Chem. Res. Toxicol. 1988,1,128-131
128
Metabolic Studies on Phencyclidine: Characterization of a Phencyclidine Iminium Ion Metabolite Marie K. P. Hoag, Michael Schmidt-Peetz, Peter Lampen, Anthony Trevor, and Neal Castagnoli, Jr.* Division of Toxicology and D e p a r t m e n t of Pharmacology, University of Califonia Schools of P h a r m a c y and Medicine, San Francisco, California 94143-0446 Received Januar.y 18,1988
Studies on the metabolic bioactivation of the psychotomimetic amine phencyclidine have been pursued through the characterization of a new metabolite which is formed via initial cytochrome P-450 catalyzed oxidation of the parent drug t o the corresponding iminium species. CI mass spectrometric and diode array UV and 'H NMR spectral analyses provided evidence for the Confirmation conjugated amino enone compound, l-(l-phenylcyclohexyl)-2,3-dihydro-4-pyridone. of the proposed structure was achieved by comparing the 'H NMR and high-resolution E1 mass spectral properties of the metabolic isolate with the corresponding spectra of an authentic synthetic sample. Possible intermediates involved in the formation of the dihydropyridone metabolite from the phencyclidine iminium ion are discussed in terms of structural analogies to reactive intermediates formed in the bioactivation of the nigrostriatal toxin 1-methyl-4phenyl-l,2,3,64etrahydropyridine(MPTP).
Introduction The cyclic tertiary amine phencyclidine [1-(1-phenyl1-cyclohexy1)piperidine(l), PCP] is a potent psychotomimetic agent which, in susceptible individuals, causes long lasting psychotic behavior that is difficult to distinguish clinically from that associated with idiopathic schizophrenia (I).We (2)and others (3-5)have examined the metabolic fate of phencyclidine with particular interest in the possible formation of chemically reactive species that may cause biochemical lesions relating to these neurotoxic effects. The reported metabolism dependent formation of covalent adducts between radioactive phencyclidine and biomacromolecules (6, 7) is consistent with the formation of such reactive metabolites. We have provided evidence that the cytochrome P-450 catalyzed a-carbon oxidation of phencyclidine to the corresponding iminium species 2 is linked to the bioalkylating properties of this piperidine derivative (8). This metabolic pathway also appears to be responsible for the phencyclidine mediated inactivation and destruction of cytochrome P-450 (9). Recent results obtained from studies with radiolabeled 2, however, indicate that the iminium metabolite must undergo further NADPH dependent microsomal transformation(s) to elicit these effects (10). Preliminary characterization of the metabolic fate of 2 in rabbit liver microsomal preparations have provided evidence for the cytochrome P-450 catalyzed formation of a product which by diode array analysis possesses a chromophore with ,A, 314 nm (10). This product also is formed from phencyclidine although at a slower rate which is consistent with a pathway proceeding through the iminium ion intermediate (10).The present paper describes the isolation and structural characterization of this product. Experimental Section All chemicals were reagent grade or, in the case of solvents, HPLC grade. Phencyclidine iminium perchlorate was synthesized as previously described (9). Proton NMR spectra were obtained on a custom built 240-MHz or a GE 500-MHz instrument, both 0893-228~/88/2701-0128$01.50/0
