Optimizing chemistry at the surface of prodrug-loaded cellulose nanofibrils with MAS-DNP.

Studying the surface chemistry of functionalized cellulose nanofibrils at atomic scale is an ongoing challenge, mainly because FT-IR, NMR, XPS and RAMAN spectroscopy are limited in sensitivity or resolution. Herein, we show that dynamic nuclear polarization (DNP) enhanced 13C and 15N solid-state NMR is a uniquely suited technique to optimize the drug loading on nanocellulose using aqueous heterogenous chemistry. We compare the efficiency of two conventional coupling agents (DMTMM vs EDC/NHS) to bind a complex prodrug of ciprofloxacin designed for controlled drug release. Besides quantifying the drug grafting, we also evidence the challenge to control the concurrent prodrug adsorption and to optimize washing procedures. We notably highlight the presence of an unexpected prodrug cleavage mechanism triggered by carboxylates at the surface of the cellulose nanofibrils.

Interest of novel N-alkylpyridinium-indolizine hybrids in the field of Alzheimer's disease: Synthesis, characterization and evaluation of antioxidant activity, cholinesterase inhibition, and amyloid fibrillation interference.

A small library of molecules combining indolizine and N-alkyl pyridinium was synthesized and evaluated in a multi-target-directed-ligand strategy for Alzheimer's disease (AD) treatment. The new compounds were classified in three series depending on the number of methylene residues linking the two heterocycles (Ind-PyCx with x = 0, 2 or 3). The molecules were synthesized from the corresponding bis-pyridines by two-step formation of the indolizine core including mono-alkylation of pyridine and 1,3-dipolar cycloaddition with an alkylpropiolate. Their activities against AD's key-targets were evaluated in vitro: acetyl- and butyrylcholinesterase (AChE and BChE) inhibition, antioxidant properties and inhibition of amyloid fibril formation. None of the three series showed significant activities against all the targets. The Ind-PyC2 and Ind-PyC3 series are active on eeAChE and hAChE (µM IC50 values). Most of the positively charged molecules from these two series also appeared active against eqBChE, however they lost their activity on hBChE. Comparative molecular modeling of 13 and 15 docked in hAChE and hBChE highlighted the importance of the substituent (p-methoxybenzoyl or methyloxycarbonyl, respectively) located on the indolizine C-3 for the binding. The larger molecule 13 fits more tightly at the active site of the two enzymes than 15 that shows a larger degree of freedom. The Ind-PyC2 and Ind-PyC3 hybrids displayed some antioxidant activity when tested at 750 µg/mL (up to 95% inhibition of DPPH radical scavenging for 10). In both series, most hybrids were also able to interact with amyloid fibers, even if the inhibitory effect was observed at a high 100 µM concentration. The Ind-PyC0 molecules stand out completely due to their spectroscopic properties which prevent their evaluation by Ellman's and ThT assays. However, these molecules showed interesting features in the presence of preformed fibers. In particular, the strong increase in fluorescence of 3 in the presence of amyloid fibers is very promising for its use as a fibrillation fluorescent reporter dye.

Two-step immobilization of metronidazole prodrug on TEMPO cellulose nanofibrils through thiol-yne click chemistry for in situ controlled release.

Nowadays, drug encapsulation and drug release from cellulose nanofibrils systems are intense research topics, and commercial grades of cellulose nanomaterials are currently available. In this work we present an ester-containing prodrug of metronidazole that is covalently bound to cellulose nanofibrils in aqueous suspension through a two-step immobilization procedure involving green chemistry principles. The presence of the drug is confirmed by several characterization tools and methods such as Raman spectroscopy, elemental analysis, Dynamic Nuclear Polarization enhanced NMR. This technique allows enhancing the sensitivity of NMR by several orders of magnitude. It has been used to study cellulose nanofibrils substrates and it appears as the ultimate tool to confirm the covalent nature of the binding through thiol-yne click chemistry. Moreover, the ester function of the immobilized prodrug can be cleaved by specific enzyme activity thus allowing controlled drug release.

