Soluble TIM-3 as a biomarker of progression and therapeutic response in cancers and other of human diseases.

Immune checkpoints inhibition is a privileged approach to combat cancers and other human diseases. The TIM-3 (T cell immunoglobulin and mucin-domain containing-3) inhibitory checkpoint expressed on different types of immune cells is actively investigated as an anticancer target, with a dozen of monoclonal antibodies in (pre)clinical development. A soluble form sTIM-3 can be found in the plasma of patients with cancer and other diseases. This active circulating protein originates from the proteolytic cleavage by two ADAM metalloproteases of the membrane receptor shared by tumor and non-tumor cells, and extracellular vesicles. In most cancers but not all, overexpression of mTIM-3 at the cell surface leads to high level of sTIM-3. Similarly, elevated levels of sTIM-3 have been reported in chronic autoimmune diseases, inflammatory gastro-intestinal diseases, certain viral and parasitic diseases, but also in cases of organ transplantation and in pregnancy-related pathologies. We have analyzed the origin of sTIM-3, its methods of dosage in blood or plasma, its presence in multiple diseases and its potential role as a biomarker to follow disease progression and/or the treatment response. In contrast to sPD-L1 generated by different classes of proteases and by alternative splicing, sTIM-3 is uniquely produced upon ADAM-dependent shedding, providing a more homogenous molecular entity and a possibly more reliable molecular marker. However, the biological functionality of sTIM-3 remains insufficiently characterized. The review shed light on pathologies associated with an altered expression of sTIM-3 in human plasma and the possibility to use sTIM-3 as a diagnostic or therapeutic marker.

Specification and Evaluation of Plasticizer Migration Simulants for Human Blood Products: A Delphi Study.

Potentially toxic plasticizers are commonly added to polyvinyl chloride medical devices for transfusion in order to improve their flexibility and workability. As the plasticizers are not chemically bonded to the PVC, they can be released into labile blood products (LBPs) during storage. Ideally, LBPs would be used in laboratory studies of plasticizer migration from the medical device. However, short supply (i.e., limited stocks of human blood in collection centres) has prompted the development of specific simulants for each type of LBP in the evaluation of new transfusion devices. We performed a Delphi study with a multidisciplinary panel of 24 experts. In the first (qualitative) phase, the panel developed consensus definitions of the specification criteria to be met by each migration simulant. Next, we reviewed the literature on techniques for simulating the migration of plasticizers into LBPs. A questionnaire was elaborated and sent out to the experts, and the replies were synthesized in order to obtain a consensus. The qualitative study established specifications for each biological matrix (whole blood, red blood cell concentrate, plasma, and platelet concentrate) and defined the criteria required for a suitable LBP simulant. Ten criteria were suggested: physical and chemical characteristics, opacity, form, stability, composition, ability to mimic a particular clinical situation, ease and safety of use, a simulant-plastic interaction correlated with blood, and compatibility with analytical methods. The questionnaire data revealed a consensus on the use of natural products (such as pig's blood) to mimic the four LBPs. Opinions diverged with regard to synthetic products. However, an isotonic solution and a rheological property modifier were considered to be of value in the design of synthetic simulants. Consensus reached by the Delphi group could be used as a database for the development of simulants used to assess the migration of plasticizers from PVC bags into LBPs.

Hinokiflavone and Related C-O-C-Type Biflavonoids as Anti-cancer Compounds: Properties and Mechanism of Action.

