Crystal structures of agonist-bound human cannabinoid receptor CB1.

The cannabinoid receptor 1 (CB1) is the principal target of the psychoactive constituent of marijuana, the partial agonist Δ9-tetrahydrocannabinol (Δ9-THC). Here we report two agonist-bound crystal structures of human CB1 in complex with a tetrahydrocannabinol (AM11542) and a hexahydrocannabinol (AM841) at 2.80 Å and 2.95 Å resolution, respectively. The two CB1-agonist complexes reveal important conformational changes in the overall structure, relative to the antagonist-bound state, including a 53% reduction in the volume of the ligand-binding pocket and an increase in the surface area of the G-protein-binding region. In addition, a 'twin toggle switch' of Phe2003.36 and Trp3566.48 (superscripts denote Ballesteros-Weinstein numbering) is experimentally observed and appears to be essential for receptor activation. The structures reveal important insights into the activation mechanism of CB1 and provide a molecular basis for predicting the binding modes of Δ9-THC, and endogenous and synthetic cannabinoids. The plasticity of the binding pocket of CB1 seems to be a common feature among certain class A G-protein-coupled receptors. These findings should inspire the design of chemically diverse ligands with distinct pharmacological properties.

Novel Δ8-Tetrahydrocannabinol Vaporizers Contain Unlabeled Adulterants, Unintended Byproducts of Chemical Synthesis, and Heavy Metals.

Cannabis e-cigarettes containing Δ8-tetrahydrocannabinol (Δ8-THC) produced synthetically from hemp-derived cannabidiol (CBD) have recently risen in popularity as a legal means of cannabis consumption, but questions surrounding purity and unlabeled additives have created doubts of their safety. Herein, NMR, GC-MS, and ICP-MS were used to analyze major components of 27 products from 10 brands, and it was determined none of these had accurate Δ8-THC labeling, 11 had unlabeled cutting agents, and all contained reaction side-products including olivetol, Δ4(8)-iso-tetrahydrocannabinol, 9-ethoxyhexahydrocannabinol, Δ9-tetrahydrocannabinol (Δ9-THC), heavy metals, and a novel previously undescribed cannabinoid, iso-tetrahydrocannabifuran.

LC-MS/MS Assay for the Measurement of Cannabidiol Profiling in CBD Oil from Japanese Market and Application for Convertible Tetrahydrocannabinol in Acetic Acid Condition.

Cannabidiol (CBD), a major non-psychoactive cannabinoid, has a lot of attention due to its potential relaxing properties and led the trend in commercial CBD aroma/oral hemp seed oil from the Japanese market. In this study, a routine assay for evaluating CBD oil samples was performed using LC coupled with tandem mass spectrometry (LC-MS/MS) and was used to apply the convertible tetrahydrocannabinol (THC) in acetic acid conditions. Based on the electrospray positive ion mode, the detection of cannabidiolic acid (CBDA; m/z 359 > 219), cannabigerolic acid (CBGA; m/z 361 > 343), cannabigerol (CBG; m/z 317 > 193), CBD (m/z 315 > 193), THC (m/z 315 > 193) and cannabinol (CBN; m/z 311 > 223) was performed by satisfying separation with high density of C18 column. Oil samples (50 mg) were diluted with isopropanol (5 mL), to which stable isotope internal standards were added by dilution with methanol/water (50/50), and accuracy rates ranged from 97.8 to 102.2%. This method was used to evaluate the CBD oil products (5 kinds) from the Japanese market. Our survey found obvious counterfeit (non-detectable CBD) CBD oil from Japanese market. Following that, we investigated the conversion of THC in CBD oil samples in simple conditions such as 10% acetic acid and 70 °C for 6 h and discovered that converts THC proportions are approximately 5% ((THC content/CBD content) × 100) and <1.0%. Thus, our developed LC-MS/MS assay could be applied to monitor the CBD concentration and convertible THC from CBD oil.

Enantioselective Total Synthesis of Potent 9ß-11-Hydroxyhexahydrocannabinol.

