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.

Synthesis, antioxidant and anti-hypoxia activities of 6-hydroxygenistein and its methylated derivatives. / 6-ç¾ŸåŸºæŸ“æ–™æœ¨ç´ åŠå ¶ç”²åŸºåŒ–è¡ç”Ÿç‰©çš„åˆæˆå’ŒæŠ—æ°§åŒ–ä¸ŽæŠ—ç¼ºæ°§æ´»æ€§.

OBJECTIVES: Hypoxia is a common pathological phenomenon, usually caused by insufficient oxygen supply or inability to use oxygen effectively. Hydroxylated and methoxylated flavonoids have significant anti-hypoxia activity. This study aims to explore the synthesis, antioxidant and anti-hypoxia activities of 6-hydroxygenistein (6-OHG) and its methoxylated derivatives. METHODS: The 6-OHG and its methoxylated derivatives, including 4',6,7-trimethoxy-5-hydroxyisoflavone (compound 3), 4',5,6,7-tetramethoxyisoflavone (compound 4), 4',6-imethoxy-5,7-dihydroxyisoflavone (compound 6), and 4'-methoxy-5,6,7-trihydroxyisoflavone (compound 7), were synthesized by methylation, bromination, methoxylation, and demethylation using biochanin A as raw material. The structure of these products were characterized by 1hydrogen-nuclear magnetic resonance spectroscopy (1H-NMR) and mass spectrometry (MS). The purity of these compounds was detected by high pressure chromatography (HPLC). The antioxidant activity in vitro was investigated by 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) free radical scavenging assay. PC12 cells were divided into a normal group, a hypoxia model group, rutin (1×10-9-1×10-5 mol/L) groups, and target compounds (1×10-9-1×10-5 mol/L) groups under normal and hypoxic conditions. Cell viability was detected by cell counting kit-8 (CCK-8) assay, the target compounds with excellent anti-hypoxia activity and the drug concentration at the maximum anti-hypoxia activity were screened. PC12 cells were treated with the optimal concentration of the target compound or rutin with excellent anti-hypoxia activity, and the cell morphology was observed under light microscope. The apoptotic rate was determined by flow cytometry, and the expressions of hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) were detected by Western blotting. RESULTS: The structure of 6-OHG and its 4 methylated derivatives were correct, and the purity was all more than 97%. When the concentration was 4 mmol/L, the DPPH free radical removal rates of chemical compounds 7 and 6-OHG were 81.16% and 86.94%, respectively, which were higher than those of rutin, the positive control. The removal rates of chemical compounds 3, 4, and 6 were all lower than 20%. Compared with the normal group, the cell viability of the hypoxia model group was significantly decreased (P<0.01). Compared with the hypoxia model group, compounds 3, 4, and 6 had no significant effect on cell viability under hypoxic conditions. At all experimental concentrations, the cell viability of the 6-OHG group was significantly higher than that of the hypoxia model group (all P<0.05). The cell viability of compound 7 group at 1×10-7 and 1×10-6 mol/L was significantly higher than that of the hypoxia model group (both P<0.05). The anti-hypoxia activity of 6-OHG and compound 7 was excellent, and the optimal drug concentration was 1×10-6 and 1×10-7 mol/L. After PC12 cells was treated with 6-OHG (1×10-6 mol/L) and compound 7 (1×10-7 mol/L), the cell damage was reduced, the apoptotic rate was significantly decreased (P<0.01), and the protein expression levels of HIF-1α and VEGF were significantly decreased in comparison with the hypoxia model group (both P<0.01). CONCLUSIONS: The optimized synthesis route can increase the yield of 6-OHG and obtain 4 derivatives by methylation and selective demethylation. 6-OHG and compound 7 have excellent antioxidant and anti-hypoxia activities, which are related to the structure of the A-ring ortho-triphenol hydroxyl group in the molecule.

Total Synthesis of the Neuroprotective Agent Cudraisoflavone J.

Cudraisoflavone J (1), isolated from Cudrania tricuspidata, is a potent neuroprotective compound with a chiral center. Herein, we report the first total synthesis of racemic cudraisoflavone J (1) using a Claisen rearrangement and a Suzuki coupling reaction as the key steps. Racemic secondary alcohol was kinetically resolved to give (+)- and (-)-cudraisoflavone J with up to 97 and 88% enantiomeric excess, respectively. The modified Mosher's method was used to elucidate the absolute configuration of naturally occurring cudraisoflavone J.

