The E551 food additive is composed of synthetic amorphous silica particles. The current regulation does not mention any specifications regarding their size and granulometric distribution, thus allowing the presence of silica nanoparticles despite their potential toxicity. The digestion process could modify their physicochemical properties and then influence their toxicological profile. After physicochemical characterization, subacute toxicity of engineered silica nanoparticles from 20 to 200 nm, native and digested E551 additives were evaluated from in vitro models of the intestinal barrier. Single cultures and a co-culture of enterocytes and mucus-secreting cells were established to investigate the mucus role. Toxicological endpoints including cytotoxicity, ROS production, intestinal permeability increase, and actin filament disruption were addressed after a 7-day exposure. The results showed a size-dependent effect of silica nanoparticles on cytotoxicity and intestinal permeability. A time-dependent disruption of actin filaments was observed in Caco-2 cells. The mucus layer spread on the HT29-MTX single culture acted as an efficient protective barrier while in the co-culture, small nanoparticles were able to cross it to reach the cells. From a hydrodynamic diameter of 70 nm, nanoparticles were not internalized in the intestinal cells, even in mucus-free models. Digestion did not affect the physicochemical properties of the additive. Due to a mean hydrodynamic diameter close to 200nm, both native and digested E551 additives did not induce any toxic effect in intestinal barrier models. This study emphasized a cutoff size of 70nm from which the interactions of the E551 additive with intestinal cells would be limited.

The discovery of the TLRs family and more precisely its functions opened a variety of gates to modulate immunological host responses. TLRs 7/8 are located in the endosomal compartment and activate a specific signaling pathway in a MyD88-dependant manner. According to their involvement into various autoimmune, inflammatory and malignant diseases, researchers have designed diverse TLRs 7/8 ligands able to boost or block the inherent signal transduction. These modulators are often small synthetic compounds and most act as agonists and to a much lesser extent as antagonists. Some of them have reached preclinical and clinical trials, and only one has been approved by the FDA and EMA, imiquimod. The key to the success of these modulators probably lies in their combination with other therapies as recently demonstrated. We gather in this review more than 360 scientific publications, reviews and patents, relating the extensive work carried out by researchers on the design of TLRs 7/8 modulators, which are classified firstly by their biological activities (agonist or antagonist) and then by their chemical structures, which total syntheses are not discussed here. This review also reports about 90 clinical cases, thereby showing the biological interest of these modulators in multiple pathologies.

The Toll-like receptors (TLRs) 7 and 8 play an important role in the immune system activation, and their agonists may therefore serve as promising candidate vaccine adjuvants. However, the chronic immune activation by excessive TLR stimulation is a hallmark of several clinically important infectious and autoimmune diseases, which warrants the search for TLR antagonists. In this study, we have synthesized and characterized a variety of compounds belonging to three heterocyclic chemical series: imidazo[1,2-a]pyrazine, imidazo[1,5-a]quinoxaline, and pyrazolo[1,5-a]quinoxaline. These compounds have been tested for their TLR7 or TLR8 agonistic and antagonistic activities. Several of them are shown to be selective TLR7 antagonists without any TLR7 or TLR8 agonistic activity. The selectivity was confirmed by a comparative ligand-docking study in TLR7 antagonist pocket. Two compounds of the pyrazolo[1,5-a]quinoxaline series (10a and 10b) are potent selective TLR7 antagonists and may be considered as promising starting points for the development of new therapeutic agents.

The Suzuki–Miyaura cross coupling is a versatile and powerful reaction for carbon–carbon bond formation which nevertheless requires optimization of conditions. While the coupling of arylboron derivatives with aryl or alkenyl halides has been widely explored, alkylation has attracted less attention. We describe herein the methylation of bromo derivatives of imidazopyrazine, imidazoquinoxaline, and pyrazoloquinoxaline having biologically interesting structures. As the challenging Suzuki–Miyaura reaction is highly substratedependent, the choices of the base, solvent, and additive are also discussed.

of of multiple cellular processes, including immune signaling, development, proliferation and survival.

