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A practical method for synthesizing chiral α-amino phosphonic acid derivatives was developed. Readily available and stable N-o-nitrophenylsulfenyl (Nps) imino phosphonate was utilized as a substrate for a highly enantioselective Friedel-Crafts-type addition of indole or pyrrole nucleophiles catalyzed by chiral phosphoric acid. The resulting adduct was easily converted into N-9-fluorenylmethyloxycarbonyl (Fmoc) amino phosphonic acid, which is useful for synthesizing peptides containing an amino phosphonic acid., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Androgen receptor (AR) is a validated target in all clinical states of prostate cancer. Androprostamines A and B, the new inhibitors of androgen receptor, were isolated from Streptomyces sp. MK932-CF8. Their structures were determined by the spectroscopic analysis, degradation studies and synthesis. Androprostamines showed potent inhibitory effect against androgen-dependent growth of human prostate cancer cells without cytotoxicity and repressed the androgen-induced expression of AR-regulated genes.

Quinofuracins A-E, novel anthraquinone derivatives containing β-D-galactofuranose that were isolated from the fungus Staphylotrichum boninense PF1444, induced p53-dependent cell death in human tumor cells. The structures of quinofuracins A-E, including absolute configurations, were elucidated by extensive spectroscopic analysis and chemical transformation studies. Quinofuracins were classified into three groups according to the aglycone moieties. 5'-Oxoaverantin was present in quinofuracins A-C, whereas averantin and versicolorin B were identified in quinofuracins D and E, respectively. These quinofuracins induced p53-dependent growth suppression in human glioblastoma LNZTA3 cells.

A new trehalose analog, lentztrehalose [4-O-(2,3-dihydroxy-3-methylbutyl)trehalose], was isolated from an actinomycete Lentzea sp. Lentztrehalose is only weakly hydrolyzed by the trehalose-hydrolyzing enzyme, trehalase, so can be regarded as an enzyme-stable analog of trehalose. Although lentztrehalose does not show apparent toxicity to mammalian cells and microbes, it has antitumor activity in mice bearing S-180 sarcoma and Ehrlich carcinoma cells. In ovariectomized mice, lentztrehalose displayed a bone reinforcement effect in the femur that was superior to trehalose and induced non-morbid suppression of weight gain comparable with trehalose. These results indicate that enzyme-stable analogs of trehalose, such as lentztrehalose, may be more beneficial for human health and thus have potential as substitutes for trehalose as a sweetener.

In Japan, various types of MRI equipment, having varying magnetic field strengths, are widely used. However, the biggest problem encountered while utilizing an MRI is the scarcity of information and guidelines pertaining to implants, internal, and external metallic objects. This leads to uncertainty when an unspecified object is encountered during an examination andcreates the possibility of performing an ambiguous MRI. Therefore, this study classified a range of objects into 12 categories using database management software. An attempt was made to create an environment where reference and comparison of products could be performed. This study also investigated the ways and extent to which medical equipment package inserts reference the MRI. With the co-operation of various corporations and the use of information such as medical equipment package inserts, product information was collected and an environment for the reference and comparison of products became available. In addition, it became apparent while examining these package inserts that orthopedic products had the least information available. It is likely that this information will be useful in medical settings and this kind of database will become increasingly necessary in the future.

Prostate stroma can regulate the growth and metastasis of prostate cancer through the tumor-stromal cell interactions. Thus, small molecules that modulate the tumor-stromal cell interactions will have a chance to become new antitumor drugs. In the course of our screening of the modulators, we isolated three new natural compounds, NBRI16716A (1), NBRI16716B (2) and NBRI16716C (3), from the fermentation broth of Perisporiopsis melioloides Mer-f16716, although compound 2 was already reported as a chemical degradation product of isotriornicin. Compounds 1 and 2 inhibited the growth of human prostate cancer DU-145 cells in the coculture with human prostate stromal cells (PrSCs) more strongly than that of DU-145 cells alone. Furthermore, both compounds showed antitumor effect against xenograft models of DU-145 cells and PrSCs in vivo.

