Re-designing environmentally persistent pharmaceutical pollutant through programmed inactivation: The case of methotrexate.

Environmental emission of pharmaceutical pollutants notably causes the contamination of aquatic ecosystems and drinking water. Typically, reduction of these pollutants in the environment is mostly managed by ameliorated wastewater treatments. Here, we report a method for the eco-design of drugs through the introduction within the molecular structure of a sensitive chemical group responsive to water treatments. The new drugs are thus programmed to fragment more easily and quickly than the original drugs. In this "retro catabolic drug design" strategy, methotrexate was used as drug model and an ether analog displaying a similar pharmacological profile was selected. Using photo-irradiation experiments at 254 nm, a representative drinking water treatment process, the identified transformation products were predominantly obtained from the expected molecular scission. Moreover, a faster kinetics of degradation was measured for the ether analog as compared to methotrexate and its transformation products were far less cytotoxic.

Photodegradation of methotrexate in aqueous solution: degradation kinetics and identification of transformation products.

Methotrexate is an antineoplastic folate analog of high environmental concern, due to its low biodegradability and toxicological properties. This study focused on its photodegradation under two irradiation conditions, aiming to be representative of environment (300-450 nm) and drinking water treatment (254 nm). The photodegradation experiments were conducted at two pH, to vary the methotrexate ionization state and to produce a large variety of transformation products (TPs). The degradation kinetics determined through LC-UV monitoring were contrasted according to pH and irradiation wavelength. However, the quantum yields were independent of ionization state at 254 nm and the changes in kinetics at higher wavelengths were attributed to a change in the degradation mechanism. The TPs formed during the reactions were identified by UHPLC-MS/MS, using both the positive and negative modes. Among the eleven proposed structures, five were described as methotrexate TPs for the first time. The TPs result from N-demethylation, glutamic acid oxidation, and C-N cleavage, all of them leading to further degraded photoproducts presenting modified or lost glutamic acid part. This was made possible thanks to the negative mode, which allowed the exploration of the glutamic acid moiety modifications. Cytotoxicity assessment on A549 cancer cells demonstrated that all photoproducts formed at pH 7 were less toxic than the parent compound.

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.

Anticancer boron-containing prodrugs responsive to oxidative stress from the tumor microenvironment.

Boronic acid (and ester) prodrugs targeting the overexpressed level of reactive oxygen species within tumor microenvironment represent a promising area for the discovery of new selective anticancer chemotherapy. This strategy that emerged only ten years ago is exponentially growing and could demonstrate its clinical usefulness in the near future. Herein, the previously described small-molecule and macromolecular anticancer prodrugs activated by carbon-boron oxidation are gathered. This review reports on the most interesting derivatives mentioned in the literature based on the in vitro and in vivo activity when available. Eventually, the pharmacological applicability of this strategy is discussed, in particular, the kinetic aspect of the prodrug oxidation and the selectivity of this reaction towards certain ROS from the tumor microenvironment are specified.

Anti-Methicillin-Resistant Staphylococcus aureus Nanoantibiotics.

Nanoparticle-based antibiotic constructs have become a popular area of investigation in the biomedical sciences. Much of this work has pertained to human diseases, largely in the cancer therapy arena. However, considerable research has also been devoted to the nanochemistry for controlling infectious diseases. Among these are ones due to bacterial infections, which can cause serious illnesses leading to death. The onset of multi-drug-resistant (MDR) infections such as those caused by the human pathogen Staphylococcus aureus has created a dearth of problems such as surgical complications, persistent infections, and lack of available treatments. In this article, we set out to review the primary literature on the design and development of new nanoparticle materials for the potential treatment of S. aureus infections, and areas that could be further expanded upon to make nanoparticle antibiotics a mainstay in clinical settings.

Arylboronate prodrugs of doxorubicin as promising chemotherapy for pancreatic cancer.

This study describes the synthesis of arylboronate-based ROS-responsive prodrugs of doxorubicin and their biological evaluation as anticancer agents. The determination of the most sensitive cancer type toward arylboronate prodrugs is crucial for further consideration of these molecules in clinical phase. To address this goal, an arylboronate-based profluorescent probe was used to compare the capacity of various cancer cell lines to efficiently convert the precursor into the free fluorophore. On the selected MiaPaCa-2 pancreatic cancer cells, a benzeneboronate prodrug exhibited 67% of the cytotoxicity obtained with the free doxorubicin. The prodrug was also able to induce tumor regression on MiaPaCa-2 pancreatic tumor model in ovo. Using this model, the amount of free doxorubicin liberated from this prodrug into the tumor was equivalent to the quantity measured after direct intratumoral injection of the same concentration of doxorubicin.

