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.
1. [2-(14)C]Indole has been synthesized from [(14)C]formate and o-toluidine via N[(14)C]-formyltoluidine. 2. When fed to rats, the (14)C of [(14)C]indole (dose 70-80mg./kg. body wt.) is fairly rapidly excreted, and in 2 days an average of 81% appears in the urine, 11% in the faeces and 2.4% as carbon dioxide in the expired air. 3. Radioactivity is excreted in the urine as indoxyl sulphate (50% of the dose), indoxyl glucuronide (11%), oxindole (1.4%), isatin (5.8%), 5-hydroxyoxindole conjugates (3.1%), N-formylanthranilic acid (0.5%) and unchanged indole (0.07%). The faeces contain indoxyl sulphate (0.4% of the dose) and indole (0.2%), but the major metabolites have not been identified. 4. Fed to rats with biliary cannulae an average of 5.6% of a dose of [(14)C]indole (20-60mg./kg. body wt.) is excreted in the bile in 2 days. Radioactivity is present as indoxyl sulphate (0.8% dose) and 5-hydroxyoxindole conjugates (0.6%). 5. Rats further metabolize indoxyl into N-formylanthranilic acid and anthranilic acid, and oxindole into 5-hydroxyoxindole. 6. With rat-liver microsomes plus supernatant under aerobic conditions, indole gives indoxyl, oxindole, possibly isatin, N-formylanthranilic acid and anthranilic acid, but under anaerobic conditions gives only oxindole. Similarly, under aerobic conditions, oxindole gives 5-hydroxyoxindole, anthranilic acid and o-aminophenylacetic acid. 7. Indole is metabolized by two pathways, one via indoxyl to isatin, N-formylanthranilic acid and anthranilic acid, and the other via oxindole to 5-hydroxyoxindole and possibly to o-aminophenylacetic and anthranilic acid. 8. The following new compounds are described: 4-hydroxy-2-nitrophenylacetic acid, 3-, 4- and 5-benzyloxy-2-nitrophenylacetic acid, 5- and 7-hydroxyoxindole and 5-aminoacridine indoxyl sulphate.
Umbelliferone (7-hydroxycoumarin) and its conjugates appear to be metabolites of coumarin in the rabbit (Mead, Smith & Williams, 1955). It was observed that, although umbelliferone fluoresced strongly in ultraviolet light at pH 9-10, its conjugates showed little or no fluorescence. The glucuronide and ethereal sulphate of umbelliferone were therefore made and the former was found to be practically non-fluorescent. The possibility that these conjugates could be used as substrates for the determination of ,-glucuronidase and arylsulphatase was therefore investigated. Since umbelliferone is highly fluorescent it appeared likely that these conjugates could be used for the deternination of the enzymes in very small amounts of material. The present communication deals with the use of the glucuronides of umbelliferone and the cheaper 4-methylumbelliferone (7-hydroxy-4methylcoumarin) for the determination of f,glucuronidase. (This work has been briefly reported by Mead, Smith & Williams, 1954.) The ethereal sulphates of -umbelliferone and 4-methylumbelliferone show a fluorescence which is considerably less than the corresponding hydroxycoumarins. We have attempted to use these compounds for the fluorimetric assay of arylsulphatase, but we do not consider them satisfactory because their fluorescence is sufficient to give relatively high blank values. The ,B-glucosides of umbelliferone and 4methylumbelliferone, however, have been found to be satisfactory for the determination of ,-glucosidase, and an account of these compounds will be given later. EXPERIMENTAL Materials. Umbelliferone, m.p. 224-226°, purchased from British Drug Houses Ltd., had a pale-reddish colour, and paper chromatography showed that it was not pure.In n-butanol-acetic acid-water (4:1:5 by vol.), besides a main fluorescent spot due to umbelliferone, three other much weaker fluorescent spots were present. As a standard for fluorescent purposes umbelliferone was purified by way of the sulphuric ester. The umbelliferone (4.5 g.) was * Part 66: Jondorf, Parke & Williams (1955).dissolved in pyridine (10 ml.) and a solution of chlorosulphonic acid (3 g.) in pyridine (10 ml.) added carefully with cooling. The mixture was then kept at room temp. for 24 hr. Water (100 ml.) was then added, followed by a slight excess of KHCO3. The precipitate which formed was collected by filtration, dissolved in the minimum of hot water (charcoal) and the solution filtered. On cooling the potassium umbeUiferone sulphate (potassium 2-oxo-1:2benzopyran-7-yl sulphate) separated and was recrystallized from 80 % (v/v) aqueous ethanol as colourless needles
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.