Synthesis and pharmacology of 1-alkyl-3-(1-naphthoyl)indoles: steric and electronic effects of 4- and 8-halogenated naphthoyl substituents

To develop SAR at both the cannabinoid CB(1) and CB(2) receptors for 3-(1-naphthoyl)indoles bearing moderately electron withdrawing substituents at C-4 of the naphthoyl moiety, 1-propyl and 1-pentyl-3-(4-fluoro, chloro, bromo and iodo-1-naphthoyl) derivatives were prepared. To study the steric and electronic effects of substituents at the 8-position of the naphthoyl group, the 3-(4-chloro, bromo and iodo-1-naphthoyl)indoles were also synthesized. The affinities of both groups of compounds for the CB(1) and CB(2) receptors were determined and several of them were evaluated in vivo in the mouse. The effects of these substituents on receptor affinities and in vivo activity are discussed and structure-activity relationships are presented. Although many of these compounds are selective for the CB(2) receptor, only three JWH-423, 1-propyl-3-(4-iodo-1-naphthoyl)indole, JWH-422, 2-methyl-1-propyl-3-(4-iodo-1-naphthoyl)indole, the 2-methyl analog of JWH-423 and JWH-417, 1-pentyl-3-(8-iodo-1-naphthoyl)indole, possess the desirable combination of low CB(1) affinity and good CB(2) affinity.     

1-Alkyl-2-aryl-4-(1-naphthoyl)pyrroles: new high affinity ligands for the cannabinoid CB1 and CB2 receptors

Two series of 1-alkyl-2-aryl-4-(1-naphthoyl)pyrroles were synthesized and their affinities for the cannabinoid CB(1) and CB(2) receptors were determined. In the 2-phenyl series (5) the N-alkyl group was varied from n-propyl to n-heptyl. A second series of 23 1-pentyl-2-aryl-4-(1-naphthoyl)-pyrroles (6) was also prepared. Several compounds in both series have CB(1) receptor affinities in the 6-30nM range. The high affinities of these pyrrole derivatives relative to JWH-030 (1, R=C(5)H(11)) support the hypothesis that these pyrroles interact with the CB(1) receptor primarily by aromatic stacking.     

N-Alkylidenearylcarboxamides as new potent and selective CB(2) cannabinoid receptor agonists with good oral bioavailability

A novel series of N-alkylidenearylcarboxamides 4, a CB(2) receptor agonist, were synthesized and evaluated for activity against the human CB(2) receptor. In a previous paper, we reported that sulfonamide derivative 1 acted as a potent CB(2) receptor agonist (IC(50)=65 nM, EC(50)=19 nM, E(max)=90%). However, compound 1 also exhibited poor metabolic stability in human liver microsomes. During the structural modification of 1, we found that a novel series of N-alkylidenearylcarboxamide, 4-1, had a moderate affinity for the CB(2) receptor (IC(50)=260 nM, EC(50)=86 nM, E(max)=100%) and good metabolic stability in human liver microsomes. We explored its analogues to discover compounds with a high affinity for the CB(2) receptor and with good oral bioavailability. Among them, compounds 4-9 and 4-27 had high affinities for the human CB(2) receptor (CB(2) IC(50)=13 nM and 1.2 nM) and a high selectivity for CB(2) (CB(1) IC(50)/CB(2) IC(50)=270 and 1600); furthermore, significant plasma levels were observed following oral administration in rats (C(max)=233 ng/mL and 148 ng/mL, respectively, after a dose of 10 mg/kg). Furthermore, compound 4-9 had good oral bioavailability (F=52%, 3mg/kg).     

