Fragment-based drug discovery and biological evaluation of novel cannabinol-based inhibitors of oxytosis/ferroptosis for neurological disorders.

The oxytosis/ferroptosis regulated cell death pathway is an emerging field of research owing to its pathophysiological relevance to a wide range of neurological disorders, including Alzheimer's and Parkinson's diseases and traumatic brain injury. Developing novel neurotherapeutics to inhibit oxytosis/ferroptosis offers exciting opportunities for the treatment of these and other neurological diseases. Previously, we discovered cannabinol (CBN) as a unique, potent inhibitor of oxytosis/ferroptosis by targeting mitochondria and modulating their function in neuronal cells. To further elucidate which key pharmacophores and chemical space are essential to the beneficial effects of CBN, we herein introduce a fragment-based drug discovery strategy in conjunction with cell-based phenotypic screens using oxytosis/ferroptosis to determine the structure-activity relationship of CBN. The resulting information led to the development of four new CBN analogs, CP1-CP4, that not only preserve the sub-micromolar potency of neuroprotection and mitochondria-modulating activities seen with CBN in neuronal cell models but also have better druglike properties. Moreover, compared to CBN, the analog CP1 shows improved in vivo efficacy in the Drosophila model of mild traumatic brain injury. Together these studies identify the key molecular scaffolds of cannabinoids that contribute to neuroprotection against oxytosis/ferroptosis. They also highlight the advantageous approach of combining in vitro cell-based assays and rapid in vivo studies using Drosophila models for evaluating new therapeutic compounds.

CBG, CBD, Δ9-THC, CBN, CBGA, CBDA and Δ9-THCA as antioxidant agents and their intervention abilities in antioxidant action.

Positive effect of some cannabinoids in the treatment and prophylaxis of a wide variety of oxidation-associated diseases and growing popularity of supplements containing cannabinoids, mainly cannabinoid oils (e.g. CBD oil, CBG oil), in the self-medication of humans cause a growing interest in the antioxidant properties of these compounds, especially those not showing psychotropic effects. Herein, we report the antioxidant activity of cannabigerol (CBG), cannabidiol (CBD), Δ9-tetrahydrocannabinol (Δ9-THC), cannabinol (CBN), cannabigerolic acid (CBGA), cannabinolic acid (CBDA) and Δ9-tetrahydrocannabinolic acid (Δ9-THCA) estimated by spectrophotometric methods: ABTS, DPPH, ORAC, beta-carotene CUPRAC and FRAP. The presented data prove that all the examined cannabinoids exhibit antioxidant activity manifested in their ability to scavenge free radicals, to prevent the oxidation process and to reduce metal ions. Although the intensity of these activities is not the same for the individual cannabinoids it is comparable for all of them with that of E vitamin. As results from the research, the significance of the two types of electron sources presenting in examined cannabinoids, phenolic groups and double bonds transferring electrons, depends on the type of electron-accepting species - radicals/metal ions.

Antioxidants help favorably regulate the kinetics of lipid peroxidation, polyunsaturated fatty acids degradation and acidic cannabinoids decarboxylation in hempseed oil.

