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Chem. Res. Toxicol. 1996, 9, 866-870
Further Branching of Valproate-Related Carboxylic Acids Reduces the Teratogenic Activity, but Not the Anticonvulsant Effect Ursula Bojic, Mohamed M. A. Elmazar,† Ralf-Siegbert Hauck,‡ and Heinz Nau* Institute of Toxicology and Embryopharmacology, D-14195 Berlin, Germany Received December 29, 1995X
In the present study, compounds derived from the anticonvulsant drug valproic acid (VPA, 2-n-propylpentanoic acid) and analogues known to be teratogenic were synthesized with an additional carbon-branching in one of the side chains. The substances were tested for their ability to induce anticonvulsant activity and sedation in adult mice, and neural tube defects (exencephaly) in the offspring of pregnant animals (Han:NMRI mice). In all cases, the rates of exencephaly, embryolethality, and fetal weight retardation induced by the methyl-branched derivatives were very low when compared to those of the parent compounds. These novel compounds exhibited anticonvulsant activity which was not significantly different from that of VPA. Neurotoxicity was considerably lower for some compounds as compared to VPA. Anticonvulsant activity and neurotoxicity of branched short chain fatty acids are far less structure-dependent and not related to teratogenic potency. Within this series of compounds, (()-4-methyl-2-n-propyl-4-pentenoic acid and (()-2-isobutyl-4-pentenoic acid exhibited the most favorable profile in regard to high anticonvulsant effect, low sedation, and teratogenicity. Valproic acid analogues with additional methyl branching may be valuable antiepileptic agents with low teratogenic potential.
Introduction After the discovery by Meunier et al. (1) of the anticonvulsant properties of valproic acid (2-n-propylpentanoic acid, VPA),1 the drug quickly found widespread use as a valuable therapeutic agent in the treatment of several forms of epilepsy. Although originally considered to be of low toxicity, VPA unfortunately proved to have considerable teratogenic potential in the human. The most apparent and severe teratogenic effect observed is spina bifida, a posterior neural tube defect (2). VPA is also teratogenic in a variety of experimental animals (3, 4). In mice, posterior (5) as well as anterior (3, 4) neural tube defects (exencephaly and spina bifida, respectively) can be induced in the offspring depending on the time and frequency of application during pregnancy. Experimental studies in vivo and in vitro suggest that the parent drug VPA is very likely the proximate teratogen (6); metabolites play a minor role in this regard because teratogenic metabolites such as 4-en-VPA (see below) are present at low concentrations only. Studies with a number of analogues and metabolites of VPA indicate that the teratogenic potential is strictly related to the structure of the administered compound: VPA, its metabolite 4-en-VPA [(()-2-n-propyl-4-pentenoic acid] (7), and several analogues, i.e., (()-2-ethylpentanoic acid (7), (()-2-ethylhexanoic acid (8, 9), (()-2-butylhexanoic acid (7), and 4-yn-VPA [(()-2-n-propyl-4-pen* Address correspondence to Dr. Heinz Nau, Institute of Toxicology and Embryopharmacology, Free University Berlin, Garystrasse 5, D-14195 Berlin, Germany. Tel: +4930-838 5053/6312; Fax: +4930831 6139. † Department of Pharmacology, College of Pharmacy, King Saud University, Riyadh 11451, P.O. Box 2457, Saudi Arabia. ‡ Presently at Schering AG, Berlin, Germany. X Abstract published in Advance ACS Abstracts, June 15, 1996. 1 Abbreviations: VPA, valproic acid, 2-n-propylpentanoic acid; 4-enVPA, (()-2-n-propyl-4-pentenoic acid; 4-yn-VPA, (()-2-n-propyl-4pentynoic acid; PTZ, pentylenetetrazole.
S0893-228x(95)00216-5 CCC: $12.00
tynoic acid] (10), were teratogenic, while many structural analogues of VPA such as (E)-2-n-propyl-2-pentenoic acid [(E)-2-en-VPA] exhibited low, if any teratogenic potential (7, 11). These studies suggest that several structural elements are required for the expression of teratogenicity by VPA congeners: (a) a free carboxylic group; valpromide, the amide of VPA, is not teratogenic; (b) branching on carbon-2; (c) presence of a hydrogen atom on carbon2; additional alkylation at carbon-2 [1-methyl-1-cyclohexanoic acid; (()-2-methyl-2-ethylhexanoic acid; 2methyl-VPA] or unsaturation on carbon-2 [(E)-2-en-VPA] abolishes teratogenicity. But the structure specificity goes even further. In studies examining the enantiomers of chiral VPA analogues, it was found that the teratogenic potential is enantioselective: the pairs of enantiomers tested exhibited differing teratogenic potencies, but similar pharmacokinetic profiles and embryonic exposure (8-12). For example, (S)-4-yn-VPA proved to be 8 times more potent than the R-enantiomer in regard to the induction of exencephaly in mice (10). The high stereoselectivity of the teratogenic response was also observed in whole rodent embryo cultures (13). In contrast, other pharmacodynamic end points such as neurotoxicity and anticonvulsant activity did not show such a strict structure dependence: these activities were related more to lipophilicity and volume rather than their stereochemistry (11, 14, 15). Consequently, pairs of enantiomers had comparable neurotoxic and anticonvulsant properties. This strict stereochemical requirement of teratogenic action called for further investigation aimed at elucidating the structural elements in the molecule of VPA-type carboxylic acids which are related to teratogenic potency. Such structural information may also be used in studies of the mechanism of teratogenic action of VPA-type carboxylic acids (16, 17). The present study was based on the observation that compound 1b (Scheme 1), which © 1996 American Chemical Society
Low Teratogenicity of Valproate Analogues Scheme 1. Teratogenic Compounds (Left) and Congeners with Low Teratogenic Potency (Right)
possesses an additional branching compared to VPA, was not teratogenic (11). To test the hypothesis that the methyl group was responsible for the observed loss of teratogenic potency, we synthesized further methyl derivatives of VPA and other teratogenic carboxylic acids (Scheme 1). The following pharmacological properties were determined: teratogenicity as exencephaly, embryotoxicity (embryolethality and weight retardation), neurotoxicity, and anticonvulsant activity.
