Designing chemical systems for precision deuteration of medicinal building blocks.

Methods are lacking that can prepare deuterium-enriched building blocks, in the full range of deuterium substitution patterns at the isotopic purity levels demanded by pharmaceutical use. To that end, this work explores the regio- and stereoselective deuteration of tetrahydropyridine (THP), which is an attractive target for study due to the wide prevalence of piperidines in drugs. A series of d0-d8 tetrahydropyridine isotopomers were synthesized by the stepwise treatment of a tungsten-complexed pyridinium salt with H-/D- and H+/D+. The resulting decomplexed THP isotopomers and isotopologues were analyzed via molecular rotational resonance (MRR) spectroscopy, a highly sensitive technique that distinguishes isotopomers and isotopologues by their unique moments of inertia. In order to demonstrate the medicinal relevance of this approach, eight unique deuterated isotopologues of erythro-methylphenidate were also prepared.

Identification of three unexpected new psychoactive substances at an Australian drug checking service.

Drug checking is a harm reduction measure that provides people with the opportunity to confirm the identity and purity of substances before consumption. The CanTEST Health and Drug Checking Service is Australia's first fixed-site drug checking service, where clients can learn about the contents of the samples they provide while receiving tailored harm reduction and health advice. Three samples were recently presented to the service with the expectation of 4-fluoromethylphenidate (4F-MPH) 1, methoxetamine (MXE) 2 and 3-methylmethcathinone (3-MMC) 3. The identity of all three samples did not meet these expectations and remained unknown on-site, as no high confidence identifications were obtained. However, further analysis by nuclear magnetic resonance spectroscopy, high resolution gas chromatography-electron ionisation-mass spectrometry and liquid chromatography-electrospray ionisation-mass spectrometry at the nearby Australian National University allowed for the structure elucidation of the three samples as 4-fluoro-α-pyrrolidinoisohexanophenone (4F-α-PiHP) 4, 1-(4-fluorobenzyl)-4-methylpiperazine (4F-MBZP) 5 and N-propyl-1,2-diphenylethylamine (propylphenidine) 6, respectively. Given all three samples were not of the expected identity and have not yet been described as new psychoactive substances in the literature, this study presents a full characterisation of each compound. As exemplified by this rapid identification of three unexpected new psychoactive substances, drug checking can be used as an effective method to monitor the unregulated drug market.

Development and validation of a LC-PDA method for methylphenidate analysis in sewage.

Methylphenidate (MPH) is an important emerging pollutant found in effluents and wastewater. Thus, we aimed to develop and validate a method for detection and quantitation of MPH residues in sewage through high performance liquid chromatography coupled with photodiode array detector (LC-PDA). Here we describe a selective, accurate, precise, and valid method for determination of MPH in sewage with a total running time of 10 min, with limits of detection and quantification of 0.27 and 0.92 µg/mL, respectively. MPH retention peak was observed at 5 min. The method was applied to MPH analysis in a sewage sample pretreated with solid phase extraction, obtaining a result of 2.8 µg/L of MPH. Thus, the developed method can be considered feasible to be applied to MPH residual contamination analysis in sewage using a widely available apparatus.

Design and Synthesis of Fluorescent Methylphenidate Analogues for a FRET-Based Assay of Synapsin III Binding.

We previously described synapsin III (Syn III) as a synaptic phosphoprotein that controls dopamine release in cooperation with α-synuclein (aSyn). Moreover, we found that in Parkinson's disease (PD), Syn III also participates in aSyn aggregation and toxicity. Our recent observations point to threo-methylphenidate (MPH), a monoamine re-uptake inhibitor that efficiently counteracts the freezing-gait characteristic of advanced PD, as a ligand for Syn III. We have designed and synthesised two different fluorescently labelled MPH derivatives, one with Rhodamine Red (RHOD) and one with 5-carboxytetramethylrhodamine (TAMRA), to be used for assessing MPH binding to Syn III by FRET. TAMRA-MPH exhibited the ideal characteristics to be used as a FRET acceptor, as it was able to enter into the SK-N-SH cells and could interact specifically with human green fluorescent protein (GFP)-tagged Syn III but not with GFP alone. Moreover, the uptake of TAMRA-MPH and co-localization with Syn III was also observed in primary mesencephalic neurons. These findings support that MPH is a Syn III ligand and that TAMRA-conjugated drug molecules might be valuable tools to study drug-ligand interactions by FRET or to detect Syn III in cytological and histological samples.

