Distinctive Nucleic Acid Recognition by Lysine-Embedded Phenanthridine Peptides.

Three new phenanthridine peptide derivatives (19, 22, and 23) were synthesized to explore their potential as spectrophotometric probes for DNA and RNA. UV/Vis and circular dichroism (CD) spectra, mass spectroscopy, and computational analysis confirmed the presence of intramolecular interactions in all three compounds. Computational analysis revealed that compounds alternate between bent and open conformations, highlighting the latter's crucial influence on successful polynucleotide recognition. Substituting one glycine with lysine in two regioisomers (22, 23) resulted in stronger binding interactions with DNA and RNA than for a compound containing two glycines (19), thus emphasizing the importance of lysine. The regioisomer with lysine closer to the phenanthridine ring (23) exhibited a dual and selective fluorimetric response with non-alternating AT and ATT polynucleotides and induction of triplex formation from the AT duplex. The best binding constant (K) with a value of 2.5 × 107 M-1 was obtained for the interaction with AT and ATT polynucleotides. Furthermore, apart from distinguishing between different types of ds-DNA and ds-RNA, the same compound could recognize GC-rich DNA through distinct induced CD signals.

Antitumoral activity of different Amaryllidaceae alkaloids: In vitro and in silico assays.

ETHNOPHARMACOLOGY RELEVANCE: The plants of Amaryllidaceae family, such as Amaryllis belladonna L., have been used as herbal remedies for thousands of years to address various disorders, including diseases that might today be identified as cancer. AIM OF THE STUDY: The objective of this work was to evaluate the potential of three Amaryllidaceae alkaloids against four cancer cell lines. MATERIAL AND METHODS: The alkaloids lycorine, 1-O-acetylcaranine, and montanine were evaluated in vitro against colon adenocarcinoma cell line (HCT-116) and breast carcinoma cell lines (MCF-7, MDAMB231, and Hs578T). Computational experiments (target prediction and molecular docking) were conducted to gain a deeper comprehension of possible interactions between these alkaloids and potential targets associated with these tumor cells. RESULTS: Montanine presented the best results against HCT-116, MDAMB231, and Hs578T cell lines, while lycorine was the most active against MCF-7. In alignment with the target prediction outcomes and existing literature, four potential targets were chosen for the molecular docking analysis: CDK8, EGFR, ER-alpha, and dCK. The docking scores revealed two potential targets for the alkaloids with scores similar to co-crystallized inhibitors and substrates: CDK8 and dCK. A visual analysis of the optimal docked configurations indicates that the alkaloids may interact with some key residues in contrast to the other docked compounds. This observation implies their potential to bind effectively to both targets. CONCLUSIONS: In vitro and in silico results corroborate with data literature suggesting the Amaryllidaceae alkaloids as interesting molecules with antitumoral properties, especially montanine, which showed the best in vitro results against colorectal and breast carcinoma. More studies are necessary to confirm the targets and pharmaceutical potential of montanine against these cancer cell lines.

Click-Capable Phenanthriplatin Derivatives as Tools to Study Pt(II)-Induced Nucleolar Stress.

It is well established that oxaliplatin, one of the three Pt(II) anticancer drugs approved worldwide, and phenanthriplatin, an important preclinical monofunctional Pt(II) anticancer drug, possess a different mode of action from that of cisplatin and carboplatin, namely, the induction of nucleolar stress. The exact mechanisms that lead to Pt-induced nucleolar stress are, however, still poorly understood. As such, studies aimed at better understanding the biological targets of both oxaliplatin and phenanthriplatin are urgently needed to expand our understanding of Pt-induced nucleolar stress and guide the future design of Pt chemotherapeutics. One approach that has seen great success in the past is the use of Pt-click complexes to study the biological targets of Pt drugs. Herein, we report the synthesis and characterization of the first examples of click-capable phenanthriplatin complexes. Furthermore, through monitoring the relocalization of nucleolar proteins, RNA transcription levels, and DNA damage repair biomarker γH2AX, and by investigating their in vitro cytotoxicity, we show that these complexes successfully mimic the cellular responses observed for phenanthriplatin treatment in the same experiments. The click-capable phenanthriplatin derivatives described here expand the existing library of Pt-click complexes. Significantly they are suitable for studying nucleolar stress mechanisms and further elucidating the biological targets of Pt complexes.

