Iron(III) chloride-based mild synthesis of phenanthrene and its application to total synthesis of phenanthroindolizidine alkaloids

Iron(III) chloride has been used to prepare polymethoxy-substituted phenanthrene-9-carboxylic acid via intramolecular oxidative coupling at room temperature in excellent yields. Mild reaction conditions and the use of environmentally friendly FeCl₃ provide a novel practical route for the synthesis of the important phenanthrene ring. The further application of this protocol as the key step to total synthesis of tylophorine, deoxytylophorinine, and antofine was achieved starting from readily available pyrrole in 48%, 44%, and 46% overall yields, respectively. This new and efficient strategy enjoys a number of advantages. The experimental procedure is simple under mild conditions, atom economy is very high without any protecting-group, starting materials are cheap or easily prepared. Hence this short and practical method is applicable to large-scale production.     

牛心朴子草植物农药的化学成分与生物活性研究

在分离鉴定化学组分基础上结合普筛农药活性,发现牛心朴子草提取物中生物碱部位显示抗植物病毒极高活性。通过生物活性跟踪与色谱分离、结构鉴定,确定它是菲并吲哚里西啶生物碱,包括7-脱甲氧基娃儿藤碱(Antofine)(1)和N-氧代-7-脱甲氧基娃儿藤碱(N-Oxideantofine)(2)。其中2是首次从该草分离得到。Antofine(1)是该草抑制植物病毒主要成份;半中枯斑法测定它在10^-^6g/mL浓度的枯斑抑制率达60%,比对照的常用植物病毒抑制剂活性高1～2个数量级。用多种生测方法验证该草生物碱试样抗植物病毒如烟草花叶病毒(TMV)、马铃薯Y病毒(PVY)、芜菁花叶病毒(TuMV)等的生物活性。     

Determination of Cytotoxic Activity of Sanguinaria canadensis Extracts against Human Melanoma Cells and Comparison of Their Cytotoxicity with Cytotoxicity of Some Anticancer Drugs

Melanoma is an enormous global health burden, and should be effectively addressed with better therapeutic strategies. Therefore, new therapeutic agents are needed for the management of this disease. The aim of this study was the investigation of cytotoxic activity of some isoquinoline alkaloid standards and extracts obtained from Sanguinaria canadensis—collected before, during, and after flowering—against three different human melanoma cells (A375, G361, SK-MEL-3). The cytotoxicity of these extracts was not previously tested on these melanoma cell lines. Determination of alkaloid contents was performed by HPLC-DAD using Polar RP column and mobile phase containing acetonitrile, water, and 1-butyl-3-methylimidazolium tetrafluoroborate. The cytotoxicity of alkaloid standards was investigated by determination of cell viability and calculation of IC₅₀ values. Significant differences were observed in the alkaloids content and cytotoxic activity of the extracts, depending on the season of collection of the plant material. In the Sanguinaria canadensis extracts high contents of sanguinarine (from 4.8543 to 9.5899 mg/g of dry plant material) and chelerythrine (from 42.7224 to 6.8722 mg/g of dry plant material) were found. For both of these alkaloids, very high cytotoxic activity against the tested cell lines were observed. The IC₅₀ values were in the range of 0.11–0.54 µg/mL for sanguinarine and 0.14 to 0.46 µg/mL for chelerythrine. IC₅₀ values obtained for Sanguinaria canadensis extracts against all tested cell lines were also very low (from 0.88 to 10.96 µg/mL). Cytotoxic activity of alkaloid standards and Sanguinaria canadensis extracts were compared with the cytotoxicity of anticancer drugs—etoposide, cisplatin, and hydroxyurea. In all cases except the one obtained for cisplatin against A375, which was similar to that obtained for Sanguinaria canadensis after flowering against the same cell line, IC₅₀ values obtained for anticancer drugs were higher than the IC₅₀ values obtained for sanguinarine, chelerythrine, and Sanguinaria canadensis extracts. Our results showed that Sanguinaria canadensis extracts and isoquinoline alkaloids, especially sanguinarine and chelerythrine, could be recommended for further in vivo experiments in order to confirm the possibility of their application in the treatment of human melanomas.     

