Cytotoxic Alkaloids Derived from Marine Sponges: A Comprehensive Review.

Marine sponges (porifera) have proved to be a prolific source of unique bioactive secondary metabolites, among which the alkaloids occupy a special place in terms of unprecedented structures and outstanding biological activities. Identification of active cytotoxic alkaloids extracted from marine animals, particularly sponges, is an important strive, due to lack of knowledge on traditional experiential and ethnopharmacology investigations. In this report, a comprehensive survey of demospongian bioactive alkaloids in the range 1987-2020 had been performed with a special emphasis on the potent cytotoxic activity. Different resources and databases had been investigated, including Scifinder (database for the chemical literature) CAS (Chemical Abstract Service) search, web of science, Marin Lit (marine natural products research) database. More than 230 representatives of different classes of alkaloids had been reviewed and classified, different genera belonging to the phylum porifera had been shown to be a prolific source of alkaloidal molecules, including Agelas sp., Suberea sp., Mycale sp., Haliclona sp., Epipolasis sp., Monanchora sp., Crambe sp., Reniera sp., and Xestospongia sp., among others. The sufficient production of alkaloids derived from sponges is a prosperous approach that requires more attention in future studies to consider the constraints regarding the supply of drugs, attained from marine organisms.

Investigation of the inhibitory effect of protostanes on human carboxylesterase 2 and their interaction: Inhibition kinetics and molecular stimulations.

Carboxylesterase 2 (CES 2), plays a pivotal role in endobiotic homeostasis and xenobiotic metabolism. Protostanes, the major constituents of the genus Alisma, display a series of pharmacological activities. Despite the extensive studies of pharmacological activities, the investigation on inhibitory effects of protostanes against CES 2 is rarely reported. In this study, the inhibitory activities of a library of protostanes (1-25) against human CES 2 were investigated for the first time, using 6,8-dichloro-9,9-dimethyl-7-oxo-7,9-dihydroacridin-2-yl benzoate (DDAB) as the specific fluorescent probe for human CES 2. Compounds 1, 2, 7, 8, 12, 13, 18, 19, and 25 showed strong inhibitory effects towards CES 2. For the most potent compounds 1, 7, 13, and 25, the inhibition kinetics were further investigated, and these four protostanes were all uncompetitive inhibitors against human CES 2 with the inhibition constant (Ki) values ranging from 0.89 µM to 2.83 µM. In addition, molecular docking and molecular dynamics stimulation were employed to analyze the potential interactions between these protostanes and CES 2, and amino acid residue Gln422 was identified to play a crucial role in the strong inhibition of protostanes towards CES 2.

Synthesis and biological evaluation of acridine-based histone deacetylase inhibitors as multitarget agents against Alzheimer's disease.

Multitarget agents simultaneously trigger molecules in functionally complementary pathways, and are therefore considered to have potential in effectively treating Alzheimer's disease (AD), which has a complex pathogenetic mechanism. In this study, the HDAC inhibitor core is incorporated into the acetylcholine esterase (ACE) inhibitor acridine-derived moiety and resulted in compounds that exhibited higher class IIa HDAC (4, 5, 7, and 9)- and class IIb HDAC6-inhibiting activity when compared to the pan-HDAC inhibitor SAHA in clinical practice. One of these compounds, 11b, displayed greater selectivity toward HDAC6 than other isoform enzymes. In contrast, the activity of compound 6a was selective toward class IIa HDAC and HDAC6. These two compounds exhibited strong activity against Aß-aggregation as well as significantly disrupted Aß-oligomer. Additionally, 11b and 6a strongly inhibited AChE. These experimental findings demonstrate that compounds 11b and 6a are HDAC-Aß-aggregation-AChE inhibitors. Notably, they can enhance neurite outgrowth, but with no significant neurotoxicity. Further biological evaluation revealed the various cellular effects of multitarget compounds 11b and 6a, which have the potential to treat AD.

'Acridines' as New Horizons in Antifungal Treatment.

