Structure-activity studies of 6-(tetrazolylalkyl)-substituted decahydroisoquinoline-3-carboxylic acid AMPA receptor antagonists. 1. Effects of stereochemistry, chain length, and chain substitution.

A series of 6-substituted decahydroisoquinoline-3-carboxylic acids were prepared as excitatory amino acid (EAA) receptor antagonists. These compounds are antagonists at the N-methyl-D-aspartate (NMDA) and 2-amino-3-(5-methyl-3-hydroxyisoxazol-4-yl) propanoic acid (AMPA) subclasses of ligand gated ion channel (ionotropic) EAA receptors. (3S,4aR, 6R,8aR)-6-(2-(1H-tetrazol-5-yl)ethyl)- 1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid (9) is a potent, selective and systemically active AMPA antagonist. Other analogs from this series, including (3S,4aR,6S,8aR)-6-((1H-tetrazol-5-yl)methyl)- 1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid (32) and (3S,4aR,6S,8aR)-6- (phosphonomethyl)-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-ca rboxylic acid (61) are potent, selective, and systemically active NMDA antagonists. This and the subsequent publication look at the AMPA antagonist aspects of this SAR. Herein we report the effects of varying stereochemistry around the hydroisoquinoline ring; of tetrahydro-versus decahydroisoquinoline; of having the carboxylic acid at C-1 versus C-3; of varying the length of the carbon chain connecting a tetrazole to the bicyclic nucleus; and of holding the connecting chain constant at two atoms, the effect of heteroatom substitution in the position adjacent to the bicyclic nucleus and substitution with methyl or phenyl on the chain. Compounds were evaluated on rat cortical tissue for their ability to inhibit the binding of radioligands selective for AMPA ([3H]AMPA), NMDA ([3H]CGS 19755), and kainic acid ([3H]-kainic acid) receptors and for their ability to inhibit depolarizations induced by AMPA (40 microM), NMDA (40 microM), and kainic acid (10 microM). Our findings revealed that the optimal stereochemical array was the same for both NMDA (32 and 61) and AMPA (9) antagonists identified in this series and that the tetrahydroisoquinoline (25) and the C-1 carboxy (30) analogs of 9 are inactive. With a tetrazole in the distal acid position, an ethylene spacer (9) is optimal; substitution with oxygen or nitrogen on the chain in the position adjacent to the bicyclic nucleus significantly reduced activity, while substitution with a methyl or phenyl group on the chain was well tolerated.

Structure-activity studies of 6-substituted decahydroisoquinoline-3-carboxylic acid AMPA receptor antagonists. 2. Effects of distal acid bioisosteric substitution, absolute stereochemical preferences, and in vivo activity.

We have explored the excitatory amino acid antagonist activity in a series of decahydroiso-quinoline-3-carboxyic acids, and within this series found the potent and selective AMPA antagonist (3SR,4aRS,6RS,8aRS)-6-(2-(1H-tetrazol-5-yl )ethyl) decahydroisoquinoline-3-carboxylic acid (1). In this and the preceding paper, we looked at the structure-activity relationships for AMPA antagonist activity in this series of compounds. We have already shown that 1 had the optimal stereochemical array and that AMPA antagonist activity was maximized for a two-carbon spacer separating a tetrazole from the bicyclic nucleus. In this paper, we explored the effects of varying the distal acid and the absolute stereochemical preferences of many of these analogs. We looked at a variety of different acid bioisosteres, including 5-membered hetereocyclic acids such as tetrazole, 1,2,4-triazole, and 3-isoxazolone; carboxylic,phosphonic, and sulfonic acid; and acyl sulfonamides. Compounds were evaluated in rat cortical tissue for their ability to inhibit the binding of radioligands selective for AMPA ([3H]AMPA), NMDA ([3H]CGS 19755), and kainic acid ([3H]kainic acid) receptors and for their ability to inhibit depolarizations induced by AMPA (40 microM), NMDA (40 microM), and kainic acid (10 microM). A number of compounds from this and the preceding paper were also evaluated in mice for their ability to block maximal electroshock-induced convulsions and ATPA-induced rigidity in mice.