New natural noncannabinoid ligands for cannabinoid type-2 (CB2) receptors

Since the discovery that Delta 9-tetrahydrocannabinol and related cannabinoids from Cannabis sativa L. act on specific physiological receptors in the human body and the subsequent elucidation of the mammalian endogenous cannabinoid system, no other natural product class has been reported to mimic the effects of cannabinoids. We recently found that N-alkyl amides from purple coneflower (Echinacea spp.) constitute a new class of cannabinomimetics, which specifically engage and activate the cannabinoid type-2 (CB2) receptors. Cannabinoid type-1 (CB1) and CB2 receptors belong to the family of G protein-coupled receptors and are the primary targets of the endogenous cannabinoids N-arachidonoyl ethanolamine and 2-arachidonoyl glyerol. CB2 receptors are believed to play an important role in distinct pathophysiological processes, including metabolic dysregulation, inflammation, pain, and bone loss. CB2 receptors have, therefore, become of interest as new targets in drug discovery. This review focuses on N-alkyl amide secondary metabolites from plants and underscores that this group of compounds may provide novel lead structures for the development of CB2-directed drugs.     

Highly selective CB(1) cannabinoid receptor ligands and novel CB(1)/VR(1) vanilloid receptor "hybrid" ligands

Anandamide and the metabolically stabler analogs, (R)-1'-methyl-2'-hydroxy-ethyl-arachidonamide (Met-AEA) and N-(3-methoxy-4-hydroxy-benzyl)-arachidonamide (arvanil), are CB(1) cannabinoid and VR(1) vanilloid receptors agonists. We synthesized 1',1'-dimethylheptyl-arvanil (O-1839) and six other AEA analogs obtained by addition of either a hydroxy, cyano, or bromo group on the C-20 atom of 1,1'-dimethylpentyl-Met-AEA (O-1811, O-1812 and O-1860, respectively) or 1,1'-dimethylpentyl-arvanil (O-1856, O-1895 and O-1861, respectively). The compounds were tested for their (i) affinity for CB(1) and CB(2) receptors, (ii) capability to activate VR1 receptors, (iii) inhibitory effect on the anandamide hydrolysis and on the anandamide membrane transporter, and (iv) cannabimimetic activity in the mouse 'tetrad' of in vivo assays. O-1812 is the first ligand ever proven to be highly (500- to 1000-fold) selective for CB(1) vs both VR(1) and CB(2) receptors, while O-1861 is the first true "hybrid" agonist of CB(1)/VR(1) receptors and a compound with potential therapeutic importance. The activities of the seven compounds in vivo did not correlate with their activities at either CB(1) or VR(1) receptors, thus suggesting the existence of other brain sites of action mediating some of their neurobehavioral actions in mice.     

7-Azaindolequinuclidinones (7-AIQD): A novel class of cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptor ligands

A series of N-benzyl-7-azaindolequinuclidinone (7-AIQD) analogs have been synthesized and evaluated for affinity toward CB1 and CB2 cannabinoid receptors and identified as a novel class of cannabinoid receptor ligands. Structure–activity relationship (SAR) studies indicate that 7-AIQD analogs are dual CB1/CB2 receptor ligands exhibiting high potency with somewhat greater selectivity towards CB2 receptors compared to the previously reported indolequinuclidinone (IQD) analogs. Initial binding assays showed that 7-AIQD analogs 8b, 8d, 8f, 8g and 9b (1 μM) produced more that 50% displacement of the CB1/CB2 non-selective agonist CP-55,940 (0.1 nM). Furthermore, Ki values determined from full competition binding curves showed that analogs 8a, 8b and 8g exhibit high affinity (110, 115 and 23.7 nM, respectively) and moderate selectivity (26.3, 6.1 and 9.2-fold, respectively) for CB2 relative to CB1 receptors. Functional studies examining modulation of G-protein activity demonstrated that 8a acts as a neutral antagonist at CB1 and CB2 receptors, while 8b exhibits inverse agonist activity at these receptors. Analogs 8f and 8g exhibit different intrinsic activities, depending on the receptor examined. Molecular docking and binding free energy calculations for the most active compounds (8a, 8b, 8f, and 8g) were performed to better understand the CB2 receptor-selective mechanism at the atomic level. Compound 8g exhibited the highest predicted binding affinity at both CB1 and CB2 receptors, and all four compounds were shown to have higher predicted binding affinities with the CB2 receptor compared to their corresponding binding affinities with the CB1 receptor. Further structural optimization of 7-AIQD analogs may lead to the identification of potential clinical agents.     

