Mechanistic Studies on the Monoamine Oxidase B Catalyzed

Oct 1, 1994 - Toxicol. 1994, 7, 740-744. Mechanistic Studies on the Monoamine Oxidase B. Catalyzed Oxidation of 1,4=Disubstituted. Tetrahydrop yridine...
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Chem. Res. Toxicol. 1994, 7, 740-744

740

Mechanistic Studies on the Monoamine Oxidase B Catalyzed Oxidation of 1,4=Disubstituted Tetrahydropyridines Simon Kuttab,$Amit Kalgutkar, and Neal Castagnoli, Jr.* Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0212 Received June 3, 1994@

Previous studies have established that l-cyclopropyl-4-phenyl-1,2,3,6-tetrahydropyridine (6) is an efficient time and concentration dependent inhibitor of the flavin containing enzyme monoamine oxidase B (MAO-B). This behavior is consistent with a proposed mechanism based inactivation pathway initiated by transfer of one of the nitrogen nonbonding pairs of electrons to the oxidized flavin cofactor to generate a n amine radical cation intermediate. Subsequent opening of the strained cyclopropylamine ring is thought to lead to a primary carbon centered radical that inactivates the enzyme by covalent modification of the flavin or a n essential active site functionality. We now have examined the MAO-B inactivator and substrate properties of 4-benzyl-l-cyclopropyl-l,2,3,6-tetrahydropyridine (11). This compound also is a time and concentration dependent inhibitor of MAO-B. Unexpectedly, however, compound 11 proved to be a n excellent MAO-B substrate. These results are discussed in terms of possible catalytic pathways for the MAO-B catalyzed oxidation of 1,4-disubstituted-l,2,3,6-tetrahydropyridines.

Introduction The flavin adenine dinucleotide (FAD)l containing monoamine oxidases (MAO)A and B catalyze the a-carbon oxidation of a variety of primary and secondary amines including the biogenic amine neurotransmitters (1-3) and various xenobiotics (4-6). It has been shown that the two forms of the enzyme are encoded by different genes located on the X-chromosome (7-9). Although the primary amino acid sequences are known, the structural features of the active sites that are responsible for the similarities and differences in their catalytic activities remain to be elucidated. Proposed mechanisms include a two-electron,polar pathway involving an intermediate substrate-flavin adduct (10). A more generally accepted view of the catalytic mechanism for MAO-B (Scheme 1) involves an initial single electron transfer (SET) step from the nitrogen nonbonding electrons of the substrate (1)to the oxidized FAD to yield an amine radical cation (2) and a flavin radical (FADH). Subsequent conversion of intermediate 2 is thought to proceed via a-proton loss to form the allylic radical 3 (pathway a), which undergoes a second one-electron transfer to yield the iminium product 4 and the reduced flavin FADH2. An alternative pathway (b) involving hydrogen atom transfer from the radical intermediate 2 to form FADHz and 4 also has been proposed (1.2). The formation of the radical cation 2 is supported by Silverman's studies on the inactivation of MAO-B by a variety of amines (12-15), all of which are thought t o form covalent adducts with the enzyme via reactive intermediates derived from the initially

* Address correspondence to this author at Virginia Tech, Department of Chemistry, 107 Davidson Hall, Blacksburg, VA 24061-0212. Present address: Department of Chemistry, Birzeit University, Birzeit, West Bank. Abstract published in Advance ACS Abstracts, October 1, 1994. Abbreviations: ESR,electron spin resonance; FAD,flavin adenine dinucleotide; GCEIMS, gas chromatography/electron ionization mass spectrometry; HP, Hewlett Packard; MAO, monoamine oxidase; MPTP, l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine; SET, single electron transfer.

