Probing the Mechanism of Bioactivation of MPTP Type Analogs by

Chem. Res. Toxicol. 1995, 8, 703-710

703

Probing the Mechanism of Bioactivation of MPTP Type Analogs by Monoamine Oxidase B: Structure-Activity Studies on Substituted 4=Phenoxy-,4-Phenyl-,and 4-Thiophenoxy - I-cyclopropyl-1,2,3,6=tetrahydropyridines John M. Rimoldi,' You-Xion Wang,' Sandeep K. Nimkar,+ Simon H. Kuttab,; Andrea H. Anderson, Heidi Burch,? and Neal Castagnoli, Jr.*>'

9

Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24861-0212, and Department of Chemistry, Birzeit University, Birzeit, West Bank Received December 27, 1994@

Previous studies have shown that 4-benzyl-l-cyclopropyl-1,2,3,6-tetrahydropyridine is a n excellent monoamine oxidase B (MAO-B) substrate (K,,JKM= 1538 min-l mM-') although the corresponding 4-phenyl analog displays MAO-B inactivating properties only. This behavior led us to speculate that the pathway for the MAO-B catalyzed oxidation of these tetrahydropyridines may not necessarily proceed via a n initial single electron transfer step as proposed by others but rather through an initial a-carbon hydrogen atom abstraction step. In the present studies we have examined the interactions of various 4-phenoxy-, 4-phenyl-, and 4-thiophenoxyl-cyclopropyl-1,2,3,6-tetrahydropyridine derivatives, some of which bear substituents on the phenyl ring. The 4-thiophenoxy- and all of the 4-phenoxytetrahydropyridinederivatives proved to be substrates but not inactivators of MAO-B, while several of the 4-phenyltetrahydropyridine derivatives were inactivators but not substrates. A case of particular interest was l-cyclopropyl4-(2-methylphenyl)-l,2,3,6-tetrahydropyridine, which displayed only substrate properties. The results are discussed in terms of two catalytic pathways, one of which involves partitioning of the proposed cyclopropylaminyl radical cation intermediate between cyclopropyl ring opening and proton loss while the second involves partitioning of the parent amine between an initial single electron transfer step, leading to cyclopropylaminyl radical cation formation and enzyme inactivation, and an initial a-carbon hydrogen atom abstraction step, leading to an allylic radical and dihydropyridinium product formation.

Introduction The flavoenzymes monoamine oxidase (MA0)l A and B catalyze the oxidative deamination of various neurotransmitters and related acyclic primary and secondary amines ( I ) . The parkinsonian inducing nigrostriatal neurotoxin MPTP [l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (111 and its structurally related analogs provide a unique opportunity to examine the catalytic pathways for these enzymes because, to the best of our knowledge, they belong to the only class of cyclic tertiary amines known to be good MA0 substrates. This reaction also is of neurotoxicological interest since the MAO-B catalyzed two-electron oxidation of MPTP leads to the formation of a dihydropyridinium intermediate [MPDP+(2)1, which autoxidizes to the pyridinium species MPP+ (31,the ultimate nigrostriatal toxin (Scheme 1) ( 2 ) . Although the mechanism of MA0 catalysis has not been unambiguously established, the reaction pathway (Scheme 2) is thought to proceed via an initial single electron transfer (SET) step from the lone pair electrons of the amine substrate (4) to the oxidized flavin (FAD)

* Address correspondence to this author at Virginia Tech, Depart-

ment of Chemistry, 107 Davidson Hall, Blacksburg, VA 24061-0212. Virginia Polytechnic Institute and State University. Birzeit University. @Abstractpublished in Advance ACS Abstracts, May 15, 1995. Abbreviations: MAO, monoamine oxidase; MPTP, l-methyl-4phenyl-l,2,3,64etrahydropyridine; SET, single electron transfer; FAD, flavin adenine dinucleotide; ESR, electron spin resonance; THF, tetrahydrofuran; GC-EIMS, gas chromatography-electron ionization mass spectrometry; HR-EIMS, high-resolutionelectron ionization mass spectrometry.