The surface chemistry of a nanocellulose drug carrier unravelled by MAS-DNP.

Cellulose nanofibrils (CNF) are renewable bio-based materials with high specific area, which makes them ideal candidates for multiple emerging applications including for instance on-demand drug release. However, in-depth chemical and structural characterization of the CNF surface chemistry is still an open challenge, especially for low weight percentage of functionalization. This currently prevents the development of efficient, cost-effective and reproducible green synthetic routes and thus the widespread development of targeted and responsive drug-delivery CNF carriers. We show in this work how we use dynamic nuclear polarization (DNP) to overcome the sensitivity limitation of conventional solid-state NMR and gain insight into the surface chemistry of drug-functionalized TEMPO-oxidized cellulose nanofibrils. The DNP enhanced-NMR data can report unambiguously on the presence of trace amounts of TEMPO moieties and depolymerized cellulosic units in the starting material, as well as coupling agents on the CNFs surface (used in the heterogeneous reaction). This enables a precise estimation of the drug loading while differentiating adsorption from covalent bonding (∼1 wt% in our case) as opposed to other analytical techniques such as elemental analysis and conductometric titration that can neither detect the presence of coupling agents, nor differentiate unambiguously between adsorption and grafting. The approach, which does not rely on the use of 13C/15N enriched compounds, will be key to further develop efficient surface chemistry routes and has direct implication for the development of drug delivery applications both in terms of safety and dosage.

Tuning the Electron Storage Potential of a Charge-Photoaccumulating RuII Complex by a DFT-Guided Approach.

Molecular photosensitizers that are able to store multiple reducing equivalents are of great interest in the field of solar fuel production, where most reactions involve multielectronic reduction processes. In order to increase the reducing power of a ruthenium tris-diimine charge-photoaccumulating complex, two structural modifications on its fused dipyridophenazine-pyridoquinolinone ligand were computationally investigated. Addition of an electron-donating oxime group was calculated to substantially decrease the reduction potentials of the complex, thus guiding the synthesis of a pyridoquinolinone-oxime derivative. Its spectroscopic and (spectro)electrochemical characterization experimentally confirmed the DFT predictions, with the first and second reduction processes cathodically shifted by -0.24 and -0.14 V, respectively, compared to the parent complex. Moreover, the ability of this novel artificial photosynthetic system to store two photogenerated electrons at a more reducing potential, via a proton-coupled electron-transfer mechanism, was demonstrated.

Photophysics of a Ruthenium Complex with a π-Extended Dipyridophenazine Ligand for DNA Quadruplex Labeling.

The light-switch mechanism of the complex [Ru(bpy)2(Br-dpqp)](PF6)2 (1, bpy = 2,2'-bipyridine, Br-dpqp = 12-bromo-14-ethoxydipyrido[3,2- a:2',3'- c]quinolino[3,2- h]phenazine), i.e., a light-up probe for the selective labeling of G-quadruplexes, is investigated by time-resolved transient absorption and emission spectroscopy. We show that, in contrast to the prototypical light-switch complex [Ru(bpy)2(dppz)](PF6)2 (2, dppz = dipyrido[3,2- a:2',3'- c]phenazine), a 3ππ* state localized on the π-extended ligand is the state determining the excited-state properties in both protic and aprotic environments. In aprotic environments, emission originates from a bright 3MLCTphen state, which is thermally accessible from the 3ππ* state at ambient temperature. In the presence of water, i.e., in environments resembling in cellulo situations, the thermally accessible 3MLCT state is altered and becomes close in energy to the 3ππ* state, which induces a rapid excited-state deactivation of the 3ππ* state and a comparably weak emission.

Enzymatically Activated Glyco-Prodrugs of Doxorubicin Synthesized by a Catalysis-Free Diels-Alder Reaction.