The interaction between the receptor for advanced glycation end-product (RAGE) and amphoterin has an important role in tumor growth and metastasis. Because the abrogation of the interaction results in the inhibition of the tumor growth and metastasis, we designed a screening system for an inhibitor of the interaction between RAGE and amphoterin. In the course of our screening of the inhibitor, we isolated a novel natural compound NBRI17671 (1) from the fermentation broth of Acremonium sp. CR17671. We also modified 1 into a more active NBRI17671al (2). Although 1 at 50 g ml(-1) weakly inhibited binding of various cells to amphoterin, 2 at 50 g ml(-1) inhibited it by >50% of control. Compound 2 effectively inhibited the tumor growth of glioma and lung tumor xenografts in mice at 25 mg kg(-1). Furthermore, 2 was found to downregulate mitogen-activated protein kinase (MAPK) activity in the tumor cells.

Although cytostatin analog protein phosphatase 2A (PP2A)-specific inhibitors are promising candidates of a new type of anticancer drug, their development has been hindered because of their liability. To find new classes of PP2A-specific inhibitors, we conducted a screening with microbial metabolites and found that rubratoxin A, a classical mycotoxin, is a highly specific and potent inhibitor of the enzyme. While rubratoxin A inhibits PP2A at Ki = 28.7 nm, it hardly inhibited any other phosphatases examined. Rubratoxin B, a close analog, also specifically but weakly inhibits PP2A at Ki = 3.1 microM. The inhibition of intracellular PP2A in cultured cells is obviously observed with 20 microM rubratoxin A treatment for 3 h, inducing the overphosphorylation in PP2A substrate proteins. Although rubratoxins and cytostatin differ in the apparent structures, these compounds share similarities in the structures in detail and PP2A-binding manners. Rubratoxin A showed higher suppression of tumor metastasis and reduction of the primary tumor volume than cytostatin in mouse experiments. As a successor of cytostatin analogs, rubratoxin A should be a good compound leading to the development of antitumor drugs targeting PP2A.

The growth and metastasis of prostate cancer are regulated by prostate stroma through the tumor-stromal cell interactions. Small molecules that modulate the tumor-stromal cell interactions will be new anticancer drugs. In the course of our screening of the modulators, we isolated two new atpenins, NBRI23477 A (4) and B (5), from the fermentation broth of Penicillium atramentosum PF1420. Compounds 4 and 5 as well as atpenin A4 (1), A5 (2) and B (3) inhibited the growth of human prostate cancer DU-145 cells in the coculture with human prostate stromal cells more strongly than that of DU-145 cells alone.

Decrease of ceramide in the skin is one of the aggravating factors of atopic dermatitis. The skin is often infected by ceramidase-producing bacteria, such as Pseudomonas aeruginosa. The bacterial ceramidase then degrades ceramide in the skin. To develop anti-atopic dermatitis drugs, we searched for ceramidase inhibitors, which led to the discovery of ceramidastin, a novel inhibitor of bacterial ceramidase, from the culture broth of Penicillium sp. Mer-f17067. Ceramidastin inhibited the bacterial ceramidase with an IC(50) value of 6.25 microg ml(-1). Here we describe the isolation, physicochemical properties, structure determination and biological activity of ceramidastin.

Angiogenesis is a critical step for the tumor therapy. Many angiogenic factors are involved in the tumor angiogenesis. In the course of our screening for inhibitors of angiogenin secretion, one of angiogenic factors, we have isolated a new terrein glucoside (1) and terrein (2) from the fermentation broth of fungal strain Aspergillus sp. PF1381. The structure and absolute stereochemistry of 1 was determined to be (4S,5R)-5-[(alpha-D-glucopyranosyl)oxy]-4-hydroxy-3-(E-1-propenyl)-2-cyclopenten-1-one on the basis of spectral and enzymatic analyses. Compounds 1 and 2 equally inhibited angiogenin secretion from androgen-dependent prostate cancer cells, LNCaP-CR, with IC50 values of 13 microM. However, both compounds did not affect VEGF secretion, another angiogenic factor. Furthermore, both compounds inhibited tube formation of human umbilical vein endothelial cells (HUVEC). These results suggested that 1 and 2 act as angiogenesis inhibitors through the inhibition of angiogenin secretion.

