Acetylcarnitine and Cholinergic Receptors
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(2) P. L. Schiff, Jr., and R. W. Doskotch, Lloydia, 33,404 (1970). ACKNOWLEDGMENTS (3) C.-H. Chen and J. Wu, Taiwan Yao Hsueh Tsa Chih, 28, 121 (1976). Adapted in part from the theses submitted by T.-M. Chen and C. Lee (4) M. Shamma, M. J. Hillman, and C.D. Jones, Chem. Reu., 69,779 to the National Taiwan University in partial fulfillment of the Master (1969). of Science degree requirements. (5) V.Simanek, V. Preininger, and J. Lasovsky, Coll. Czech. Chem. Supported by the National Science Council of the Republic of Commun., 41,1050 (1976). China. (6) G.Habermehl, J. Schunk, and G. Schaden, Justus Liebigs Ann. The authors thank Dr. Edward H. Fairchild and Dr. Jinn Wu, College Chem., 742, 138 (1970). of Pharmacy, Ohio State University, for measurement of some PMR and (7) C. Moulis, J. Gleye, and E. Stanislas, Phytochemistry, 16,1283 mass spectra and Dr. Kuo-Hsiung Lee, School of Pharmacy, University (1977). of North Carolina at Chapel Hill, for measurement of the high-resolution (8) H.Suguna and B. R. Pai, Coll. Czech. Chem. Commun., 41,1219 mass spectra. They also thank Dr. B. R. Pai, Department of Chemistry, (1976). Presidency University, Madras, India, for an authentic sample of 3- (9) J. Schmutz, Helu. Chim. Acta, 42,335 (1959). hydroxy-2-methoxy-l0,ll-methylenedioxyberbineand Dr. C. Moulis, (10) T. Kametani, M. Takeshita, and S. Takano, J. Chem. SOC.Perkin Faculty of Pharmaceutical Sciences, University of Toulouse, France, for I, 1972,2834. authentic samples of pseudocolumbamine and dehydropseudocheilan- (11) T. Kametani, Y.Hirai, F. Satoh, K. Ogasawara, and K. Fukumoto, thifoline. C.-H. Chen also expresses his gratitude to the late Professor Chem. Pharm. Bull., 21,907 (1973). Taito 0. Soine for his encouragement and interest in this work. Acetylcarnitine and Cholinergic Receptors K. W. REED, W. J. MURRAY", and E. B. ROCHE Received April 2, 1979, from the Department of Biomedicinal Chemistry, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68105. Accepted for publication April 28,1980. + I Abstract o Acetylcarnitine, a naturally occurring compound found in (CH,),NCH,-CH~H,COOHI, high concentration in heart and skeletal muscle of vertebrates, bears structural resemblance to acetylcholine, and studies have shown that it has slight cholinergic properties. Acetylcarnitine was subjected to con- formational analysis by extended Hiickel theory (EHT) and complete neglect of differential overlap (CNDOD) molecular orbital methods. The preferred conformations were examined with respect to their similarity to the Kier and Chothia-Pauling models of cholinergic receptor patterns. The preferred conformations of both isomers did not fit the receptor pattern described by Kier's model, although energy barriers to rotation I1 are low enough to permit accommodation. The Chothia-Pauling model prompted investigations into its pharmacological prop- predicts activity for the S-isomer only. These studies partially explain erties as either a cholinomimetic or an anticholinergic the low cholinergic activity found for acetylcarnitine and the higher ac- compound (2-11). tivity of (S)-acetylcarnitine compared to the R-isomer. Keyphrases Acetylcarnitine-conformational analysis, comparison BACKGROUND to cholinergic receptor patterns 0 Cholinergic activity-acetylcarnitine, It was reported that high doses of acetylcarnitine exerted a cholinergic conformational analysis, comparison to cholinergic receptor patterns 0 effect on the heart which was blocked by atropine (2). Strack and Structure-activity relationships-acetylcarnitine, conformational Forsterling reported that carnitine had little biological activity (3). analysis, comparison to cholinergic receptor patterns However, Dallemagne et al. (4) found that carnitine and acetylcarnitine depolarized motor end plates. They reported that (R,S)-carnitine was L-(-)-Carnitine [(R)-carnitine,I] is a naturally occurring l/lOOO as potent as acetylcholine on frog rectus abdominis muscle and that (R)-acetylcarnitine was 10-25% as potent as acetylcholine (4). substance found in high concentrations in plants, micro- In contrast, Blum et al. (5) found (R)-carnitine to have 1/10,000 and organisms, and animals. The muscles of vertebrates and (S)-carnitineto have 1/4000 the activity of acetylcholine on rectus ab- invertebrates have especially high levels. Its principal dominis muscle. They also reported that (R)-and (S)-acetylcarnitine physiological function is to transport free fatty acids into were 1/700 as potent as acetylcholine in lowering arterial blood pressure in dogs (5). They concluded that acetylcarnitine acts at the cholinergic the mitochondria prior to their oxidation uia the 0-ox- site rather than by blocking acetylcholinesterase. Two