Activation and Signaling Mechanism Revealed by Cannabinoid Receptor-Gi Complex Structures.

Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with Gi, as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric modulating role.

Synthon-based ligand discovery in virtual libraries of over 11 billion compounds.

Structure-based virtual ligand screening is emerging as a key paradigm for early drug discovery owing to the availability of high-resolution target structures1-4 and ultra-large libraries of virtual compounds5,6. However, to keep pace with the rapid growth of virtual libraries, such as readily available for synthesis (REAL) combinatorial libraries7, new approaches to compound screening are needed8,9. Here we introduce a modular synthon-based approach-V-SYNTHES-to perform hierarchical structure-based screening of a REAL Space library of more than 11 billion compounds. V-SYNTHES first identifies the best scaffold-synthon combinations as seeds suitable for further growth, and then iteratively elaborates these seeds to select complete molecules with the best docking scores. This hierarchical combinatorial approach enables the rapid detection of the best-scoring compounds in the gigascale chemical space while performing docking of only a small fraction (<0.1%) of the library compounds. Chemical synthesis and experimental testing of novel cannabinoid antagonists predicted by V-SYNTHES demonstrated a 33% hit rate, including 14 submicromolar ligands, substantially improving over a standard virtual screening of the Enamine REAL diversity subset, which required approximately 100 times more computational resources. Synthesis of selected analogues of the best hits further improved potencies and affinities (best inhibitory constant (Ki) = 0.9 nM) and CB2/CB1 selectivity (50-200-fold). V-SYNTHES was also tested on a kinase target, ROCK1, further supporting its use for lead discovery. The approach is easily scalable for the rapid growth of combinatorial libraries and potentially adaptable to any docking algorithm.

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.

Differential Enantiomer-Specific Signaling of Cannabidiol at CB1 Receptors.

The two main constituents of cannabis are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). While Δ9-THC pharmacology has been studied extensively, CBD-long considered inactive-is now the subject of vigorous research related to epilepsy, pain, and inflammation and is popularly embraced as a virtual cure-all. However, our understanding of CBD pharmacology remains limited, although CBD inhibits cannabinoid CB1 receptor signaling, likely as a negative allosteric modulator. Cannabis synthesizes (-)-CBD, but CBD can also exist as an enantiomer, (+)-CBD. We enantioselectively synthesized both CBD enantiomers using established conditions and describe here a new, practical, and reliable, NMR-based method for confirming the enantiomeric purity of two CBD enantiomers. We also investigated the pharmacology of (+)-CBD in autaptic hippocampal neurons, a well-characterized neuronal model of endogenous cannabinoid signaling, and in CHO-K1 cells. We report the inhibition constant for displacing CP55,940 at CB1 by (+)-CBD, is 5-fold lower than (-)-CBD. We find that (+)-CBD is ∼10 times more potent at inhibiting depolarization-induced suppression of excitation (DSE), a form of endogenous cannabinoid-mediated retrograde synaptic plasticity. (+)-CBD also inhibits CB1 suppression of cAMP accumulation but with less potency, indicating that the signaling profiles of the enantiomers differ in a pathway-specific manner. In addition, we report that (+)-CBD stereoselectively and potently activates the sphingosine-1 phosphate (S1P) receptors, S1P1 and S1P3 These results provide an attractive method for synthesizing and distinguishing enantiomers of CBD and related phytocannabinoids and provide further evidence that these enantiomers have their own unique and interesting signaling properties. SIGNIFICANCE STATEMENT: Cannabidiol (CBD) is the subject of considerable scientific and popular interest, but we know little of the enantiomers of CBD. We find that the enantiomer (+)-CBD is substantially more potent inhibitor of cannabinoid CB1 receptors and that it activates sphingosine-1-phosphate receptors in an enantiomer-specific manner; we have additionally developed an improved method for the synthesis of enantiomers of CBD and related compounds.

Evidence for spontaneous cannabinoid withdrawal in mice.