Biflavonoids are divided in two classes: C-C type compounds represented by the dimeric compound amentoflavone and C-O-C-type compounds typified by hinokiflavone (HNK) with an ether linkage between the two connected apigenin units. This later sub-group of bisflavonyl ethers includes HNK, ochnaflavone, delicaflavone and a few other dimeric compounds, found in a variety of plants, notably Selaginella species. A comprehensive review of the anticancer properties and mechanism of action of HNK is provided, to highlight the anti-proliferative and anti-metastatic activities of HNK and derivatives, and HNK-containing plant extracts. The anticancer effects rely on the capacity of HNK to interfere with the ERK1-2/p38/NFκB signaling pathway and the regulation of the expression of the matrix metalloproteinases MMP-2 and MMP-9 (with a potential direct binding to MMP-9). In addition, HNK was found to function as a potent modulator of pre-mRNA splicing, inhibiting the SUMO-specific protease SENP1. As such, HNK represents a rare SENP1 inhibitor of natural origin and a scaffold to design synthetic compounds. Oral formulations of HNK have been elaborated to enhance its solubility, to facilitate the compound delivery and to enhance its anticancer efficacy. The review shed light on the anticancer potential of C-O-C-type biflavonoids and specifically on the pharmacological profile of HNK. This compound deserves further attention as a regulator of pre-mRNA splicing, useful to treat cancers (in particular hepatocellular carcinoma) and other human pathologies.

Simultaneous determination of di(2-ethylhexyl) phthalate and diisononylcyclohexane-1,2-dicarboxylate and their monoester metabolites in four labile blood products by liquid chromatography tandem mass spectrometry.

Di(2-ethylhexyl) phthalate (DEHP) is a common plasticizer that is largely used for PVC blood bags. The migration of DEHP from medical devices into labile blood products (LBP) is a well-known situation. While DEHP has beneficial effects on the storage of red blood cells, it can have toxicological impact due to its potential reprotoxic effects (classified group 1B). Since July 1st, 2015, the French law prohibits the use of tubing made in DEHP-plasticized PVC in paediatric, neonatal and maternity wards. This provision, which could extend in several years more widely to medical devices used for drugs infusion, dialysis, feeding and blood bags, has led manufacturers to replace DEHP to alternative plasticizers such as diisononylcyclohexane-1,2-dicarboxylate (DINCH). In this paper, a liquid chromatography-tandem mass spectrometry (LCMS/MS) method has been developed and validated for the determination of DEHP, DINCH and their corresponding monoester metabolites (MEHP and MINCH) in four labile blood products (LBP): whole blood (WB), red cells concentrate (RCC), plasma and platelet concentrate (PC). Due to strong contamination of blank LBP by DEHP because of its ubiquitous presence in working environment and despite the attention paid to avoid contamination of solvents and glassware, a trap chromatographic column was implemented between the solvent mixing chamber and the injector of the LC system. This set-up permitted to discriminate DEHP present in the sample to DEHP brought by the environmental contamination. In the optimized conditions, all compounds were separated in less than 10 min. The analytes were extracted from LBP samples using a liquid-liquid extraction. After optimization, recoveries were ranged from 47 to 96 %, depending on the analytes and the nature of LBP. Except for DEHP which exhibited RSD values of intermediate precision higher than 20 % at a concentration of 25 nM, all the precision results (repeatability and intermediate precision) were lower than 16 % and trueness values ranged from -16.2-19.8%. Using the validated method, the leachability of DEHP and DINCH from corresponding PVC-blood bags was investigated and the concentrations of their corresponding metabolites, MEHP and MINCH, were determined in whole blood, red cells concentrate, plasma and platelet concentrate.

DNA Methylation Targeting: The DNMT/HMT Crosstalk Challenge.

Chromatin can adopt a decondensed state linked to gene transcription (euchromatin) and a condensed state linked to transcriptional repression (heterochromatin). These states are controlled by epigenetic modulators that are active on either the DNA or the histones and are tightly associated to each other. Methylation of both DNA and histones is involved in either the activation or silencing of genes and their crosstalk. Since DNA/histone methylation patterns are altered in cancers, molecules that target these modifications are interesting therapeutic tools. We present herein a vast panel of DNA methyltransferase inhibitors classified according to their mechanism, as well as selected histone methyltransferase inhibitors sharing a common mode of action.

Chiral separation of new sulfonamide derivatives and evaluation of their enantioselective affinity for human carbonic anhydrase II by microscale thermophoresis and surface plasmon resonance.