Fractionation of Lycopodiaceae Alkaloids and Evaluation of Their Anticholinesterase and Cytotoxic Activities

In view of the abundant evidence that Lycopodiaceae alkaloids, including the well-known huperzine A (HupA), are among the potent acetylcholinesterase (AChE) inhibitors, an attempt was made to search for new compounds responsible for this property. For this purpose, three plant species belonging to the Lycopodiaceae family, commonly found in the Euro-Asia region, were subjected to the isolation of bioactive compounds, their identification and subsequent evaluation of their anticholinesterase and cytotoxic activities. Methanolic extracts of two Lycopodium and one Hupezia species were obtained via optimized pressurized liquid extraction (PLE) and then pre-purified using innovative gradient vacuum liquid chromatography (gVLC). For the first time, three sorbents of different porosity packed in polypropylene cartridges and mobile phase systems of different polarity were used to elute the target compounds. This technique proved to be a rapid tool for the obtainment of alkaloid fractions and allowed one to select the appropriate process conditions to yield potent AChE inhibitors in each of the species studied. More than 100 collected fractions were analyzed via HPLC/ESI-QTOF-MS, which enabled one to detect more than 50 compounds, including several new ones previously unreported. Some of them were present in high purity fractions (60–90% of the established purity). TLC bioautography assays proved that the analyzed species are rich sources of AChE inhibitors, but H. selago showed the highest anti-AChE activity. Additionally, the modified silanized silica gel sorbent used allowed one to isolate L. clavatum alkaloids more efficiently using an aqueous reversed-phase solvent system. Furthermore, the tested extracts from the three plant extracts were found to be safe, as they did not exhibit cytotoxicity to skin fibroblasts.     

Directed metalation of tertiary benzamides. Abbreviated syntheses of phenanthro-quinolizidine and -indolizidine alkaloids

Short synthesis of the alkaloids cryptopleurine ($\text{1}$) and antofine ($\text{2}$) via directed metalation of the common phenanthrene amide $\text{3}$ are described.     

Cobalt(III) Chaperone Complexes of Curcumin: Photoreduction,Cellular Accumulation and Light‐Selective Toxicity towards Tumour Cells

Light‐activated prodrugs offer the potential for highly selective tumour targeting. However, the application of many photoactivated chemotherapeutics is limited by a requirement for oxygen, or for short activation wavelengths that can damage surrounding tissue. Herein, we present a series of cobalt(III)‐curcumin prodrugs that can be activated by visible light under both oxygenated and hypoxic conditions. Furthermore, the photoproduct can be controlled by the activation wavelength: green light yields free curcumin, whereas blue light induces photolysis of curcumin to a phototoxic product. Confocal fluorescence microscopy and phototocytotoxicity studies in DLD‐1 and MCF‐7 tumour cells demonstrated that the cobalt(III) prodrugs are nontoxic in the dark but accumulate in significant concentrations in the cell membrane. When cells were treated with light for 15 min, the cytotoxicity of the cobalt complexes increased by up to 20‐fold, whereas free curcumin exhibited only a two‐fold increase in cytotoxicity. The nature of the ancillary ligand and cobalt reduction potential were found to strongly influence the stability and biological activity of the series.     

Facile preparation of pH-responsive PEGylated prodrugs for activated intracellular drug delivery

PEGylated prodrug, covalent attaching polyethylene glycol (PEG) polymer chains to therapeutic drugs, is one of the most promising techniques to improve the water-solubility, stability, and therapeutic effect of drugs. In this study, three PEGylated acid-sensitive prodrugs DOX-PEG-DOX with different molecular weights, were prepared via Schiff-base reaction between aldehyde-modified PEG and the amino groups of doxorubicin (DOX). This kind of amphiphilic polymeric prodrug could be self-assemble into nanoparticles in aqueous solution. The average particle size and morphologies of the prodrug nanoparticles under different pH conditions were observed by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. It turned out that the nanoparticles could be kept stable in the physiological environment, but degraded in acidic medium. Subsequently, we also investigated in vitro drug release behavior and found that the prodrug had acid-sensitive property. The cytotoxicity and intracellular uptake assays revealed that the prodrugs could rapidly internalized by HeLa or HepG2 cells to release DOX and effectively inhibited the proliferation of the tumor cells, which have the potential for use in cancer therapy.     

Optimization of Pressurized Liquid Extraction of Lycopodiaceae Alkaloids Obtained from Two Lycopodium Species