Although the behavioral effects of acute and chronic exposure to cannabinoids have been extensively studied in mice, spontaneous withdrawal following exposure to cannabinoids has not been well characterized in this species. To address this issue, different groups of mice were treated for 5 days with saline, 20-36 mg/kg/day of the CB partial agonist Δ9-tetrahydrocannabinol (Δ9-THC), or 0.06-0.1 mg/kg/day of the CB high-efficacy agonist AM2389. Initial studies assessed changes in observable behavior (paw tremors) that were scored from the recordings taken at 4 or 24 h after the last injection. Subsequently, radiotelemetry was used to continuously measure body temperature and locomotor activity before (baseline), during, and after the 5-day dosing regimens. Results show that increases in paw tremors occurred following 5-day exposure to AM2389 or Δ9-THC. In telemetry studies, acute AM2389 or THC decreased both temperature and activity. Rapid tolerance occurred to the hypothermic effects of the cannabinoids, whereas locomotor activity continued to be suppressed following each drug injection. In contrast, increases in locomotor activity were evident 12-72 h after discontinuing daily injections of either 0.06 or 0.1 mg/kg/day AM2389. Increases in locomotor activity were also noted in mice treated daily with 30 or 36, but not 20 mg/kg/day Δ9-THC; these effects were smaller and appeared later than effects seen in AM2389-treated mice. These results indicate that the discontinuation of daily treatment with a CB high-efficacy agonist will yield evidence of spontaneous withdrawal that may reflect prior dependence, and that the degree of cannabinoid dependence may vary in relation to the dose or efficacy of the agonist injected daily.

Altered sleep during spontaneous cannabinoid withdrawal in male mice.

Cessation of cannabinoid use in humans often leads to a withdrawal state that includes sleep disruption. Despite important health implications, little is known about how cannabinoid abstention affects sleep architecture, in part because spontaneous cannabinoid withdrawal is difficult to model in animals. In concurrent work we report that repeated administration of the high-efficacy cannabinoid 1 (CB1) receptor agonist AM2389 to mice for 5 days led to heightened locomotor activity and paw tremor following treatment discontinuation, potentially indicative of spontaneous cannabinoid withdrawal. Here, we performed parallel studies to examine effects on sleep. Using implantable electroencephalography (EEG) and electromyography (EMG) telemetry we examined sleep and neurophysiological measures before, during, and after 5 days of twice-daily AM2389 injections. We report that AM2389 produces decreases in locomotor activity that wane with repeated treatment, whereas discontinuation produces rebound increases in activity that persist for several days. Likewise, AM2389 initially produces profound increases in slow-wave sleep (SWS) and decreases in rapid eye movement (REM) sleep, as well as consolidation of sleep. By the third AM2389 treatment, this pattern transitions to decreases in SWS and total time sleeping. This pattern persists following AM2389 discontinuation and is accompanied by emergence of sleep fragmentation. Double-labeling immunohistochemistry for hypocretin/orexin (a sleep-regulating peptide) and c-Fos (a neuronal activity marker) in lateral hypothalamus revealed decreases in c-Fos/orexin+ cells following acute AM2389 and increases following discontinuation, aligning with the sleep changes. These findings indicate that AM2389 profoundly alters sleep in mice and suggest that sleep disruption following treatment cessation reflects spontaneous cannabinoid withdrawal.

Blockade of CB1 or Activation of CB2 Cannabinoid Receptors Is Differentially Efficacious in the Treatment of the Early Pathological Events in Streptozotocin-Induced Diabetic Rats.