*

+

0893-228x/95/2708-0703$09.00/0

Scheme 1. Oxidation of MPTP by Monoamine Oxidase R

Ph

I

I

I

CH3 1: R = Ph (MPTP) 13: R=CH2Ph

CH3

CH3

(MPDP+)

3 (MPP')

Scheme 2. MA0 Catalyzed Oxidation of Amines Ha

7

I

I

4

I

fb7

FADH* FADH2

to yield an aminyl radical cation ( 5 ) and a flavin radical (FADH)(3). Subsequent processing of the highly reactive intermediate 5 is proposed to proceed via aproton loss to form the radical 6 (pathway a) which undergoes a second one-electron transfer t o yield the iminium product 7 and the reduced flavin FADH2. This SET mechanism is supported by results from studies on

0 1995 American Chemical Society

Rimoldi et al.

704 Chem. Res. Toxicol., Vol. 8, No. 5, 1995 Scheme 3. MAO-B Catalyzed Oxidation of l-Cyclopropyl-l,2,3,6-tetrahydropyriridines 8 and 14 R R R R

LA-().-@ N

A 8: R = P h 14: R = CHpPh

A 9 15

A

A

11 16

12 17

10: R = P h 18: R = CHzPh

cyclopropylamine containing mechanism-based MAO-B inactivators and model photochemical (4)and ESR studies (5). An alternative pathway (b)invoking hydrogen atom transfer from the radical intermediate 6 to form FADH2 and product 7 also appears to be consistent with the available experimental evidence (6). A third catalytic pathway ( c ) involving hydrogen atom transfer from the amine substrate to yield the carbon centered radical intermediate 6 directly has been suggested by Edmondson on the basis of deuterium isotope effects observed with a series of benzylamine analogs (7). Previously, we (8-10) and others (11-13) have examined the MA0 substrate and inhibitor properties of a variety of MPTP analogs in which the nature of the substituents at C-4 and N-1 vary. Substituent changes at C-4 have been shown to alter substrate properties [when N(l)=CHB] as manifested by variance in KM and ItCat values. With the exception of the cyclopropyl group (see below), reported modifications of the N-methyl substituent invariably lead to diminished substrate properties (14).Although the data are scant, functional group modifications around the periphery of the tetrahydropyridine ring system give rise to analogs devoid of substrate properties (15,161,suggesting adverse steric interactions between substrate and enzyme. As part of our studies we have shown that l-cyclopropy1-4-phenyl-l,2,3,64etrahydropyridine (8)is a timeand concentration-dependent inhibitor of MAO-B (Itma& = 0.7 min-l, KI = 0.182 mM at 37 "C), a finding which is consistent with the proposed SET pathway (17).The cyclopropyl aminyl radical cation 9 generated from the SET should ring open spontaneously to afford the primary carbon centered radical 10,the species which could mediate the inactivation of the enzyme (Scheme 3). The fact that no detectable levels of the dihydropyridinium product 12 were observed under these conditions argues that the rate of cylcopropyl ring opening must be faster than a-deprotonation to form 11, the precursor to 12. These findings are consistent with the immeasurably fast rates of ring opening of cyclopropylaminyl radical cations derived from tertiary N-alkylcylclopropylamines(18)and the behavior of N-benzylcyclopropylamines (19)and related compounds (20)that act as MAO-B inactivators.

In an effort to design a more efficient tetrahydropyridine based inactivator of MAO-B, we chose to exam(14) ine 4-benzyl-l-cyclopropyl-l,2,3,6-tetrahydropyridine since the corresponding 4-benzyl-1-methyl-1,2,3,6-tetrahydropyridine (13)is a better substrate than its C-4phenyl congener MPTP (211. Compound 14 proved to be a weak time- and concentration-dependent MAO-B inhibitor. To our surprise, however, 14 displayed excellent MAO-B substrate properties. The K,,JKMvalue (1538 min-l mM-', 37 "C) for the conversion of 14 to the corresponding dihydropyridinium metabolite 17 is comparable to that observed for the conversion of MPTP to MPDP+ (1400 min-l mM-l, 37 "C) (22). These results suggested that the partitioning of the proposed cyclopropylaminyl radical cation 15 must favor the a-deprotonation pathway, leading to the allylic radical 16 and product 17, rather than the ring opening pathway, leading to the primary radical 18 and enzyme inactivation. In an effort to assess possible steric and stereoelectronic factors which might contribute to the unexpected MAO-B substrate properties of 1-cyclopropyl-1,2,3,6tetrahydropyridine derivatives such as 14, we have synthesized a series of structurally related analogs substituted at C-4 with aryl, aryloxy, and thiophenoxy groups and have examined their interactions with MAOB.