The seed of the hemp plant (Cannabis sativa L.) has been revered as a nutritional resource in Old World Cultures. This has been confirmed by contemporary science wherein hempseed oil (HSO) was found to exhibit a desirable ratio of omega-6 and omega-3 polyunsaturated fatty acids (PUFAs) considered optimal for human nutrition. HSO also contains gamma-linoleic acid (GLA) and non-psychoactive cannabinoids, which further contribute to its' potential bioactive properties. Herein, we present the kinetics of the thermal stability of these nutraceutical compounds in HSO, in the presence of various antioxidants (e.g. butylated hydroxytoluene, alpha-tocopherol, and ascorbyl palmitate). We focussed on oxidative changes in fatty acid profile and acidic cannabinoid stability when HSO was heated at different temperatures (25 °C to 85 °C) for upto 24 h. The fatty acid composition was evaluated using both GC/MS and 1H-NMR, and the cannabinoids profile of HSO was obtained using both HPLC-UV and HPLC/MS methods. The predicted half-life (DT50) for omega-6 and omega-3 PUFAs in HSO at 25 °C was about 3 and 5 days, respectively; while that at 85 °C was about 7 and 5 hours respectively, with respective activation energies (Ea) being 54.78 ± 2.36 and 45.02 ± 2.87 kJ/mol. Analysis of the conjugated diene hydroperoxides (CDH) and p-Anisidine value (p-AV) revealed that the addition of antioxidants significantly (p < 0.05) limited lipid peroxidation of HSO in samples incubated at 25-85 °C for 24 h. Antioxidants reduced the degradation constant (k) of PUFAs in HSO by upto 79%. This corresponded to a significant (p < 0.05) increase in color stability and pigment retention (chlorophyll a, chlorophyll b and carotenoids) of heated HSO. Regarding the decarboxylation kinetics of cannabidiolic acid (CBDA) in HSO, at both 70 °C and 85 °C, CBDA decarboxylation led to predominantly cannabidiol (CBD) production. The half-life of CBDA decarboxylation (originally 4 days) could be increased to about 17 days using tocopherol as an antioxidant. We propose that determining acidic cannabinoids decarboxylation kinetics is a useful marker to measure the shelf-life of HSO. The results from the study will be useful for researchers looking into the thermal treatment of hempseed oil as a functional food product, and those interested in the decarboxylation kinetics of the acidic cannabinoids.

Potent Anti-Inflammatory and Pro-Resolving Effects of Anabasum in a Human Model of Self-Resolving Acute Inflammation.

Anabasum is a synthetic analog of Δ8 -tetrahydrocannabinol (THC)-11-oic acid that in preclinical models of experimental inflammation exerts potent anti-inflammatory actions with minimal central nervous system (CNS) cannabimimetic activity. Here we used a novel model of acute inflammation driven by i.d. UV-killed E. coli in healthy humans and found that anabasum (5 mg) exerted a potent anti-inflammatory effect equivalent to that of prednisolone in terms of inhibiting neutrophil infiltration, the hallmark of acute inflammation. These effects arose from the inhibition of the neutrophil chemoattractant LTB4 , while the inhibition of antiphagocytic prostanoids (PGE2 , TxB2 , and PGF2 α) resulted in enhanced clearance of inflammatory stimulus from the injected site. Anabasum at the higher dose of 20 mg possessed the additional properties of triggering the biosynthesis of specialized pro-resolving lipid mediators including LXA4 , LXB4 , RvD1, and RvD3. Collectively, we demonstrate for the first time a striking anti-inflammatory and pro-resolution effects of a synthetic analog of THC in healthy humans.

Cannabinoid CB1 Discrimination: Effects of Endocannabinoids and Catabolic Enzyme Inhibitors.

An improved understanding of the endocannabinoid system has provided new avenues of drug discovery and development toward the management of pain and other behavioral maladies. Exogenous cannabinoid type 1 (CB1) receptor agonists such as Δ9-tetrahydrocannabinol are increasingly used for their medicinal actions; however, their utility is constrained by concern regarding abuse-related subjective effects. This has led to growing interest in the clinical benefit of indirectly enhancing the activity of the highly labile endocannabinoids N-arachidonoylethanolamine [AEA (or anandamide)] and/or 2-arachidonoylglycerol (2-AG) via catabolic enzyme inhibition. The present studies were conducted to determine whether such actions can lead to CB1 agonist-like subjective effects, as reflected in CB1-related discriminative stimulus effects in laboratory subjects. Squirrel monkeys (n = 8) that discriminated the CB1 full agonist AM4054 (0.01 mg/kg) from vehicle were used to study, first, the inhibitors of fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MGL) alone or in combination [FAAH (URB597, AM4303); MGL (AM4301); FAAH/MGL (JZL195, AM4302)] and, second, the ability of the endocannabinoids AEA and 2-AG to produce CB1 agonist-like effects when administered alone or after enzyme inhibition. Results indicate that CB1-related discriminative stimulus effects were produced by combined, but not selective, inhibition of FAAH and MGL, and that these effects were nonsurmountably antagonized by low doses of rimonabant. Additionally, FAAH or MGL inhibition revealed CB1-like subjective effects produced by AEA but not by 2-AG. Taken together, the present data suggest that therapeutic effects of combined, but not selective, enhancement of AEA or 2-AG activity via enzyme inhibition may be accompanied by CB1 receptor-mediated subjective effects.