The aim of this study was to develop a method combining chiral separation and biophysical techniques to evaluate the enantioselective affinity of original sulfonamide derivatives towards their therapeutic target, the human carbonic anhydrase II (hACII). The first step consisted in the preparation of the enantiomers by chromatographic separation. The performances of HPLC and Supercritical Fluid Chromatography (SFC) were studied at the analytical scale by optimization of various experimental conditions using adsorbed polysaccharide chiral stationary phases (amylose AD-H and cellulose OD-H). Since SFC allowed obtaining higher enantioresolutions per time unit, it was selected for the semi-preparative scale and successfully used to isolate each enantiomer with a satisfactory enantiomeric purity (>98%). Secondly, microscale thermophoresis (MST) method and surface plasmon resonance (SPR) used as reference method were developed to measure potential enantioselective affinities of these enantiomers towards the hACII. The optimizations of both methods were performed using a reference compound, i.e. acetazolamide, which affinity for hCAII has previously been demonstrated. For all compounds, KD values obtained using MST and SPR were in good agreement, leading to similar affinity scales despite both approaches totally differ (labeling for MST versus immobilization of the protein for SPR). The equilibrium dissociation constants of our original compounds for the hCAII were in the range 100-1000nM and an enantioselectivity was observed using the MST and SPR methods for the diarylpyrazole 2. Finally, by comparing the MST and SPR techniques, MST appears especially adapted for further screening of a series of sulfonamide derivatives due to the lower time required to estimate a binding constant while consuming as little hCAII as SPR.

Amine coupling versus biotin capture for the assessment of sulfonamide as ligands of hCA isoforms.

This work was dedicated to the development of a reliable SPR method allowing the simultaneous and quick determination of the affinity and selectivity of designed sulfonamide derivatives for hCAIX and hCAXII versus hCAII, in order to provide an efficient tool to discover drugs for anticancer therapy of solid tumors. We performed for the first time a comparison of two immobilization approaches of hCA isoforms. First one relies on the use of an amine coupling strategy, using a CM7 chip to obtain higher immobilization levels than with a CM5 chip and consequently the affinity with an higher precision (CV% < 10%). The second corresponds to a capture of proteins on a streptavidin chip, named CAP chip, after optimization of biotinylation conditions (amine versus carboxyl coupling, biotin to protein ratio). Thanks to the amine coupling approach, only hCAII and hCAXII isoforms were efficiently biotinylated to reach relevant immobilization (3000 RU and 2700 RU, respectively) to perform affinity studies. For hCAIX, despite a successful biotinylation, capture on the CAP chip was a failure. Finally, concordance between affinities obtained for the three derivatives to CAs isozymes on both chips has allowed to valid the approaches for a further screening of new derivatives.

Synthesis and biological evaluation of di-aryl urea derivatives as c-Kit inhibitors.

Inhibition of receptor tyrosine kinases (RTKs) continued to be a successful approach for the treatment of many types of human cancers and many potent small molecules kinase inhibitors have been discovered the last decade. In the present study, we describe the synthesis of thienopyrimidine derivatives and their pharmacological evaluation against nine kinases (EGFR, PDGFR-ß, c-Kit, c-Met, Src, Raf, VEGFR-1, -2 and -3). Most of the synthesized compounds showed from moderate to potent activities against c-Kit with IC50 values in the nanomolar range. Among them, 4-anilino(urea)thienopyrimidine analogs showed selectivity and potent c-Kit inhibition with IC50 values less than 6 nM. Docking simulation was performed for the most promising compound 9 into the c-Kit active site to determine the potential binding mode. This study reveal that the 4-anilino(urea)thienopyrimidine is an interesting scaffold to design novel potent and selective c-Kit inhibitors which may make promising candidates for cancers where c-Kit receptors are overexpressed.

Quinazoline derivatives as anticancer drugs: a patent review (2011 - present).