*

@

Scheme 1. Proposed Pathway for the MAO-B Catalyzed Oxidation of Amines H+ FAD FADH-

H+

H+ FADH. FADHo

formed amine radical cation. Results obtained with model photochemical (16) and ESR studies (17) are consistent with this proposal. Our examination of the MA0 catalytic pathway has focused on 1,4-disubstituted-l,2,3,6-tetrahydropyridine derivatives (18-23), analogs of the Parkinsonian inducing neurotoxin l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP, 5). In addition to their value as tools to probe chemically induced neurodegenerative processes, members of this class of compounds are unique in that they are the only known cyclic tertiary amines which are substrates of MAO. As part of these studies, we have found that the MPTP analog 1-cyclopropyl-4-phenyl1,2,3,6-tetrahydropyridine(6) is an efficient concentration and time dependent inhibitor of this enzyme with kinact = 0.7min-l and KI = 182pM at 37 "C (18). The MAO-B inactivation properties of 6 may be rationalized in terms of the SET pathway (Scheme 2)) which is analogous to that described by Silverman for N-benzylcyclopropylamine and related systems (13-15, 24). Initial SET generates the unstable radical cation intermediate 7, which undergoes spontaneous ring opening (pathway a) to give the reactive primary carbon centered radical 8. Covalent bond formation between 8 and the enzyme leads to enzyme inactivation. The absence of detectable levels of the dihydropyridinium metabolite 10 under these conditions suggests that the deprotonation of 7 to form 9 (pathway b) does not compete effectively with the ring opening pathway (a). This may be a reasonable outcome

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Chem. Res. Toxicol., Vol. 7, No. 6, 1994 741