linked to a Nicolet 1180 computer, or on a Varian FT 80 instrument. Chemical shifts (6) are reported in parts per million (ppm) relative to tetramethylsilane (Me,Si). Spin multiplicity is given as (s) singlet, (d) doublet, (t)triplet, (9) quartet, or (m) multiplet. Infrared spectra were taken on a 50X Nicolet FT-IR and UV spectra on a Beckman DU-50. HPLC separations were performed on a Beckman Model 330 liquid chromatographic system. An Alltech Ekonosil silica column (5" particle diameter; 4.6 mm x 25 cm) and an Upchurch precolumn packed with Alltech silica pellicular guard column refill was used. The mobile phase was 0.5% propylamine in acetonitrile at a flow rate of 1.5 mL/min. Preparative centrifugal chromatographic separations were performed on Model 7924 T Chromatotron (Harrison Research, Palo Alto, CA) using a rotor plate with a 1-mm layer of silica gel PFm with CaS04.1/zHz0(E. Merck). UV characterization of the eluent was performed on the above HPLC system with an on-line diode array detector (Hewlett-Packard Model 1040A). Capillary oncolumn, GC analyses were performed on a HewlettrPackard Model 5890 instrument using a 5% cross-linked phenylmethylsilicone Ultra 2 column (0.17-mm film thickness; 12.5 m X 0.32 mm) and helium carrier gas (30 mL/min) with a nitrogen-phosphorous detector maintained at 250 "C. The initial oven temperature (50 " C ) was increased to 190 "C (40 "C/min) and then to 230 "C (10 OC/min), where it was held for 1 min. Low-resolution probe CI mass spectra were run on a modified AEI 902s at 8 kV with 2-methylpropane (ca. 1Torr) as reagent gas. The high-resolution E1 mass spectrum of the biological isolate was obtained at the University of Washington on a VG 70-70; the corresponding spectrum of the synthetic sample was obtained at the Mass Spectrometry Laboratory of the University of California,Berkeley, on a Kratos MS 50s. Melting points were obtained on a Thomas-Hoover melting point apparatus and are uncorrected. Microanalyses were performed by the Microanalytical Laboratory, University of California, Berkeley, CA. Microsomal Metabolism of Phencyclidine Iminium Perchlorate. Liver microsomes were prepared from rabbits treated with phenobarbital as described previously (9). For isolation and purification of the metabolite, phencyclidine iminium perchlorate (0.5 mM) was incubated with liver microsomes (1.5 mg of protein/mL) in 200 mL 0.1 M HEPES buffer, pH 7.6, containing an NADPH generating system (0.5 mM NADP', 8 mM glucose-6-phosphate, 1unit/mL glucose-&phosphatedehydrogenase, and 4 mM MgCl2)and 1 mM EGTA. After 30 min at 37 OC, the metabolic reaction was stopped by addition of 100 mL of ice-cold 0.2 M potassium hydrogen phthalate. The resulting mixture (pH 0 1988 American Chemical Society
Metabolic Studies on Phencyclidine 5) was eaturated with NaC1, and the aqueous phase was extracted three times with 100 mL of heptane containing 1.5% isoamyl alcohol. The combined extracts were dried over MgSO, and concentrated under vacuum to yield a crude isolate which displayed one major and symmetrical peak on HPLC with a retention time of 4.5 min and diode array W characteristics corresponding to the 314 nm absorbing metabolite. Separation of the metabolite from the other components present in the extract was achieved by centrifugal chromatography eluting with the following solvents at 5 mL/min in the indicated order of increasing polarity: 100 mL ethyl acetate/hexane (50/50), 100 mL ethyl acetate/hexane (75/50), and 100 mL pure ethyl acetate. Fractions of approximately 10 mL were collected and were monitored by HPLC and on-column GC. The fractions containing the metabolite were combined and the solvent evaporated under vacuum. This sample gave the high-resolution E1 mass spectrum shown in Figure 1. The sample was dried overnight at room temperature and 0.1 Torr to give a solvent free product which displayed the 500-MHz 'H NMR spectrum shown in Figure 2. 