Hypoxia-inducible factor-1 (HIF-1) is a central mediator of cellular responses to low oxygen and has recently become an important therapeutic target for solid tumor therapy. To identify small molecule inhibitors of the HIF-1 transcriptional activation, we have established a high through-put assay system using a stable transformant of mammalian cells that express a luciferase reporter gene construct containing a HIF-1 binding site. Using this system, we screened 5000 cultured broths of microorganisms, and we found that fermentation broth produced by Streptomyces strain 1759-27 showed significant inhibition of the reporter activity induced by hypoxic conditions. The active substance NBRI759-27 was purified and determined to be tartrolone C by several methods including X-ray crystallography. In the reporter gene assay, tartrolone C inhibited the HIF-1 transcriptional activity under hypoxic conditions with an IC50 value of 0.17 microg/ml.

Deficiency of Fas-mediated apoptosis is one of the mechanisms involved in the immune evasion by tumors. Thus, it might be a practical approach for cancer treatment that Fas-mediated apoptosis in tumor cells is modified by drugs. In the course of screening, we have isolated two new naphthoquinones, f13102A and B, from the culture broth of fungus strain F-13102. Coumpound f13102A sensitizes Fas-resistant human lung adenocarcinoma A549 cells to apoptosis.

The stereochemistry of kigamicins A (1), C (2) and D (3) were elucidated by a combination of X-ray crystallographic analysis and degradation studies. The absolute structures of kigamicins thus determined were depicted as shown in Fig. 2.

Stromal cells play an important role in regulating epithelial malignancies through diffusible factors and adhesion. Modulation of the tumor-stromal cell interaction is an attractive target for new antitumor strategies. To screen for a modulator of the interaction, we have now developed a quantitative colorimetric assay for measurement of tumor cell growth in coculture with stromal cells using rhodanile blue dye. Rhodanile blue specifically stained cytokeratin-positive tumor cells in the coculture. When human prostate carcinoma cells LNCaP, PC-3 and DU-145 were cocultured with normal prostate stromal cells (PrSC) in a microplate, growth of the prostate cancer cells in the coculture was selectively measured by the rhodanile blue staining method. Using this system, we searched for a modulator of the tumor-stromal cell interaction among clinically used drugs and natural products. As a result, we found that 5-fluorouracil, bleomycin and phthoxazolin A inhibit prostate cancer cell growth more strongly in coculture with PrSC than that in monoculture. Without need to pre-label cells and transfect a marker gene, our new method is simple, rapid and thus useful for screening for modulators of the tumor-stromal cell interaction. Furthermore, our results suggest that low molecular weight compounds modulate the tumor-stromal cell interaction.

ICM0301A (1), B (2) and their congeners (3 approximately 8) were isolated from a culture broth of Aspergillus sp. F-1491 as new angiogenesis inhibitors. Their structures were elucidated by spectroscopic analyses. ICM0301A and B have a substituted decalin skeleton containing two oxirane rings.

In the course of screening program for inhibitors of angiogenesis, novel substances designated as ICM0301A approximately H (1 approximately 8) were isolated from the culture broth of Aspergillus sp. F-1491. ICM0301s inhibited the growth of human umbilical vein endothelial cells (HUVECs) induced by basic fibroblast growth factor (bFGF) with IC50 values of 2.2 approximately 9.3 microg/ml. ICM0301A (1) showed significant anti-angiogenic activity at lower than 10 microg/ml in the angiogenesis model using rat aorta cultured in fibrin gel. ICM0301s showed very low cytotoxicity against various tumor cells. Furthermore, 1CM0301A did not show any toxic symptom in mice by intraperitoneal injection at 100 mg/kg.