MAO-B Catalyzed Oxidation of MPTP Analogs

ployed unless otherwise noted: 125 "C for 1 min, followed by a ramp of 10 "C/min for 18 min. Normalized peak heights are reported as percentages of the base peak. Microanalyses were b performed by Atlantic Microlab, Inc. (Norcross, GA). Melting 1 points were determined using a Thomas-Hoover melting point apparatus and are uncorrected. N,iV-Bis[(ethoxycarbonyl)ethylJcyclopmpyl~ine (13). A mixture of ethyl acrylate (92.3 g, 923 mmol) and cyclopropyla amine (13.2 g, 230 mmol) in absolute ethanol (200 mL) was stirred a t room temperature for 5 days. The ethanol and excess ethyl acrylate were removed in vacuo,and the resulting yellow oil was distilled under vacuum to afford pure 13 (55 g, 92%): bp 112-113 OCA50 TOIT; 'H NMR (CDCl3) 6 4.15 (q,OCH2,4H), 2.93 (t, NCH2, 4H), 2.51 (t, NCH2CH2, 4H), 1.75 (m, NCHCHZ, 5: 1H), 1.25 (t, CH3, 6H), 0.46 (m, NCHCHz, 4H); GC/EIMS (mlz 10 8 9 6: re1 intensity) 257 (14, M+), 184 (551,170 (1001, 142 (81, 128 (101, 96 (20), 73 (18). Anal. Calcd for C13H23N04: C, 60.66; H, 9.01; N, 5.45. Found: C, 60.56; H, 8.97; N, 5.49. since cyclopropyl groups attached to a radical bearing 1-Cyclopropyl-4-piperidone (15). A solution of 13 (4.6 g, atom undergo very rapid ring opening (25). For example, 18 mmol) in 60 mL of anhydrous THF was added dropwise to a the cyclopropylmethyl carbinyl radical ring opens with suspension of NaH (1.08 g of 60% oil dispersion, 27 mmol) in a rate constant of 1.2 x lo8 s-l (26) while compounds THF. Absolute ethanol (1 mL) was added, and the resulting such as the aminyl radical derived from N-ethylcyclomixture was heated under reflux for 36 h. The solution obtained propylamine ring open, even at 135 K, at rates too rapid after adding 50%aqueous acetic acid to adjust the pH to 7 was to measure (27). extracted with ethyl acetate (4 x 30 mL). The combined extracts As part of our ongoing studies to characterize the were dried over Na2S04, and the solvent was removed in uacuo catalytic mechanism of MAO-B and its potential role in t o yield crude 14 as a reddish oil. The crude product was then the bioactivation of protoxins capable of causing neuronal heated under reflux in 60 mL of 18%aqueous HC1 for 5 h. After basification with 1 N NaOH, the product was extracted into degeneration, we have examined the interactions of this enzyme with 4-benzyl-l-cyclopropyl-1,2,3,6-tetrahydro- ethyl acetate (4 x 30 mL). The combined extracts were dried over NazS04, and the solvent was removed in uacuo to afford pyridine (11,Scheme 3). We were interested in compar15 (1.9 g, 75%) as a yellow oil: lH NMR (CDC13) 6 2.8 (t, NCHzing the inhibition properties of the 4-benzyl analog 11 CH2, 4H), 2.25 (t, NCHzCH2, 4H), 1.65 (m, NCHCH2, lH), 0.4 with those of the 4-phenyl analog 6 because of the (m, NCHCHz, 4H); GC/EIMS (mlz, re1 intensity) 139 (12, M+), significantly improved substrate properties obtained 124 (81, 111 (261, 96 (loo), 82 (661, 68 (go), 54 (64). The free when the phenyl group of MPTP (5) is substituted with base in diethyl ether (25 mL) was treated with oxalic acid (1.80 the benzyl group to give 12 (28). At 37 "C we find that g, 20 mmol) in 15 mL of diethyl ether to precipitate the oxalate, the k c a J Kvalue ~ for the MAO-B catalyzed oxidation of 5 which was recrystallized from 2-propanol-diethyl ether to afford is 1400 min-l mM-l while the corresponding value for the corresponding white crystalline oxalate salt as its mono6 3.08 (t,NCH2hydrate: mp 122-124 "C; lH NMR (MezS0-d~) 12 at 30 "Cis reported to be 2700 min-l mM-' (28). We CH2, 4H), 2.40 (t,NCH2CH2,4H), 2.15 (m, NCHCH2, lH), 0.59 anticipated that substitution of the phenyl group present (m, NCHCH2,4H); GC/EIMS (mlz,re1 intensity) 139 (12, M+), in 6 with the benzyl group present in 11would lead to a 124 (8), 111(25), 96 (go), 82 (68), 68 (loo), 54 (70). Anal. Calcd more efficient inactivator. The results are discussed in for C10H15NOgH20: C, 48.58; H, 6.93; N, 5.66. Found: C, 48.97; terms of the currently proposed mechanisms of catalysis H, 6.61; N, 5.87. and inactivation of MAO-B. 4-Benzyl-l-cyclopropyl-4-piperidinol(16). A solution of the piperidone 15 as its free base (2.4 g, 17.26 mmol) in dry Experimental Section THF was added dropwise to a solution of benzylmagnesium chloride (13mL of a 2 M solution, 26 mmol) in dry THF. After Caution: 1-Methyl-sC-phenyl-l,2,3,6-tetrahydropyridine (5)is heating under reflux for 4 h, the cooled mixture was made a known nigrostriatal neurotoxin and should be handled using strongly basic by the careful addition of 1 N NaOH and then disposable gloves in a properly ventilated hood. Detailed was extracted into diethyl ether (3 x 30 mL). Workup of this procedures for the safe handling of MPTP have been reported extract afforded a thick orange oil that was dissolved in (29). anhydrous diethyl ether and treated with an ethereal solution Chemistry. Solvents and reagents were obtained from (20 mL) of oxalic acid (1.69 g, 28 mmol) t o yield a hygroscopic MPTP, used to monitor the inactivation commercial sources. yellow solid. This product was recrystallized from acetonitrileof MAO-B, was purchased from Research Biochemicals Inc. ethyl acetate to afford 16 (4.2 g, 76%) as its oxalate salt mp (12) (Natick, MA); 4-benzyl-l-methyl-l,2,3,6-tetrahydropyridine and the perchlorate salt of the 4-benzyl-l-methyl-l,2-dihydro- 70-71 "C; 1H NMR (Me2SO-ddCDCls)6 7.2 (m, ArH, 5H), 3.37 (m, NCH2CH2,4H) 2.67 (8, ArCH2,2H), 2.60 (m, NCHCH2, lH), pyridinium species 22 (23)were prepared according to the cited 1.71 (m, piperidine NCH&H,,H,, 2H), 1.54 (d, NCH2CHe&?,, literature. THF and diethyl ether were distilled from LiAlH4. 2H), 0.90 (bs, cyclopropyl ethylene protons trans to N, 2H), 0.67 Synthetic reactions were conducted under a dry nitrogen (m, cyclopropyl ethylene protons cis to N, 2H); GC/EIMS (mlz, atmosphere. UV-vis absorption spectra were recorded on a re1 intensity) 231 (18, M+), 202 (221, 140 (20), 97 (301, 91 (1001, Beckman DU Series 50 spectrophotometer and lH NMR spectra 82 ( 9 9 , 70 (40). Anal. Calcd for C17H~3N05:C, 63.52; H, 7.22; on a Bruker WP 270 or 200 MHz spectrometer. Chemical shifts N, 4.36. Found: C, 63.38; H, 7.27; N, 4.34. ( 6 ) are reported in parts per million (ppm) relative to the Oxalate Salt of l-Cyclopropyl-4-benzy1-1,2,3,6-tetrahyinternal standard tetramethylsilane or, with CD3OD as solvent, dropyridine (11). The above oxalate salt of 16 (0.7 g, 2.18 Gas chromatogsodium 3-(trimethylsilyl)propionate-2,2,3,4-d4. "01) andp-tolucnesulfonic acid (0.51 g, 2.7 mmol) were heated raphy/electron ionization mass spectrometry (GCEIMS) was under reflux in dry benzene (70 mL) for 20 h, and the water performed on a Hewlett Packard (HP) 5890 capillary GC coupled formed in the reaction was separated by azeotropic distillation to a HP 5970 mass selective detector. Data were acquired using using a Dean Stark trap. The reaction mixture was cooled and an HP 5970 MS ChemStation. The capillary column used in extracted with 1N NaOH (3 x 20 mL) followed by H2O (2 x 20 all cases was an HP-1 (12.5 m x 200 pm x 0.33 pm film mL). The organic layer was dried (NazSOd, filtered, and thickness). The following GC temperature program was em-

