Chemical Targeting of Voltage Sensitive Dyes to Specific Cells and Molecules in the Brain.

Voltage sensitive fluorescent dyes (VSDs) are important tools for probing signal transduction in neurons and other excitable cells. The impact of these highly lipophilic sensors has, however, been limited due to the lack of cell-specific targeting methods in brain tissue or living animals. We address this key challenge by introducing a nongenetic molecular platform for cell- and molecule-specific targeting of synthetic VSDs in the brain. We employ a dextran polymer particle to overcome the inherent lipophilicity of VSDs by dynamic encapsulation and high-affinity ligands to target the construct to specific neuronal cells utilizing only native components of the neurotransmission machinery at physiological expression levels. Dichloropane, a monoamine transporter ligand, enables targeting of dense dopaminergic axons in the mouse striatum and sparse noradrenergic axons in the mouse cortex in acute brain slices. PFQX in conjunction with ligand-directed acyl imidazole chemistry enables covalent labeling of AMPA-type glutamate receptors in the same brain regions. Probe variants bearing either a classical electrochromic ANEP dye or state-of-the-art VoltageFluor-type dye respond to membrane potential changes in a similar manner to the parent dyes, as shown by whole-cell patch recording. We demonstrate the feasibility of optical voltage recording with our probes in brain tissue with one-photon and two-photon fluorescence microscopy and define the signal limits of optical voltage imaging with synthetic sensors under a low photon budget determined by the native expression levels of the target proteins. This work demonstrates the feasibility of a chemical targeting approach and expands the possibilities of cell-specific imaging and pharmacology.

The DARK Side of Total Synthesis: Strategies and Tactics in Psychoactive Drug Production.

Humankind has used and abused psychoactive drugs for millennia. Formally, a psychoactive drug is any agent that alters cognition and mood. The term "psychotropic drug" is neutral and describes the entire class of substrates, licit and illicit, of interest to governmental drug policy. While these drugs are prescribed for issues ranging from pain management to anxiety, they are also used recreationally. In fact, the current opioid epidemic is the deadliest drug crisis in American history. While the topic is highly politicized with racial, gender, and socioeconomic elements, there is no denying the toll drug mis- and overuse is taking on this country. Overdose, fueled by opioids, is the leading cause of death for Americans under 50 years of age, killing ca. 64,000 people in 2016. From a chemistry standpoint, the question is in what ways, if any, did organic chemists contribute to this problem? In this targeted review, we provide brief historical accounts of the main classes of psychoactive drugs and discuss several foundational total syntheses that ultimately provide the groundwork for producing these molecules in academic, industrial, and clandestine settings.

Cocaethylene, the in vivo product of cocaine and ethanol, is a narcotic more potent than its precursors.

The molecular conformation and supramolecular architecture of cocaethylene [systematic name: ethyl (1R,2R,3S,5S)-3-benzoyloxy-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylate], C18H23NO4, have been determined for the first time. Cocaethylene is a narcotic produced in vivo when cocaine and ethanol are administered concomitantly. The intra- and intermolecular features of cocaethylene and its less potent narcotic precursor cocaine are very similar. The only molecular difference is in the conformation of the methyl group of the ethoxycarbonyl group. Similar to cocaine, the carboxylate atoms and the α-C atom are coplanar in cocaethylene, but the methyl C atom of the ethyl group is bent by ca 90° away from this plane in the narcotic reported here. The main supramolecular motif is a one-dimensional chain stabilized by weak C-H...O contacts.

Essential/precursor chemicals and drug consumption: impacts of US sodium permanganate and Mexico pseudoephedrine controls on the numbers of US cocaine and methamphetamine users.

