Formation of halogenated forms of bisphenol A (BPA) in water: Resolving isomers with ion mobility - mass spectrometry and the role of halogenation position in cellular toxicity.

Halogenated BPA (XBPA) forms resulting from water chlorination can lead to increased toxicity and different biological effects. While previous studies have reported the occurrence of different XBPAs, analytical limitation have hindered the analysis and differentiation of the many potential isomeric forms. Using online solid-phase extraction - liquid chromatography - ion-mobility - high-resolution mass spectrometry (OSPE-LC-IM-HRMS), we demonstrated a rapid analysis method for the analysis of XBPA forms after water chlorination, with a total analysis time of less than 10 min including extraction and concentration and low detection limits (∼5-80 ng/L range). A multi in-vitro bioassay testing approach for the identified products revealed that cytotoxicity and bioenergetics impacts were largely associated with the presence of halogen atoms at positions 2 or 2' and the overall number of halogens incorporated into the BPA molecule. Different XBPA also showed distinct impacts on oxidative stress, peroxisome proliferator-activated receptor gamma - PPARγ, and inflammatory response. While increased DNA damage was observed for chlorinated water samples (4.14 ± 1.21-fold change), the additive effect of the selected 20 XBPA studied could not explain the increased DNA damage observed, indicating that additional species or synergistic effects might be at play.

Characterization of pregnant women exposure to halogenated parabens and bisphenols through water consumption.

Exposure of pregnant women to endocrine disruptor compounds, such as parabens and bisphenol A is of concern for fetal transition. Their halogenated degradation products, mainly coming from water treatment plans, could be problematic as well, depending on their occurrence in drinking water in the first place. Thus, 25 halogenated compounds were synthesised in order to investigate 60 substances (Bisphenols, parabens and their degradation products) in 325 drinking water samples coming from a French cohort study of pregnant women. Analysis was performed by tandem mass spectrometry coupled to gas chromatography (GC-MS/MS) after SPE extraction and derivation of the contaminants. Results indicate that parabens (methylparaben, n-propylparaben, ethylparaben and n-butylparaben), bisphenols S, A and F, and their degradation product, 4-hydroxybenzoic acid, were detected up to several hundred ng/L in drinking water, with detection frequencies between 16% and 88%. Regarding halogenated degradation products, the highest detection frequencies were found for monochlorinated products (about 50% for 2-chlorobisphenol A), which were quantified up to several tens of ng/L. Such analytical approaches with broader spectrum of monitoring (i.e. chemical hazards and their degradation products) constitute in the beginning of a solution to exhaustively answer the questions related to the characterization of the human chemical exposome.

Quantification of the Conjugated Forms of Dichlorobisphenol A (3,3'-Cl 2 BPA) in Rat and Human Plasma Using HPLC-MS/MS.

BACKGROUND: Bisphenol A (BPA) is a ubiquitous contaminant that has endocrine-disrupting effects. Chlorinated derivatives of BPA are formed during chlorination of drinking water and have higher endocrine-disrupting activity. Dichlorobisphenol A (Cl 2 BPA) is the most abundant chlorinated BPA derivative found in several human biological matrices. Recent in vitro experiments have shown that Cl 2 BPA is metabolized in sulpho- and glucuro-conjugated compounds. To date, no assay has been developed to quantify the sulfo- and glucuro-conjugates of 3,3'-Cl 2 BPA (3,3'-Cl 2 BPA-S and 3,3'-Cl 2 BPA-G, respectively). METHODS: A high-performance liquid chromatography-tandem mass spectrometry assay for the determination of 3,3'-Cl 2 BPA conjugated forms in plasma samples was developed and validated according to the European Medicines Agency guidelines. Quantification was performed in the multiple reaction monitoring mode for all target analytes using a SCIEX 6500 + tandem mass spectrometer with an electrospray source operating in the negative ionization mode. Chromatographic separation was achieved using a C18 column maintained at 40°C and a binary mobile phase delivered in the gradient mode at a flow rate of 0.35 mL/min. Sample was prepared via simple precipitation using acetonitrile. The assay was validated and applied to rat and human plasma samples. RESULTS: Linearity was demonstrated over the range of 0.006-25 ng/mL for 3,3'-Cl 2 BPA-G and 0.391-100 ng/mL for 3,3'-Cl 2 BPA-S. Intraday and interday bias values were in the 95%-109% range, and the imprecision <9%. Internal standard corrected matrix effects were also investigated. This method enabled quantification of the conjugated forms of 3,3'-Cl 2 BPA in plasma samples. CONCLUSIONS: This is the first report on the development and validation of an analytical method for the quantification of 3,3'-Cl 2 BPA-G and 3,3'-Cl 2 BPA-S in the plasma matrix. This study is also the first report on the in vivo occurrence of these metabolites.