1-(1,4-Dioxa-8-azaspiro[4.S]dec-8-yl)cyclohexane-lcarbonitrile (11). To an ice bath cooled, stirred mixture of 1,4-dioxa-8-azaspiro[4.5]decane(10,5 g, 90 mmol) was added 9 mL of concentrated HCl to give a final pH of 3-4. Following the and a solution rapid addition of cyclohexanone(9,8.8 g, 90"01) of KCN (5.85 gm, 90 mmol) in 70 mL of HzO, the mixture was allowed to warm to room temperature and stirring was continued for 3 h. The resulting emulsion was extracted with dichloromethane (3 X 80 mL), the combined organic layers were dried (CaC12)and liltered, and the solvent was evaporated under reduced pressure. The oily residue was purified by flash chromatography on a silica column (30x 200 mm, Kieaelgel60) using toluene/ethyl acetate (43) as mobile phase. Recrystallization from diisopropyl ether gave 13.3 g (60.8%) of colorless crystals: mp 108 "C; 240MHz 'H NMR (CDC13)6 1.1-2.5 (m, 10 H, cyclohexyl), 1.22 (t, 4 H, J = 6 Hz, H-3,5), 2.7 (t, 4 H, J = 6 Hz, H-2,6), 3.95 (8, 4 H, OCH,CH,O); IR (CC14)Y 2214 cm-'; CIMS, m/z 224 [(MH HCN)+]. AnaL Calcd for Cl4HZ2N2O2: C, 67.15; H, 8.87; N, 11.19. Found C, 67.08; H, 8.79; N, 11.12. 8 4l-Phenylcyclohexy1)-1,4-dioxa-8-azaspiro[ 4.5ldecane (12). To a solution of the above a-cyano amine (5.5 g, 22 mmol) in 80 mL of toluene/dry ether (82) was added with vigorous stirring under a nitrogen atmosphere a 3 M ethereal solution of phenylmagnesium bromide (11 mL, 33 mmol). The resulting mixture was heated under reflux for 6 h and then was cooled in an ice bath and treated with 50 mL of saturated aqueous NH40H. The separated aqueotls layer was extracted twice with ether, and the combined organic layers were extracted with ice-cold 2 N HzSO4 (2 X 80 mL). The acidic extracts were poured quickly into a mixture of cold 2 N NaOH/ether. The separated aqueous layer (pH 10) was extracted with ether (2 X 80 mL), the combined organic extracts were dried (CaC1,) and filtered, and the solvent was removed under reduced pressure. The residue was recrystallized from diisopropyl ether to give 3.54 g (53%) of colorless crystals: mp 118 "C; 240-MHz 'H NMR (CDC13)6 1.25-1.9 (m, 6 H, cyclohexyl H-3,4,5), 1.65 (t,4 H, J = 5.5 Hz, H-3,5), 2.05 (t, 4 H, J = 5.5 Hz, H-2,6), 2.3-2.47 (m, 4 H, cyclohexyl H-2,6), 3.87 (s,4 H, OCH2CH20),7.3-7.5 (m, 5 H, Ar); CIMS, m/z 302 (MH+). Anal. Calcd for C19HnN02: C, 75.69; H, 9.05; N, 4.65. Found C, 75.82; H, 9.12; N, 4.68. 1-(l-Phenylcyclohexyl)-2,3-dihydro-4-pyridone (3). To a heated (90 "C) and rapidly stirred suspension of mercuric acetate (8.3 g, 26 mmol) in 60 mL of 5% acetic acid was added 2 g (6.6 "01) of the above product. Stirring and heating were continued for 5 h. The reaction mixture was cooled to 10 "C, and the grey mercurous acetate precipitate was filtered and carefully washed with 5% acetic acid. The filtrate was saturated with hydrogen suKde, and the resulting precipitate was filtered and washed with 5% acetic acid. The combined filtrates and washes were treated with 5 N NaOH in the cold, and the resulting mixture was extracted (4 x 80 mL) with dichloromethane. The combined organic layers were dried (CaC12)and filtered, and the solvent was removed under reduced pressure. Recrystallization of the residue from diisopropyl ether/ethanol (82) gave 380 mg (22.3%)of colorless crystals: mp 122 "C; 5W-MHz 'H NMR (CDCl,) 6 1.35-1.8 (m, 6 H, cyclohexyl H-3,4,5), 1.95-2.3 (m, 4 H, cyclohexyl H-2,6),2.33
Chem. Res. Toxicol., Vol. 1, No. 2, 1988 129
