Relationship Between the Structure of Alkylpsoralens and Their Antiproliferative Activity in HL60 Cells.

BACKGROUND/AIM: As part of our continuing investigation in coumarin derivatives as potential anticancer substances, a series of alkylpsoralens were synthesized, and their antiproliferative activity was evaluated in leukemic HL60 cells. MATERIALS AND METHODS: Alkylpsoralens were systematically synthesized from the combination of several chloroketones and 7-hydroxycoumarin derivatives. RESULTS: Among the compounds synthesized, 4,4',8-trimethylpsoralen demonstrated the most potent activity (IC50=6.6 µM). CONCLUSION: The correlation between the alkylation pattern and antiproliferative activity showed the importance of the C4-methyl and C8-methyl moieties in the psoralen nucleus as well as the importance of lipophilicity for their antiproliferative activity.

Relationship Between the Structure of Methoxylated and Hydroxylated Flavones and Their Antiproliferative Activity in HL60 Cells.

As part of our continuing investigation on flavonoid derivatives as potential anticancer substances, a series of methoxylated and hydroxylated flavones was synthesized, and their cytotoxic and anti-proliferative activity was evaluated in leukemic HL60 cells. Their structure-activity relationship was also investigated. The correlation between the methoxylation/hydroxylation pattern and antiproliferative activity revealed the importance of the 5,4'- and 3',4'-dihydroxyl moieties in flavone nucleus.

Protodioscin, Isolated from the Rhizome of Dioscorea tokoro Collected in Northern Japan is the Major Antiproliferative Compound to HL-60 Leukemic Cells.

BACKGROUND: The rhizome of Oni-dokoro (a wild yam, Dioscorea tokoro) has extremely bitter taste and is not generally regarded edible;, however, in northern part of Japan, such as Iwate and a part of Aomori, it is used as health promoting food. To clarify the reason, we examined the biologically active compounds in the rhizome collected at Iwate and compared them from the other area in literature. METHODS: The acetonitrile extract from northern part of Japan was purified by bioassay-guided separation using antiproliferative activity to human leukemia HL-60 cell, and protodioscin (PD) was isolated and identified by instrumental analyses as the major active compound. RESULTS: PD known as a saponin with four sugar moieties, an inhibitor for platelet aggregation, and a low density lipoprotein (LPL) lowering agent, displayed strong growth inhibitory effect to HL-60. The literature search suggested that the rhizome from other area contained dioscin and other saponins with three sugar moieties as their major component. We assume that the edible and health promoting effect of the rhizome in the particular area is partially derived from these different components. CONCLUSION: We were interested in the differences of utilization in the rhizome of wild yam Dioscorea tokoro, and examined the chemical composition in the rhizome to find protodioscin as antiproliferative compound to HL-60. In the report from other area, the rhizome exhibited dioscin as the major compound. Our study indicated that the protodioscin/dioscin composition varied regionally, although the reason is still needs to be investigated.

Gene expression analysis of enzymes of the carotenoid biosynthesis pathway involved in ß-cryptoxanthin accumulation in wild raspberry, Rubus palmatus.

ß-cryptoxanthin (ß-Cry), a xanthophyll, is unlike other abundant carotenoids, such as α-carotene, ß-carotene, lycopene, lutein, and zeaxanthin. It is not found in most fruits or vegetables but is found only in specific fruits, such as hot chili pepper, persimmon, and citrus fruits. Because recent reports suggest that ß-Cry intake is beneficial to human health, the xanthophyll requires further investigation. Although ß-Cry accumulates in the fruit of wild raspberry, Rubus palmatus, it is not present in cultivated raspberry. In the present study, two wild raspberry species were studied-R. palmatus, which accumulates ß-Cry in the fruit, and R. crataegifolius, which does not accumulate ß-Cry. Four carotenoid biosynthetic enzymes derived from these two species were analyzed-phytoene synthase (PSY), lycopene ß-cyclase (LCYb), ß-carotene hydroxylase (HYb), and zeaxanthin epoxidase (ZEP). Expression levels of their genes were also assessed to elucidate mechanism underlying ß-Cry accumulation. Partial gene sequences of RubPSY, RubLCYb, RubHYb, and RubZEP, isolated from immature raspberry fruits of R. palmatus, were used as probes for Northern blot analysis. RubZEP expression ceased as the fruits matured, possibly because of reduced production of zeaxanthin. ß-Cry is considered to be an intermediate compound that accumulates in the mature fruits of R. palmatus. High expression of RubPSY was detectable in the mature fruits of R. crataegifolius, and the expression of RubLCYb, RubHYb, and RubZEP was detectable during all stages of fruit maturation. In contrast, ß-Cry was absent in the mature fruits of R. crataegifolius.

