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Migraine affects ∼15% of the world's population greatly diminishing their quality of life. Current preventative treatments are effective in only a subset of migraine patients, and although cannabinoids seem beneficial in alleviating migraine symptoms, central nervous system side effects limit their widespread use. We developed peripherally restricted cannabinoids (PRCBs) that relieve chronic pain symptoms of cancer and neuropathies, without appreciable central nervous system side effects or tolerance development. Here, we determined PRCB effectiveness in alleviating hypersensitivity symptoms in mouse models of migraine and medication overuse headache. Long-term glyceryl trinitrate (GTN, 10 mg/kg) administration led to increased sensitivity to mechanical stimuli and increased expression of phosphorylated protein kinase A, neuronal nitric oxide synthase, and transient receptor potential ankyrin 1 proteins in trigeminal ganglia. Peripherally restricted cannabinoid pretreatment, but not posttreatment, prevented behavioral and biochemical correlates of GTN-induced sensitization. Low pH-activated and allyl isothiocyanate-activated currents in acutely isolated trigeminal neurons were reversibly attenuated by PRCB application. Long-term GTN treatment significantly enhanced these currents. Long-term sumatriptan treatment also led to the development of allodynia to mechanical and cold stimuli that was slowly reversible after sumatriptan discontinuation. Subsequent challenge with a previously ineffective low-dose GTN (0.1-0.3 mg/kg) revealed latent behavioral sensitization and increased expression of phosphorylated protein kinase A, neuronal nitric oxide synthase, and transient receptor potential ankyrin 1 proteins in trigeminal ganglia. Peripherally restricted cannabinoid pretreatment prevented all behavioral and biochemical correlates of allodynia and latent sensitization. Importantly, long-term PRCB treatment alone did not produce any behavioral or biochemical signs of sensitization. These data validate peripheral cannabinoid receptors as potential therapeutic targets in migraine and medication overuse headache.

5-Aminoimidazole-4-carboxamide-1-β-D-[13 C5 ] ribofuranosyl 5'-monophosphate ([13 C5 ribose] AICAR-PO3 H2 ) (6) has been synthesized from [13 C5 ]adenosine. Incorporation of the mass-label into [13 C5 ribose] AICAR-PO3 H2 provides a useful standard to aid in metabolite identification and quantification in monitoring metabolic pathways. A synthetic route to the 13 C-labeled compound has not been previously reported. Our method employs a hybrid enzymatic, and chemical synthesis approach that applies an enzymatic conversion from adenosine to inosine followed by a ring-cleavage of the protected inosine. A direct phosphorylation of the resulting 2',3'-isopropylidine acadesine (5) was developed to yield the title compound in 99% purity following ion exchange chromatography.

Many malignant cancers, including breast cancer, have a propensity to invade bones, leading to excruciating bone pain. Opioids are the primary analgesics used to alleviate this cancer-induced bone pain (CIBP) but are associated with numerous severe side effects, including enhanced bone degradation, which significantly impairs patients’ quality of life. In contrast, agonists activating only peripheral CB1 receptors (CB1Rs) have been shown to effectively alleviate multiple chronic pain conditions with limited side effects, yet no studies have evaluated their role(s) in CIBP. Here, we demonstrate for the first time that a peripherally selective CB1R agonist can effectively suppress CIBP. Our studies using a syngeneic murine model of CIBP show that both acute and sustained administration of a peripherally restricted CB1R agonist, 4-{2-[−(1E)-1[(4-propylnaphthalen-1-yl)methylidene]-1H-inden-3-yl]ethyl} morpholine (PrNMI), significantly alleviated spontaneous pain behaviors in the animals. This analgesic effect by PrNMI can be reversed by a systemic administration but not spinal injection of SR141716, a selective CB1R antagonist. Additionally, the cancer-induced bone loss in the animals was not exacerbated by a repeated administration of PrNMI. Furthermore, catalepsy and hypothermia, the common side effects induced by cannabinoids, were measured at the supra-therapeutic doses of PrNMI tested. PrNMI induced mild sedation, yet no anxiety nor a decrease in limb movements were detected. Overall, our studies demonstrate that CIBP can be effectively managed by using a peripherally restricted CB1R agonist, PrNMI, without inducing dose-limiting central side effects. Thus, targeting peripheral CB1Rs could be an alternative therapeutic strategy for the treatment of CIBP.

Chemotherapy-induced peripheral neuropathy (CIPN) is a severe and dose-limiting side effect of cancer treatment that affects millions of cancer survivors throughout the world and current treatment options are extremely limited by their side effects. Cannabinoids are highly effective in suppressing pain symptoms of chemotherapy-induced and other peripheral neuropathies but their widespread use is limited by central nervous system (CNS)-mediated side effects. Here, we tested one compound from a series of recently developed synthetic peripherally restricted cannabinoids (PRCBs) in a rat model of cisplatin-induced peripheral neuropathy. Results show that local or systemic administration of 4-{2-[-(1E)-1[(4-propylnaphthalen-1-yl)methylidene]-1H-inden-3-yl]ethyl}morpholine (PrNMI) dose-dependently suppressed CIPN mechanical and cold allodynia. Orally administered PrNMI also dose-dependently suppressed CIPN allodynia symptoms in both male and female rats without any CNS side effects. Co-administration with selective cannabinoid receptor subtype blockers revealed that PrNMI's anti-allodynic effects are mediated by CB1 receptor (CB1R) activation. Expression of CB2Rs was reduced in dorsal root ganglia from CIPN rats, whereas expression of CB1Rs and various endocannabinoid synthesizing and metabolizing enzymes was unaffected. Daily PrNMI treatment of CIPN rats for two weeks showed a lack of appreciable tolerance to PrNMI's anti-allodynic effects. In an operant task which reflects cerebral processing of pain, PrNMI also dose-dependently suppressed CIPN pain behaviors. Our results demonstrate that PRCBs exemplified by PrNMI may represent a viable option for the treatment of CIPN pain symptoms.

The photolabile analogue of the broad-spectrum opioid antagonist naloxone, 3-O-(4,5-dimethoxy-2-nitrophenyl)carboxymethyl naloxone (also referred to as “caged naloxone”, 3-O-(α-carboxy-6-nitroveratryl)naloxone, CNV-NLX), has been found to be a valuable biochemical probe. While the synthesis of CNV-NLX is simple, its characterization is complicated by the fact that it is produced as a mixture of αR,5R,9R,13S,14S and αS,5R,9R,13S,14S diastereomers. Using long-range and heteronuclear NMR correlations, the 1H NMR and 13C NMR resonances of both diastereomers have been fully assigned, confirming the structures. Monitoring of solutions of CNV-NLX in saline buffer, in methanol, and in DMSO has shown CNV-NLX to be stable for over a week under fluorescent laboratory lights at room temperature. Exposure of such solutions to λ 365 nm from a hand-held UV lamp led to the formation of naloxone and CNV-related breakdown products.

