Water-soluble Polyester-based Amino Acids-modified Dendrimers loaded with Ursolic and Oleanolic Acids as promising Prodrugs suitable for Intravenous Administration

Dendrimers, macromolecules characterized by high controlled size, shape and architecture, presence of inner cavities able to accommodate small molecules and many peripheral functional groups, are of eminent interest for biomedical applications, including gene transfection, tissue engineering, imaging, drug delivery.[1] The well-known pharmacological activities of Ursolic and Oleanolic acids[2] are limited by low water solubility, non-specific cells distribution, poor bioavailability and pharmacokinetics. Thus, the research for new formulations of UA and OA that overcome such drawbacks is very extensive. The strategy most applied concerns the use of carriers, such as liposomes or PAMAM dendrimers which are among the most investigated vectors because endowed with very efficient transfection and delivery activities but also with remarkable cytotoxicity. In this work we described the physical incorporation of the two triterpene acids extracted from Salvia Corrugata inside amino acids-modified polyester-based dendrimers.[3,4] IR, NMR, zeta potential, mean size of particles and buffer capacity of prepared materials were reported. Figure 1. An eye-catching representation of the prepared complexes The achieved water soluble complexes harmonize a polycationic character and a buffer capacity which presuppose efficient cells penetration and increased residence time with a biodegradable scaffold thus appearing as a promising team of new non-toxic prodrugs for safe intravenous administration of Ursolic and Oleanolic acids (Figure 1). [1] S. N. Goonewardena, J. D. Kratz, H. Zong, A. M. Desai, S. Tang, S. Emery, J. R. Jr. Baker, B. Huang, Design considerations for PAMAM dendrimer therapeutics, Biorg. Med. Chem. lett., 23, 2872 (2013). [2] J. Liu, Pharmacology of oleanolic acid and ursolic acid, J. Ethnopharmacol., 49, 57 (1995). [3] S. Alfei, S. Castellaro, Synthesis and Characterization of Polyester-Based Dendrimers Containing Peripheral Arginine or Mixed Amino Acids as Potential Vectors for Gene and Drug Delivery, Macromol. Res., DOI 10.1007/s13233-017-5160-3 (2017). [4] S. Alfei, S. Castellaro, G. B. Taptue, Synthesis and NMR characterization of dendrimers based on 2, 2-bis-(hydroxymethyl)-propanoic acid (bis-HMPA) containing peripheral amino acid residues for gene transfection, Org. Commun., 10, 144 (2017)

Recommendations to Synthetize Old and New β-Lactamases Inhibitors: A Review to Encourage Further Production

open2The increasing emergence of bacteria producing β-lactamases enzymes (BLEs), able to inactivate the available β-lactam antibiotics (BLAs), causing the hydrolytic opening of their β-lactam ring, is one of the global major warnings. According to Ambler classification, BLEs are grouped in serine-BLEs (SBLEs) of class A, C, and D, and metal-BLEs (MBLEs) of class B. A current strategy to restore no longer functioning BLAs consists of associating them to β-lactamase enzymes inhibitors (BLEsIs), which, interacting with BLEs, prevent them hydrolyzing to the associated antibiotic. Worryingly, the inhibitors that are clinically approved are very few and inhibit only most of class A and C SBLEs, leaving several class D and all MBLEs of class B untouched. Numerous non-clinically approved new molecules are in development, which have shown broad and ultra-broad spectrum of action, some of them also being active on the New Delhi metal-β-lactamase-1 (NDM-1), which can hydrolyze all available BLAs except for aztreonam. To not duplicate the existing review concerning this topic, we have herein examined BLEsIs by a chemistry approach. To this end, we have reviewed both the long-established synthesis adopted to prepare the old BLEsIs, those proposed to achieve the BLEsIs that are newly approved, and those recently reported to prepare the most relevant molecules yet in development, which have shown high potency, providing for each synthesis the related reaction scheme.openAlfei, Silvana; Zuccari, GuendalinaAlfei, Silvana; Zuccari, Guendalin

An optimized and very detailed, grams scale synthesis of CTEP, through a complete characterization of all the isolated and purified intermediates

Glutamate is the major excitatory neurotransmitter in the brain. 2-Chloro-4-[2,5-dimethyl-1-(4-trifluoromethoxyphenyl)-1H-imidazol-4-ylethynyl]pyridine (5) (CTEP) is the first reported negative allosteric modulator of the metabotropic glutamate receptor 5 (mGlu5NAM) with a biological half-life of 48 hours in rodents and therefore considered an ideal tool for chronic studies in rats and mice. In this work an optimized protocol for the synthesis and purification of CTEP is reported. Through the developed new work up scrupulously described in detail, CTEP was obtained in 63 % yield, with a significant improvement in comparison with the methods reported in literature (27%) and in a tripled overall yield (27% versus 9%). Furthermore, all the intermediates between which the unreported multifunctional and appealing compound 8 as well as the final compound 5 were isolated, purified and fully characterized by IR, NMR (1H NMR and 13C NMR), melting point, except for the oily 7, and Elemental analysis