The transcription nuclear factor NF-κB plays a pivotal role in chronic and acute inflammatory diseases. Among the several and diverse strategies for inhibiting NF-κB, one of the most effective approach considered by the pharmaceutical industry seems to be offered by the development of IKK inhibitors. In a former study, two potential IKK2 inhibitors have been highlighted among a series of imidazo[1,2-a]quinoxaline derivatives. In order to enhance this activity, we present herein the synthesis of twenty-one new compounds based on the imidazo[1,2-a]pyrazine, imidazo[1,5-a]quinoxaline or pyrazolo[1,5-a]quinoxaline structures. Their potential to inhibit IKK1 and IKK2 activities is also tested.

The inhibition of the NF-κB-dependent pathways by IKK inhibitors plays an important role in immunity, inflammation, and cancer. New imidazoquinoxalines tricyclic derivatives are prepared using microwave assistance and their biological activities as IKK inhibitors are described. Compounds 6a present a potent inhibition activity and selectivity for IKK2. Docking studies in the IKK2 binding site allowed identification of residues most likely to interact with theses inhibitors and explain their potent IKK2 inhibition activity and selectivity.

Microtubules are considered as important targets of anticancer therapy. EAPB0503 and its structural imidazo[1,2-a]quinoxaline derivatives are major microtubule-interfering agents with potent anticancer activity. In this study, the synthesis of several new derivatives of EAPB0503 is described, and the anticancer efficacy of 13 novel derivatives on A375 human melanoma cell line is reported. All new compounds show significant antiproliferative activity with IC50 in the range of 0.077-122μM against human melanoma cell line (A375). Direct inhibition of tubulin polymerization assay in vitro is also assessed. Results show that compounds 6b, 6e, 6g, and EAPB0503 highly inhibit tubulin polymerization with percentages of inhibition of 99%, 98%, 90%, and 84% respectively. Structure-activity relationship studies within the series are also discussed in line with molecular docking studies into the colchicine-binding site of tubulin.

Antimalarial activities and pharmacokinetics of the bis-alkylamidine, M64, and its amidoxime, M64-AH, and O-methylsulfonate, M64-S-Me, derivatives were investigated. M64 and M64-S-Me had the most potent activity against the Plasmodium falciparum growth (IC(50)<12nM). The three compounds can clear the Plasmodium vinckei infection in mice (ED(50)<10mg/kg). A liquid chromatography-mass spectrometry method was validated to simultaneously quantify M64 and M64-AH in human and rat plasma. M64 is partially metabolized to M64-monoamidoxime and M64-monoacetamide by rat and mouse liver microsomes. The amidoxime M64-AH undergoes extensive metabolism forming M64, M64-monoacetamide, M64-diacetamide and M64-monoamidoxime. Strong interspecies differences were observed. The pharmacokinetic profiles of M64, M64-AH and M64-S-Me were studied in rat after intravenous and oral administrations. M64 is partially metabolized to M64-AH; while M64-S-Me is rapidly and totally converted to M64 and M64-AH. M64-AH is mostly oxidized to the inactive M64-diacetamine while its N-reduction to the efficient M64 is a minor metabolic pathway. Oral dose of M64-AH was well absorbed (38%) and converted to M64 and M64-diacetamide. This study generated substantial information about the properties of this class of antimalarial drugs. Other routes of synthesis will be explored to prevent oxidative transformation of the amidoxime and to favour the N-reduction.