Frequent fungal infections in immunocompromised patients and mortality due to invasive mycosis are important clinical problems. Opportunistic pathogenic Candida species remain one of the leading causes of systemic mycosis worldwide. The repertoire of antifungal chemotherapeutic agents is very limited. Although new antifungal drugs such as lanosterol 14α-demethylase and ß-glucan synthase inhibitors have been introduced into clinical practice, the development of multidrug resistance has become increasingly significant. The urgency to expand the range of therapeutic options for the treatment of fungal infections has led researchers in recent decades to seek alternative antifungal targets to the conventional ones currently used. Among them, many compounds containing an acridine scaffold have been synthesized and tested. In this review, the applicability of acridines and their functional analogues acridones as antifungal agents is described. Acridine derivatives usage in photoantifungal chemotherapy, interactions with fungal transporters resulting in modulation of efflux/influx pumps and the effect of acridine derivatives on fungal topoisomerases are discussed. This article explores new perspectives on the mechanisms of antifungal acridine-peptide conjugates and acridine-based hybrid molecules to effectively combat fungal infections.

On the use of acridinium indicators for the chemiluminescent determination of the total antioxidant capacity of dietary supplements.

Acridinium salts, due to their chemiluminogenic properties, have found several applications in biomedical analysis as labels and indicators, where the assessment of emission intensity is used for the end-point detection. This work presents the use of chemiluminescent indicators in the form of selected acridinium esters in order to determine the antioxidant properties of exemplary formulations, namely quercetin, vitamin C and the dietary supplement, Apiextract. The principle of measurements is based on a change in the kinetics of emission decay derived from the acridinium cations in alkaline solutions of hydrogen peroxide in the presence of an antioxidant (the analyte). The proposed system makes a beneficial alternative to related methods, which mostly rely on the assessment of emission efficiency and use the luminometric standard luminol - due to superior parameters of acridinium chemiluminescence, among others - high temporary emission efficiency. The features of the proposed method are manifested by a shorter time period of analysis and lower background signals associated with the environmental influences, as compared to typical approaches. The chromatographic (RP-HPLC) analyses of the substrates and products generated during chemiluminogenic oxidation of acridinium cations under assay conditions are also presented.

Intercalating Electron Dyes for TEM Visualization of DNA at the Single-Molecule Level.

Staining compounds containing heavy elements (electron dyes) can facilitate the visualization of DNA and related biomolecules by using TEM. However, research into the synthesis and utilization of alternative electron dyes has been limited. Here, we report the synthesis of a novel DNA intercalator molecule, bis-acridine uranyl (BAU). NMR spectroscopy and MS confirmed the validity of the synthetic strategy and gel electrophoresis verified the binding of BAU to DNA. For TEM imaging of DNA, two-dimensional DNA origami nanostructures were used as a robust microscopy test object. By using scanning transmission electron microscopy (STEM) imaging, which is favored over conventional wide-field TEM for improved contrast, and therefore, quantitative image analysis, it is found that the synthesized BAU intercalator can render DNA visible, even at the single-molecule scale. For comparison, other staining compounds with a purported affinity towards DNA, such as dichloroplatinum, cisplatin, osmium tetroxide, and uranyl acetate, have been evaluated. The STEM contrast is discussed in terms of the DNA-dye association constants, number of dye molecules bound per base pair, and the electron-scattering capacity of the metal-containing ligands. These findings pave the way for the future development of electron dyes with specific DNA-binding motifs for high-resolution TEM imaging.

Discovery of 1,8-acridinedione derivatives as novel GCN5 inhibitors via high throughput screening.

The general control nonrepressed protein 5 (GCN5) plays a crucial role in many biological processes. Dysregulation of GCN5 has been closely related to various human diseases, especially cancers. Hence, the exploitation of small molecules targeting GCN5 is essential for drug design and academic research. Based on the amplified luminescent proximity homogeneous assay screen methodology, we performed high throughput screening and discovered a novel GCN5 inhibitor DC_G16 with 1,8-acridinedione scaffold. Structure optimization led to the identification of a highly potent inhibitor, namely DC_G16-11 with the half-maximal inhibitory concentration (IC50) value of 6.8 µM. The binding between DC_G16-11 and GCN5 was demonstrated by NMR and SPR with a KD of 4.2 µM. It could also inhibit proliferation and induce cell cycle arrest and apoptosis in cancer cells while it presented minimal effects on normal cells. Herein, DC_G16-11 could be applied as a validated chemical probe for GCN5-related biological function research and presented great potential for clinical disease treatment.

Incorporation of lipolysis in monolayer permeability studies of lipid-based oral drug delivery systems.