Experimental Section Caution! 1-Methyl-l-pheny1-1,2,3,6-tetrahydropyridine (1) is a Known nigrostriatal neurotoxin and should be handled using disposable gloves i n a properly ventilated hood. Detailed procedures for the safe handling of MPTP have been reported (23). General Methods. All nonaqueous reactions were carried out using glassware that had been flame dried under an inert atmosphere of nitrogen. Diethyl ether and tetrahydrofuran (THF) were distilled from sodium benzophenone ketyl, and CH2Clz and CH&N were distilled from CaHz. Thionyl chloride was distilled from linseed oil (5% v/v) prior to use. y-Pyrone and 1-cyclopropyl-4-piperidone were prepared according to the cited literature. All reagents (with the exception of solvents) were purchased from Aldrich Chemical Co (Milwaukee, WI). UVvis absorption spectra were recorded on a Beckman DU Series 50 spectrophotometer. Proton NMR spectra were recorded on a Bruker WP 270-MHz spectrometer. Chemical shifts ( 6 ) are reported relative to tetramethylsilane as an internal standard. Spin multiplicities are given as s (singlet), bs (broad singlet), d (doublet), t (triplet), q (quartet), or m (multiplet). Coupling (J) values are given in hertz (Hz). Gas chromatography-electron ionization mass spectrometry (GC-EIMS) was performed on a Hewlett Packard 5890 GC fitted with an HP-1 capillary column (20 m x 200 pm x 0.33 pm film thickness) which was coupled to a Hewlett Packard 5870 mass-selective detector. Data were acquired using a n HP 5970 ChemStation. Normalized peak heights are reported as a percentage of the base peak. Highresolution electron ionization mass spectrometry (HR-EIMS) was performed on a VG 7070 HF instrument using methane as the reagent gas. Melting points were performed on a ThomasHoover melting point apparatus and are uncorrected. Microanalyses were performed by Atlantic Microlab, Inc.,Norcross, GA. 1-Cylclopropyl-4-pyridone(20). To y-pyrone (6.lg, 63.5 mmol) in 30 mL of water was added cyclopropylamine (4.35 g, 76.2 mmol). The solution was heated under reflux for 3 h and cooled to room temperature. After saturating the aqueous layer with KzC03, the product was extracted with CHC13 (6 x 30 mL). The combined extracts were dried (Na2S04) and concentrated i n vacuo to yield the crude product as a yellow oil which was flash-vacuum filtered over basic alumina with 10% 2-propanol/

