Investigation of the Aquatic Photolytic and Photocatalytic Degradation of Citalopram

This study investigated the direct and indirect photochemical degradation of citalopram (CIT), a selective serotonin reuptake inhibitor (SSRI), under natural and artificial solar radiation. Experiments were conducted in a variety of different operating conditions including Milli-Q (MQ) water and natural waters (lake water and municipal WWT effluent), as well as in the presence of natural water constituents (organic matter, nitrate and bicarbonate). Results showed that indirect photolysis can be an important degradation process in the aquatic environment since citalopram photo-transformation in the natural waters was accelerated in comparison to MQ water both under natural and simulated solar irradiation. In addition, to investigate the decontamination of water from citalopram, TiO₂-mediated photocatalytic degradation was carried out and the attention was given to mineralization and toxicity evaluation together with the identification of by-products. The photocatalytic process gave rise to the formation of transformation products, and 11 of them were identified by HPLC-HRMS, whereas the complete mineralization was almost achieved after 5 h of irradiation. The assessment of toxicity of the treated solutions was performed by Microtox bioassay (Vibrio fischeri) and in silico tests showing that citalopram photo-transformation involved the formation of harmful compounds.     

Enantiomeric separation of citalopram analogues by HPLC using macrocyclic glycopeptide and cyclodextrin based chiral stationary phases

The enantiomeric separation of a novel series of twenty-eight racemic mixtures of citalopram analogues was performed by high performance liquid chromatography (HPLC). Due to the effectiveness of citalopram as an antidepressant drug, the development of new compounds based on its chemical structure is interesting, and their enantiomeric separation is needed to allow further pharmacokinetic studies. Several bonded cyclodextrin (both native and derivatized) and macrocyclic glycopeptide based chiral stationary phases (CSPs) were evaluated for their ability to separate this set of compounds via HPLC. Polar ionic, polar organic, and reversed phase modes were tested. Twenty-five of the racemic mixtures were separated with resolutions and enantiomeric selectivities up to 2.9 and 1.33, respectively. A total of eighteen baseline separations were achieved, while seven compounds were partially separated. Vancomycin based columns operated in the polar ionic mode resulted in the greatest number of separations. Lastly, the chromatographic behaviors of similar compounds were compared based on their chemical structure and also on the chiral selectors used.     

Improved Syntheses of N‐Desmethylcitalopram and N,N‐Didesmethylcitalopram

An improved and efficient synthesis of N‐desmethylcitalopram (2) and N,N‐didesmethylcitalopram (3) is presented. The method involved N‐demethylation of citalopram (1) using 1‐chloroethyl chloroformate to give 2 in 87% yield. Synthesis of 3 was accomplished by alkylation of 8 with 1‐(3‐bromopropyl)‐2,2,5,5‐tetramethyl‐1‐aza‐2,5‐disilacyclopentane (9).     

有机相酶催化转酯化反应拆分西酞普兰中间体

利用脂肪酶催化的不对称转酯反应成功远程拆分具有季碳手性中心的西酞普兰中间体4-[4-(二甲基氨基)-1-(4’-氟苯基)-1-羟基丁基]-3-(羟基甲基)苄腈(1). 以乙酸乙烯酯作为酰基供体, 通过筛选酶和溶剂, 确定最佳脂肪酶及溶剂分别为Candida antarctica lipase B (Novozym 435)和乙腈; 并在该反应体系中考察了反应温度、二醇1与酰基供体的比例、酶浓度和摇床转速等对反应的影响, 确定优化的反应条件为: 温度30 ℃, 二醇1与酰基供体的物质的量比为1∶5, 酶浓度为10 mg/mL, 摇床转速200 r/min. 分别考察二醇1浓度为60和180 mmol/L的反应情况, 均具有较高的选择性和反应速度. 实验结果表明酶能够多次重复利用.     