Lipid-based drug delivery systems, a well-tolerated class of formulations, have been evaluated extensively to enhance the bioavailability of poorly soluble drugs. However, it has been difficult to predict the in vivo performance of lipid dosage forms based on conventional in vitro techniques such as cell monolayer permeability studies because of the complexity of the gastrointestinal processing of lipid formulations. In the current study, we explored the feasibility of coupling Caco-2 and Madin-Darby canine kidney monolayer permeability studies with lipolysis, a promising in vitro technique to evaluate lipid systems. A self-emulsifying lipid delivery system was formulated using a blend of oil (castor oil), surfactant (Labrasol® or PL497), and co-surfactant (lecithin). Formulations demonstrating high drug solubility and rapid self-emulsification were selected to study the effect of lipolysis on in vitro cell permeability. Lipolysis of the formulations was carried out using pancreatin as the digestive enzyme. All the digested formulations compromised monolayer integrity as indicated by lowered trans-epithelial electrical resistance (TEER) and enhanced Lucifer yellow (LY) permeability. Further, the changes in TEER value and LY permeability were attributable to the digestion products of the formulation rather than the individual lipid excipients, drug, digestion enzyme, or the digestion buffer. The digested formulations were fractionated into pellet, oily phase, and aqueous phase, and the effect of each of these on cell viability was examined. Interestingly, the aqueous phase, which is considered important for in vivo drug absorption, was responsible for cytotoxicity. Because lipid digestion products lead to disruption of cell monolayer, it may not be appropriate to combine lipolysis with cell monolayer permeability studies. Additional in vivo studies are needed to determine any potential side effects of the lipolysis products on the intestinal permeability barrier, which could determine the suitability of lipid-based systems for oral drug delivery.

Effects of a detergent micelle environment on P-glycoprotein (ABCB1)-ligand interactions.

P-glycoprotein (P-gp) is a multidrug transporter that uses energy from ATP hydrolysis to export many structurally dissimilar hydrophobic and amphipathic compounds, including anticancer drugs from cells. Several structural studies on purified P-gp have been reported, but only limited and sometimes conflicting information is available on ligand interactions with the isolated transporter in a dodecyl-maltoside detergent environment. In this report we compared the biochemical properties of P-gp in native membranes, detergent micelles, and when reconstituted in artificial membranes. We found that the modulators zosuquidar, tariquidar, and elacridar stimulated the ATPase activity of purified human or mouse P-gp in a detergent micelle environment. In contrast, these drugs inhibited ATPase activity in native membranes or in proteoliposomes, with IC50 values in the 10-40 nm range. Similarly, a 30-150-fold decrease in the apparent affinity for verapamil and cyclic peptide inhibitor QZ59-SSS was observed in detergent micelles compared with native or artificial membranes. Together, these findings demonstrate that the high-affinity site is inaccessible because of either a conformational change or binding of detergent at the binding site in a detergent micelle environment. The ligands bind to a low-affinity site, resulting in altered modulation of P-gp ATPase activity. We, therefore, recommend studying structural and functional aspects of ligand interactions with purified P-gp and other ATP-binding cassette transporters that transport amphipathic or hydrophobic substrates in a detergent-free native or artificial membrane environment.

An acridine derivative, [4,5-bis{(N-carboxy methyl imidazolium)methyl}acridine] dibromide, shows anti-TDP-43 aggregation effect in ALS disease models.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease associated with aggregation of TAR DNA-binding protein-43 (TDP-43) in neuronal cells and manifests as motor neuron dysfunction &muscle atrophy. The carboxyl-terminal prion-like domain of TDP-43 can aggregate in vitro into toxic ß-sheet rich amyloid-like structures. So far, treatment options for ALS are very limited and Riluzole, which targets glutamate receptors, is the only but highly ineffective drug. Therefore, great interest exists in developing molecules for ALS treatment. Here, we have examined certain derivatives of acridine containing same side chains at position 4 &5, for inhibitory potential against TDP-43 aggregation. Among several acridine derivatives examined, AIM4, which contains polar carboxyl groups in the side arms, significantly reduces TDP-43-YFP aggregation in the powerful yeast model cell and also abolishes in vitro amyloid-like aggregation of carboxyl terminal domain of TDP-43, as observed by AFM imaging. Thus, AIM4 can be a lead molecule potentiating further therapeutic research for ALS.

Design and cellular studies of a carbon nanotube-based delivery system for a hybrid platinum-acridine anticancer agent.