BACKGROUND AND AIMS: In December 2006 the United States regulated sodium permanganate, a cocaine essential chemical. In March 2007 Mexico, the United States' primary source for methamphetamine, closed a chemical company accused of illicitly importing 60+ tons of pseudoephedrine, a methamphetamine precursor chemical. US cocaine availability and methamphetamine availability, respectively, decreased in association. This study tested whether the controls had impacts upon the numbers of US cocaine users and methamphetamine users. DESIGN: Auto-regressive integrated moving average (ARIMA) intervention time-series analysis. Comparison series-heroin and marijuana users-were used. SETTING: United States, 2002-14. PARTICIPANTS: The National Survey on Drug Use and Health (n = 723 283), a complex sample survey of the US civilian, non-institutionalized population. MEASUREMENTS: Estimates of the numbers of (1) past-year users and (2) past-month users were constructed for each calendar quarter from 2002 to 2014, providing each series with 52 time-periods. FINDINGS: Downward shifts in cocaine users started at the time of the cocaine regulation. Past-year and past-month cocaine users series levels decreased by approximately 1 946 271 (-32%) (P < 0.05) and 694 770 (-29%) (P < 0.01), respectively-no apparent recovery occurred through 2014. Downward shifts in methamphetamine users started at the time of the chemical company closure. Past-year and past-month methamphetamine series levels decreased by 494 440 (-35%) [P < 0.01; 95% confidence interval (CI) = -771 897, -216 982] and 277 380 (-45%) (P < 0.05; CI = -554 073, -686), respectively-partial recovery possibly occurred in 2013. The comparison series changed little at the intervention times. CONCLUSIONS: Essential/precursor chemical controls in the United States (2006) and Mexico (2007) were associated with large, extended (7+ years) reductions in cocaine users and methamphetamine users in the United States.

Underexplored Opportunities for Natural Products in Drug Discovery.

The importance of natural products in the treatment of human disease is well documented. While natural products continue to have a profound impact on human health, chemists have succeeded in generating semisynthetic analogues that sometimes overshadow the original natural product in terms of clinical significance. Synthetic efforts based on natural products have primarily focused on improving their drug-like features while targeting utility in the same biological space. A less documented phenomenon is that natural products can serve as powerful starting materials to generate drug substances with novel therapeutic utility that is unrelated to the biological space of the natural product starting material. In this Perspective, examples of natural product derived marketed drugs with therapeutic utility in clinical space that is different from the biological profile of the starting material are presented, demonstrating that this is not merely a theoretical concept but both a clinical reality and an underexplored opportunity.

Economical synthesis of 13C-labeled opiates, cocaine derivatives and selected urinary metabolites by derivatization of the natural products.

The illegal use of opiates and cocaine is a challenge world-wide, but some derivatives are also valuable pharmaceuticals. Reference samples of the active ingredients and their metabolites are needed both for controlling administration in the clinic and to detect drugs of abuse. Especially, (13)C-labeled compounds are useful for identification and quantification purposes by mass spectroscopic techniques, potentially increasing accuracy by minimizing ion alteration/suppression effects. Thus, the synthesis of [acetyl-(13)C4]heroin, [acetyl-(13)C4-methyl-(13)C]heroin, [acetyl-(13)C2-methyl-(13)C]6-acetylmorphine, [N-methyl-(13)C-O-metyl-(13)C]codeine and phenyl-(13)C6-labeled derivatives of cocaine, benzoylecgonine, norcocaine and cocaethylene was undertaken to provide such reference materials. The synthetic work has focused on identifying (13)C atom-efficient routes towards these derivatives. Therefore, the (13)C-labeled opiates and cocaine derivatives were made from the corresponding natural products.

Headspace-gas chromatographic-mass spectrometric analysis of South American commercial solvents and their use in the illicit conversion of cocaine base to cocaine hydrochloride.

