XOB: A novel phenylalkylamine antagonist of 5-HT2A receptors and voltage-gated sodium channels.

Bipolar disorder impacts millions of patients in the United States but the mechanistic understanding of its pathophysiology and therapeutics is incomplete. Atypical antipsychotic serotonin2A (5-HT2A) receptor antagonists, such as quetiapine and olanzapine, and mood-stabilizing voltage-gated sodium channel (VGSC) blockers, such as lamotrigine, carbamazepine, and valproate, show therapeutic synergy and are often prescribed in combination for the treatment of bipolar disorder. Combination therapy is a complex task for clinicians and patients, often resulting in unexpected difficulties with dosing, drug tolerances, and decreased patient compliance. Thus, an unmet need for bipolar disorder treatment is to develop a therapeutic agent that targets both 5-HT2A receptors and VGSCs. Towards this goal, we developed a novel small molecule that simultaneously antagonizes 5-HT2A receptors and blocks sodium current. The new compound, N-(4-bromo-2,5-dimethoxyphenethyl)-6-(4-phenylbutoxy)hexan-1-amine (XOB) antagonizes 5-HT-stimulated, Gq-mediated, calcium flux at 5-HT2A receptors at low micromolar concentrations while displaying negligible affinity and activity at 5-HT1A, 5-HT2B, and 5-HT2C receptors. At similar concentrations, XOB administration inhibits sodium current in heterologous cells and results in reduced action potential (AP) firing and VGSC-related AP properties in mouse prefrontal cortex layer V pyramidal neurons. Thus, XOB represents a new, proof-of-principle tool that can be used for future preclinical investigations and therapeutic development. This polypharmacology approach of developing a single molecule to act upon two targets, which are currently independently targeted by combination therapies, may lead to safer alternatives for the treatment of psychiatric disorders that are increasingly being found to benefit from the simultaneous targeting of multiple receptors. Significance Statement We synthesized a novel small molecule (XOB) that simultaneously antagonizes two key therapeutic targets of bipolar disorder, 5-HT2A receptors and voltage-gated sodium channels (VGSCs), in heterologous cells, and inhibits the intrinsic excitability of mouse prefrontal cortex layer V pyramidal neurons in brain slices. XOB represents a valuable new proof-of-principle tool for future preclinical investigations and provides a novel molecular approach to the pharmacological treatment of complex neuropsychiatric disease, which often requires a combination of therapeutics for sufficient patient benefit.

The origin of 2,5-dimethoxy-4-methylamphetamine (DOM, STP).

The story of the 1967 appearance of the powerful psychedelic 2,5-dimethoxy-4-methylamphetamine (DOM, STP) commonly omits details and often includes hyperbole and inaccuracies. It is well known how and when the drug was first distributed to the public for free by Owsley Stanley, but the role that Alexander Shulgin played in providing that material is not as well understood. In the interest of transparency and historical accuracy, this article attempts to present an accurate account of this well-known but inadequately detailed event. It follows DOM's development as an experimental substance believed to hold potential promise in psychotherapeutic applications through its appearance as a street drug generating bad press and a lasting bad impression among the public. One of the more interesting questions is why Shulgin would have taken such an immense risk in releasing this material to clandestine operators. While DOM was still legal it was also Dow's intellectual property, so discovery of his involvement could have jeopardized his career. The escape is especially curious as all fingers would logically first point towards Shulgin as the source. Drawing from published and unpublished sources, the authors attempt to suggest answers. DOM rapidly faded into oblivion before human pharmacodynamics and pharmacokinetics could be established. In this account, the reader is informed of the potential value that the compound played in non-clinical molecular neuroscience, elucidating receptor specificity of new drugs, and how mistaken warnings about combining DOM with chlorpromazine led to better non-pharmacological drug crisis response.

Synthesis and characterization of high-purity N,N-dimethyltryptamine hemifumarate for human clinical trials.