Structural elucidation of phenidate analogues via the ESI-MS/MS spectra of their sodium adduct ions.

The identification of phenidate new psychoactive substances (NPS) by implementing MS (Mass spectrometry) techniques is a challenging task. Phenidate analogues present information-poor mass spectra, both in GC-EI-MS and LC-ESI-MS/MS of the protonated molecules [M+H]+, with a high abundance fragment/product ion representing the secondary amine-containing residue. This lack of EI-MS and ESI-MS/MS information is attributed to the strong tendency of the amine residue to stabilize the positive charge and leads to unavoidable ambiguity in the identification process. Moreover, thermal decomposition of these compounds occurs in the injection port and/or on the column under standard GC conditions. Herein, we demonstrate how structural information can be attained instantaneously through the LC-ESI-MS/MS fragmentation of the accompanied sodium adducts [M+Na]+. The sodium cation alters the charge distribution during ESI-MS/MS fragmentation, generating a major product ion corresponding to the Na+ adduction of the carbonyl group, providing new structural information of the main core of phenidate derivatives (alkylaryl acetate/acetic acid), enabling their reliable structural elucidation. This quick, simple and easy technique can be implemented to confirm the identity or identify various structurally related phenidate analogues in forensic toxicology and doping analysis without the need for sample handling.

Synthetic Cathinone Analogues Structurally Related to the Central Stimulant Methylphenidate as Dopamine Reuptake Inhibitors.

Synthetic cathinones are, primarily, stimulant drugs of abuse that act at monoamine transporters (e.g., the dopamine transporter or DAT) as releasing agents or as reuptake inhibitors. In the past few years, the emergence of >150 new synthetic cathinones has attracted considerable attention from medical and law enforcement communities. threo-Methylphenidate (tMP), used clinically for the treatment of ADHD and narcolepsy, is also a DAT reuptake inhibitor. tMP is somewhat structurally similar to abused cathinone stimulants, and the structure-activity relationships (SAR) of tMP have been well-defined. Hence, available tMP literature might assist in understanding the SAR of synthetic cathinones, about which less is known. In the present study, we synthesized and examined eight 2-benzoylpiperidine analogues (4, 6-12) to determine if tMP SAR might be applicable to cathinone SAR. The benzoylpiperidine analogues were evaluated in a competition assay using live-cell imaging against APP+ in HEK293 cells stably expressing hDAT and in cells coexpressing DAT and voltage-gated Ca2+ channels. All compounds were found to be DAT reuptake inhibitors, and a significant correlation was obtained between the potency of the benzoylpiperidines and tMP binding data (r = 0.91), suggesting that the SAR of tMP analogues might be directly applicable to certain synthetic cathinones as DAT reuptake inhibitors.

Novel and rapid LC-MS/MS method for quantitative analysis of methylphenidate in dried blood spots.

AIM: Development and validation of a novel, sensitive, specific and rapid dried blood spots (DBS)-LC-MS/MS method for methylphenidate (MPH), an attention-deficit hyperactivity disorder drug. Methodology & results: Protein precipitation with acetonitrile was used to extract MPH from the DBS cards. Chromatographic separation was achieved on a Zorbax C18 column using an isocratic mobile phase composed of acetonitrile and 5 mM ammonium formate buffer (20:80, v/v) at a flow rate of 0.5 ml/min. MPH was quantified over a linear range of 200-25,000 pg/ml. CONCLUSION: The clinical DBS-LC-MS/MS method was successfully validated as per the US FDA's Bioanalytical Method Validation Guidance to support an ongoing pediatric pharmacokinetic study.

Rapid quantitative analysis of methylphenidate and ritalinic acid in oral fluid by liquid chromatography triple quadrupole mass spectrometry (LC-QqQ-MS).

