3 [1 alpha, 25(OH)(2)D-3], suggesting that 25(OH)-19-nor-D-3 might be converted to 1 alpha,25-dihydroxy-19-nor-vitamin D-3 [1 alpha,25(OH)(2)-19-nor-D-3] by CYP27B1 before exerting its antiproliferative activity. Using an in vitro cell-free model to study the kinetics of CYP27B1-dependent 1 alpha-hydroxylation of 25(OH)-19-nor-D-3 and 25-hydroxyvitamin D-3 [25(OH) D3] and CYP24A1-dependent hydroxylation of 1 alpha,25(OH)-19-nor-D3 and 1 alpha, 25(OH)(2)D-3, we found that k(cat)/ Km for 1 alpha-hydroxylation of 25(OH)-19-nor-D-3 was less than 0.1% of that for 25(OH) D-3, and the kcat/ Km value for 24-hydroxylation was not significantly different between 1 alpha,25(OH)219- nor-D-3 and 1 alpha, 25( OH)(2)D-3. The data suggest a much slower formation and a similar rate of degradation of 1 alpha,25(OH)(2)-19-nor-D-3 compared with 1 alpha,25(OH)(2)D-3. We then analyzed the metabolites of 25(OH)D-3 and 25(OH)-19-nor-D-3 in PZ-HPV-7 cells by high-performance liquid chromatography. We found that a peak that comigrated with 1 alpha, 25(OH)(2)D-3 was detected in cells incubated with 25(OH)D-3, whereas no 1 alpha,25(OH)(2)-19-nor-D-3 was detected in cells incubated with 25(OH)-19-nor-D-3. Thus, the present results do not support our previous hypothesis that 25(OH)-19-nor-D-3 is converted to 1 alpha,25(OH)2-19-nor-D-3 by CYP27B1 in prostate cells to inhibit cell proliferation. We hypothesize that 25(OH)-19-nor-D-3 by itself may have a novel mechanism to activate vitamin D receptor or it is metabolized in prostate cells to an unknown metabolite with antiproliferative activity without 1 alpha-hydroxylation. Thus, the results suggest that 25(OH)-19-nor-D-3 has potential as an attractive agent for prostate cancer therapy.

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Synthesis, determination of stereochemistry, and evaluation of new bisindole alkaloida from the myxomycete Arcyria ferruginea: An approach for Wnt signal inhibitor

Kouken Kaniwa, Midori A. Arai, Xiaofan Li, Masami Ishibashi

BIOORGANIC & MEDICINAL CHEMISTRY LETTERS (PERGAMON-ELSEVIER SCIENCE LTD) 17 ( 15 ) 4254 - 4257 2007.08

Accepted, ISSN 0960-894X

To determine the stereochemistry of dihydroarcyriarubin C (1), new bisindole alkaloid isolated from the myxomycete Arcyriajerruginea, cis- (2) and trans-dihydroarcyriarubin C (3) were synthesized. Comparison of their NMR characteristics allowed the trans stereochemistry of the natural product to be confirmed. Moreover, the Writ signal inhibitory activities of 2 and 3 were compared with that of arcyriaflavin C (4), which is a natural product containing a bond between C-2 and C-T. The cis-dihydroar-cyriarubin C (2) showed moderate inhibition of Wnt signal transcription, which suggests that bisindole frameworks might be useful as small-molecule Writ signal inhibitors. (c) 2007 Elsevier Ltd. All rights reserved.

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Design and synthesis of chiral hybrid spiro (isoxazole-isoxazoline) ligands

Priti S. Koranne, Tetsuya Tsujihara, Midori A. Arai, Gan B. Bajracharya, Takeyuki Suzuki, Kiyotaka Onitsuka, Hiroaki Sasai

TETRAHEDRON-ASYMMETRY (PERGAMON-ELSEVIER SCIENCE LTD) 18 ( 8 ) 919 - 923 2007.05

Accepted, ISSN 0957-4166

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High-throughput system for analyzing ligand-induced cofactor recruitment by vitamin D receptor

Midori A. Arai, Ken-ichi Takeyama, Saya Ito, Shigeaki Kato, Tai C. Chen, Atsushi Kittaka

BIOCONJUGATE CHEMISTRY (AMER CHEMICAL SOC) 18 ( 3 ) 614 - 620 2007.05

Accepted, ISSN 1043-1802

A high-throughput screening system for analyzing small molecule-induced coactivator (CoA) recruitment by the vitamin D receptor (VDR) has been developed. The vitamin D-induced protein - protein interactions between VDR and fluorophore (Cy3 or Cy5)-labeled TIF2 or SRC-1 were successfully detected by using a new HCHO fixing method of the protein complex on microplates. The results obtained from this screening of our synthetic vitamin D analogues suggest that the CoA-recruiting activities play an important role in determining the biological activity of various vitamin D analogues and explain the discrepancies between the VDR binding affinity and their biological activity.