Metabolomics analysis depends on the identification and validation of specific metabolites. This task is significantly hampered by the absence of well-characterized reference standards. The one-carbon carrier 10-formyltetrahydrofolate acts as a donor of formyl groups in anabolism where it is a substrate in formyltransferase reactions in purine biosynthesis. It has been reported as an unstable substance and is currently unavailable as a reference standard for metabolomics analysis.The current study was undertaken to provide the metabolomics community thoroughly characterized 10-formyltetrahydrofolate along with analytical methodology and guidelines for its storage and handling.Anaerobic base treatment of 5,10-methenyltetrahydrofolate chloride in the presence of anti-oxidant was utilized to prepare 10-formyltetrahydrofolate.Pure 10-formyltetrahydrofolate has been prepared and physicochemically characterized. Conditions toward maintaining the stability of a solution of the dipotassium salt of 10-formyltetrahydrofolate in solution have been determined.This study describes the facile preparation of pure (90%) 10-formyltetrahydrofolate, its qualitative physicochemical characterization, as well as conditions to enable its use as a reference standard in physiologic samples.

PIMSR is among the first neutral antagonists for the CB1R and was demonstrated pharmacologically to bind to the CB1R, yet not alter calcium flux. It was further shown computationally to be able to stabilize both the active and inactive states of CB1R revealing the molecular interactions that mechanistically afford the property of neutral antagonism. PIMSR shows dramatic positive effects in reducing weight, food intake, and adiposity as well as in improving glycemic control and lipid homeostasis in high-fat diet-induced obese mice, but also shows increased ALT and liver weight as markers of liver injury with chronic administration. Further, in a separate study, 3-day administration of PIMSR in C57BL/6J mice, hepatic steatosis from an acute administration of high of ethanol was significantly reduced. Also, it partially prevented alcohol-induced increases in ALT, AST, and LDH. The differences in ALT levels in obese and nonobese mice under different test paradigms are unlikely to be due to neutral antagonism itself since other neutral antagonists (AM6545) do not exhibit liver injury. The brain levels of low micromolar would support significant brain CB1 receptor occupancy (re: Ki = 17nM), thus potentially including both CNS and peripheral influences on the observed weight loss. Overall, these studies suggest that marked improvements in aspects of metabolic disease and alcoholic steatosis can be realized with CB1R neutral antagonists and hence warrants the exploration of further members of this class of cannabinoid ligands.

The cannabinoid 1 (CB1) allosteric modulator, 5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)-ethyl]-amide) (ORG27569), has the paradoxical effect of increasing the equilibrium binding of [3H](−)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl]cyclohexan-1-ol (CP55,940, an orthosteric agonist) while at the same time decreasing its efficacy (in G protein-mediated signaling). ORG27569 also decreases basal signaling, acting as an inverse agonist for the G protein-mediated signaling pathway. In ligand displacement assays, ORG27569 can displace the CB1 antagonist/inverse agonist, N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide(SR141716A). The goal of this work was to identify the binding site of ORG27569 at CB1. To this end, we used computation, synthesis, mutation, and functional studies to identify the ORG27569-binding site in the CB1 TMH3-6-7 region. This site is consistent with the results of K3.28192A, F3.36200A, W5.43279A, W6.48356A, and F3.25189A mutation studies, which revealed the ORG27569-binding site overlaps with our previously determined binding site of SR141716A but extends extracellularly. Additionally, we identified a key electrostatic interaction between the ORG27569 piperidine ring nitrogen and K3.28192 that is important for ORG27569 to act as an inverse agonist. At this allosteric site, ORG27569 promotes an intermediate conformation of the CB1 receptor, explaining ORG27569's ability to increase equilibrium binding of CP55,940. This site also explains ORG27569's ability to antagonize the efficacy of CP55,940 in three complementary ways. 1) ORG27569 sterically blocks movements of the second extracellular loop that have been linked to receptor activation. 2) ORG27569 sterically blocks a key electrostatic interaction between the third extracellular loop residue Lys-373 and D2.63176. 3) ORG27569 packs against TMH6, sterically hindering movements of this helix that have been shown to be important for receptor activation.

GPR55 is a class A G protein-coupled receptor (GPCR) that has been implicated in inflammatory pain, neuropathic pain, metabolic disorder, bone development, and cancer. Initially deorphanized as a cannabinoid receptor, GPR55 has been shown to be activated by non-cannabinoid ligands such as l-α-lysophosphatidylinositol (LPI). While there is a growing body of evidence of physiological and pathophysiological roles for GPR55, the paucity of specific antagonists has limited its study. In collaboration with the Molecular Libraries Probe Production Centers Network initiative, we identified a series of GPR55 antagonists using a β-arrestin, high-throughput, high-content screen of ~300000 compounds. This screen yielded novel, GPR55 antagonist chemotypes with IC50 values in the range of 0.16-2.72 μM [Heynen-Genel, S., et al. (2010) Screening for Selective Ligands for GPR55: Antagonists (ML191, ML192, ML193) (Bookshelf ID NBK66153; PMID entry 22091481)]. Importantly, many of the GPR55 antagonists were completely selective, with no agonism or antagonism against GPR35, CB1, or CB2 up to 20 μM. Using a model of the GPR55 inactive state, we studied the binding of an antagonist series that emerged from this screen. These studies suggest that GPR55 antagonists possess a head region that occupies a horizontal binding pocket extending into the extracellular loop region, a central ligand portion that fits vertically in the receptor binding pocket and terminates with a pendant aromatic or heterocyclic ring that juts out. Both the region that extends extracellularly and the pendant ring are features associated with antagonism. Taken together, our results provide a set of design rules for the development of second-generation GPR55 selective antagonists.

Despite the therapeutic promise of the subnanomolar affinity cannabinoid CB2 antagonist, 5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-N-[(1S,2S,4R)-1,3,3-trimethylbicyclo[2.2.1]hept-2-yl]...

Alleviation of neuropathic pain by cannabinoids is limited by their central nervous system (CNS) side effects. Indole and indene compounds were engineered for high hCB1R affinity, peripheral selectivity, metabolic stability, and in vivo efficacy. An epithelial cell line assay identified candidates with

There are numerous medicinal chemistry reports in the literature describing the pharmacological properties of thousands of narcotics, stimulants, hallucinogens, sedative-hypnotic drugs, cannabinoids, and other psychoactive substances as well as synthetic methods for their preparations. This information, while essential for the advancement of science, has been used by clandestine chemists to manufacture and market an endless variety of analogs of so-called designer drugs. In this review, we describe how clandestine chemists used the principles of medicinal chemistry to design molecules, referred to as designer drugs, that elicit the effects of opioids, amphetamine and analogs, cannabinoids, and phencyclidine analogs while circumventing the law.