Synergistic Antiproliferative Effects of Zoledronic Acid and Fluvastatin on Human Pancreatic Cancer Cell Lines: An in Vitro Study.

Bisphosphonates and statins are known to have antitumor activities against different types of cancer cell lines. In the present study, we investigated the antiproliferative effects of the combination of zoledronic acid (ZOL), a bisphophosphonate, and fluvastatin (FLU), a statin, in vitro on two types of human pancreatic cancer cell lines, Mia PaCa-2 and Suit-2. The pancreatic cancer cell lines were treated with ZOL and FLU both individually and in combination to evaluate their antiproliferative effects using WST-8 cell proliferation assay. In this study, we demonstrated a potent synergistic antiproliferative effect of both drugs when used in combination in both cell lines. Moreover, we studied the molecular mechanism behind this synergistic effect, which was inhibited by the addition of the mevalonate pathway products, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Furthermore, we aimed to determine the effect of ZOL and FLU combination on RhoA and Ras guanosine 5'-triphosphate (GTP)-proteins. The combination induced a marked accumulation in RhoA and unprenylated Ras. GGPP and FPP reversed the increase in the amount of both proteins. These results indicated that the combination treatment impaired RhoA and Ras signaling pathway by the inhibition of geranylgeranylation and/or farnesylation. This study provides a potentially effective approach for the treatment of pancreatic cancer using a combination treatment of ZOL and FLU.

In vitro and in vivo evidence for the inhibition of brassinosteroid synthesis by propiconazole through interference with side chain hydroxylation.

We carried out the biochemical evaluation of the target site of propiconazole in BR biosynthesis. Applying BR biosynthesis intermediates to Arabidopsis seedlings grown in the presence of propiconazole under dark condition, we found that the target site of propiconazole in BR biosynthesis can be identified among the C22 and C23 side chain hydroxylation steps from campestanol to teasterone. Using differential spectra techniques to determine the binding affinity of propiconazole to CYP90D1, which is responsible for C23 hydroxylation of BR, we found that propiconazole induced typical type II binding spectra in response to purified recombinant CYP90D1 and the Kd value was found approximately 0.76 µM.

Fenarimol, a Pyrimidine-Type Fungicide, Inhibits Brassinosteroid Biosynthesis.

The plant steroid hormone brassinosteroids (BRs) are important signal mediators that regulate broad aspects of plant growth and development. With the discovery of brassinoazole (Brz), the first specific inhibitor of BR biosynthesis, several triazole-type BR biosynthesis inhibitors have been developed. In this article, we report that fenarimol (FM), a pyrimidine-type fungicide, exhibits potent inhibitory activity against BR biosynthesis. FM induces dwarfism and the open cotyledon phenotype of Arabidopsis seedlings in the dark. The IC50 value for FM to inhibit stem elongation of Arabidopsis seedlings grown in the dark was approximately 1.8 ± 0.2 µM. FM-induced dwarfism of Arabidopsis seedlings could be restored by brassinolide (BL) but not by gibberellin (GA). Assessment of the target site of FM in BR biosynthesis by feeding BR biosynthesis intermediates indicated that FM interferes with the side chain hydroxylation of BR biosynthesis from campestanol to teasterone. Determination of the binding affinity of FM to purified recombinant CYP90D1 indicated that FM induced a typical type II binding spectrum with a Kd value of approximately 0.79 µM. Quantitative real-time PCR analysis of the expression level of the BR responsive gene in Arabidopsis seedlings indicated that FM induces the BR deficiency in Arabidopsis.

YCZ-18 is a new brassinosteroid biosynthesis inhibitor.