Alkaloids of the Lycopodiaceae family are of great interest to researchers due to their numerous properties and wide applications in medicine. They play a very important role mainly due to their potent antioxidant, antidepressant effects and a reversible ability to inhibit acetylcholinesterase (AChE) enzyme activity. This property is of immense importance due to the growing problem of an increasing number of patients with neurodegenerative diseases in developed countries and a lack of effective and efficient treatment for them. Numerous studies have shown that Lycopodiaceae alkaloids are a rich source of AChE inhibitors. In the obtaining of new therapeutic phytochemicals from plant material, the extraction process and its efficiency is crucial. Therefore, the aim of this work was to optimize the conditions of modern PLE to obtain bioactive alkaloids from two Lycopodium species: L. clavatum L. and L. annotinum L. Five different solvents of different polarity were used for prepared plant extracts in order to compare the alkaloid content in and thereby effectiveness of the entire extraction. PLE parameters were used based on multiple studies conducted that gave the highest alkaloids recovery. Crude extracts were purified using solid-phase extraction (SPE) on Oasis HLB cartridge and examined by HPLC/ESI-QTOF–MS of the highly abundant alkaloids. To the best of our knowledge, this is the first time such high recoveries have been obtained for known Lycopodiaceae alkaloids. The best extraction results of alkaloid-lycopodine were detected in the dichloromethane extract from L. clavatum, where the yield exceeded 45%. The high recovery of annotinine above 40% presented in L. annotinum was noticed in dichloromethane and ethyl acetate extracts. Moreover, chromatograms were obtained with all isolated alkaloids and the best separation and quality of the bands in methanolic extracts. Interestingly, no alkaloid amounts were detected in cyclohexane extracts belonging to the non-polar solvent. These results could be helpful for understanding and optimizing the best conditions for isolating potent AChE inhibitors.     

Expedient synthesis and structure-activity relationships of phenanthroindolizidine and phenanthroquinolizidine alkaloids

The total synthesis of alkaloids phenanthroindolizidine 1a, tylophorine 1b, and phenanthroquinolizidine 1c, has been achieved in 46%, 49%, and 42% overall yield, respectively, starting from the corresponding phenanthrene-9-carboxaldehyde. Compound exhibited potent inhibition activity in three human cancer cell lines, with IC(50) values ranging from 104 to 130 nM. The structure-activity relations of these alkaloids and some of their synthetic intermediates against the three cell lines were also described.     

Isolation and structure determination of lysiformine from bacteria associated with marine sponge Halichondria okadai

Marine sponge Halichondria okadai is a source of numerous toxic secondary metabolites, which are considered as products of the symbiotic species associated with the sponge. In this study, we tested 720 culture conditions and purified 48 sponge-associated microbes. The ethyl acetate extract from the liquid culture of each species was tested for cytotoxicity, hemolytic activity, and brine shrimp lethal activity. Lysiformine (1) was isolated from Lysinibacillus fusiformis and its structure was determined by HRMS and NMR.     

Oligoglycol carbonate prodrugs of 5-modified 2'-deoxyuridines: synthesis and antibacterial activity

In order to develop a new generation of antibacterial nucleosides, a representative set of novel 3'- and 5'-tri- or tetraethylene glycol prodrug forms of 5-alkyloxymethyl-2'- deoxyuridines was synthesized. These compounds were at least two orders of magnitude more soluble than the parent nucleosides, possessed significant inhibitory activity against a set of bacteria including resistant strains of Staphylococcus aureus and Mycobacterium smegmatis, and showed low cytotoxicity. The obtained data indicate that glycol carbonates are convenient and prospective for usage in prodrugs of nucleoside derivatives with antibacterial activity.     

Erufosine (ErPC3) Cationic Prodrugs as Dual Gene Delivery Reagents for Combined Antitumor Therapy

Sixteen cationic prodrugs of the antitumor alkylphospholipid (APL) erufosine were rationally synthesized to provide original gene delivery reagents with improved cytotoxicity profile. The DNA complexation properties of these cationic lipids were determined and associated transfection rates were measured. Furthermore, the self-assembly properties of the pro-erufosine compounds were investigated and their critical aggregation concentration was determined. Their hydrolytic stability under pH conditions mimicking the extracellular environment and the late endosome milieu was measured. Hemolytic activity and cytotoxicity of the compounds were investigated. The results obtained in various cell lines demonstrate that the prodrugs of erufosine display antineoplastic activity similar to that of the parent antitumor drug but are not associated with hemolytic toxicity, which is a dose-limiting side effect of APLs and a major obstacle to their use in anticancer therapeutic regimen. Furthermore, by using lipoplexes prepared from a prodrug of erufosine and a plasmid DNA encoding a pro-apoptotic protein (TRAIL), evidence was provided for selective cytotoxicity towards tumor cells while nontumor cells were resistant. This study demonstrates that the combination approach involving well tolerated erufosine cationic prodrugs and cancer gene therapy holds significant promise in tumor therapy.     