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Creative synthesis of novel vitamin D analogs for health and disease

Atsushi Kittaka, Nozomi Saito, Shinobu Honzawa, Kazuya Takenouchi, Seiichi Ishizuka, Tai C. Chen, Sara Peleg, Shigeaki Kato, Midori A. Arai

JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY (PERGAMON-ELSEVIER SCIENCE LTD) 103 ( 3-5 ) 269 - 276 2007.03

Accepted, ISSN 0960-0760

We report new analogs of 1 alpha,25-dihydroxyvitamin D-3 (1) in three categories. First, design and synthesis of ligands for a mutant vitamin D receptor (VDR)(Arg274Leu), which possess proper functional groups at both C1 alpha and C2 alpha positions of 1 to study the biological activity of the mutant VDR. Among our synthetic analogs, 1 alpha-methyl-2 alpha-(3-hydroxypropyl)-25-hydroxyvitamin D-3 (8) showed 7.3-fold greater transcriptional activity for the VDR(Arg274Leu) than that of 1. Next, we examined the anti proliferative activity of 2-substituted 19-norvitamin D3 analogs on an immortalized normal prostate cell line, PZ-HPV-7, and we found MART 10 (14) showed the activity even at very low concentration of 10(-10) to 10(-11) M. We also synthesized 25-hydroxy-19-norvitamin D-3 (13) using Julia-type olefination to connect between the C5 and C6 positions, effectively, to test it as a prohormone type agent for antiprostate diseases. Synthesized compound 13 showed potent antiproliferative activity in PZ-HPV-7, which has high 1 alpha-hydroxylase activity. Finally, we describe design and synthesis of a new TEI-9647 analog, 2 alpha-(3-hydroxypropoxy)-24-propyl-25-dehydro-1 alpha-hydroxyvitamin D-3-26,23-lactone (17), which showed the strongest VDR antagonism. Its IC50 value is 7.4 pM to inhibit differentiation of HL-60 cells induced by 10 nM of 1. (c) 2006 Elsevier Ltd. All rights reserved.

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Evaluation of C-2-substituted 19-nor-1α,25-dihydroxyvitamin D3 analogs as therapeutic agents for prostate cancer

T. C. Chen, K. S. Persons, S. Zheng, J. Mathieu, M. F. Holick, Y. F. Lee, B. Bao, M. A. Arai, A. Kittaka

Journal of Steroid Biochemistry and Molecular Biology 103 ( 3-5 ) 717 - 720 2007.03

ISSN 0960-0760

1α,25-Dihydroxyvitamin D3 (1α,25(OH)2D3) is known to inhibit the proliferation and invasiveness of prostate cancer cells. However, 1α,25(OH)2D3 can cause hypercalcemia and is not suitable as a therapeutic agent. 19-Nor-vitamin D derivatives are known to be less calcemic when administered systemically. In order to develop more potent anti-cancer agents with less calcemic side effect, we therefore utilized 3H-thymidine incorporation as an index for cell proliferation and examined the antiproliferative activities of nine C-2-substituted 19-nor-1α,25(OH)2D3 analogs in the immortalized PZ-HPV-7 normal prostate cell line. Among the nine analogs we observed that the substitution with 2α- or 2β-hydroxypropyl group produced two analogs having antiproliferative potency that is approximately 500- to 1000-fold higher than 1α,25(OH)2D3. The 3H-thymidine incorporation data were supported by the cell counting data after cells were treated with 1α,25(OH)2D3, 19-nor-2α-(3-hydroxypropyl)-1α,25(OH)2D3 or 19-nor-2β-(3-hydroxypropyl)-1α,25(OH)2D3 for 7 days. 19-Nor-2α-(3-hydroxypropyl)-1α,25(OH)2D3 and 19-nor-2β-(3-hydroxypropyl)-1α,25(OH)2D3 were also shown to be about 10-fold more active than 1α,25(OH)2D3 in cell invasion studies using prostate cancer cells. In conclusion, a substitution at the C-2 position of 19-nor-1α,25(OH)2D3 molecule with a hydroxypropyl group greatly increased the antiproliferative and anti-invasion potencies. Thus, these two analogs could be developed to be effective therapeutic agents for treating early and late stages of prostate cancer. © 2006 Elsevier Ltd. All rights reserved.