A three-component drug-delivery system has been developed consisting of multi-walled carbon nanotubes (MWCNTs) coated with a non-classical platinum chemotherapeutic agent ([PtCl(NH3)2(L)]Cl (P3A1; L=N-(2-(acridin-9-ylamino)ethyl)-N-methylproprionimidamide) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-5000] (DSPE-mPEG). The optimized P3A1-MWCNTs are colloidally stable in physiological solution and deliver more P3A1 into breast cancer cells than treatment with the free drug. Furthermore, P3A1-MWCNTs are cytotoxic to several cell models of breast cancer and induce S-phase cell cycle arrest and non-apoptotic cell death in breast cancer cells. By contrast, free P3A1 induces apoptosis and allows progression to G2/M phase. Photothermal activation of P3A1-MWCNTs to generate mild hyperthermia potentiates their cytotoxicity. These findings suggest that delivery of P3A1 to cancer cells using MWCNTs as a drug carrier may be beneficial for combination cancer chemotherapy and photothermal therapy.

Sagitol D, a New Thiazole Containing Pyridoacridine Alkaloid from a Vietnamese Ascidian.

A new thiazole containing pyridoacridine alkaloid, named sagitol D (1), and five known alkaloids kuanoniaminesA (2), C (3), D (4), E (5), and F (6), have been isolated from an unidentified Vietnamese ascidian. The structure of the new compound was established from NMR spectroscopic data. Kuanoniamines C, D, E, and F showed moderate antioxidant activity in the DPPH (IC50 36 µM) and ABTS assays (TE = 0.5), while sagitol D showed weak activity (IC50 92 M;TE = 0.10), and kuanoniamine A was inactive.

Harmonization of QSAR Best Practices and Molecular Docking Provides an Efficient Virtual Screening Tool for Discovering New G-Quadruplex Ligands.

Telomeres and telomerase are key players in tumorogenesis. Among the various strategies proposed for telomerase inhibition or telomere uncapping, the stabilization of telomeric G-quadruplex (G4) structures is a very promising one. Additionally, G4 stabilizing ligands also act over tumors mediated by the alternative elongation of telomeres. Accordingly, the discovery of novel compounds able to act on telomeres and/or inhibit the telomerase enzyme by stabilizing DNA telomeric G4 structures as well as the development of approaches efficiently prioritizing such compounds constitute active areas of research in computational medicinal chemistry and anticancer drug discovery. In this direction, we applied a virtual screening strategy based on the rigorous application of QSAR best practices and its harmonized integration with structure-based methods. More than 600,000 compounds from commercial databases were screened, the first 99 compounds were prioritized, and 21 commercially available and structurally diverse candidates were purchased and submitted to experimental assays. Such strategy proved to be highly efficient in the prioritization of G4 stabilizer hits, with a hit rate of 23.5%. The best G4 stabilizer hit found exhibited a shift in melting temperature from FRET assay of +7.3 °C at 5 µM, while three other candidates also exhibited a promising stabilizing profile. The two most promising candidates also exhibited a good telomerase inhibitory ability and a mild inhibition of HeLa cells growth. None of these candidates showed antiproliferative effects in normal fibroblasts. Finally, the proposed virtual screening strategy proved to be a practical and reliable tool for the discovery of novel G4 ligands which can be used as starting points of further optimization campaigns.

Cytotoxic compounds from the fruits of Uapaca togoensis towards multifactorial drug-resistant cancer cells.

Cancer cells may rapidly acquire multidrug resistance, mainly due to the presence of adenosine triphosphate-binding cassette transporters, epidermal growth factor receptor, or mutations in the p53 tumor suppressor gene. This work was designed to assess the cytotoxicity of the methanol crude extracts and compounds from the fruits of Uapaca togoensis, namely, ß-amyryl acetate (1), 11-oxo-α-amyryl acetate (2), lupeol (3), pomolic acid (4), futokadsurin B (5), arborinin (6), and 3-O-ß-D-glucopyranosyl sitosterol (7) against nine drug sensitive and multidrug-resistant cancer cell lines. The resazurin reduction assay was used to evaluate the cytotoxicity of the fruits of U. togoensis and compounds, whilst the caspase-Glo assay was used to detect the activation of caspase enzymes by the fruits of U. togoensis and compound 6. Cell cycle, mitochondrial membrane potential, and levels of reactive oxygen species were all analyzed via flow cytometry. The acridone alkoid 6 and the crude extract from the fruits of U. togoensis were active on all of the nine tested cancer lines with IC50 values below 32 µM and 30 µg/mL, respectively. Compounds 2 and 5 showed selective activities and IC50 values below 99 µM or 42 µM, respectively, which were obtained towards 3/9 and 6/9 tested cancer cell lines. Compound 6 displayed IC50 values below 10 µM towards seven of the nine tested cancer cell lines. The IC50 values ranged from 3.55 µM (against CEM/ADR5000 cells) to 31.77 µM (against CCRF-CEM cells) for alkaloid 6 and from 0.20 µM (against CCRF-CEM cells) to 195.12 µM (against CEM/ADR5000 cells) for doxorubicin. The crude extract of the fruits of U. togoensis induced apoptosis in the CCRF-CEM leukemia cells, which was mediated by the disruption of the mitochondrial membrane potential. Compound 6 also strongly induced apoptosis in CCRF-CEM cells and cell cycle arrest in the G0/G1 and S phases. The crude extract from the fruits of this plant as well as aborinin are potential antiproliferative natural products that deserve further investigation to develop novel cytotoxic drugs to fight sensitive and otherwise drug-resistant phenotypes.