Structure-activity relationships of 2,4-diphenyl-1H-imidazole analogs as CB2 receptor agonists for the treatment of chronic pain

A series of 2,4-diphenyl-1H-imidazole analogs have been synthesized and displayed potent human CB2 agonist activity. Many of these analogs showed high functional selectivity over human CB1 receptors. The syntheses, structure-activity relationships, and selected pharmacokinetic data of these analogs are described.     

3-Indolyl-1-naphthylmethanes: new cannabimimetic indoles provide evidence for aromatic stacking interactions with the CB(1) cannabinoid receptor

A series of 1-pentyl-1H-indol-3-yl-(1-naphthyl)methanes (9-11) and 2-methyl-1-pentyl-1H-indol-3-yl-(1-naphthyl)methanes (12-14) have been synthesized to investigate the hypothesis that cannabimimetic 3-(1-naphthoyl)indoles interact with the CB(1) receptor by hydrogen bonding to the carbonyl group. Indoles 9-11 have significant (K(i)=17-23nM) receptor affinity, somewhat less than that of the corresponding naphthoylindoles (5, 15, 16). 2-Methyl-1-indoles 12-14 have little affinity for the CB(1) receptor, in contrast to 2-methyl-3-(1-naphthoyl)indoles 17-19, which have affinities comparable to those of 5, 15, 16. A cannabimimetic indene hydrocarbon (26) was synthesized and found to have K(i)=26+/-4nM. Molecular modeling and receptor docking studies of naphthoylindole 16, its 2-methyl congener (19) and indolyl-1-naphthylmethanes 11 and 14, combined with the receptor affinities of these cannabimimetic indoles, strongly suggest that these cannabinoid receptor ligands bind primarily by aromatic stacking interactions in the transmembrane helix 3-4-5-6 region of the CB(1) receptor.     

Discovery of N-(3-(morpholinomethyl)-phenyl)-amides as potent and selective CB2 agonists

Recently sulfamoyl benzamides were identified as a novel series of cannabinoid receptor ligands. Replacing the sulfonamide functionality and reversing the original carboxamide bond led to the discovery of N-(3-(morpholinomethyl)-phenyl)-amides as potent and selective CB₂ agonists. Selective CB₂ agonist 31 (K_i = 2.7; CB₁/CB₂ = 190) displayed robust activity in a rodent model of postoperative pain.     

Evaluation of (Z)-2-((1-benzyl-1H-indol-3-yl)methylene)-quinuclidin-3-one analogues as novel,high affinity ligands for CB1 and CB2 cannabinoid receptors

A small library of N-benzyl indolequinuclidinone (IQD) analogs has been identified as a novel class of cannabinoid ligands. The affinity and selectivity of these IQDs for the two established cannabinoid receptor subtypes, CB1 and CB2, was evaluated. Compounds 8 (R = R² = H, R¹ = F) and 13 (R = COOCH₃, R¹ = R² = H) exhibited high affinity for CB2 receptors with K_i values of 1.33 and 2.50 nM, respectively, and had lower affinities for the CB1 receptor (K_i values of 9.23 and 85.7 nM, respectively). Compound 13 had the highest selectivity of all the compounds examined, and represents a potent cannabinoid ligand with 34-times greater selectivity for CB2R over CB1R. These findings are significant for future drug development, given recent reports demonstrating beneficial use of cannabinoid ligands in a wide variety of human disease states including drug abuse, depression, schizophrenia, inflammation, chronic pain, obesity, osteoporosis and cancer.     

