2-aminocyclic and Bicyclic Alkane Carboxylic Acids: Differential Effects on Selected Amino Acids of Rat

Thc intraperitoneal administration or I -aminocyclopentane carboxylic acid, I -aminocyelohexane carhoxylic acid. I-aminocycloheptane carboxylic acid. I-aminocyclooctane carboxylic acid, uxo-2aminohicyclo(2.2. I )heptdne-2-carboxylic acid. enrlo-2-aminobicyclo( 2.21 )heptane-2-carboxylic acid. 2aminohicyclo(2.2.2~octane-2-earboxylic acid and 2-aminobicyclo(3.2. I )octane-2qrboxylic acid to I8-dayold male rats selectively perturbed the levcls of neutral amino acids in the cerebral cortex. While the effect of the above compounds war rather diversified and usually resulted in a reduction of amino acid levels. marked elevations of the levels of valinc and isoleucinr were also noted. I-Aminocycloheptane and cyclooctane carhoxylic acids were particularly noteworthy. in that they elicited a marked reduction of the levels of cortical phenylalanine. RESFARCH in our and other laboratories has shown that several non-metabolizable analogues of natural amino acids possess unique biochemical properties in that they affect amino acid transport a s well as endogenous brain amino acid levels (CHRISTFNSEK rr a/.. & WATANABE. 1972). I t was of interest to cxtend these studies to see if some newly synthesized cyclic and bicyclic amino acids perturb the amino acid pool of brain tissue selectively. Previous reports in the literature have, for the most part. measurcd the effects of the administration of large doses of natural amino acids on the brain amino acid pool. focusing on the identification of specific regulatory mechanisms for their transport across the blood-brain barrier. Thus L0wi)i.b & L A R A M W (1969) have shown that the hranched chain amino acids decrease in thc rat brain as ;L rcsult of the administration of large doscs of phcnqlalanine. whilc thc findings of R I C H T ~ R & WAINER (1971) provide evidence for the existence of separate systems for the transport of neutral and basic amino acids across the blood-brain barrier. For a review of thc earlier literature in this field see NEAM~. 1969;ZANI>ef u/.. I ~ ~ ~ ; S E L L I N G E R f'l [i/.. 1972; SCHAlN (1968). Regional differences in amino acid distribution and movement have been examined by BArnsrlN & LAITHA (1970), while more recently PlCCoLl er a/. (1971)noted changes in the pattern of inhibition of the transport of glycine. alanine and leucine into rat brain slices by 2-aminobicyclo(2.21)heptane-2-carboxylic acid (BCH). The extent of the inhibition was highest for the uptake of leucine and was age-dependent confirming a previous report by one of us in which similar effects of BCH on amino acid transport in rirro were observed in rat liver. pigeon erythrocytes and ascites tumor cells (CHRISTENSXN ('I ol.. 1969). The present study reports the results of experiments in which the effects of the administration in tin) of both geometric isomers of BCH and of four monocyclic and two new bicyclic amino acids were investigated. In particular. the effect of these compounds on the Ieveh of the neutral and aromntic amino acids of rat cerebral cortcx was cxarnined. The rcsultz show that the levels of some amino acids were selectively and specifically perturbed while those of others rcmained unchanged. A preliminary report of some of the results has appeared (ZANI) e l ni.. 1 9 7 2 ) . I This work was supported bq grants from the National Sciencr Foundation(GB 2143.5, R.Z.) and the United Stater Puhlic Health Service (NS 06294. O.Z.S.). METHODS Thc txperimental design utilized in this \tiid! consistcd of thc intrapcritonual administration of 4.7 nimol bg ol the 1201 I202 R ZAHV. 0.2 St.LLlhCtR. R. WATER and R. HARRIS c)cIic ; i d hicyclic amino acids. a dose indcpendentlq determined to be free of o\sr t toxic effects. Thc amino acid pool uas prepared (StLLIXGtR ct ul.. 19721 from the cerebral cortex of the injected rats I and 3 h after the admmistration of the two BCH isomers and 1 h after the administration of the other qnthctic amino acids. the 3 h analyses not being performed as maximum alterations from control levels occurred already at I h (unpublished observations). Amino acid analyses were performed on a Spinco Model I lo(' amino acid analyser using the standard two column met hod Syllthrllc The cyclic amino acids were prepared via their respective hydantoins combining the procedure of BUCHERER & LIEB (1934) and the more recent modifications of CONNORS & Ross (1960). 