Mechanistic Studies on MAO-B Catalysis

Chem. Res. Toxicol., Vol. 8, No. 5, 1995 705

2H, NCHzCH), 3.11 (t, J = 5.8, 2H, NCH~CHZ), 2.34 (m, 2H, 90% CHzC12. Recrystallization from cold ethyl acetate and NCH~CHZ), 2.25 (m, l H , NCH), 0.61 (m, 4H, NHCH2). Anal. filtration under a Nz atmosphere yielded 4.2 g (60%) of 20 a s Calcd for C16H17C12N05: C, 51.35; H, 4.58; N, 3.74. Found: C, pale yellow hydroscopic needles: mp 62-64 "C; GC (tempera51.48; H, 4.65; N, 3.70. ture program: 100 "C for 1 min, then 25 "Clmin up to 290 "C; t R 7.55 min)-EIMS m l z (%), 135 (65, M+), 107 (251, 106 (loo), 4-(2,4-Dichlorophenoxy)-l-cyclopropyl1,2,3,6-tetra80 (28), 67 (151, 54 (40); 'H NMR (CDC13) 6 7.45 (dd, J = 7.7, hydropyridins(CO0H)z (23d). Recrystallized from CH30H1.8,2H, NCH-CH), 6.32 (dd, J = 7.7, 1.8,2H, NCH=CH), 3.41 diethyl ether (0.68 g, 58%): mp 156-157 "C; 'H NMR (DMSO(m, l H , NCH), 0.98-1.12 (m, 4H, NCHCHz). Due to the d6) 6 7.70 (d, J = 2.0, l H , PhH), 7.40 (dd, J = 8.7,2.0, l H , PhH), hydroscopic nature of the pyridone, elemental analysis was 7.20 (d, J = 8.7, lH, PhH), 4.73 (bs, lH, NCHzCH), 3.38 (bs, performed on the corresponding hydrochloride salt (mp 1842H, NCHzCH), 3.11 (t, J = 5.9, 2H, NCHZCHZ),2.40 (m, 2H, 186 "C): Anal. Calcd for CBHgNO.HC1: C, 55.99; H, 5.87; N, NCHZCH~), 2.24 (m, l H , NCH), 0.57-0.63 (m, 4H, NCHCHz). 8.16. Found: C, 56.09; H, 5.92; N, 8.13. Anal. Calcd for C16H17C12N05: c, 51.35; H, 4.58; N, 3.74. Found: C, 51.22; H, 4.70; N, 3.67. 4-Chloro-l-cyclopropylpyridinium Chloride (21). A mixl-Cyclopropyl-4-(4-nitrophenoxy)-1,2,3,6-tetrature of thionyl chloride (12 mL, 150 mmol) and pyridone 20 (2.2 hydropyridine.(COOH)z (23e). Recrystallized from CH&N g, 16.3 mmol) was heated under reflux for 4 h. The light brown (0.53 g, 48%): mp 173-174 "C; GC (temperature program: 100 residue obtained following removal of the excess thionyl chloride "C for 1min, then 25 "Clmin to 290 "C; t~ 7.33 mid-EIMS m l z by rotary evaporation was dissolved in CH2C12, and the crude (%) 260 (25, M+), 245 (loo), 199 (171,138 (131,106 (25), 68 (60); product was precipitated out with the addition of diethyl ether. 'H NMR (DMSO-d6)8.23 ( d d , J = 9.3, 2.2, 2H,PhH), 7.22 (dd, Recrystallization from anhydrous CH3CN yielded 2.3 g (75%) 9.3,2.2,2H,PhH), 5.36 (bs, lH,NCHzCH),3.43 (bs,2H,NCH2of 21 as hygroscopic, pale yellow needles: mp 218-220 "C dec; CH), 3.10 (t,J = 5.8, 2H, NCHZCHZ),2.33 (m, 2H, NCHZCHZ), 'H NMR (DMSO-&) 6 9.16 (d, J = 7.0, 2H, NCH=CH), 8.31 (d, 2.19 (m, l H , NCH), 0.60 (m, 4H, NCHCHz). Anal. Calcd for J = 7.0, 2H, NCH=CH), 4.38 (m, l H , NCH), 1.41 (m, 2H, NCHCH2), 1.25 (m, 2H, NCHCHZ). Anal. Calcd for C~~HI~NZ C,O54.86; ~ : H, 5.18; N, 8.00. Found: C, 54.79; H, 5.22; N, 8.08. C~HgCl2N(1/6)H20:C, 49.77; H, 4.87; N, 7.25. Found: C, 49.80; H, 5.04; N, 7.18. l-Cyclopropyl-4-thiophenoxy-l,2,3,6-tetrahydropyridine.