This study presents data that establish the makeup of solvents utilized in illicit cocaine hydrochloride production, as determined via the identification of the occluded solvents in the crystal matrix of the final product. The occluded solvent ratios can differ dramatically from the ratios of the original processing solvents. Additionally, the presented data suggest the diversion of commercial solvents to illicit cocaine hydrochloride laboratories. Thirty-five commercial solvents were obtained from five chemical manufacturing companies in South America. Each solvent was qualitatively and quantitatively analyzed using static headspace-gas chromatography-mass spectrometry (HS-GC-MS). After obtaining the chemical profile for each commercial solvent, solvents and/or solvent mixtures were prepared to be comparable in composition to several of the commercial products. Over 90 individual batches of cocaine hydrochloride were prepared from cocaine base using these solvents or solvent mixtures, which match those most commonly employed in clandestine laboratories. Additionally, a number of unique manufacturing by-products produced from processing solvents were identified, and their significance is discussed.

Synthesis, radiosynthesis and first in vitro evaluation of novel PET-tracers for the dopamine transporter: [(11)C]IPCIT and [(18)F]FE@IPCIT.

INTRODUCTION: Present data indicate that merging beneficial structural elements from previously published DAT-ligands highest DAT affinity, selectivity and a suitable metabolic profile should be achieved. This combination led to the development of IPCIT and FE@IPCIT. METHODS: Precursor synthesis was done starting from cocaine in a six step reaction. O-[(11)C]-methylation was established using [(11)C]methyl iodide, optimized and subsequently automated. Small scale (18)F-fluroroethylation as well as optimization of reaction parameters and automation were performed. Affinity and selectivity of the candidate substances were tested in standard binding experiments on human membranes. Metabolic stability and blood-brain-barrier (BBB) penetration were determined. RESULTS: Precursor compound, IPCITacid, and reference compounds, IPCIT and FE@IPCIT, were obtained in 4.9%, 12.7% and 4.1% yield, respectively. Automated radiosynthesis of [(11)C]IPCIT yielded 1.9 ± 0.7 GBq (12.5 ± 4%, corrected for decay). Optimum parameters for (18)F-fluoroethylation were 110 °C for 15 min under TBAH catalysis, yielding 67 ± 16 % radiochemical incorporation. Affinity was determined as 1.7 ± 0.6 nM for IPCIT, 1.3 ± 0.2 nM for FE@IPCIT and 37 ± 13 nM for the precursor molecule, IPCIT-acid. Results from in vitro and in silico evaluations revealed high stability but also high lipophilicity. CONCLUSION: Present data indicate high affinity and stability of both IPCIT and FE@IPCIT. Radiolabelling, optimization of reaction parameters and automation succeeded. On the other hand, data concerning BBB-penetration are not promising.

Two-step radiosynthesis of 18)F]FE-ß-CIT and [18F]PR04.MZ.

The cocaine-derived dopamine reuptake inhibitors FE-ß-CIT (8-(2-fluoroethyl)-3-(4-iodophenyl)-8-azabicyclo[3.2.1]octane-2-carboxylic acid methyl ester) (1) and PR04.MZ(8-(4-fluorobut-2-ynyl)-3-p-tolyl-8-azabicyclo[3.2.1]octane-2-carboxylic acid methyl ester) (2) were labelled with (18)F-fluorine using a two-step route. 2-[(18)F]Fluoroethyltosylate and 4-[(18)F]fluorobut-2-yne-1-yl tosylate were used as labelling reagents, respectively. Radiochemically pure (>98%) [(18)F]FE-ß-CIT and [(18)F]PRD04.MZ (32-86 GBq/µmol) were obtained after a synthesis time of 100 min in about 25% non-decay-corrected overall yield.

Asymmetric synthesis of the tropane alkaloid (+)-pseudococaine via ring-closing iodoamination.

Ring-closing iodoamination of tert-butyl 2-hydroxy-7-[N-methyl-N-(α-methyl-p-methoxybenzyl)amino]cyclohept-3-ene-1-carboxylates proceeds with concomitant loss of the N-α-methyl-p-methoxybenzyl group to give the corresponding 8-azabicyclo[3.2.1]octane scaffolds in >99:1 dr. Subsequent elaboration of one of these templates provided access to (+)-pseudococaine hydrochloride, in seven steps and 31% overall yield from commercially available starting materials.