C1'-Azacycloalkyl Hexahydrocannabinols.

We report the design, synthesis, and biological evaluation of a novel class of cannabinergic ligands, namely C1'-azacycloalkyl hexahydrocannabinols. Our synthetic approaches utilize an advanced common chiral intermediate triflate from which all analogues could be derived. Key synthetic steps involve microwave-assisted Liebeskind-Srogl C-C cross-coupling and palladium-catalyzed decarboxylative coupling reactions. The C1'-N-methylazetidinyl and C1'-N-methylpyrrolidinyl analogues were found to be high affinity ligands for the CB1 and CB2 cannabinoid receptors.

Novel C-Ring-Hydroxy-Substituted Controlled Deactivation Cannabinergic Analogues.

In pursuit of safer controlled-deactivation cannabinoids with high potency and short duration of action, we report the design, synthesis, and pharmacological evaluation of novel C9- and C11-hydroxy-substituted hexahydrocannabinol (HHC) and tetrahydrocannabinol (THC) analogues in which a seven atom long side chain, with or without 1'-substituents, carries a metabolically labile 2',3'-ester group. Importantly, in vivo studies validated our controlled deactivation approach in rodents and non-human primates. The lead molecule identified here, namely, butyl-2-[(6aR,9R,10aR)-1-hydroxy-9-(hydroxymethyl)-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl]-2-methylpropanoate (AM7499), was found to exhibit remarkably high in vitro and in vivo potency with shorter duration of action than the currently existing classical cannabinoid agonists.

A high efficacy cannabinergic ligand (AM4054) used as a discriminative stimulus: Generalization to other adamantyl analogs and Δ(9)-THC in rats.

In addition to endogenous lipids, the two main cloned receptors (CB1R and CB2R) of the endocannabinoid signaling system (ECS) can be activated (and blocked) by various exogenous ligands. A relatively novel template for CB1R activators contains an adamantyl moiety as a key structural subunit, the first being the cannabinergic AM411. Additional chemical optimization efforts using the classical tricyclic scaffold led to AM4054. Here we explored the in vivo consequences of novel adamantyl analogs in rats trained to recognize the effects of the potent adamantyl cannabinergic AM4054. Rats were trained to discriminate between AM4054 (0.1mg/kg) and vehicle. Three AM4054 analogs and Δ(9)-THC were tested for generalization (substitution) and antagonism was assessed with rimonabant. We found that all cannabinergics resulted in response generalization to the target stimulus AM4054. The order of potency was: AM4054≥AM4083≥AM4050>AM4089>Δ(9)-THC. The CB1R antagonist/inverse agonist rimonabant blocked the discriminative stimulus effects of AM4054. Thus the examined structural modifications affected binding affinities but did not markedly change potencies with the exception of AM4089. In vitro (cAMP assay) functional data have suggested that AM4089 behaves as a partial rather than as a full agonist at CB1R which could explain its lower potency compared to AM4054 (Thakur et al., 2013). The 9ß-formyl functionality at C-9 position was identified as an important pharmacophore yielding high in vivo potency. Antagonism by rimonabant suggested CB1R mediation.

Tolerance to the Diuretic Effects of Cannabinoids and Cross-Tolerance to a κ-Opioid Agonist in THC-Treated Mice.

