Synthesis of Aminobisphosphinates through a Cascade Reaction between Hypophosphorous Acid and Bis(trimethylsilyl)imidates Mediated by ZnI2.

Among phosphorylated derivatives, phosphinates occupy a prominent place due to their ability to be bioisosteres of phosphates and carboxylates. These properties imply the necessity to develop efficient methodologies leading to phosphinate scaffolds. In recent years, our team has explored the nucleophilic potential of silylated phosphonite towards various electrophiles. In this paper, we propose to extend our study to other electrophiles. We describe here the implementation of a cascade reaction between (trimethylsilyl)imidates and hypophosphorous acid mediated by a Lewis acid allowing the synthesis of aminomethylenebisphosphinate derivatives. The present study focuses on methodological development including a careful NMR monitoring of the cascade reaction. The optimized conditions were successfully applied to various aliphatic and aromatic substituted (trimethylsilyl)imidates, leading to the corresponding AMBPi in moderate to good yields.

One-Pot Synthesis of Phosphinylphosphonate Derivatives and Their Anti-Tumor Evaluations.

This paper reports on the synthesis of new hydroxymethylene-(phosphinyl)phosphonates (HMPPs). A methodology has been developed to propose an optimized one-pot procedure without any intermediate purifications. Various aliphatic and (hetero)aromatic HMPPs were synthesized in good to excellent yields (53-98%) and the influence of electron withdrawing/donating group substitution on aromatic substrates was studied. In addition, the one-pot synthesis of HMPP was monitored by 31P NMR spectroscopy, allowing effective control of the end of the reaction and identification of all phosphorylated intermediate species, which enabled us to propose a reaction mechanism. Optimized experimental conditions were applied to the preparation of biological relevant aminoalkyl-HMPPs. A preliminary study of the complexation to hydroxyapatite (bone matrix) was carried out in order to verify its lower affinity towards bone compared to bisphosphonate molecules. Moreover, in vitro anti-tumor activity study revealed encouraging antiproliferative activities on three human cancer cell lines (breast, pancreas and lung).

Formation of 1-Hydroxymethylene-1,1-bisphosphinates through the Addition of a Silylated Phosphonite on Various Trivalent Derivatives.

An easily handled one-pot synthetic procedure was previously developed for the synthesis of bisphosphinates starting from acyl chlorides. Herein, other trivalent derivatives as acid anhydrides and activated esters were tested to form various bisphosphinates. This modulation of the reactivity can be controlled according to the nature of the acid derivative for the use of sensitive and functionalized substrates.

Liquid-Crystalline Suspensions of Photosensitive Paramagnetic CeF3 Nanodiscs.

The design of high-performance energy-converting materials is an essential step for the development of sensors, but the production of the bulk materials currently used remains costly and difficult. Therefore, a different approach based on the self-assembly of nanoparticles has been explored. We report on the preparation by solvothermal synthesis of highly crystalline CeF3 nanodiscs. Their surface modification by bisphosphonate ligands led to stable, highly concentrated, colloidal suspensions in water. Despite the low aspect ratio of the nanodiscs (∼6), a liquid-crystalline nematic phase spontaneously appeared in these colloidal suspensions. Thanks to the paramagnetic character of the nanodiscs, the nematic phase was easily aligned by a weak (0.5 T) magnetic field, which provides a simple and convenient way of orienting all of the nanodiscs in suspension in the same direction. Moreover, the more dilute, isotropic, suspensions displayed strong (electric and magnetic) field-induced orientation of the nanodiscs (Kerr and Cotton-Mouton effects), with fast enough response times to make them suitable for use in electro-optic devices. Furthermore, an emission study showed a direct relation between the luminescence intensity and magnetic-field-induced orientation of the colloids. Finally, with their fast radiative recombination decay rates, the nanodiscs show luminescence properties that compare quite favorably with those of bulk CeF3. Therefore, these CeF3 nanodiscs are very promising building blocks for the development and processing of photosensitive materials for sensor applications.

A convenient synthetic route towards H-bisphosphinates.

A practical generalisable procedure to synthesize hydroxymethylene H-bisphosphinates has been optimised. Unlike previous reports, numerous alkyl (including an alendronate bisphosphinate analogue) or (hetero)aryl compounds were rapidly obtained in satisfactory to excellent yields. A side product could have been identified as a phosphino-phosphonate isomer and plausible mechanistic pathways are proposed here. Moreover to check the literature data, a pKa value study was also performed.

Bifunctional Tripeptide with a Phosphonic Acid as a Brønsted Acid for Michael Addition: Mechanistic Insights.