N,N,N-Tris(tert-butoxycarbonyl)-l-arginine: five isoforms whose obtainment depends on procedure and scrupulous NMR confirmation of their structures

L-arginine is often covalently linked to vectors for gene or drug delivery as a means of increasing their transfection activity and reducing toxicity. This strategy relies on the protection of basic nitrogen atoms, for example, by employing the tert-butoxycarbonyl group. Our aim in the present work was to prepare the widely described\uce\ub1N,\ucf\u89N,\ucf\u89\ue2\u80\ub2N-tris(tert-butyloxycarbonyl)-l-arginine as a single isomer in high yield and with high levels of purity for use in the esterification of dendrimers with several peripheral hydroxyl groups. Following three reported protocols which assured this goal, we observed the unexpected formation of four additional isomers. Using the first procedure,\uce\ub1N,\ucf\u89N,\ucf\u89\ue2\u80\ub2N-tris(tert-butyloxycarbonyl)-l-arginine was never obtained. The second procedure delivered the desired compound as a mixture of geometric isomers (E/Z), while the third protocol led to a single isomer in high yield and purity, but with an unreported symmetrical structure. Since Boc protection is transient, this discovery would seem to be of little interest, but preliminary results from an ongoing investigation of the behavior of each of the isomers obtained in the esterification reactions of interest has shown that their reactivity depends on their structure. Although this research is ongoing, here we report a detailed description of these unexpected results, along with an NMR investigation focusing on the double-bond geometry and position which enabled confirmation of the structures

Biodegradable and biocompatible spherical dendrimer nanoparticles with a gallic acid shell and a double-acting strong antioxidant activity as potential device to fight diseases from \uaboxidative stress\ubb

Gallic acid (GA) is a natural polyphenol with remarkable antioxidant power present in several vegetables and fruits. A normal feeding regime leads to a daily intake of GA which is reasonably regarded as \u2018\u2018natural\u2019\u2019 and \u2018\u2018safe\u2019\u2019 for humans. It owns strong potentials as alternative to traditional drugs to treat several diseases triggered by oxidative stress (OS), but poor gastrointestinal absorbability, pharmacokinetic drawbacks and fast metabolism limit its clinical application. In this work, a fifth generation polyester-based dendrimer was firstly prepared as a better absorbable carrier to protect and deliver GA. Then, by its peripheral esterification with GA units, a GA-enriched delivering system (GAD) with remarkable antioxidant power and high potential against diseases from OS, was achieved. Scanning Electron Microscopy results and Dynamic Light Scattering analysis, revealed particles with an average size around 387 and 375 nm respectively and an extraordinarily spherical morphology. These properties, by determining a large particles surface area, typically favor higher systemic residence time and bio-efficiency. Z-potential of -25 mV suggests satisfactory stability in solution with tendency to form megamers and low PDI. GAD showed intrinsic antioxidant power, higher than GA by 4 times and like prodrugs, it can carry contemporary several bioactive GA units versus cells. In physiological condition, the action of Pig Liver Esterase (PLE), selected as a model of cells esterase, hydrolyzes GAD to non-cytotoxic small molecules, thus setting free the bioactive GA units, for further antioxidant effects. Cytotoxicity studies performed on two cell lines demonstrated a high cell viability

By connecting a synthetic scaffold and a natural shell of Gallic acid: an innovative double-acting antioxidant device to figh \uaboxidative stress\ubb.

Oxidative stress (OS) is involved in the onset and developing of most degenerative diseases hard to fight with exsisting synthetic drugs without adverse side effects. Gallic acid (GA) (Figure 1), a natural triphenolic acid present in several plants, fruits and common foodstuffs, is provided both with the basic nutritional values and with several extra health benefits such as a remarkable antioxidant power. GA exhibited abilities in protecting cells from OS via a number of pathways without triggering unpleasant side effects.1 GA several potentials could be exploited both in industry and in medicine but unfortunately its clinical application is limited by its pharmacokinetic drawbacks, poor bioavailability, slow GIT absorption, fast metabolism and short half-life. Dendrimer nanoparticles, thanks to their nonpareil physicochemical properties are extensively exploited in nanomedicine to control molecular weight, hydrophilicity, solubility,2-4 bioavailability and pharmacokinetic behaviour of drugs as well as to protect them from early degradation or fast metabolism. With the aim at minimizing GA's limitations for medical purposes, in this study, a G5 polyester-based dendrimer was prepared and subsequently, it was peripherally further esterified with bioactive GA

Synthesis and NMR characterization of dendrimers based on 2, 2-bis-(hydroxymethyl)-propanoic acid (bis-HMPA) containing peripheral amino acid residues for gene transfection

Dendrimers, the emerging man made, highly branched, star-shaped macromolecules with nanometer-scale dimensions are well known for their well defined and high controlled architecture, their versatility and high functionality and are of eminent interest in nanomedical applications such as drug delivery, gene transfection, and imaging. In this paper, versatile protocols for the synthesis of polyester-based, hydrolysable, polycationic dendrimers have been setup. A fourth generation dendrimer equipped with 48 peripheral hydroxyl groups was prepared from 2,2-bis(hydroxymethyl)propanoic acid and was used for grafting BOC-amino acids or as "hypercores" on which dendrons functionalized with BOC-amino acids were attached. A library of 15 polycationic homo- and hetero-dendrimers in the form of hydrochloride was obtained. Their structures and composition were confirmed by NMR analysis and by experimental molecular weight computed by volumetric titration. Their buffer capacity and results obtained from cytotoxicity assays and tests of binding with both pDNA and siRNA were very satisfactory

