Constitutive activity of cannabinoid-2 (CB2) receptors plays an essential role in the protean agonism of (+)AM1241 and L768242.

BACKGROUND AND PURPOSE: Cannabinoid-2 (CB(2)) receptor-selective agonists have shown anti-nociceptive activity in models of neuropathic and inflammatory pain, and the two agonists most widely used, (+/-)AM1241 [(2-iodo-5-nitrophenyl)-[1-(1-methylpiperidin-2-ylmethyl)-1H-indol-3-yl-methanone] and L768242 [(2,3-dichloro-phenyl)-[5-methoxy-2-methyl-3-(2-morpholin-4-yl-ethyl)-indol-1-yl]-methanone] (GW405833), have been suggested to be protean agonists. Here we investigated the role of the constitutive activity of CB(2) receptors in (+)AM1241 and L768242 protean agonism. EXPERIMENTAL APPROACH: Pharmacological profiles of CB(2) receptor ligands were evaluated in Chinese hamster ovary cells expressing recombinant human (hCB(2)) or rat (rCB(2)) receptors, by measuring modulation of cAMP. To assess the influence of constitutive activity on pharmacological profile, constitutive activity was abolished by pretreatment with AM630 [(6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl) methanone)], followed by extensive washing. KEY RESULTS: In cell lines expressing either hCB(2) or rCB(2) receptors, (+)AM1241 did not reverse forskolin stimulation of cAMP levels. Conversely, L768242 was an inverse agonist at both hCB(2) and rCB(2) receptors. Abolition of constitutive activity disclosed (+)AM1241 and L768242 agonist activity, while activity of CP55940 [5-(1,1-dimethylheptyl)-2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxy-propyl)-cyclohexyl]-phenol] was unaffected and AM630 became a neutral antagonist. In presence of constitutively active CB(2) receptors, (+)AM1241 antagonized CP55940, but when constitutive activity was abolished, it acted as a partial agonist with additive or antagonistic behaviour, depending on concentration. CONCLUSIONS AND IMPLICATIONS: These results show that (+)AM1241 and L768242 are protean agonists at both hCB(2) and rCB(2) receptors. Abolition of constitutive activity reveals the agonist activity of these compounds. Thus, differences between in vivo and in vitro profiles of CB(2) receptor agonists could be due to different levels of constitutive activity in recombinant versus native CB(2) receptors.

Examination of the D2/5-HT2 affinity ratios of resolved 5,6,7,8,9,10-hexahydro-7,10-iminocyclohept[b]indoles: an enantioselective approach toward the design of potential atypical antipsychotics.

Enantiomers of several N-substituted 5,6,7,8,9,10-hexahydro-7,10-iminocyclohept[b]indoles were obtained by the resolution of 2-fluoro-5,6,7,8,9,10-hexahydro-7,10-iminocyclohept[b]indole and 5,6,7,8,9,10-hexahydro-7,10-iminocyclohept[b]indole followed by N-alkylation. These, as well as the racemates, were evaluated for their affinity for the 5-HT2 and D2 receptors. Those compounds possessing the 7S,10R stereochemistry were consistently recognized by the 5-HT2 and D2 receptors as the eutomer. 2-Fluoro-11-[4-(4-fluorophenyl)-4-oxobutyl]-5,6,7,8,9,10-hexahydro-7S,10 R- iminocyclohept[b]indole [(7S,10R)-8] had the highest affinity for the 5-HT2 receptor (Ki = 0.80 nM), while its distomer (7R,10S)-8 was the most selective member of this class of bridged gamma-carbolines (D2/5-HT2 = 562). Incorporation of a benzoyl or isosteric benzisoxazole moiety tethered by a four-carbon spacer to a bridged gamma-carboline nucleus, possessing the 7S,10R absolute configuration, produced high affinity ligands for the 5-HT2 and D2 receptors.

Synthesis and reaction of potential alternate substrates and mechanism-based inhibitors of clavaminate synthase.

Clavaminate synthase is an FeII/alpha-ketoglutarate-dependent enzyme central to the biosynthesis of the beta-lactamase inhibitor clavulanic acid. In the presence of dioxygen it catalyzes the oxidative cyclization/desaturation of proclavaminic acid to clavaminic acid in a two-step process. Samples of (4'R)- and (4'S)-D,L-[4'-2H]proclavaminic acid have been prepared and used to demonstrate that oxazolidine ring formation occurs with retention of configuration. The stereochemical course of oxygen insertion from substrate that takes place in this oxidative cyclization is the same as that observed from molecular oxygen in several hydroxylation reactions catalyzed by other FeII/alpha-ketoglutarate-dependent enzymes. The ferryl (FeIV = O) species thought to be transiently involved in each of these processes was investigated in the present work with clavaminate synthase and three structural analogues of proclavaminic acid bearing vinyl or ethynyl groups at C-4' or a cyclopropyl at C-4. In the synthesis of the former two derivatives and proclavaminic acid stereoselectively labeled with deuterium at C-4', introduction of the unsaturated substituents in a stereochemically defined manner at C-4' relied upon ready access to (4R)-4-thiophenyl-2-azetidinone. Trimethylsilyl substitution could be easily achieved at C-3 of the optically pure starting material to give the readily separable cis and trans diastereomers. In radical chain reactions in which the thiophenyl was replaced by deuterium or in anionic reactions in which the thiophenyl was eliminated as its sulfone and replaced by addition of carbanions, the steric bulk of the trimethylsilyl group at C-3 governed the approach of incoming reagents to give the trans product. The enzymatic fate, however, of these derivatives was disappointing, yielding neither detectable reaction nor hoped-for inactivation of clavaminate synthase. Finally, as mixed competitive/noncompetitive inhibitors of catalysis, they gave unexceptional inhibition constants in the range 2-10 mM.

Bridged gamma-carbolines and derivatives possessing selective and combined affinity for 5-HT2 and D2 receptors.

A series of 5,6,7,8,9,10-hexahydro-7,10-iminocyclohept[b]indoles and 6,7,8,9,10,11-hexahydro-7,11-imino-5H-cyclooct[b]indoles was prepared. Structural modifications of the lead compound, 11-[4-(4-fluorobenzoyl)propyl]-5,6,7,8,9,10-hexahydro-7,10- iminocyclohept[b]indole (5, Ki = 0.82 nM vs [3H]ketanserin) enabled the identification of the functionality necessary for high affinity at serotonin 5-HT2 and dopamine D2 receptors in ligand binding studies. The indole ring, as well as the benzoyl or isosteric benzisoxazole moiety, were essential for high affinity. Variations of the length of the side chains resulted in ligands having either selective affinity for the 5-HT2 receptor or a combination of 5-HT2 and D2 affinity. In vivo binding studies were performed on selected members in this series. The most potent member, 2-fluoro-11-[4-(4-fluorobenzoyl)butyl]-5,6,7,8,9,10-hexahydro-7,10- iminocyclohept[b]indole (36) had an ED50 of < 1 mg/kg at the 5-HT2 and D2 receptors following oral administration.