Salvinorin A, the primary psychoactive derivative of the hallucinogenic herb Salvia divinorum, is a potent and highly selective kappa-opioid receptor (KOR) agonist. Several recent studies, however, have suggested endocannabinoid system mediation of some of its effects.This study represents a systematic examination of this hypothesis.Salvinorin A was isolated from S. divinorum and was evaluated in a battery of in vitro and in vivo procedures designed to detect cannabinoid activity, including CB(1) receptor radioligand and [(35)S]GTPgammaS binding, calcium flux assay, in vivo cannabinoid screening tests, and drug discrimination.Salvinorin A did not bind to nor activate CB(1) receptors. In vivo salvinorin A produced pronounced hypolocomotion and antinociception (and to a lesser extent, hypothermia). These effects were blocked by the selective KOR antagonist, JDTic, but not by the CB(1) receptor antagonist rimonabant. Interestingly, however, rimonabant attenuated KOR activation stimulated by U69,593 in a [(35)S]GTPgammaS assay. Salvinorin A did not substitute for Delta(9)-tetrahydrocannabinol (THC) in mice trained to discriminate THC.These findings suggest that similarities in the pharmacological effects of salvinorin A and those of cannabinoids are mediated by its activation of KOR rather than by any direct action of salvinorin A on the endocannabinoid system. Further, the results suggest that rimonabant reversal of salvinorin A effects in previous studies may be explained in part by rimonabant attenuation of KOR activation.

Antagonists or inverse agonists of the cannabinoid CB1 receptor (CB1R) reported from the 2006–2007 period are reviewed. The compounds are either variations of the archetypical cannabinoid CB1 inverse agonist rimonabant (SR141716) or are other structures less obviously related to the archetype. Many of the former class replace the core pyrazole ring of rimonabant with other ring systems to display/arrange the appended binding/activity elements. A structure-activity relationship evolves from bioassays of these compounds that also provides a more detailed understanding of the molecular mechanism of the binding to and stabilization of the active and inactive states of the constitutively active CB1R. In addition to diverse variations of the core ring and appended substituents, conformationally constrained analogs with high affinity were revealed, a number of which do not fit a one-to-one atom correspondence with the putatively active conformations of the rimonabant template and other active analogs. Diphenylmethyl analogs were a repeated motif in structures where core rings were either absent or played less restrictive roles in structures pursuing higher conformational freedom. Bicyclic nitrogen heterocyclic ring systems as the central core structures were reported, as were linear structures including phenyl ureas and taranabant analogs. Finally, peptides in the hemopressin class were presented as CB1 antagonists. Drug Dev Res 70:601–615, 2009. © 2009 Wiley-Liss, Inc.

Type 1 cannabinoid receptor (CB1) antagonists might be useful for treating obesity, liver disease, metabolic syndrome, and dyslipidemias. Unfortunately, inhibition of CB1 in the central nervous system (CNS) produces adverse effects, including depression, anxiety and suicidal ideation in some patients, which led to withdrawal of the pyrazole inverse agonist rimonabant (SR141716A) from European markets. Efforts are underway to produce peripherally selective CB1 antagonists to circumvent CNS-associated adverse effects. In this study, novel analogues of rimonabant (1) were explored in which the 1-aminopiperidine group was switched to a 4-aminopiperidine, attached at the 4-amino position (5). The piperidine nitrogen was functionalized with carbamates, amides, and sulfonamides, providing compounds that are potent inverse agonists of hCB1 with good selectivity for hCB1 over hCB2. Select compounds were further studied using in vitro models of brain penetration, oral absorption and metabolic stability. Several compounds were identified with predicted minimal brain penetration and good metabolic stability. In vivo pharmacokinetic testing revealed that inverse agonist 8c is orally bioavailable and has vastly reduced brain penetration compared to rimonabant.

The nociceptin/orphanin FQ opioid peptide (NOP) receptor is a widely expressed GPCR involved in the modulation of pain, anxiety, and motor behaviors. Dissecting the functional properties of this receptor is limited by the lack of systemically active ligands that are brain permeant. The small molecule NOP receptor-selective, full agonist 8-[(1S,3aS)-2,3,3a,4,5,6-hexahydro- 1H-phenalen-1-yl]-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (Ro 64-6198) hydrochloride is an active, brain penetrant ligand, but its difficult and cost-prohibitive synthesis limits its widespread use and availability for animal studies. Here, we detail a more efficient and convenient method of synthesis, and use both in vitro and in vivo pharmacological assays to fully characterize this ligand. Specifically, we characterize the pharmacodynamics of Ro 64-6198 in cAMP and G-protein coupling in vitro and examine, for the first time, the effects of nociceptin/orphanin FQ and Ro 64-6198 in arrestin recruitment assays. Further, we examine the effects of Ro 64-6198 on analgesia, anxiety, and locomotor responses in vivo. This new synthesis and pharmacological characterization provide additional insights into the useful, systemically active, NOP receptor agonist Ro 64-6198.

The development of an easy and inexpensive immunoassay to measure the limited quantities of endogenous cannabinoids found in the body would be beneficial for both cannabinoid researchers and clinicians. This report describes the hapten design and carrier molecule strategy that we used to generate a panel of monoclonal antibodies (mAB) to the endogenous cannabinoid anandamide (N-arachidonylethanolamide, AEA). We designed and successfully prepared a hapten, N-arachidonyl-7-amino-6-hydroxy-heptanoic acid (AHA), which retained the basic characteristic features of anandamide--the carboxamide, the hydroxyl and the lipophilic arachidonyl moiety with its skipped double bond system, while still allowing attachment to protein. In addition, a secondary alcohol structure was added to reduce the potential for biological hydrolysis of the hapten. Because of the diverse responses obtained after coupling this hapten to four different carriers, we determined that the type of carrier molecule used was particularly important for generating anti-anandamide antibodies. Described in this report are the characteristics of a panel of 11 mAB, generated from four separate fusions, with a range of relative affinities and cross reactivities. Excellent selectivity for anandamide vs. two other endogenous cannabinoids and arachidonic acid was achieved this strategy (cross-reactivities5%). In addition, at least one mAB maintained specificity for anandamide compared to two very closely related fatty acid amide molecules. However, the IC50 values in a standard enzyme-linked immunosorbent assay (ELISA) format (ca. 2-3 microM) indicate that improvement in antibody affinities or assay format will be required for an immunoassay to measure endogenous levels. Such work is underway.