Daily treatment with cannabinoids results in tolerance to many, but not all, of their behavioral and physiologic effects. The present studies investigated the effects of 7-day exposure to 10 mg/kg daily of Δ(9)-tetrahydrocannabinol (THC) on the diuretic and antinociceptive effects of THC and the synthetic cannabinoid AM4054. Comparison studies determined diuretic responses to the κ-opioid agonist U50,488 and furosemide. After determination of control dose-response functions, mice received 10 mg/kg daily of THC for 7 days, and dose-response functions were re-determined 24 hours, 7 days, or 14 days later. THC and AM4054 had biphasic diuretic effects under control conditions with maximum effects of 30 and 35 ml/kg of urine, respectively. In contrast, antinociceptive effects of both drugs increased monotonically with dose to >90% of maximal possible effect. Treatment with THC produced 9- and 7-fold rightward shifts of the diuresis and antinociception dose-response curves for THC and, respectively, 7- and 3-fold rightward shifts in the AM4054 dose-response functions. U50,488 and furosemide increased urine output to >35 ml/kg under control conditions. The effects of U50,488 were attenuated after 7-day treatment with THC, whereas the effects of furosemide were unaltered. Diuretic effects of THC and AM4054 recovered to near-baseline levels within 14 days after stopping daily THC injections, whereas tolerance to the antinociceptive effects persisted longer than 14 days. The tolerance induced by 7-day treatment with THC was accompanied by a 55% decrease in the Bmax value for cannabinoid receptors (CB1). These data indicate that repeated exposure to THC produces similar rightward shifts in the ascending and descending limbs of cannabinoid diuresis dose-effect curves and to antinociceptive effects while resulting in a flattening of the U50,488 diuresis dose-effect function.

Effects of a novel CB1 agonist on visual attention in male rats: role of strategy and expectancy in task accuracy.

The effects of cannabinoid CB1 agonists (including Δ9-tetrahydrocannabinol, the main psychoactive component of marijuana) on attention are uncertain, with reports of impairments, no effects, and occasionally performance enhancements. To better understand these effects, we sought to uncover a role of changing online (within-session) strategy as a possible mediator of the effects of the novel, potent CB1 agonist AM 4054, on a model of sustained attention in male Sprague-Dawley rats. In this operant, two-choice reaction time (RT) task, AM 4054 decreased accuracy in an asymmetric manner; that is, performance was spared on one lever but impaired on the other. Furthermore, this pattern was enhanced by the outcome of the previous trial such that AM 4054 strengthened a win-stay strategy on the "preferred" lever and a lose-shift strategy on the "nonpreferred" lever. This pattern is often found in tests of expectancy; therefore, in a second experiment AM 4054 enhanced expectancy that we engendered by altering the probability of the two stimulus cues. Accuracy was impaired in reporting the less frequent cue, but only after two or more presentations of the more frequent cue. Taking the results of the experiments together, AM 4054 engendered expectancy by increasing the role of previous trial location and outcome on performance of future trials, diminishing stimulus control (and therefore, accuracy). This novel effect of CB1 receptor agonism may contribute to the deleterious effects of cannabinoids on attention.

Behavioral effects of the novel potent cannabinoid CB1 agonist AM 4054.

Due to the ubiquity of the CB1 cannabinoid receptor throughout the nervous system, as well as the many potential therapeutic uses of CB1 agonist-based interventions, it is desirable to synthesize novel probes of the CB1 receptor. Here, the acute behavioral effects of systemic (i.p.) administration of the putative novel CB1 full agonist AM 4054 were tested in rats. In Experiment 1, a dose range (0.15625-1.25 mg/kg) of AM 4054 produced effects consistent with CB1 agonism in the cannabinoid tetrad of tasks in rats, including induction of analgesia, catalepsy, hypothermia, and locomotor suppression. These effects were reversed with the CB1-selective inverse agonist AM 251 in Experiment 2, indicating that AM 4054 produced CB1 receptor-mediated effects. Analysis of open-field activity indicated that the reduction in locomotion is more consistent with general motor slowing than anxiogenesis. AM 4054 (0.0625-0.5 mg/kg) also dose-dependently reduced fixed-ratio 5 (FR5) operant responding for food in Experiment 3, and microanalysis of the timing and rate of lever pressing indicated a pattern of suppression similar to other CB1 agonists. Minimum doses of AM 4054 (0.125-0.3125 mg/kg) required to produce significant effects in these behavioral assays were lower than those of many CB1 agonists. It is likely that AM 4054 is a potent pharmacological tool for assessment of cannabinoid receptor function.