Tricyclic analogues of epidithiodioxopiperazine alkaloids with promising in vitro and in vivo antitumor activity

Epipolythiodioxopiperazine (ETP) alkaloids are structurally elaborate alkaloids that show potent antitumor activity. However, their high toxicity and demonstrated interactions with various biological receptors compromises their therapeutic potential. In an effort to mitigate these disadvantages, a short stereocontrolled construction of tricyclic analogues of epidithiodioxopiperazine alkaloids was developed. Evaluation of a small library of such structures against two invasive cancer cell lines defined initial structure–activity relationships (SAR), which identified 1,4-dioxohexahydro-6H-3,8a-epidithiopyrrolo[1,2-a]pyrazine 3c and related structures as particularly promising antitumor agents. ETP alkaloid analogue 3c exhibits low nanomolar activity against both solid and blood tumors in vitro. In addition, 3c significantly suppresses tumor growth in mouse xenograft models of melanoma and lung cancer, without obvious signs of toxicity, following either intraperitoneal (IP) or oral administration. The short synthesis of molecules in this series will enable future mechanistic and translational studies of these structurally novel and highly promising clinical antitumor candidates.     

Determination of Cinchona alkaloids and Vitamin B6 by high-performance liquid chromatography with fluorescence detection

A simple and specific method has been developed for the simultaneous determination of the four major Cinchona alkaloids and their dihydroderivatives and pyridoxine hydrochloride (Vitamin B₆) by high-performance liquid chromatography (HPLC) with fluorescence detection (λ_em=420 nm with λ_ex=330 nm). The chromatographic separation was performed on a Phenomenex Prodigy ODS column (5 μm,  mm i.d.), recommended for basic compounds, under isocratic reversed-phase conditions. The method allowed a good peak shape and an effective resolution of the tested compounds. The extraction of alkaloids from the Cinchona succirubra bark was carried out in mild and fast conditions (ambient temperature, 20 min) by ultrasonication. The procedure showed to be advantageous respect to a reference method, which involved Soxhlet extraction. The results were compared statistically by means of the Student’s t-test and the variance ratio F-test; no significant difference was found. The method was reproducible (relative standard deviations in the range of 1.0-5.0% for the different alkaloids) and gave quantitative recovery of alkaloids added to bark samples (97.8-105%). For additional informations a photoreactor was arranged between the analytical column and the detector and the online post-column photochemical conversion (irradiation=254 nm) was investigated. Vitamin B₆ was shown to be highly photosensitive, giving significantly different fluorescence spectra with and without UV irradiation. The proposed method was successfully applied to the quality control of Cinchona bark, liquid extract and cosmetics.     

Hydroxypropylcellulose-aceclofenac conjugates: high covalent loading design, structure characterization, nano-assemblies and thermal kinetics

This article presents synthesis of novel macromolecular prodrugs of aceclofenac (an anti-inflammatory drug) onto hydroxypropylcellulose (HPC). The HPC-aceclofenac conjugates were prepared using an acylating agent 1,1′-carbonyldiimidazole (CDI) under homogenous reaction conditions. Aceclofenac was first activated by using CDI to form its N-acylimidazole. The N-acylimidazole of aceclofenac was then reacted with HPC polymer at 80 °C for 24 h. Highly pure prodrugs of aceclofenac were synthesized with a wide range of moderate to high degree of substitution (DS 0.41–2.12) as calculated by ¹H NMR spectroscopy. The UV spectroscopic analysis has also revealed that the active drug aceclofenac was found in different conjugates from 28 to 67 mg/100 mg of HPC-aceclofenac conjugates which are in good agreement with DS calculated by ¹H NMR spectroscopy. The gel permeation chromatography showed unimodal absorption that indicates no significant degradation in polymer chains during the reaction. The macromolecular prodrugs of aceclofenac were characterized using different spectroscopic and chromatographic techniques. The thermal analysis has revealed that HPC-aceclofenac conjugates (prodrugs) are 92 and 96 °C more stable than pure aceclofenac regarding their initial (Tdi) and maximum degradation temperatures (Tdm), respectively. The activation energy (Ea) and frequency factor (Z) of the degradation reactions were evaluated using Friedman, Broido and Chang methods. Degradation followed first order (n) kinetics. Transmission electron microscopy has revealed the formation of sponge like nano aggregates with population size distribution of around 80–150 nm.     