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.

Semisynthesis of prunetin, a bioactive O-methylated isoflavone from naringenin, by the sequential deacetylation of chalcone intermediates and oxidative rearrangement.

Prunetin (4',5-dihydroxy-7-methoxyisoflavone) was semisynthesized in 8 steps from readily available naringenin in 26% total yield. The key reaction was chemoenzymatic sequential deacetylation to 6'-acetoxy-2',4″-dihydroxy-4'-methoxychalcone, the in situ-formed precursor for thallium(III) nitrate-mediated oxidative rearrangement.

Total Syntheses of Prenylated Isoflavones from Erythrina sacleuxii and Their Antibacterial Activity: 5-Deoxy-3'-prenylbiochanin A and Erysubin F.

The prenylated isoflavones 5-deoxyprenylbiochanin A (7-hydroxy-4'-methoxy-3'-prenylisoflavone) and erysubin F (7,4'-dihydroxy-8,3'-diprenylisoflavone) were synthesized for the first time, starting from mono- or di-O-allylated chalcones, and the structure of 5-deoxy-3'-prenylbiochanin A was corroborated by single-crystal X-ray diffraction analysis. Flavanones are key intermediates in the synthesis. Their reaction with hypervalent iodine reagents affords isoflavones via a 2,3-oxidative rearrangement and the corresponding flavone isomers via 2,3-dehydrogenation. This enabled a synthesis of 7,4'-dihydroxy-8,3'-diprenylflavone, a non-natural regioisomer of erysubin F. Erysubin F (8), 7,4'-dihydroxy-8,3'-diprenylflavone (27), and 5-deoxy-3'-prenylbiochanin A (7) were tested against three bacterial strains and one fungal pathogen. All three compounds are inactive against Salmonella enterica subsp. enterica (NCTC 13349), Escherichia coli (ATCC 25922), and Candida albicans (ATCC 90028), with MIC values greater than 80.0 µM. The diprenylated natural product erysubin F (8) and its flavone isomer 7,4'-dihydroxy-8,3'-diprenylflavone (27) show in vitro activity against methicillin-resistant Staphylococcus aureus (MRSA, ATCC 43300) at MIC values of 15.4 and 20.5 µM, respectively. In contrast, the monoprenylated 5-deoxy-3'-prenylbiochanin A (7) is inactive against this MRSA strain.

One-pot bi-enzymatic cascade synthesis of puerarin polyfructosides.

Enzymatic glycosylation is an efficient way to increase the water solubility and the bioavailability of flavonoids. Levansucrases from Bacillus subtilis (Bs_SacB), Gluconacetobacter diazotrophicus (Gd_LsdA), Leuconostoc mesenteroides (Lm_LevS) and Zymomonas mobilis (Zm_LevU) were screened for puerarin (daidzein-8-C-glucoside) fructosylation. Gd_LsdA transferred the fructosyl unit of sucrose onto the glucosyl unit of the acceptor forming ß-d-fructofuranosyl-(2→6)-puerarin (P1a), while Bs_SacB, Lm_LevS and Zm_LevU synthesized puerarin-4'-O-ß-D-fructofuranoside (P1b) and traces of P1a. The Gd_LsdA product P1a was purified and assayed as precursor for the synthesis of puerarin polyfructosides (PPFs). Bs_SacB elongated P1a more competently forming a linear series of water-soluble PPFs reaching at least 21 fructosyl units, as characterized by HPLC-UV-MS, HPSEC and MALDI-TOF-MS. Simultaneous or sequential Gd_LsdA/Bs_SacB reactions yielded PPFs directly from puerarin with the acceptor conversion ranging 82-92 %. The bi-enzymatic cascade synthesis of PPFs in the same reactor avoided the isolation of the intermediate product P1a and it is appropriate for use at industrial scale.

Halogenated Flavones and Isoflavones: A State-of-Art on their Synthesis.