Enamine catalysis is a widespread activation mode in the field of organocatalysis and is often encountered in bifunctional organocatalysts. We previously described H-Pro-Pro-pAla-OMe as a bifunctional catalyst for Michael addition between aldehydes and aromatic nitroalkenes. Considering that opposite selectivities were observed when compared to H-Pro-Pro-Glu-NH2 , an analogue described by Wennemers, the activation mode of H-Pro-Pro-pAla-OMe was investigated through kinetic, linear effect studies, NMR analyses, and structural modifications. It appeared that only one bifunctional catalyst was involved in the catalytic cycle, by activating aldehyde through an (E)-enamine and nitroalkene through an acidic interaction. A restrained tripeptide structure was optimal in terms of distance and rigidity for better selectivities and fast reaction rates. Transition-state modeling unveiled the particular selectivity of this phosphonopeptide.

Targeting VEGF-A with a vaccine decreases inflammation and joint destruction in experimental arthritis.

OBJECTIVES: Inflammation and angiogenesis are two tightly linked processes in arthritis, and therapeutic targeting of pro-angiogenic factors may contribute to control joint inflammation and synovitis progression. In this work, we explored whether vaccination against vascular endothelial growth factor (VEGF) ameliorates collagen-induced arthritis (CIA). METHODS: Anti-VEGF vaccines were heterocomplexes consisting of the entire VEGF cytokine (or a VEGF-derived peptide) linked to the carrier protein keyhole limpet hemocyanin (KLH). Two kinds of vaccines were separately tested in two independent experiments of CIA. In the first, we tested a kinoid of the murine cytokine VEGF (VEGF-K), obtained by conjugating VEGF-A to KLH. For the second, we selected two VEGF-A-derived peptide sequences to produce heterocomplexes (Vpep1-K and Vpep2-K). DBA/1 mice were immunized with either VEGF-K, Vpep1-K, or Vpep2-K, before CIA induction. Clinical and histological scores of arthritis, anti-VEGF, anti-Vpep Ab titers, and anti-VEGF Abs neutralizing capacity were determined. RESULTS: Both VEGF-K and Vpep1-K significantly ameliorated clinical arthritis scores and reduced synovial inflammation and joint destruction at histology. VEGF-K significantly reduced synovial vascularization. None of the vaccines reduced anti-collagen Ab response in mice. Both VEGF-K and Vpep1-K induced persistently high titers of anti-VEGF Abs capable of inhibiting VEGF-A bioactivity. CONCLUSION: Vaccination against the pro-angiogenic factor VEGF-A leads to the production of anti-VEGF polyclonal Abs and has a significant anti-inflammatory effect in CIA. Restraining Ab response to a single peptide sequence (Vpep1) with a peptide vaccine effectively protects immunized mice from joint inflammation and destruction.

Towards potential nanoparticle contrast agents: Synthesis of new functionalized PEG bisphosphonates.

The use of nanotechnologies for biomedical applications took a real development during these last years. To allow an effective targeting for biomedical imaging applications, the adsorption of plasmatic proteins on the surface of nanoparticles must be prevented to reduce the hepatic capture and increase the plasmatic time life. In biologic media, metal oxide nanoparticles are not stable and must be coated by biocompatible organic ligands. The use of phosphonate ligands to modify the nanoparticle surface drew a lot of attention in the last years for the design of highly functional hybrid materials. Here, we report a methodology to synthesize bisphosphonates having functionalized PEG side chains with different lengths. The key step is a procedure developed in our laboratory to introduce the bisphosphonate from acyl chloride and tris(trimethylsilyl)phosphite in one step.

Synthesis and biological evaluation of new bisphosphonate-dextran conjugates targeting breast primary tumor.

Bisphosphonates (BPs) have interesting antitumor effects as well in vitro as in vivo, despite their poor bioavailability in the organism after oral ingestion. To overcome this problem and reduce drug doses and secondary effects, we report the chemical synthesis of new bioconjugates. They were built with a nitrogen-containing BP as the drug covalently coupled to the carboxymethyldextran. This polysaccharide was used as a carrier, in order to increase BP lifetime in bloodstream and to target tumor cells which have a strong affinity with dextran. The efficiency of our vectorization system was biologically proved in vitro and in vivo on mammalian carcinoma models in mice.

Synthesis and preliminary anticancer evaluation of photo-responsive prodrugs of hydroxymethylene bisphosphonate alendronate.

