Brain Research, 457 (1988) 371-375 371 Elsevier BRE 23028 Marked stereospecificity in a new class of anticonvulsants Harold Kohn 1, Judith D. Conley I and J. David Leander 2 I Department of Chemistry, University of Houston, Houston, TX 77004 (U.S.A.) and 2Central Nervous System Pharmacology, Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285 (U.S.A.) (Accepted 26 April 1988) Key words: Anticonvulsant; Stereoselectivity; Maximal electric shock; Mouse N-Acetyl-D,L-alanine-N-benzylamide and N-acetyl-D.L-phenyiglycine-N-benzylamide are two novel anticonvulsants that selective- ly block maximal electric shock-induced tonic extensor seizures in mice. For both compounds, the anticonvulsant activity is due to the D-stereoisomer, and the L-stereoisomer is virtually inactive as an anticonvulsant. The marked stereoselectivity of these anticonvul- sants may make them very useful pharmacological tools for the study of the mechanism(s) of anticonvulsants that selectively inhibit maximal electric shock-induced seizures. The prototypical anticonvulsant drugs for the treatment of partial and generalized tonic-clonic sei- zures are phenobarbital, phenytoin and carbamaze- pine 14. In mice and rats, these prototypical anticon- vulsants effectively prevent maximal electric shock (MES)-induced seizures at doses lower than those which produce neurological impairment 18. Recently, a novel series of anticonvulsants, the functionalized amino acid derivatives, has been discovered with a similar selectivity for MES-induced seizures 4'5. Two of the most active compounds from this series are the N-acetyl-D,L-alanine-N-benzylamide (D,L-AAB) and the N-acetyl-D,L-phenylglycine-N-benzylamide (D,L- APB) (referred to as la and ld, respectively, in ref. 4). The D,L-AAB had an EDs0 of 76.54 mg/kg (i.p.) in mice against MES seizures, was inactive at 600 mg/kg (i.p.) against Metrazol-induced clonic seizures, and had an EDs0 for producing neurological impairment of 453.86 mg/kg (i.p.). The D,L-APB was more po- tent, with an MES TDs0 of 20.31 mg/kg (i.p.), was also inactive against Metrazol-induced clonic sei- zures, and produced an TDs0 for neurological impair- ment of 96.92 mg/kg (i.p.). Thus, these two com- pounds had similar anticonvulsant profiles to pheny- toin; that is, they were selective for MES seizures, compared to Metrazol-induced seizures, and had re- spectable P.I.'s (protective index = neurological im- pairment TDs0 divided by MES EDs0 ). A number of racemic anticonvulsant agents have been resolved and the anticonvulsant activities of the individual stereoisomers have been studied 1. Typi- cally, qualitatively similar anticonvulsant activities were seen with both stereoisomers, with only small differences in potencies. Nirvanol, the demethylated metabolite of mephenytoin, has been reported to dis- play the largest difference in activity between its two stereoisomers. The (R)-stereoisomer was 3.8 times more potent than the (S)-stereoisomer against sei- zures induced by electroshock in mice 1. Recently, the stereoisomers of a novel anticonvulsant, LY188544 (S,R-4-amino-N-(a-methylbenzyl)benzamide), have been studied for anticonvulsant activity 1°. After oral administration, the (S)-stereoisomer was 2.2 times more potent than the (R)-stereoisomer in the MES test. However, after i.v. administration the two stereoisomers were approximately equal in potency. The purpose of the present research was to evaluate the stereoisomers of D,L-AAB and D,L-APB tO deter- mine if there was isomeric selectivity within this se- ries of novel anticonvulsants. All of the compounds used in this study were synthesized from the appro- priate chiral or D,L-amino acid, using procedures Correspondence: H. Kohn, Department of Chemistry, University of Houston, University Park, Houston, TX 77004, U.S.A. 0006-8993/88/$03.50 (~) 1988 Elsevier Science Publishers B.V. (Biomedical Division)
