Total Syntheses and Antibacterial Studies of Natural Isoflavones: Scandenone, Osajin, and 6,8-Diprenylgenistein.

Isoflavones are a class of natural products that exhibit a wide range of interesting biological properties, including antioxidant, hepatoprotective, antimicrobial, and anti-inflammatory activities. Scandenone (1), osajin (2), and 6,8-diprenylgenistein (3) are natural prenylated isoflavones that share the same polyphenol framework. In this research, the key intermediate 15 was used for the synthesis of the natural isoflavones 1-3, establishing a stereoselective synthetic method for both linear and angular pyran isoflavones. The antibacterial activities of 1-3 were also evaluated, and all of them displayed good antibacterial activity against Gram-positive bacteria. Among them, 2 was the most potent one against MRSA, with a MIC value of 2 µg/mL, and the SEM assay indicated that the bacterial cell membranes of both MRSA and E. faecalis could be disrupted by 2. These findings suggest that this type of isoflavone could serve as a lead for the development of novel antibacterial agents for the treatment of Gram-positive bacterial infections.

An Efficient Regioselective Synthesis of 8-Formylhomoisoflavonoids with Neuroprotective Activity by Enhancing Autophagy.

6-Formylisoophiopogonone B (7a) and 8-formylophiopogonone B (7b), two natural products isolated from Ophiopogon japonicus, represent a subgroup of rare 6/8-formyl/methyl-homoisoflavonoid skeletons. Herein we report an efficient method for the synthesis of these formyl/methyl-homoisoflavonoids. The synthesized compounds were evaluated for their neuroprotective effects on the MPP+-induced SH-SY5Y cell injury model and showed marked activity. Exploration of the neuroprotective mechanisms of compound 7b led to an increased expression of autophagy marker LC3-II and down-regulation of autophagy substrate p62/SQSTM1. Molecular docking studies showed that 7b may prevent the inhibition of the classic PI3K-AKT-mTOR signaling pathway by interfering with the human HSP90AA1.

Natural Isoflavones and Semisynthetic Derivatives as Pancreatic Lipase Inhibitors.

Obesity, now widespread all over the world, is frequently associated with some chronic diseases. Thus, there is a growing interest in the prevention and treatment of obesity. To date, the only antiobesity drug is orlistat, a natural product-derived pancreatic lipase (PL) inhibitor with some undesired side effects. In the last decades, many natural compounds or derivatives have been evaluated as potential PL inhibitors, and natural polyphenols are among the most promising for possible exploitation as antiobesity agents. However, few studies have been devoted to isoflavones. In this work, we report a study on the PL inhibitory properties of a small library of semisynthetic isoflavone derivatives together with the natural leads daidzein (1), genistein (2), and formononetin (3). In vitro lipase inhibition assay showed that 2 is the most promising PL inhibitor. Among synthetic isoflavones, the hydroxylated and brominated derivatives were more potent than their natural leads. Detailed studies through fluorescence measurements and kinetics of lipase inhibition showed that 2 and the bromoderivatives 10 and 11 have the greatest affinity for PL. Docking studies corroborated these findings highlighting the interactions between isoflavones and the enzyme, confirming that hydroxylation and bromination are useful modifications.

Molecular Design, Synthesis and Docking Study of Alkyl and Benzyl Derivatives of Robustic Acid as Topoisomerase I Inhibitors.

Robustic acid is reported to be a bioactive compound, isolated from the medicinal plant Dalbergia benthamii Prain. Ten alkyl and benzyl derivatives (2a-2j) of robustic acid were designed and synthesized based on molecular docking approaches. The biological activities of most of the synthesized compounds (such as 2g, 2h, and 2i) were closely consistent with the docking results. In particular, 4-O-phenylpropyl substituted compound 2g displayed potent topoisomerase I inhibitory activity as well as cytotoxicity against SMMC-7721, HepG2, and HeLa cell lines. Further biological testing suggests that compound 2g acted mainly by an arrest of the tumor cells in G1 phase of the cell cycle and suppressed cell proliferation by inducing apoptosis. The findings of this study are encouraging with respect to potential utilization of these compounds as new topoisomerase I inhibitors.

In vitro antiproliferative activity of glabridin derivatives and their in silico target identification.

