α-[Amino(4-aminophenyl)thio]methylene-2-(trifluoromethyl)benzeneacetonitrile; Configurational equilibria in solution.

Inconsistent results have been reported for the effects of the mitogen-activating extracellular kinase (MEK) inhibitor α-[amino(4-aminophenyl)thio]methylene-2-(trifluoromethyl)benzeneacetonitrile (SL 327) on ethanol-induced conditioned place preference (EtOH-CPP). Since such inconsistencies may be due to the configurational composition of administered SL 327, the interconvertibility of the geometric isomers of this class of compounds has been investigated. This study provides conditions for determination of configurational composition of this class of compounds by HPLC and by 1H NMR and reports details of configurational equilibria as a function of medium and time in solution along with solubility data for SL 327 in aqueous DMSO. The results suggest that the apparently inconsistent results reported for CPP-EtOH may be due to the administration of suspension vs. solutions, as well as to different configurational compositions of SL 327.

Synthesis of 13 C-labeled 5-aminoimidazole-4-carboxamide-1-ß-D-[13 C5 ] ribofuranosyl 5'-monophosphate.

5-Aminoimidazole-4-carboxamide-1-ß-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.

Pyrazole antagonists of the CB1 receptor with reduced brain penetration.

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 analogs 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.

Allosteric modulation of a cannabinoid G protein-coupled receptor: binding site elucidation and relationship to G protein signaling.

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 [(3)H](-)-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 K3.28(192)A, F3.36(200)A, W5.43(279)A, W6.48(356)A, and F3.25(189)A 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 K3.28(192) 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 D2.63(176). 3) ORG27569 packs against TMH6, sterically hindering movements of this helix that have been shown to be important for receptor activation.

Identification of the GPR55 antagonist binding site using a novel set of high-potency GPR55 selective ligands.

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 ß-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.

Isolation of Hemiketal and Hydroxy Ketone Tautomers of Synthetic Intermediates Each Affording 18-Hydroxycortisol-1,6,7-d3.

During the synthesis of deuterated 18-hydroxycortisol, two of the synthetic intermediates have been found to exist in tautomeric forms as the acyclic 18-hydroxy 20-ketone and the cyclic 18,20-hemiketal corresponding to the previously identified less polar (L) and more polar (M) forms of C-18 hydroxylated steroids, respectively. Specifically, p-chloranil oxidation of 18-hydroxycortisol-17,21-acetonide afforded two isomers of the 6,7-dehydro analogue; separate catalytic reduction of each isomer under deuterium gave a single isomer of acetonide-protected 18-hydroxycortisol-1,6,7-d3 for each, with the more polar isomer giving a more polar product and the less polar isomer giving a less polar product. The more polar product (corresponding to M) was characterized as 18,20-hemiketal; 18-hydroxycortisol-17,21-acetonide-18,20-hemiketal-1,6,7-d3: in the deuterochloroform solution, it was found to slowly convert to a substance consistent with the hydroxy ketone structure with features resembling those of the isolated less polar isomer (corresponding to L). Deacetonidization of each gave 18-hydroxycortisol as a single product, which was characterized as the 18,20-hemiketal. The issues associated with the existence of 18-hydroxysteroids as hydroxy ketones and hemiketals, both in solution and as isolable solids, are discussed.

Therapeutic potential of PIMSR, a novel CB1 receptor neutral antagonist, for cocaine use disorder: evidence from preclinical research.

Cannabinoid CB1 receptors (CB1Rs) have been major targets in medication development for the treatment of substance use disorders. However, clinical trials with rimonabant, a CB1R antagonist/inverse agonist, failed due to severe side effects. Here, we evaluated the therapeutic potential of PIMSR, a neutral CB1R antagonist lacking an inverse agonist profile, against cocaine's behavioral effects in experimental animals. We found that systemic administration of PIMSR dose-dependently inhibited cocaine self-administration under fixed-ratio (FR5), but not FR1, reinforcement, shifted the cocaine self-administration dose-response curve downward, decreased incentive motivation to seek cocaine under progressive-ratio reinforcement, and reduced cue-induced reinstatement of cocaine seeking. PIMSR also inhibited oral sucrose self-administration. Importantly, PIMSR alone is neither rewarding nor aversive as assessed by place conditioning. We then used intracranial self-stimulation (ICSS) to explore the possible involvement of the mesolimbic dopamine system in PIMSR's action. We found that PIMSR dose-dependently attenuated cocaine-enhanced ICSS maintained by electrical stimulation of the medial forebrain bundle in rats. PIMSR itself failed to alter electrical ICSS, but dose-dependently inhibited ICSS maintained by optical stimulation of midbrain dopamine neurons in transgenic DAT-Cre mice, suggesting the involvement of dopamine-dependent mechanisms. Lastly, we examined the CB1R mechanisms underlying PIMSR's action. We found that PIMSR pretreatment attenuated Δ9-tetrahydrocannabinol (Δ9-THC)- or ACEA (a selective CB1R agonist)-induced reduction in optical ICSS. Together, our findings suggest that the neutral CB1R antagonist PIMSR deserves further research as a promising pharmacotherapeutic for cocaine use disorder.

Identification of the GPR55 agonist binding site using a novel set of high-potency GPR55 selective ligands.