Kigamicin A (1), B (2), C (3), D (4) and E (5) are novel antitumor antibiotics. Their structures were determined by spectroscopic analyses including various NMR measurements. Kigamicins have a unique aglycone of fused octacyclic ring system containing seven of six-membered rings and one oxazolidine. The aglycone links a sugar chain composed of one to four deoxysugars. These sugars were found to be amicetose and oleandrose.

Imbalances in the epithelial-stromal interactions are important in the pathogenesis of prostate cancer. However, we know little about androgenic regulation in the stroma of prostate cancer. We examined the cancer-stromal interaction paying attention to androgen responsiveness of stromal side. In co-culture, PC3 and LNCaP cells did not affect dihydrotestosterone (DHT)-dependent growth of prostate stromal cells (PrSCs), but DU145 cells significantly reduced it. Conditioned medium from DU145 cells (DU145-CM) also inhibited DHT-dependent PrSCs growth, androgen receptor (AR) expression, and prostate specific antigen transcription. Although the inhibitory effect of DU145-CM was not affected by neutralizing antibody against EGF, FGF-2, or TNF-alpha, pretreatment with testosterone-Sepharose partially reduced the inhibitory ability of DU145-CM. These results suggest that DU145 cells produce inhibitory factors for androgen responsiveness, including steroid-binding protein(s), and these may participate in crosstalk between DU145 cells and PrSCs as modulators of androgen.

ICM0201 (1), a new inhibitor of murine osteoclastogenesis in culture was isolated from a fermentation broth of Cunninghamella sp. F-1490. The structure of ICM0201 was determined to be (3S,10aR)-3,4a-dihydroxy-2,3,4,4a-tetrahydro-2H-pyrano[3,2-b]benzo[e]morpholine-9-carboxylic acid by spectroscopic analyses and chemical studies. The structure of 1 is unique in that the tricycle ring system is composed of aminal and hemiacetal bonds.

In the course of screening for inhibitors of osteoclastogenesis, a new substance designated as ICM0201 was isolated from a fermentation broth of Cunninghamella sp. F-1490. ICM0201 inhibited the formation of osteoclasts in mouse bone marrow cells with an IC50 value of 0.78 microg/ml and showed weak cytotoxicity against bone marrow cells.

Hygrolidin family antibiotics showed selective cytotoxicity against both cyclin E- and cyclin A-overexpressing cells. Among them, hygrolidin was the most potent and inhibited growth of solid tumor-derived cell lines such as DLD-1 human colon cancer cells efficiently more than that of hematopoietic tumor cells and normal fibroblasts. FACS analysis revealed that hygrolidin increased cells in G1 and S phases in DLD-1 cells. While hygrolidin decreased amounts of cyclin-dependent kinase (cdk) 4, cyclin D, and cyclin B, it increased cyclin E and p21 levels. Hygrolidin-induced p21 bound to and inhibit cyclin A-cdk2 complex more strongly than cyclin E-cdk2 complex. Furthermore, hygrolidin was found to increase p21 mRNA in DLD-1 cells, but not in normal fibroblasts. Thus, hygrolidin inhibited tumor cell growth through induction of p21. In respect to p21 induction, inhibition of vacuolar-type (H+)-ATPase by hygrolidin was suggested to be involved.