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`372 common for the preparation of peptides 2. The D- and L-enantiomers of AAB were prepared from the cor- responding optically pure alanylmethyl ester hydro- chlorides using the conditions employed for the race- mate 4. An alternative route was employed for the synthesis of the D- and L-enantiomers of APB. In this series, the appropriate chiral phenylglycine was con- verted to the N-t-butoxycarbonyl derivative and then coupled with benzylamine using the mixed anhydride method (ethyl chloroformate, triethylamine). De- protection (trifluoroacetic acid), followed by acetyla- tion (acetyl chloride, triethylamine) afforded the de- sired product. The racemates have been previously delineated 4. The enantiomeric purity of the isomeric final products was supported by their observed opti- cal rotations [for D-AAB, [Ct] 23 = +36.2 (2.5% in MeOH); L-AAB, [a] 23 = -35.2 (2.5% in MeOH); D- APB, [a] 23 = -103.0 (1% in EtOH); L-APB, [Ct] 23 = +105.1 (1% in EtOH)] as well as by NMR studies using the chiral shift reagent, (+)-2,2,2-trifluoro-1- (9-anthryl)ethanoll3. The D- and L-stereoisomers of both compounds were evaluated for anticonvulsant activity using the MES test. Some of the data were generated by the Antiepileptic Drug Development (ADD) program of the Epilepsy Branch of the National Institute of Neurological and Communicative Disorders and Stroke s.~5, whereas the rest of the data were gener- ated at Lilly Research Laboratories. Both laborato- ries used male albino mice (CF-1 strain, 18-25 g; Charles River Breeding Laboratories, Portage, MI) as experimental subjects. The mice were allowed free access to food and water, except when they were removed from their colony cages for the experi- mental procedures. For i.p. administration, the com- pounds were administered in 30% polyethylene gly- col 400/water mixture in a volume of 0.01 ml/g b. wt. The MES test measures the ability of the test drug to abolish the hind limb extensor component of maxi- mal seizures induced by 50 mA of 60-cycle current delivered for 0.2 s via corneal electrodes. This amount of stimulation is approximately 6 times the threshold and reveals the ability of the compound to prevent seizure spread. To determine selectivity for anticonvulsant effects, the compounds were also studied for their effects to neurologically impair the mice. At the Epilepsy Branch, the neurologically im- pairing effect was studied using the rotorod proce- dure 7. When a normal mouse is placed on a knurled rod 1 inch in diameter rotating at a speed of 6 rpm, it can maintain its equilibrium for at least 1 min. Inabili- ty to do this was defined as 'neurological impair- ment'. At Lilly Research Laboratories, the hori- zontal screen test was used to determine neurological impairment 6. Previously trained mice were dosed with the compound and placed individually on top of a square (13 cm × 13 cm) wire screen (no. 4 mesh) which was mounted on a metal rod. The rod was ro- tated 180 ° , and the number of mice that returned to the top of the screen was determined. Inability to climb to the top within 1 min was defined as 'neuro- logical impairment'. Each compound was tested at various time inter- vals to determine the time of peak effect. The time of peak effect after i.p. administration for these com- pounds was 0.5 h and that is the time at which dose- response data were generated. From the dose-res- ponse curves (with at least 4 doses from no-effect to complete protection), the dose of compound which was estimated by computer probit analysis to protect 50% of the mice from MES-induced tonic extensor seizures was defined as the MES-EDs0. The dose es- timated to produce neurological impairment in 50% of the mice was defined as the toxic dose50 (TDs0). The two racemates, AAB and APB, and the two stereoisomers of each were also studied on the MES test at 5 min after i.v. administration in an attempt to determine intrinsic activity without the potential problems of absorption, distribution and metabolism obscuring the pharmacological effects. For the i.v. study, the test compounds were administered in the tail vein in a solution of 5% emulphor, 5% ethanol and 90% water 3. Table I shows the MES-EDs0 and TDs0 values for the various compounds. At the Epilepsy Branch, O,L- AAB had an MES-ED50 of 76.54 mg/kg. The D- stereoisomer was more potent, with an EDs0 of 54.80 mg/kg, and the L-stereoisomer was 10-fold less po- tent than the D-isomer. D-AAB was also more potent than both the racemate and the L-stereoisomer in producing neurological impairment; however, the ra- cemate and D-AAB maintained P.I.'s that indicated they had selective anticonvulsant effects. The P.I. for L-AAB of 1.53 indicates much less of a selective anti- convulsant effect than that seen with the racemate and D-AAB. Note that the D-stereoisomer was 1.4