In vitro stability of free and glucuronidated cannabinoids in blood and plasma following controlled smoked cannabis.

BACKGROUND: Blood and plasma cannabinoid stability is important for test interpretation and is best studied in authentic rather than fortified samples. METHODS: Low and high blood and plasma pools were created for each of 10 participants after they smoked a cannabis cigarette. The stabilities of Δ(9)-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), 11-nor-9-carboxy-THC (THCCOOH), cannabidiol (CBD), cannabinol (CBN), THC-glucuronide, and THCCOOH-glucuronide were determined after 1 week at room temperature; 1, 2, 4, 12, and 26 (±2) weeks at 4 °C; and 1, 2, 4, 12, 26 (±2), and 52 (±4) weeks at -20 °C. Stability was assessed by Friedman test. RESULTS: Numbers of THC-glucuronide and CBD-positive blood samples were insufficient to assess stability. In blood, 11-OH-THC and CBN were stable for 1 week at room temperature, whereas THC and THCCOOH-glucuronide decreased and THCCOOH increased. In blood, THC, THCCOOH-glucuronide, THCCOOH, 11-OH-THC, and CBN were stable for 12, 4, 4, 12, and 26 weeks, respectively, at 4 °C and 12, 12, 26, 26, and 52 weeks at -20 °C. In plasma, THC-glucuronide, THC, CBN, and CBD were stable for 1 week at room temperature, whereas THCCOOH-glucuronide and 11-OH-THC decreased and THCCOOH increased. In plasma, THC-glucuronide, THC, THCCOOH-glucuronide, THCCOOH, 11-OH-THC, CBN, and CBD were stable for 26, 26, 2, 2, 26, 12, and 26 weeks, respectively, at 4 °C and 52, 52, 26, 26, 52, 52, and 52 weeks, respectively, at -20 °C. CONCLUSIONS: Blood and plasma samples should be stored at -20 °C for no more than 3 and 6 months, respectively, to assure accurate cannabinoid quantitative results.

Diuretic effects of cannabinoids.

In vivo effects of cannabinoid (CB) agonists are often assessed using four well-established measures: locomotor activity, hypothermia, cataleptic-like effects, and analgesia. The present studies demonstrate that doses of CB agonists that produce these effects also reliably increase diuresis. Diuretic effects of several CB agonists were measured in female rats over 2 hours immediately after drug injection, and results were compared with hypothermic effects. Direct-acting CB1 agonists, including Δ(9)-tetrahydrocannabinol, WIN 55,212 [R-(1)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate], AM2389 [9ß-hydroxy-3-(1-hexyl-cyclobut-1-yl)-hexahydrocannabinol], and AM4054 [9ß-(hydroxymethyl)-3-(1-adamantyl)-hexahydrocannabinol], produced dose-dependent increases in diuresis and decreases in colonic temperature, with slightly lower ED(50) values for diuresis than for hypothermia. The highest doses of cannabinoid drugs yielded, on average, 26-32 g/kg urine; comparable effects were obtained with 10 mg/kg furosemide and 3.0 mg/kg trans-(-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U50-488). Methanandamide (10.0 mg/kg) had lesser effect than other CB agonists, and the CB2 agonist AM1241 [1-(methylpiperidin-2-ylmethyl)-3-(2-iodo-5-nitrobenzoyl)indole], the anandamide transport inhibitor AM404, and the CB antagonist rimonabant did not have diuretic effects. In further studies, the diuretic effects of the CB1 agonist AM4054 were similar in male and female rats, displayed a relatively rapid onset to action, and were dose-dependently antagonized by 30 minutes pretreatment with rimonabant, but not by the vanilloid receptor type I antagonist capsazepine, nor were the effects of WIN 55,212 antagonized by the CB2 antagonist AM630 [(6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl) methanone)]. These data indicate that cannabinoids have robust diuretic effects in rats that are mediated via CB1 receptor mechanisms.