Upon irradiation with a 450 W high-pressure mercury lamp, the CB1 cannabinoid antagonist N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR14-1716; 1) undergoes a photocyclization reaction to yield a single reaction product. This product, 2, the structure of which is based on a pyrazolo[1,5-f]phenanthridine ring system, was established by two-dimensional NMR techniques (COSY, HSQC, HMBC and ROESY), and was later confirmed by single-crystal x-ray diffraction analysis. The crystal structure shows two independent molecules of 3 and a half molecule of the 1,2-dichloroethane solvate. Compound 2 has reasonably high affinity for the CB1 receptor (Ki = 48.0 ± 2.7 nM). Copyright © 2003 John Wiley & Sons, Ltd.

Analogues of the biaryl pyrazole N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716; 5) were synthesized to investigate the structure−activity relationship (SAR) of the aminopiperidine region. The structural modifications include the substitution of alkyl hydrazines, amines, and hydroxyalkylamines of varying lengths for the aminopiperidinyl moiety. Proximity and steric requirements at the aminopiperidine region were probed by the synthesis of analogues that substitute alkyl hydrazines of increasing chain length and branching. The corresponding amide analogues were compared to the hydrazides to determine the effect of the second nitrogen on receptor binding affinity. The N-cyclohexyl amide 14 represents a direct methine for nitrogen substitution for 5, reducing the potential for heteroatom interaction, while the morpholino analogue 15 adds the potential for an additional heteroatom interaction. The series of hydroxyalkyl amides of increasing chain length ...

A WIN 35,065-2 analog, 3β-(4-iodophenyl)tropane-2β-pyrrolidine carboxamide (RTI-229), has been radiolabelled with iodine-125 by radioiododestannylation of the corresponding trimethyltin derivative using carrier-free sodium iodide-125 as the isotope source. Purification by reversed-phase HPLC gives [125I]RTI-229 in good yield (89·4%) with high radiochemical purity (>99%) and high specific activity (2125 mCi/μmol, 78·6 GBq/μmol, based on the specific activity of the Na125I used). Copyright © 1999 John Wiley & Sons, Ltd.

The t-butyldimethylsilylation of the allylic alcohol of 1-iodomorphine and 1-iodocodeine protects the double bond of these molecules from catalytic reduction while allowing the reduction of the aryl iodide. This selectivity has been applied to the preparation of tritiated morphine and codeine without complicating over reduction to the dihydromorphine and dihydrocodeine. © 1998 John Wiley & Sons, Ltd.

(Dimethylheptyl)anandamide [(16,16-dimethyldocosa-cis-5,8,11,14-tetraenoyl)ethanolamine ] (17a) and its amide analogs were synthesized by Wittig coupling of a ylide derived from a fragment of arachidonic acid. These amides were compared to the endogenous cannabinoid receptor ligand arachidonylethanolamide (anandamide, 2a) and its amide analogs in pharmacological assays for potential enhancement of cannabimimetic activities. The receptor affinity to rat brain membranes of the dimethylheptyl (DMH) analogs increased by an order of magnitude in most comparisons to the corresponding anandamides in displacement assays versus the cannabinoid agonist [3H]CP 55,940 or antagonist [3H]SR141716A, for which rank order differences in affinity were observed. An order of magnitude enhancement of potency with comparable or higher efficacy in behavioral assays in the mouse tetrad of tests of cannabinoid activity was observed in 17a versus 2a. In contrast, no enhancement in potency for the pentyl to DMH side chain exchange was seen in the mouse vas deferens assay. The data indicate a structural equivalence between classical plant cannabinoids and 2a as well as different receptor-ligand interactions that characterize multiple receptor sites or binding modes.

Antagonists of the CB1 receptor can be useful in the treatment of several diseases including obesity, diabetes, and liver disease. However, to date, the only clinically approved CB1 receptor antagonist, rimonabant, was withdrawn due to adverse CNS related side effects such as depression and suicidal ideation. Since rimonabant’s withdrawal, several groups have begun pursuing peripherally selective CB1 antagonists. These compounds are expected to be devoid of undesirable CNS related effects but maintain efficacy through antagonism of peripherally expressed CB1 receptors within target tissues. Reported here are our latest results toward development of a peripherally selective analog of the diphenyl purine CB1 antagonist otenabant 1. Compound 9 (N-{1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-yl}pentanamide) is a potent, orally absorbed antagonist of the CB1 receptor that is >50-fold selective for CB1 over CB2, highly selective for the periphery in a rodent model, and without efficacy in a series of in vivo assays designed to evaluate its ability to mitigate the central effects of Δ9-THC through the CB1 receptor.

A selective ligand for the dopamine transporter 3β-(4-iodophenyl)tropan-2β-carboxylic acid isopropyl ester (RTI-121) has been labeled with iodine-125 by electrophilic radioiododestannylation. The [ 125 I]RTI-121 was obtained in good yield (86 ± 7%, n = 3) with high radiochemical purity (>99%) and specific radioactivity (1210–1950 mCi/μmol). After i.v. administration of [ 125 I]RTI-121 to mice, the rank order of regional brain tissue radioactivity (striatum > olfactory tubercles ⪢> cortex, hippocampus, thalamus, hypothalamus, cerebellum) was consistent with dopamine transporter labeling. Specific in vivo binding in striatum and olfactory tubercles was saturable, and was blocked by the dopamine transporter ligands GBR 12,909 and (±)-nomifensine. By contrast, binding was not reduced by paroxetine, a serotonin transporter inhibitor, or desipramine, a norepinephrine transporter inhibitor. A variety of additional drugs having high affinities for recognition sites other than the neuronal dopamine transporter also had no effect. The [ 125 I]RTI-121 binding in striatum and olfactory tubercles was inhibited by d -amphetamine in dose-dependent fashion. Nonmetabolized radioligand represents 85% of the signal observed in extracts of whole mouse brain. Thus, [ 125 I]RTI-121 is readily prepared, and is a useful tracer for dopamine transporter studies in vivo .