The influence of residual nor-ß-CFT in 11C CFT injection on the Parkinson disease diagnosis: a 11C CFT PET study.

PURPOSE: Fully automated synthesis of 11C-labeled 2ß-carbomethoxy-3ß-(4-fluorophenyl)tropane (11C CFT) as a dopamine transporter positron emission tomography (PET) tracer is performed with Sep-Pak purification, which cannot separate 11C CFT from nor-ß-CFT and will result in the residual precursor nor-ß-CFT in the final 11C CFT injection. The aim of this study is to estimate the influence of the residual precursor nor-ß-CFT in the 11C CFT injection on the Parkinson disease (PD) diagnosis results. METHODS: Automated synthesis of 11C CFT was performed using the different chemical amounts (0.10, 0.20, 0.25, and 0.30 mg) of nor-ß-CFT with Sep-Pak purification. According to the given different amounts of nor-ß-CFT in the radiosynthesis, clinically suspected 25 PD patients were randomly divided into the following 4 groups: 0.10 mg, 0.20 mg, 0.25 mg, and 0.30 mg, which had 5, 9, 5, and 6 cases, respectively. A normal control group with 0.10 mg of nor-ß-CFT included 2 volunteers. After the brain PET images of the subjects were acquired, the regions of interests of striatum and cerebellum were drawn, and the standard uptake values of these regions were calculated. Finally, comparing the 18F FDG PET and clinical diagnosis, the coincidence rates of 11C CFT PET imaging for PD patients were determined. RESULTS: Given 0.25 mg of the precursor nor-ß-CFT, high radiochemical yield (59.4%) and high radiochemical purity of 11C CFT were obtained using Sep-Pak purification within a short synthesis time. The 11C CFT standard uptake value ratios of striatum to cerebellum had no statistically significant difference (P > 0.05) between the 4 suspected PD groups. However, there was statistically significant difference (P < 0.05) between the suspected PD groups and the control group. Also, the coincidence rates between the PD diagnosis using 11C CFT PET imaging for different dose groups and the final clinical diagnosis result were greater than 80%, but difference between the coincidence rates was not statistically significant (P = 0.955). CONCLUSIONS: A simple, rapid, and efficient automated synthesis of 11C CFT using 0.25 mg of nor-ß-CFT with Sep-Pak purification is afforded, providing enough radioactivity for PD PET imaging routinely. The residual nor-ß-CFT in the 11C CFT injection is not inhibit 11C CFT binding to dopamine transporter, and also has no influence on PET diagnosis results of PD.

Let it snow! Let it snow! Let it snow! Estimating cocaine production using a novel dataset based on reported seizures of laboratories in Colombia.

BACKGROUND: Data on the cocaine market appear inconsistent, as they tend to show declining prices vis-a-vis steady or increasing demand and a declining supply. This paper proposes an explanation for this trend by providing evidence of an under-estimation of the supply of cocaine. METHODS: We propose a conservative estimate of cocaine production in Colombia for 2008, using data based on all reported seizures from 328 laboratories made by the counteracting organisations operating within the Colombian territory. RESULTS: Our conservative estimate of 935 tons from the seized laboratories is at least twice the estimate declared in official statistics of 295-450 tons. We are careful to keep all variables to their minimum boundary values. Our methodology could prove to be a useful tool, especially if used in parallel with the standard tools. Moreover, its characteristics (affordability, ease of use and potential for worldwide adoption) make it a powerful instrument to counteract cocaine production.

Synthesis and applications of masked oxo-sulfinamides in asymmetric synthesis.

This short perspective reports on the synthesis and applications of a class of chiral amino carbonyl compounds, masked oxo-sulfinamides where the amine is protected with an N-sulfinyl moiety and the carbonyl group is protected as the ketal or 1,3-dithiane. These polyfunctionalized chiral building blocks are prepared by addition of organometallic reagents to masked oxo-sulfinimines (N-sulfinyl imines) or the addition of oxo-organometallic reagents and lithio-1,3-dithianes to sulfinimines. Because unmasking of the amino and carbonyl groups results in cyclic imines, these chiral building blocks are particularly useful for the asymmetric synthesis of functionalized nitrogen heterocycles, including prolines, pipecolic acids, pyrrolidines, homotropinones, tropinones, and tropane alkaloids such as cocaine and C-1 cocaine analogues.