BACKGROUND: Flavonoid is a family of compounds present in the everyday consumption plants and fruits, contributing to a balanced diet and beneficial health effects. Being a scaffold for new drugs and presenting a wide range of applicability in the treatment of illnesses give them also an impact in medicine. Among the several types of flavonoids, flavone and isoflavone derivatives can be highlighted due to their prevalence in nature and biological activities already established. The standard synthetic route to obtain both halogenated flavones and isoflavones is through the use of already halogenated starting materials. Halogenation of the flavone and isoflavone core is less common because it is more complicated and involves some selectivity issues. OBJECTIVE: Considering the importance of these flavonoids, we aim to present the main and more recent synthetic approaches towards their halogenation. METHODS: The most prominent methodologies for the synthesis of halogenated flavones and isoflavones were reviewed. A careful survey of the reported data, using mainly the Scopus database and halogenation, flavones and isoflavones as keywords, was conducted. RESULTS: Herein, a review is provided on the latest and more efficient halogenation protocols of flavones and isoflavones. Selective halogenation and the greener methodologies, including enzymatic and microbial halogenations, were reported. Nevertheless, some interesting protocols that allowed the synthesis of halogenated flavone and isoflavone derivatives in specific positions using halogenated reagents are also summarized. CONCLUSION: Halogenated flavones and isoflavones have risen as noticeable structures; however, most of the time, the synthetic procedures involve toxic reagents and harsh reaction conditions. Therefore, the development of new synthetic routes with low environmental impact is desirable.

Facile Synthesis of Kwakhurin, a Marker Compound of Pueraria mirifica and Its Quantitative NMR Analysis for Standardization as a Reagent.

The side effects of kwao keur dietary supplements (obtained from the tuberous root of Pueraria mirifica) have recently been reported by the Ministry of Health, Labour and Welfare, Japan. To control the quality of kwao keur products, its ingredients need to be maintained by characteristic marker compounds, such as miroestrol, deoxymiroestrol, and kwakhurin (KWA). In this study, we described the facile synthesis of KWA, a marker compound of P. mirifica. Our revised synthetic method produced KWA with shorter steps and higher yield than the reported method. Furthermore, the absolute purity of KWA was determined by quantitative NMR analysis for standardization as a reagent, and its purity was 92.62 ± 0.12%.

Ionic Liquids and Ohmic Heating in Combination for Pd-catalyzed Cross-coupling Reactions: Sustainable Synthesis of Flavonoids.

In order to meet the increasing demand for environmentally benign chemical processes, we developed a Suzuki-Miyaura reaction protocol based on the combination of ohmic heating (ΩH) and supported ionic liquid phase catalysis (SILPC) in aqueous media. This methodology was applied to the synthesis of a series of flavonoid derivatives, including isoflavones, styrylisoflavones, and diarylalkenylisoflavones.

Fabrication of Fine Puerarin Nanocrystals by Box-Behnken Design to Enhance Intestinal Absorption.

Puerarin is widely used as a therapeutic agent to cardiovascular diseases in clinics in China through intravenous administration, which could elicit adverse drug reactions caused by cosolvents, hindering its application in clinics. Therefore, the development of oral dosage is urgently needed. In our previous studies, we proved that the bioavailability of puerarin increased as particle sizes of nanocrystals decreased; however, we have not optimized the best process parameters for nanocrystals. In this study, we aim to fabricate fine nanocrystals (with smallest particle size) by Box-Behnken design and study the intestinal permeability of puerarin and its nanocrystals via employing everted gut sac model and in situ perfusion model. The results showed that the Box-Behnken design could be used to optimize the producing parameters of puerarin nanocrystals, and the particle sizes of fine nanocrystals were about 20 nm. Results of everted gut sacs showed that the polyvinylpyrrolidone (PVP) and verapamil had no influence on the absorption of puerarin and nanocrystals, and the nanocrystals could increase the Papp of puerarin for 2.2-, 2.9-, and 2.9-folds, respectively, in duodenum, jejunum, and ileum. Enhanced Ka and Peff were observed on the nanocrystal group, compared with puerarin, and PVP and verapamil had no influence on the absorption of nanocrystals, while the absorption of puerarin was influenced by P-gp efflux. Combining the results mentioned above, we can conclude that the Box-Behnken design benefits the optimization for preparation of nanocrystals, and the nanocrystals could enhance the intestinal absorption of puerarin by enhanced permeability and inhibited P-gp efflux.

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).

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.

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.

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.