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In spite of the development of new anticancer drugs by the pharmaceutical industry, melanoma and T lymphomas are diseases for which medical advances remain limited. Thus, there was an urgent need of new therapeutics with an original mechanism of action. Since several years, our group develops quinoxalinic compounds. In this paper, the first preclinical results concerning one lead compound, EAPB0203, are presented. This compound exhibits in vitro cytotoxic activity on A375 and M4Be human melanoma cell lines superior to that of imiquimod and fotemustine. A liquid chromatography-mass spectrometry method was first validated to simultaneously quantify EAPB0203 and its metabolite, EAPB0202, in rat plasma. Thereafter, the pharmacokinetic profiles of EAPB0203 were studied in rat after intravenous and intraperitoneal administrations. After intraperitoneal administration the absolute bioavailability remains limited (22.7%). In xenografted mouse, after intraperitoneal administration of 5 and 20mg/kg, EAPB0203 is more potent than fotemustine. The survival time was increased up to 4 and 2 weeks compared to control mice and mice treated by fotemustine, respectively. The results of this study demonstrate the relationship between the dose of EAPB0203 and its effects on tumor growth. Thus, promising efficacy, tolerance and pharmacokinetic data of EAPB0203 encourage the development towards patient benefit.

New imidazo[1,2-a]quinoxaline analogues have been synthesized in good yields via a bimolecular condensation of 2-imidazole carboxylic acid, followed by a coupling with ortho-fluoroaniline and subsequent substitution on the imidazole ring by Suzuki Cross-coupling reaction using microwave assistance. Antitumor activities of these derivatives were evaluated by growth inhibition of A375 cells in vitro. All compounds exhibited high activities compared to imiquimod and fotemustine used as references.

Since several years, our group developed quinoxalinic compounds. Among the synthesized compounds, in the imidazo[1,2-a]quinoxaline series, EAPB0203 has shown interesting activities both on melanoma and lymphoma. The structure of EAPB0203 has been modulated and a new compound, EAPB0503, exhibits an in vitro cytotoxic activity on melanoma cancer cell line 7-9 times higher than EAPB0203. We validated an LC/ESI-MS method to simultaneously quantify EAPB0503 and its metabolite EAPB0603 in human and rat plasma. Chromatography was performed on a C8 Zorbax eclipse XDB column with a mobile phase consisting of acetronitrile and formate buffer gradient elution. LC-MS data were acquired in SIM mode at m/z 305, 291, and 303 for EAPB0503, EAPB0603, and the internal standard, respectively. The drug/internal standard peak area ratios were linked via quadratic relationships to concentrations (low range: 5-300 microg/L, high range: 100-1000 microg/L). The method is precise (precision, < or = 14%) and accurate (recovery, 92-113%). Mean extraction efficiencies, > 72% for each analyte, were obtained. The lower LOQs were 5 microg/L. This highly specific and sensitive method was successfully used to investigate plasma concentrations of EAPB0503 and EAPB0603 in a pharmacokinetic study carried out in rat and would also be useful in clinical trials at a later stage.

Imidazoquinoxaline and pyrazoloquinoxaline derivatives, analogues of imiquimod, were synthesized, and their in vitro cytotoxic and pharmacodynamic activities were evaluated. In vitro cytotoxicity studies were assessed against melanoma (A375, M4Be, RPMI-7591), colon (LS174T), breast (MCF7), and lymphoma (Raji) human cancer cell lines. In vivo studies were carried out in M4Be xenografted athymic mice. EAPB0103, EAPB0201, EAPB0202, and EAPB0203 showed significant in vitro activities against A375 compared to fotemustine and imiquimod used as references. These compounds were 6-110 and 2-45 times more active than fotemustine and imiquimod, respectively. EAPB0203 bearing phenethyl as substituent at position 1 and methylamine at position 4 showed the highest activity. EAPB0203 has also a more potent cytotoxic activity than imiquimod and fotemustine in M4Be and RPMI-7591 and interesting cytotoxic activity in other tumor cell lines tested. In vivo, EAPB0203 treatment schedules caused a significant decrease in tumor size compared to vehicle control and fotemustine treatments. (c) 2008 Elsevier Ltd. All rights reserved.