Scheme 2. Potential MAO-B Catalyzed Pathways for Cyclopropylamine 6

r

-

QA

Kuttab et al.

742 Chem. Res. Toxicol., Vol. 7, No. 6, 1994

Scheme 3. Synthetic Pathway to 4-benzyl-l-cyclopropyl-l,2,3,6-tetrahydropyridine (11)

13

15

14

A 16

RR

= 17 =A A A z3

11: R 12: R = CH3

Scheme 4. Possible Reaction Pathways for the MAO-B Catalyzed Oxidation of 1-C yclopropyl-4-benzyl-1,2,3,64etrahydropyidine(11) H+

J -

b

. concentrated to 50% of the original volume. Treatment of this concentrate with oxalic acid (0.45 g, 5 mmol) in 15 mL of diethyl ether provided a solid that was recrystallized from acetone to afford a mixture of 11 and the isomeric l-cyclopropyl-4-(phenylmethy1ene)piperidine(1'7)as white crystals. lH N M R and GC/ EIMS analyses indicated a product ratio of 2.5:l (11:1'7). Several recrystallizations from acetone gave essentially pure 11: mp 148-149 "C; 'H NMR (MezSO-d6)6 7.15-7.31 (m, h H , 5H), 5.43 (bs, NCHzCH, lH), 3.56 (bs, NCHzCH, 2H), 3.30 (s, ArCH2, 2H), 3.13 (t, NCHZCHZ,2H), 2.49 (m, NCHCHZ, lH), 2.12 (m, NCHzCH2, 2H), 0.62-0.77 (m, NCHCHz, 4H); GC ( t ~ = 9.7 min)/EIMS of free base (mlz,re1 intensity) 213 (581, 198 (100, M - 15), 155 (81, 122 (lo), 106 (30), 91 (65),68(25). Anal. Calcd for C17HzlN04: C, 67.32; H, 6.93; N, 4.62. Found: C, 67.24; H, 6.97; N, 4.62. The isomeric 17 was obtained in partially purified form: lH NMR 7.15-7.33 (m, ArH, 5H), 6.40 (s, ArCH, lH), 3.02 (t, NCHz, 4H), 2.58 (t, NCHzCHz, 4H), 2.40 (m, NCHCHz, lH), 0.62-0.70 (m, NCHCHz, 4H); GC ( t =~ 10 min)/EIMS of free base (m/z,re1 intensity) ( t R = 10.0 min) 213 (25), 198 (40), 184 (20), 155 (201, 141 (lo), 115 (251, 91 (lo), 70 (100).

