Novel pyridine-and pyrimidine-based allosteric inhibitors are reported that achieve PDE4D subtype selectivity through recognition of a single amino acid difference on a key regulatory domain, known as UCR2, that opens and closes over the catalytic site for cAMP hydrolysis. The design and optimization of lead compounds was based on iterative analysis of X-ray crystal structures combined with metabolite identification. Selectivity for the activated, dimeric form of PDE4D provided potent memory enhancing effects in a mouse model of novel object recognition with improved tolerability and reduced vascular toxicity over earlier PDE4 inhibitors that lack subtype selectivity. The lead compound, 28 (BPN14770), has entered midstage, human phase 2 clinical trials for the treatment of Fragile X Syndrome.

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Dimethylnitrosamine has been shown to be selectively hepatotoxic for several species of mammals, producing demonstrable necrosis within 24 hr. (Barnes & Magee, 1954) and, on prolonged administration, to produce malignant liver tumours (Magee & Barnes, 1956). It is rapidly metabolized (Dutton & Heath, 1956) and Magee (1956) demonstrated that the liver is the main, and probably the only, site of its metabolism. Magee (1957) also found that the incorporation of labelled amino acids into liver protein is impaired as early as 6 hr. after treatment.The rapid necrotic effect on liver cells suggests that dimethylnitrosamine, or a metabolite, might interfere with respiratory metabolism of the liver cells, and this paper reports an investigation of this. MATERIALS AND METHODS Animal8The animals belonged to a strain of Sprague-Dawley rats inbred by brother-sister matings to the stage of successful homologous skin grafting. The diet consisted of Poultry Growers' Pellets (Barastoc Products, Melbourne), fresh green vegetables and water. Rats were used irrespective of sex when they weighed between 150 and 250 g. As far as possible, litter mates were used for each series of experiments. Dimethylnitrosamine was injected intraperitoneally in aqueous solution. ReagentsInorganic reagents were of analytical grade; glassdistilled water was used throughout. Cytochrome c was prepared by the method of Keilin & Hartree (1937) and dialysed against water. Diphosphopyridine nucleotide (DPN), adenosine 5'-phosphoric acid, adenosine 5'-tri-phosphoric acid (ATP), coenzyme A, L-malic acid and sodium pyruvate were obtained from Nutritional Biochemicals Corp., Cleveland, Ohio, U.S.A. Thiamine pyrophosphate, flavinadenine dinucleotide (FAD), thioctic acid, a-oxoglutaric acid, fi-hydroxybutyric acid and choline chloride were products of L. Light and Co. Ltd. Liver concentrate 202-20 was obtained from Sigma Chemical Co., St Louis, Mo., U.S.A. Vitamin B13 was a product of The Distillers Co. (Biochemicals) Ltd. Sodium citrate (A.R.), sucrose (A.R.), sodium succinate, octanoic acid, nicotinamide and the disodium salt of ethylenediaminetetraacetic acid (EDTA) were obtained from British Drug Houses Ltd. Octanoic acid was purified by redistillation in vacuo.Dimethylnitrosamine was prepared by the method of Hatt (1946).The quantity of DPN required for each experiment was weighed shortly before use and dissolved in 0-4M-nicotinamide to a concentration of 5 mg. of DPN/ml. of solution. This precaution aimed at preventing non-enzymic hydrolysis of DPN, which is otherwise rapid at neutral or acid pH, before the beginning of the incubation. A volume of 0-2 ml. of the mixture was added to each flask immediately before addition of the enzyme. In experiments in which four runs of twelve flasks had to be made in rapid succession, volumetric addition of the DPN was the most practical method.Tissue preparations Animals were killed by stunning and exsanguination, and the whole liver was rapidly excised and placed immediately in aqueous 0 25M-sucrose solution at 0°. Aft...