Photoactive monofunctional platinum(II) anticancer complexes of multidentate phenanthridine-containing ligands: photocytotoxicity and evidence for interaction with DNA.

Pt(II) complexes supported by chelating, multidentate ligands containing π-extended, planar phenanthridine (benzo[c]quinoline) donors (RLPtCl) exhibit a promising in vitro therapeutic index compared with phenanthriplatin, a leading preclinical anticancer complex containing a monodentate phenanthridine ligand. Here, we report evidence for non-specific interactions of CF3LPtCl with DNA through intercalation-mediated turn-on luminescence in O2-saturated aqueous buffer. Brief irradiation with visible light (490 nm) was also found to drastically increase the activity of CF3LPtCl, with photocytotoxicity increased up to 87% against a variety of human cancer cell lines. Mechanistic studies highlight significantly improved cellular uptake of CF3LPtCl compared with cisplatin, with localization in the nucleus and mitochondria triggering effective apoptosis. Photosensitization experiments with 1,3-diphenylisobenzofuran demonstrate that CF3LPtCl efficiently mediates the generation of singlet dioxygen (1O2), highlighting the potential of RLPtCl in photodynamic therapy.

Conversion of Natural Narciclasine to Its C-1 and C-6 Derivatives and Their Antitumor Activity Evaluation: Some Unusual Chemistry of Narciclasine.

During the search for a general, efficient route toward the synthesis of C-1 analogues of narciclasine, natural narciclasine was protected and converted to its C-1 enol derivative using a novel semi-synthetic route. Attempted conversion of this material to its triflate in order to conduct cross-coupling at C-1 resulted in a triflate at C-6 that was successfully coupled with several functionalities. Four novel compounds were fully deprotected after seven steps and subjected to evaluation for cytotoxic activity against three cancer cell lines. Only one derivative showed moderate activity compared to that of narciclasine. Spectral and physical data are provided for all new compounds.

Electrochemical Decarboxylative Cyclization of α-Amino-Oxy Acids to Access Phenanthridine Derivatives.

Phenanthridines are a class of useful heterocycles in the field of drug development. In this work, a method via electrochemical decarboxylative cyclization of α-amino-oxy acids to access phenanthridine derivatives was developed. This reaction proceeded through iminyl radical formation cascade intramolecular cyclization from readily available materials under environmentally friendly conditions. A wide range of phenanthridine derivatives were obtained in moderate to high yields.

Novel nucleocapsid protein-targeting phenanthridine inhibitors of SARS-CoV-2.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unprecedented in human history. As a major structural protein, nucleocapsid protein (NPro) is critical to the replication of SARS-CoV-2. In this work, 17 NPro-targeting phenanthridine derivatives were rationally designed and synthesized, based on the crystal structure of NPro. Most of these compounds can interact with SARS-CoV-2 NPro tightly and inhibit the replication of SARS-CoV-2 in vitro. Compounds 12 and 16 exhibited the most potent anti-viral activities with 50% effective concentration values of 3.69 and 2.18 µM, respectively. Furthermore, site-directed mutagenesis of NPro and Surface Plasmon Resonance (SPR) assays revealed that 12 and 16 target N-terminal domain (NTD) of NPro by binding to Tyr109. This work found two potent anti-SARS-CoV-2 bioactive compounds and also indicated that SARS-CoV-2 NPro-NTD can be a target for new anti-virus agents.

Design, synthesis and structure-activity relationship optimization of phenanthridine derivatives as new anti-vitiligo compounds.