The first total synthesis of potent cannabinoid, 9ß-11-hydroxyhexahydrocannabinol, is achieved through a proline-catalyzed inverse-electron-demand Diels-Alder reaction. Using this asymmetric catalysis, the cyclohexane ring is constructed with two chiral centers as a single diastereomer with 97% ee. The creation of the third chiral center and benzopyran ring is demonstrated with the elegant synthetic strategies. This mild and efficient synthetic methodology provides a new route for the asymmetric synthesis of the other potent hexahydrocannabinols.

Thermolytic Degradation of Synthetic Cannabinoids: Chemical Exposures and Pharmacological Consequences.

Synthetic cannabinoids are manufactured clandestinely with little quality control and are distributed as herbal "spice" for smoking or as bulk compound for mixing with a solvent and inhalation via electronic vaporizers. Intoxication with synthetic cannabinoids has been associated with seizure, excited delirium, coma, kidney damage, and other disorders. The chemical alterations produced by heating these structurally novel compounds for consumption are largely unknown. Here, we show that heating synthetic cannabinoids containing tetramethylcyclopropyl-ring substituents produced thermal degradants with pharmacological activity that varied considerably from their parent compounds. Moreover, these degradants were formed under conditions simulating smoking. Some products of combustion retained high affinity at the cannabinoid 1 (CB1) and CB2 receptors, were more efficacious than (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55,940) in stimulating CB1 receptor-mediated guanosine 5'-O-(3-thiotriphosphate) (GTPγS) binding, and were potent in producing Δ9-tetrahydrocannabinol-like effects in laboratory animals, whereas other compounds had low affinity and efficacy and were devoid of cannabimimetic activity. Degradants that retained affinity and efficacy also substituted in drug discrimination tests for the prototypical synthetic cannabinoid 1-pentyl-3-(1-naphthoyl)indole (JWH-018), and are likely to produce psychotropic effects in humans. Hence, it is important to take into consideration the actual chemical exposures that occur during use of synthetic cannabinoid formulations to better comprehend the relationships between dose and effect.

Synthetic cannabinoids: the hidden side of Spice drugs.

Spice drugs are herbal mixtures sprayed with synthetic cannabinoids designed to mimic the psychoactive ingredient in marijuana [Δ-tetrahydrocannabinol (Δ-THC)] and synthesized by introducing modifications to the chemical structure of parental compounds aiming to circumvent legal regulations. Synthetic cannabinoid use/abuse can be devastating as toxicological effects and adverse reactions cannot be entirely predicted and may vary with the dose, route of administration, individual vulnerability and concomitant intake with other drugs. The absence of validated testing procedures in the clinical field makes difficult the adoption of a therapeutic approach effective in coping with the synthetic cannabinoid phenomenon, posing a significant challenge for prevention, treatment and public health in general. The aim of this review is to gain insights into the epidemiological, pharmacological and toxicological properties of synthetic cannabinoids, aiming to provide a reliable background needed for the management of synthetic cannabinoid-related adverse effects. Consumers, competent authorities and medical care professionals should be aware of the risks associated with synthetic cannabinoid use.

Activation of Marginally Reactive Boron Enolates by MeLi for the Formation of Enol Phosphates and Synthesis of the Δ(9)-THC Intermediate.

The addition of MeLi to boron enolates produced by the 1,4-addition of Ar2Cu(CN)Li2 to BF3·OEt2-activated enones was followed by the reaction with ClP(O)(OEt)2 to afford the corresponding enol phosphates in moderate to good yields. The scope of this method was examined with sterically hindered or electronically biased enones and/or reagents. This activation of boron enolates was successfully applied to the synthesis of the methyl ether of Δ(9)-tetrahydrocannabinol.

Short and Divergent Total Synthesis of (+)-Machaeriol†B, (+)-Machaeriol†D, (+)-Δ(8)-THC, and Analogues.

Short and highly efficient stereoselective syntheses provide machaeriols and cannabinoids in a divergent approach starting from a common precursor, commercially available (S)-perillic acid. Key features of the novel strategy are a stereospecific palladium-catalyzed decarboxylative arylation and a one-pot sequence comprising a stereoselective hydroboration followed by oxidation or reduction of the corresponding intermediary boranes. The divergent approach is convincingly demonstrated by the five-step syntheses of (+)-machaeriol B, (+)-machaeriol D, and related analogues, and the four-step synthesis of (+)-Δ(8)-THC and an analogue.