Discovery of Compounds That Selectively Repress the Amyloidogenic Processing of the Amyloid Precursor Protein: Design, Synthesis and Pharmacological Evaluation of Diphenylpyrazoles.

The rationale to define the biological and molecular parameters derived from structure-activity relationships (SAR) is mandatory for the lead selection of small drug compounds. Several series of small molecules have been synthesized based on a computer-assisted pharmacophore design derived from two series of compounds whose scaffold originates from chloroquine or amodiaquine. All compounds share similar biological activities. In vivo, Alzheimer's disease-related pathological lesions are reduced, consisting of amyloid deposition and neurofibrillary degeneration, which restore and reduce cognitive-associated impairments and neuroinflammation, respectively. Screening election was performed using a cell-based assay to measure the repression of Aß1-x peptide production, the increased stability of APP metabolites, and modulation of the ratio of autophagy markers. These screening parameters enabled us to select compounds as potent non-competitive ß-secretase modulators, associated with various levels of lysosomotropic or autophagy modulatory activities. Structure-activity relationship analyses enabled us to define that (1) selectively reducing the production of Aß1-x, and (2) little Aßx-40/42 modification together with (3) a decreased ratio of p62/(LC3-I/LC3-II) enabled the selection of non-competitive ß-secretase modulators. Increased stability of CTFα and AICD precluded the selection of compounds with lysosomotropic activity whereas cell toxicity was associated with the sole p62 enhanced expression shown to be driven by the loss of nitrogen moieties. These SAR parameters are herein proposed with thresholds that enable the selection of potent anti-Alzheimer drugs for which further investigation is necessary to determine the basic mechanism underlying their mode of action.

Genotoxic effects of chlorinated disinfection by-products of 1,3-diphenylguanidine (DPG): Cell-based in-vitro testing and formation potential during water disinfection.

1,3-diphenylguanidine (DPG) is a commonly used rubber and polymer additive, that has been found to be one of the main leachate products of tire wear particles and from HDPE pipes. Its introduction to aquatic environments and potentially water supplies lead to further questions regarding the effects of disinfection by-products potentially formed. Using different bioassay approaches and NGS RNA-sequencing, we show that some of the chlorinated by-products of DPG exert significant toxicity. DPG and its chlorinated by-products also can alter cell bioenergetic processes, affecting cellular basal respiration rates and ATP production, moreover, DPG and its two chlorination products, 1,3-bis-(4-chlorophenyl)guanidine (CC04) and 1-(4-chlorophenyl)-3-(2,4-dichlorophenyl)guanidine (CC11), have an impact on mitochondrial proton leak, which is an indicator of mitochondria damage. Evidence of genotoxic effects in the form of DNA double strand breaks (DSBs) was suggested by RNA-sequencing results and further validated by an increased expression of genes associated with DNA damage response (DDR), specifically the canonical non-homologous end joining (c-NHEJ) pathway, as determined by qPCR analysis of different pathway specific genes (XRCC6, PRKDC, LIG4 and XRCC4). Immunofluorescence analysis of phosphorylated histone H2AX, another DSB biomarker, also confirmed the potential genotoxic effects observed for the chlorinated products. In addition, chlorination of DPG leads to the formation of different chlorinated products (CC04, CC05 and CC15), with analysed compounds representing up to 42% of formed products, monochloramine is not able to effectively react with DPG. These findings indicate that DPG reaction with free chlorine doses commonly applied during drinking water treatment or in water distribution networks (0.2-0.5 mg/L) can lead to the formation of toxic and genotoxic chlorinated products.