The novel cocaine analog RTI-121[3β-(4-iodophenyl)tropane-2β-carboxylic acid isopropyl ester] was evaluated as a probe for the in vitro labeling and localization of the dopamine transporter in the human brain. Saturation binding experiments conducted in sucrose-phosphate buffer (10 mM sodium phosphate, pH 7.4, 0.32 M sucrose) revealed high- and low-affinity binding components with affinity values (KD) of 0.25 ± 0.04 and 4.9 ± 1.6 nM (mean ± SE) and densities (Bmax) of 56.8 ± 13.8 and 147.7 ± 23.4 pmol/g tissue, respectively. In contrast, when saturation binding experiments were performed in phosphate-buffered saline (10 mM Na2HP04, 1.8 mM KH2Po4, 136 mM NaCl,2.8 mM KC1, 10 mM NaI, pH 7.4), a 9-fold decrease in the density of the low-affinity component was noted, suggesting that the low-affinity RTI-121 binding site is associated with the region of the transporter involved in the ionic dependence of substrate recognition andor uptake. The rank order. of potency for inhibition of [1251]RTI-121 binding to human caudate membranes demonstrates that the radioligand selectively labels the dopamine transporter (GBR 12909 > RTI-121 > mazindol > nomifensine > (−)cocaine > desipramine > citalopram). Autoradiograpliic mapping of [1251]RTI-121 revealed very high densities of cocaine recognition sites over areas known to be rich in dopaminergic innervation, including the caudate, putamen, and nucleus accumbens. Moderate densities were also observed over the substantia nigra and the ventral tegmental area. Low-to-background labeling of [1251]RTI- 121 was seen throughout the cerebral cortex, amygdaloid nuclei, globus pallidus, and thalamus. In comparison with the autoradiographic distribution of the cocaine analogs L3H1WIN 35,428 (or CFT) and [1251]RTI-55 (or β-CIT), the labeling pattern for [1251]RTI-121 was more restricted. These studies demonstrate that [1251]RTI-121 labels dopamine-rich brain regions with greater selectivity than other currently available cocaine analogs, which makes it a potentially superior imaging probe for mapping the dopamine transporter in the human brain. © 1995 WiIey-Liss, Inc.

Cannabidiol CBD, a non-psychoactive constituent of marihuana, has been reported to possess essentially no affinity for cannabinoid CB1 receptor binding sites in the brain. Our hypothesis concerning CBD's lack of affinity for the cannabinoid CB1 receptor is that CBD is not capable of clearing a region of steric interference at the CB1 receptor and thereby not able to bind to this receptor. We have previously characterized this region of steric interference at the CB1 receptor [P.H. Reggio, A.M. Panu, S. Miles J. Med. Chem. 36, 1761-1771 (1993)] in three dimensions using the Active Analog Approach. We report here a conformational analysis of CBD which, in turn, led to the design of a new analog, desoxy-CBD. Modeling results for desoxy-CBD predict that this compound is capable of clearing the region of steric interference by expending 3.64 kcal/mol of energy in contrast to the 12.39 kcal/mol expenditure required by CBD. Desoxy-CBD was synthesized by condensation of 3-pentylphenol with p-mentha-2,8-dien-1-ol mediated by DMF-dineopental acetal. Desoxy-CBD was found to behave as a partial agonist in the mouse vas deferens assay, an assay which is reported to detect the presence of cannabinoid receptors. The compound produced a concentration related inhibition of electrically-evoked contractions of the mouse vas deferens, possessing an IC50 of 30.9 nM in this assay. Taken together, these results support the hypothesis of the existence of a region of steric interference at the CB1 receptor. While the energy expenditure to clear this region was too high for the parent compound, CBD, the removal of the C6' hydroxyl of CBD produced a molecule (desoxy-CBD) able to clear this region and produce activity, albeit at a reduced level.

A new method is presented for the tritiation of aryl compounds with tritium gas which is conducted on the underivatized substrate. Combined directed ortho metallation and facile reduction of the carbonpotassium bond affords incorporation of tritium at high specific activity under mild conditions. The metallation/reduction method is demonstrated for the preparation of [3H]ibogaine.

CB1 receptor antagonists that are peripherally restricted were targeted. Compounds with permanent charge as well as compounds that have increased polar surface area were made and tested against CB1 for binding and activity. Sulfonamide and sulfamide with high polar surface area and good activity at CB1 were rationally designed and pharmacologically tested. Further optimization of these compounds and testing could lead to the development of a new class of therapeutics to treat disorders where the CB1 receptor system has been implicated.

Known agonists of the orphan receptor GPR35 are kynurenic acid, zaprinast, 5-nitro-2-(3-phenylproplyamino) benzoic acid, and lysophosphatidic acids. Their relatively low affinities for GPR35 and prominent off-target effects at other pathways, however, diminish their utility for understanding GPR35 signaling and for identifying potential therapeutic uses of GPR35. In a screen of the Prestwick Library of drugs and drug-like compounds, we have found that pamoic acid is a potent GPR35 agonist. Pamoic acid is considered by the Food and Drug Administration as an inactive compound that enables long-acting formulations of numerous drugs, such as the antihelminthics oxantel pamoate and pyrantel pamoate; the psychoactive compounds hydroxyzine pamoate (Vistaril) and imipramine pamoate (Tofranil-PM); and the peptide hormones triptorelin pamoate (Trelstar) and octreotide pamoate (OncoLar). We have found that pamoic acid induces a Gi/o-linked, GPR35-mediated increase in the phosphorylation of extracellular signal-regulated kinase 1/2, recruitment of β-arrestin2 to GPR35, and internalization of GPR35. In mice, it attenuates visceral pain perception, indicating an antinociceptive effect, possibly through GPR35 receptors. We have also identified in collaboration with the Sanford-Burnham Institute Molecular Libraries Probe Production Center new classes of GPR35 antagonist compounds, including the nanomolar potency antagonist methyl-5-[(tert-butylcarbamothioylhydrazinylidene)methyl]-1-(2,4-difluorophenyl)pyrazole-4-carboxylate (CID2745687). Pamoic acid and potent antagonists such as CID2745687 present novel opportunities for expanding the chemical space of GPR35, elucidating GPR35 pharmacology, and stimulating GPR35-associated drug development. Our results indicate that the unexpected biological functions of pamoic acid may yield potential new uses for a common drug constituent.

Dimerization or oligomerization of many G-protein-coupled receptors (GPCRs), including the cannabinoid 1 (CB1) receptor, is now widely accepted and may have significant implications for medications development targeting these receptor complexes. A library of bivalent ligands composed of two identical CB1 antagonist pharmacophores derived from SR141716 linked by spacers of various lengths were developed. The affinities of these bivalent ligands at CB1 and CB2 receptors were determined using radiolabeled binding assays. Their functional activities were measured using GTP-γ-S accumulation and intracellular calcium mobilization assays. The results suggest that the nature of the linker and its length are crucial factors for optimum interactions of these ligands at CB1 receptor binding sites. Finally, selected bivalent ligands (5d and 7b) were able to attenuate the antinociceptive effects of the cannabinoid agonist CP55,940 (21) in a rodent tail-flick assay. These novel compounds may serve as probes that will enable further characterization of CB1 receptor dimerization and oligomerization and its functional significance and may prove useful in the development of new therapeutic approaches to G-protein-coupled receptor mediated disorders.