INTRODUCTION: Quinazoline is one of the most studied moieties in medicinal chemistry due to the wide range of biological properties such as the anticancer, antibacterial, anti-inflammatory, antimalarial and antihypertensive activities. During the past decades, several patents and articles have been published in international peer-reviewed literature regarding the discovery and development of original and promising quinazoline derivatives for cancer treatment. Although quinazolines are well known to inhibit EGFR, there is also a large panel of other therapeutic protein targets. AREAS COVERED: This review summarized the new patents and articles published about quinazoline derivatives as anticancer drugs since 2011. EXPERT OPINION: Since 2011, a lot of quinazoline compounds have shown EGFR inhibition. Unlike the first-generation EGFR inhibitors, they inhibit both wild-type and mutated EGFR. In recent years, a number of studies on quinazoline synthesis have been reported and used by several medicinal chemistry groups for better and easier development of new derivatives. Therefore, several patents have been approved for the use of quinazoline compounds as inhibitors of other kinases, histone deacetylase, Nox and some metabolic pathways. Because of the large number of proteins targeted, some high structural diversity is observed in patented quinazoline compounds. Due to the vast applications of quinazoline derivatives, development of novel quinazoline compounds as anticancer drugs remains a promising field.

NMR investigation of the complexation and chiral discrimination of pyrazole sulfonamide derivatives with cyclodextrins.

The complexes formed between six original chiral diaryl-pyrazole sulfonamide derivatives, displaying poor solubility, and various CDs (native α-, ß- and γ-CDs, hydroxypropylated HP-ß-CD, methylated Me-ß-CD or amino NH2-ß-CD) were studied by 1D and 2D (1)H NMR at physiological pH in order to determine their apparent binding constant, stoichiometry and structure of the supramolecular assembly. For some complexes, the spectra obtained for free racemic compound and for racemic compound in presence of CD indicate a splitting of signal(s). Additional experiments with pure enantiomer and enriched enantiomer allow us to attribute this behavior to chiral discrimination. The complexing ability of the native ß-CD towards our compounds appears the most promising since binding values around 7×10(2)M(-1) are obtained. The two-dimensional ROESY ((1)H-(1)H) experiments prove the inclusion of the aliphatic part of the compound in the CD cavity. It is noteworthy that this inclusion occurs via the smaller opening of the cavity.

Inhibition of tumor cell growth and angiogenesis by 7-aminoalkoxy-4-aryloxy-quinazoline ureas, a novel series of multi-tyrosine kinase inhibitors.

Several regulatory and signaling molecules governing angiogenesis are targets of interest for the development of drugs in the cancer, including growth factors such as Vascular Endothelial Growth Factor (VEGF) and Platelet-Derived Growth Factor (PDGF). A series of 4-aryloxy-6,7-dimethoxyquinazolines, previously synthesized in our laboratory, has shown a nanomolar inhibition of kinase enzymatic activity of VEGFR, PDGFR and c-Kit. We have therefore studied the impact of the variation in the 7-position substitution of the quinazoline core. Substitution by aminoalkoxy chains led to new highly potent ATP-competitive inhibitors of VEGFR, PDGFR and c-Kit enzyme with IC50 values in the nanomolar range and this substitution has increased greatly antiproliferative activity on cancer cell lines (PC3, MCF7, HT29) and HUVEC (human umbilical vein endothelial cells). One of the most promising compounds (36) was assessed for its ability to limit the induction of web like network of capillary tubes by the human umbilical vascular endothelial cells (HUVEC) and for its ability to inhibit invasion.

Optimization of the enantioseparation of a diaryl-pyrazole sulfonamide derivative by capillary electrophoresis in a dual CD mode using experimental design.