Novel hexahydrocannabinol analogs as potential anti-cancer agents inhibit cell proliferation and tumor angiogenesis.

Both natural and synthetic cannabinoids have been shown to suppress the growth of tumor cells in culture and in animal models by affecting key signaling pathways including angiogenesis, a pivotal step in tumor growth, invasion, and metastasis. In our search for cannabinoid-like anticancer agents devoid of psychoactive side effects, we synthesized and evaluated the anti-angiogenic effects of a novel series of hexahydrocannabinol analogs. Among these, two analogs LYR-7 [(9S)-3,6,6,9-tetramethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol] and LYR-8 [(1-((9S)-1-hydroxy-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-2-yl)ethanone)] were selected based on their anti-angiogenic activity and lack of binding affinity for cannabinoid receptors. Both LYR-7 and LYR-8 inhibited VEGF-induced proliferation, migration, and capillary-like tube formation of HUVECs in a concentration-dependent manner. The inhibitory effect of the compounds on cell proliferation was more selective in endothelial cells than in breast cancer cells (MCF-7 and tamoxifen-resistant MCF-7). We also noted effective inhibition of VEGF-induced new blood vessel formation by the compounds in the in vivo chick chorioallantoic membrane (CAM) assay. Furthermore, both LYR analogs potently inhibited VEGF production and NF-κB transcriptional activity in cancer cells. Additionally, LYR-7 or LYR-8 strongly inhibited breast cancer cell-induced angiogenesis and tumor growth. Together, these results suggest that novel synthetic hexahydrocannabinol analogs, LYR-7 and LYR-8, inhibit tumor growth by targeting VEGF-mediated angiogenesis signaling in endothelial cells and suppressing VEGF production and cancer cell growth.

Novel 1',1'-chain substituted hexahydrocannabinols: 9ß-hydroxy-3-(1-hexyl-cyclobut-1-yl)-hexahydrocannabinol (AM2389) a highly potent cannabinoid receptor 1 (CB1) agonist.

In pursuit of a more detailed understanding of the structural requirements for the key side chain cannabinoid pharmacophore, we have extended our SAR to cover a variety of conformationally modified side chains within the 9-keto and 9-hydroxyl tricyclic structures. Of the compounds described here, those with a seven-atom long side chain substituted with a cyclopentyl ring at C1' position have very high affinities for both CB1 and CB2 (0.97 nM < K(i) < 5.25 nM), with no preference for either of the two receptors. However, presence of the smaller cyclobutyl group at the C1' position leads to an optimal affinity and selectivity interaction with CB1. Thus, two of the C1'-cyclobutyl analogues, namely, (6aR,10aR)-3-(1-hexyl-cyclobut-1-yl)-6,6a,7,8,10,10a-hexahydro-1-hydroxy-6,6-dimethyl-9H-dibenzo[b,d]pyran-9-one and (6aR,9R,10aR)-3-(1-hexyl-cyclobut-1-yl)-6a,7,8,9,10,10a-hexahydro-6,6-dimethyl-6H-dibenzo[b,d]pyran-1,9 diol (7e-ß, AM2389), exhibited remarkably high affinities (0.84 and 0.16 nM, respectively) and significant selectivities (16- and 26-fold, respectively) for CB1. Compound 7e-ß was found to exhibit exceptionally high in vitro and in vivo potency with a relatively long duration of action.

Acute effects of Delta9-tetrahydrocannabinol and standardized cannabis extract on the auditory evoked mismatch negativity.