Plant hormone brassinosteroids (BRs) are a group of polyhydroxylated steroids that play critical roles in regulating broad aspects of plant growth and development. The structural diversity of BRs is generated by the action of several groups of P450s. Brassinazole is a specific inhibitor of C-22 hydroxylase (CYP90B1) in BR biosynthesis, and the application use of brassinazole has emerged as an effective way of complementing BR-deficient mutants to elucidate the functions of BRs. In this article, we report a new triazole-type BR biosynthesis inhibitor, YCZ-18. Quantitative analysis the endogenous levels of BRs in Arabidopsis indicated that YCZ-18 significantly decreased the BR contents in plant tissues. Assessment of the binding affinity of YCZ-18to purified recombinant CYP90D1 indicated that YCZ-18 induced a typical type II binding spectrum with a Kd value of approximately 0.79 µM. Analysis of the mechanisms underlying the dwarf phenotype associated with YCZ-18 treatment of Arabidopsis indicated that the chemically induced dwarf phenotype was caused by a failure of cell elongation. Moreover, dissecting the effect of YCZ-18 on the induction or down regulation of genes responsive to BRs indicated that YCZ-18 regulated the expression of genes responsible for BRs deficiency in Arabidopsis. These findings indicate that YCZ-18 is a potent BR biosynthesis inhibitor and has a new target site, C23-hydroxylation in BR biosynthesis. Application of YCZ-18 will be a good starting point for further elucidation of the detailed mechanism of BR biosynthesis and its regulation.

Synthesis and structure-activity relationship studies of furan-ring fused chalcones as antiproliferative agents.

As part of our continuing investigation of flavonoid derivatives as potential anticancer substances, the synthesis of 25 cinnamoyl derivatives of benzofuran as furan-fused chalcones was carried-out and these compounds were further evaluated for their antiproliferative activity towards HL60 promyelocytic leukemia cells. In comparison with 2',4'-dihydroxychalcone, attachment of a furan moiety on the A-ring enhanced activity by more than twofold. Benzofurans may be useful in the design of biologically active flavonoids.

Conversion of nicotinic acid to trigonelline is catalyzed by N-methyltransferase belonged to motif B' methyltransferase family in Coffea arabica.

Trigonelline (N-methylnicotinate), a member of the pyridine alkaloids, accumulates in coffee beans along with caffeine. The biosynthetic pathway of trigonelline is not fully elucidated. While it is quite likely that the production of trigonelline from nicotinate is catalyzed by N-methyltransferase, as is caffeine synthase (CS), the enzyme(s) and gene(s) involved in N-methylation have not yet been characterized. It should be noted that, similar to caffeine, trigonelline accumulation is initiated during the development of coffee fruits. Interestingly, the expression profiles for two genes homologous to caffeine synthases were similar to the accumulation profile of trigonelline. We presumed that these two CS-homologous genes encoded trigonelline synthases. These genes were then expressed in Escherichiacoli, and the resulting recombinant enzymes that were obtained were characterized. Consequently, using the N-methyltransferase assay with S-adenosyl[methyl-(14)C]methionine, it was confirmed that these recombinant enzymes catalyzed the conversion of nicotinate to trigonelline, coffee trigonelline synthases (termed CTgS1 and CTgS2) were highly identical (over 95% identity) to each other. The sequence homology between the CTgSs and coffee CCS1 was 82%. The pH-dependent activity curve of CTgS1 and CTgS2 revealed optimum activity at pH 7.5. Nicotinate was the specific methyl acceptor for CTgSs, and no activity was detected with any other nicotinate derivatives, or with any of the typical substrates of B'-MTs. It was concluded that CTgSs have strict substrate specificity. The K(m) values of CTgS1 and CTgS2 were 121 and 184µM with nicotinic acid as a substrate, and 68 and 120µM with S-adenosyl-L-methionine as a substrate, respectively.

Asymmetric synthesis and effect of absolute stereochemistry of YCZ-2013, a brassinosteroid biosynthesis inhibitor.

The four stereoisomers of 2RS,4RS-1-[[2-(2,4-dichlorophenyl)-4-(2-(2-propenyloxy)phenoxymethyl)-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole (YCZ-2013), a novel brassinosteroid biosynthesis inhibitor, were prepared. The diastereomers of 2RS,4R-5 and 2RS,4S-5 were prepared by using the corresponding optically pure R and S toluene-4-sulfonic acid 2,3-dihydroxypropyl ester (R-4,S-4). The enatiomerically and diastereomerically pure acetonide (5) was obtained by a method involving diastereoselective crystallisation of the tosylate salt, followed by re-equilibration with the mother liquor and chromatography. The optical purity of four target compounds (YCZ-2013) was confirmed by chiral high-performance liquid chromatography (HPLC) and NMR. The effects of these stereoisomers on Arabidopsis stem elongation indicated that the cis isomers of 2S,4R-YCZ-2013 and 2R,4S-YCZ-2013 exhibited potent inhibitory activity with IC50 values of approximately 24 ± 3 and 24 ± 2 nM, respectively. The IC50 values of the trans isomers of 2S,4S-YCZ-2013 and 2R,4R-YCZ-2013 are approximately 1510 ± 50 and 3900 ± 332 nM, respectively. Co-application of brassinolide (10nM), the most potent BR, and GA3 (1 µM) to Arabidopsis seedlings grown in the dark with 2R,4S-YCZ-2013 and 2S,4R-YCZ-2013 revealed that brassinolide recovered the induced dwarfism of Arabidopsis seedlings, whereas GA3 showed no effect.