A CE method using dual cationic and neutral cyclodextrins (CD) was optimized for the enantiomeric separation of a compound presenting a diaryl sulfonamide group. Preliminary studies were made to select the optimal CDs and pH of the BGE. Two CDs (amino-ß-CD and ß-CD) were selected to separate the enantiomers in a 67 mM phosphate buffer at pH 7.4. However, the repeatability of the analyses obtained on bare-fused silica capillary was not acceptable owing to the adsorption of the amino-ß-CD to the capillary. To prevent this, a dynamic coating of the capillary was used employing five layers of ionic-polymer (poly(diallyldimethylammonium) chloride (PDADMAC) and poly(sodium 4-styrenesulfonate). The efficiency of the coating was assessed by measuring the EOF stability. Repeatability of the injections was obtained when intermediate coating with PDADMAC was performed between each run. Secondly, this enantioseparation method was optimized using a central composite circumscribed design including three factors: amino-ß-CD and ß-CD concentrations and the percentage of methanol. Under the optimal conditions (i.e. 16.6 mM of amino-ß-CD, 2.6 mM of ß-CD, 0% MeOH in 67 mM phosphate buffer (pH 7.4) as BGE, cathodic injection 0.5 psi, 5 s, separation voltage 15 kV and a temperature of 15°C), complete enantioresolution of the analyte was obtained. It is worth mentioning that the design of experiments (DOE) protocol employed showed a significant interaction between CDs, highlighting the utility of DOE in method development. Finally, small variations in the ionic-polymer concentrations did not significantly influence the EOF, confirming the robustness of the coating method.

Label-free characterization of carbonic anhydrase-novel inhibitor interactions using surface plasmon resonance, isothermal titration calorimetry and fluorescence-based thermal shift assays.

This work describes the development of biophysical unbiased methods to study the interactions between new designed compounds and carbonic anhydrase II (CAII) enzyme. These methods have to permit both a screening of a series of sulfonamide derivatives and the identification of a lead compound after a thorough study of the most promising molecules. Interactions data were collected using surface plasmon resonance (SPR) and thermal shift assay (TSA). In the first step, experiments were performed with bovine CAII isoform and were extended to human CAII. Isothermal titration calorimetry (ITC) experiments were also conducted to obtain thermodynamics parameters necessary for the processing of the TSA data. Results obtained with this reference methodology demonstrate the effectiveness of SPR and TSA. KD values obtained from SPR data were in perfect accordance with ITC. For TSA, despite the fact that the absolute values of KD were quite different, the same affinity scale was obtained for all compounds. The binding affinities of the analytes studied vary by more than 50 orders of magnitude; for example, the KD value determined by SPR were 6 ± 4 and 299 ± 25 nM for compounds 1 and 3, respectively. This paper discusses some of the theoretical and experimental aspects of the affinity-based methods and evaluates the protein consumption to develop methods for the screening of further new compounds. The double interest of SPR, that is, for screening and for the quick thorough study of the interactions parameters (ka , kd , and KD ), leads us to choose this methodology for the study of new potential inhibitors.

New selective carbonic anhydrase IX inhibitors: synthesis and pharmacological evaluation of diarylpyrazole-benzenesulfonamides.

Carbonic anhydrase (CA) IX expression is increased upon hypoxia and has been proposed as a therapeutic target since it has been associated with poor prognosis, tumor progression and pH regulation. We report the synthesis and the pharmacological evaluation of a new class of human carbonic anhydrase (hCA) inhibitors, 4-(5-aryl-2-hydroxymethyl-pyrazol-1-yl)-benzenesulfonamides. A molecular modeling study was conducted in order to simulate the binding mode of this new family of enzyme inhibitors within the active site of hCA IX. Pharmacological studies revealed high hCA IX inhibitory potency in the parameters nanomolar range. This study showed that the position of sulfonamide group in meta of the 1-phenylpyrazole increase a selectivity hCA IX versus hCA II of our compounds. An in vitro antiproliferative screening has been performed on the breast cancer MDA-MB-231 cell using doxorubicin as cytotoxic agent and in presence of selected CA IX inhibitor. The results shown that the cytotoxic efficiency of doxorubicin in an hypoxic environment, expressed in IC50 value, is restored at 20% level with 1µM CA IX inhibitor.