(t, 2 H, J = 7 Hz, H-3), 3.21 (t, 2 H, J = 7 Hz, H-2), 5.07 (d, 1 H, J = 8 Hz, s after decoupling at 7.56, H-5), 7.25-7.4 (m, 5 H, Ar), 7.56 (d, 1H, J = 8 Hz, s after decoupling at 5.07, H-6); UV (CH3CN)X, 314 nm (e 9140); IR (CCl,) u 1630,1564 cm-'. The high-resolution EIMS and 500-MHz 'H NMR spectrum were essentially identical to those observed for the phencyclidine iminium ion metabolite shown in Figure 1 and 2, respectively. Anal. Calcd for C1,HZ1NO C, 79.94; H, 8.30; N, 5.49. Found C, 79.88; H, 8.22; N, 5.40. Reaction of Dihydropyridone 3 with NaCNBH3. A mixture of the dihydropyridone 3 (2.0 mmol) and NaCNBH3 (2.0 mmol) in 50 mL of methanol was treated with 2 drops of concentrated HC1, and the resulting mixture was warmed to 50 "C with stirring for 6 h. After cooling to room temperature 50 mL of water was added, the pH of the resulting mixture was adjusted to 1 2 with 1N NaOH, and the products were extracted with ether (3 X 30 mL). The combined ethereal solutions were dried (CaC12),fdtered, and evaporated to dryness under reduced pressure. Centrifugally accelerated,radial thin-layer chromatography using toluene/ethyl acetate (1:2) as mobile phase led to the isolation of three products. The fiist fraction (12 mL) contained a neutral compound (35%), which was identified as l-phenylcyclohexene(16) by comparison of its 'H NMR spectrum with the spectrum reported in the literature (11). Fraction 2 (8 mL) contained a compound (50%) with the following properties: mp 81 "C; 80-MHz 'H NMR (CDCl,) 6 1.05-1.75 (m, 6 H, cyclohexyl H-3,4,5), 1.85-2.17 (m, 4 H, cyclohexyl H-2,6), 2.35 (t, 4 H, J = 6 Hz, H-3,5), 2.55 (t, 4 H, J = 6 Hz, H-2,6), 7.28 (s,5 H, Ar). These 'H NMR data and comparison with the spectrum for 1-methyl-4-piperidone (12) permit the assignment of the isolate as l-(l-phenylcyclohexyl)-2,3,5,6-tetrahydro-4-pyridone(19). Fraction 3 (25 mL) contained l-(l-phenylcyclohexyl)-4-piperidinol(20,10%), which was characterized by comparison of its 'H NMR spectrum with the corresponding spectrum of an authentic sample prepared according to the literature (13).
Results and Discussion A preparative-scale rabbit liver microsomal incubation was carried o u t in order to isolate adequate quantities of the 314 nm absorbing product for spectral characterization. Initial efforts to purify t h e metabolite by preparative HPLC were only partially successful as revealed by the complexity of the 'H NMR spectrum of the material which eluted as a symmetrical peak. An alternative approach involving initial concentration by solvent extraction at pH 5 followed by centrifugally accelerated, radial thin-layer chromatography provided a product that was homogeneous b y HPLC and also b y on-column capillary GC chromatography. Direct insertion probe chemical ionization mass spectral analysis of this isolate showed a protonated molecular ion at m/z 256. A proposed empirical formula, C17H21N0,requires the alteration of the structure of t h e starting material by one additional degree of unsaturation and the introduction of one oxygen atom. High-resolution probe E1 mass spectral analysis (Figure 1) confirmed t h e empirical formula of the parent ion a n d also revealed a fragment ion at m/z 159.1174 (C12H15). As t h e phenylcyclohexyl cationic species i, this fragment rules o u t structural modifications of the phenylcyclohexyl moiety present in the iminium ion substrate. A second informative fragment ion ii was observed at m/z 98.0607 (C5H8NO) which may be represented by a protonated dihydropyridone structure. T h e base peak at m / z 91.0548 corresponds t o the expected tropylium ion iii. Collectively the data suggest t h e formation of a n N-(phenylcyclohexy1)dihydropyridone system involving t h e net introduction of one carbonyl group into t h e starting tetrahydropyridinium moiety. Six isomeric compounds (3-8) are consistent with t h e mass spectral data. T h e 500-MHz 'H N M R spectrum of t h e purified metabolite (Figure 2) displayed two coupled doublets, each integrating for l-proton, centered at 6 5.07 a n d 7.56 and