Design, synthesis and evaluation of acridine and fused-quinoline derivatives as potential anti-tuberculosis agents.

The synthesis of twelve acridine and polycyclic acridine derivatives prepared via the Friedländer reaction is described. The one-pot reactions of 2-amino-5-chloro or 5-nitro-benzophenones and a variety of cyclanones and indanones were carried out in a MW oven under TFA catalysis in good yields. The products were designed according natural antituberculosis products and were evaluated for growth inhibitory activity towards Mycobacterium tuberculosis H37Rv (Mtb) through the National Institute of Allergy and Infectious Diseases (NIAID, USA). Three of them underwent additional testings. The cyclopenta[b]quinoline derivative 9 and the acridine derivative 13 showed remarkable MIC values against the rifampin resistant strain. The former exhibited bactericidal activity at 50 µg/mL, its intracellular activity is similar to rifampin and it was not cytotoxic at low concentrations so it can be considered a new lead compound.

Two new acridone alkaloids from the branch of Atalantia buxifolia and their biological activity.

Two new acridone alkaloids, 3-methoxy-1,4,5-trihydroxy-10-methylacridone (1) and 2,3-dimethoxy-1,4,5-trihydroxy-10-methylacridone (2), were isolated from the ethanol extract of the branch of Atalantia buxifolia. Their structures were elucidated by spectroscopic methods including 1D and 2D NMR. Compounds 1 and 2 exhibited significant antibacterial activity against Staphylococcus aureus and weak inhibitory effect on acetylcholinesterase.

A novel acridone akaloid from Atalantia buxifolia.

Study of the chemical constituents of ethanol extract from the aerial parts of Atalantia buxifolia led to the identification of a new acridone alkaloid named as buxifoliadine (1), along with known compounds citrusinine--I (2), N-methylatalaphylline (3), Severinolid (4) and cycloseverinolide (5). Structural elucidation of compound 1 was carried out by a combination of mass spectrometry and (1)H and (13)C NMR spectroscopy analyses.

New acridone from the wood of Citrus reticulata Blanco.

A new acridone, named citruscridone (1) together with five known compounds were isolated from the wood of Citrus reticulata Blanco. Their structures were established based on spectroscopic evidence. The antibacterial and antifungal activities of the wood extracts and pure compounds were evaluated.

Synthesis and biological evaluation of acridine derivatives as antimalarial agents.

New N-alkylaminoacridine derivatives attached to nitrogen heterocycles were synthesized, and their antimalarial potency was examined. They were tested in vitro against the growth of Plasmodium falciparum, including chloroquine (CQ)-susceptible and CQ-resistant strains. This biological evaluation has shown that the presence of a heterocyclic ring significantly increases the activity against P. falciparum. The best compound shows a nanomolar IC(50) value toward parasite proliferation on both CQ-susceptible and CQ-resistant strains. The antimalarial activity of these new acridine derivatives can be explained by the two mechanisms studied in this work. First, we showed the capacity of these compounds to inhibit heme biocrystallization, a detoxification process specific to the parasite and essential for its survival. Second, in our search for alternative targets, we evaluated the in vitro inhibitory activity of these compounds toward Sulfolobus shibatae topoisomerase VI-mediated DNA relaxation. The preliminary results obtained reveal that all tested compounds are potent DNA intercalators, and significantly inhibit the activity of S. shibatae topoisomerase VI at concentrations ranging between 2.0 and 2.5 µM.

Two new acridone alkaloids from Glycosmis macrantha.

Extraction and chromatographic separation of the extracts of dried stem barks of Glycosmis macrantha lead to isolation of two new acridone alkaloids, macranthanine and 7-hydroxynoracronycine, and a known acridone, atalaphyllidine. The structures of these alkaloids were determined by detailed spectral analysis and also by comparison with reported data.