Effect of 6-Benzoyl-benzothiazol-2-one scaffold on the pharmacological profile of α-alkoxyphenylpropionic acid derived PPAR agonists.

A series of nitrogen heterocycles containing α-ethoxyphenylpropionic acid derivatives were designed as dual PPARα/γ agonist ligands for the treatment of type 2 diabetes (T2D) and its complications. 6-Benzoyl-benzothiazol-2-one was the most tolerant of the tested heterocycles in which incorporation of O-methyl oxime ether and trifluoroethoxy group followed by enantiomeric resolution led to the (S)-stereoisomer 44 b displaying the best in vitro pharmacological profile. Compound 44 b acted as a very potent full PPARγ agonist and a weak partial agonist on the PPARα receptor subtype. Compound 44 b showed high efficacy in an ob/ob mice model with significant decreases in serum triglyceride, glucose and insulin levels but mostly with limited body-weight gain and could be considered as a selective PPARγ modulator (SPPARγM).

New piperazine multi-effect drugs prevent neurofibrillary degeneration and amyloid deposition, and preserve memory in animal models of Alzheimer's disease.

Alzheimer's Disease is a devastating dementing disease involving amyloid deposits, neurofibrillary tangles, progressive and irreversible cognitive impairment. Today, only symptomatic drugs are available and therapeutic treatments, possibly acting at a multiscale level, are thus urgently needed. To that purpose, we designed multi-effects compounds by synthesizing drug candidates derived by substituting a novel N,N'-disubstituted piperazine anti-amyloid scaffold and adding acetylcholinesterase inhibition property. Two compounds were synthesized and evaluated. The most promising hybrid molecule reduces both the amyloid pathology and the Tau pathology as well as the memory impairments in a preclinical model of Alzheimer's disease. In vitro also, the compound reduces the phosphorylation of Tau and inhibits the release of Aß peptides while preserving the processing of other metabolites of the amyloid precursor protein. We synthetized and tested the first drug capable of ameliorating both the amyloid and Tau pathology in animal models of AD as well as preventing the major brain lesions and associated memory impairments. This work paves the way for future compound medicines against both Alzheimer's-related brain lesions development and the associated cognitive impairments.

Benzo[d]thiazol-2(3H)-ones as new potent selective CB2 agonists with anti-inflammatory properties.

The high distribution of CB2 receptors in immune cells suggests their important role in the control of inflammation. Growing evidence offers this receptor as an attractive therapeutic target: selective CB2 agonists are able to modulate inflammation without triggering psychotropic effects. In this work, we report a new series of selective CB2 agonists based on a benzo[d]thiazol-2(3H)-one scaffold. This drug design project led to the discovery of compound 9, as a very potent CB2 agonist (Ki = 13.5 nM) with a good selectivity versus CB1. This compound showed no cytotoxicity, acceptable ADME-Tox parameters and demonstrates the ability to counteract colon inflammatory process in vivo.

A phenotypic approach to the discovery of compounds that promote non-amyloidogenic processing of the amyloid precursor protein: Toward a new profile of indirect ß-secretase inhibitors.