Humans have been suffering from vitiligo for a long time. Target vitiligo drugs have yet been approved. Activation of Wnt/ß-catenin signalling has potential in the therapeutic use of vitiligo, so exploring new drugs that specifically directly activate Wnt is worthwhile to obtain new anti-vitiligo agents. In this work, two portions design and synthesis were put into effect. firstly, 17 phenanthridine derivatives with C-4 substitutes were designed and synthesized, which compounds 4, 6, 12, 13 served as H-acceptor with protein showed enhance melanogenesis activity; Secondly, 7 hybrid new scaffolds of compounds were designed and synthesized, scaffold hopping compound 36 that aromatic benzene was replaced pyrazole on ring C showed enhance melanogenesis and tyrosinase activity; The last and most important, a comprehensive optimization and SARs of compound 36 were carried out, compounds 41 and 43 shared phenolic hydroxyl or 3-methyl-pyridine substitutes at C-7 position remarkably improved the capacity of melanogenesis and tyrosinase activity. Compound 43 were identified as new anti-vitiligo agents that specifically activate the Wnt/ß-catenin signalling pathway by targeting Axin. Structure-activity relationship analysis implied that H-acceptor substitutions at the C-4 position and phenolic hydroxyl or pyridine substitutions at the C-7 position would improve the activities of the compounds. These findings reveal a new therapeutic strategy for vitiligo, and compounds 41 and 43 may represent potential compounds for vitiligo treatment.

Novel phenanthridine amide analogs as potential anti-leishmanial agents: In vitro and in silico insights.

In the current work, sixteen novel amide derivatives of phenanthridine were designed and synthesized using 9-fluorenone, 4-Methoxy benzyl amine, and alkyl/aryl acids. The characterization of the title compounds was performed using LCMS, elemental analysis, 1HNMR, 13CNMR and single crystal XRD pattern was also developed for compounds A8. All the final analogs were screened in vitro for anti-leishmanial activity against promastigote form of L. infantum strain. Among the tested analogs, four compounds (A-06, A-11, A-12, and A-15) exhibited significant anti-leishmanial activity with EC50 value ranges from 8.9 to 21.96 µM against amastigote forms of tested L. infantum strain with SI ranges of 1.0 to 4.3. From the activity results it was found that A-11 was the most active compound in both promastigote and amastigotes forms with EC50 values 8.53 and 8.90 µM respectively. In-silico ADME prediction studies depicted that the titled compounds obeyed Lipinski's rule of five as that of the approved marketed drugs. The predicted in-silico toxicity profile also confirmed that the tested compounds were non-toxic. Finally, molecular docking and molecular dynamics study was also performed for significantly active compound (A-11) in order to study it's putative binding pattern at the active site of the selected leishmanial trypanothione reductase target as well as to understand the stability pattern of target-ligand complex for 100 ns. Single crystal XRD of compound A-08 revealed that the compound crystallizes in monoclinic C2/c space group and showed interesting packing arrangements.

Cyclin-dependent kinase 1 as a potential target for lycorine against hepatocellular carcinoma.

The pathological changes and possible underlying molecular mechanisms of hepatocellular carcinoma (HCC) are currently unclear. Effective treatment of this pathological state remains a challenge. The purpose of this study is to obtain some key genes with diagnostic and prognostic meaning and to identify potential therapeutic agents for HCC treatment. Here, CDK1, CCNB1 and CCNB2 were found to be highly expressed in HCC patients and accompanied by poor prognosis, and knockdown of them by siRNA drastically induced autophagy and senescence in hepatoma cells. Simultaneously, the anti-HCC effect of lycorine was comparable to that of interfering with these three genes, and lycorine significantly promoted the decrease both in protein and mRNA expression of CDK1. Molecular validation mechanistically demonstrated that lycorine might attenuate the degradation rate of CDK1 via interaction with it, which had been confirmed by cellular thermal shift assay and drug affinity responsive targets stability assay. Taken together, these findings suggested that CDK1, CCNB1 and CCNB2 could be regarded as potential diagnostic and prognostic biomarkers for HCC, and CDK1 might serve as a promising therapeutic target for lycorine against HCC.

In-Cell Generation of Anticancer Phenanthridine Through Bioorthogonal Cyclization in Antitumor Prodrug Development.

Pharmacological inactivation of antitumor drugs toward healthy cells is a critical factor in prodrug development. Typically, pharmaceutical chemists graft temporary moieties to existing antitumor drugs to reduce their pharmacological activity. Here, we report a platform able to generate the cytotoxic agent by intramolecular cyclization. Using phenanthridines as cytotoxic model compounds, we designed ring-opened biaryl precursors that generated the phenanthridines through bioorthogonal irreversible imination. This reaction was triggered by reactive oxygen species, commonly overproduced in cancer cells, able to convert a vinyl boronate ester function into a ketone that subsequently reacted with a pendant aniline. An inactive precursor was shown to engender a cytotoxic phenanthridine against KB cancer cells. Moreover, the kinetic of cyclization of this prodrug was extremely rapid inside living cells of KB cancer spheroids so as to circumvent drug action.