L’imiquimod (Aldara®), premier medicament de type immunomodulateur, fait l’objet de nombreuses etudes en chimiotherapie anticancereuse. Il exerce un effet antitumoral de deux types : direct par apoptose et indirect par stimulation du systeme immunitaire. Cette molecule est decrite comme efficace sur le melanome humain, pathologie qui presente comme seul traitement de chimiotherapie l’utilisation de la fotemustine. La synthese, la reactivite chimique et l’activite anticancereuse de nouveaux derives tricycliques de type imidazoquinoxaline et pyrazoloquinoxaline, analogues de l’imiquimod, ont ete etudiees grâce a des approches chimiques et biologiques. Des voies de synthese innovantes, permettant une modulation chimique importante grâce a des reactions de Suzuki, ont ete mises au point et ont permis d’obtenir une vingtaine de molecules appartenant a trois series tricycliques differentes. L’activite antitumorale des molecules synthetisees a ete evaluee in vitro sur la lignee cellulaire A375 de melanome humain. Deux molecules ont presente une activite cytotoxique importante et superieure a celle de l’imiquimod et la fotemustine, utilises comme references. Ces resultats ont ete confirmes sur d’autres lignees cellulaires de melanome humain. L’activite cytotoxique du compose le plus actif a aussi ete mesuree sur des lignees tumorales de colon, de sein, de lymphome B et de lymphocytes T malins (ATL). Dans ce dernier cas, a partir de modeles cellulaires et moleculaires, le mecanisme d’action de la molecule chef de file (EAPB0203) a ete etudie. EAPB0203, a des concentrations de l’ordre du micro-molaire, induit a la fois l’inhibition de la proliferation et la mort par apoptose des cellules cancereuses, de plus ces effets s’accompagnent d’un arret du cycle cellulaire en phase G2/M. Il est sans effet sur les lymphocytes normaux isoles d’un sujet normal, actives ou non par la PHA. La mort cellulaire est dependante de l’activation des caspases. Ces etudes preliminaires montrent que ce compose induit la mort cellulaire par apoptose par l’intermediaire de la voie intrinseque mitochondriale. Parallelement, des etudes in vivo sur modele animal (souris nude SWISS xenogreffees par une lignee cancereuse de melanome humain, les M4Be) ont ete realisees. Un suivi de l’evolution tumorale a ete etudie apres administration de EAPB0203 a differentes doses. A 20mg/kg, les souris traitees par EAPB0203 ont montre un retard significatif dans la croissance de la tumeur par rapport aux souris temoin et aux souris fotemustine. Aujourd’hui, la modulation chimique des molecules actives nous permet de donner naissance a de nouveaux leaders. L’ensemble de ces composes pourrait jouer un role majeur dans le developpement d’une nouvelle strategie anticancereuse.

Imiquimod is an immune response modifier currently used as a topical treatment of genital warts, basal cell carcinoma, cutaneous metastasis of malignant melanoma, and vascular tumors. We developed more efficient killers from the same family of compounds that can induce apoptosis without the prominent pro-inflammatory response associated with imiquimod. Among these new products, tk;4EAPB0203, a member of the imidazo[1,2-a]quinoxalines, exhibits an important cytotoxic activity in vitro. HTLV-I-associated adult T-cell leukemia (ATL) and HTLV-I-negative peripheral T-cell lymphomas are associated with poor prognosis. Using potentially achievable concentrations of EAPB0203, we demonstrate inhibition of cell proliferation, G2/M cell- cycle arrest, and induction of apoptosis in HTLV-I-transformed and HTLV-I-negative malignant T cells and fresh ATL cells, whereas normal resting or activated T lymphocytes were resistant. EAPB0203 treatment significantly down-regulated the antiapoptotic proteins c-IAP-1 and Bcl-XL and resulted in a significant loss of mitochondrial membrane potential, cytoplasmic release of cytochrome c, and caspase-dependent apoptosis. Moreover, in HTLV-I-transformed cells only, EAPB0203 treatment stabilized p21 and p53 proteins but had no effect on NF-kappaB activation. These results support a potential therapeutic role for EAPB0203 in ATL and HTLV-I-negative T-cell lymphomas, either as a systemic or topical therapy for skin lesions.