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`373 TABLE I Pharmacological evaluation of the o- and L-stereoisomers of AAB and APB after i.p. administration MES-EDso TDao a P. 1. Epilepsy Branch D.L-AAB 76.54 (66.58-89.04) b 453.86 (416.56-501.01) b D-AAB 54.80 (50.32-59.65) 213.82 (147.71-261.56) L-AAB 548.37 (462.57-740.50) 841.38 (691.25-953.59) Lilly Research Laboratories D,L-AAB 51.0 (44.6-58.6) >100 D-AAB 32.0 (27.49-40.19) >80 D,L-APB 32.1 (27.5-40.2) >40 D-APB 26.4 (21.1-32.0) >80 L-APB >300 >100 <300 5.93 3.90 1.53 a TDs0 from the Epilepsy Branch determined from rotorod; from Lilly Research Laboratories determined from horizontal screen. b 95% Confidence limits. times more potent than the racemate in the anticon- vulsant assay, but 2.1 times more potent in the neuro- logical impairment assay. The fact that it was not twice as potent in the anticonvulsant assay suggests that the presence of the relatively inactive L-stereo- isomer in the racemic mixture somehow enhanced the pharmacological activity of the racemate. D,L-AAB and D-AAB were also compared for anti- convulsant effects at Lilly Research Laboratories. The EDs0'S were 51.0 and 32.0 mg/kg, respectively. As was seen with the Epilepsy Branch data, the 19- stereoisomer was more potent as an anticonvulsant than the racemate by a factor of 1.6. Since the Epi- lepsy Branch data had already shown that D,L-AAB and D-AAB had good P.I.'s, no effort was expended to determine an actual TDs0 on the horizontal screen test at Lilly Research Laboratories. When the racemate and stereoisomers of APB were studied at Lilly Research Laboratories, a simi- lar picture was seen (lower part of Table I). D,L-APB and D-APB had MES-EDs0's of 32.1 and 26.4 mg/kg, respectively, whereas the L-APB was completely without effect at doses as high as 300 mg/kg. Both D,L-APB and D-APB produced their MES protective effects at doses lower than those which produced neurological impairment, but, because of limitations on compound supply, no TDs0'S or P.I.'s were deter- mined. The fact that the o-stereoisomer is not twice as potent as the racemate again suggests that the presence of the inactive L-stereoisomer in the race- mate somehow enhances the pharmacological action of the D-stereoisomer. Fig. 1 shows the comparison of both the D- and L- enantiomers with their racemates at 5 min after i.v. administration. D-APB and D,L-APB produced dose- related anticonvulsant effects with MES-EDs0's of 2.86 (95% confidence interval = 1.93-3.4) and 5.13 (4.29-6.17) mg/kg, respectively. Note that the O- stereoisomer was approximately 1.8 times more po- tent than the racemate. By contrast, L-APB did not produce protection against MES, but rather a dose- related lethal effect at 10 and 20 mg/kg. Likewise, 19- AAB and D,L-AAB produced dose-related anticon- vulsant effects, with EDs0's of 31.18 (26.66-38.65) and 70.01 (60.58-88.76), respectively. In contrast, L- AAB was inactive at 80 mg/kg. The EDs0 of D-APB (2.86 mg/kg) was administered at 5, 10, 20 and 40 min before challenge with MES to determine the dura- tion of activity. The percentages of the 12 animals
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`20
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`40
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`Fig. 1. Effects of i.v. administration of the D-, L-, and D,L-forms of APB and AAB 5 min before MES challenge. Data shown are the % of 12 (except 6 for L-APB) animals protected for each dose.