Synthesis of [13C4]-labeled ∆9-tetrahydrocannabinol and 11-nor-9-carboxy-∆9-tetrahydrocannabinol as internal standards for reducing ion suppressing/alteration effects in LC/MS-MS quantification.

(-)-∆9-Tetrahydrocannabinol is the principal psychoactive component of the cannabis plant and also the active ingredient in some prescribed drugs. To detect and control misuse and monitor administration in clinical settings, reference samples of the native drugs and their metabolites are needed. The accuracy of liquid chromatography/mass spectrometric quantification of drugs in biological samples depends among others on ion suppressing/alteration effects. Especially, 13C-labeled drug analogues are useful for minimzing such interferences. Thus, to provide internal standards for more accurate quantification and for identification purpose, synthesis of [13C4]-∆9-tetrahydro-cannabinol and [13C4]-11-nor-9-carboxy-∆9-tetrahydrocannabinol was developed via [13C4]-olivetol. Starting from [13C4]-olivetol the synthesis of [13C4]-11-nor-9-carboxy-∆9-tetrahydrocannabinol was shortened from three to two steps by employing nitromethane as a co-solvent in condensation with (+)-apoverbenone.

Stereodivergent total synthesis of Δ9-tetrahydrocannabinols.

All four stereoisomers of Δ(9)-tetrahydrocannabinol (Δ(9)-THC) were synthesized in concise fashion using stereodivergent dual catalysis. Thus, following identical synthetic sequences and applying identical reaction conditions to the same set of starting materials, selective access to the four stereoisomers of THC was achieved in five steps.

High-pressure access to the Δ9-cis- and Δ9-trans-tetrahydrocannabinols family.

Diels-Alder reactions of a range of 1-(alkoxy/alkyl-substituted phenyl)buta-1,3-dienes with methyl vinyl ketone and methyl acrylate carried out in ethanol as the reaction medium under 9 kbar pressure were investigated. The use of high pressure as the activating method of the Diels-Alder reactions allows the efficient and endodiastereoselective generation of a series of cis-cyclohexenyl-benzene cycloadducts, which are selectively converted into their trans-epimers. The cis-cyclohexenyl-benzenes and trans-cyclohexenyl-benzenes produced are useful precursors for accessing substituted privileged cis-6a,7,8,10a-tetrahydro-6H-benzo[c]chromene and trans-6a,7,8,10a-tetrahydro-6H-benzo[c]chromene skeletons. The total syntheses of Δ(9)-cis-tetrahydrocannabinol (THC) and Δ(9)-trans-THC, through the use of selected Diels-Alder adducts, are described. Finally, a route for obtaining Δ(9)-trans-THC in both enantiomeric pure forms based on the (S)-(-)-1-amino-2-(methoxymethyl)pyrrolidine (SAMP)-hydrazone method is also reported.

The Diels-Alder Approach towards Cannabinoid Derivatives and Formal Synthesis of Tetrahydrocannabinol (THC).

Based on the Diels-Alder reaction of vinylchromenes with electron-poor dienophiles, we developed a strategy for the synthesis of tetrahydrocannabinol derivatives. Substituted vinyl chromenes could be converted with several dienophiles to successfully isolate several complex molecules. These molecules already contain the cannabinoid-like base structure and further processing of one such derivative led to a precursor of Δ9 -tetrahydrocannabinol. The most challenging step towards this precursor was an epoxidation step that was ultimately achieved via dimethyl dioxirane.

The synthetic CB1 cannabinoid receptor selective agonists: Putative medical uses and their legalization.