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Enantioselective glyoxylate-ene reaction using a novel spiro bis(isoxazoline) ligand in copper catalysis

Kazuhiko Wakita, Gan B. Bajracharya, Midori A. Arai, Shinobu Takizawa, Takeyuki Suzuki, Hiroaki Sasai

TETRAHEDRON-ASYMMETRY (PERGAMON-ELSEVIER SCIENCE LTD) 18 ( 3 ) 372 - 376 2007.02

Accepted, ISSN 0957-4166

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Novel 2-alkyl-1 alpha,25-dihydroxy-19-norvitamin D-3: Efficient synthesis with Julia olefination, evaluation of biological activity and development of new analyzing system for co-activator recruitment

Midori A. Arai, Atsushi Kittaka

ANTICANCER RESEARCH (INT INST ANTICANCER RESEARCH) 26 ( 4A ) 2621 - 2631 2006.07

Accepted, ISSN 0250-7005

New 2-alkyl-1a,25-dihydroxy-19-norvitamin D-3 analogs were efficiently synthesized. The C2-alkyl-A-ring precursors were prepared as (3R,5R)-4-alkyl-3,5-dihydroxycyclohexanones from (-)-quinic acid based on radical allylation at the C4 position of methyl (-)-quinicate. The novel CD-ring coupling partner, with the elongated two carbon unit, was synthesized from 25-hydroxy Grundmann's ketone, and applied to modified Julia olefination to construct a diene unit between the A-ring and the CD-ring. The coupling yields, including a deprotection step, were moderate (47-62%). After the separation of the diastereomers based on C2 stereochemistry using HPLC, the structure (2 alpha or 2 beta) was determined by H-1 NMR studies including NOE (nuclear Overhauser effect) experiments. It was also found that 1H NMR chemical shifts on the A-ring showed good correlation with DeLuca's 2-methyl- and 2-ethyl-1 alpha,25-dihydroxy-19-norvitamin D-3 analogs. Among the synthesized 19-norvitamin D-3 analogs, 2 alpha-(3-hydroxypropyl)-1a,25-dihydroxy-19-norvitamin D-3 (8a) showed almost the same potency in binding to the bovine thymus vitamin D receptor (VDR) as the natural hormone (1), while its beta-isomer had only a 3% affinity. Both 2 alpha-allyl- and 2 alpha-propyl-1 alpha, 25-dihydroxy-19-norvitamin D-3 and their 2 beta-analogs possessed a weak affinity for the VDR. The strong VDR ligand 8a was ca. 36-fold more potent in induction of HL-60 cell differentiation than 1 and, interestingly, even the weaker ligand, 2 beta-(3-hydroxypropyl)-1 alpha,25-dihydroxy-19-norvitamin D-3 (8b), showed a 67-fold higherpotency in cell differentiation activity than that of 1. Next, a novel rapid analyzing system of ligand-induced co-factor recruitment on VDR was developed using fluorophore labelled CoA and HCHO fixing of the complex. The efficiency of recruitment of co-activators would explain the discrepancy between the biological activity and the VDR affinity of the ligand.

Design and synthesis of oligosaccharides that interfere with glycoprotein quality-control systems

MA Arai, Matsuo, I, S Hagihara, K Totani, J Maruyama, K Kitamoto, Y Ito

CHEMBIOCHEM (WILEY-V C H VERLAG GMBH) 6 ( 12 ) 2281 - 2289 2005.12

Accepted, ISSN 1439-4227

Calnexin (CNX) and its soluable homologue calreticulin (CRT) are lectin-like molecular chaperones that help newly synthesized glycoproteins to fold correctly in the rough endoplasmic reticulum (ER). To investigate the mechanism of glycoprotein-quality control, we have synthesized structurally defined high mannose-type oligosaccharides related to this system. The paper describes the synthesis of the non-natural undecsaccharide 2 and heptasaccharide 16, designed as potential inhibitors of the ER quality-control system. Each possesses the key tetrasaccharide element (Glc(1)Man(3)) critical for the CNX/CRT binding, while lacking the pentamannosyl branch required for glucosidase II recognition. These oligosaccharides were evaluated for their ability to bind CRT by isothermal titration colorimetry (ITC). As expected, each of them had a significant affinity towards CRT In addition, these compounds were shown to be resistant to glucosidose II digestion. Their activities in blocking the chaperone function of CRT wet I e next measured by using malate dehydrogenose (MDH) as a substrate. Their inhibitory effects were shown to correlate well with their CRT-binding affinities, both being critically dependent upon the presence of the terminal glucose (Glc) residue.

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Synthesis of 25-Hydoxy-19-norvitamin D3 Analogs and Their Antiproliferative Activities on Prostate Cells

Midori A. Arai, Ryuji Tsutsumi, Hideki Hara, Tai C. Chen, Toshiyuki Sakaki, Naoko Urushino, Kuniyo Inouye, Atsushi Kittaka

Heterocycles 66 ( 1 ) 469 - 479 2005

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