(COOH)z (230. Recrystallized from CH3CN (0.52 g, General Procedure for Synthesis of the Oxalate Salts of the l-Cyclopropyl-4-phenoxy-l,2,3,6-tetrahydrop~- 52%): mp 148-149 "C; GC (temperature program: 100 "C for 1 min then 20 "Clmin, to 290 "C; t~ 8.46 min)-EIMS m l z (%) dine Derivatives 23a-f. A solution of 4-chloro-1-cyclopropy231 (50, M+), 216 (loo), 198 (8), 147 (91, 122 (221, 106 (191, 80 lpyridinium chloride (21,3.13 mmol), the appropriate arenol (17), 53 (30);'H NMR (DMSO-&) 7.33 (m, 5H, PhH), 5.88 (bs, (3.44 mmol), and triethylamine (4.70 mmol) in 30 mL of l H , NCHzCH), 3.52 (bd, J = 2.8, 2H, NCHzCH), 3.08 (t, J = anhydrous CH3CN was stirred a t room temperature for 24 h. 5.8,2H, NCH2CH2),2.28 (m, 3H, NCH2CH2 and NCH), 0.61 (m, The reaction mixture then was evaporated to dryness, and the 4H, NCHCH2). Anal. Calcd for C16H1gN04S: C, 59.8; H, 5.96; residue in a stirred solution of CH30H (20 mL) was treated a t N, 4.36. Found: C, 59.6; H, 5.92; N, 4.47. 0 "C portionwise with NaBHp (1.5 g, 4.1 mmol). After stirring an additional 30 min at room temperature, the solvent was General Procedure for the Synthesis of 1-Cyclopropylremoved under vacuum and the residue in CHzClz was washed 4.phenyl-l,2,3,6-tetrahydropyridinew(COOH)~(26a-e). with dilute aqueous NaHC03. The organic layer was washed Stirred solutions of the appropriate substituted bromobenzene successively with water and brine, dried over NazS04, and (10 mmol) and Mg turnings (11 mmol) in anhydrous diethyl evaporated to yield the crude product. "he oxalate salt was then ether (20 mL) were treated with a crystal of IZ to initiate the prepared by adding an ethereal solution of oxalic acid (1.5 equiv) reaction. After stirring a t room temperature for 1 h, the to the tetrahydropyridine in ether. The analytic sample was Grignard reagents were added dropwise to a solution of l-cyrecrystallized from the indicated solvent system. clopropyl-4-piperidone (20,10.5 mmol) in anhydrous diethyl l-Cyclopropyl-4-phenoxy-1,2,3,6-tetrahydropyridine~ ether (20 mL) a t room temperature. The reaction mixtures were stirred for an additional 1h a t room temperature and then were (COOH)2(23a). Recrystallized from CH3CN (0.52 g, 55%): mp treated with saturated aqueous NH4Cl followed by 10% HC1 (to 148-150 "C; GC (temperature program: 100 "C for 1min, then pH l), and the resulting solutions were washed with diethyl 25 "Clmin up to 290 "C; t~ 7.35 mid-EIMS mlz (%) 215 (30, M+), ether. The aqueous phases were basified with 40% NaOH and 200 (loo), 138 (lo), 122 (25), 94 (47), 77 (371, 68 (52); 'H NMR extracted with CHzClz to afford the crude piperidinols which, (DMSO-&) 6 7.36 (t, J = 7.7, 2H, PhH), 7.11 (t, J = 7.3, l H , without purification, were heated under reflux for 15-20 h in PhH), 