Synthetic Strategies in the Preparation of Phenanthridinones.

Phenanthridinones are important heterocyclic frameworks present in a variety of complex natural products, pharmaceuticals and displaying wide range of pharmacological actions. Its structural importance has evoked a great deal of interest in the domains of organic synthesis and medicinal chemistry to develop new synthetic methodologies, as well as novel compounds of pharmaceutical interest. This review focuses on the synthesis of phenanthridinone scaffolds by employing aryl-aryl, N-aryl, and biaryl coupling reactions, decarboxylative amidations, and photocatalyzed reactions.

Development of a UPLC-MS/MS method for the determination of narciclasine and 7-deoxynarciclasine in mouse blood and its application in pharmacokinetics.

In this study, we used ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to measure the concentration of narciclasine and 7-deoxynarciclasine in mouse blood after intravenous (i.v.) and oral administration (p.o.), and we used this method to investigate their pharmacokinetics profiles in mice. Chromatographic separation of the analytes was achieved using a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 µm) with a mobile phase consisting of acetonitrile-water (0.1% formic acid) by gradient elution. Electrospray ionization (ESI positive-ion mode)-tandem mass spectrometry in multiple reaction monitoring (MRM) mode was employed for quantitative analysis of the analytes in mouse blood samples. Twelve mice were administered narciclasine and 7-deoxynarciclasine (2 mg/kg) intravenously (iv), while the other twelve mice were administered narciclasine and 7-deoxynarciclasine (10 mg/kg) orally. The mouse blood was withdrawn from the caudal vein to be processed, after which the blood was analyzed by UPLC-MS/MS, and the corresponding data were fitted using the Drug and Statistics (DAS) software. Standard curves of narciclasine and 7-deoxynarciclasine were generated over the concentration range of 5-5000 ng/mL. The intra-day accuracy of narciclasine and 7-deoxynarciclasine was 90-105%, and the corresponding inter-day accuracy was 87-108%. The intra-day precision was less than 13%, while the inter-day precision was less than 14%. Matrix effects were also observed (between 94% and 104%), and the recovery calculated was higher than 70%. The developed and validated UPLC-MS/MS method was then successfully applied in determining the mouse pharmacokinetics of narciclasine and 7-deoxynarciclasine. From this, thebioavailabilityofnarciclasine and 7-deoxynarciclasinewasdetermined to be 10.3%and35.4%, respectively.

Assessment of Mitochondrial Reactive Oxygen Species and Redox Regulation in Stem Cells.

Mitochondrial reactive oxygen species (mtROS) and redox regulation play an important role in stem cell maintenance and cell fate decisions. Although changes in mtROS and redox homeostasis represent a physiological mechanism to drive stem cell commitment and differentiation, dysregulation of this system can lead to defects in stem cell maintenance and regenerative capacity. This chapter explains the methods used to assess mitochondrial superoxide levels and redox regulation in stem cell populations.

A flow-cytometry-based protocol for detection of mitochondrial ROS production under hypoxia.

Hypoxia is known to stimulate mitochondrial reactive oxygen species (mROS) in cells. Here, we present a detailed protocol to detect mROS using MitoSOX staining in live cells under normoxia and hypoxia. Flow cytometry allows sensitive and reliable quantification of mROS by FlowJo software. We optimized several aspects of the procedure including hypoxic treatment, working concentrations of the staining buffer, and quantitative analyses. Here, we use HepG2 cells, but the protocol can be applied to other cell lines. For complete details on the use and execution of this protocol, please refer to Yang et al. (2020).

Selective formation of a phenathridine derivative by photodegradation of azilsartan.