The antitumoral activity of hydroxymethylene bisphosphonates (HMBP) such as alendronate or zoledronate is hampered by their exceptional bone-binding properties and their short plasmatic half-life which preclude their accumulation in non-skeletal tumors. In this context, the use of lipophilic prodrugs represents a simple and straightforward strategy to enhance the biodistribution of bisphosphonates in these tissues. We describe in this article the synthesis of light-responsive prodrugs of HMBP alendronate. These prodrugs include lipophilic photo-removable nitroveratryl groups which partially mask the highly polar alendronate HMBP scaffold. Photo-responsive prodrugs of alendronate are stable in physiological conditions and display reduced toxicity compared to alendronate against MDA-MB-231 cancer cells. However, the antiproliferative effect of these prodrugs is efficiently restored after cleavage of their nitroveratryl groups upon exposure to UV light. In addition, substitution of alendronate with such photo-responsive substituents drastically reduces its bone-binding properties, thereby potentially improving its biodistribution in soft tissues after i.v. administration. The development of such lipophilic photo-responsive prodrugs is a promising approach to fully exploit the anticancer effect of HMBPs on non-skeletal tumors.

Synthesis and preliminary anticancer evaluation of new triazole bisphosphonate-based isoprenoid biosynthesis inhibitors.

The synthesis of a new set of triazole bisphosphonates 8a-d and 9a-d presenting an alkyl or phenyl substituent at the C-4 or C-5 position of the triazole ring is described. These compounds have been evaluated for their antiproliferative activity against MIA PaCa-2 (pancreas), MDA-MB-231 (breast) and A549 (lung) human tumor cell lines. 4-hexyl- and 4-octyltriazole bisphosphonates 8b-c both displayed remarkable antiproliferative activities with IC50 values in the micromolar range (0.75-2.4 µM) and were approximately 4 to 12-fold more potent than zoledronate. Moreover, compound 8b inhibits geranylgeranyl pyrophosphate biosynthesis in MIA PaCa-2 cells which ultimately led to tumor cells death.

Hybrid multimodal contrast agent for multiscale in vivo investigation of neuroinflammation.

Neuroinflammation is a process common to several brain pathologies. Despites its medical relevance, it still remains poorly understood; there is therefore a need to develop new in vivo preclinical imaging strategies to monitor inflammatory processes longitudinally. We here present the development of a hybrid imaging nanoprobe named NP3, that was specifically designed to get internalized by phagocytic cells and imaged in vivo with MRI and bi-photon microscopy. NP3 is composed of a 16 nm core of gadolinium fluoride (GdF3), coated with bisphosphonate polyethylene glycol (PEG) and functionalized with a Lemke-type fluorophore. It has a hydrodynamic diameter of 28 ± 8 nm and a zeta potential of -42 ± 6 mV. The MR relaxivity ratio at 7 T is r1/r2 = 20; therefore, NP3 is well suited as a T2/T2* contrast agent. In vitro cytotoxicity assessments performed on four human cell lines revealed no toxic effects of NP3. In addition, NP3 is internalized by macrophages in vitro without inducing inflammation or cytotoxicity. In vivo, uptake of NP3 has been observed in the spleen and the liver. NP3 has a prolonged vascular remanence, which is an advantage for macrophage uptake in vivo. The proof-of-concept that NP3 may be used as a contrast agent targeting phagocytic cells is provided in an animal model of ischemic stroke in transgenic CX3CR1-GFP/+ mice using three complementary imaging modalities: MRI, intravital two-photon microscopy and phase contrast imaging with synchrotron X-rays. In summary, NP3 is a promising preclinical tool for the multiscale and multimodal investigation of neuroinflammation.

Fragmentation patterns of new esterified and unesterified aromatic 1-hydroxymethylene-1, 1-bisphosphonic acids by ESI-MSn.

1-Hydroxymethylene-1,1-bisphosphonic acids (HMBPs) are compounds that have interesting pharmacological applications. Unfortunately few studies exist on their analyses by mass spectrometry (MS). In this work, we have analyzed new aromatic HMBPs and their prodrugs with electrospray tandem mass spectrometry (ESI-MS(n)). We describe, for the first time, a complete study of fragmentation patterns, in both positive and negative-ion modes. In positive mode, the cation dissociations are mainly elimination of water and phosphorus fragments. In negative mode, losses of ROH (R==H, C(6)H(5), CH(3)OC(6)H(5)) and HPO(2) were observed. The results have revealed specific structural fingerprints for the screening of these compounds in complex biological mixtures.

Polyphosphonate ligands: From synthesis to design of hybrid PEGylated nanoparticles toward phototherapy studies.