Since 2006, there has been a resurgent interest in the pharmacology and therapeutics of psychedelic drugs. Psilocybin, the 4-phosphoryl ester of N,N-dimethyltryptamine (DMT), has been studied most often, but DMT itself is also appealing because of its brief but profound psychological effects and its presence as an endogenous substance in mammalian brain. Although there have been a few studies of ayahuasca, a DMT-containing water infusion, only one human study with pure DMT has been reported since the early 2000s. Newly planned clinical trials to assess the safety and efficacy of DMT in humans with major depressive disorders require high-purity water-soluble DMT for intravenous administration. Accordingly, we synthesized and characterized DMT hemifumarate for these upcoming studies. The synthetic approach of Speeter and Anthony was slightly modified to gain some efficiency in time. In particular, this is the first known report to use aluminum hydride, generated in situ from lithium aluminum hydride, to reduce the intermediate 2-(1H-indol-3-yl)-N,N-dimethyl-2-oxoacetamide to DMT. A quench protocol was developed to produce a good yield of exceptionally pure free base DMT upon workup, which was then converted to the hemifumarate salt. Analysis of the final product included differential scanning calorimetry, thermogravimetric analysis, gas chromatography-mass spectrometry (GC-MS), 1 H and 13 C nuclear magnetic resonance spectroscopy, high-performance liquid chromatography, residual solvent analysis by GC headspace sampling, X-ray powder diffraction analysis, and residual lithium analysis by inductively coupled plasma-mass spectrometry. The DMT hemifumarate was minimally 99.9% pure, with no significant impurities or residual solvents, thus meeting regulatory standards for administration to humans.

Receptor binding profiles and behavioral pharmacology of ring-substituted N,N-diallyltryptamine analogs.

Substantial effort has been devoted toward understanding the psychopharmacological effects of tryptamine hallucinogens, which are thought to be mediated by activation of 5-HT2A and 5-HT1A receptors. Recently, several psychoactive tryptamines based on the N,N-diallyltryptamine (DALT) scaffold have been encountered as recreational drugs. Despite the apparent widespread use of DALT derivatives in humans, little is known about their pharmacological properties. We compared the binding affinities of DALT and its 2-phenyl-, 4-acetoxy-, 4-hydroxy-, 5-methoxy-, 5-methoxy-2-methyl-, 5-fluoro-, 5-fluoro-2-methyl-, 5-bromo-, and 7-ethyl-derivatives at 45 receptor and transporter binding sites. Additionally, studies in C57BL/6 J mice examined whether these substances induce the head twitch response (HTR), a 5-HT2A receptor-mediated response that is widely used as a behavioral proxy for hallucinogen effects in humans. Most of the test drugs bound to serotonin receptors, σ sites, α2-adrenoceptors, dopaminergic D3 receptors, histaminergic H1 receptors, and the serotonin transporter. DALT and several of the ring-substituted derivatives were active in the HTR assay with the following rank order of potency: 4-acetoxy-DALT > 5-fluoro-DALT > 5-methoxy-DALT > 4-hydroxy-DALT > DALT > 5-bromo-DALT. 2-Phenyl-DALT, 5-methoxy-2-methyl-DALT, 5-fluoro-2-methyl-DALT, and 7-ethyl-DALT did not induce the HTR. HTR potency was not correlated with either 5-HT1A or 5-HT2A receptor binding affinity, but a multiple regression analysis indicated that 5-HT2A and 5-HT1A receptors make positive and negative contributions, respectively, to HTR potency (R2 = 0.8729). In addition to supporting the established role of 5-HT2A receptors in the HTR, these findings are consistent with evidence that 5-HT1A activation by tryptamine hallucinogens buffers their effects on HTR. This article is part of the Special Issue entitled 'Psychedelics: New Doors, Altered Perceptions'.

Receptor binding profiles and quantitative structure-affinity relationships of some 5-substituted-N,N-diallyltryptamines.