Methylphenidate (MPH), which is metabolized into ritalinic acid (RA), is an amphetamine derivative largely used in the treatment of attention-deficit hyperactivity disorder, a neurological condition commonly diagnosed in early childhood. Ensuring that patients comply with clinical treatment is crucial and compliance is generally monitored in blood or urine specimens which, especially in the case of children, can be challenging to obtain on a repetitive basis. Here we report validation of a specific, non-invasive, and rapid dilute-and-shoot analytical method for the detection and quantitation of MPH and RA in oral fluid (OF). The method is based on liquid chromatography coupled to triple quadrupole MS with electrospray ionization utilizing dynamic MRM mode. Subject OF specimens were collected using a Quantisal™ device, processed, and diluted for analysis with seven-point quadratic calibration curves (weighting of 1/x) using MPH-d9 and (±)-threo-RA-d10 as internal standards. QC samples and diluted specimens showed intra- and inter-day bias and imprecision values no greater than ±12%. The LOD and LOQ for MPH were 0.1 and 0.5 ng/mL, respectively, and 0.2 ng/mL and 0.5 ng/mL for RA, respectively, indicating the validity of the method for identification and confirmation at low concentrations. Selectivity was specific for the analytes of interest and matrix effects were minimized through the use of internal standard based quantitation.

Pharmacological profile of methylphenidate-based designer drugs.

BACKGROUND: Methylphenidate-based designer drugs are new psychoactive substances (NPS) that are used outside medical settings and their pharmacology is largely unexplored. The aim of the present study was to characterize the pharmacology of methylphenidate-based substances in vitro. METHODS: We determined the potencies of the methylphenidate-based NPS N-benzylethylphenidate, 3,4-dichloroethylphenidate, 3,4-dichloromethylphenidate, ethylnaphthidate, ethylphenidate, 4-fluoromethylphenidate, isopropylphenidate, 4-methylmethylphenidate, methylmorphenate, and propylphenidate and the potencies of the related compounds cocaine and modafinil with respect to norepinephrine, dopamine, and serotonin transporter inhibition in transporter-transfected human embryonic kidney 293 cells. We also investigated monoamine efflux and monoamine receptor and transporter binding affinities. Furthermore, we assessed the cell integrity under assay conditions. RESULTS: All methylphenidate-based substances inhibited the norepinephrine and dopamine transporters 4 to >1000-fold more potently than the serotonin transporter. Similar to methylphenidate and cocaine, methylphenidate-based NPS did not elicit transporter-mediated efflux of monoamines. Besides binding to monoamine transporters, several test drugs had affinity for adrenergic, serotonergic, and rat trace amine-associated receptors but not for dopaminergic or mouse trace amine-associated receptors. No cytotoxicity was observed after drug treatment at assay concentrations. CONCLUSION: Methylphenidate-based substances had pharmacological profiles similar to methylphenidate and cocaine. The predominant actions on dopamine transporters vs. serotonin transporters may be relevant when considering abuse liability. This article is part of the Special Issue entitled 'Designer Drugs and Legal Highs.'

The impact of methylphenidate and its enantiomers on dopamine synthesis and metabolism in vitro.

Methylphenidate (MPH), a psychostimulant, is an effective first-line treatment for the symptoms associated with Attention-Deficit/Hyperactivity Disorder (ADHD). Although most MPH formulations are composed of the racemic 1:1 mixture of the two enantiomers (d- and l-threo), converging lines of evidence indicate that d-threo MPH seems to be superior to the l-isomer. We aimed to investigate whether MPH racemic mixture or pure enantiomers influence the enzyme activity of tyrosine hydroxylase (TH), monoamine oxidase B (MAO-B), catechol-O-methyltransferase (COMT), and aldehyde dehydrogenase (ALDH) in vitro in homogenates of rat PC12 cells incubated with racemic, d- and l-threo MPH (1nM up to 100µM), or a vehicle for control. We could observe dose dependent enhancement of TH activity with d-threo MPH, probably due to its higher affinity to the enzyme, which we could confirm for d-threo versus l-threo MPH via docking and molecular dynamic simulations analysis. MAO-B enzyme activity was found to be enhanced when incubated with both d- and l-isomers but not with the racemic mixture. This conflicting result was hypothesized to be due to possible aggregation of the two enantiomers or other molecular conformations. Such a possible interaction was observed indirectly, when TH was incubated with constant d-threo MPH while increasing l-isomer (increasing total MPH concentrations). Hence, TH activity was slightly decreased with increased l-isomer. In conclusion, the current in vitro investigation points to the stereoselectivity of the investigated enzymes and pharmacological effects of MPH enantiomers.