Materials and Methods Chemistry. Melting points were determined using a Dr. Tottoli (Bu¨chi, Switzerland) capillary melting point apparatus and are uncorrected. NMR spectra were recorded with a Bruker AC 250-MHz spectrometer using tetramethylsilane as an internal standard. Chemical purity was assessed by titration with standardized 1 N NaOH (Merck, Germany) and by GC/MS. GC/ MS was performed by previously published methods (18) with some modifications: A Hewlett Packard system (type HP 5890A gas chromatograph, type 5971 MSD mass spectrometer operated by a MS Chem Station). Compounds of interest were dissolved in acetonitrile and treated with MSTFA [N-methyl-N-(trimethylsilyl)trifluoroacetamide] (Pierce, Bender & Hobein GmbH, Mu¨nchen, Germany), 2 h, room temperature. GC separations were achieved using a 50 m × 0.2 mm i.d. HP-1 capillary column (0.11 µm film thickness) with helium as carrier gas (0.5 mL/ min). The initial temperature of 70 °C was held for 1 min, and then raised by 10 °C/min to 170 °C. The injector temperature was 250 °C. The mass spectrometer (electron impact) was operated in scan mode (m/z: 60-270). The relative intensities of the various ions were related to the TMS ion (m/z ) 73). Thin layer chromatography (TLC) was performed using silica gel plates (Merck, Kieselgel 60F-254) with n-hexane/ethyl acetate ) 7/1 plus 5% acetic acid (v/v) as the mobile phase. After drying, the plates were sprayed with 50% sulfuric acid and heated for 15 min at 150 °C. VPA analogues and precursors appeared as
Chem. Res. Toxicol., Vol. 9, No. 5, 1996 867 dark spots. Preparative column chromatography was performed with silica gel (Merck, 0.040-0.063 mm, 230-400 mesh) by flash chromatography (19) with eluents as described for TLC. Dialkylated malonic acid diethyl esters were prepared by three different synthetic procedures (all reaction vessels had to be dried by heating and were flushed with argon or nitrogen during reactions): (a) Sodium (0.1 mol) was dissolved in 50 mL of dry ethanol. Malonic acid diethyl ester or monoalkylmalonic acid diethyl ester (0.1 mol) diluted with 20 mL of ethanol was added slowly while stirring to the hot solution. Alkyl halide (0.12 mol) was added in such a way as to keep the mixture at a steady boil. After the addition of the alkyl halide was completed, the mixture was kept boiling by external heating for 2-8 h until TLC showed no starting material. Ethanol was distilled off and water added to dissolve salts. The mixture was extracted three times with ether and dried over anhydrous sodium sulfate. Evaporation of the combined ether extracts yielded crude product. (b) To a stirred mixture of 0.1 mol of malonic ester (or monoalkyl derivative) and 0.12 mol of alkyl halide was added a solution of 0.1 mol of sodium in 50 mL of dry ethanol to keep the mixture at a slow boil. The mixture was heated until TLC indicated completion of reaction. Workup was carried out as described for (a). (c) A suspension of 0.1 mol of sodium hydride in 35 mL of dry dimethylformamide was prepared, and monoalkylmalonic ester was added slowly such that the temperature remained below 50 °C. After hydrogen gas evolution subsided, the mixture was stirred for another 15 min. Alkyl halide (0.12 mol) was added very slowly such that the temperature remained below 40 °C. After addition was completed, the mixture was stirred at room temperature for 2 h. The mixture was poured into 300 mL of water and extracted three times with ether. Drying over anhydrous sodium sulfate and evaporation of solute afforded crude product. Products gained from procedures a-c were distilled under reduced pressure. The dialkylated malonic esters were heated in a solution of 20.3 g (0.35 mol) of potassium hydroxide, 50 mL of water, and 100 mL of ethanol (5-12 h). After saponification was completed, ethanol was evaporated. The remaining residue was diluted with water and washed with ether. The water layer was acidified with concentrated HCl (pH