Novel Mannich base derivatives of glabridin were synthesized and their antiproliferative activity were performed along with our previously reported glabridin-chalcone hybrids molecules (GCHMs) against various human cell lines MDA-MB-231 (breast adenocarcinoma), HEK-293 (embryonic kidney cell line), K562 (leukemia), MCF-7 (breast adenocarcinoma), HeLa (cervix adenocarcinoma), HepG2 (hepatocellular carcinoma) and WRL-68 (hepatic carcinoma). The result showed that the glabridin significantly reduced cell proliferation with IC50 ranges from 3.67 to 58.30 µM against all the tested cell lines. The remarkable reduction in antiproliferative activity 2',4'-dimethoxyglabridin and GCHMs compounds with phenolic OH groups protected by methoxy (OCH3) groups suggested that the free OH groups are essential factor for the antiproliferative activity of glabridin and its derivatives. The Mannich base derivatives of glabridin showed moderate activity IC50 (2.20->95.78 µM). Furthermore, in silico target identification analysis revealed that AKT1, DECR1 and NOS1 are the potential targets for glabridin and their derivatives.

Synthesis of Isoflavones by Tandem Demethylation and Ring-Opening/Cyclization of Methoxybenzoylbenzofurans.

The unexpected conversion of benzoylbenzofurans into isoflavones through an intramolecular cascade that involves deprotection and ring-opening/cyclization is described. This was discovered in an investigation of the possible transformation of benzoylbenzofurans into coumaronochromones. This route affords isoflavones in two major steps from acetophenones and benzoquinones. The transformation was validated by synthesizing differently substituted isoflavone derivatives and further applied to a concise synthesis of a potential anticancer lead compound, glaziovianin A (1).

Enantioselective Synthesis of Homoisoflavanones by Asymmetric Transfer Hydrogenation and Their Biological Evaluation for Antiangiogenic Activity.

Neovascular eye diseases are a major cause of blindness. Excessive angiogenesis is a feature of several conditions, including wet age-related macular degeneration, proliferative diabetic retinopathy, and retinopathy of prematurity. Development of novel antiangiogenic small molecules for the treatment of neovascular eye disease is essential to provide new therapeutic leads for these diseases. We have previously reported the therapeutic potential of anti-angiogenic homoisoflavanone derivatives with efficacy in retinal and choroidal neovascularization models, although these are racemic compounds due to the C3-stereogenic center in the molecules. This work presents asymmetric synthesis and structural determination of anti-angiogenic homoisoflavanones and pharmacological characterization of the stereoisomers. We describe an enantioselective synthesis of homoisoflavanones by virtue of ruthenium-catalyzed asymmetric transfer hydrogenation accompanying dynamic kinetic resolution, providing a basis for the further development of these compounds into novel experimental therapeutics for neovascular eye diseases.

Synthesis of Daidzein Glycosides, α-Tocopherol Glycosides, Hesperetin Glycosides by Bioconversion and Their Potential for Anti-Allergic Functional-Foods and Cosmetics.

Daidzein is a common isoflavone, having multiple biological effects such as anti-inflammation, anti-allergy, and anti-aging. α-Tocopherol is the tocopherol isoform with the highest vitamin E activity including anti-allergic activity and anti-cancer activity. Hesperetin is a flavone, which shows potent anti-inflammatory effects. These compounds have shortcomings, i.e., water-insolubility and poor absorption after oral administration. The glycosylation of bioactive compounds can enhance their water-solubility, physicochemical stability, intestinal absorption, and biological half-life, and improve their bio- and pharmacological properties. They were transformed by cultured Nicotiana tabacum cells to 7-ß-glucoside and 7-ß-gentiobioside of daidzein, and 3'- and 7-ß-glucosides, 3',7-ß-diglucoside, and 7-ß-gentiobioside of hesperetin. Daidzein and α-tocopherol were glycosylated by galactosylation with ß-glucosidase to give 4'- and 7-ß-galactosides of daidzein, which were new compounds, and α-tocopherol 6-ß-galactoside. These nine glycosides showed higher anti-allergic activity, i.e., inhibitory activity toward histamine release from rat peritoneal mast cells, than their respective aglycones. In addition, these glycosides showed higher tyrosinase inhibitory activity than the corresponding aglycones. Glycosylation of daidzein, α-tocopherol, and hesperetin greatly improved their biological activities.

An investigation on the metabolic pathways of synthetic isoflavones by gas chromatography coupled to high accuracy mass spectrometry.