N,N-Diallyltryptamine (DALT) and 5-methoxy-N,N-diallyltryptamine (5-MeO-DALT) are two tryptamines synthesized and tested by Alexander Shulgin. In self-experiments, 5-MeO-DALT was reported to be psychoactive in the 12-20mg range, while the unsubstituted compound DALT had few discernible effects in the 42-80 mg range. Recently, 5-MeO-DALT has been used in nonmedical settings for its psychoactive effects, but these effects have been poorly characterized and little is known of its pharmacological properties. We extended the work of Shulgin by synthesizing additional 5-substituted-DALTs. We then compared them to DALT and 5-MeO-DALT for their binding affinities at 45 cloned receptors and transporter proteins. Based on in vitro binding affinity, we identified 27 potential receptor targets for the 5-substituted-DALT compounds. Five of the DALT compounds had affinity in the 10-80 nM range for serotonin 5-HT1A and 5-HT2B receptors, while the affinity of DALT itself at 5-HT1A receptors was slightly lower at 100 nM. Among the 5-HT2 subtypes, the weakest affinity was at 5-HT2A receptors, spanning 250-730 nM. Five of the DALT compounds had affinity in the 50-400 nM range for serotonin 5-HT1D, 5-HT6, and 5-HT7 receptors; again, it was the unsubstituted DALT that had the weakest affinity at all three subtypes. The test drugs had even weaker affinity for 5-HT1B, 5-HT1E, and 5-HT5A subtypes and little or no affinity for the 5-HT3 subtype. These compounds also had generally nanomolar affinities for adrenergic α2A, α2B, and α2C receptors, sigma receptors σ1 and σ2, histamine H1 receptors, and norepinephrine and serotonin uptake transporters. They also bound to other targets in the nanomolar-to-low micromolar range. Based on these binding results, it is likely that multiple serotonin receptors, as well as several nonserotonergic sites are important for the psychoactive effects of DALT drugs. To learn whether any quantitative structure-affinity relationships existed, we evaluated correlations among physicochemical properties of the congeneric 5-substituted-DALT compounds. The descriptors included electronic (σp), hydrophobic (π), and steric (CMR) parameters. The binding affinity at 5-HT1A, 5-HT1D, 5-HT7, and κ opioid receptors was positively correlated with the steric volume parameter CMR. At α2A, α2B, and α2C receptors, and at the histamine H1 receptor, binding affinity was correlated with the Hammett substituent parameter σp; higher affinity was associated with larger σp values. At the σ2 receptor, higher affinity was correlated with increasing π. These correlations should aid in the development of more potent and selective drugs within this family of compounds.

Pharmacological examination of trifluoromethyl ring-substituted methcathinone analogs.

Cathinones are a class of drugs used to treat various medical conditions including depression, obesity, substance abuse, and muscle spasms. Some "designer" cathinones, such as methcathinone, mephedrone, and methylone, are used nonclinically for their stimulant or entactogenic properties. Given the recent rise in nonmedical use of designer cathinones, we aimed to improve understanding of cathinone pharmacology by investigating analogs of methcathinone with a CF(3) substituent at the 2-, 3-, or 4-position of the phenyl ring (TFMAPs). We compared the TFMAPs with methcathinone for effects on monoamine uptake transporter function in vitro and in vivo, and for effects on locomotor activity in rats. At the serotonin transporter (SERT), 3-TFMAP and 4-TFMAP were 10-fold more potent than methcathinone as uptake inhibitors and as releasing agents, but 2-TFMAP was both a weak uptake inhibitor and releaser. At the norepinephrine and dopamine transporters (NET and DAT), all TFMAP isomers were less potent than methcathinone as uptake inhibitors and releasers. In vivo, 4-TFMAP released 5-HT, but not dopamine, in rat nucleus accumbens and did not affect locomotor activity, whereas methcathinone increased both 5-HT and dopamine and produced locomotor stimulation. These experiments reveal that TFMAPs are substrates for the monoamine transporters and that phenyl ring substitution at the 3- or 4-position increases potency at SERT but decreases potency at NET and DAT, resulting in selectivity for SERT. The TFMAPs might have a therapeutic value for a variety of medical and psychiatric conditions and may have lower abuse liability compared to methcathinone due to their decreased DAT activity.