Oxidative stress, neurodegeneration, neuroinflammation, and vascular leakage are believed to play a key role in the early stage of diabetic retinopathy (ESDR). The aim of this study was to investigate the blockade of cannabinoid receptor 1 (CB1R) and activation of cannabinoid receptor 2 (CB2R) as putative therapeutics for the treatment of the early toxic events in DR. Diabetic rats [streptozotocin (STZ)-induced] were treated topically (20 µL, 10 mg/mL), once daily for fourteen days (early stage DR model), with SR141716 (CB1R antagonist), AM1710 (CB2R agonist), and the dual treatment SR141716/AM1710. Immunohistochemical-histological, ELISA, and Evans-Blue analyses were performed to assess the neuroprotective and vasculoprotective properties of the pharmacological treatments on diabetes-induced retinal toxicity. Activation of CB2R or blockade of CB1R, as well as the dual treatment, attenuated the nitrative stress induced by diabetes. Both single treatments protected neural elements (e.g., RGC axons) and reduced vascular leakage. AM1710 alone reversed all toxic insults. These findings provide new knowledge regarding the differential efficacies of the cannabinoids, when administered topically, in the treatment of ESDR. Cannabinoid neuroprotection of the diabetic retina in ESDR may prove therapeutic in delaying the development of the advanced stage of the disease.

Oxa-adamantyl cannabinoids.

As a continuation of earlier work on classical cannabinoids bearing bulky side chains we report here the design, synthesis, and biological evaluation of 3'-functionalized oxa-adamantyl cannabinoids as a novel class of cannabinergic ligands. Key synthetic steps involve nucleophilic addition/transannular cyclization of aryllithium to epoxyketone in the presence of cerium chloride and stereoselective construction of the tricyclic cannabinoid nucleus. The synthesis of the oxa-adamantyl cannabinoids is convenient, and amenable to scale up allowing the preparation of these analogs in sufficient quantities for detailed in vitro evaluation. The novel oxa-adamantyl cannabinoids reported here were found to be high affinity ligands for the CB1 and CB2 cannabinoid receptors. In the cyclase assay these compounds were found to behave as potent and efficacious CB1 receptor agonists. Isothiocyanate analog AM10504 is capable of irreversibly labeling both the CB1 and CB2 receptors.

Antiemetic Effects of Cannabinoid Agonists in Nonhuman Primates.

Attenuating emesis elicited by both disease and medical treatments of disease remains a critical public health challenge. Although cannabinergic medications have been used in certain treatment-resistant populations, Food and Drug Administration-approved cannabinoid antiemetics are associated with undesirable side effects, including cognitive disruption, that limit their prescription. Previous studies have shown that a metabolically stable analog of the endocannabinoid anandamide, methanandamide (mAEA), may produce lesser cognitive disruption than that associated with the primary psychoactive constituent in cannabis, Δ9-tetrahydrocannabinol (Δ9-THC), raising the possibility that endocannabinoids may offer a therapeutic advantage over currently used medications. The present studies were conducted to evaluate this possibility by comparing the antiemetic effects of Δ9-THC (0.032-0.1 mg/kg) and mAEA (3.2-10.0 mg/kg) against nicotine- and lithium chloride (LiCl)-induced emesis and prodromal hypersalivation in squirrel monkeys. Pretreatment with 0.1 mg/kg Δ9-THC blocked nicotine-induced emesis and reduced hypersalivation in all subjects and blocked LiCl-induced emesis and reduced hypersalivation in three of four subjects. Pretreatment with 10 mg/kg mAEA blocked nicotine-induced emesis in three of four subjects and LiCl-induced emesis in one of four subjects and reduced both nicotine- and LiCl-induced hypersalivation. Antiemetic effects of Δ9-THC and mAEA were reversed by rimonabant pretreatment, providing verification of cannabinoid receptor type 1 mediation. These studies systematically demonstrate for the first time the antiemetic effects of cannabinoid agonists in nonhuman primates. Importantly, although Δ9-THC produced superior antiemetic effects, the milder cognitive effects of mAEA demonstrated in previous studies suggest that it may provide a favorable treatment option under clinical circumstances in which antiemetic efficacy must be balanced against side effect liability. SIGNIFICANCE STATEMENT: Emesis has significant evolutionary value as a defense mechanism against ingested toxins; however, it is also one of the most common adverse symptoms associated with both disease and medical treatments of disease. The development of improved antiemetic pharmacotherapies has been impeded by a paucity of animal models. The present studies systematically demonstrate for the first time the antiemetic effects of the phytocannabinoid Δ9-tetrahydrocannabinol and endocannabinoid analog methanandamide in nonhuman primates.