Dysregulation of the Amyloid Precursor Protein (APP) processing leading to toxic species of amyloid ß peptides (Aß) is central to Alzheimer's disease (AD) etiology. Aß peptides are produced by sequential cleavage of APP by ß-secretase (BACE-1) and γ-secretase. Lysosomotropic agent, chloroquine (CQ), has been reported to inhibit Aß peptide production. However, this effect is accompanied by an inhibition of lysosome-mediated degradation pathways. Following on from the promising activity of two series of APP metabolism modulators derived from CQ, we sought to develop new series of compounds that would retain the inhibitory effects on Aß production without altering lysosome functions. Herein, we applied a ligand-based pharmacophore modeling approach coupled with de novo design that led to the discovery of a series of biaryl compounds. Structure-activity relationship studies revealed that minor modifications like replacing a piperidine moiety of compound 30 by a cyclohexyl (compound 31) allowed for the identification of compounds with the desired profile. Further studies have demonstrated that compounds 30 and 31 act through an indirect mechanism to inhibit ß-secretase activity. This work shows that it is possible to dissociate the inhibitory effect on Aß peptide secretion of CQ-derived compounds from the lysosome-mediated degradation effect, providing a new profile of indirect ß-secretase inhibitors.

Chlorinated and brominated bisphenol A derivatives: Synthesis, characterization and determination in water samples.

Bisphenol A (BPA) has been used in the plastics industry for several decades. During the treatment of drinking water with chlorine reagent, the formation of chlorinated derivatives of BPA (ClxBPA) but also bromoBPA and bromochloroBPA is to be expected. Some of these compounds are considered to have an estrogenic effect and could induce major risks for human health by targeting different organs and systems in the body. In this paper, we describe the synthesis of chloro- and bromobisphenol A (ClxBPA, BrxBPA, BrxClxBPA)and their analytical characterization. These derivatives could be used as analytical standards in LC-MS/MS or evaluated in in vitro biological tests for their potential as endocrine disruptors. In this study, we evaluated the presence of BPA, ClxBPA in a pilot study from water samples. Range values found for BPA, ClxBPA were respectively 2.8-4169.3 ng/L and 0.8-11.3 ng/L.

New phenylaniline derivatives as modulators of amyloid protein precursor metabolism.

The chloroquinoline scaffold is characteristic of anti-malarial drugs such as chloroquine (CQ) or amodiaquine (AQ). These drugs are also described for their potential effectiveness against prion disease, HCV, EBV, Ebola virus, cancer, Parkinson or Alzheimer diseases. Amyloid precursor protein (APP) metabolism is deregulated in Alzheimer's disease. Indeed, CQ modifies amyloid precursor protein (APP) metabolism by precluding the release of amyloid-beta peptides (Aß), which accumulate in the brain of Alzheimer patients to form the so-called amyloid plaques. We showed that AQ and analogs have similar effects although having a higher cytotoxicity. Herein, two new series of compounds were synthesized by replacing 7-chloroquinolin-4-amine moiety of AQ by 2-aminomethylaniline and 2-aminomethylphenyle moieties. Their structure activity relationship was based on their ability to modulate APP metabolism, Aß release, and their cytotoxicity similarly to CQ. Two compounds 15a, 16a showed interesting and potent effect on the redirection of APP metabolism toward a decrease of Aß peptide release (in the same range compared to AQ), and a 3-10-fold increased stability of APP carboxy terminal fragments (CTFα and AICD) without obvious cellular toxicity at 100 µM.

Validation of a probe for assessing deconjugation of glucuronide and sulfate phase II metabolites assayed through LC-MS/MS in biological matrices.

LC-MS/MS has been proposed in various areas such as Therapeutic Drug Monitoring (TDM), Human Biomonitoring (HBM), disease diagnosis, clinical toxicology and doping control to identify and quantify chemical parents and their metabolites in biological matrices. To determine the total content of a xenobiotic (unconjugated+conjugated forms), an enzymatic hydrolysis step is required. Most studies in the literature have not controlled the effectiveness of the deconjugation process because no method has been described for that purpose. Therefore the aim of this study was to develop and validate a deconjugation probe using a LC-MS/MS method. In order to estimate deconjugation using ß-glucuronidase and/or sulfatase, 4-methyl-umbelliferone (MU) and its conjugates were used as markers. Glucuronidase/sulfatase was added to plasma or urine spiked with 4-methylumbelliferyl-ß-d-glucuronide (MUG) and 4-methylumbelliferyl sulfate (MUS) and umbelliferone, which was used as the internal standard. After incubation at 37°C during 90min, MU appears as a result of the deconjugation of MUG and MUS. The concentrations of the 3 markers were determined using LC-MS/MS. Trueness and precision of the LC-MS/MS method were determined by quality control analysis at three different levels of concentration covering the whole range of calibration. In both matrices, the analytical method allows quantification of the different compounds, with good linearity, trueness and precision and negligible matrix effects. The method was applied with success to deconjugation assay using active glucuronidase/sulfatase in plasma and urine. The probe developed in this study allows to ensure that enzymatic preparation is working properly in the frame of a quality system.