7.00 (d, J = 7.7, 2H, PhH), 4.80 (bs, l H , NCHzCH), 3.38 HC1-HOAc (1:3 vlv, 30 mL). These reaction mixtures were (bs, 2H, NCHzCH), 3.10 (t, J = 5.9, 2H, NCH2CH21, 2.36 (bs, cooled to room temperature, basified with 40% NaOH, and then 2H, NCH2CH2), 2.24 (m, l H , NCH), 0.61 (bs, 4H, NCHCHz). extracted with CH2Clz to afford the crude tetrahydropyridine Anal. Calcd for C16H19N05: C, 62.94; H, 6.27; N, 4.59. Found: products which were characterized as the corresponding oxalate C, 62.75; H, 6.33; N, 4.51. 4-(3-Chlorophenoxy)-l-cyclopropyl-1,2,3,6-tetra- salts (prepared in diethyl ether). Recrystallization from an appropriate solvent afforded pure products. hydropyridine*(COOH)2(23b). Recrystallized from CH30H4-(4-Chlorophenyl)-l-cyclopropyl1,2,3,6-tetrahydropydiethyl ether (0.62 g, 59%): mp 147-148 "C; GC (temperature ridine (26a). Recrystallized from CH~CN-HZOfollowed by program: 100 "C for 2 min, then 25 "Clmin to 275 "C; t~ 7.86 sublimation (75%): mp 115-116 "C; GC (temperature promin)-EIMS mlz (%) 251 (11,M+, 37Cl),249 (36, M+, 35Cl),236 gram: 125 "C for 1 min, then 10 "Clmin up to 290 "C; t~ 5.81 (351, 234 (loo), 158 (8), 128 (15), 106 (261, 68 (35); lH NMR mid-EIMS m l z (%) 235 (13, M+, 37C1),233 (40, M+, 35C1),218 (DMSO-&) 6 7.37 (t, J = 8.0, l H , PhH), 7.17 (d, J = 7.4, l H , (loo), 177 (lo), 149 (81, 128 (251, 115 (201, 75 (81, 54 (38); 'H PhH), 7.10 (d, J = 1.9, l H , PhH), 7.01 (d, J = 8.2, lH, PhH), NMR (oxalate salt; DMSO-&) 6 7.44 (d, J = 8.7,2H, PhH), 7.40 4.98 (bs, l H , NCHzCH), 3.47 (bs, 2H, NCH2CH), 3.16 (t, J = (d, J = 8.7, 2H, PhH), 6.19 (bs, lH, NCHzCH), 3.59 (bd, 2H, 5.9, 2H, NCH~CHZ), 2.38 (m, 3H, NCHzCHz, NCH), 0.61-0.68 NCH2CH), 3.15 (t, J = 5.8, 2H, NCHZCH~),2.58 (m, 2H, C,O56.56; ~ : H, (m, 4H, NCHCH2). Anal. Calcd for C I ~ H ~ B C ~ N NCHzCHz), 2.31 (m, l H , NCH), 0.64 (m, 4H, NCHCHz). Anal. 5.34; N, 4.12. Found: C, 56.66; H, 5.38; N, 4.10. 4-(3,S-Dichlorophenoxy)-l-cyclopropyl-l,2,3,6-tetra- Calcd for C14H16CN C, 71.94; H, 6.9; N, 5.99. Found: C, 72.22; H, 7.01; N, 5.87. hydropyridine(CO0H)z (23~).Recrystallized from CH30Hl-Cyclopropyl-4-(4-trifluoromethyl)phenyll-l,2,3,6diethyl ether (0.83 g, 72%): mp 167-168 "C; GC (temperature tetrahydropyridine(CO0H)z (26b). Recrystallized from CH3program: 100 "C for 2 min, then 25 "Clmin up to 275 "C; t~ OH-diethyl ether (0.67 g, 32%): mp 179-180 "C; GC (temper8.54 mid-EIMS m l z (%) 285 (4, M+, 35,37C1),283 (7, M+, 35,35C1), ature program: 100 "C for 2 min, then 25 "C/min to 275 "C; t~ 270 (lo), 268 (23), 147 (9), 145 (141, 111(291, 110 (291, 106 (331, 6.97 mid-EIMS m l z (%) 267 (36, M+),252 (loo), 224 (131, 191 68 (100); 'H NMR (DMSO-&) 6 7.33 (d, J = 1.7, l H , PhH), 7.11 (13), 177 (lo), 159 (201,128 (22), 54 (58); 'H NMR (DMSO-&) 6 (dd, J = 1.2, 0.51, 2H, PhH), 5.13 (bs, l H , NCHzCH), 3.44 (bs,

