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 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.

Synthesis, antiviral and antitumor activities investigations of a series of Ribavirin C-nucleoside analogue prodrugs.

Phosphoramidates obtained according to the ProTide strategy are known for their ability to increase the biological activity of various nucleosides. A series of such prodrugs of SRO-91, a non-natural ribofuranosyl-1,2,3-triazole C-nucleoside obtained by a synthetic sequence involving an indium mediated alkynylation and a Huisgen cycloaddition, was prepared and the antitumor activity on 3 strains of tumor cells was investigated. Two compounds 9a and 9c exhibited interesting cell proliferative inhibitions (IC50 = 2.5-12.1 µM) on two cell lines (pancreas and lung). Moreover, concerning the antiviral activity, another phosphoramidate 14 bearing a different aryl masking group exhibited an IC50 of 5 µM on Crimean-Congo Hemorrhagic Fever orthonairovirus. In both cases, free SRO-91 presented no activity on these cell lines.

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).

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.

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.

Synthesis and antitumor activities investigation of a C-nucleoside analogue of ribavirin.

SRO-91 is a non-natural ribofuranosyl-1,2,3-triazole C-nucleoside obtained by a synthetic sequence involving a C-alkynyl glycosylation mediated by metallic indium and a Huisgen cycloaddition for the construction of the triazole. Its structure is close to the one of ribavirin, a drug presenting a broad-spectrum against viral infections. SRO-91 antitumor activities were investigated on 9 strains of tumor cells and IC50 of the order of 1 µM were obtained on A431 epidermoid carcinoma cells and B16F10 skin melanoma cells. In addition, studies of ovarian tumor cell inhibitions show an interesting activity in regard to the need for new drugs for this pathology. Finally, cytotoxicity and mouse toxicity studies reveal a favorable therapeutic index for SRO-91.

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.

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.

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.

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.

In vitro assessment of liposomal neridronate on MDA-MB-231 human breast cancer cells.

Bisphosphonates have been used for decades in the standard therapy of bone-related diseases, including bone metastasis of various malignancies, and they might as well be toxic on early cancer cells themselves. In order to allow a better delivery of neridronate (a N-containing bisphosphonate with relatively poor activity), liposomes were evaluated in vitro on cancer cell lines (MDA-MB-231, U87-MG and Caco2). After chemical synthesis, this water-soluble molecule was encapsulated into liposomes containing DOPC:DOPG:Chol (72:27:1 molar ratio). The influence of neridronate (free or liposomal) on cell viability or proliferation after treatment was evaluated using the MTT method, as well as cell migration and invasion assays; these techniques showed a drastic improvement of the action of neridronate on MDA-MB-231 cells with an EC(50) 50 times lower when neridronate was encapsulated. Internalization of liposomes was followed by flow cytometry and fluorescence microscopy, demonstrating internalization via the endocytic pathway. Furthermore, since overexpression of matrix metalloproteinases (particularly MMP-2 and MMP-9) has been correlated to poor prognosis in many cancer types, detection of MMP expression is a satisfactory indication of the therapy efficiency and was then performed on treated cells. On MDA-MB-231 cells, MPPs expression was also significantly reduced by neridronate while entrapped in liposomes.

Impact of alendronate and VEGF-antisense combined treatment on highly VEGF-expressing A431 cells.

Bisphosphonates, and more specially nitrogen-containing bisphosphonates, which are in current use for the treatment of bone diseases, demonstrate proapoptotic, antiproliferative, antiangiogenic and anti-invasive properties on tumor cells. The amino-bisphosphonate alendronate is considered as a potential anticancer drug. In the case of A431 cells, which express high levels of VEGF, it had a two-step effect. At 24h, the antitumor properties of alendronate were counterbalanced by a survival process, which consisted of an enhancement of VEGF expression (mRNA and protein secretion) and TGF alpha secretion. It was only at 48 h that alendronate displayed the expected antiproliferative and antiangiogenic properties. The first step, in which the PI3K pathway was engaged, could be prevented by the use of a VEGF-antisense oligonucleotide. The combination of such an antisense with small concentrations of alendronate (approximately 2 microM), which is of the order of clinically used concentrations, was shown to have an antiangiogenic effect as soon as 12h.

Methyl [hydr-oxy(phen-yl)phosphono-meth-yl]phospho-nate methanol solvate.

The title compound, C(8)H(12)O(7)P(2)·CH(4)O, is a monoesterified bis-phospho-nate (or 1-hydroxy-methyl-ene-1,1-bis-phospho-nic acid). These synthetic compounds are widely used in medicine to inhibit bone resorption in diseases like osteoporosis, and are characterized by a stable P-C-P group and are thus analogs of inorganic pyrophosphate. By masking one or several ionizable groups, introduced as phosphono-ester, it was anti-cipated the formation of prodrugs with higher lipophilicity that could facilitate the drug delivery and metabolization. Mol-ecules are paired by inter-molecular hydrogen bonds involving the phospho-nic groups. In addition, dimers are connected side-by-side, building infinite ribbons along the a-axis direction; these ribbons are cross-linked perpendicularly along the b-axis direction via a methanol solvent mol-ecule (disordered over two sites with occupancy factors ca 0.6 and 0.4), forming an extended inter-molecular hydrogen-bonded network. The H atoms of the methyl group in the main molecule are disordered equally over two positions.

Adaptation of the base-paired double-helix molecular architecture to extreme pressure.

The behaviour of the d(GGTATACC) oligonucleotide has been investigated by X-ray crystallography at 295 K in the range from ambient pressure to 2 GPa (approximately 20,000 atm). Four 3D-structures of the A-DNA form (at ambient pressure, 0.55, 1.09 and 1.39 GPa) were refined at 1.60 or 1.65 A resolution. In addition to the diffraction pattern of the A-form, the broad meridional streaks previously explained by occluded B-DNA octamers within the channels of the crystalline A-form matrix were observed up to at least 2 GPa. This work highlights an important property of nucleic acids, their capability to withstand very high pressures, while keeping in such conditions a nearly invariant geometry of base pairs that store and carry genetic information. The double-helix base-paired architecture behaves as a molecular spring, which makes it especially adapted to very harsh conditions. These features may have contributed to the emergence of a RNA World at prebiotic stage.