Synthesis and pharmacological evaluation of benzamide derivatives as potent and selective sigma-1 protein ligands.

A series of novel benzamide-derived compounds was designed, synthesized and pharmacologically evaluated. Among all 37 synthesized compounds, two series were developed with the modulation of the nature, the position of atoms or groups on the benzamide scaffold, but also the nature of the amine group separated from the benzamide with 2, 3 or 4 methylene groups. In vitro competition binding assays against sigma proteins (sigma-1 S1R and sigma-2 S2R) revealed that most of them conferred S2R/S1R selectivity toward without cytotoxic effects on SY5Y cells, especially with the first series with compounds 7a-z. Some selected compounds were also evaluated for their agonist and antagonist activities on a panel of 40 receptors. Results showed the importance of the nature and the position with halogeno atom on the benzamide scaffold, the length chain but also the contribution of the hydrophobic part on the amine group. Among them, compounds 7i, w, y with Cl, CN or NO2 groups at the 4-position of the benzamide scaffold showed excellent affinity for S1R (Ki = 1.2-3.6 nM), selectivity for S2R (Ki up to 1400 nM) and high selectivity index (IC50(SY5Y)/Ki(S1R) ratio from 28 000 to 83 000). Futhermore, these compounds presented an excellent safety profile over 40 other receptors. These derivatives will be selected for further biological investigations.

6-Sulfonylbenzothiazolones as potential scaffolds for the design of 5-HT6 ligands.

5-HT6 Receptors are relatively recently discovered receptors that interact with cholinergic, glutamatergic, GABAergic and dopaminergic transmission systems. These receptors have been implicated in the CNS system as therapeutic targets in applications such as psychosis, reduction of body weight or Alzheimer's disease. As part of our efforts to develop 5-HT6 antagonists, we explored the benzothiazolone scaffold substituted in position 3 or 6 respectively with ethylamino chains and an aromatic ring connected through a sulfonyl linker. Final compounds were evaluated in radioligand binding assays for their ability to interact with 5-HT6 receptors. Their potential cytotoxic effects were determined on the human neuroblastoma cell line SY5Y. They showed very low cytotoxicity, and one of them has submicromolar affinity for 5-HT6 receptors.

Synthesis and pharmacological evaluation of benzannulated derivatives as potent and selective sigma-1 protein ligands.

The σ1 proteins are considered to be a new class of target structures for several central nervous system disorders, including depression, anxiety, psychosis, and Parkinson's and Alzheimer's diseases. Recently, the involvement of these receptors in neuropathic pain and cancer has also been observed. So far, only a few ligands are in clinical trials. In a continuation of our previous studies on the development of σ1 ligands, a new series of benzannulated heterocycles was designed and synthesised. In vitro competition binding assays showed that many of them possessed high σ1 receptor affinity (Ki = 0.6-10.3 nM), and good σ2/σ1 subtype selectivity, without cytotoxic effects on SY5Y cells (human neuroblastoma cell line).

Carboline- and phenothiazine-derivated heterocycles as potent SIGMA-1 protein ligands.