Reduced amplitudes of auditory evoked mismatch negativity (MMN) have often been found in schizophrenic patients, indicating deficient auditory information processing and working memory. Cannabis-induced psychotic states may resemble schizophrenia. Currently, there are discussions focusing on the close relationship between cannabis, the endocannabinoid and dopaminergic system, and the onset of schizophrenic psychosis. This study investigated the effects of cannabis on MMN amplitude in 22 healthy volunteers (age 28+/-6 years, 11 male) by comparing Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and standardized cannabis extract containing Delta(9)-THC and cannabidiol (CBD) in a prospective, double-blind, placebo-controlled cross-over design. The MMNs resulting from 1000 auditory stimuli were recorded by 32 channel EEG. The standard stimuli were 1000 Hz, 80 dB SPL, and 100 ms duration. The deviant stimuli differed in frequency (1500 Hz). Significantly greater MMN amplitude values at central electrodes were found under cannabis extract, but not under Delta(9)-THC. There were no significant differences between MMN amplitudes at frontal electrodes. MMN amplitudes at central electrodes were significantly correlated with 11-OH-THC concentration, the most important psychoactive metabolite of Delta(9)-THC. Since the main difference between Delta(9)-THC and standardized cannabis extract is CBD, which seems to have neuroprotective and anti-psychotic properties, it can be speculated whether the greater MMN amplitude that may imply higher cortical activation and cognitive performance is related to the positive effects of CBD. This effect may be relevant for auditory cortex activity in particular because only MMN amplitudes at the central, but not at the frontal electrodes were enhanced under cannabis.

Synthesis and spectroscopic characterization of cannabinolic acid.

Cannabinoids, the main constituents of the cannabis plant, are being increasingly studied for their medicinal properties. Cannabinolic acid (CBNA; 1) was synthesized from tetrahydrocannabinolic acid (THCA; 2), a major constituent of the cannabis plant, by aromatization using selenium dioxide mixed with trimethylsilyl polyphosphate as catalyst in chloroform. Purification was achieved by centrifugal partition chromatography, and the final product had a purity of over 96% by GC analysis. Spectroscopic data on CBNA such as 1H-NMR and IR, and molar extinction coefficients, as well as chromatographic data are presented as useful references for further research on CBNA. The developed method allows production of CBNA on a preparative scale, making it available for further studies on its biological activities as well as use as a reference standard for analytical procedures.

Synthesis and pharmacology of 11-nor-1-methoxy-9-hydroxyhexahydrocannabinols and 11-nor-1-deoxy-9-hydroxyhexahydrocannabinols: new selective ligands for the cannabinoid CB2 receptor.

Fourteen novel CB2 receptor selective cannabinoids were synthesized via initial Lewis acid catalyzed rearrangement of resorcinol precursors to obtain the cannabinoid moiety. These are the 1-methoxy-9-hydroxyhexahydrocannabinols and the 1-deoxy-9-hydroxyhexahydrocannabinols, with 1',1'-dimethylalkyl side chains of four to seven carbon atoms at C-3 of the cannabinoid nucleus. The cannabinols synthesized and described in this paper all exhibit greater affinity for the CB2 receptor than for the CB1 receptor. Exceptionally high CB2 affinity was observed for 1-deoxy-9beta-hydroxy-dimethylhexylhexahydrocannabinol (JWH-361, 9, n = 3) K(i) = 2.7 nM and 1-deoxy-9beta-hydroxydimethylpentylhexahydrocannabinol (JWH-300, 9, n = 2) K(i) = 5.3 nM. In general, the stereochemistry of the 9-hydroxy group is important and the beta-orientation enhances both CB2 receptor affinity and selectivity.

(-)-7'-Isothiocyanato-11-hydroxy-1',1'-dimethylheptylhexahydrocannabinol (AM841), a high-affinity electrophilic ligand, interacts covalently with a cysteine in helix six and activates the CB1 cannabinoid receptor.