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`374 protected at each of those intervals were 58, 42, 17 and 0, respectively. Thus, after i.v. administration, 5 min was the peak time of activity and the protection became inadequate within 40 min after administra- tion. This suggested that the parent compound, and not a metabolite, was the active compound. These data suggest that the stereospecificity ex- hibited after i.p. administration may be a true phar- macodynamic stereospecificity, and would not ap- pear to be due to differences in absorption, distribu- tion or metabolism between the stereoisomers. After i.v. administration, both D-enantiomers were ap- proximately twice as potent as their respective race- mates, and both L-enantiomers have no anticonvul- sant activity. Thus, this stereospecific anticonvulsant effect differs from that observed with the (S)- and (R)-stereoisomers of LY188544. The 2.2-fold stereo- specificity of the (S)-stereoisomer compared to the (R)-stereoisomer of LY188544 was present after oral administration, but there was no stereospecificity af- ter i.v. administration1°. The marked stereospecificity of these function- alized amino acid derivatives demonstrated by these present data are far superior to stereoselective ef- fects shown for other MES-selective anticonvul- sants I. Thus, these sets of 'active' and 'inactive' stereoisomers may have great value in studying the biochemical mechanisms of MES-selective anticon- vulsants, such as the prototypes phenytoin and carba- mazepine. The clearly less than 2-fold difference in potency in the anticonvulsant assays between the 'active' D- stereoisomers and their respective racemates after i.p. administration suggest that the presence of the L- stereoisomer enhances the pharmacological action of the D-stereoisomer. Such an effect has previously been observed for the D- and L-stereoisomers of pro- poxyphene after oral, but not subcutaneous adminis- tration 12. That effect was suggested to be due to the inactive isomer saturating the uptake sites for the compound upon first-pass of the liver, which resulted in higher amounts of the active stereoisomer being in the plasma after combined administration of both isomers, than after only administration of the active stereoisomer. Such a mechanism could be acting in this situation since i.p.-administered compounds do undergo a first-pass of the liver before reaching the systemic circulation 11. The fact that the i.v. compari- son with D-APB and O,L-APB was closer to the ideal value of 2 supports this hypothesis. Also, the obser- vation that the ratio of the TD50's for D-AAB and D,L- AAB was 2.1 is supportive since this hypothesis of liver saturation predicts that, as the amount of drug reaching the liver increases, the percentage of the to- tal taken up by the liver decreases n. Other studies have shown that D-APB and L-APB have no protective effects against pentylenetetrazol- induced (85 mg/kg s.c.) clonic seizures 16'17 or N- methyl-o-aspartic acid-induced lethality 9. These re- suits indicate that the benzodiazepine receptor and the N-methyl-D-aspartic acid-defined glutamate re- ceptor are probably not involved in the mechanism of action of this class of very interesting anticonvul- sants. The chemical synthesis of these compounds was supported in part by a fellowship awarded to J.D.C. by the American Association of University Women. We thank Dr. D.W. Robertson and Mr. E. Beedle of Lilly Research Laboratories for helpful discussions and Mr. G.D. Gladding, Mr. J.P. Stables, Dr. H.J. Kupferberg and Dr. E.A. Swinyard of the ADD pro- gram of the Epilepsy Branch, NINCD, NIH, and Mr. R.R. Lawson of Lilly Research Laboratories for the pharmacological data. Research samples of these compounds can be obtained from Dr. J. David Leander, Central Nervous System Research, Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, U.S.A. The Lilly serial numbers are: D,L-APB = LY193326; D-APB = LY248319; L-APB = LY248320; D,L-AAB = LY258499; D-AAB = LY258843; and L-AAB = LY248510. 