Spermine-NBD as fluorescent probe for studies of the polyamine transport system in Leishmania donovani.

This study describes the synthesis of fluorescent probes as potential substrates for the polyamine transport system (PTS) of Leishmania donovani. A competitive radioassay was used to determine the most efficient probe. We observed that the conjugate spermine-nitrobenzofurazan (Spm-NBD) was able to compete with [3H]-spermidine in L. donovani at a potent IC50 of 60 µM.

A chemically encoded timer for dual molecular delivery at tailored ranges and concentrations.

Spatiotemporal control of molecular distribution is much in demand in many fields of chemistry. To address this goal, we exploit a low molecular weight branched self-immolative architecture, which acts as a triggerable chemically encoded timer for autonomous sequential release of two chemicals. Using a light-activated model liberating two distinct fluorophores, we generated a tunable spatially contrasted molecular distribution.

Synthesis and antikinetoplastid evaluation of bis(benzyl)spermidine derivatives.

This study describes the synthesis and the biological evaluation of twenty-four original bis(benzyl)spermidines. Structural modifications of the polyamine scaffold were performed in order to avoid easily metabolized bonds. Some bis(benzyl)polyamine derivatives have demonstrated promising activity in vitro against Trypanosoma brucei gambiense and Leishmania donovani. From the enzymatic experiments on trypanothione reductase, we observed that this enzyme was not targeted by our compounds. In vivo evaluation on Swiss mice model infected by T. b. gambiense or L. donovani was done with the most interesting compound of the series.

Polyamine-based analogs and conjugates as antikinetoplastid agents.

Naturally occurring polyamines: putrescine, spermidine and spermine are crucial for Kinetoplastid growth and persistence. These aliphatic polycations are either biosynthesized or internalized into Kinetoplastid by active transport. Impairing the polyamine metabolism using polyamine derivatives is an interesting path in the search of new antikinetoplastid chemotherapy. In the past 30 years, research interest in this field has been constantly expanding and recent results demonstrated that the discovery of a polyamine-based antikinetoplastid drug is undoubtedly possible. In this paper, all the polyamine derivatives previously described to present an antikinetoplastid activity are reported. This review is organized around three main parts which are diamine, triamine and tetramine derivatives. Each part includes the description of the series of molecules and, their in vitro and in vivo activity when available. Structure-activity relationships of these derivatives are discussed and the most promising structures for a positive outcome are eventually highlighted.

Synthesis and in vitro antikinetoplastid activity of polyamine-hydroxybenzotriazole conjugates.

Thirteen new polyamine derivatives coupled to hydroxybenzotriazole have been synthesized and evaluated for their in vitro antikinetoplastid activity. Trypanosoma Trypanothione reductase (TryR) was envisioned as a potential target. Among all tested molecules, only one compound, a N3-spermidine-benzotriazole derivative, displayed relevant inhibitory activity on this enzyme but was not active on parasites. The corresponding Boc-protected spermidine-benzotriazole was however trypanocidal against Trypanosoma brucei gambiense with an IC50 value of 1µM and was completely devoid of cytotoxicity. On the intramacrophage amastigotes of Leishmania donovani, a N2-spermidine conjugate of this series, exhibited an interesting IC50 value of 3µM associated with both low cytotoxicity against axenic Leishmania donovani. These new compounds are promising leads for the development of antikinetoplastid agents and their targets have to be deciphered.

Self-immolative spacers: kinetic aspects, structure-property relationships, and applications.

Self-immolative spacers are covalent assemblies tailored to correlate the cleavage of two chemical bonds after activation of a protective part in a precursor: Upon stimulation, the protective moiety is removed, which generates a cascade of disassembling reactions leading to the temporally sequential release of smaller molecules. Originally introduced to overcome limitations for drug delivery, self-immolative spacers have gained wide interest in medicinal chemistry, analytical chemistry, and material science. For most applications, the kinetics of the disassembly of the activated self-immolative spacer governs functional properties. This Review addresses kinetic aspects of self-immolation. It provides information for selecting a particular self-immolative motif for a specific demand. Moreover, it should help researchers design kinetic experiments and fully exploit the rich perspectives of self-immolative spacers.

Design, synthesis and in vitro antikinetoplastid evaluation of N-acylated putrescine, spermidine and spermine derivatives.

A structure-activity relationship study on polyamine derivatives led to the synthesis and the determination of antikinetoplastid activity of 17 compounds. Among them, a spermidine derivative (compound 13) was specifically active in vitro against Leishmania donovani axenic amastigotes (IC50 at 5.4µM; Selectivity Index >18.5) and a spermine derivative (compound 28) specifically active against Trypanosoma brucei gambiense (IC50 at 1.9µM; Selectivity Index >52).