other groups found idation cycle (1). that synthetic (R,S)-acetylcarnitine possessed only weak to no nicotinic (R)-Acetylcarnitine (II), a derivative of carnitine, also activity (6,7). is found in high concentrations in the tissues of vertebrates. Hosein and Proulx (8) claimed to have isolated a cholinergic active Enveloped within the structure of acetylcarnitine is the fraction of brain synaptosomes and identified this fraction as containing the coenzyme A ester derivatives of y-butyrobetaine, crotonbetaine, structure of the parasympathetic neurotransmitter ace- carnitine, and acetylcarnitine. Six groups of investigators were unable tylcholine (indicated by the dashed lines in 11).The close to verify Hosein's isolation and identification of the active cholinergic structural similarity of acetylcarnitine to acetylcholine has substance of the brain, and the substance isolated by Hosein et al. ap- + parently was acetylcholine itself (7). Falchetto et a/. (9) reported that (CH,),NCH,-CH-CH&OOH cortical neurons excited by acetylcholine also are excited by carnitine and acetylcarnitine. Carnitine was found to be more potent than acetylcar- I nitine. OH Fritz (10) injected (R,S)-acetylcarnitine intracisternally into rat brains I and evoked pronounced motor activity, which was not observed with 0022-35491801 0900-1065$0 1.0010 Journal of Pharmaceutical Sciences / 1065 @ 1980, American Pharmaceutical Association Vol. 69, No. 9. September 1980 c,- c*+c3eCq-N5-cI n CCL7\ O8 a Figure 1-Torsional angles for acetylcarnitine. Key: $1, C1-C2-C3-06; 62. CYC~-O~-C~;$3, CZ-C~-C~-N~; and $4, c&&7-08. (R.S)-carnitine. (R,S)-Acetylcarnitine injected intraventricularly caused phonation, pupillary dilatation, and markedly increased motor activity. Walker et al. (11) reported carnitine and acetylcarnitine to be less than 1/5000 as active as acetylcholine on excitatory neurons from Helix as- pera . Overall, the evidence shows that (R)- and (S)-acetylcarnitines possess low cholinergic activity. Since atropine is reported to block the cholinergic action of acetylcarnitine, its activity may be primarily at muscarinic re- ceptors (5). Many models of muscarinic and nicotinic receptors have been proposed based on the activity of potent cholinergic compounds. Therefore, it is of interest to examine the preferred conformations of (R)- and (S)- acetylcarnitines and to compare their fit to these proposed models. If the models are useful in predicting a pattern for an active cholinergic com- b pound, then low activity compounds should not be expected to fit the Figure %-Preferred conformationsfor acetylcarnitine. Key: a, folded pattern. conformation;and b, extended conformation. EXPERIMENTAL (Fig. 1). In addition, torsional angles for the preferred orientation of the quaternary ammonium group and carboxylate anion were determined. To determine the preferred conformations, two quantum chemical The optimal conformations of the isolated torsional angles, $1, $2, and methods were used: the extended Huckel theory (EHT)' (12) and com- $4, were determined first and then were used to find $:<.With the whole plete neglect of differential overlap (CND0/2)2 (13). Although EHT molecule, slight variations in each torsional angle were examined to op- usually exaggerates the energy barriers to rotation, it does correctly timize all parameters. predict preferred rotational conformers. The CND0/2 method relies less The bond angles and bond lengths were derived from crystallographic on empirical parameters than does EHT and uses a self-consistent field data (15,16). The three-dimensional Cartesian coordinates for each atom technique to estimate electronic interactions (13). Compared to EHT, in the differing conformations were calculated with PROXYZ, QCPE CNDO/2 gives more realistic estimations of electronic parameters such Program 94, using X-ray data (15,16). as electron populations and charge densities and also gives reasonable estimations of energy barriers to rotation. The CNDO/2 method was used RESULTS to verify the EHT calculations for preferred conformations and allowed the determination of the effect of electrostatic interactions between the Kier's (14) partitioning method for determining the lowest energy quaternary ammonium group and the carboxylate anion. conformer for each torsional angle as the molecule is constructed was The conformational problem concerns the steric and electronic effects followed. For (R)-acetylcarnitine,the torsional angle for the ester group, of the C1-carboxylate anion and the Ns-quaternary ammonium cation $4 (C3-06-c7-08), using 111 was determined first using 30" increments. and the effect of the acetyl group in determining the rotational and conformational properties of the carnitine molecule. The numbering scheme and dihedral angles considered are given in Fig.