Ultraviolet/ultrasound-activated persulfate for degradation of drug by zinc selenide quantum dots: Catalysis and microbiology study.

In this study, wet chemical method used for ZnSe quantum dots (QDs) and characterized by, UV-vis, photoluminescence spectroscopy, X-ray diffraction and transmission electron microscopy. The crystallites size of ZnSe QDs was 4.0nm. The average diameters of ZnSe QDs were 3.0-5.3nm. Ritalin was degraded using the UV/ZnSe QDs/persulfate process. The several parameters investigated for the influence of Rtialin degradation were the temperature, the persulfate concentration, and the initial Ritalin concentration. The values of optimum parameters ware room temperature, concentration persulfate 5mmol/L and initial Ritalin concentration 0.09mmol/L. Comparative analyses showed the maximum degradation of Ritalin was found for ZnSe/persulfate under ultra-visible and ultra-sonic irradiation process. Comparative analysis showed the maximum degradation of Ritalin was found for ZnSe/persulfate under ultra-visible and ultra-sonic irradiation process. The values of first-order rate constants from degradation of Ritalin at 25°C were 0.96×10-2, 1.09×10-2, 1.59×10-2 and 2.19×10-2 for US/PS, UV/PS, ZnSe/US/PS and ZnSe/UV/PS system, respectively. The antibacterial activity evaluation against two bacterials, including Gram-positive bacteria Staphylococcus aureus (ATCC 43300), Bacillus megaterium (ATCC 14581) and Gram-negative bacteria Pseudomonas aeruginosa (ATCC 27853), Micrococcus luteus (ATCC 4698) was considered. It was found that the MIC values for the antibacterial assay in the presence of ZnSe QDs were around 0.30mM with 64.0, 66.0, 79.2, and 83.5% inhibition for the S. aureus, B. megaterium, P. aeruginosa and M. luteus bacterial strains, respectively. Then, results show that the ZnSe QDs have antibacterial activity.

Rationalising polymer selection for supersaturated film forming systems produced by an aerosol spray for the transdermal delivery of methylphenidate.

Film forming systems offer a number of advantages for topical and transdermal drug delivery, in particular enabling production of a supersaturated state which can greatly improve drug absorption and bioavailability. However the suitability of individual film forming polymers to stabilise the supersaturated state and optimise delivery of drugs is not well understood. This study reports the use of differential scanning calorimetry (DSC) to measure the solubility of methylphenidate both as the free base and as the hydrochloride salt in two polymethacrylate copolymers, Eudragit RS (EuRS) and Eudragit E (EuE) and relates this to the ability of films formed using these polymers to deliver methylphenidate across a model membrane. EuRS provided greater methylphenidate delivery when the drug was formulated as the free base in comparison EuE because the lower solubility of the drug in EuRS provided a higher degree of drug saturation in the polymeric film. In contrast EuE provided greater delivery of methylphenidate hydrochloride as EuRS could not prevent its crystallisation from a supersaturated state. Methylphenidate flux across the membrane could be directly related to degree of saturation of the drug in the film formulation as estimated by the drug solubility in the individual polymers demonstrating the importance of drug solubility in the polymer included in film forming systems for topical/transdermal drug delivery. In addition DSC has been demonstrated to be a useful tool for determining the solubility of drugs in polymers used in film forming systems and the approaches outlined here are likely to be useful for predicting the suitability of polymers for particular drugs in film forming transdermal drug delivery systems.

Analytical characterization and pharmacological evaluation of the new psychoactive substance 4-fluoromethylphenidate (4F-MPH) and differentiation between the (±)-threo and (±)-erythro diastereomers.