More than 500 molecules have been identified as components of Cannabis sativa (C. sativa), of which the most studied is Δ9-tetrahydrocannabinol (Δ9-THC). Several studies have suggested that Δ9-THC exerts diverse biological effects, ranging from fragmentation of DNA to behavioral disruptions. Currently, it is accepted that most of the pharmacological properties of Δ9-THC engage the activation of the cannabinoid receptors, named CB1 and CB2. Interestingly, multiple pieces of evidence have suggested that the cannabinoid receptors play an active role in the modulation of several diseases leading to the design of synthetic cannabinoid-like compounds. Advances in the development of synthetic CB1 cannabinoid receptor selective agonists as therapeutical approaches are, however, limited. This review focuses on available evidence searched in PubMed regarding the synthetic CB1 cannabinoid receptor selective agonists such as AM-1235, arachidonyl-2' chloroethylamide (ACEA), CP 50,556-1 (Levonantradol), CP-55,940, HU-210, JWH-007, JWH-018, JWH-200 (WIN 55,225), methanandamide, nabilone, O-1812, UR-144, WIN 55,212-2, nabiximols, and dronabinol. Indeed, it would be ambitious to describe all available evidence related to the synthetic CB1 cannabinoid receptor selective agonists. However, and despite the positive evidence on the positive results of using these compounds in experimental models of health disturbances and preclinical trials, we discuss evidence in regards some concerns due to side effects.

Synthesis and structure-activity relationship of substitutions at the C-1 position of Delta9-tetrahydrocannabinol.

A novel series of Delta9-tetrahydrocannabinol (Delta9-THC) analogues were synthesized to determine their potential as cannabinoid receptor modulators. Chemistry focused on conversion of the phenol of Delta9-THC to other functionality through palladium catalyzed reactions with an intermediate triflate 2. Two analogues with sub 100 nM affinity for the CB1 and CB2 receptors were identified.

1-Bromo-3-(1',1'-dimethylalkyl)-1-deoxy-Δ(8)-tetrahydrocannabinols: New selective ligands for the cannabinoid CB(2) receptor.

Δ(8)-Tetrahydrocannabinol (26), 3-(1',1'-dimethylbutyl)- (12), 3-(1',1'-dimethylpentyl)- (13), 3-(1',1'-dimethylhexyl)- (14) and 3-(1',1'-dimethylheptyl)-Δ(8)-tetrahydrocannabinol (15) have been converted into the corresponding 1-bromo-1-deoxy-Δ(8)-tetrahydrocannabinols (25, 8-11). This was accomplished using a protocol developed in our laboratory in which the trifluoromethanesulfonate of a phenol undergoes palladium mediated coupling with pinacolborane. Reaction of this dioxaborolane with aqueous-methanolic copper(II) bromide provides the aryl bromide. The affinities of these bromo cannabinoids for the cannabinoid CB(1) and CB(2) receptors were determined. All of these compounds showed selectivity for the CB(2) receptor and one of them, 1-bromo-1-deoxy-3-(1',1'-dimethylhexyl)-Δ(8)-tetrahydrocannabinol (10), exhibits 52-fold selectivity for this receptor with good (28nM) affinity.

Preparation and characterization of inclusion complexes of a hemisuccinate ester prodrug of delta9-tetrahydrocannabinol with modified beta-cyclodextrins.

Delta(9)-Tetrahydrocannabinol hemisuccinate (THC-HS), an ester prodrug of Delta(9)-tetrahydrocannabinol (THC) has been investigated for its potential to form inclusion complexes with modified synthetic beta-cyclodextrins (CDs). Phase solubility studies were performed to determine the stoichiometric ratio of complexation of THC-HS with random methylated beta-cyclodextrin (RAMEB) and 2-hydroxypropyl beta-cyclodextrin (HPBCD). THC-HS/RAMEB and THC-HS/HPBCD solid systems were prepared by lyophilization and the lyophilized complexes were characterized by Fourier transform infrared (FT-IR) spectroscopy, proton nuclear magnetic spectroscopy, and molecular modeling techniques. The formation of inclusion complexes of THC-HS/RAMEB and THC-HS/HPBCD was demonstrated by an A(L) type curve with the slopes less than unity by the phase solubility method. The association constants for THC-HS/RAMEB and THC-HS/HPBCD were found to be 562.48 and 238.83 M(-1), respectively. The stoichiometry of both of the complexes was found to be 1:1 as determined from the Job's plot. This was confirmed by (1)H NMR and FT-IR techniques. The results obtained from the molecular modeling studies were in accordance with the data obtained from nuclear magnetic resonance and FT-IR. The docking studies revealed the most probable mode of binding of THC-HS with RAMEB in which the alkyl chain was submerged in the hydrophobic pocket of the CD molecule and hydrogen bonding interactions were observed between the hemisuccinate ester side chain of THC-HS and the rim hydroxy groups of RAMEB. The solubility of THC-HS was significantly higher in RAMEB compared to HPBCD. Solid dispersions of THC-HS with CDs will be further utilized to develop oral formulations of THC-HS with enhanced bioavailability.

