128 Chem. Res. Toxicol. 1988,1, 128-131

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 Depar tment of Pharmacology, University of Califonia Schools of

Received Januar.y 18, 1988

Pharmacy and Medicine, San Francisco, California 94143-0446

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 to the corresponding iminium species. CI mass spectrometric and diode array UV and 'H NMR spectral analyses provided evidence for the conjugated amino enone compound, l-(l-phenylcyclohexyl)-2,3-dihydro-4-pyridone. Confirmation 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-4- phenyl-l,2,3,64etrahydropyridine (MPTP).

Introduction The cyclic tertiary amine phencyclidine [ 1-(1-phenyl-

1-cyclohexy1)piperidine (l), PCP] is a potent psychotom- imetic agent which, in susceptible individuals, causes long lasting psychotic behavior that is difficult to distinguish clinically from that associated with idiopathic schizo- phrenia (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 bioalky- lating 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 de- pendent microsomal transformation(s) to elicit these ef- fects (1 0). 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 characteriza- tion 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 0

linked to a Nicolet 1180 computer, or on a Varian FT 80 in- strument. 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 per- formed 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 on- column, 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. 1 Torr) 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 Thom- as-Hoover melting point apparatus and are uncorrected. Mi- croanalyses were performed by the Microanalytical Laboratory, University of California, Berkeley, CA.

Microsomal Metabolism of Phencyclidine Iminium Per- chlorate. 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 pro- tein/mL) in 200 mL 0.1 M HEPES buffer, pH 7.6, containing an NADPH generating system (0.5 mM NADP', 8 mM glu- cose-6-phosphate, 1 unit/mL glucose-&phosphate dehydrogenase, 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

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 dis- played 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 a t 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 approxi- mately 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-l- carbonitrile (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 rapid addition of cyclohexanone (9,8.8 g, 90 "01) and a solution 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 dichloro- methane (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 (30 x 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; 240- MHz 'H NMR (CDC13) 6 1.1-2.5 (m, 10 H, cyclohexyl), 1.22 (t,

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 4 l-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 recrys- tallized 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 ex- tracted (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

4 H, J = 6 Hz, H-3,5), 2.7 (t, 4 H, J = 6 Hz, H-2,6), 3.95 (8, 4

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, 1 H, 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 im- inium 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 12 with 1 N 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-phenylcyclo- hexyl)-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 out in order to isolate adequate quantities of the 314 nm absorbing product for spectral characterization. Initial efforts to purify the 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 by HPLC and also b y on-column capillary GC chroma- tography. Direct insertion probe chemical ionization mass spectral analysis of this isolate showed a protonated mo- lecular ion at m/z 256. A proposed empirical formula, C17H21N0, requires the alteration of the structure of t he 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 the empirical formula of the parent ion a n d also revealed a fragment ion at m/z 159.1174 (C12H15). As t h e phenyl- cyclohexyl cationic species i, this fragment rules ou t structural modifications of the phenylcyclohexyl moiety present in the iminium ion substrate. A second informa- tive fragment ion ii was observed at m/z 98.0607 (C5H8NO) which may be represented by a protonated dihydro- pyridone structure. T h e base peak at m / z 91.0548 cor- responds t o the expected tropylium ion iii. Collectively the data suggest t he formation of a n N-(phenylcyclo- hexy1)dihydropyridone system involving the net intro- duction of one carbonyl group into the starting tetra- hydropyridinium 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 the purified me- tabolite (Figure 2) displayed two coupled doublets, each integrating for l-proton, centered at 6 5.07 and 7.56 and

130 Chem. Res. Toxicol., Vol. 1, No. 2, 1988 Hoag et al.

R R R R 5 6 7 8

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 func- tionality present in 3 also is consistent with the 314-nm chromophore of the metabolite (1 4-1 6).

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 corre- sponding a-cyano amine 11. Reaction of 11 with phe- nylmagnesium 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~ed in 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-de- methylase activity compared to controls which were run in the absence of substrate. This modest effect on cyto- chrome P-450 activity and the relative stability of 3 (0.5 mM) in microsomal incubation mixtures (only 10% de- crease 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 car- bon monoxide ( lo) , it is likely to be catalyzed by cyto- chrome 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 in- volved in the bioalkylation reactions observed with phen- cyclidine and ita iminium ion metabolite.

100

80

I I I (C7H7) Calcd. 91.0545

Ynd 91 .0552

11 (C5H8NO) Calcd. 98.0604

;ound 98.0607

3 E 6o

8 40

VI E

E 0 -

20

0

1 0 I (C IP tJ Calcd. 159.1175

159.1173 M'+ ( C I 7 Y W )

FOund255.1621 Calcd 255.1618

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

0 P-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 cyano- borohydride in the presence of HC1, the 314-nm chromo- phore 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-

Metabolic Studies on Phencyclidine Chem. Res. Toxicol., Vol. 1, No. 2, 1988 131

Scheme 111. Sodium Cyanoborohydride Reduction of Enamine Ketone 3"

16 3 17 18 19 20

"R = 1-phenylcyclohexyl.

matography, however, led to the characterization only of 1-phenylcyclohexene (16), l-(l-phenylcyclohexy1)-2,3,5,6- tetrahydro-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-phenyl- cyclohexene (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 puta- tive ultimate toxin MPP+ (23) (18). The cytochrome

,,,G \NI Lh/ Q

I I CH3 CH3 CH3 21 22 23

P-450 catalyzed oxidation of the iminium ion of phency- clidine to the amino enone 3 may involve the intermediacy of a dihydropyridinium species (14) with structural fea- tures 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 in- termediates 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.

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