Synthesis and pharmacological characterization of functionalized 6-piperazin-1-yl-purines as cannabinoid receptor 1 (CB1) inverse agonists

Antagonists of peripheral type 1 cannabinoid receptors (CB1) may have utility in the treatment of obesity, liver disease, metabolic syndrome and dyslipidemias. We have targeted analogues of the purine inverse agonist otenabant (1) for this purpose. The non-tissue selective CB1 antagonist rimonabant (2) was approved as a weight-loss agent in Europe but produced centrally mediated adverse effects in some patients including dysphoria and suicidal ideation leading to its withdrawal. Efforts are now underway to produce compounds with limited brain exposure. While many structure-activity relationship (SAR) studies of 2 have been reported, along with peripheralized compounds, 1 remains relatively less studied. In this report, we pursued analogues of 1 in which the 4-aminopiperidine group was switched to piperazine group to enable a better understanding of SAR to eventually produce compounds with limited brain penetration. To access a binding pocket and modulate physical properties, the piperazine was functionalized with alkyl, heteroalkyl, aryl and heteroaryl groups using a variety of connectors, including amides, sulfonamides, carbamates and ureas. These studies resulted in compounds that are potent antagonists of hCB1 with high selectivity for hCB1 over hCB2. The SAR obtained led to the discovery of 65 (Ki?=?4?nM, >1,000-fold selective for hCB1 over hCB2), an orally bioavailable aryl urea with reduced brain penetration, and provides direction for discovering peripherally restricted compounds with good in vitro and in vivo properties.     

Structure-activity relationships of 1-alkyl-5-(3,4-dichlorophenyl)-5-{2-[3-(substituted)-1-azetidinyl]-ethyl}-2-piperidones. Part 2: Improving oral absorption

A series of piperidone analogues of 1b-q, seeking replacements for the polar sulfamide moiety in clinical candidate UK-224,671 1a, possessing reduced H-bonding potential as a strategy to improve oral absorption, were prepared. These studies led to the successful identification of 1n, which demonstrated equivalent pharmacology and metabolic stability to 1a, and greatly improved oral absorption as assessed in rat PK studies.     

Structure-activity relationships of non-imidazole H(3) receptor ligands. Part 1

SAR studies for novel non-imidazole containing H(3) receptor antagonists with high potency and selectivity for rat H(3) receptors are described. A high throughput screening lead, A-923, was further elaborated in a systematic manner to clarify a pharmacophore for this class of aryloxyalkyl piperazine based compounds.     

Synthesis and evaluation of N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-aminopropanamide as human cannabinoid-1 receptor (CB1R) inverse agonists

Obesity is a chronic medical condition that is affecting large population throughout the world. CB1 as a target for treatment of obesity has been under intensive studies. Taranabant was discovered and then developed by Merck as the 1st generation CB1R inverse agonist. Reported here is part of our effort on the 2nd generation of CB1R inverse agonist from the acyclic amide scaffold. We replaced the oxygen linker in taranabant with nitrogen and prepared a series of amino heterocyclic analogs through a divergent synthesis. Although in general, the amine linker gave reduced binding affinity, potent and selective CB1R inverse agonist was identified from the amino heterocycle series. Molecular modeling was applied to study the binding of the amino heterocycle series at CB1 binding site. The in vitro metabolism of representative members was studied and only trace glucuronidation was found. Thus, it suggests that the right hand side of the molecule may not be the appropriate site for glucuronidation.     

The discovery of novel indole-2-carboxamides as cannabinoid CB(1) receptor antagonists

The discovery and structure-activity relationship of a novel series of indole-2-carboxamide antagonists of the cannabinoid CB(1) receptor is disclosed. Compound 26i was found to be a high potency, selective cannabinoid CB(1) antagonist.     

Expression, purification, and isotope labeling of cannabinoid CB2 receptor fragment, CB2(180-233)

To develop an approach to obtain milligram quantities of purified isotope-labeled seven transmembrane G-protein coupled cannabinoid (CB) receptor for NMR structural analysis, we chose a truncated CB receptor fragment, CB2(180-233), spanning from the fifth transmembrane domain (TM5) to the associated loop regions of cannabinoid CB2 receptor. This highly hydrophobic membrane protein fragment was pursued for developmental studies of membrane proteins through expression and purification in Escherichia coli. The target peptide was cloned and over-expressed in a preparative scale as a fusion protein with a modified TrpDeltaLE1413 (TrpLE) leader sequence and a nine-histidine tag at its N-terminal. An experimental protocol for enzyme cleavage was developed by using Factor Xa to remove the TrpLE tag from the fusion protein. A purification process was also established using a nickel affinity column and reverse-phase HPLC, and then monitored by SDS-PAGE and MS. This expression level is one of the highest reported for a G-protein coupled receptor and fragments in E. Coli, and provided a sufficient amount of purified protein for further biophysical studies.     