Cannabinoid Antagonist Drug Discrimination in Nonhuman Primates.

Despite a growing acceptance that withdrawal symptoms can emerge following discontinuation of cannabis products, especially in high-intake chronic users, there are no Food and Drug Administration (FDA)-approved treatment options. Drug development has been hampered by difficulties studying cannabis withdrawal in laboratory animals. One preclinical approach that has been effective in studying withdrawal from drugs in several pharmacological classes is antagonist drug discrimination. The present studies were designed to examine this paradigm in squirrel monkeys treated daily with the long-acting CB1 agonist AM2389 (0.01 mg/kg) and trained to discriminate the CB1 inverse agonist/antagonist rimonabant (0.3 mg/kg) from saline. The discriminative-stimulus effects of rimonabant were both dose and time dependent and, importantly, could be reproduced by discontinuation of agonist treatment. Antagonist substitution tests with the CB1 neutral antagonists AM4113 (0.03-0.3 mg/kg), AM6527 (0.03-1.0 mg/kg), and AM6545 (0.03-1.0 mg/kg) confirmed that the rimonabant discriminative stimulus also could be reproduced by CB1 antagonists lacking inverse agonist action. Agonist substitution tests with the phytocannabinoid ∆9-tetrahydrocannabinol (0.1-1.0 mg/kg), synthetic CB1 agonists nabilone (0.01-0.1 mg/kg), AM4054 (0.01-0.03 mg/kg), K2/Spice compound JWH-018 (0.03-0.3 mg/kg), FAAH-selective inhibitors AM3506 (0.3-5.6 mg/kg), URB597 (3.0-5.6 mg/kg), and nonselective FAAH/MGL inhibitor AM4302 (3.0-10.0 mg/kg) revealed that only agonists with CB1 affinity were able to reduce the rimonabant-like discriminative stimulus effects of withholding daily agonist treatment. Although the present studies did not document physiologic disturbances associated with withdrawal, the results are consistent with the view that the cannabinoid antagonist drug discrimination paradigm provides a useful screening procedure for examining the ability of candidate medications to attenuate the interoceptive stimuli provoked by cannabis discontinuation. SIGNIFICANCE STATEMENT: Despite a growing acceptance that withdrawal symptoms can emerge following the discontinuation of cannabis products, especially in high-intake chronic users, there are no FDA-approved pharmacotherapies to assist those seeking treatment. The present studies systematically examined cannabinoid antagonist drug discrimination, a preclinical animal model that is designed to appraise the ability of candidate medications to attenuate the interoceptive effects that accompany abrupt cannabis abstinence.

Synthesis of Functionalized Cannabilactones.

A new approach to synthesize cannabilactones using Suzuki cross-coupling reaction followed by one-step demethylation-cyclization is presented. The two key cannabilactone prototypes AM1710 and AM1714 were obtained selectively in high overall yields and in a lesser number of synthetic steps when compared to our earlier synthesis. The new approach expedited the synthesis of cannabilactone analogs with structural modifications at the four potential pharmacophoric regions.

Cannabinoid CB2 Agonist AM1710 Differentially Suppresses Distinct Pathological Pain States and Attenuates Morphine Tolerance and Withdrawal.