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2.32 (s, 3H, CH3), 1.50 and 0.61 (m, 4H, NCHCH2). A solution of trifluoroacetic anhydride (0.41 g, 1.95 mmol) in anhydrous CHC13 (10 mL) was added dropwise to a solution of the crude N-oxide 28 (0.09 g, 0.39 mmol) in CHC13 (10 mL) at room temperature with stirring under nitrogen. The reaction l-Cyclopropyl-4-(4-methoxyphenyl)-l,2,3,6-tetra-was complete after 20 min at room temperature, and the reaction mixture was evaporated to give the dihydropyridinium hydropyridine(CO0H)z (26c). Recrystallized from CH30H350 nm; GC (temperature intermediate 29: TJV (CHC13) A,, diethyl ether (0.947 g, 37%): mp 184-185 "C; GC (temperature program: 100 "C for 2 min, then 25 "Clmin up to 275 "C; t~ program: 100 "C for 2 min, then 25 "Clmin up to 275 "C; t~ 6.67 min)-EIMS of the dihydropyridine conjugate base of 29 m l z 8.21 min)-EIMS m l z (%) 229 (33, M-1, 214 (loo), 172 (181, 145 (%) 211 (M+);'H NMR (CDC13)6 8.58 (d, lH, NCH=CH), 7.20(20), 121 (18), 115 (27), 91 (151, 54 (22); 'H NMR ( D M s 0 - d ~6) 7.38 (m, 4H, PhH), 6.50 (d, l H , NCH=CH), 4.10 (t, 2H, NCHz), 7.37 (d, J = 8.7, 2H, PhH), 6.89 (d, J = 8.7, 2H, PhH), 6.03 (bs, 3.15 (t, 2H, NCHZCH~), 2.64 (m, l H , NCH), 2.39 (s, 3H, CH3), lH, NCHzCH), 3.74 ( s , 3H, OCH3), 3.64 (bs, 2H, NCHzCH), 3.21 1.23 (m, 4H, NCHCH2). The crude dihydropyridinium species 2.59 (bs, 2H, NCHZCH~), 2.41 (m, (t, J = 5.8, 2H, N-CHZCH~), 29 in CH30H (10 mL) was treated at 0 "C with a solution of l H , NCH), 0.64-0.72 (m, 4H, NCHCH2). Anal. Calcd for NaBD, (20 mg) in CH30H (2 mL). After a n additional 20 min C17H21N06:C, 63.93; H, 6.63; N, 4.39. Found: C, 64.00; H, 6.69; at 0 "C, aqueous workup of the reaction mixture gave the crude N, 4.47. l-Cyclopropyl-4-(2-methylphenyl)-1,2,3,6-tetra- product, which was chromatographed on a short silica column (EtOAc) to give l-cyclopropyl-4-(2-methylphenyl)-1,2,3,6-tethydropyridineHC104 (26d). Due to the hydroscopic nature rahydropyridine-6-dl. (30): GC (temperature program: 100 "C of the oxalate salt, the perchlorate salt was prepared by the for 2 min, then 25 "Clmin up to 275 "C; t~ 6.60 mid-EIMS mlz addition of 10% methanolic HC104 to the free base followed by (SI214 (M+); IH NMR ( D M s 0 - d ~6) 7.08-7.17 (m, 4H, PhH), recrystallization from CH30H (0.35 g, 15%): mp 214-215 "C; 5.59 (bs, l H , NCHDCH), 3.49 (m, l H , NCHD), 3.09 (t, 2H, GC (temperature program: 100 "C for 2 min, then 25 "Clmin NCH~CHZ), 2.48 (t, 2H, NCH~CHZ), 2.28 (s, 3H, CH3), 2.02 (m, up to 275 "C; t~ 6.88 min)-EIMS mlz (9%)213 (48, M+),198 (loo), l H , NCH), 0.83 and 0.63 (m, 4H, NCHCH2). Anal. Calcd for 142 (21), 129 (52),91(17),54 (37); 'H NMR (oxalate salt; DMSOC14Hl~DClN04.HC104:C, 57.23; WD, 6.72; N, 4.45. Found: C, &) d 7.05-7.15 (m, 4H, PhH), 5.49 (bs, l H , NCHzCH), 3.75 (bs, 56.99; H/D, 6.36; N, 4.43. 2H, NCH&Hj, 3.34 (bt, 2H, NCHzCHz), 2.68 (m, lH, NCHCHz), Enzymology. MAO-B was isolated from bovine liver mito2.50 (m, 2H, NCHzCHz), 2.19 ( s , 3H, CH3), 0.71-0.95 (m, 4H, chondria according to the method of Salach and Weyler ( 2 4 ) . NCHCH2); W (0.1mM phosphate buffer) ,Imap208 nm, E 8100 The activity was determined spectrophotometrically at 30 "C M-I). Anal. Calcd for C15H2oClN04: C, 57.42; H, 6.42; N, 4.46. on a Beckman DU 50 spectrophotometer using 5 mM MPTP as Found: C, 57.15; H, 6.41; N, 4.48. substrate and recording initial rates (120 s) of formation of the l-Cyclopropyl-4-(3,5-dichlorophenyl)1,2,3,6-tetradihydropyridinium metabolite (,Imap343 nm, e 16 000 M-I) as hydropyridine(CO0H)z(26e). Recrystallized