Nanopharmacological Force Sensing to Reveal Allosteric Coupling in Transporter Binding Sites

Controversy regarding the number and function of ligand binding sites in neurotransmitter/sodium symporters arose from conflicting data in crystal structures and molecular pharmacology. Here, we have designed novel tools for atomic force microscopy that directly measure the interaction forces between the serotonin transporter (SERT) and the S‐ and R‐enantiomers of citalopram on the single molecule level. This approach is based on force spectroscopy, which allows for the extraction of dynamic information under physiological conditions thus inaccessible via X‐ray crystallography. Two distinct populations of characteristic binding strengths of citalopram to SERT were revealed in Na⁺‐containing buffer. In contrast, in Li⁺‐containing buffer, SERT showed only low force interactions. Conversely, the vestibular mutant SERT‐G402H merely displayed the high force population. These observations provide physical evidence for the existence of two binding sites in SERT when accessed in a physiological context. Competition experiments revealed that these two sites are allosterically coupled and exert reciprocal modulation.     

Static and Hydrodynamic Monitoring of Citalopram Based on its Electro-oxidation Behavior at a Glassy-Carbon Surface

Abstract

The electrooxidative behavior of citalopram (CTL) in aqueous media was studied by cyclic voltammetry (CV) and square-wave voltammetry (SWV) at a glassy-carbon electrode. The electrochemical behaviour of CTL involves two electrons and two protons in the irreversible and diffusion controlled oxidation of the tertiary amine group. The maximum analytical signal was obtained in a phosphate buffer (pH = 8.2). For analytical purposes, an SWV method and a flow-injection analysis (FIA) system with amperometric detection were developed. The optimised SWV method showed a linear range between 1.10 × 10^?5–1.20 × 10^?4 mol L^?1, with a limit of detection (LOD) of 9.5 × 10^?6 mol L^?1. Using the FIA method, a linear range between 2.00 × 10^?6–9.00 × 10^?5 mol L^?1 and an LOD of 1.9 × 10^?6 mol L^?1 were obtained. The validation of both methods revealed good performance characteristics confirming applicability for the quantification of CTL in several pharmaceutical products.     

Simultaneous Determination of Escitalopram Impurities including the R-enantiomer on a Cellulose tris(3,5-Dimethylphenylcarbamate)-Based Chiral Column in Reversed-Phase Mode

A high-performance liquid chromatographic method was developed for the simultaneous determination of the related substances—three potential synthesis-related chemical impurities and the distomer—of escitalopram. The separation capacity of seven different polysaccharide-type chiral columns, including three amylose-based (Lux Amylose-1, Lux i-Amylose-1, Lux Amylose-2) and four cellulose-based columns (Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-3, and Lux Cellulose-4) were screened in the polar organic and reversed-phase modes. Lux Cellulose-1, based on cellulose tris(3,5-dimethylphenylcarbamate) as the chiral selector with an acetonitrile-water mixture containing 0.1% diethylamine was identified as the most promising separation system. Using the “one factor at a time” optimization approach, the effect of column temperature, flow rate, and mobile phase constituents on separation performance was evaluated, and the critical resolution values were determined. A U-shaped retention pattern was obtained when plotting the retention factors of the citalopram enantiomers versus the water content of the binary mobile phases on the Lux Cellulose-1 column. A thermodynamic analysis revealed enthalpy-driven enantioseparation in both the polar organic and reversed-phase modes. For further method optimizations, an L9 orthogonal array table was employed. Using the optimized parameters (Lux Cellulose-1 column with 0.1% (v/v) diethylamine in water/acetonitrile 55/45 (v/v); 0.8 mL/min flow rate at 25 °C), baseline separations were achieved between all compounds. Our newly developed HPLC method was validated according to the ICH guidelines and its application was tested with a commercially available pharmaceutical formulation. The method proved to be suitable for routine quality control of related substances and the enantiomeric purity of escitalopram.     

Allosteric Binding of MDMA to the Human Serotonin Transporter (hSERT) via Ensemble Binding Space Analysis with ΔG Calculations,Induced Fit Docking and Monte Carlo Simulations