The severe side effects associated with the use of anthracycline anticancer agents continues to limit their use. Herein we describe the synthesis and preliminary biological evaluation of three enzymatically activatable doxorubicin-oligosaccharide prodrugs. The synthetic protocol allows late stage variation of the carbohydrate and is compatible with the use of disaccharides such as lactose as well as more complex oligosaccharides such as xyloglucan oligomers. The enzymatic release of doxorubicin from the prodrugs by both protease (plasmin) and human carboxylesterases (hCE1 and 2) was demonstrated in vitro and the cytotoxic effect of the prodrugs was assayed on MCF-7 breast cancer cells.

An artificial photosynthetic system for photoaccumulation of two electrons on a fused dipyridophenazine (dppz)-pyridoquinolinone ligand.

Increasing the efficiency of molecular artificial photosynthetic systems is mandatory for the construction of functional devices for solar fuel production. Decoupling the light-induced charge separation steps from the catalytic process is a promising strategy, which can be achieved thanks to the introduction of suitable electron relay units performing charge accumulation. We report here on a novel ruthenium tris-diimine complex able to temporarily store two electrons on a fused dipyridophenazine-pyridoquinolinone π-extended ligand upon visible-light irradiation in the presence of a sacrificial electron donor. Full characterization of this compound and of its singly and doubly reduced derivatives thanks to resonance Raman, EPR and (TD)DFT studies allowed us to localize the two electron-storage sites and to relate charge photoaccumulation with proton-coupled electron transfer processes.

Selective Luminescent Labeling of DNA and RNA Quadruplexes by π-Extended Ruthenium Light-Up Probes.

A series of RuII complexes exhibiting π-extended, acridine-based ancillary chelating heterocycles display high affinity and selectivity for DNA and RNA quadruplexes. The most promising candidates (3, 4) possess remarkable light-up luminophore properties (up to 330-fold luminescence enhancement upon interaction with quadruplexes), enabling them to discriminate quadruplexes from genomic DNA owing to a photochemical mechanism involving DNA protection against non-radiative decay (DAND), thus deviating from the other complexes of this series of ligands that exhibit an excited-state intramolecular proton transfer (ESIPT) that quenches their luminescence. The in vitro and preliminary in cellulo results shown here confirm the interest of this new family of fluorophores as invaluable molecular tools to detect G-quadruplexes in cells.

Indolizine-Based Scaffolds as Efficient and Versatile Tools: Application to the Synthesis of Biotin-Tagged Antiangiogenic Drugs.

We describe the design and optimization of polyfunctional scaffolds based on a fluorescent indolizine core derivatized with various orthogonal groups (amines, esters, oximes, alkynes, etc.). To show one application as tools in biology, the scaffold was used to prepare drug-biotin conjugates that were then immobilized onto avidin-agarose for affinity chromatography. More specifically, the antiangiogenic drug COB223, whose mechanism of action remained unclear, was chosen as a proof-of-concept drug. The drug-selective discrimination of proteins observed after elution of the cell lysates through the affinity columns, functionalized either with the biologically active COB223 or a structurally related inactive analogue (COB236), is a clear indication that the presence of the indolizine core does not limit drug-protein interaction and confirms the usefulness of the indolizine scaffold. Furthermore, the separation of COB223-interacting proteins from human placental extracts unveiled unanticipated protein targets belonging to the family of regulatory RNA-binding proteins, which opens the way to new hypotheses on the mode of action of this antiangiogenic drug.

Synthesis of three series of ruthenium tris-diimine complexes containing acridine-based π-extended ligands using an efficient "chemistry on the complex" approach.