130 Chem. Res. Toxicol., Vol. 1, No. 2, 1988
Hoag et al. 100
80
3
I II (C7H7) Calcd. 91.0545
Ynd
91 .0552
E VI
1
0
6o
E
R 5
R 6
R 7
R 8
-80E
40 11 (C5H8NO) Calcd. 98.0604
R = 1-phenylcyclohexyl moiety
two coupled triplets, each integrating for two protons, centered at S 2.33 and 3.21. Of the structures 3-8 proposed on the basis of the mass spectral data, compound 3 fits beat the 'H NMR spectrum with the olefinic protons at C-3 and C-2 corresponding to the two downfield doublets and the methylene protons at C-5 and C-6 corresponding to the two upfield triplets. The conjugate amino enone functionality present in 3 also is consistent with the 314-nm chromophore of the metabolite (14-16). The spectral data summarized above prompted us to attempt the synthesis of the dihydropyridone derivative 3 by the route outlined in Scheme I. Condensation of cyclohexanone (9) with the dioxalane 10 of 4-piperidone in the presence of potassium cyanide led to the corresponding a-cyano amine 11. Reaction of 11 with phenylmagnesium bromide gave intermediate 12, which upon treatment with mercuric acetate (17) in the presence of acetic acid yielded the desired product 3. The W, CI, and high-resolution E1 mass spectral features of the synthetic compound and the metabolic isolate were essentially identical. Except for the absence of the minor upfield impurity obse~edin the spectrum of the metabolite at 1.2 ppm, the 500-MHz lH NMR spectrum of synthetic 3 was identical with that shown in Figure 2. The possible contribution which this new metabolite may make to the phencyclidine dependent inhibition of cytochrome P-450 (9) was examined. Incubation of 3 in the presence of an NADPH generating system resulted in a decrease of approximately 10% in microsomal N-demethylase activity compared to controls which were run in the absence of substrate. This modest effect on cytochrome P-450 activity and the relative stability of 3 (0.5 mM) in microsomal incubation mixtures (only 10% decrease following a 30-min incubation period) suggest that this metabolite is unlikely to participate in the inhibition of liver microsomal N-demethylase activity caused by phencyclidine and its iminium ion metabolite. The conversion of the phencyclidine iminium species 2 to this dihydropyridone metabolite involves an overall four-electron oxidation which will proceed via an initial two-electron oxidation step. Since this biotransformation requires NADPH and is inhibited by SKF 525A and carbon monoxide ( l o ) ,it is likely to be catalyzed by cytochrome P-450. We propose that the oxidative metabolism of 2 proceeds through the conjugate eneamine free base 13 of the iminium ion substrate which then is oxidized to the allyl alcohol 14 (Scheme 11). This intermediate then must undergo a second two-electron oxidation to yield the final product 3. Mass balance studies with tritium-labeled phencyclidine iminium perchlorate indicate that only about 40% of the substrate metabolized can be accounted for by the dihydropyridone metabolite. Therefore, it is possible that the intermediate allyl alcohol, perhaps via the electrophilic 2,3-dihydropyridinium species 15, is involved in the bioalkylation reactions observed with phencyclidine and ita iminium ion metabolite.