Sigma 1 receptors are associated with neurodegenerative and psychiatric disorders. These receptors, via their chaperoning functions that counteract endoplasmic reticulum stress and block neurodegeneration, may serve as a target for a new generation of antidepressants or neuroprotective agents. The involvement of these receptors has also been observed in neuropathic pain and cancer. Only a few ligands, such as Igmesine and Anavex 2-73, have been involved in clinical trials. Thus the development of sigma 1 ligands is of interest to a new generation of drugs. Previous work in our lab underlined the potency of benzannulated bicyclic compounds as interesting ligands. Herein the work was extended to a series of novel tricyclic compounds. Carboline- and phenothiazine-derivated compounds were designed and synthesized. In vitro competition binding assays for sigma 1 and 2 receptors showed that most of them have high affinity for sigma 1 receptor (Ki = 2.5-18 nM), and selectivity toward sigma 2 receptor, without cytotoxic effects on SY5Y cells.

Antioxydant activity of ß-carboline derivatives in the LDL oxidation model.

A series of ß-carboline compounds were synthesized, starting from compound GWC22, their antioxidant activity was determined by inhibition of lipid peroxidation. The oxidation of LDL was induced in the presence of CuSO4 or 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). The protective actions of these compounds against the cytotoxicity were evaluated with lactate dehydrogenase (LDH) activity in bovine aortic endothelial cells (BAECs) and cellular vitality by measuring mitochondrial activity in the presence of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Most of compounds showed an higher antioxidant activity than GWC22 derivative (R=1.6 for 5 µM CuSO4). The best antioxidant activities are phenolic and benzyloxy derivatives with ratio R=1.9 to 2.8 for 1 µM CuSO4. These substances have protective actions and increase significantly the cell viability.

Synthesis, biological evaluation and docking studies of 4-amino-tetrahydroquinazolino[3,2-e]purine derivatives.

The synthesis of new 4-amino-tetrahydroquinazolino[3,2-e]purine derivatives along with their activity in cell-free enzymatic assays on Src is reported. Some compounds emerged as moderately active inhibitors of the enzyme and showed antiproliferative effects on the murine leukemia L1210 cell line. Docking studies have been also performed to analyze the binding mode of compounds under study and to identify the structural determinants of their interaction. Therefore, this study provides a new promising scaffold with moderate enzymatic inhibitory activities for further development of new anticancer drugs targeting Src tyrosine kinase.

Antioxidant activity of benzoxazolinonic and benzothiazolinonic derivatives in the LDL oxidation model.

A series of benzazolone compounds were synthesized utilizing benzoxazolinonic and benzothiazolinonic heterocycles as the building unit. The antioxidant activity of these compounds was determined by inhibition of lipid peroxidation. The oxidation of LDL was induced in the presence of CuSO(4) or 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). The protective action of these compounds against the cytotoxicity was evaluated with lactate dehydrogenase (LDH) activity in bovine aortic endothelial cells (BAECs) and cellular vitality by measuring mitochondrial activity in the presence of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The best antioxidant activities were observed for phenolic compounds 13 and 14b with ratio R=2.5, 3.2 (5microM). Both of these test substances increased the cell viability significantly as indicated by MTT assay and LDH release assay.

Benzopyridooxathiazepine derivatives as novel potent antimitotic agents.

Herein, we describe the structure-activity relationship study of a new 1-(arylalkyl)-11H-benzo[f]-1,2-dihydropyrido[3,2,c][1,2,5]oxathiazepine 5,5-dioxide series of antimitotic agents. The pharmacological results obtained from previous works allowed us to identify compound 1 as a new cytotoxic agent inhibiting tubulin polymerization. We have undertaken the synthesis of its non-methylated analogue 7 and have extended our investigations to a novel, structurally related benzopyridooxathiazepine dioxide series. Among all analogues synthesized in this study, compound 10b was the most promising, being 12-fold more potent than compound 1. Its activity over a panel of five tumoral cell lines was in the nanomolar range for all of the histological types tested and flow cytometric studies performed on L1210 cells showed an accumulation of the cells in the G2/M phases of the cell cycle with a significant percentage of tetraploid cells (8N DNA content). This interesting pharmacological profile, resulting from inhibition of tubulin polymerization, encouraged us to perform preliminary in vivo studies.