Despite the recent promising results of MDMA (3,4-methylenedioxy-methamphetamine) as a psychotherapeutic agent and its history of misuse, little is known about its molecular mode of action. MDMA enhances monoaminergic neurotransmission in the brain and its valuable psychoactive effects are associated to a dual action on the 5-HT transporter (SERT). This drug inhibits the reuptake of 5-HT (serotonin) and reverses its flow, acting as a substrate for the SERT, which possesses a central binding site (S1) for antidepressants as well as an allosteric (S2) one. Previously, we characterized the spatial binding requirements for MDMA at S1. Here, we propose a structure-based mechanistic model of MDMA occupation and translocation across both binding sites, applying ensemble binding space analyses, electrostatic complementarity, and Monte Carlo energy perturbation theory. Computed results were correlated with experimental data (r = 0.93 and 0.86 for S1 and S2, respectively). Simulations on all hSERT available structures with Gibbs free energy estimations (ΔG) revealed a favourable and pervasive dual binding mode for MDMA at S2, i.e., adopting either a 5-HT or an escitalopram-like orientation. Intermediate ligand conformations were identified within the allosteric site and between the two sites, outlining an internalization pathway for MDMA. Among the strongest and more frequent interactions were salt bridges with Glu494 and Asp328, a H-bond with Thr497, a π-π with Phe556, and a cation-π with Arg104. Similitudes and differences with the allosteric binding of 5-HT and antidepressants suggest that MDMA may have a distinctive chemotype. Thus, our models may provide a framework for future virtual screening studies and pharmaceutical design and to develop hSERT allosteric compounds with a unique psychoactive MDMA-like profile.     

Enantiomeric determination, validation and robustness studies of racemic citalopram in pharmaceutical formulations by capillary electrophoresis

A chiral capillary electrophoresis (CE) method has been developed allowing the enantiomeric separation of racemic citalopram (R-(-) and S-(+) citalopram) using as chiral selector carboxymethyl-gamma-cyclodrextrin (CM-gamma-CD). The influence of chemical and instrumental parameters on the separation such as cyclodextrin (CD) and buffer concentrations, buffer pH, voltage, injection pressure, ..., was investigated. Good chiral separation of the racemic mixture was achieved in less than 4 min using a fused-silica capillary and as background electrolyte (BGE) a phosphate buffer solution (20 mM, pH 7) containing 0.15% (w/v) of CM-gamma-CD as chiral selector. The separation was driven in normal polarity mode at 15 degrees C, 30 kV and hydrodynamic injection. In order to validate the method, the stability of the solutions, precision (repeatability, reproducibility and F-Snedecor test), linearity (Lack of Fit and ANOVA tests) accuracy (98-101%), detection and quantitation limits (0.06 and 0.2 mg L(-1), respectively), on a selected analytical placebo, were examined. Besides, a robustness test was performed using the Plackett-Burman fractional factorial experimental design using a matrix of 15 experiments for seven factors (internal parameters) with a statistical treatment suggested by Youden and Steinner. The proposed method is fast, sensitive, inexpensive and, besides, it has been evaluated by means of an extensive validation study and an exhaustive robustness test. The scope of this validated and robust method has been proved in the analysis of four pharmaceutical formulations; two of them (recently available in Spain), which just contained S-(+)-citalopram (escitalopram) as active principle. Recoveries between 101 and 103%, with regard to their nominal contents were obtained. In the other two pharmaceutical ones, the method provided the separation and quantification of both chiral isomers in the existing racemic mixture.     

Quantitative ROESY analysis of computational models: structural studies of citalopram and β‐cyclodextrin complexes by 1H‐NMR and computational methods

Complexation of racemic citalopram with β‐cyclodextrin (β‐CD) in aqueous medium was investigated to determine atom‐accurate structure of the inclusion complexes. ¹H‐NMR chemical shift change data of β‐CD cavity protons in the presence of citalopram confirmed the formation of 1 : 1 inclusion complexes. ROESY spectrum confirmed the presence of aromatic ring in the β‐CD cavity but whether one of the two or both rings was not clear. Molecular mechanics and molecular dynamic calculations showed the entry of fluoro‐ring from wider side of β‐CD cavity as the most favored mode of inclusion. Minimum energy computational models were analyzed for their accuracy in atomic coordinates by comparison of calculated and experimental intermolecular ROESY peak intensities, which were not found in agreement. Several least energy computational models were refined and analyzed till calculated and experimental intensities were compatible. The results demonstrate that computational models of CD complexes need to be analyzed for atom‐accuracy and quantitative ROESY analysis is a promising method. Moreover, the study also validates that the quantitative use of ROESY is feasible even with longer mixing times if peak intensity ratios instead of absolute intensities are used. Copyright © 2015 John Wiley & Sons, Ltd.