AM1710 (3-(1,1-dimethyl-heptyl)-1-hydroxy-9-methoxy-benzo(c) chromen-6-one), a cannabilactone cannabinoid receptor 2 (CB2) agonist, suppresses chemotherapy-induced neuropathic pain in rodents without producing tolerance or unwanted side effects associated with CB1 receptors; however, the signaling profile of AM1710 remains incompletely characterized. It is not known whether AM1710 behaves as a broad-spectrum analgesic and/or suppresses the development of opioid tolerance and physical dependence. In vitro, AM1710 inhibited forskolin-stimulated cAMP production and produced enduring activation of extracellular signal-regulated kinases 1/2 phosphorylation in human embryonic kidney (HEK) cells stably expressing mCB2. Only modest species differences in the signaling profile of AM1710 were observed between HEK cells stably expressing mCB2 and hCB2. In vivo, AM1710 produced a sustained inhibition of paclitaxel-induced allodynia in mice. In paclitaxel-treated mice, a history of AM1710 treatment (5 mg/kg per day × 12 day, i.p.) delayed the development of antinociceptive tolerance to morphine and attenuated morphine-induced physical dependence. AM1710 (10 mg/kg, i.p.) did not precipitate CB1 receptor-mediated withdrawal in mice rendered tolerant to Δ9-tetrahydrocannabinol, suggesting that AM1710 is not a functional CB1 antagonist in vivo. Furthermore, AM1710 (1, 3, 10 mg/kg, i.p.) did not suppress established mechanical allodynia induced by complete Freund's adjuvant (CFA) or by partial sciatic nerve ligation (PSNL). Similarly, prophylactic and chronic dosing with AM1710 (10 mg/kg, i.p.) did not produce antiallodynic efficacy in the CFA model. By contrast, gabapentin suppressed allodynia in both CFA and PSNL models. Our results indicate that AM1710 is not a broad-spectrum analgesic agent in mice and suggest the need to identify signaling pathways underlying CB2 therapeutic efficacy to identify appropriate indications for clinical translation.

Chain Substituted Cannabilactones with Selectivity for the CB2 Cannabinoid Receptor.

In earlier work, we reported a novel class of CB2 selective ligands namely cannabilactones. These compounds carry a dimethylheptyl substituent at C3, which is typical for synthetic cannabinoids. In the current study with the focus on the pharmacophoric side chain at C3 we explored the effect of replacing the C1'-gem-dimethyl group with the bulkier cyclopentyl ring, and, we also probed the chain's length and terminal carbon substitution with bromo or cyano groups. One of the analogs synthesized namely 6-[1-(1,9-dihydroxy-6-oxo-6H-benzo[c]chromen-3-yl) cyclopentyl] hexanenitrile (AM4346) has very high affinity (Ki = 4.9 nM) for the mouse CB2 receptor (mCB2) and 131-fold selectivity for that target over the rat CB1 (rCB1). The species difference in the affinities of AM4346 between the mouse (m) and the human (h) CB2 receptors is reduced when compared to our first-generation cannabilactones. In the cyclase assay, our lead compound was found to be a highly potent and efficacious hCB2 receptor agonist (EC50 = 3.7 ± 1.5 nM, E(max) = 89%). We have also extended our structure-activity relationship (SAR) studies to include biphenyl synthetic intermediates that mimic the structure of the phytocannabinoid cannabinodiol.

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.

13-Methylarachidonic acid is a positive allosteric modulator of endocannabinoid oxygenation by cyclooxygenase.

Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonylethanolamide to prostaglandins, prostaglandin glyceryl esters, and prostaglandin ethanolamides, respectively. A structural homodimer, COX-2 acts as a conformational heterodimer with a catalytic and an allosteric monomer. Prior studies have demonstrated substrate-selective negative allosteric regulation of 2-AG oxygenation. Here we describe AM-8138 (13(S)-methylarachidonic acid), a substrate-selective allosteric potentiator that augments 2-AG oxygenation by up to 3.5-fold with no effect on AA oxygenation. In the crystal structure of an AM-8138·COX-2 complex, AM-8138 adopts a conformation similar to the unproductive conformation of AA in the substrate binding site. Kinetic analysis suggests that binding of AM-8138 to the allosteric monomer of COX-2 increases 2-AG oxygenation by increasing kcat and preventing inhibitory binding of 2-AG. AM-8138 restored the activity of COX-2 mutants that exhibited very poor 2-AG oxygenating activity and increased the activity of COX-1 toward 2-AG. Competition of AM-8138 for the allosteric site prevented the inhibition of COX-2-dependent 2-AG oxygenation by substrate-selective inhibitors and blocked the inhibition of AA or 2-AG oxygenation by nonselective time-dependent inhibitors. AM-8138 selectively enhanced 2-AG oxygenation in intact RAW264.7 macrophage-like cells. Thus, AM-8138 is an important new tool compound for the exploration of allosteric modulation of COX enzymes and their role in endocannabinoid metabolism.