Analytical characterization of three trifluoromethyl-substituted methcathinone isomers.

Cathinone derivatives display a wide range of pharmacological activities and uses; some of them are used as prescription medicines, while others are encountered within a recreational context and are available without a prescription over the Internet and in retail shops around the world. One of the difficulties involved in the unambiguous identification of these new psychoactive substances is the lack of suitable reference standards, particularly when dealing with unreported derivatives and positional isomers. In order to address this need, three trifluoromethyl analogues of the psychostimulant methcathinone, with a CF(3) substituent at the 2-, 3- and 4-position of the phenyl ring (2-TFMAP 1, 3-TFMAP 2 and 4-TFMAP 3), have been prepared for analytical characterization using ATR-FTIR, (1)H and (13) C NMR, and GC-(EI/CI)-ion trap-MS. Differentiation among isomers was feasible by IR, for example when assessing the carbonyl stretch at 1711 (1), 1693 (2) and 1688 (3) cm(-1) , respectively. In addition to the expected iminium base peak at m/z 58, EI-MS displayed key ions at m/z 173, 145, 125, 95, and 75. Separation of isomers was possible under GC conditions. A characteristic feature under CI conditions was the loss of water from the [M + H](+) yielding m/z 214 in addition to m/z 58. Studies currently underway show that the three CF(3) -methcathinone analogues have central nervous system effects and that the 4-CF(3) isomer 3 is more potent as a serotonin uptake inhibitor and releasing agent than the 3-CF(3) and 2-CF(3) counterparts.

Synthesis and characterization of 5-methoxy-2-methyl-N,N-dialkylated tryptamines.

The absence of reference material is a commonly experienced difficulty among medical and forensic professionals tasked with identifying new psychoactive substances that are encountered for the first time. The identification of newly emerging substances lies at the heart of forensic and clinical analysis, and a proactive public health policy calls for a thorough analysis of the properties of new psychoactive substances before they appear in the emergency clinic, where they may be noticed because of adverse reactions or toxicity. For example, a wide range of N,N-dialkyltryptamines show psychoactive properties in humans and these tryptamines are sometimes encountered as intoxicants. However, most of the existing reference data on new psychoactive tryptamines have been obtained retrospectively, after reports of acute toxicities. To address the need for reference standards for new tryptamines, thirteen 5-methoxy-2-methyl-N,N-dialkyltryptamines were prepared. Analytical characterization was based on ¹H and ¹³C nuclear magnetic resonance (NMR), gas chromatography-electron ionization ion-trap mass spectrometry (GC-EI-IT-MS) and chemical ionization-ion-trap tandem mass spectrometry (CI-IT-MS/MS), respectively. Differentiation among isomers was feasible by NMR and MS. In addition to the expected iminium ion base peak, indole-related key ions were detected under EI-IT-MS conditions at m/z 174, 159, 131, 130, and 103. CI-IT-MS/MS analysis of the 5-methoxy-2-methyl derivatives revealed the presence of m/z 188 in addition to [M+H]+ and the iminium species. This study served as an extension from previous work on isomeric 5-ethoxylated counterparts and confirmed the ability to differentiate between the two groups. The data provided here add to the existing body of literature and aim to serve both forensic and clinical communities.

The designer methcathinone analogs, mephedrone and methylone, are substrates for monoamine transporters in brain tissue.