RATIONALE: Isoflavones are a group of flavonoids that may be of interest in sport doping because they can be used by athletes in the recovery periods after the administration of anabolic steroids, with the aim of increasing the natural production of luteinizing hormone (LH) and, consequently, the biosynthesis of endogenous androgens. METHODS: The in vivo metabolism of methoxyisoflavone (5-methyl-7-methoxyisoflavone) and ipriflavone (7-isopropoxyisoflavone), respectively present in a dietary supplement and in a pharmaceutical preparation, was investigated. The study was carried out by the analysis of urinary samples collected from male Caucasian subjects before, during and after the oral administration of methoxyisoflavone or ipriflavone. After enzymatic hydrolysis and liquid-liquid extraction, all urinary samples were analyzed by gas chromatography/quadrupole time-of-flight (qTOF MS system/qTOF) electron ionization mass spectrometry (EI-MS). RESULTS: Eight metabolites of methoxyisoflavone and six metabolites of ipriflavone were isolated. The corresponding accurate mass spectra are specific for isoflavone structures and revealed also a retro-Diels-Alder fragmentation. CONCLUSIONS: When excreted in large amounts, the urinary metabolites of methoxyisoflavone and ipriflavone can be traced to potential confounding factors in doping analysis. As methoxyisoflavone and ipriflavone have been shown to inhibit the enzyme aromatase, thus interfering with the normal metabolic pathways of testosterone, the detection of their intake, by screening for the presence of their main metabolites in urine, might be helpful in routine doping control analysis.

Synthesis, characterization, and formulation of poly-puerarin as a biodegradable and biosafe drug delivery platform for anti-cancer therapy.

Nano-drug delivery systems are widely used in medical diagnoses, tumour drug delivery and other fields due to their unique advantages. Thus, the preparation of more biocompatible nanocarriers by modifying natural materials has become a research hotspot in recent years. As a type of abundant and environment-friendly natural material, puerarin has been proven to be effective in the treatment of many diseases. However, its low solubility and low oral utilization limit its use. In this study, a novel biocompatible nanomaterial was developed. Puerarin was modified with an unsaturated olefin via acryloyl chloride and the amphiphilic polymer poly-puerarin was finally obtained through free radical polymerization, which was used in the preparation of a drug delivery system. Poly-puerarin nanoparticles (PPue NPs) were prepared by nanoprecipitation and used as a platform to load paclitaxel (PPue@PTX NPs). Physicochemical characterization showed that the nanoparticle size was around 70 nm and the drug loading efficiency of PTX was up to 23.8%. The cytotoxicity test revealed that the modified puerarin derivatives, PPue, exhibited good biocompatibility even at large doses of 100 µg mL-1 and the PPue@PTX NPs still maintained the excellent anti-cancer effect of PTX. The in vitro cellular uptake assay demonstrated that the PPue@PTX NPs were rapidly uptaken by CT26 cells. In the in vivo experiments, the PPue@PTX NPs featured rapid aggregation and slow clearance and the most significant effect of inhibiting tumour growth among all the treatment groups. Therefore, our work provides a new strategy for the modification of natural drugs and PPue is expected to become a new safe and reliable nano-drug delivery platform.

Semi-synthetic isoflavones as BACE-1 inhibitors against Alzheimer's disease.

BACE-1 is considered to be one of the targets for prevention and treatment of Alzheimer's disease (AD). We here report a novel class of semi-synthetic derivatives of prenylated isoflavones, obtained from the derivatization of natural flavonoids from Maclura pomifera. In vitro anti-AD effect of the synthesized compounds were evaluated via human recombinant BACE-1 inhibition assay. Compound 7, 8 and 13 were found to be the most active candidates which demonstrates good correlation between the computational docking and pharmacokinetic predictions. Moreover, cytotoxic studies demonstrated that the compounds are not toxic against normal and cancer cell lines. Among these three compounds, compound 7 enhance the activity of P-glycoprotein (P-gp) on A549 cancer cells and increases the activity of P-gp ATPase with a possible role on the efflux of amyloid-ß across the blood- brain barrier. In conclusion, the present findings may pave the way for the discovery of a novel class of compounds to prevent and/or treat AD.

Novel hybrids of podophyllotoxin and formononetin inhibit the growth, migration and invasion of lung cancer cells.

In this study, three hybrids of podophyllotoxin and formononetin were synthesized and evaluated for anticancer efficacy. Some of the derivatives exhibited potent cytotoxicity against a panel of human and mouse cancer cell lines, with IC50 values in the low micromolar to submicromolar range. Evaluation against A549 lung tumor cell line identified that the IC50 value of compound 10a was 0.753 µM, indicating that 10a was 2.568-fold more efficacious than parent podophyllotoxin. Mechanistic studies revealed that 10a induced A549 cell apoptosis mainly via caspase pathway, as well as disrupted the microtubule organization by occupying the colchicine binding site of the tubulin. Moreover, wound healing assay and transwell invasion assay indicated that 10a displayed potent inhibitory effects on invasion and migration in A549 cancer cells. In additiona, a decrease in vimentin immunostaining was also observed in A549 cells after treatment with 10a. Overall, hybrid 10a might be a promising candidate for the potential treatment of human lung carcinoma.