Ascidian eggs are surrounded by a noncellular layer and two cellular layers, which are penetrated by sperm. Three sperm surface proteases are essential for fertilization of eggs from the stolidobranch ascidian Halocynthia: spermosin, acrosin, and the proteasome. In the phlebobranch Ciona, a chymotrypsin-like protease and the proteasome are essential in fertilization. Sperm from the phlebobranch ascidians Phallusia mammillata, Ascidia (=Phallusia) nigra, and Ascidia columbiana, all express spermosin, acrosin, and the proteasomal chymotrypsin activities on their surfaces. Chymostatin blocks cleavage in phlebobranchs, but inhibitors of spermosin and acrosin only delay it by several minutes. Protease inhibitors have little effect upon sperm binding in Phallusia but strongly affect the rate of sperm passage through the vitelline coat. Peptide substrates and inhibitors to spermosin and acrosin cause a significant decline in the number of eggs undergoing pre-meiotic contractions at 3 min after fertilization. Thus while chymotrypsin activity is essential for penetration of the vitelline coat, spermosin and acrosin both function to increase the rate of fertilization. A crucial step in the divergence of the phlebobranchs and stolidobranchs may have been the conversion of spermosin and acrosin to essential proteases in the stolidobranchs, or, perhaps, their essential function was lost in the evolution of phlebobranchs. Aplousobranch ascidians are all colonial with very small zooids. Sperm from Aplidium californicum, Aplidium solidum (Polyclinidae), and Distaplia occidentalis (Holozoidae) have acrosin and chymotrypsin activities but lack spermosin activity. This enzyme is also missing from sperm of colonial phlebobranch and stolidobranch ascidians, suggesting that spermosin is not necessary for small zooids with internal fertilization., (Copyright 2002 Wiley-Liss, Inc.)

15-Deoxyspergualin (DSG) inhibited growth of mouse EL-4 lymphoma cells with an IC50 0.02 microg/ml. Even though the cells were treated with DSG for only 4 hours and then washed, the antiproliferative effect lasted long with an IC50 0.4 microg/ml. DSG has spermidine and guanidine moieties in its structure. One decomposed element containing guanidine moiety inhibited the growth at higher doses than DSG, but the effect did not last long unlike DSG. While another element containing spermidine moiety did not affect the growth, it diminished the long-lasting antiproliferative effect of DSG by pretreatment of the cells. Pretreatment with polyamines such as putrescine, spermidine, and spermine also diminished the effect of DSG. Furthermore, N-alkylation of spermidine moiety in DSG abolished the antiproliferative effect. These results suggested that DSG binds to the cells through its spermidine moiety and exerts its long-lasting antiproliferative effect.

IC202B (1) and C (2) were isolated from the culture filtrate of Streptoalloteichus sp. 1454-19. The structures were elucidated by various NMR spectral analyses including 1H-15N HMBC and FAB-MS experiments. IC202B and C showed immunosuppressive activity on a mixed lymphocyte culture reaction at the same IC50 value of 1.6 microg/ml.

IC202A, a new immunosuppressive compound, was isolated from the culture filtrate of Streptoalloteichus sp. 1454-19. It showed a suppressive effect on mixed lymphocyte culture reaction with an IC50 value of 3.6 microg/ml and mitogen induced lymphocyte blastogenesis in vitro.

IC202A (1) was isolated from the culture filtrate of Streptoalloteichus sp. 1454-19. The structure of 1 was determined by spectral analysis including a variety of two-dimentional NMR and FAB-MS experiments. IC202A is a ferrioxamine-related compound containing a butylidene N-oxide function.

In order to investigate systematically the substrate or subsite specificity of two sperm proteases, acrosin and spermosin (a novel trypsin-like protease) of the ascidian, Halocynthia roretzi, the effects of peptidyl-argininals on the purified enzymes as well as on fertilization were examined. Among four benzyloxycarbonyl (Z)-Leu-X-argininals (X = Pro, Leu, Ser, and Gly), Z-Leu-Pro-argininal showed the strongest inhibition toward the spermosin activity. On the P3 site specificity, Val-Pro-argininal derivatives showed a stronger inhibition than a Leu-Pro-argininal derivative, suggesting the preference of Val rather than Leu residue at the P3 position. Similar results were obtained by analyzing the hydrolyzing activity of the fluorogenic peptide substrates: it hydrolyzed Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide (MCA) most efficiently, and Boc-Asp(O-benzyl)-Pro-Arg-MCA was the next best substrate, but Gly-Pro-Arg (or Lys)-MCAs were hardly hydrolyzed. On the other hand, acrosin was found to prefer Leu or Pro residue rather than Gly or Ser residue at the P2 position as revealed by comparing the Ki values of peptidyl-argininals. Detailed kinetic analysis on the inhibitory abilities of peptidyl-argininals toward the purified enzymes and the ascidian fertilization suggested that both acrosin and spermosin are involved in ascidian fertilization.