The nonmedical use of 'designer' cathinone analogs, such as 4-methylmethcathinone (mephedrone) and 3,4-methylenedioxymethcathinone (methylone), is increasing worldwide, yet little information is available regarding the mechanism of action for these drugs. Here, we employed in vitro and in vivo methods to compare neurobiological effects of mephedrone and methylone with those produced by the structurally related compounds, 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine. In vitro release assays using rat brain synaptosomes revealed that mephedrone and methylone are nonselective substrates for plasma membrane monoamine transporters, similar to MDMA in potency and selectivity. In vivo microdialysis in rat nucleus accumbens showed that i.v. administration of 0.3 and 1.0 mg/kg of mephedrone or methylone produces dose-related increases in extracellular dopamine and serotonin (5-HT), with the magnitude of effect on 5-HT being greater. Both methcathinone analogs were weak motor stimulants when compared with methamphetamine. Repeated administrations of mephedrone or methylone (3.0 and 10.0 mg/kg, s.c., 3 doses) caused hyperthermia but no long-term change in cortical or striatal amines, whereas similar treatment with MDMA (2.5 and 7.5 mg/kg, s.c., 3 doses) evoked robust hyperthermia and persistent depletion of cortical and striatal 5-HT. Our data demonstrate that designer methcathinone analogs are substrates for monoamine transporters, with a profile of transmitter-releasing activity comparable to MDMA. Dopaminergic effects of mephedrone and methylone may contribute to their addictive potential, but this hypothesis awaits confirmation. Given the widespread use of mephedrone and methylone, determining the consequences of repeated drug exposure warrants further study.

Microwave-accelerated preparation and analytical characterization of 5-ethoxy-N,N-dialkyl-[α,α,ß,ß-H(4) ]- and [α,α,ß,ß-D(4) ]-tryptamines.

The increased interest in N,N-dialkyl tryptamines is a reflection of their diverse range of biologically active properties. Deuterated derivatives are of interest for use as internal standards in bioanalytical or pharmacological assays. The present study reports on the synthesis of twelve novel 5-ethoxy-N,N-dialkyl-[α,α,ß,ß-H(4) ]-tryptamines and their [α,α,ß,ß-D(4) ]-counterparts following the Speeter and Anthony procedure. The normally time-consuming reduction step was carried out in 5 min under microwave-accelerated conditions. Good yields were obtained using tetrahydrofuran as the solvent at 150 °C. The resulting 24 tryptamines have been characterized by 1D/2D nuclear magnetic resonance spectroscopy and gas chromatography ion trap mass spectrometry. Differential fragmentation of side-chain-related iminium ions has been observed as a key principle. Because many N,N-dialkyltryptamines are available outside of traditional pharmaceutical supply chains as so-called 'research chemicals', the availability, as standards, of these new N,N-dialkyltryptamines will aid in identifiying novel tryptamines arising from these other souces. They should therefore be of immediate value within forensic, research, and public health contexts.

Dimethyltryptamine and other hallucinogenic tryptamines exhibit substrate behavior at the serotonin uptake transporter and the vesicle monoamine transporter.

N,N-dimethyltryptamine (DMT) is a potent plant hallucinogen that has also been found in human tissues. When ingested, DMT and related N,N-dialkyltryptamines produce an intense hallucinogenic state. Behavioral effects are mediated through various neurochemical mechanisms including activity at sigma-1 and serotonin receptors, modification of monoamine uptake and release, and competition for metabolic enzymes. To further clarify the pharmacology of hallucinogenic tryptamines, we synthesized DMT, N-methyl-N-isopropyltryptamine (MIPT), N,N-dipropyltryptamine (DPT), and N,N-diisopropyltryptamine. We then tested the abilities of these N,N-dialkyltryptamines to inhibit [(3)H]5-HT uptake via the plasma membrane serotonin transporter (SERT) in human platelets and via the vesicle monoamine transporter (VMAT2) in Sf9 cells expressing the rat VMAT2. The tryptamines were also tested as inhibitors of [(3)H]paroxetine binding to the SERT and [(3)H]dihydrotetrabenazine binding to VMAT2. Our results show that DMT, MIPT, DPT, and DIPT inhibit [(3)H]5-HT transport at the SERT with K ( I ) values of 4.00 +/- 0.70, 8.88 +/- 4.7, 0.594 +/- 0.12, and 2.32 +/- 0.46 microM, respectively. At VMAT2, the tryptamines inhibited [(3)H]5-HT transport with K ( I ) values of 93 +/- 6.8, 20 +/- 4.3, 19 +/- 2.3, and 19 +/- 3.1 muM, respectively. On the other hand, the tryptamines were very poor inhibitors of [(3)H]paroxetine binding to SERT and of [(3)H]dihydrotetrabenazine binding to VMAT2, resulting in high binding-to-uptake ratios. High binding-to-uptake ratios support the hypothesis that the tryptamines are transporter substrates, not uptake blockers, at both SERT and VMAT2, and also indicate that there are separate substrate and inhibitor binding sites within these transporters. The transporters may allow the accumulation of tryptamines within neurons to reach relatively high levels for sigma-1 receptor activation and to function as releasable transmitters.