Two rotationally restricted tetrahydrocannabinol (THC) ehters were synthesized to test the concept that the psychopharmacological activity of cannabinoids derives, in part, from the orientation of the lone pairs of electrons of the phenolic hydroxyl oxygen. These compounds, O,2-propano-Δ 8 -THC (3) and O,10-methano-Δ 9 -THC (12), lock the orientation of the lone pairs of electrons toward and away from the cyclohexene ring, respectively, by restricting bond rotation through the formation of cyclic ethers

In this decade, classical cannabinoid SAR has increasingly been interpreted in terms of ligand-receptor interaction. Membrane receptor binding affinity, inhibition of receptor coupled processes, functional in vitro assays that distinguish between agonists and antagonists, transfected receptor systems, and distinction among receptor subtypes have been applied to cannabinoid pharmacology studies. These studies have revealed information about the pivotal receptor interaction that mediates the neurochemical system characterized by the receptor. The positive correlation between binding affinity and behavioral effects validates ligand-receptor interaction as a focus for developing new cannabinoids with the potential for selective pharmacological effects. Added to these direct receptor studies are the computational methods that compare active and inactive ligands for steric, electronic, and conformational similarities that reveal an SAR for activity with a sophistication and a perspective not possible before computer methodology. Merging these studies with knowledge of the receptor sequence, consequences of mutations of the receptor, and a calculated structure of the receptor are evolving a physical picture of the interaction of cannabinoids with its receptor(s). This picture of a ligand-receptor interaction guides medicinal chemists in their interpretation of classical cannabinoid SAR and the design of new analogs for selective pharmacological activity and as probes of the cannabinoid receptor. It is from a receptor perspective that this review of the SAR of classical cannabinoids is presented.

Trimethylsilyl bromide (TMSBr) in the presence of catalytic ZnI2 induces facile substitution of allylic acetates to the bromides with or without rearrangement as governed by product stability. This was applied to the preparation of a key intermediate employed in the synthesis of important cannabinoid metabolites.

In the last few decades, our knowledge of cannabinoid structure–activity relationships (SARs) has increased dramatically. Computational methods have allowed us to compare active and inactive ligands for steric, electronic, and conformational similarities and dissimilarities. Knowing that there are specific G-protein-coupled cannabinoid receptors with which cannabinoids interact, we increasingly interpret changes in structure to specific alterations in ligand–receptor interaction. With additional knowledge of the CB1 and CB2 receptors’ sequence, the consequences of mutations and chimeric constructs of the receptor, and the development of rhodopsin-based homology models of the cannabinoid receptors, we continue to gain insight into the interaction of cannabinoids with their receptors. Finally, our emerging appreciation for cannabinoid homo- and heterodimerization/oligomerization provides for further understanding of ligand–receptor interactions, the interaction of receptors with each other, and the interaction of G-protein-coupled receptors with their signal transduction components and other molecules affecting each receptor’s dynamics. In the following review, we have attempted to characterize the emerging SARs of an important class of cannabinoid ligands: the CB1 receptor antagonists and inverse agonists. This class of compounds is showing therapeutic potential for a variety of indications, and is therefore of interest for both basic research and pharmaceutical development. Throughout this work, we have used molecular modeling, particularly quenched molecular dynamics, to illustrate particular structural modifications that have been reported for CB1 antagonists, their conformational properties, and the potential implications for receptor interaction. In this way, we hope this review serves to further guide medicinal chemists in their understanding of CB1 receptor antagonist SAR and the design of new analogs.

Two rotationally restricted tetrahydrocannabinol (THC) ehters were synthesized to test the concept that the psychopharmacological activity of cannabinoids derives, in part, from the orientation of the lone pairs of electrons of the phenolic hydroxyl oxygen. These compounds, O,2-propano-Δ 8 -THC (3) and O,10-methano-Δ 9 -THC (12), lock the orientation of the lone pairs of electrons toward and away from the cyclohexene ring, respectively, by restricting bond rotation through the formation of cyclic ethers

Structure-activity relationships (SARs) of 1 (SR141716) have been extensively documented, however, the conformational properties of this class have received less attention. In an attempt to better understand ligand conformations optimal for receptor recognition, we have designed and synthesized a number of derivatives of 1, including a four-carbon-bridged molecule (11), to constrain rotation of the diaryl rings. Computational analysis of 11 indicates approximately 20 kcal/mol energy barrier for rotation of the two aryl rings. NMR studies have determined the energy barrier to be approximately 18 kcal/mol and suggested atropisomers could exist. Receptor binding and functional studies with these compounds displayed reduced affinity and potency when compared to 1. This indicates that our structural modifications either constrain the ring systems in a suboptimal orientation for receptor interaction or the introduction of steric bulk leads to disfavored steric interactions with the receptor, and/or the relatively modest alterations in the molecular electrostatic potentials results in disfavored Coulombic interactions.

We have shown previously that neonatal exposure to the cannabinoid CB1 receptor antagonist/inverse agonist rimonabant (SR141716) interfered with suckling and development. However, it was not clear whether the developmental deficiencies were induced by neutral CB1 receptor blockade, thereby inhibiting endogenous cannabinoid "tone," or by inverse agonist reduction of constitutive CB1 receptors. CB1 receptor blockade supports our hypothesis that low CB1 receptor concentrations and/or reduced endocannabinoid levels underlie infant nonorganic failure to thrive (NOFTT). Inverse agonism implies that lower constitutive CB1 receptor activity may be responsible for impaired food intake in newborns. In the present study, we injected the neutral CB1 receptor antagonist 5-(4-chlorophenyl)-3-[(E)-2-cyclohexylethenyl]-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole (VCHSR1) to 1-d-old mouse pups and recorded weight gain, gastric milk contents (milkbands), axillary temperature, and survival between age 1 and 10 d. The results showed a dose-related interference with all measures. These data show that (1) growth failure induced by rimonabant is generalized to another CB1 antagonist and (2) cannabinoid CB1 receptor activation by endocannabinoids is essential for normal milk ingestion and development in mice. This supports our hypothesis that endocannabinoid deficiency and perhaps CB1 receptor dysfunction represents the uncharacterized biologic vulnerability, which underlies NOFTT.