Photodegradation of azilsartan produces a phenanthridine derivative, with its molecular structure determined by 1H and 13C NMR spectroscopy. This structure is confirmed by single-crystal X-ray diffraction and alternative synthesis. The phenanthridine ring formation is explained through the ring closure of an imidoylnitrene intermediate produced by decarboxylation of the 5-oxo-1,2,4-oxadiazole ring (oxadiazolone).

Is the cytotoxic activity of phenanthriplatin dependent on the specific size of the phenanthridine ligand π system?

The monofunctional Pt(II) drug phenanthriplatin is a leading preclinical anticancer drug, whose main characteristic is the presence of the extended aromatic system of the phenanthridine ligand, which allows intercalation. Intercalation, in turn, induces DNA unwinding and facilitates DNA binding. Aiming at verifying to what extent the peculiar cytotoxic activity of phenanthriplatin depends on the specific size of the aromatic system, two phenanthriplatin derivatives have been designed increasing the number of the rings in the N-heterocyclic ligand, and their reactivity has been computationally investigated. Both quantum mechanical DFT computations and molecular dynamics (MD) simulations have been employed to investigate some of the aspects that are considered important for the activity of Pt(II) monofunctional complexes. In particular, the substitution of the chlorido ligand with water, subsequent interaction of the aquated complexes with guanine as a model, eventual deactivation by the model N-acetyl methionine as well as intercalation into, binding to and distortion of DNA have been examined. The outcomes of such analysis have been compared with the analogous ones for the phenanthriplatin complex in order to highlight how the addition of one more ring to the phenanthridine ligand and, eventually, its identity influence the reactivity and, consequently, the cytotoxic profile of the complexes.

Prodrug Activation by Gold Artificial Metalloenzyme-Catalyzed Synthesis of Phenanthridinium Derivatives via Hydroamination.

An emerging approach in the field of targeted drug delivery is the establishment of abiotic metal-triggered prodrug mechanisms that can control the release of bioactive drugs. Currently, the design of prodrugs that use abiotic metals as a trigger relies heavily on uncaging strategies. Here, we introduce a strategy based on the gold-catalyzed activation of a phenanthridinium-based prodrug via hydroamination under physiological conditions. To make the prodrug strategy biocompatible, a gold artificial metalloenzyme (ArM) based on human serum albumin, rather than the free gold metal complex, was used as a trigger for prodrug activation. The albumin-based gold ArM protected the catalytic activity of the bound gold metal even in the presence of up to 1 mM glutathione in vitro. The drug synthesized via the gold ArM exerted a therapeutic effect in cell-based assays, highlighting the potential usefulness of the gold ArM in anticancer applications.

Discovery of potent histone deacetylase inhibitors with modified phenanthridine caps.

In discovery of novel HDAC inhibitory with anticancer potency, pharmacophores of phenanthridine were introduced to the structure of HDAC inhibitors. Fatty and aromatic linkers were evaluated for their solubility and activity. Both enzyme inhibitory and in vitro antiproliferative (against U937 cells) screening results revealed better activities of compounds with aromatic linker than molecules with fatty linker. Compared with SAHA (IC50 values of 1.34, 0.14, 2.58, 0.67 and 18.17 µM), molecule Fb-4 exhibited 0.87, 0.09, 0.32, 0.34 and 17.37 µM of IC50 values against K562, U266, MCF-7, U937 and HEPG2 cells, respectively. As revealed by cell cycle and apoptotic analysis, induction of G2/M phase arrest and apoptosis plays an important role in the inhibition of MCF-7 cells by Fb-4. Generally, a potent HDAC inhibitor was developed in the present study which could be utilised as a lead compound for further anticancer drug design.

The Chemical Synthesis of the Crinine and Haemanthamine Alkaloids: Biologically Active and Enantiomerically-Related Systems that Serve as Vehicles for Showcasing New Methodologies for Molecular Assembly.

The title alkaloids, often referred to collectively as crinines, are a prominent group of structurally distinct natural products with additional members being reported on a regular basis. As such, and because of their often notable biological properties, they have attracted attention as synthetic targets since the mid-1950s. Such efforts continue unabated and more recent studies on these alkaloids have focused on using them as vehicles for showcasing the utility of new synthetic methods. This review provides a comprehensive survey of the nearly seventy-year history of these synthetic endeavors.