Interpreting velocities from heat-based flow sensors by numerical simulation.

We have carried out numerical simulations of three-dimensional nonisothermal flow around an in situ heat-based flow sensor to investigate how formation heterogeneities can affect the interpretation of ground water flow velocities from this instrument. The flow sensor operates by constant heating of a 0.75-m-long, 5-cm-diameter cylindrical probe, which contains 30 thermistors in contact with the formation. The temperature evolution at each thermistor can be inverted to obtain an estimate of the ground water flow velocity vector using the standard interpretive method, which assumes that the formation is homogeneous. Analysis of data from heat-based flow sensors installed in a sand aquifer at the Former Fort Ord Army Base near Monterey, California, suggested an unexpected component of downward flow. The magnitudes of the vertical velocities were expected to be much less than those of the horizontal velocities at this site because the sensors were installed just above a clay aquitard. Numerical simulations were conducted to examine how differences in thermal conductivities may lead to spurious indications of vertical flow velocities. We found that a decrease in the thermal conductivity near the bottom of the sensor can perturb the temperature profiles along the instrument in such a manner that analyses assuming homogeneous thermal conductivity could indicate a vertical flow component even though flow is actually horizontal. This work demonstrates how modeling can be used to simulate instrument response to formation heterogeneity and shows that caution must be used in interpreting data from such devices.

Intelligent signal processing for detection system optimization.

A wavelet-neural network signal processing method has demonstrated approximately 10-fold improvement over traditional signal processing methods for the detection limit of various nitrogen and phosphorus compounds from the output of a thermionic detector attached to a gas chromatograph. A blind test was conducted to validate the lower detection limit. All 14 of the compound spikes were detected when above the estimated threshold, including all 3 within a factor of 2 above the threshold. In addition, two of six spikes were detected at levels of half the concentration of the nominal threshold. Another two of the six would have been detected correctly if we had allowed human intervention to examine the processed data. One apparent false positive in five nulls was traced to a solvent impurity, whose presence was subsequently identified by analyzing a solvent aliquot evaporated to 1% residual volume, while the other four nulls were properly classified. We view this signal processing method as broadly applicable in analytical chemistry, and we advocate that advanced signal processing methods should be applied as directly as possible to the raw detector output so that less discriminating preprocessing and postprocessing does not throw away valuable signal.

Assessment of in-situ bioremediation at a refinery waste-contaminated site and an aviation gasoline contaminated site.

A combination of geochemical, microbiological and isotopic methods were used to evaluate in-situ bioremediation of petroleum hydrocarbons at one site contaminated with refinery waste and a second site contaminated with aviation gasoline at Alameda Point, California. At each site, geochemical and microbiological characteristics from four locations in the contaminated zone were compared to those from two uncontaminated background locations. At both sites, the geochemical indicators of in-situ biodegradation included depleted soil gas and groundwater oxygen, elevated groundwater alkalinity, and elevated soil gas carbon dioxide and methane in the contaminated zone relative to the background. Radiocarbon content of methane and carbon dioxide measured in soil gas at both sites indicated that they were derived from hydrocarbon contaminant degradation. Direct microscopy of soil core samples using cell wall stains and activity stains, revealed elevated microbial numbers and enhanced microbial activities in contaminated areas relative to background areas, corroborating geochemical findings. While microbial plate counts and microcosm studies using soil core samples provided laboratory evidence for the presence of some microbial activity and contaminant degradation abilities, they did not correlate well with either contaminant location, geochemical, isotopic, or direct microscopy data.