Synthesis and biological evaluation of novel azole derivatives as selective potent inhibitors of brassinosteroid biosynthesis.

Brassinosteroids (BRs) are phytohormones that control several important agronomic traits, such as flowering, plant architecture, seed yield, and stress tolerance. To manipulate the BR levels in plant tissues using specific inhibitors of BR biosynthesis, a series of novel azole derivatives were synthesized and their inhibitory activity on BR biosynthesis was investigated. Structure-activity relationship studies revealed that 2RS, 4RS-1-[4-(2-allyloxyphenoxymethyl)-2-(4-chlorophenyl)-[1,3]dioxolan-2-ylmethyl]-1H-[1,2,4]triazole (G(2)) is a highly selective inhibitor of BR biosynthesis, with an IC(50) value of approximately 46 ± 2 nM, which is the most potent BR biosynthesis inhibitor observed to date. Use of gibberellin (GA) biosynthesis mutants and BR signaling mutants to analyze the mechanism of action of this synthetic series indicated that the primary site of action is BR biosynthesis. Experiments feeding BR biosynthesis intermediates to chemically treated Arabidopsis seedlings suggested that the target sites of this synthetic series are CYP90s, which are responsible for the C-22 and/or C-23 hydroxylation of campesterol.

New Compounds Induce Brassinosteroid Deficient-like Phenotypes in Rice.

Brassinosteroids (BRs) are steroidal plant hormones with potent plant growth promoting activity. Because BR-deficient mutants of rice exhibit altered plant architecture and important agronomic traits, we conducted a systemic search for specific inhibitors of BR biosynthesis to manipulate the BR levels in plant tissues. Although previous studies have been conducted with BR biosynthesis inhibitors in dicots, little is known regarding the effects of BR biosynthesis inhibition in monocot plants. In this work, we used potent inhibitors of BR biosynthesis in Arabidopsis, and we performed a hydroponic culture of rice seedlings to evaluate the effects of BR biosynthesis inhibition. Among the test compounds, we found that 1-[[2-(4-Chlorophenyl)-4-(phenoxymethyl)-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole (1) is a potent inhibitor that could induce phenotypes in rice seedlings that were similar to those observed in brassinosteroid deficient plants. The IC50 value for the retardation of plant growth in rice seedlings was approximately 1.27 ± 0.43 µM. The IC50 value for reducing the bending angle of the lamina joint was approximately 0.55 ± 0.15 µM.

Relationship between structure and antiproliferative activity of polymethoxyflavones towards HL60 cells.

As part of our continuing investigation of polymethoxyflavone (PMF) derivatives as potential anticancer substances, a series of PMF derivatives was synthesized. The synthesized compounds were evaluated for cytotoxicity against the promyelocytic leukemic HL60 cell line, and structure-activity relationship correlations were investigated along with previously isolated PMFs from the peel of king orange (Citrus nobilis). 7,3'-Dimethoxyflavone demonstrated the most potent activity among the synthetic PMFs. Consideration of correlation between the methoxylation pattern and antiproliferative activity revealed the importance of the 3'-methoxyl group and the higher degree of methoxylation on the A-ring moiety of PMFs.

Relationship between structure and antiproliferative activity of 1-azaflavanones.

The synthesis of 19 derivatives of 2-phenyl-3,4-dihydroquinolin-4(1H)-one, as aza analogs of flavanones, was carried out and these compounds were further screened for their antiproliferative activity toward HL60 promyelocytic leukemia cells. In comparison with flavanone the replacement of C-ring ether oxygen atom with a nitrogen atom potentiated activity by more than 100-fold. It was suggested that the aromaticity of the B-ring contributes greatly to the activity of 1-azaflavanones.