The preparation and characterization of three series of novel ruthenium(ii) complexes are reported, each series differing by the nature of the ancillary ligands (2,2'-bipyridine - bpy, 1,10-phenanthroline - phen or 1,4,5,8-tetraazaphenanthrene - TAP). The third ligand was either the heptacyclic heterocycle dipyrido[3,2-a:2',3'-c]quinolino[3,2-h]phenazine (dpqp) substituted at position 12 by an hydroxyl (oxo), 2,2-dimethoxyethylamine (DMEA) or halogeno (Cl or Br) substituent, or the octacyclic dipyrido[3,2-a:2',3'-c]pyrido[2,3,4-de]quinolino[3,2-h]phenazine (dppqp), prepared by a multi-step "chemistry on the complex" strategy from [RuL2(oxo-dpqp)](PF6)2. The three steps, halogenation, substitution by a dimethoxyethylamino group and cyclization in trifluoroacetic acid, were performed in reasonable to high yields depending on the nature of the ancillary ligands. Isolation and purification processes were facilitated by the ability to switch the solubility of the complex from aqueous to organic solvents, depending on the counter-ion. All new complexes were fully characterized; in particular their absorption properties were compared by UV-vis spectroscopy. Finally, π-stacking properties induced by these extended ligands were studied by 1H NMR studies and quantum chemical calculations.

Investigation of the Pyridinium Ylide--Alkyne Cycloaddition as a Fluorogenic Coupling Reaction.

The cycloaddition of pyridinium ylides with alkynes was investigated under mild conditions. A series of 13 pyridinium salts was prepared by alkylation of 4-substituted pyridines. Their reactivity with propiolic ester or amide in various reaction conditions (different temperatures, solvents, added bases) was studied, and 11 indolizines, with three points of structural variation, were, thus, isolated and characterized. The highest yields were obtained when electron-withdrawing groups were present on both the pyridinium ylide, generated in situ from the corresponding pyridinium salt, and the alkyne (X, Z = ester, amide, CN, carbonyl, etc.). Electron-withdrawing substituents, lowering the acid dissociation constant (pKa) of the pyridinium salts, allow the cycloaddition to proceed at pH 7.5 in aqueous buffers at room temperature.

A new chemical inhibitor of angiogenesis and tumorigenesis that targets the VEGF signaling pathway upstream of Ras.

The efficacy of anti-angiogenic therapies on cancer patients is limited by the emergence of drug resistance, urging the search for second-generation drugs. In this study, we screened an academic chemical library (DCM, University of Grenoble-Alpes) and identified a leader molecule, COB223, that inhibits endothelial cell migration and proliferation. It inhibits also Lewis lung carcinoma (LLC/2) cell proliferation whereas it does not affect fibroblast proliferation. The anti-angiogenic activity of COB223 was confirmed using several in vitro and in vivo assays. In a mouse LLC/2 tumor model, ip administration of doses as low as 4 mg/kg COB223 efficiently reduced the tumor growth rate. We observed that COB223 inhibits endothelial cell ERK1/2 phosphorylation induced by VEGF, FGF-2 or serum and that it acts downstream of PKC and upstream of Ras. This molecule represents a novel anti-angiogenic and anti-tumorigenic agent with an original mechanism of action that deserves further development as an anti-cancer drug.

COB231 targets amyloid plaques in post-mortem human brain tissue and in an Alzheimer mouse model.

Previous works have shown the interest of naturally fluorescent proflavine derivatives to label Abeta deposits in vitro. This study aimed to further characterize the properties of the proflavine 3-acetylamino-6-[3-(propargylamino)propanoyl]aminoacridine (COB231) derivative as a probe. This compound was therefore evaluated on human post-mortem and mice brain slices and in vivo in 18-month-old triple transgenic mice APPswe, PS1M146V and tauP301L (3xTgAD) mice presenting the main characteristics of Alzheimer's disease (AD). COB231 labelled amyloid plaques on brain slices of AD patients, and 3xTgAD mice at 10 and 0.1 µM respectively. However, no labelling of the neurofibrillary tangle-rich areas was observed either at high concentration or in the brain of fronto-temporal dementia patients. The specificity of this mapping was attested in mice using Thioflavin S and IMPY as positive controls of amyloid deposits. After intravenous injection of COB231 in old 3xTgAD mice, fluorescent amyloid plaques were detected in the cortex and hippocampus, demonstrating COB231 blood­brain barrier permeability. We also controlled the cellular localization of COB231 on primary neuronal cultures and showed that COB231 accumulates into the cytoplasm and not into the nucleus. Finally, using a viability assay, we only detected a slight cytotoxic effect of COB231 (< 10%) for the highest concentration (100 µM).