1 Andrews, P.R. and Mark, L.C., Structural specificity of barbiturates and related drugs, Anesthesiology, 57 (1982) 314-320. 2 Bodanzsky, M., Klausner, Y.S. and Ondetti, M.A., Pep- tide Synthesis, 2nd edn., Wiley, New York, 1976. 3 Carney, J.M., Uwaydah, I.M. and Balster, R.L., Evalu- ation of a suspension system for intravenous self-adminis- tration studies of water-insoluble compounds in the rhesus monkey, Pharmacol. Biochem. Behav., 7 (1977) 357-364. 4 Conley, J.D. and Kohn, H., Functionalized O.L-amino acid
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`derivatives. Potent new agents for the treatment of epilep- sy, J. Med. Chem., 30 (1987a) 567-574. 5 Conley, J.D. and Kohn, H., Functionalized amino acid de- rivatives. Potent new agents for the treatment of epilepsy. 193rd National Meeting of the American Chemical Society, Denver, CO, April 1987, Abstr. Med. Chem., (1987) 25. 6 Coughenour, L.L., McLean, R.R. and Parker, R.B., A new device for the rapid measurement of impaired motor function in mice, Pharmacol. Biochem. Behav., 6 (1977) 351-353. 7 Dunham, N.W. and Miya, T.S., A note on a simple appara- tus for detecting neurological deficit in rats and mice, J. Am. Pharm. Assoc., 46 (1957) 208-209. 8 Krall, R.L., Penry, J.K., White, B.G., Kupferberg, H.J. and Swinyard, E.A., Antiepileptic drug development. II. Anticonvulsant drug screening, Epilepsia, 19 (1978) 409-428. 9 Leander, J.D., Lawson, R.R., Ornstein, P.L. and Zimmer- man, D.M., N-Methyl-D-aspartic acid-induced lethality in mice: selective antagonism by phencyclidine-like drugs, Brain Research, 448 (1988) 115-120. 10 Leander, J.D., Robertson, D.W., Clark, C.R., Lawson, R.R. and Rathbun, R.C., Pharmacological effects of enan- tiomers of 4-amino-N-(a-methylbenzyl)benzamide, a chemically novel anticonvulsant, Epilepsia, 29 (1988) 83-90. 11 Lukas, G., Brindle, S.D. and Greengard, P., The route of 375 absorption of intraperitoneally administered compounds, J. Pharmacol. Exp. Ther., 178 (1971)562-566. 12 Murphy, P.J., Nickander, R.C., Bellamy, G.M. and Kurtz, W.L., Effect of L-propoxyphene on plasma levels and anal- gesic activity of o-propoxyphene in the rat, J. Pharmacol. Exp. Ther., 199 (1976) 415-422. 13 Pirkle, W.H. and Rinaldi, P,L., Nuclear magnetic reso- nance determination of enantiomeric compositions of oxa- zindines using chiral solvating agents, J. Org. Chem., 41 (1977) 3217-3219. 14 Porter, R.J., Antiepileptic drugs: efficacy and inadequacy. In B.S. Meldrum and R.J. Porter (Eds.), New Anticonvul- sant Drugs, Libbey, London, 1986, pp. 3-15. 15 Porter, R.J., Cereghino, J.J,, Gladding, G.D., Hessie, B.J., Kupferberg, H.J., Scoville, B. and White, B.G., An- tiepileptic drug development program, Cleveland Clin. Q., 51 (1984) 293-305. 16 Skolnick, P. and Paul, S.M., The mechanism(s) of action of the benzodiazepines, Med. Res. Rev., 1 (1981) 3-22. 17 Swinyard, E.A. and Castellion, A.W., Anticonvulsant properties of some benzodiazepines, J. Pharmacol. Exp. Ther., 151 (1966) 369-375. 18 Swinyard, E.A. and Woodhead, J.H., General principles: experimental detection, quantification and evaluation of anticonvulsants. In D.M. Woodbury, J.K. Penry and C.E. Pippenger (Eds.), Antiepileptic Drugs, Raven, New York, 1982, pp. 111-126.
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