Synthesis and Characterization of Pyrazole-Enriched Cationic Nanoparticles as New Promising Antibacterial Agent by Mutual Cooperation

A pyrazole derivative (CB1) was previously evaluated in vivo for various pharmacological activities (with the exception of antimicrobial effects), using DMSO as the administrative medium, mainly due to its water insolubility. Considering the global necessity for new antimicrobial agents, CB1 attracted our attention as a candidate to meet this need, mainly because the secondary amine group in its structure would make it possible to obtain its hydrochloride salt (CB1H), thus effortlessly solving its water-solubility drawbacks. In preliminary microbiologic investigations on Gram-negative and Gram-positive bacteria, CB1H displayed weak antibacterial effects on MDR isolates of Gram-positive species, nonetheless better than those displayed by the commonly-used available antibiotics. Therefore, aiming at improving such activity and extending the antibacterial spectrum of CB1H to Gram-negative pathogens, in this first work CB1 was strategically formulated in nanoparticles using a cationic copolymer (P7) previously developed by us, possessing potent broad-spectrum bactericidal activity. Using the nanoprecipitation method, CB1H-loaded polymer nanoparticles (CB1H-P7 NPs) were obtained, which were analyzed by attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to confirm the successful loading. Additionally, CB1H-P7 NPs were fully characterized in terms of morphology, size, polydispersity indices, surface charge, DL%, and EE%, as well as release and potentiometric profiles

SYNTHESIS OF WATER-SOLUBLE, POLYESTER-BASED DENDRIMER PRODRUGS FOR EXPLOITING THERAPEUTIC PROPERTIES OF TWO TRITERPENOID ACIDS

Dendrimers are macromolecules characterized by high controlled size, shape and architecture, presence of inner cavities able to accommodate small molecules and many peripheral functional groups to bind target entities and are of eminent interest for biomedical applications, including gene transfection, tissue engineering, imaging, drug delivery. The well-known pharmacological activities of Ursolic and Oleanolic acids are limited by their small water solubility, non-specific cells distribution, low bioavailability, poor pharmacokinetics and their direct administration could result in the release of thrombi. To overcome such problems, in this paper we described their physical incorporation inside amino acids-modified polyester-based dendrimers which made them highly water-soluble. IR, NMR, zeta potential, mean size of particles, buffer capacity and drugs release profiles of prepared materials were reported. The achieved water-soluble complexes harmonize a polycationic character and a buffer capacity which presuppose efficient cells penetration and increased residence time with a biodegradable cells respectful scaffold thus appearing a promising team of not toxic prodrugs for safe administration of Ursolic and Oleanolic acids

Pyrazole-Enriched Cationic Nanoparticles Induced Early- and Late-Stage Apoptosis in Neuroblastoma Cells at Sub-Micromolar Concentrations

Neuroblastoma (NB) is a severe form of tumor occurring mainly in young children and originating from nerve cells found in the abdomen or next to the spine. NB needs more effective and safer treatments, as the chance of survival against the aggressive form of this disease are very small. Moreover, when current treatments are successful, they are often responsible for unpleasant health problems which compromise the future and life of surviving children. As reported, cationic macromolecules have previously been found to be active against bacteria as membrane disruptors by interacting with the negative constituents of the surface of cancer cells, analogously inducing depolarization and permeabilization, provoking lethal damage to the cytoplasmic membrane, and cause loss of cytoplasmic content and consequently, cell death. Here, aiming to develop new curative options for counteracting NB cells, pyrazole-loaded cationic nanoparticles (NPs) (BBB4-G4K and CB1H-P7 NPs), recently reported as antibacterial agents, were assayed against IMR 32 and SHSY 5Y NB cell lines. Particularly, while BBB4-G4K NPs demonstrated low cytotoxicity against both NB cell lines, CB1H-P7 NPs were remarkably cytotoxic against both IMR 32 and SHSY 5Y cells (IC50 = 0.43–0.54 µM), causing both early-stage (66–85%) and late-stage apoptosis (52–65%). Interestingly, in the nano-formulation of CB1H using P7 NPs, the anticancer effects of CB1H and P7 were increased by 54–57 and 2.5–4-times, respectively against IMR 32 cells, and by 53–61 and 1.3–2 times against SHSY 5Y cells. Additionally, based on the IC50 values, CB1H-P7 was also 1-12-fold more potent than fenretinide, an experimental retinoid derivative in a phase III clinical trial, with remarkable antineoplastic and chemopreventive properties. Collectively, due to these results and their good selectivity for cancer cells (selectivity indices = 2.8–3.3), CB1H-P7 NPs represent an excellent template material for developing new treatment options against NB