Enzymology. MAO-B was isolated from bovine liver mitochondria following the method of Salach and Weyler (30). The activity was determined spectrophotometrically at 30 "C on a Beckman DU 50 spectrophotometer using 5 mM MPTP as substrate and recording initial rates (120 s ) of formation of the dihydropyridinium metabolite (Imm = 343 nm) as described previously (18). The final enzyme concentration was calculated to be 8 nmol/mL. The MAO-B inactivating properties of 11 were examined following published procedures (19).Due to its good substrate activity, attempts to obtain useful KI and kinact values were unsuccessful. In preliminary experiments, the potential MAO-B substrate properties of 11 and 12 were examined by recording repeated scans (500-250 nm) of a 500 pM solution of each tetrahydropyridine in the presence of 0.08 pM MAO-B. These scans established the I,, value for both of the resulting dihydropyridinium metabolites 21 and 22 (see Scheme 4) as 296 nm. Subsequent kinetic studies employed concentrations of 11 of 500, 250, 125, 100, and 60 pM. A 490 pL aliquot of each solution,

21: R = 22: R = CH,

A

23: R = 24: R = CH3

pre-equilibrated to 30 "C, was added to a sample cuvette, which was then placed in the spectrophotometer that was maintained at 30 "C. m e r a 30 s equilibration period, 10 pL of the MAO-B preparation (final concentration 0.16 pM) was added and the initial rates of oxidation were determined by monitoring the increment in absorbance of the expected dihydropyridinium metabolite 21 a t 296 nm formed during the enzyme catalyzed reactions every 3 s for 2 min.

Results and Discussion Chemistry. The synthesis of 4-benzyl-1-cyclopropyl1,2,3,64etrahydropyridine(11,Scheme 3) was achieved via condensation of ethyl acrylate with cyclopropylamine, which afforded the bis-Michael adduct 13. Treatment of 13 with NaH in THF gave the cyclic P-ketoester 14 which, when heated in 18% HC1, provided l-cyclopropyldpiperidone (15). Reaction of 16 with benzylmagnesium chloride afforded the tertiary carbinol 16,which underwent acid catalyzed dehydration to yield a mixture of isomeric olefins in an approximate ratio of 2.5:l as determined by G C N S and 'H NMR analysis. Repeated crystallizations of the corresponding mixture of oxalate salts from acetone led to the isolation of the major isomer, which proved to be the desired tetrahydropyridine 11. The characteristic signals for the C-5 olefinic proton (bs at 5.43 ppm) and the benzylic methylene protons (s at 3.30 ppm) readily distinguished this endocyclic olefin from 17,which displayed its proton olefinic signal at 6.40 ppm and no benzylic proton signals. Enzymology. As anticipated, compound 11 proved to be an efficient inhibitor of MAO-B. The kinetics of inhibition were examined by assessing the rate of loss of enzyme activity with timed aliquots of the incubation mixture (as measured by the initial rates of oxidation of 5 mM MPTP) at several inhibitor concentrations. Although it was clear that 11 was both a concentration and time dependent inhibitor of MAO-B, attempts to determine the KI and kinactfor the inactivation were unsuc-