The use of phosphonate ligands to modify the nanoparticle (NPs) surface has attracted a strong interest in the last years for the design of highly functional hybrid materials. Here, we applied a methodology to synthesize bisphosphonates having functionalized PEG side chains with a specific length in order to design a novel class of hybrid nanomaterials composed by tetraphosphonate-complex-gold COOH-terminated PEG-coated NPs (Bis-PO-PEG-AuNPs). The synthetic approach consist in three steps: (1) Complexation between new phosphonate ligands (Bis PO) and tetrachloroauric acid (HAuCl4) to form gold clusters; (2) adsorption of COOH-terminated PEG molecules (PEG) onto Bis PO-Au complex; (3) reduction of metal ions in that vicinity, growth of gold particles and colloidal stabilization. The obtained snow-shape-like hybrid nanoparticles, have been characterized by ultra-violet/visible, Raman spectroscopies, and electron microscopy imaging, involving their optical properties and photothermal activity in pancreatic adenocarcinoma cancer cells (PDAC).

Cyclodextrins: A promising drug delivery vehicle for bisphosphonate.

Bisphosphonates are well established pharmaceutical drugs with wide applications in medicine. Nevertheless, the side chain and the nature of phosphorous groups could induce a poor aqueous solubility and act on their bioavailability. At the same time, cyclodextrins are cage molecules that facilitate transport of hydrophobic molecules to enhance the intestinal drug absorption of these molecules by forming inclusion complexes. Here we demonstrate that cyclodextrins could be used as a bisphosphonate carrier. The formation of cyclodextrins-bisphosphonate complexes was characterized by 1D and 2D NMR spectroscopy, Isothermal Titration Calorimetry and UV-vis spectroscopy. The results revealed that only the side chain of bisphosphonate was involved in the inclusion phenomenon and its length was a crucial parameter in the control of affinity. Findings from this study suggest that cyclodextrin will be a useful carrier for bisphosphonates.

Novel approach to nucleoside-5'-(1-hydroxymethylene-1, 1-bisphosphonates): synthesis of new AZT analogues.

An efficient synthetic method of nucleoside-5'-(1-hydroxymethylene-1,1-bisphosphonates) is reported here. The procedure was optimized with 3'-protected thymidine and then applied to synthesis of new AZT analogues.

Structural requirements for bisphosphonate binding on hydroxyapatite: NMR study of bisphosphonate partial esters.

Eighteen different bisphosphonates, including four clinically used bisphosphonate acids and their phosphoesters, were studied to evaluate how the bisphosphonate structure affects binding to bone. Bisphosphonates with weak bone affinity, such as clodronate, could not bind to hydroxyapatite after the addition of one ester group. Medronate retained its ability to bind after the addition of one ester group, and hydroxy-bisphosphonates could bind even after the addition of two ester groups. Thus, several bisphosphonate esters are clearly bone binding compounds. The following conclusions about bisphosphonate binding emerge: (1) a hydroxyl group in the geminal carbon takes part in the binding process and increases the bisphosphonate's ability to bind to bone; (2) the bisphosphonate's ability to bind decreases when the amount of ester groups increases; and (3) the location of the ester groups affects the bisphosphonate's binding ability.

Bisphosphonate prodrugs: synthesis and biological evaluation in HuH7 hepatocarcinoma cells.

We investigated the biological effects of new synthesized bisphosphonates (BPs) on HuH7 hepatocarcinoma cells. BPs containing p-bromophenyl (R1 = p-Br, Ph, 2) in their side chain were the more potent to inhibit HuH7 cell viability. In addition, phenyl diesterified analogues (R2 = R3 = Ph, 2a) were more potent than methyl (R2 = R3 = Me, 2b) or non-esterified BPs (2) inducing more necrosis suggesting that they better entered into cells. Phosphodiesterase inhibitor (IBMX) reversed the effect of the esterified BPs and not that of non-esterified ones suggesting role of cell phosphodiesterases to release active BPs. BP analogues inhibited HuH7 cell migration but esterified ones had no effect on invasion due to the hiding of phosphonic groups. All together, these results indicated the therapeutic interest of these new BP prodrugs.

Aqueous suspensions of GdPO4 nanorods: a paramagnetic mineral liquid crystal.

Colloidal suspensions of rod-like nanoparticles are well-known to readily form liquid-crystalline phases. Using mineral nanoparticles for this purpose may impart their liquid-crystalline suspensions with original physical properties. We synthesized GdPO(4) nanorods whose aqueous suspensions spontaneously organize in a nematic phase at high concentrations. The nematic phase is very well aligned by small magnetic fields, and the isotropic phase displays a very large field-induced birefringence. Moreover, the nanorods migrate to regions of high magnetic field. On the basis of magnetization measurements, we show that this unusual behavior is due to the fact that GdPO(4) nanorods are actually paramagnetic. Such a paramagnetic mineral liquid crystal, easily synthesized and little sensitive to temperature, may be an interesting alternative to organometallic thermotropic liquid crystals for applications where magnetic field alignment would be more suitable than electric field alignment.