Synthesis and structure-activity relationships of (aryloxy)quinazoline ureas as novel, potent, and selective vascular endothelial growth factor receptor-2 inhibitors.

In our continuing search for medicinal agents to treat proliferative diseases, quinazoline derivatives were synthesized and evaluated pharmacologically as epithelial growth factor receptor and vascular endothelial growth factor receptor 2 (VEGFR-2) tyrosine kinase inhibitors. A quantitative structure-activity relationship analysis was conducted to rationalize the structure-activity relationship and to predict how similar the inhibitor-binding profiles of two protein kinases are likely to be on the basis of the docking of lead coumpounds into the ATP-binding site. This model was used to direct the synthesis of new compounds. A series of N-(aromatic)-N'-{4-[(6,7-dimethoxyquinazolin-4-yl)oxy]phenyl}urea were identified as potent and selective inhibitors of the tyrosine kinase activity of VEGFR-2 (fetal liver kinase 1, kinase insert domain-containing receptor). An efficient route was developed that enabled the synthesis of a wide variety of analogues with substitution on several positions of the template. Substitution of diarylurea, competitive with ATP, afforded several analogues with low nanomolar inhibition of enzymatic activity of VEGFR-2. In this paper, we describe the synthesis, structure-activity relationships, and pharmacological characterization of the series.

Impact of aryloxy-linked quinazolines: a novel series of selective VEGFR-2 receptor tyrosine kinase inhibitors.

Three series of 6,7-dimethoxyquinazoline derivatives substituted in the 4-position by aniline, N-methylaniline and aryloxy entities, targeting EGFR and VEGFR-2 tyrosine kinases, were designed and synthesized. Pharmacological activities of these compounds have been evaluated for their enzymatic inhibition of VEGFR-2 and EGFR and for their antiproliferative activities on various cancer cell lines. We have studied the impact of the variation in the 4-position substitution of the quinazoline core. Substitution by aryloxy groups led to new compounds which are selective inhibitors of VEGFR-2 enzyme with IC(50) values in the nanomolar range in vitro.

Preparative enantiomeric separation of new selective CB2 receptor agonists by liquid chromatography on polysaccharide-based chiral stationary phases: determination of enantiomeric purity and assignment of absolute stereochemistry by X-ray structure analysis.

The development of high-performance liquid chromatography (HPLC) methods using derivatized amylose chiral stationary phases has permitted preparative enantioseparations of substituted 4-oxo-1,4-dihydroquinoline-3-carboxamide derivatives with satisfactory yields. These compounds constitute new potent selective agonists of the cannabinoid CB(2) receptor. Analytical enantioseparation methods using UV detection were validated to determine the enantiomeric purity of these compounds. Linear calibration curves in the range from 0.18 to 0.40 mM were obtained; repeatability, limits of detection (LOD), and quantification (LOQ) were determined: LOD varied, for the various solutes, from 0.5 to 1.2 µM. All the separated compounds were prepared with high enantiomeric purities superior to 99.3% Absolute configuration of the enantiomers was unequivocally established by single crystal X-ray diffraction method and correlated to the chiroptical properties of isolated enantiomers.

Design, synthesis, and DNA-binding of N-alkyl(anilino)quinazoline derivatives.