Enantioselective synthesis of cocaine C-1 analogues using sulfinimines (N-sulfinyl imines).

The first examples of cocaine analogues having substituents (methyl, ethyl, n-propyl, n-pentyl, and phenyl) at the C-1 position of the cocaine tropane skeleton were prepared by heating sulfinimine-derived α,ß-unsaturated pyrrolidine nitrones. In the presence of the Lewis acid Al(O(t)Bu)(3) the nitrones undergo an intramolecular [3 + 2] cycloaddition to give tricyclic isoxazolidines that were transformed in three steps to the cocaine analogues. In the absence of the Lewis acid, lactams were formed resulting from rearrangement of the nitrone to an oxaziridine. A novel Pd- and base-promoted rearrangement of methanesulfonate salts of isoxazolidine to bridge bicyclic[4.2.1]isoxazolidines was discovered.

The syntheses and characterization 3ß-(4-fluorobenzoyloxy)tropane (fluorotropacocaine) and its 3α isomer.

3ß-(4-Fluorobenzoyloxy)tropane (3ß-FBT, fluorotropacocaine) was first reported by Finnish authorities to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) via the Early Warning System (EWS) in 2008 and our own laboratory tentatively identified it in 2010 in several products purchased from head shops. Very little is known about this cocaine-like drug and, as no reference standards were available, we have synthesized and characterized both 3ß-FBT and its 3α isomer for use as reference standards. The two compounds are separable by gas chromatography (GC) but their electron-impact (EI) mass spectra were found to be almost identical. ¹9F NMR spectroscopy was also found to be a useful technique for distinguishing the two isomers.

Detección de Benzoilecgonina en orina de consumidores de té de coca mediante métodos de inmunoensayo / Detection of Benzoilecgonine in urine of consumers of coca tea by immunoassay methods

Con el fin de detectar la presencia de benzoilecgonina en orina de consumidores de té de coca, se realizó un estudio piloto analizando muestras de orina a 10 voluntarios sanos, no consumidores de cocaína, antes de ingerir la infusión de té de coca (Nasa Esh´s Coca Nasa), y las recolectadas hasta las 48 horas después de la ingestión, de una toma única de 100 mL el mismo día. El análisis se realizó por métodos de inmunoensayo, cualitativo, mediante pruebas rápidas Acu-check, y semicuantitativo AxSYM Cocaine Metabolite, basado en In munoensayo de Fluorescencia Polarizada (FPIA). Antes de la ingestión de la infusión por los métodos cualitativos y semicuantitativos las muestras recolectadas resultaron negativas, después de haber ingerido la infusión, por ambos métodos se detectó concentraciones de benzoilecgonina desde la primera hasta las 48 horas con diversas variaciones entre las muestras, observándose excelente concordancia entre los métodos para la determinación de benzoilecgonina en orina. En este estudio, se concluyó que existe la presencia de benzoilecgonina en muestras de orina de consumidores de té de coca. Los métodos utilizados sólo proporcionan resultados preliminares, se recomienda utilizar unos más específicos a fin de encontrar parámetros que permitan discriminar individuos que hayan ingerido infusión de té de coca, de aquellos que son adictos a la cocaína. Además, es importante prevenir a los consumidores de té de coca sobre el riesgo de detección de benzoilecgonina en orina dentro de las primeras 24 a 48 horas y las implicaciones que trae consigo tales hallazgos de laboratorio.