Structure-activity relationships of selective estrogen receptor modulators: modifications to the 2-arylbenzothiophene core of raloxifene.

The 2-arylbenzothiophene raloxifene, 1, is a selective estrogen receptor modulator which is currently under clinical evaluation for the prevention and treatment of postmenopausal osteoporosis. A series of raloxifene analogs which contain modifications to the 2-arylbenzothiophene core have been prepared and evaluated for the ability to bind to the estrogen receptor and inhibit MCF-7 breast cancer cell proliferation in vitro. Their ability to function as tissue-selective estrogen agonists in vivo has been assayed in a short-term, ovariectomized (OVX) rat model with end points of serum cholesterol lowering, uterine weight gain, and uterine eosinophil peroxidase activity. These studies have demonstrated that (1) the 6-hydroxy and, to a lesser extent, the 4'-hydroxy substituents of raloxifene are important for receptor binding and in vitro activity, (2) small, highly electronegative 4'-substituents such as hydroxy, fluoro, and chloro are preferred both in vitro and in vivo, (3) increased steric bulk at the 4'-position leads to increased uterine stimulation in vivo, and (4) additional substitution of the 2-aryl moiety is tolerated while additional substitution at the 4-, 5-, or 7-position of the benzothiophene results in reduced biological activity. In addition, compounds in which the 2-aryl group is replaced by alkyl, cycloalkyl, and naphthyl substituents maintain a profile of in vitro and in vivo biological activity qualitatively similar to that of raloxifene. Several novel structural variants including 2-cyclohexyl, 2-naphthyl, and 6-carbomethoxy analogs also demonstrated efficacy in preventing bone loss in a chronic OVX rat model of postmenopausal osteopenia, at doses of 0.1-10 mg/kg.

Evaluation of the major metabolites of raloxifene as modulators of tissue selectivity.

Raloxifene (LY139481 HCl) is a selective estrogen receptor modulator (SERM) which blocks the effects of estrogen on some tissues, such as the breast and uterus, while mimicking estrogen in other tissues, such as bone. To study the origins of this unique pharmacology, we have prepared the major metabolites of raloxifene as chemical probes for examining the estrogen receptor function in vitro and in vivo. In human breast cancer cell (MCF-7) related assays, these glucuronide conjugates show little affinity for the estrogen receptor and are more than two orders of magnitude less potent at inhibiting cell proliferation than raloxifene. In non-traditional estrogen target tissue, such as bone, these metabolites are less effective than the parent at inhibiting cytokine-stimulated bone resorbing activity in rat osteoclasts or producing transforming growth factor beta-3 (TGF-beta3). In animal models, tissue distribution studies with radiolabelled metabolite indicate that conversion to raloxifene occurs readily in a variety of tissues including the liver, lung, spleen, kidney, bone and uterus. Differential conversion of metabolite in target organs, such as bone and the uterus, is not observed indicating that the origin of raloxifene's pharmacology does not result from tissue-selective deconjugation of metabolite to parent.

Development of analytical and preparative chromatographic separations of novel growth hormone secretagogue compounds.

Chromatographic separations of new growth hormone secretagogue compounds were developed to support structure-activity relationship (SAR) studies in conjunction with lead optimization. These new compounds differed from Merck's MK-677 by having two chiral centers and thus diastereomeric mixtures were generated. Separation of initial compounds in the SAR was achieved on a Kromasil C18 column using an ammonium acetate buffer and acetonitrile. However, additional candidates were not separable on C18 columns and a chiral Kromasil CHI-DMB column was used to resolve the diastereomeric compounds. The Kromasil CHI-DMB packing was also used in a preparative chromatographic system to resolve multigram quantities of secretagogue candidates for testing. Chiral separations of different intermediates were also developed in support of evolution of an asymmetric synthetic route. This report summarizes development of the preparative chromatographic system used to purify diastereomeric mixtures and chiral separations of intermediates in the synthesis.