Several pyrrolidine-containing analogues of poststatin were synthesized and examined for their inhibitory activity against prolyl endopeptidase and cathepsin B in vitro. Replacement of the postine residue with 2-oxo-2-(2-pyrrolidinyl)acetic acid increased the selectivity and inhibitory activity against prolyl endopeptidase. Benzyloxycarbonyl-L-phenylalanyl-(S)-2-oxo-2- (2-pyrrolidinyl)acetyl-D-phenylalanine was about 46 times as active to propyl endopeptidase as natural poststatin.

The structure of cytostatin was determined to be 5,6-dihydro-5-methyl-6-(6-hydroxy,1,5-dimethyl-4-phosphonooxy-7,9, 11-tridecatrienyl)-2H-pyran-2-one sodium salt on the basis of physico-chemical properties and NMR studies.

In our search for inhibitors of cell adhesion to components of extracellular matrix (ECM), fibronectin, laminin and collagen type IV, we succeeded in finding a novel cyclic hexadepsipeptide antibiotic, named IC101, which was isolated from cultured mycelium of Streptomyces albulus MJ202-72F3. It was purified by centrifugal partition chromatography, preparative reverse phase HPLC and Sephadex LH-20 and was obtained as a white powder. IC101 strongly inhibited cell adhesion to ECM components, suppressed immune responses in vitro and in vivo, and exhibited antimicrobial activity on Gram-positive bacteria.

Among various protease inhibitors, chymostatin (an inhibitor of sperm chymotrypsin-like protease) strongly inhibited the binding of sperm to the vitelline coat of glycerinated eggs of the ascidian Halocynthia roretzi, whereas leupeptin (an inhibitor for sperm acrosin), antipain, and soybean trypsin inhibitor had no significant inhibitory effects. Dansyl-Val-Pro-argininal (an inhibitor of the sperm trypsin-like protease, spermosin) had an inhibitory effect on the binding of sperm that was much smaller than its effects on fertilization. Since the sperm chymotrypsin-like protease that is involved in ascidian fertilization has been identified as a proteasome (multicatalytic proteinase complex), we tested the effects of several peptidyl argininals, inhibitors of the activities of proteasomes, on this binding process. The ranking of the inhibitory effects of these compounds on the binding of sperm was the same as that of their effects on the chymotrypsin-like activity of the proteasome, reported previously. The potent inhibitors of binding used in these studies had no or minimal effects on sperm motility. These results suggest that a sperm chymotrypsin-like protease (most probably the chymotrypsin-like protease in the proteasome) plays a key role in binding of sperm to the vitelline coat of the ascidian egg.

The biosynthesis of delaminomycin A, produced by Streptomyces albulus MJ202-72F3, was investigated by feeding 13C-labeled compounds followed by 13C NMR analyses. The results indicate that delaminomycin A is derived from six acetate units, five propionate units and one glycine unit.

Delaminomycins A, B, C and their derivatives were prepared and investigated biological activities of them. Among these compounds, spiro compounds (A2, B2 and C2) showed stronger inhibitory activity than natural products (A1, B1 and C1) on B16 melanoma cells adhesion assay and Con A-induced proliferation of murine splenic lymphocytes assay. In MLCR and antimicrobial assay, however, A1, B1 and C1 showed more potent inhibitory activity than spiro compounds (A2, B2 and C2). On the other hand, as to C-5' substituents of pyrrolidine ring, the order of inhibitory activity was R = OH > R = OCH3 > R = H on Con A-induced proliferation of murine splenic lymphocytes assay. In MLCR and antimicrobial assay, however, the order of inhibitory activities were R = H > R = OCH3 > R = OH. Inhibitory activities of A4 which was lacked pyrrolidine ring were reduced on B16 melanoma cells adhesion assay and on cytotoxicity against tumor cells in vitro in comparison with those of A1.