In order to detect the presence of benzoylecgonine in urine of consumers of coca tea, a pilot study was conducted by analyzing urine samples from 10 volunteers not cocaine users, be fore drinking the coca tea infusion, and collected until 48 hours after ingestión of a single shot (100 mL) the same day, The analysis was performed by immunoassay qualitative methods, using fast Acu-check tests, and semi quantitative automated equipment Abbott Axsym System, based on fluorescence polarization immunoassay (FPIA). Before ingestion of the infusion for qualitative and semiquantitative methods for samples collected were negative, and after drinking the tea, for both methods, concentrations of benzoylecgonine was detected from the first to 48 hours with several variations between samples, observed excellent agreement between the methods for the determination of benzoilecgonine in urine. In this study, we concluded that there is the presence of benzoylecgonine in urine samples from consumers of coca tea. According to the methods used provide only preliminary results, we recommend using a more specific in order to find parameters to discriminate individuals who have ingested coca tea infusions of dose that are cocaine addicts, In turn, it is important to prevent cosumers coca tea on the risk of detection of benzoylecgonine in urine within the first 24 to 48 hours and the implications it brings such laboratory findings.

Facile and enantiospecific syntheses of (6S,7R)-6-chloro-7-benzyloxy-, (7S)-halo-, and (7S)-hydroxy-cocaine and natural (-)-cocaine from D-(-)-ribose.

First syntheses of C6,7 and C7 enantiopure cocaine analogues were achieved from D-(-)-ribose via a trans-acetonide controlled endo-selective intramolecular nitrone-alkene cycloaddition (INAC) as the key step. This synthetic scheme allows practical preparation of cocaine analogues for bioevaluation as potential candidates for the treatment of cocaine addiction and as potential conjugates for immunotherapy.

Total synthesis of (-)-cocaine and (-)-ferruginine.

Total synthesis of tropane alkaloids (-)-cocaine and (-)-ferruginine were accomplished in nine steps each and in 55% and 46% overall yields, respectively, starting from the known Betti base derivative (+)-(7aR,10R,12S)-10-(1H-benzotriazol-1-yl)-7a,8,9,10-tetrahydro-12-phenyl-12H-naphtho[1,2-e]pyrrolo[2,1-b][1,3]oxazine. In this novel route, RCM reaction and 1,3-dipolar cycloaddition were employed as key steps for the enantioselective construction of tropane skeleton and the regioselective introduction of 3-bromo-2-isoxazoline ring as masked cis-2,3-disubstituents. To obtain the desired precursor (2S,5R)-2-allyl-5-vinylpyrrolidine for RCM reaction, we developed a general and practical method for the preparation of enantiopure cis-2,5-disubstituted pyrrolidines bearing alkene- and/or alkyne-containing substituents. We also offered two highly efficient pathways for the conversion of the 3-bromo-2-isoxazoline ring into the desired cis-2,3-disubstituted groups in (-)-cocaine and (-)-ferruginine.

Visualization of the cocaine-sensitive dopamine transporter with ligand-conjugated quantum dots.

The presynaptic dopamine (DA) transporter is responsible for DA inactivation following release and is a major target for the psychostimulants cocaine and amphetamine. Dysfunction and/or polymorphisms in human DAT (SLC6A3) have been associated with schizophrenia, bipolar disorder, Parkinson's disease, and attention-deficit hyperactivity disorder (ADHD). Despite the clinical importance of DAT, many uncertainties remain regarding the transporter's regulation, in part due to the poor spatiotemporal resolution of conventional methodologies and the relative lack of efficient DAT-specific fluorescent probes. We developed a quantum dot-based labeling approach that uses a DAT-specific, biotinylated ligand, 2-ß-carbomethoxy-3-ß-(4-fluorophenyl)tropane (IDT444), that can be bound by streptavidin-conjugated quantum dots. Flow cytometry and confocal microscopy were used to detect DAT in stably and transiently transfected mammalian cells. IDT444 is useful for quantum-dot-based fluorescent assays to monitor DAT expression, function, and plasma membrane trafficking in living cells as evidenced by the visualization of acute, protein-kinase-C (PKC)-dependent DAT internalization.