Misuse of (±)-threo-methylphenidate (methyl-2-phenyl-2-(piperidin-2-yl)acetate; Ritalin®; MPH) has long been acknowledged, but the appearance of MPH analogs in the form of 'research chemicals' has only emerged in more recent years. 4-Fluoromethylphenidate (4F-MPH) is one of these recent examples. This study presents the identification and analytical characterization of two powdered 4F-MPH products that were obtained from an online vendor in 2015. Interestingly, the products appeared to have originated from two distinct batches given that one product consisted of (±)-threo-4F-MPH isomers whereas the second sample consisted of a mixture of (±)-threo and (±)-erythro 4F-MPH. Monoamine transporter studies using rat brain synaptosomes revealed that the biological activity of the 4F-MPH mixture resided with the (±)-threo and not the (±)-erythro isomers based on higher potencies determined for blockage of dopamine uptake (IC50 4F-MPHmixture  = 66 nM vs. IC50 (±)-threo = 61 nM vs. IC50 (±)-erythro = 8,528 nM) and norepinephrine uptake (IC50 4F-MPHmixture  = 45 nM vs. (±)-threo = 31 nM vs. IC50 (±)-erythro = 3,779 nM). In comparison, MPH was three times less potent than (±)-threo-4F-MPH at the dopamine transporter (IC50  = 131 nM) and around 2.5 times less potent at the norepinephrine transporter (IC50  = 83 nM). Both substances were catecholamine selective with IC50 values of 8,805 nM and >10,000 nM for (±)-threo-4F-MPH and MPH at the serotonin transporter. These findings suggest that the psychostimulant properties of (±)-threo-4F-MPH might be more potent in humans than MPH. Copyright © 2017 John Wiley & Sons, Ltd.

Analysis of six 'neuro-enhancing' phenidate analogs.

Six collected phenidates, i.e., 4-methylmethylphenidate, 3,4-dichloromethylphenidate, ethylphenidate, 3,4-dichloroethylphenidate, ethylnaphthidate, and N-benzyl-ethylphenidate were fully characterized by means of X-ray, nuclear magnetic resonance (NMR), gas chromatography-mass spectrometry (GC-MS), electrospray ionization-tandem mass spectrometry (ESI-MS/MS), attenuated total reflectance Fourier transform infrared (ATR-FTIR) and GC solid-state IR analysis. Crystallography revealed the exclusive presence of the threo-configuration. Steric crowding induced by N-benzyl substitution at the piperidine moiety prompted an adoption of an unexpected axial positioning of substituents on the piperidine moiety in the crystal state as opposed to the exclusive equatorial positioning encountered in N-unsubstituted phenidate analogues. Gas phase computations of the relative lowest energy conformers confirm that the axial positioning appears to be favoured over the equatorial positioning; in solution, however, equatorial positioning is predominant according to nuclear Overhauser effect experiments. All samples, mainly originating from China, had a good to very good degree of purity indicative of their professional chemical synthesis. Routine analysis of these drugs by GC-MS revealed thermal decomposition of phenidate analogues in the injection port and/or on column to 2-aryl-ethyl-acetates and 2,3,4,5-tetrahydropyridines. The decomposition pathway was suggested to proceed via a 6-membered transition state which was supported by density functional theory (DFT) computations. Fragmentation pathways of decomposition products as well as the corresponding electron ionization (EI) mass spectra are provided. The thermal instability might thus render smoking or 'vaping' of these drugs a less effective route of administration. The analytical fingerprints of six structurally diverse phenidate analogues provide a helpful reference to forensic chemists in charge of identifying new psychoactive substances. Copyright © 2017 John Wiley & Sons, Ltd.

Classics in Chemical Neuroscience: Methylphenidate.