Synthesis and in vitro antimicrobial evaluation of new N-heterocyclic diquaternary pyridinium compounds.

A series of bis-pyridinium quaternary ammonium salts (bis-PyQAs) with different aryl and heteroaryl moieties were synthesized and their antimicrobial activity investigated. The inhibition effect of the compounds was evaluated against bacteria, molds and yeasts; the activities were expressed as the minimum inhibitory concentrations (MIC). The relationships between the structure descriptors (logP, polarizability, polar surface area (2D), van der Waals area (3D)) and the biological activity of the tested bis-PyQAs are discussed.

Electrochemical transduction of DNA hybridization at modified electrodes by using an electroactive pyridoacridone intercalator.

A synthetic redox probe structurally related to natural pyridoacridones was designed and electrochemically characterised. These heterocycles behave as DNA intercalators due to their extended planar structure that promotes stacking in between nucleic acid base pairs. Electrochemical characterization by cyclic voltammetry revealed a quasi-reversible electrochemical behaviour occurring at a mild negative potential in aqueous solution. The study of the mechanism showed that the iminoquinone redox moiety acts similarly to quinone involving a two-electron reduction coupled with proton transfer. The easily accessible potential region with respect to aqueous electro-inactive window makes the pyridoacridone ring suitable for the indirect electrochemical detection of chemically unlabelled DNA. Its usefulness as electrochemical hybridization indicator was assessed on immobilised DNA and compared to doxorubicin. The voltamperometric response of the intercalator acts as an indicator of the presence of double-stranded DNA at the electrode surface and allows the selective transduction of immobilised oligonucleotide hybridization at both macro- and microscale electrodes.

Novel one-pot green synthesis of indolizines biocatalysed by Candida antarctica Lipases.

Marine microorganisms are of considerable interest as a promising source of enzymes with unsuspected potentials as catalysts for chemical synthesis. We describe here an efficient method for one-pot indolizine synthesis that has been developed using lipase A and lipase B from Candida antarctica as biocatalysts. As showed by HPLC/MS analysis, the yield in indolizines was higher in the presence of the biocatalyst than in absence of enzyme. Lipase A, from Candida antarctica, showed high catalytic activity and selectivity for the cycloaddition reactions. When the reactions were performed under ultrasound irradiation, the Candida antarctica lipase catalyzed reactions yielded pure indolozines, in good yields and in very short time.

Survey of recent literature related to the biologically active 4(3H)-quinazolinones containing fused heterocycles.

The present review focuses on the synthesis and biological evaluation of polycyclic 4(3H)-quinazolinones containing fused aromatic or heteroaromatic rings. The first part of the review is related to compounds with ring fused to the pyrimidine part of the quinazoline core. Most of the quinazolinone alkaloids belong to this class of molecules. The second part presents molecules bearing extra ring(s) fused to the benzo moiety of the quinazolinone skeleton. Their structural diversity opens new fields in the search of active molecules.

Catalyst-free synthesis of quinazolin-4-ones from (hetero)aryl-guanidines: application to the synthesis of pyrazolo[4,3-f]quinazolin-9-ones, a new family of DYRK1A inhibitors.

A small library of heterocycle-fused quinazolin-4-ones was prepared and evaluated as kinase inhibitors. The key step of the two-step process involves the environmental friendly thermolysis of N-ethoxycarbonyl-N'-(hetero) arylguanidines at 130 °C in water. The cyclization is fully regioselective. The most active molecules, 7-(2-hydroxyethylamino)- and 7-(3-hydroxypropylamino)-pyrazolo[4,3-f]quinazolin-9-ones, inhibit DYRK1A and CLK1 at submicromolar concentrations, indicating the potential interest of this new heterocycle in drug design.