2-Arylimino-5,6-dihydro-4H-1,3-thiazines as a new class of cannabinoid receptor agonists. Part 1: discovery of CB2 receptor selective compounds

2-Arylimino-5,6-dihydro-4H-1,3-thiazines have been identified as a novel class of cannabinoid agonists. A lead structure with moderate activity was discovered through a high throughput screening assay. Structure-activity relationships led to the discovery of potent agonists of CB(2) receptor. The most potent compound 13 displays K(i) values of >5000 and 9 nM to CB(1) and CB(2) receptors, respectively.     

Synthesis and structure-activity relationships of (R)-1-alkyl-3-[2-(2-amino)phenethyl]-5-(2-fluorophenyl)-6-methyluracils as human GnRH receptor antagonists

The synthesis of a series of (R)-1-alkyl-3-[2-(2-amino)phenethyl]-5-(2-fluorophenyl)-6-methyluracils is discussed. SAR around N-1 of the uracil was explored, which led to the discovery that an electron-deficient 2,6-disubstituted benzyl group is required for optimal receptor binding. The best compound from the series had binding affinity of 0.7 nM (K(i) for the human GnRH receptor, which was 8-fold better than the 2,6-difluorobenzyl analog.     

Development and characterization of immobilized cannabinoid receptor (CB1/CB2) open tubular column for on-line screening

Cannabinoid receptors, CB1 and CB2, are therapeutic targets in the treatment of anxiety, obesity, movement disorders, glaucoma, and pain. We have developed an on-line screening method for CB1 and CB2 ligands, where cellular membrane fragments of a chronic myelogenous leukemia cell line, KU-812, were immobilized onto the surface of an open tubular (OT) capillary to create a CB1/CB2–OT column. The binding activities of the immobilized CB1/CB2 receptors were established using frontal affinity chromatographic techniques. This is the first report that confirms the presence of functional CB1 and CB2 receptors on KU-812 cells. The data from this study confirm that the CB1/CB2–OT column can be used to determine the binding affinities (K_i values) for a single compound and to screen individual compounds or a mixture of multiple compounds. The CB1/CB2–OT column was also used to screen a botanical matrix, Zanthoxylum clava-herculis, where preliminary results suggest the presence of a high-affinity phytocannabinoid.     

3-(1',1'-Dimethylbutyl)-1-deoxy-delta8-THC and related compounds: synthesis of selective ligands for the CB2 receptor

The synthesis and pharmacology of 15 1-deoxy-delta8-THC analogues, several of which have high affinity for the CB2 receptor, are described. The deoxy cannabinoids include 1-deoxy-11-hydroxy-delta8-THC (5), 1-deoxy-delta8-THC (6), 1-deoxy-3-butyl-delta8-THC (7), 1-deoxy-3-hexyl-delta8-THC (8) and a series of 3-(1',1'-dimethylalkyl)-1-deoxy-delta8-THC analogues (2, n = 0-4, 6, 7, where n = the number of carbon atoms in the side chain-2). Three derivatives (17-19) of deoxynabilone (16) were also prepared. The affinities of each compound for the CB1 and CB2 receptors were determined employing previously described procedures. Five of the 3-(1',1'-dimethylalkyl)-1-deoxy-delta8-THC analogues (2, n = 1-5) have high affinity (Ki = < 20 nM) for the CB2 receptor. Four of them (2, n = 1-4) also have little affinity for the CB1 receptor (Ki = > 295 nM). 3-(1',1'-Dimethylbutyl)-1-deoxy-delta8-THC (2, n = 2) has very high affinity for the CB2 receptor (Ki = 3.4 +/- 1.0 nM) and little affinity for the CB1 receptor (Ki = 677 +/- 132 nM).