High-affinity glycine transport in neurons and glial cells is a primary means of inactivating synaptic glycine. We have synthesized a potent selective inhibitor of glycine transporter 1 (GlyT1), and characterized its activity using a quail fibroblast cell line (QT6). The glycine transporters GlyT1A, GlyT1B, GlyT1C, and GlyT2 were stably expressed in QT6 cells. The transporters expressed in these cells exhibited appropriate characteristics as described previously for these genes: Na(+)/Cl(-) dependence, appropriate K(m) values for glycine uptake, and appropriate pharmacology, as defined in part by the ability of N-methyl glycine (sarcosine) to competitively inhibit glycine transport. Furthermore, the characteristics of the transporters in the cell lines recapitulate the characteristics of glycine transporters observed in tissue preparations. We developed a sarcosine derivative, (R)-(N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl])sarcosine (ALX 5407), and examined its activity against the cloned glycine transporters. ALX 5407 completely inhibited glycine transport in the GlyT1 cells, with an IC(50) value of 3 nM, but had little or no activity at the human GlyT2 transporter, at other binding sites for glycine, or at other neurotransmitter transporters. The inhibition of glycine transport was essentially irreversible. ALX 5407 represents a novel tool in the investigation of N-methyl-D-aspartate-receptor function. This class of drug may lead to novel therapies in the treatment of schizophrenia.

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A series of 7,8-disubstituted guanosine derivatives was designed and prepared as potential B-cell-selective activators of the humoral immune response. These compounds were evaluated for their ability to act as B-cell mitogens and to augment the antibody response of B cells to sheep red blood cell (SRBC) challenge (adjuvanticity). In addition, they were tested for their ability to stimulate the natural killer (NK) cell response in murine in vitro cell assays. Certain of the compounds demonstrated in vivo activity when administered either intravenously, subcutaneously, or orally. Analogues with a medium-length alkyl chain (2-4 carbons, 5-7) on the 7-position of 7-alkyl-8-oxoguanosines were found to be particularly potent. Compounds bearing hydroxyalkyl, aminoalkyl, or substituted aminoalkyl substituents on this 7-position were weakly active. However, benzyl groups, including those substituted with heteroatoms (e.g., p-nitrobenzyl, 14), were active. Oxo, thioxo, and seleno groups on C-8 of the guanosine ring all imparted strong activity, whereas other larger substituents did not (e.g., N = CN). Stereochemical inversion of the 2'-hydroxyl on the ribose ring in this series, giving arabinose analogue 70, lessened activity. However, removal of the 2'-hydroxyl, either with (64) or without (73) removal of the 3'-hydroxyl, resulted in excellent activity and improved solubility; 64 also displayed good oral in vivo activity as well. A series of ketals involving the 2',3'-hydroxyls were prepared; certain of the nonpolar ketals (e.g., 48) were remarkably active, pointing to an ancillary hydrophobic binding region that can augment activity. 5'-Phosphate derivative 57 was fairly active, and acyclovir analogue 90 displayed good NK-selective activity: other N-9 sugar mimetics were also active (97-104), although this activity did not carry over into the human B-cell assay. A total of 80 compounds were prepared and evaluated for their immunostimulating activity. Within this group, compounds could be divided into those that were active in all three assays, those that displayed some measure of selectivity for the adjuvanticity assay, and those that preferentially activated NK responses. Because of its overall biological profile and ease of synthesis, 7-allyl-8-oxoguanosine (6; loxoribine, RWJ-21757) was chosen for further development. It is among the most potent compounds evaluated in the three biological assays.

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The syntheses of 2-oxo-1,8-naphthyridine-3-carboxylic acid derivatives having potent gastric antisecretory properties in the pyloric-ligated (Shay) rat model are described. Two of the more potent compounds tested that were selected for more detailed dose-response evaluation were 4-amino-1-ethyl-1,2-dihydro-2-oxonaphthyridine-3-carboxylic acid ethyl ester (35) and 1-ethyl-1,2-dihydro-7-methyl-4-(4-methyl-1-piperazinyl)-2- oxo-1,8-naphthyridine-3-carboxylic acid ethyl ester (77). These compounds lowered total acid output in the rat in a dose-related fashion. Both compounds were more potent than cimetidine when tested in the rat. Both 35 and 77 showed inhibitory activity in food-stimulated acid secretion in the Pavlov-pouch, conscious dog. The mechanism of action for this series is not known. Details of structure-activity relationships are described.

2-(Diethylamino)-6-nitrobenzonitrile (72). To a mixture of 3.82 g (0.02 mol) of 2-(ethylamino)-6-nitrobenzonitrile (73) and 3.2 g (0.02 mol) of ethyl iodide in 100 mL of DMF was added 0.96 g (0.02 mol) of 50% NaH (in oil) in portions. The mildly exothermic reaction was stirred for 10 min, poured into ice-water, and extracted into methylene chloride. Evaporation gave an oil that was passed through a short column of silica gel (20 g) with methylene chloride. The bright orange oil was collected and triturated with hexane to give 3.1 g of pure product, mp 39-41 °C.2-Nitro-5-(dimethylamino)benzoic Acid (75). Sodium cyanoborohydride (57 g) was added to a mixture of 54.6 g (0.21 mol) of commercial (70%) 2-nitro-5-aminobenzoic acid and 240 mL of formalin in 1200 mL of acetonitrile. The reaction mixture was stirred overnight and evaporated to one-third volume. The product crystallized as the hemihydrate: mp 187-189 °C; yield 50 g (100%). Anal. (C9H10N2O4-0.5H2O) C, , N. 5-(Dimethylamino)anthranilic Acid (76). A mixture of 10.5 g (0.05 mol) of the nitrobenzoic acid 75,5 g of 10% Pd on charcoal, and 25 g (0.3 mol) of cyclohexene in 200 mL of ethanol was heated at reflux for 3 h. The hot reaction mixture was filtered through Celite, the Celite pad was extracted three times with hot ethanol, and the combined ethanol filtrates were evaporated to one-third volume. Water was added to cloudiness, whereupon the pure product crystallized: yield 8.0 g (93%); mp 234-238 °C (lit.28 234-236 °C).

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