The sedative, neurotoxic and embryotoxic effects of (±)-a-phthalimidoglutarimide or thalidomide are now well known. Any of these effects could be due to thalidomide, or to its metabolites, and it is therefore important to identify these metabolites and to study their biological properties. Our studies on thalidomide began with an investigation of the urinary metabolites excreted by rabbits dosed orally with thalidomide and, as will be described in the succeeding paper (Schumacher, Smith & Williams, 1965), it was soon found that a large number of metabolites occurred. In fact, we succeeded in isolating or detecting by colour reactions and paper chromatography all twelve of its possible hydrolysis products.This caused us to suspect that thalidomide might be unstable in solution and in this paper we shall describe the conditions for the spontaneous hydrolysis of thalidomide in aqueous solution at various pH values.In describing the spontaneous hydrolysis and metabolites of thalidomide, it will be useful to refer to Fig. 1, which shows how thalidomide could break down by simple hydrolysis of its substituted amide bonds. METHODS AND RESULTS Reference compoundsThese compounds were either obtained as gifts or prepared in this laboratory. A list is given in Table 1 of the sources of reference compounds, their melting points and any other relevant data. The following compounds were synthesized and their properties are described.2-and 4-(o-carboxybenzamido)glutaramic acid and 2-(o-carboxybenzamido)glutaric acid and their sodium salts. (±)-4-Phthalimidoglutaramic acid (5 g) was stirred into an amount of aqueous sodium hydroxide (40% w/v) calculated to yield the disodium salt of (±)-4-(o-carboxybenzamido)glutaramic acid. The greenish solution was allowed to stand at room temperature for 10 min and then methanol (100 ml.) was added. The mixture was kept for 4 to 5 hr. The precipitate which had formed was filtered, washed with methanol and then recrystallized five times from aqueous methanol and dried in a desiccator. The disodium salt of (±)-4-(o-carboxybenzamido)glutaramic acid was obtained as a white crystalline powder in a yield of 80

When thalidomide is administered orally to animals, only a small amount of the unchanged drug is excreted in the urine (for example, Beckman, 1962). The major portion of the compound is broken down and excreted as transformation products. After administration of the drug to man, rats and rabbits, Smith, Williams & Williams (1962) isolated 4-phthalimidoglutaramic acid from rabbit urine and detected it in human and rat urine. They also noted the presence in the urine of a fluorescent metabolite which they considered to be related to 3-hydroxyphthalic acid. Using [14C]-thalidomide, Faigle, Keberle, Riess & Schmid (1962) showed that the urine of dogs given thalidomide contained 2-and 4-phthalimidoglutaramic acid, 2-phthalimidoglutaric acid, a-(o-carboxybenzamido)glutarimide, 2-(o-carboxybenzamido)glutaric acid, phthalic acid and small amounts ofunchanged thalidomide, whilst the faeces contained only unchanged thalidomide.In order to emphasize the complexity of the problem of identifying the metabolites of thalidomide it should be pointed out that there are twelve possible hydrolysis products of (±)-thalidomide of which eleven can occur in (+)-and (-)-forms. The number of possible optically active products, including (+)-and (-)-thalidomide, is thus twenty-four. If thalidomide is hydroxylated in vivo, this could occur in the 3-or 4-position of the aromatic ring and possibly in three different positions (3', 4' and 5') in the glutarimide ring (see formula). Disregarding hydroxylation of the glutarimide ring and considering only the hydroxylation of the aromatic ring, there are twelve 3-hydroxy-and twelve 4-hydroxy-metabolites possible, and twenty-two of these can occur in (+)-and (-)-forms, the two compounds not containing an optically active carbon atom being 3-and 4-hydroxy-Q) NC H2 H phthalic acids. The number of possible metabolites of thalidomide is thus very large, for if any racemic metabolite underwent resolution the possible number of metabolites would be well over 100. In the present paper we describe the isolation and detection of some fifteen urinary metabolites and prove that rabbit urine contains derivatives of 3-and 4-hydroxyphthalic acid.

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Pregnant Wistar rats treated orally at day 9, 10, or 11 with 250, 500, 750, or 1000 mg/kg aspirin showed a teratogenic dose-response relation at each treatment time, but a decreased overall embryonic susceptibility as treatment was applied later in gestation. The types of abnormalities produced generally correIated with the state of development at the time of treatment. Treatment of animals at day 9 with 510 mg/kg benzoic acid and 2 hours later with 250 or 500 mg/kg aspirin caused a significant increase in percentage of malformations above effects observed after 250 or 500 mg/kg aspirin alone. Determination of free salicylate in embryos and maternal serum after treatment at day 11 with sodium salicylate with or without benzoic acid pretreatment indicated that benzoic acid significantly increased the concentration of salicylate in both embryos and serum 6 and 12 hours after salicylate treatment and throughout the observed period. Thin-layer chromatography of tissues from these animals revealed only salicyfic acid in both embryos and maternal serum, thus implicating salicylic acid as the causative agent in aspirin teratogenesis. ~~ 1

The prenatal effects of methotrexate (MTX) in rats and rabbits were assessed. It was found highly embryotoxic in postimplantation rat embryos; 0.3 mg/kg ip or less caused nearly total embryolethality with slight teratogenicity. Rabbit embryos were far more resistant to small doses of MTX than rats, but 19.2 mg/kg iv, when given during days 10 to 15 of gestation, produced little death and a constant spectrum of malformation in a high percentage of offspring. Cleft palate, skull defects, and severe fore- and hindlimb dysplasias, occurred with a high degree of regularity and were strongly dose and developmental-stage specific.