Marijuana is the most widely abused illegal drug, and its spectrum of effects suggests that several receptors are responsible for the activity. Two cannabinoid receptor subtypes, CB1 and CB2, have been identified, but the complex pharmacological properties of exogenous cannabinoids and endocannabinoids are not fully explained by their signaling. The orphan receptor GPR55 binds a subset of CB1 and CB2 ligands and has been proposed as a cannabinoid receptor. This designation, however, is controversial as a result of recent studies in which lysophosphatidylinositol (LPI) was identified as a GPR55 agonist. Defining a biological role for GPR55 requires GPR55 selective ligands that have been unavailable. From a ß-arrestin, high-throughput, high-content screen of 300000 compounds run in collaboration with the Molecular Libraries Probe Production Centers Network initiative (PubChem AID1965), we identified potent GPR55 selective agonists. By modeling of the GPR55 activated state, we compared the GPR55 binding conformations of three of the novel agonists obtained from the screen, CID1792197, CID1172084, and CID2440433 (PubChem Compound IDs), with that of LPI. Our modeling indicates the molecular shapes and electrostatic potential distributions of these agonists mimic those of LPI; the GPR55 binding site accommodates ligands that have inverted-L or T shapes with long, thin profiles that can fit vertically deep in the receptor binding pocket while their broad head regions occupy a horizontal binding pocket near the GPR55 extracellular loops. Our results will allow the optimization and design of second-generation GPR55 ligands and provide a means for distinguishing GPR55 selective ligands from those interacting with cannabinoid receptors.

Towards rational design of cannabinoid receptor 1 (CB1) antagonists for peripheral selectivity.

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.

Designer drugs: a medicinal chemistry perspective (II).

During 2012-2018, the clandestine manufacture of new psychoactive substances (NPS) designed to circumvent substance control regulations increased exponentially worldwide, with concomitant increase in fatalities. This review focuses on three compound classes identified as synthetic opioids, synthetic amphetamines, and synthetic cannabinoids and highlights the medicinal chemistry precedents utilized by clandestine laboratories to develop new NPS with increased brain penetration, longer duration of action, and greater potency. Chemical approaches to illicit drug abuse treatment options, particularly for opioid use disorder, are also discussed.

Selective targeting of peripheral cannabinoid receptors prevents behavioral symptoms and sensitization of trigeminal neurons in mouse models of migraine and medication overuse headache.

ABSTRACT: 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.

Targeting of the orphan receptor GPR35 by pamoic acid: a potent activator of extracellular signal-regulated kinase and ß-arrestin2 with antinociceptive activity.

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 G(i/o)-linked, GPR35-mediated increase in the phosphorylation of extracellular signal-regulated kinase 1/2, recruitment of ß-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.

Conformationally constrained analogues of N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4- dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716): design, synthesis, computational analysis, and biological evaluations.

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.

Synthetic peripherally-restricted cannabinoid suppresses chemotherapy-induced peripheral neuropathy pain symptoms by CB1 receptor activation.

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.

Caged Naloxone: Synthesis, Characterization, and Stability of 3- O-(4,5-Dimethoxy-2-nitrophenyl)carboxymethyl Naloxone (CNV-NLX).

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 α R,5 R,9 R,13 S,14 S and α S,5 R,9 R,13 S,14 S 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.

Peripherally restricted cannabinoid 1 receptor agonist as a novel analgesic in cancer-induced bone pain.

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. By 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. In addition, 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 supratherapeutic doses of PrNMI tested. PrNMI induced mild sedation, yet no anxiety or a decrease in limb movements was 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.

Metabolic Profiling of CB1 Neutral Antagonists.

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.

Synthesis and Physicochemical Characterization of the One-Carbon Carrier 10-Formyltetrahydrofolate; A Reference Standard for Metabolomics.

INTRODUCTION: 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. OBJECTIVES: 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. METHODS: Anaerobic base treatment of 5,10-methenyltetrahydrofolate chloride in the presence of anti-oxidant was utilized to prepare 10-formyltetrahydrofolate. RESULTS: 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. CONCLUSION: 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.

Biarylpyrazole inverse agonists at the cannabinoid CB1 receptor: importance of the C-3 carboxamide oxygen/lysine3.28(192) interaction.

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, 3,1<5,4<2<6 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.

Peripherally Selective Cannabinoid 1 Receptor (CB1R) Agonists for the Treatment of Neuropathic Pain.

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 <1% blood-brain barrier penetration for testing in a rat neuropathy induced by unilateral sciatic nerve entrapment (SNE). The SNE-induced mechanical allodynia was reversibly suppressed, partially or completely, after intraperitoneal or oral administration of several indenes. At doses that relieve neuropathy symptoms, the indenes completely lacked, while the brain-permeant CB1R agonist HU-210 (1) exhibited strong CNS side effects, in catalepsy, hypothermia, and motor incoordination assays. Pharmacokinetic findings of ∼0.001 cerebrospinal fluid:plasma ratio further supported limited CNS penetration. Pretreatment with selective CB1R or CB2R blockers suggested mainly CB1R contribution to an indene's antiallodynic effects. Therefore, this class of CB1R agonists holds promise as a viable treatment for neuropathic pain.