Synthesis of 2RS,4RS-1-[2-phenyl-4-[2-(2-trifluromethoxy-phenoxy)-ethyl]-1,3-dioxolan-2-yl-methyl]-1H-1,2,4-triazole derivatives as potent inhibitors of brassinosteroid biosynthesis.

Brassinosteroids are important phytohormones that affect many aspects of plant growth and development. In order to manipulate brassinosteroid levels in plant tissues by using specific biosynthesis inhibitors, we have carried out a systemic search for specific inhibitors of brassinosteroid biosynthesis. Synthesis of triazole derivatives based on the ketoconazole scaffold revealed a series of novel brassinosteroid biosynthesis inhibitors (the YCZ series). To explore the structure-activity relationships of this synthetic series, we now report the synthesis of new triazole derivatives with different aromatic structures at position 2 of 1,3-dioxolane skeleton. We found that the variation of aromatic substituent significantly affect the inhibitory potency. Structure-activity relationships studies indicated that 4-chlorophenyl analogue is the most potent inhibitor of BR biosynthesis with an IC50 value approximately 0.12 ± 0.04 µM, while a bulky biphenyl group exhibited a great negative effect on promoting the inhibitory potency with an IC50 larger than 10 µM.

Synthesis of novel brassinosteroid biosynthesis inhibitors based on the ketoconazole scaffold.

Brassinosteroids (BRs) are steroidal plant hormones that control several important agronomic traits such as plant architecture, seed yield, and stress tolerance. Inhibitors that target BR biosynthesis are candidate plant growth regulators. We synthesized novel triazole derivatives, based on the ketoconazole scaffold, that function as inhibitors of BR biosynthesis. The biological activity of the test compounds was evaluated by determining their ability to induce dwarfism in Arabidopsis seedlings grown in the dark. The chemically induced dwarfism of Arabidopsis seedlings was further evaluated by a rescue experiment using the co-application of brassinolide and/or gibberellins (GA). The structure-activity relationship studies revealed a potent BR biosynthesis inhibitor, 2RS, 4RS-1-{2-(4-chlorophenyl)-4-[2-(2-ethoxyphenyl)-ethyl]-1,3-dioxolan-2-ylmethyl}-1H-1,2,4-triazole (7m), with an IC(50) value of 0.10±0.03 µM for retardation of Arabidopsis seedling stem elongation. The compound-induced hypocotyl dwarfism was counteracted by the co-application of 10nM brassinolide, but not 1 µM GA(3), which produced seedlings that resembled BR-deficient mutants. This result suggests that 7m is a potent and specific inhibitor of BR biosynthesis.

The occurrence of renin inhibitor in rice: isolation, identification, and structure-function relationship.

We found renin inhibitory activity in rice. The physico-chemical data on the isolated inhibitors were identical to those of oleic acid and linoleic acid. Oleic acid and linoleic acid competitively inhibited renin activity, with K(i) values of 15.8 and 19.8 microM respectively. Other unsaturated free fatty acids also inhibited renin activity, but saturated fatty acids had no effect on it.

Essential region for 3-N methylation in N-methyltransferases involved in caffeine biosynthesis.

The caffeine biosynthetic pathway is composed of three methylation steps, and N-methyltransferase catalyzing each step has high substrate specificity. Since the amino acid sequences among coffee 7-methylxanthosine synthase (CmXRS1), theobromine synthase, and caffeine synthase are highly homologous to each other, these substrate specificities seem to be determined in a very restricted region. The analysis of site-directed mutants for CmXRS1 that naturally acts at the initial step, i.e., 7-N methylation of xanthosine, revealed that the activity of 3-N methylation needs a histidine residue at corresponding position 161 in the CmXRS1 sequence. We succeeded in producing the mutant enzyme which can catalyze the first and second methylation steps in caffeine biosynthesis.

A lichen substance as an antiproliferative compound against HL-60 human leukemia cells: 16-O-acetyl-leucotylic acid isolated from Myelochroa aurulenta.

The lichen substance, 16-O-acetyl-leucotylic acid (1), was isolated from an acetone extract of Myelochroa aurulenta and found to exhibit antiproliferative activity against HL-60 human leukemia cells. This is the first report on its anti-leukemia activity (EC(50)=21 microM) which is greater than that of leucotylic acid (2) and the structurally related anti-tumor agent, betulinic acid (4).