5.S'-CIclopentanr spirohduritoin Cyclopentanone 84.1 g (1.0 mol) in 350 ml of ethanol was mixed with a solution of 173 g (1.8 mol) of ammonium carbonate in 500 ml ofdistilled water. The mixture was warmed to S O T in a water bath and a solution of potassium cyanide 65 g (1.0 mol) in I50 ml of distilled water was added. The flask was stoppered and placed in a water bath at 6 0 C for 6 h. The reaction mixture was then distilled until the volume had been reduced by one-half and then allowed to cool. The crystalline precipitate was collected and the mother liquor carefully acidified to pH 2 with concentrated sulphuric acid whereupon a second crop of crystals was obtained. Recrystallization of the product from water and Norite gave I45 g of hydantoin m.p. 205207'C. Reported by HENZE & SPEER (1942): 2W205 C. I -Ariiiiio~?.cloprntanr carhoxylic acid (Compound I ) The 5.5'-cyclopentane spirohydantoin 130 g (0.84 mol) and barium hydroxide octahydrate 453 g (1.44 mol) in 2 I. of distilled water were heated in an autoclave at IZO'C for 12 h. After cooling. the precipitated barium carbonate was removed by filtration and the mother liquor treated with ammonium carbonate and dry ice until no additional precipitation occurred. Concentration of the filtered solution in a stainless steel beaker on a hot plate to one-fourth the original volume and cooling to room temperature overnight yielded the amino acid. Recrystallization from ethanol water gave 85 g of product which after drying at 1 IOC had a m.p. of 3IS317'C. Reported by ZELINSKY & STADNIKOFF (191 I): 320'C. 1 -Aminocycloht.vane carhoxylic acid (Compound 2) The 5S'-cyclohexane spirohydantoin prepared as above m.p. 217-220°C. reported by CONNORS & Ross (1960) m.p. 221-225'C. was hvdrolysed by barium hydroxide at 120" giving I-aminocyclohexane carboxylic acid which after recrystallization from water had a m.p. of 334337°C dec. or 335'C in a sealed tube. Reported by ZELiNsKY & STADNIKOFF (1906): 334335'C. IA~i~ i~~oc 'yc lo l~~p tu i i~ curhoxyl c ticid (Conipourld 3 ) The amino acid prepared from the hydantoin. m.p. 214216 C by hydrolysis with barium hydroxide yielded the amino acid having a m.p. of 3 15 3 I 7 C dec. after recrystallization from water. Reported by C o ~ w a s & Ross (1960): 320 c. 1 A IllillllC~~/i~octullt~ i.iirho.ydic ircid (Ci~rllporrlld 4) The 5.5'-cyclooctane spirohydantoln m.p. 240k141 c' upon hydrolysis with barium hjdroxide yielded the amino acid. m.p. 31(& 312 c' dec. Reported b) DLOXCH cl i l l . (1964): 31CL316 C dec. 2-Ariii~~ohicyr/o(2,2.I)heptu11r-2-curho.~ylic irr i l i s o i i i t v h ( B C H h ) (Coriipowd 5 ) This amino acid was prepared by a modification of our previously reported method (CHHISlXYStN r f id.. 19691 A solution of 220g(2-0 mol) of norbornanone was dissolved in I litre of 95",, ethanol and cooled to 5 C. To this solution was added I30 g (2.0 mol) of KCN in 500 ml of distillcd water at 4 C and 25Og (2.6 mol) of (NH,),CO, in 500 ml of distilled water at 4 ~ C . The mixture was allowed to stand at 4'C for 30 days and then filtered to obtain the precipitated hydantoin. After recrystallization I SO g of spirohydantoin was obtained. The hydantoin was hydrolysed with barium hydroxide at 120°C for 48 hand the amino acid isolated and purified as described previously (CHRISTENSEK rt 01.. 1969). Amino acid analysis indicated that the product was better than 98 per cent pure b isomer. 2-Ami11ohrr~~c/o(2,2.1)/1rptt111e-2-curhos~l1c 1rcid i.wmrr 11 To a solution of l l O g (1.0mol) of norbornanone in 2501111 of methanol in a I litre round bottom flask was added 55 g ( I .O mol) of NH,CI and 65.2 g (1.0 mol) of KCN in 250 ml of distilled water. The flask was stoppered and placed in a water bath at 8CL85"C for 8 h. After cooling to room temperaturc the contents were transferred to a 3-1. beaker in a hood and concentrated HCI was added until the pH of 1.0 was reached. The solution was transfcrred to a 2-1. round bottom flask and concentrated on a rotary evapnrator until nearly dry. The collected amino nitrile hydrochloride was recrystallized rapidly from boiling HCI containing some Norite. Three recrystallizations yielded a whitc powder of m.p. 198-200 C dec. The recrystallized ammo nitrile hydrochloride was dksolved in I litre of 6 N HCI and autoclabed at I20 C for 24 h. The solution was filtered through a sintercd glasa funnel while still hot, the filtrate distilled until the original volume was reduced by 70 per cent. and the remaining liquid then removed on a rotary evaporator. The residual powder was dried at I10T overnight and triturated repeatedly (at least 6 times) in \ / I (viv) methanol-ether. The reridue was redissolved in water. taken to dryness and the trituration repeated. The combined mcthanol-ether extracts were taken to dryness and the residue dissolved in 500 mi of boiling water. filtered and cooled rapidly in an ice bath. Upon (BCH-U) ( C O I I I ~ O I I I I ~ 6) Cyclic carboxylic acids in brain 1203 adjurring the pH to 5.5 the amino acid precipitated. Analysis of the product on the amino acid analyser indicated it to be at least 99 per Cent isomer a, m.p.: 321-323'C dec. 2-Ar1iitiohic~clo(2.2.2)uc.rur~e-2-curhos)lic a id (Compound 7) The desired ketone for the preparation of the hydantoin wits prepared from bicyclo(2.2.2)octane-2ene by the procedure of KLFINFFLTER & SCHLI Y1.R (1962). The hydantoin was prepared by treating 14.5g (0. I I7 mol) of ketone with 7.6 g (0.1 17 mol) of KCN and 22.4 (0.234 mol) of (NH&CO, in 250ml of l / l (v/v) ethanolwater ;it 60 C lor 6 hand then allowing the mixture to stand at room temperature overnight. Yield: 19.53 g. m p . : 274 C. Thc hyddntoin was treated with harium hydroxide at I20 C in an autoclave for 24 h and the product isolated as in previous prcparations. Yield: 5.72 g. n1.p.: 305 C dec. l-Aiiiiriohicyclo( 3.2, I )ocraiir-2-curho.~~lic acid (Compound 8) The desired hydantoin was prepared from the ketone hy the method described for the preparation of the hicyclo(2.22bctane homologue. Yield: 72 per cent. m.p.: 258 260 C. I 2 3 4