Water-soluble derivatives of 1 -tetrahydrocannabinol.

Delta1-Tetrahydrocannabinol, which is resinous and insoluble in water and therefore difficult to study pharmacologically, can be converted to a watersoluble derivative without loss of its biological activity. This has been achieved by preparing esters bearing a nitrogen moiety with the use of carbodiimide as the condensing agent. The availability of such water-soluble derivatives will allow the evaluation of Delta1-tetrahydrocannabinol in self-administration studies in monkeys for its addiction liability potential in man. This technique of water solubilization is also applicable to other compounds of chemical and biological significance.

Single and combined effects of plant-derived and synthetic cannabinoids on cognition and cannabinoid-associated withdrawal signs in mice.

BACKGROUND AND PURPOSE: It has been suggested that the non-euphorogenic phytocannabinoid cannabidiol (CBD) can ameliorate adverse effects of Δ9 -tetrahydrocannabinol (THC). We determined whether CBD ameliorates cognitive deficits and withdrawal signs induced by cannabinoid CB1 /CB2 receptor agonists or produces these pharmacological effects on its own. EXPERIMENTAL APPROACH: The effects of THC or the CB1 /CB2 receptor full agonist WIN55212 alone, CBD alone or their combination were tested across a range of doses. Cognitive effects were assessed in C57BL/6 mice in a conditional discrimination task and in the Barnes maze. Cannabinoid withdrawal signs were assessed following precipitated withdrawal by acute administration of the CB1 receptor antagonist SR141716, the 5-HT1A receptor antagonist WAY100635, the TRPV1 receptor antagonist capsazepine or the adenosine A2A receptor antagonist SCH58261. KEY RESULTS: THC produced significant motor and cognitive impairment in the Barnes maze task, none of which were attenuated by the addition of CBD. CBD alone did not affect cognitive performance. Precipitation of withdrawal signs by SR141716 occurred in mice chronically treated with THC or WIN55,212. These withdrawal signs were not attenuated by addition of chronic CBD. Chronic treatment with CBD alone did not induce withdrawal signs precipitated by SR141716 or WAY100635. Chronic CBD treatment also produced anxiolysis, which was not altered by attempting to precipitate withdrawal-induced anxiety with a range of antagonists. CONCLUSIONS AND IMPLICATIONS: CBD as a monotherapy may prove to be a safer pharmacological agent, than CB1 receptor agonists alone or in combination with CBD, for the treatment of several disorders. LINKED ARTICLES: This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.

Stereoselective total synthesis of (-)-perrottetinene and assignment of its absolute configuration.

The first stereoselective total synthesis of the bibenzyl tetrahydrocannabinol, (-)-perrottetinene, has been achieved from readily available starting materials. The absolute stereochemistry is derived from a chiral gamma-hydroxy vinylstannane. The key reaction is the synthesis of the cis-disubstituted cyclohexene ring of perrottetinene by diastereoselective Ireland-Claisen rearrangement and a ring-closing metathesis reaction. The absolute configuration of (-)-perrottetinene is proposed.

Exploring the substituent effects on a novel series of C1'-dimethyl-aryl Delta8-tetrahydrocannabinol analogs.

The synthesis and characterization of novel C1'-phenyl-substituted Delta(8)-THC analogs were previously reported by our laboratory. Within this small series of compounds, the C1'-dimethyl phenyl group was found to impart 13.5-fold selectivity for the CB2 receptor with a K(i) 0.91 nM. The current study expands on the previous report by evaluating the effects of aromatic ring substitution on CB1 and CB2 receptor subtype binding and selectivity. The ring substituents synthesized in this study include aliphatic, halogen, nitrile, and acetamido functional groups. In addition, the isosteric replacement of the phenyl group by thiophene was evaluated. The anti-glioma activities of selected compounds were evaluated in vitro and compared to the lead compound 2.