Application of HPLC-DAD for In Vitro Investigation of Acetylcholinesterase Inhibition Activity of Selected Isoquinoline Alkaloids from Sanguinaria canadensis Extracts

Isoquinoline alkaloids may have a wide range of pharmacological activities. Some of them have acetylcholinesterase activity inhibition. Nowadays, neurodegenerative disorders such as Alzheimer’s disease have become a serious public health problem. Searching for new effective compounds with inhibited acetylcholinesterase activity is one of the most significant challenges of modern scientific research. The aim of this study was the in vitro investigation of acetylcholinesterase activity inhibition of extracts obtained from Sanguinaria canadensis collected before, during and after flowering. The acetylcholinesterase activity inhibition of these extracts has not been previously tested. The aim was also to quantify selected alkaloids in the investigated extracts by high performance liquid chromatography (HPLC). The analyses of alkaloid content were performed using HPLC in reversed phase (RP) mode using Polar RP column and mobile phase containing acetonitrile, water and ionic liquid (IL). The acetylcholinesterase activity inhibition of the tested plant extracts and respective alkaloid standards were examined using high performance liquid chromatography with diode-array detector (HPLC-DAD) for the quantification of 5-thio-2-nitro-benzoic acid, which is the product of the reaction between the thiocholine (product of the hydrolysis of acetylthiocholine reaction) with Ellman reagent. The application of the HPLC method allowed for elimination of absorption of interfering components, for example, alkaloids such as sanguinarine and berberine. It is revealed that the HPLC method can be successfully used for the evaluation of the acetylcholinesterase inhibitory activity in samples such as plant extracts, especially those containing colored components adsorbing at wavelength in the range 405–412 nm. The acetylcholinesterase inhibition activity synergy of pairs of alkaloid standards and mixture of all investigated alkaloids was also determined. Most investigated alkaloids and all Sanguinaria canadensis extracts exhibited very high acetylcholinesterase activity inhibition. IC₅₀ values obtained for alkaloid standards were from 0.36 for berberine to 23.13 µg/mL for protopine and from 61.24 to 89.14 µg/mL for Sanguinaria canadensis extracts. Our investigations demonstrated that these plant extracts can be recommended for further in vivo experiments to confirm their acetylcholinesterase activity inhibition.     

A general approach to triphenylenes and azatriphenylenes: total synthesis of dehydrotylophorine and tylophorine

A convergent and flexible synthesis of substituted triphenylenes, azatriphenylenes, and the cytotoxic alkaloids dehydrotylophorine and tylophorine has been developed.     

Evaluation of the Cytotoxicity of Ayahuasca Beverages

Ayahuasca is a beverage consumed at shamanic ceremonies and currently has gained popularity on recreational scenarios. It contains beta-carboline alkaloids and N,N-dimethyltryptamine, which possesses hallucinogenic effects. Only a few studies have elicited the psychoactive effects and the dose of such compounds on neurological dopaminergic cells or animals. In this work, we aimed to study the cytotoxic effects of these compounds present in ayahuasca beverages and on five different teas (Banisteriopsis caapi, Psychotria viridis, Peganum harmala, Mimosa tenuiflora and Dc Ab (commercial name)) preparations on dopaminergic immortalized cell lines. Moreover, a characterization of the derivative alkaloids was also performed. All the extracts were characterized by chromatographic systems and the effect of those compounds in cell viability and total protein levels were analyzed in N27 dopaminergic neurons cell line. This is the first article where cytotoxicity of ayahuasca tea is studied on neurological dopaminergic cells. Overall, results showed that both cell viability and protein contents decreased when cells were exposed to the individual compounds, as well as to the teas and to the two mixtures based on the traditional ayahuasca beverages.     

Bioactive molecules in Kalanchoe pinnata leaves: extraction, purification, and identification

Kalanchoe pinnata (Lam.) Pers. (syn. Bryophyllum pinnatum; family Crassulaceae) is a popular plant used in traditional medicine in many temperate regions of the world and particularly in South America. In Guyana, the leaves are traditionally used as an anti-inflammatory and antiseptic to treat coughs, ulcers, and sores. The purpose of this study was to implement a method for targeting and identifying molecules with antimicrobial activity, which could replace chemical preservatives in cosmetic applications. The leaves were extracted by a method based on pressurized liquid extraction (PLE), using different solvents. A study of antimicrobial activity and cytotoxicity tests were performed to select the most interesting extract. To isolate one or more active molecules, the selected crude extract was fractionated by centrifugal partition chromatography (CPC) and then antimicrobial activity and cytotoxicity of each fraction were tested under the same procedure. The last step consisted of identifying the main compounds in the most active fraction by LC-MS/MS.     

Expedient syntheses of antofine and cryptopleurine via intramolecular 1,3-dipolar cycloaddition

The practical and expedient total syntheses of the representative phenanthroindolizidine and phenanthroquinolizidine alkaloids, antofine and cryptopleurine, are described. Construction of the pyrrolidine and piperidine ring of each alkaloid was achieved by using an intramolecular 1,3-dipolar cycloaddition of an azide onto an alkene and subsequent reduction of the resulting imine and aziridine.