The biarylpyrazole, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716; 1) has been shown to act as an inverse agonist/antagonist at the cannabinoid CB1 receptor. Our previous mutant cycle study suggested that K3.28(192) is involved in a direct interaction with the C-3 substituent of 1 in wild-type (WT) CB1.(1) However, these results did not establish what part of the C-3 substituent of 1 is involved in the K3.28(192) hydrogen bond, the carboxamide oxygen or the piperidine nitrogen. Furthermore, our previous calcium channel assay results for 5-(4- chlorophenyl)-3-[(E)-2-cyclohexylethenyl]-1-(2,4-dichlorophenyl)-4- methyl-1H-pyrazole (VCHSR; 2) (an analogue of 1 that lacks hydrogen-bonding capability in its C-3 substituent) showed that this compound acts as a neutral antagonist, a result that is in contrast to 1, which acts as an inverse agonist in this same assay.(1) These results suggested a relationship between biarylpyrazole interaction with K3.28(192) at CB1 and inverse agonism, but these results were for a single pair of compounds (1 and 2). The work presented here was designed to test two hypotheses derived from our modeling and mutant cycle results. The hypotheses are as follows: (1) it is the carboxamide oxygen of the C-3 substituent of 1 that interacts directly with K3.28(192) and (2) the interaction with K3.28(192) is crucial for the production of inverse agonism for biarylpyrazoles such as 1. To determine whether the carboxamide oxygen or the piperidine nitrogen of the C-3 substituent may be the interaction site for K3.28(192), we designed, synthesized, and evaluated a new set of analogues of 1 (3-6, Chart 1) in which modifications only to the C-3 substituent of 1 have been made. In each case, the modifications that were made preserved the geometry of this substituent in 1. The absence of the piperidine nitrogen was not found to affect affinity, whereas the absence of the carboxamide oxygen resulted in a reduction in affinity. CB1 docking studies in an inactive state model of CB1 resulted in the trend, 3,15,426 for ligand/CB1 interaction energies. This trend was consistent with the trend in WT CB1 Ki values versus [3H]CP55,940 reported here. In calcium channel assays, all analogues with carboxamide oxygens (1, 3, and 4) were found to be inverse agonists, whereas those that lacked this group (2, 5, and 6) were found to be neutral antagonists. Taken together, these results support the hypothesis that it is the carboxamide oxygen of the C-3 substituent of 1 that engages in a hydrogen bond with K3.28(192) in WT CB1. Furthermore, functional results for 1-6 support the hypothesis that the interaction of 1 with K3.28(192) may be key to its inverse agonism.

The CB1 allosteric modulator, ORG27569, has the paradoxical effect of increasing the equilibrium binding of CP55,940 (an orthosteric agonist), while at the same time decreasing its efficacy. ORG27569 also acts as an inverse agonist. We have previously used computational methods, synthesis, mutation, and functional studies to identify ORG27569's binding site in the THM3/TMH6/TMH7 region (Shore et al., ICRS, 2012). In this site, ORG27569 promotes an active-like conformation of the CB1 receptor, explaining ORG27569's ability to increase CP55,940's equilibrium binding. This site explains ORG27569's ability to antagonize CP55,940's efficacy in three complimentary ways: 1) ORG27569 sterically blocks movements of the second extracellular loop that have been linked to receptor activation, 2) ORG27569 sterically blocks a key electrostatic interaction between the third extracellular loop residue K373 and D2.63(176), and 3) ORG27569 packs against TMH6, sterically hindering movements of TMH6 that the Farrens lab have shown to be important to receptor activation. Additionally, we identified a key interaction between ORG27569's piperidine ring nitrogen and K3.28(192) that is required for ORG27569 to act as an inverse agonist.Using our model of ORG27569 docked in our active state model (in the presence of CP55,940), we designed, synthesized, and functionally characterized 4 analogs of ORG27569 that were designed to test our model and have improved interactions with the receptor. The analogs were functionalized with 3 different goals: 1) to form electrostatic interactions with D6.58(366), 2) to form an aromatic stack with F3.25(189), or 3) to test packing with TMH6-7. Our strategy to form new interactions with D6.58(366) was the most successful, resulting in an analog that is more potent than ORG27569. Interestingly, none of the analogs acted as inverse agonists, suggesting the potential therapeutic promise of CB1's allosteric site.

An extended series of alkyl carboxamide analogs of N -(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl- 1 H -pyrazole-3-carboxamide (SR141716; 5 ) was synthesized. Each compound was tested for its ability to displace the prototypical cannabinoid ligands ([ 3 H]CP-55,940, [ 3 H] 2 ; [ 3 H]SR141716, [ 3 H] 5 ; and [ 3 H]WIN55212-2, [ 3 H] 3 ), and selected compounds were further characterized by determining their ability to affect guanosine 5′-triphosphate (GTP)-γ-[ 35 S] binding and their effects in the mouse vas deferens assay. This systematic evaluation has resulted in the discovery of novel compounds with unique binding properties at the central cannabinoid receptor (CB1) and distinctive pharmacological activities in CB1 receptor tissue preparations. Specifically, compounds with nanomolar affinity which are able to fully displace [ 3 H] 5 and [ 3 H] 2 , but unable to displace [ 3 H] 3 at similar concentrations, have been synthesized. This selectivity in ligand displacement is unprecedented, in that previously, compounds in every structural class of cannabinoid ligands had always been shown to displace each of these radioligands in a competitive fashion. Furthermore, the selectivity of these compounds appears to impart unique pharmacological properties when tested in a mouse vas deferens assay for CB1 receptor antagonism.

In superior cervical ganglion neurons, N -(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H -pyrazole-3-carboxamide (SR141716A) competitively antagonizes the Ca 2+ current effect of the cannabinoid (CB) agonist ( R )-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3- de ]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55212-2), and behaves as an inverse agonist by producing opposite current effects when applied alone. In contrast, in neurons expressing CB1 with a K→A mutation at residue 3.28(192) (i.e., K3.28A), SR141716A competitively antagonizes the effects of WIN55212-2, but behaves as a neutral antagonist by producing no current effects itself. Receptor modeling studies suggested that in the CB1 inactive (R) state, SR1417A16A stabilizes transmembrane helix 6 in its inactive conformation via aromatic stacking with F3.36/W6.48. In this binding site, SR141716A would exhibit higher affinity for CB1 R due to a hydrogen bond between the SR141716A C3 substituent and K3.28(192), a residue available to SR141716A only in R. To test this hypothesis, a “mutant thermodynamic cycle” was constructed that combined the evaluation of SR141716A affinity at WT CB1 and K3.28A with an evaluation of the wild-type CB1 and K3.28A affinities of an SR141716A analog, 5-(4-chlorophenyl)-3-[(E)-2-cyclohexylethenyl]-1-(2,4-dichlorophenyl)-4-methyl-1 H -pyrazole (VCHSR), that lacks hydrogen bonding potential at C3. Binding affinities suggested that K3.28 is involved in a strong interaction with SR141716A in WT CB1, but does not interact with VCHSR. Thermodynamic cycle calculations indicated that a direct interaction occurs between the C3 substituent of SR141716A and K3.28 in WT CB1. Consistent with these results, VCHSR acted as a neutral antagonist at WT CB1. These results support the hypothesis that hydrogen bonding of the SR141716A C3 substituent with K3.28 is responsible for its higher affinity for the inactive R state, leading to its inverse agonism.