Comparisons of Δ9-Tetrahydrocannabinol and Anandamide on a Battery of Cognition-Related Behavior in Nonhuman Primates.

The primary psychoactive ingredient of marijuana, Δ(9)-tetrahydrocannabinol (Δ(9)-THC), has medicinal value but also produces unwanted deleterious effects on cognitive function, promoting the search for improved cannabinergic therapeutics. The present studies used a battery of touchscreen procedures in squirrel monkeys to compare the effects of different types of cannabinergic drugs on several measures of performance including learning (repeated acquisition), cognitive flexibility (discrimination reversal), short-term memory (delayed matching-to-sample), attention (psychomotor vigilance), and motivation (progressive ratio). Drugs studied included the cannabinoid agonist Δ(9)-THC, fatty acid amide hydrolase (FAAH) inhibitor cyclohexylcarbamic acid 3-carbamoylbiphenyl-3-yl ester (URB597), and endocannabinoid anandamide and its stable synthetic analog methanandamide [(R)-(+)-arachidonyl-1'-hydroxy-2'-propylamide]. The effects of Δ(9)-THC and anandamide after treatment with the cannabinoid receptor type 1 inverse agonist/antagonist rimonabant [5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1Hpyrazole-3-carboxamide] and the FAAH inhibitor URB597, respectively, also were examined. The results showed the following: 1) Δ(9)-THC produced dose-related impairments of discrimination-based cognitive behavior with potency that varied across tasks (discriminative capability < learning < flexibility < short-term memory); 2) anandamide alone and URB597 alone were without effect on all endpoints; 3) anandamide following URB597 pretreatment and methanandamide had negligible effects on discriminative capability, learning, and reversal, but following large doses affected delayed matching-to-sample performance in some subjects; 4) all drugs, except anandamide and URB597, disrupted attention; and 5) progressive ratio breakpoints were generally unaffected by all drugs tested, suggesting little to no effect on motivation. Taken together, these data indicate that metabolically stable forms of anandamide may have lesser adverse effects on cognitive functions than Δ(9)-THC, possibly offering a therapeutic advantage in clinical settings.

Novel tail and head group prostamide probes.

We report the design and synthesis of novel prostaglandin-ethanolamide (PGE2-EA) analogs containing head and tail group modifications to aid in the characterization of a putative prostamide receptor(s). Our synthetic approach utilizes Horner-Wadsworth-Emmons and Wittig reactions to construct the head and the tail moieties of the key PGE2 precursor, which leads to the final products through a peptide coupling, Swern oxidation and HF/pyridine assisted desilylation. The synthesized analogs were shown not to interact significantly with endocannabinoid proteins and recombinant EP1, EP3 and EP4 receptors and suggest a yet to be identified prostamide receptor as their site(s) of action.

Design and synthesis of novel prostaglandin E2 ethanolamide and glycerol ester probes for the putative prostamide receptor(s).

Novel prostaglandin-ethanolamide (PGE2-EA) and glycerol ester (2-PGE2-G) analogs were designed and synthesized to aid in the characterization of a putative prostamide receptor. Our design incorporates the electrophilic isothiocyanato and the photoactivatable azido groups at the terminal tail position of the prototype. Stereoselective Wittig and Horner-Wadsworth-Emmons reactions install the head and the tail moieties of the PGE2 skeleton. The synthesis is completed using Mitsunobu azidation and peptide coupling as the key steps. A chemoenzymatic synthesis for the 2-PGE2-G is described for first time.