Disassembly kinetics of quinone-methide-based self-immolative spacers that contain aromatic nitrogen heterocycles.

We prepared several pyridine- and pyrimidine-based self-immolative spacer groups to evaluate the significance of the resonance energy of the spacer aromatic ring on the kinetics of 1,4- and 1,6-elimination reactions, which govern spacer disassembly. Subsequently, we relied on a photoactivation procedure to accurately analyze the disassembly kinetics. Beyond providing new results that are relevant for deriving quantitative structure-property relationships, herein, we demonstrate that pH value can be used as an efficient parameter to finely control the disassembly time of a self-immolative spacer after an initial activation.

Light activation for the versatile and accurate kinetic analysis of disassembly of self-immolative spacers.

Three procedures that rely on photoactivation are introduced to accurately analyze the disassembly kinetics of a collection of self-immolative spacer groups within the window 10(-2)-10(3) s. Our results are relevant for deriving quantitative structure-property relationships. In particular, we have been able to access 20 ms temporal resolution, which made possible the measurement of the shortest ever reported disassembly time for an activated self-immolative spacer.

Attenuating the size and molecular carrier capabilities of polyacrylate nanoparticles by a hydrophobic fluorine effect.

This study investigates the effect of introducing alkyl chain fluorination on the properties of polyacrylate nanoparticles prepared in aqueous solution by emulsion polymerization. For this, 2,2,3,3,4,4,4-heptafluorobutyl acrylate (1) and methyl trifluoroacrylate (2) were tested as monomers as a means to prepare fluorinated polyacrylate nanoparticles to evaluate how side chain fluorination may affect nanoparticle size and drug carrier properties. Our results show that as fluorine content within the polyacrylate matrix increases, the size of the nanoparticle systematically diminishes, from 45 nm (for nanoparticles containing no fluoroacrylate) to ~7 nm (for nanoparticles constructed solely of fluoroacrylate). We also observe that as fluoroacrylate content and hydrophobicity increases, the nanoparticles decrease their ability to incorporate lipophilic molecules during the process of emulsification. These findings have meaningful implications in the implementation of fluorinated nanoparticles in molecular delivery.

Design, synthesis, and biological evaluation of the first podophyllotoxin analogues as potential vascular-disrupting agents.

We designed and synthesized two novel series of azapodophyllotoxin analogues as potential antivascular agents. A linker was inserted between the trimethoxyphenyl ring E and the tetracyclic ABCD moiety of the 4-aza-1,2-didehydropodophyllotoxins. In the first series, the linker enables free rotation between the two moieties; in the second series, conformational restriction of the E nucleus was considered. We have identified several new compounds with inhibitory activity toward tubulin polymerization similar to that of CA-4 and colchicine, while displaying low cytotoxic activity against normal and/or cancer cells. An aminologue and a methylenic analogue were shown to disrupt endothelial cell cords on Matrigel at subtoxic concentrations, and an original assay of drug washout allowed us to demonstrate the rapid reversibility of this effect. These two new analogues are promising leads for the development of vascular-disrupting agents in the podophyllotoxin series.

Poly(vinyl benzoate) nanoparticles for molecular delivery: Studies on their preparation and in vitro properties.

The preparation and properties of poly(vinyl benzoate) nanoparticle suspensions as molecular carriers are described for the first time. These nanoparticles can be formed by nanoprecipitation of commercial poly(vinyl benzoate) in water using Pluronic F68 as surfactant, to create spherical nanostructures measuring 200-250nm in diameter. These nanoparticles are stable in phosphate buffer and blood serum, and only slowly degrade in the presence of esterases. Pluronic F68 stabilizes the nanoparticle and also protects it from enzymatic degradation. Up to 1.6% by weight of a lipid-soluble molecule such as coumarin-6 can be introduced into the nanoparticle during nanoprecipitation, compared to a water-soluble compound (5(6)-carboxyfluorescein) which gave almost no loading. Kinetics experiments in phosphate buffer indicate that 78% of the coumarin-6 was encapsulated within the polymer matrix of the nanoparticle, and the residual 22% of coumarin-6 was surface-bound and quickly released. The nanoparticles are non-toxic in vitro towards human epithelial cells (IC(50)>1000µg/mL) and primary bovine aortic endothelial cells (IC(50)>500µg/mL), and non-bactericidal against a selection of representative test microbes (MIC >250µg/mL). These properties suggest that the poly(vinyl benzoate) nanoparticles may be suitable carriers for molecular delivery of lipophilic small molecules such as pharmaceutical and imaging agents.