Structure-guided design and synthesis of isoflavone analogs of GW4064 with potent lipid accumulation inhibitory activities.

Our group has previously reported a series of isoflavone derivatives with antidyslipidemic activity. With this background, a series of isoflavone analogs of GW4064 were designed, synthesized and evaluated the lipid-lowering activity of analogs. As a result, most of compounds significantly reduced the lipid accumulation in 3T3-L1 adipocytes and four of them (10a, 11, 15c and 15d) showed stronger inhibitory than GW4064. The most potent compound 15d exhibited promising agonistic activity for FXR in a cell-based luciferase reporter assay. Meanwhile, 15d up-regulated FXR, SHP and BSEP gene expression and down-regulated the mRNA expression of lipogenesis gene SREBP-1c. Besides, an improved safety profile of 15d was also observed in a HepG2 cytotoxicity assay compared with GW4064. The obtained biological results were further confirmed by a molecular docking study showing that 15d fitted well in the binding pocket of FXR and interacted with some key residues simultaneously.

Synthesis and in vitro evaluation of homoisoflavonoids as potent inhibitors of nitric oxide production in RAW-264.7 cells.

Syntheses of natural homoisoflavonoids, (±)-portulacanones A-C (4, 8 and 9), portulacanone D (6), isolated from Portulaca oleracea L. (POL) and their derivatives (3, 5 and 7) have been achieved for the first time along with the synthesis of known derivatives (1 and 2) and their in vitro inhibitory effect against NO production in LPS-induced RAW-264.7 macrophages was evaluated as an indicator of anti-inflammatory activity. All the compounds tested had a concentration-dependent inhibitory effect on NO production by RAW-264.7 macrophages without obvious cytotoxicity. Compounds 3 (97.2% at 10 µM; IC50 = 1.26 µM) followed by 6 (portulacanone D) (92.5% at 10 µM; IC50 = 2.09 µM), 1 (91.4% at 10 µM; IC50 = 1.75 µM) and 7 (83.0% at 10 µM; IC50 = 2.91 µM) were the most potent from the series. This finding was further correlated with the suppressed expression of iNOS induced by LPS. Our promising preliminary results may provide the basis for the assessment of compound 3 as a lead structure for a NO production-targeted anti-inflammatory drug development and also could support the usefulness of POL as a folklore medicinal plant in the treatment of inflammatory diseases.

Developing antineoplastic agents that target peroxisomal enzymes: cytisine-linked isoflavonoids as inhibitors of hydroxysteroid 17-beta-dehydrogenase-4 (HSD17B4).

Cytisine-linked isoflavonoids (CLIFs) inhibited PC-3 prostate and LS174T colon cancer cell proliferation by inhibiting a peroxisomal bifunctional enzyme. A pull-down assay using a biologically active, biotin-modified CLIF identified the target of these agents as the bifunctional peroxisomal enzyme, hydroxysteroid 17ß-dehydrogenase-4 (HSD17B4). Additional studies with truncated versions of HSD17B4 established that CLIFs specifically bind the C-terminus of HSD17B4 and selectively inhibited the enoyl CoA hydratase but not the d-3-hydroxyacyl CoA dehydrogenase activity. HSD17B4 was overexpressed in prostate and colon cancer tissues, knocking down HSD17B4 inhibited cancer cell proliferation, suggesting that HSD17B4 is a potential biomarker and drug target and that CLIFs are potential probes or therapeutic agents for these cancers.

Synthesis of isoflavene-thiosemicarbazone hybrids and evaluation of their anti-tumor activity.

Phenoxodiol is an isoflavene with potent anti-tumor activity. In this study, a series of novel mono- and di-substituted phenoxodiol-thiosemicarbazone hybrids were synthesized via the condensation reaction between phenoxodiol with thiosemicarbazides. The in vitro anti-proliferative activities of the hybrids were evaluated against the neuroblastoma SKN-BE(2)C, the triple negative breast cancer MDA-MB-231, and the glioblastoma U87 cancer cell lines. The mono-substituted hybrids exhibited potent anti-proliferative activity against all three cancer cell lines, while the di-substituted hybrids were less active. Selected mono-substituted hybrids were further investigated for their cytotoxicity against normal MRC-5 human lung fibroblast cells, which identified two hybrids with superior selectivity for cancer cells over normal cells as compared to phenoxodiol. This suggests that mono-substituted phenoxodiol-thiosemicarbazone hybrids have promising potential for further development as anti-cancer agents.

Design and synthesis of formononetin-dithiocarbamate hybrids that inhibit growth and migration of PC-3 cells via MAPK/Wnt signaling pathways.