As the first drug to see widespread use for the treatment of attention deficit hyperactivity disorder (ADHD), methylphenidate was the forerunner and catalyst to the modern era of rapidly increasing diagnosis, treatment, and medication development for this condition. During its often controversial history, it has variously elucidated the importance of dopamine signaling in memory and attention, provoked concerns about pharmaceutical cognitive enhancement, driven innovation in controlled-release technologies and enantiospecific therapeutics, and stimulated debate about the impact of pharmaceutical sales techniques on the practice of medicine. In this Review, we will illustrate the history and importance of methylphenidate to ADHD treatment and neuroscience in general, as well as provide key information about its synthesis, structure-activity relationship, pharmacological activity, metabolism, manufacturing, FDA-approved indications, and adverse effects.

Development and validation of an UFLC-MS/MS method for enantioselectivity determination of d,l-thero-methylphenidate, d,l-thero-ethylphenidate and d,l-thero-ritalinic acid in rat plasma and its application to pharmacokinetic study.

A chiral UFLC-MS/MS method was established and validated for quantifying d-threo-methylphenidate (d-threo-MPH), l-threo-methylphenidate (l-threo-MPH), d-threo-ethylphenidate (d-threo-EPH), l-threo-ethylphenidate (l-threo-EPH) and d,l-threo-ritalinic acid (d,l-threo-RA) in rat plasma over the linearity range of 1-500ng/mL. Chiral separation was performed on an Astec Chirobiotic V2 column (5µm, 250×2.1mm) with isocratic elution using methanol containing 0.003% ammonium acetate (w/v) and 0.003% trifluoroacetic acid (v/v) at a flow of 0.3mL/min. All analytes and IS were extracted from rat plasma by a one-step liquid-liquid extraction (LLE) method. The intra- and inter-run accuracies were within 85-115%, and the intra- and inter-run precision were <10% for all analytes. Extraction recoveries were 55-62% for d-threo-MPH, 54-60% for l-threo-MPH, 55-60% for d-threo-EPH, 53-57% for l-threo-EPH and 25-30% for d,l-threo-RA. The validated UFLC-MS/MS method successfully applied to the pharmacokinetic interaction study of oral d-threo-MPH and l-threo-MPH (alone or in combination) in female Sprague Dawley rats. The EPH was not detected in rat plasma following oral administrated MPH without EtOH. As far as it is known to the authors, this study is the first one step liquid-liquid extraction method to extract and UFLC-MS/MS method to quantify d-threo-MPH, l-threo-MPH, d-threo-EPH, l-threo-EPH and d,l-threo-RA simultaneously.

A highly enantioselective Mannich reaction of aldehydes with cyclic N-acyliminium ions by synergistic catalysis.

Matched combinations of Brønsted or Lewis acids with suitable pro-electrophiles and secondary amine organocatalysts enable the novel enantioselective syntheses of carbamoyl dihydroquinoline and tetrahydropyridine derivatives with concomitant formation of two stereocenters. A short formal asymmetric synthesis of (2R,2'R)-threo-methylphenidate (Ritalin) is also described.

Sex differences in the kinetic profiles of d- and l- methylphenidate in the brains of adult rats.

OBJECTIVE: Methylphenidate is commonly used in the treatment of Attention Deficit Hyperactivity Disorder and narcolepsy. Methylphenidate is administered as a racemic mixture of the d- and l- threo enantiomers; however, the d-enantiomer is primarily responsible for the pharmacologic activity. Previous studies of the behavioral effects of methylphenidate have highlighted sex differences in the responsiveness to the drug, namely an increased sensitivity of females to its stimulatory effects. These differences may be due to differences in the uptake, distribution, and elimination of methylphenidate from male and female brains. Therefore, we compared the pharmacokinetics of d- and l- threo methylphenidate in the brains of male and female rats. MATERIALS AND METHODS: Adult male and female Sprague-Dawley rats were injected with 5 mg/kg d, l- threo methylphenidate, and whole brains were collected at various time points following injection. We measured methylphenidate concentrations utilizing chiral high pressure liquid chromatography followed by mass spectrometry. RESULTS: Females exhibited consistently higher brain concentrations of both d- and l- methylphenidate and a slower clearance of methylphenidate from brain as compared to males, particularly with the active d-enantiomer. CONCLUSIONS: The increased sensitivity of females to methylphenidate may be partially explained by an increase in total brain exposure to the drug.