New N-alkylanilinoquinazoline derivatives 5, 12, 20, and 22 have been prepared from 4-chloro-6,7-dimethoxyquinazoline 3, 4-chloro-6,7-methylenedioxyquinazoline 19, and commercially available anilines. Differents classes of compounds substituted by an aryloxygroup (6a-c, 16a,b, and 17a,b), (aminophenyl)ureas (12a,b and 13a-f), anilines (4a-m, 20a,b), N-alkyl(aniline) (5a-m, 21a,b, 22a,d), and N-aminoalkyl(aniline) (22e-g) have been synthesized. These molecules were evaluated for their cytotoxic activities and as potential DNA intercalating agents. We studied the strength and mode of binding to DNA of these molecules by DNA melting temperature measurements, fluorescence emission, and circular dichroism. The results of various spectral and gel electrophoresis techniques obtained with the different compounds, in particular compounds 5g and 22f, revealed significant DNA interaction. These experiments confirm that the N-aminoalkyl(anilino)-6,7-dimethoxyquinazoline nucleus is an efficient pharmacophore to trigger binding to DNA, via an intercalative binding process.

Design, solid-phase synthesis, and biological evaluation of novel 1,5-diarylpyrrole-3-carboxamides as carbonic anhydrase IX inhibitors.

Following previous studies we herein report the synthesis and the pharmacological evaluation of a new class of human carbonic anhydrase (hCA) inhibitors, 1,5-diarylpyrrole-3-carboxamides prepared by a solid-phase strategy involving a PS(HOBt) resin. A molecular modeling study was conducted in order to simulate the binding mode of this new family of enzyme inhibitors within the active site of hCA IX. This study revealed that the 3-position of the pyrrole was opened to the solvent, so we introduced an amino side-chain, protonated at physiological pH both to enhance the aqueous solubility and to decrease the cell membrane penetration. This strategy consisted of preparing membrane-impermeant inhibitors that may selectively target the tumor-associated hCA IX. Physico-chemical characterizations including aqueous solubility and lipophilic parameters are described. Pharmacological studies revealed high hCA IX inhibitory potency in the nanomolar range. Some compounds are selective for hCA IX displaying hCA I/hCA IX and hCA II/hCA IX ratios higher than 20 and 5, respectively.

Antispasmodic and antioxidant activities of fractions and bioactive constituent davidigenin isolated from Mascarenhasia arborescens.

ETHNOPHARMACOLOGICAL RELEVANCE: Mascarenhasia arborescens A. DC. (Apocynaceae) is used in traditional medicine in the North of Madagascar to treat intestinal disorders, intestinal spasms and diarrhoea. AIM OF THE STUDY: The main objective of this work was to evaluate the antispasmodic activity of the crude methanolic extract of Mascarenhasia arborescens and of its four partitions and to identify the effective compound responsible for this effect. MATERIALS AND METHODS: Isolation and structure elucidation techniques were performed in order to identify the bioactive constituent of Mascarenhasia arborescens and HPLC analysis was used for its quantification. Total phenolic content (TPC) of crude extracts and partitions were determined using the Folin-Ciocalteu method. Crude methanolic extract, partitions and the bioactive compound were investigated for their spasmolytic activity on several isolated organs. Their antiradical activity was also investigated by the DPPH test. RESULTS: Bioassay-guided fractionation using isolated guinea pig ileum pre-contracted with histamine 3x10(-6) M led to the isolation of davidigenin (DG), a dihydrochalcone, as the main active constituent from the most promising methylene chloride partition (McP). This partition was effective on isolated guinea pig ileum pre-contracted with 3x10(-6) M histamine, with a median effective concentration (EC(50)) of 41.19+/-3.74 microg/ml. The DG content of this partition was shown to be 26.5% by HPLC. DG induced a concentration-dependent relaxation of the histamine pre-contracted guinea pig ileum with an EC(50) of 8.04+/-0.81 microg/ml and a concentration-dependent relaxation of the acetylcholine pre-contracted rat duodenum with an EC(50) of 9.35+/-0.30 microg/ml. It inhibited in a non-competitive manner histamine-induced isolated ileum contraction and the acetylcholine-induced isolated duodenum contraction. Moreover, DG does not have any antiradical activity. CONCLUSIONS: We demonstrated for the first time antispasmodic and antioxidant effects of Mascarenhasia arborescens. This study supports its use in traditional medicine. Furthermore, we highlighted the crucial role of davidigenin in the antispasmodic activity of this plant.