I (CIPtJ Calcd. 159.1175 159.1173
M'+ ( C I 7 Y W ) Calcd 255.1618 FOund255.1621
;ound
20
98.0607
0
mie Figure 1. High-resolution probe E1 mass spectrum of the phencyclidine iminium metabolite.
,
I
d
e
Figure 2. 500-MHz lH NMR spectrum of the phencyclidine iminium metabolite. Scheme 1. Synthetic Pathway to Enamine Ketone 3 n
n
+
x LN/ I
H
9
10
11
12
Scheme 11. Proposed Metabolic Pathway for Iminium Ion 2
0P-450
2 -
13
14
15
We attempted to prepare the allyl alcohol 14 by hydride reduction of 3. As expected for a vinylogous amide, the amino enone was stable to sodium borohydride reduction. However, when treated with methanolic sodium cyanoborohydride in the presence of HC1, the 314-nm chromophore slowly disappeared. The 'H NMR spectrum of the resulting complex mixture displayed doublets centered at 6 5.48 and 6.30 (J = 7.5 Hz) consistent with the desired allyl alcohol. Further purification by centrifugal chro-
Chem. Res. Toxicol., Vol. 1, No. 2, 1988 131
Metabolic Studies on Phencyclidine Scheme 111. Sodium CyanoborohydrideReduction of Enamine Ketone 3"
16
3
17
19
18
20
"R = 1-phenylcyclohexyl.
matography, however, led to the characterization only of 1-phenylcyclohexene (16), l-(l-phenylcyclohexy1)-2,3,5,6tetrahydro-4-pyridone (19), and 1-(1-phenylcyclohexy1)4-piperidinol(20). We postulate that under acidic reaction conditions the amino enone may undergo a Hoffman elimination (pathway a, Scheme 111) to yield l-phenylcyclohexene (16) or may protonate on oxygen (pathway b) to yield intermediate 17. Subsequent reduction of the iminium moiety present in 17 yields enol 18, which, via the keto tautomer 19, generates 20. The oxidative metabolism of cyclic amines to reactive metabolic products has been well documented recently by the demonstration of the dramatic neurotoxic properties of the tetrahydropyridine derivative MPTP (21) (18). The initial step in the bioactivation of MPTP involves its MA0 B catalyzed oxidation to the dihydropyridinium species MPDP+ (22) which subsequently is oxidized to the putative ultimate toxin MPP+ (23) (18). The cytochrome
,,,G \NI
Lh/
I
I
CH3 21
CH3 22
Q CH3
23
P-450 catalyzed oxidation of the iminium ion of phencyclidine to the amino enone 3 may involve the intermediacy of a dihydropyridinium species (14) with structural features resembling those present in MPDP+. It should be recognized, however, that PCP and MPTP exert quite different toxic effects, the nature of which presumably depends on other factors in addition to their metabolic activation to form potentially reactive intermediates. Studies on the metabolism of phencyclidine in brain tissue and attempts to synthesize the proposed metabolic intermediates 14 and 15 are currently being pursued. Acknowledgment. M.K.P.H. was a recipient of an ARCS Foundation grant. M.S.-P. and P.L. were supported by grants from the Deutsche Forschungsgemeinschaft, Bonn. This research was supported by NIH Grant DA 03405.