A series of novel formononetin-dithiocarbamate derivatives were designed, synthesized and evaluated for antiproliferative activity against three selected cancer cell line (MGC-803, EC-109, PC-3). The first structure-activity relationship (SAR) for this formononetin-dithiocarbamate scaffold is explored in this report with evaluation of 14 variants of the structural class. Among these analogues, tert-butyl 4-(((3-((3-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)propyl)thio)carbonothioyl)piperazine-1-carboxylate (8i) showed the best inhibitory activity against PC-3 cells (IC50 = 1.97 µM). Cellular mechanism studies elucidated 8i arrests cell cycle at G1 phase and regulates the expression of G1 checkpoint-related proteins in concentration-dependent manners. Furthermore, 8i could inhibit cell growth via MAPK signaling pathway and inhibit migration via Wnt pathway in PC-3 cells.

Design, synthesis and biological evaluation of photoaffinity probes of antiangiogenic homoisoflavonoids.

A naturally occurring homoisoflavonoid, cremastranone (1) inhibited angiogenesis in vitro and in vivo. We developed an analogue SH-11037 (2) which is more potent than cremastranone in human retinal microvascular endothelial cells (HRECs) and blocks neovascularization in animal models. Despite their efficacy, the mechanism of these compounds is not yet fully known. In the course of building on a strong foundation of SAR and creating a novel chemical tool for target identification of homoisoflavonoid-binding proteins, various types of photoaffinity probes were designed and synthesized in which benzophenone and biotin were attached to homoisoflavanonoids using PEG linkers on either the C-3' or C-7 position. Notably, the photoaffinity probes linking on the phenol group of the C-3' position retain excellent activity of inhibiting retinal endothelial cell proliferation with up to 72nM of GI50.

Synthesis and cytotoxicity evaluation of 3-amino-2-hydroxypropoxyisoflavone derivatives.

Soy isoflavones exert a wide variety of biological activities, such as antioxidant, anti-inflammatory and anti-cancer properties. Nuclear factor erythroid 2-related factor 2 (Nrf2), a bZip transcription factor, plays a key role in soy isoflavones induced protection against oxidative stress and cancer. To obtain more effective isofavones, a series of 7,4'-bis-(3-amino-2-hydroxypropoxy), 7 or 4'-(3-amino-2-hydroxypropoxy) isoflavone derivatives have been synthesized as potential antitumor agents and Nrf2/ARE (antioxidant response element) activators. The cytotoxicity of these compounds in human cancer cell lines MDA-MB-231, HT-29, HCT116, HepG2 and 7402 was tested by MTT assay. In this study, the cytotoxicity of compound 3b exhibited highest cytotoxic activity and at the safety dose range, it also strongly up-regulated antioxidant response element (ARE)-luciferase reporter activity. In addition, compound 3b induced Nrf2 nuclear translocation and upregulated its downstream target genes NQO-1 and HO-1 at protein level. Taken together, our results suggest that compound 3b could be a potential agent for cancer themotherapy or cancer chemoprevention.

GEN-27, a Newly Synthetic Isoflavonoid, Inhibits the Proliferation of Colon Cancer Cells in Inflammation Microenvironment by Suppressing NF-κB Pathway.

Nonresolving inflammation is one of the consistent features of the tumor microenvironment in the intestine and plays a critical role in the initiation and development of colon cancer. Here we reported the inhibitory effects of GEN-27, a new derivative of genistein, on the inflammation-related colon cancer cell proliferation and delineated the mechanism of its action. The results indicated that GEN-27 inhibited the proliferation of human colon tumor HCT116 cells stimulated by culture supernatants of LPS-induced human monocytes THP-1 cells and significantly decreased LPS-induced secretion of proinflammatory cytokines interleukin-6 and interleukin-1ß in THP-1 cells. The HCT116 cell proliferation elicited by THP-1-conditioned medium could be blocked by the interleukin-1 receptor antagonist (IL-1RA). Further mechanistic study revealed that GEN-27 remarkably inhibited the nuclear translocation of NF-κB and phosphorylation of IκB and IKKα/ß in both HCT116 and THP-1 cells. In addition, GEN-27 markedly suppressed the HCT116 cell proliferation stimulated by IL-1ß treatment, which was dependent on the inhibition of NF-κB/p65 nuclear localization, as verified by p65 overexpression and BAY 11-7082, an NF-κB inhibitor. Taken together, our findings established that GEN-27 modulated NF-κB signaling pathway involved in inflammation-induced cancer cells proliferation and therefore could be a potential chemopreventive agent against inflammation-associated colon cancer.