The CB1 cannabinoid receptor has been shown to play important physiological roles in the central nervous system, as well as peripherally, and is a target for development of therapeutic medications. To gain insight on the ligand binding site(s) and structural features of activation, we designed and synthesized (-)-7'-isothiocyanato-11-hydroxy-1',1'-dimethylheptylhexahydrocannabinol (AM841), a classical cannabinoid affinity label that incorporates an isothiocyanate substituent as an electrophilic reactive group capable of interacting irreversibly with a suitably located and properly oriented nucleophilic amino acid residue at or near the binding site. To obtain evidence for the site of covalent attachment of AM841, C6.47, identified in part by interactive ligand docking, was mutated to serine, alanine, and leucine to reduce or eliminate the nucleophilic character. Wild-type (WT) and mutant CB1 receptors were evaluated for their abilities to recognize a series of cannabinergic ligands. Each bound comparably to WT, excluding C6.47L, which displayed a reduced affinity for 3H-labeled (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol (CP55940), AM841, 11-hydroxy-1',1'-dimethylheptylhexahydrocannabinol (AM4056), and (-)-7'-bromo-11-hydroxy-1',1'-dimethylheptylhexahydrocannabinol (AM4043) and an improvement in affinity for (-)-trans-delta9-tetrahydrocannabinol (delta9-THC). The affinity of 3H-labeled [2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](naphthyl)methanone (WIN55212-2) was unchanged across all mutants. It is noteworthy that AM841 was shown to bind irreversibly to WT CB1 but exhibited no covalent attachment with the mutants and behaved as an agonist suggesting irreversible attachment to C6.47 maintains CB1 in its active state. The evidence presented identifies C6.47 as the site of covalent bond formation with AM841 and combined with the binding data fully supports the molecular modeling. These studies present the first report of tandem applications of affinity labeling, site-directed mutagenesis, and interactive ligand docking for CB1.

Novel cannabinol probes for CB1 and CB2 cannabinoid receptors.

The observation that the phenolic hydroxyl of THCs was important for binding to the CB1 receptor but not as critical for binding to the CB2 receptor prompted us to extend this finding to the cannabinol (CBN) series. To study the SAR of CBN analogues, CBN derivatives with substitution at the C-1, C-3, and C-9 positions were chosen since these positions have played a key role in the SAR of THCs. CBN-3-(1',1'-dimethylheptyl) analogues were prepared by sulfur dehydrogenation of Delta(8)-THC-3-(1',1'-dimethylheptyl) analogues. 9-Substituted CBN analogues were prepared by the standard sulfur dehydrogenation of 9-substituted Delta(8)-THC analogues (Scheme 1), which in turn were prepared following our previous procedure using selenium dioxide oxidation of the corresponding Delta(8)-THCs followed by sodium chlorite oxidation to give the 9-carboxy-Delta(8)-THC derivatives. 11-Hydroxy-CBN analogues were prepared from the corresponding 9-carbomethoxy-CBN analogues by reduction with LiAlH(4). Deoxy-CBN analogue 14 was prepared from the corresponding Delta(8)-THC analogue 11 by conversion of the phenolic hydroxyl to the phosphate derivative 12, followed by lithium ammonia reduction to provide the deoxy-Delta(8)-THC analogue 13, which in turn was dehydrogenated with sulfur to provide the deoxy-CBN analogue 14 (Scheme 2). The various analogues were assayed for binding both to the brain and the peripheral cannabinoid receptors (CB1 and CB2). We have found that the binding profile differs widely between the CBN and the THC series. Specifically, in the CBN series the removal of the phenolic hydroxyl decreases binding affinity to both the CB1 and CB2 receptors, whereas in the THC series, CB1 affinity is selectively reduced. Thus, in the CBN series, the selectivity of binding observed with the removal of the hydroxy group is decreased severalfold as compared to what occurs in the THC series. Generally, high affinity for the CB2 receptor was found in analogues when the phenolic hydroxyl was present. The 3-(1', 1'-dimethylheptyl) derivatives were found to have much higher affinities than the CBN analogues, which is in complete agreement with previously reported work by Rhee et al.