References (1) Petersen, R. C., and Stillman, R. C. (1978) "Phencyclidine: an overview". Natl. Inst. Drug Abuse Res. Monogr. Ser. 21, 1-17. (2) Ward, D., Kalir, A., Trevor, A., Adams, J., Baillie, T., and Castagnoli, N., Jr., (1982) "Metabolic formation of iminium species: metabolism of phencyclidine". J. Med. Chem. 25, 491-492. (3) Wong, L. K., and Biemann, K. (1975) 'Metabolites of phencyclidine in humans". Biomed. Mass Spectrom. 2, 204-205. (4) Kammerer, R. C., Schmitz, D. A., DiStefano, E. W., and Cho, A. K. (1981) 'The metabolism of phencyclidine by rabbit liver preparations". Drug Metab. Dispos. 9,274-278. (5) Baker, J. K., Wohlford, J. G., Bradbury, B. J., and Wirth, P. W. (1981) "Mammalian metabolism of phencyclidine by rabbit liver preparations". J. Med Chem. 24, 666-669. (6) Law, F. C. P., and Farquharson, T. E. (1980) "Metabolism and irreversible binding of phencyclidine by rabbit lung and liver microsomes". In Microsomes, Drug Oxidations, and Chemical Carcinogenesis (Coon, M. J., Conney, A. H., Estabrook, R. W., Gelboin, H. V., Gillette,J. R., and O'Brien, P. J. Eds.) pp 985-988, Academic, New York. (7) Law, F. C. P. (1981) 'Metabolic disposition and irreversible binding of phencyclidine in rata". Toxicol. Appl. Pharmacol. 57, 263-272. (8) Ward, D. P., Trevor, A. J., Kalir, A., Adams, J. D., Baillie, T. A., and Castagnoli, N., Jr. (1982) 'Metabolism of phencyclidine-the role of iminium ion formation in covalent binding to rabbit microsomal protein". Drug Metab. Dispos. 10, 690-695. (9) Hoag, M. K. P., Trevor, A. J., Asscher, Y.,Weissman, J., and Castagnoli, N., Jr. (1984) 'Metabolism-dependent inactivation of liver microsomal enzymes by phencyclidine". Drug Metab. DisP O S . 12, 371-375. (10) Hoag, M. K. P., Trevor, A. J., Kalir, A,, and Castagnoli, N., Jr. (1987) "Phencyclidine iminium ion-NADPH-dependent metabolism, covalent binding to macromolecules, and inactivation of cytochrome(s) P-450". Drug Metab. Dispos. 15,485-490. (11) Pouchert, C. J. (1983) The Aldrich Library of NMR Spectra, ed. 2, Vol. I, p 754b, Aldrich, Milwaukee. (12) Pouchert, C. J. (1983) The Aldrich Library of NMR Spectra, ed. 1, Vol. I, p 408d, Aldrich, Milwaukee. (13) Lin, D. C. K., Fentiman, A. F., Jr., Foltz, R. L., Forney, R. D., Jr., and Sunshine, I. (1975) 'Quantification of phencyclidine in body fluids by gas chromatography chemical ionization mass spectrometry and identification of two metabolites". Biomed. Mass Spectrom. 2, 206-214. (14) Liberatore, F., Casini, A., Carelli, V., Amone, A., and Mondelli, R. (1975) "Borohydride reduction of pyridinium salts. V. Thermal dimerization of 1,6-dihydro-1-methylppidine-2-carbonitrile". J. Org. Chem. 40, 559-563. (15) Guerry, P., and Neier, R. (1984) "Reduktion von 4Pyridinonen". Synthesis 6,485-488. (16) Sundberg, R. J., Bukowick, P. A., and Holcombe, F. 0. (1967) "The preparation of esters of 4-alkyl-2,4-pentadienoic acids by the phosphonate modification of the Wittig reaction". J. Org. Chem. 32, 2938-2941. (17) Leonard, N. J., and Cook, A. G. (1959) "Unsaturated amines. XIV. The mercuric acetate oxidation of substituted pyrrolidines". J. Am. Chem. SOC.81, 5627-5631. (18) Markey, S. P., Castagnoli, N., Jr., Trevor, A. J., and Kopin, I. J. (1986) M P T P A Neurotoxin Producing a Parkinsonian Syndrome, Academic, Orlando, FL. (19) Chiba, K., Peterson, L. A,, Castagnoli, K. P., Trevor, A. J., and Castagnoli, N., Jr. (1985) 'Studies on the molecular mechanism of bioactivation of the selective nigrostriatal toxin l-methyl-4phenyl-1,2,3,6-tetrahydropyridine".Drug Metab. Dispos. 13, 342-347.