MAO-B Catalyzed Oxidation of MPTP Analogs

cessful since plots of In % remaining enzyme activity against time were not linear. The possibility that the nonlinear kinetic behavior observed in these activation studies was a consequence of the MAO-B catalyzed substrate properties of 11 was investigated by scanning a 500 pM solution of 11in the presence of 0.16 pM MAO-B. In fact, we did observe the time dependent formation of a species absorbing maximally a t 296 nm, which we tentatively assigned to the dihydropyridinium metabolite 21 (see Scheme 4). Formation of this species was completely blocked when the enzyme was pretreated with M deprenyl, a potent and selective inactivator of MAO-B (31). The absorption maximum of the incubation mixture shifted slowly from 296 to 342 nm during the course of the 60 min study. Upon adjusting the pH to 1, however, the A- reverted to 296 nm. At pH 10, the 342 nm absorbing chromophore reappeared. The spectroscopic behavior of incubation mixtures containing MAO-B and the corresponding 1-methyl analog 12 was examined as a model for the proposed transformation. The resulting dihydropyridinium metabolite 22 also absorbed maximally at 296 nm but was stable under the incubation conditions. This chromophore, however, did shift to 342 nm at pH 10 and back to 296 nm at pH 1. Furthermore, the synthetic perchlorate salt of the N-methyldihydropyridinium species 22 displayed identical UV spectral behavior as that observed for the enzyme generated metabolite. These results lead us to conclude that the MAO-B generated products derived from the 4-benzyltetrahydropyridine substrates 11 and 12 are the corresponding dihydropyridinium species 21 and 22, respectively, and that these 4-benzyl-2,3-dihydropyridiniummetabolites (Am= 296 nm) behave as weak carbon acids which may undergo reversible conversion to the corresponding 1,2dihydropyridine free bases 23 and 24 (A, 342 nm). The N-cyclopropyldihydropyridinium analog 21 may be a somewhat stronger acid than the N-methyl analog 22, which would account for its conversion to the conjugate base 23 at a lower pH than that observed with the N-methyl analog 24. Kinetic analyses of the MAO-B catalyzed oxidation of 11were performed at 30 "C by initial rate measurements of the formation of the dihydropyridinium metabolite 21, which was monitored at 296 nm.2 Linear semilog plots of initial rates vs concentration were obtained, and the resulting data were analyzed by a double reciprocal plot (Figure 1). The KM and kat were found to be 414 pM and 637 min-I, respectively. The value for k&/KM (1500 min-' mM-') compares favorably with the corresponding values of the N-methyl analog 22 (2700 min-l mM-'1 and MFTP (1400 min-' mM-'). The exceptionally good substrate properties of 11were unexpected. Silverman has reported that the MAO-B catalyzed oxidation of N-benzylcyclopropylamineleads to both benzaldehyde formation and enzyme inactivation with a partition ratio of about 1(32). Although we could not determine the partition ratio for the MAO-B catalyzed oxidation of 11, it is clear that it must be a relatively large number. For example, at KM the rate of product formation (v = (Eo)(kcaJ(I -I- &/[SI} = 2 Attempts to synthesize the dihydropyridinium species 21 to establish ita molar extinction coefficient were unsuccessful. Consequently, we have estimated the k,,t for this reaction by assuming a molar extinction coefficient at 296 nm of 5500 M-l, the experimentally determinedvalue for the synthetic 1-methyldihydropyridiniumspecies 22 (23).

Chem. Res. Toxicol., Vol. 7, No. 6,1994 743

0.012-

do05

0:005

0:015

1411( PM)1

Figure 1. Double reciprocal plot for the MAO-B catalyzed oxidation of 4-benzyl-l-cyclopropyl-l,2,3,6-tetrahydropyridine (11) at 30 "C.

(637)(0.16)/2 = 51 pmol/(Lmin). Consequently, after 2 min, when the rate of product formation is still linear, the ratio of the number of moles of product formed to the total number of moles of enzyme present is (2 x 51)/0.16 = 639. The rates of ring opening of cyclopropylamine radical cations such as 18 have not been estimated experimentally, but based on the behavior of many a-radical bearing cyclopropyl systems, we had anticipated that the cyclopropylamine radical cation 18 would favor the ring opening pathway (a) over the proton loss pathway (b). It is possible that the favored partitioning of 18 to yield 20 rather than 19 may be explained by conformational constraints that would lead to poor overlap of the half-filled p-orbital on the nitrogen radical cation and the p-type orbitals of the cyclopropyl group present in 18. This restriction should retard the ring opening reaction leading to enzyme inactivation and therefore would favor the proton loss step leading to the dihydropyridinium product 21. A n alternative pathway that could account for the good substrate properties of 11 involves a direct hydrogen atom transfer reaction (pathway c, Scheme 4). This pathway relies on homolytic cleavage of the relatively weak allylic carbon-hydrogen bond which might be effected by the enzyme bound radical reported to be present in MAO-B purified from beef liver (33). Furthermore, results from kinetic deuterium isotope effect studies have prompted Edmondson to consider an analogous homolytic cleavage of a benzylic carbon-hydrogen bond cleavage as a possible pathway for the MAO-B catalyzed oxidation of benzylamine based substrates (34). Additional insights into the structural features that contribute to the substrate and inactivator properties of 1,4-disubstituted-l,2,3,6-tetrahydropyridines are being pursued in an effort to gain a better understanding of the mechanism(s1 by which these compounds are processed by MAO-B.

Acknowledgment. This work was supported by the National Institute of Neurological and Communicative Disorders and Stroke Grant NS 28792 and the Harvey W. Peters Research Center for the Study of Parkinson's Disease and Disorders of the Central Nervous System.

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