The separation of the mood-altering effects of cannabinoids from their therapeutic effects has been long sought. Results reported here for a series of C-9 analogs of the cyclic ether O,2-propano-delta 8-tetrahydrocannabinol (O,2-propano-delta 8-THC) point to the C-1 position in classical cannabinoids as a position for which CB2 subtype selectivity occurs within the cannabinoid receptors. O,2-Propano-11-delta 8-THC, O,2-propano delta 9,11-THC, O,2-propano-9-oxo-11-nor-hexahydrocannabinol (O,2-propano-9-oxo-11-nor-HHC), and O,2-propano-9 alpha- and O,2-propano-9 beta-OH-11-nor-HHC were synthesized and evaluated in radioligand displacement assays for affinity at the CB1 and CB2 receptors and in the mouse vas deferens in vitro assay and the mouse tetrad in vivo assay for cannabinoid activity. Evaluation of binding affinity at the CB1 and CB2 receptors revealed that each compound possesses a modest increased affinity for the CB2 receptor. Analogs which contained an oxygen attached to C-9 (i.e., oxo and hydroxy derivatives) showed the highest affinity and selectivity for CB2 (for O,2-propano-9-oxo-11-nor-HHC, Ki(CB1) = 90 nM, Ki(CB2) = 23 nM, selectivity ratio 3.9; for O,2-propano-9 beta-OH-11-nor-HHC, Ki(CB1) = 26 nM, Ki(CB2) = 5.8 nM, selectivity ratio 4.5). Each compound was found to produce a dose-dependent inhibition of electrically-evoked contractions of the mouse isolated vas deferens when administered at submicromolar concentrations. This inhibition could readily be prevented by the selective CB1 cannabinoid receptor antagonist SR-141716A. The analogs exhibited unique in vivo profiles with O,2-propano-delta 9,11-THC exhibiting antinociception with reduced activity in three other in vivo measures and O,2-propano-9 beta-OH-HHC exhibiting lack of dose responsiveness in all measures. The CB2 selectivities in the O,2-propano analogs may be due to differences in solvation/desolvation that occur when the ligands enter the CB1 vs CB2 binding site. Alternatively, the CB2 selectivities may be a results of an amino acid change from a hydrogen bond-accepting residue in CB1 to a hydrogen bond-donating residue in CB2.

The compound 9-beta-hydroxy-hexahydrocannabinol [(−)-9β-OH-HHC] was designed to fit a combined theoretical profile of an analgesic cannabinoid (equatorial alcohol at C-9, phenol at C-1 and a C-3 side chain) with reduced psychoactivity (axial C-9 substituent which protrudes into the α face). (−)-9β-OH-HHC was synthesized by the addition of methyl Grignard to 9-oxo-11-nor-HHC. Its α epimer was obtained by the regiospecific epoxide ring opening of 9α, 10α-epoxy-HHC acetate. (−)-9β-OH-HHC and (−)-9α-OH-HHC were each evaluated in a battery of tests in mice and were found to be 10–25 times less potent than (−)-trans-Δ9-tetrahydrocannabinol (Δ9-THC) in all tests including the tail flick test for antinociception (analgesia). Molecular mechanics calculations [MMP2(85)] revealed that, in the global minimum energy conformation of (−)-9β-OH-HHC, the axial methyl at C-9 protrudes into the α face of the molecule, while the axial hydroxyl at C-9 in (−)-9α-OH-HHC protrudes into this same face. These calculations also identified a higher energy carbocyclic ring (twist) conformer of each in which there is no protrusion of a C-9 substituent of the carbocyclic ring into the α face. The minimal activity of both compounds is attributed to these higher energy forms. It is concluded that protrusion of a C-9 substituent into the α-face of the molecule is associated with reduced cannabinoid analgesia, as well as with reduced cannabinoid psychopharmacological activity.

An efficient synthesis of the cannabinoid antagonist SR141716A is presented and the structure is established by nOe experiments; tritiated SR141716A is synthesized by a novel sequence of metallation–iodination–tritiation and the labelled site is shown by tritium NMR and tritium–hydrogen nOe experiments.

The lipid composition of a membrane fraction from bovine adrenal cortex was determined. This preparation has the capacity to bind adrenocorticotropic hormone and is enriched in adenylate cyclase and 5'-nucleotidase. The adrenal plasma membranes have a significantly higher lipid content (54.8%) than bovine liver plasma membranes and a surprisingly low proportion of this lipid is cholesterol (4.2%). The phospholipids comprise 76.4% of the total lipids and their composition if very similar to that of bovine liver membranes with the exception of sphingomyelin which comprises only 4.5% of the phospholipids in the adrenal preparation.

The treatment of hypertension with guanethidine is now in widespread use (1). Investigations of its biochemical and clinical pharmacology in hypertensive patients have been severely limited due to a lack of adequate analytical methodology for its measurement in plasma. The reported techniques for its estimation in biological fluids include spectrofluorimetric detection (2,3), radioisotopic dilution (4-6), and gas chromatography with electron capture (7) or multiple ion detection (8). The in vi?ro metabolic transformation of guanethidine in rabbit and pig liver homogenates as well as its excretion in rat and human urine have been studied (9,lO). A recent review discusses its metabolism (11). There are large variations in the dose (50-300 pg/kg) necessary for the sufficient control of blood pressure in different individuals as well as many side effects during administration of guanethidine. The cause of this variability has not been extensively investigated since the existing methods for its measurement are too insensitive (7) or are based upon expensive gas chromatographic-mass spectrometric instrumentation (8). We have developed a procedure based on a two-dimensional gas-liquid chromatograph (12,15) utilizing electroncapture detection for the analysis of guanethidine. The method consists of a twostep extraction of plasma followed by hydrolysis, derivatization, and dual gas chromatography. MATERIALS AND METHODS

The design and application of a two dimensional gas-liquid chromatograph (gc) to the analysis of clofibrate in rabbit plasma is described. The assay involves three steps: extraction of plasma, derivatization with pentafluorobenzylbromide and dual gc analysis. Development time for an assay for drugs is considerably shortened as compared to conventional approaches using other chromatographic methods. Approximately 20 samples can be processed in a day.