from acetonedescribed previously (8). The final enzyme concentration was diethyl ether (69%): mp 156-157 "C; GC (temperature procalculated to be 8 nmol/mL. gram: 125 "C for 1 min, then 10 "Clmin up to 290 "C; t~ 6.70 All enzyme assays were performed at 37 "C with a Beckman 269 (30, M-, 37,35C1), min)-EIMS m l z (9%)271 (4.5, M-, 37337Cl), DU-50 spectrophotometer. In preliminary experiments, the 254 268 (141, 267 (41, M', 3s.35C1),256 (10, MT - 15, 37*37C1), potential MAO-B substrates were examined by recording re(65, M+ - 15, 3i,3sCl),252 (100, MA - 15, 35.35C1),68 (511, 54 peated scans (500-220 nm) of a 500 pM solution of each (59); 'H NMR (oxalate salt; DMSO-&) b 7.42 (s, bs, 3H, PhH), tetrahydropyridine in the presence of 0.08 pM MAO-B. In those 6.30 (bs, l H , NCHnCH), 3.59 (bd, 2H, NCHzCH), 3.15 (t, J = cases where product formation was detected, initial rates of 5.8,2H, NCHzCHz), 2.58 (m, 2H, NCH~CHZ), 2.26 (m, l H , NCH), oxidation were determined at four substrate concentrations. 0.64 (m, 4H, NCHCH2). Anal. Calcd for C ~ G H ~ ~ C ~ C, ZNO~: Stock solutions (ranging from 1000 to 37.5 pM) of the substrates 53.77; H, 4.8; N, 3.92. Found: C, 53.74; H, 4.83; N, 3.83. were prepared in 100 mM sodium phosphate buffer (pH 7.4). A l-Cyclopropyl-2,3-dihydro-4-pyridone (27). To a slurry 495 pL aliquot of each solution was added to the cuvette which of LiAlH4 (0.155 g, 4.07 mmol) in 20 mL of anhydrous THF was was placed in the spectrophotometer maintained at 37 "C. After added pyridone 20 (1.1g, 8.15 mmol) over 5 min a t 0 "C. After a 3 min equilibration period, 5 pL of the MAO-B enzyme stirring at this temperature for 1 h, the reaction was stopped preparation was added. The initial rates of oxidation of each by the careful addition of 10 mL of 15% NaOH and 5 mL of substrate were estimated by monitoring the absorbance of the HzO. The product was extracted into CH2C12, and the extract corresponding aminoenone 27 or dihydropyridinium species 29 was washed several times with water, dried over Na2S04, and every 3 s for 2 min. The KM and kcat values were calculated concentrated in uacuo to yield a yellow oil. Column chromafrom Lineweaver-Burke double-reciprocal plots. tography (neutral alumina-19 MeOH in CHC13) gave pure 27 MAO-B inactivation studies were conducted as follows: Stan(0.4 g, 3691 as a yellow oil: Amax (0.1 M Na3P04) 328 nm ( E dard solutions of substrate (ranging from 1000 to 100 pM) in 10 000 M-I); GC (temperature program: 100 "C for 1 min, then sodium phosphate buffer (100 mM, pH 7.4) were prepared. Each 25 W m i n up to 290 "C; t~ 5.3 mini-EIMS m l z (%I 137 (15, M+), solution (50 ,uL) was mixed with 50 pL of the stock MAO-B 109 (221, 108 (261, 94 (251, 82 (251, 81 (501, 80 (451, 68 (33), 54 preparation (final enzyme concentration 0.80 pM) and, the (100); 'H NMR (CDC13) 6 7.14 (d, J = 7.6, lH, NCH=CH), 4.97 resulting mixtures were incubated with gentle agitation in a (d, J = 7.6, l H , NCHeCHj, 3.50 (t, J = 7.6, 2H, NCHzCHz), water bath at 37 "C. A 10 ,uLaliquot of each incubation mixture 2.65 (m, l H , NCH), 2.44 (t, J = 7.6, 2H, NCHzCHz), 0.68-0.80 taken at 0, 5, 10, and 15 min was added to a sample cuvette (m, 4H, NCHCH2). EI-HRMS: Calcd for CsH11NO: 137.0841 containing 490 p L of a 5 mM solution of MPTP (pre-equilibrated (M+). Found: 137.0845. to 37 "C) in sodium phosphate buffer (100 mM, pH 7.4). The Synthesis of the l-Cyclopropyl-4-(2-methylphenyI)-2,3,- rate of MPTP oxidation was determined a t 37 "C by monitoring dihydropyridinium Species 29 and Subsequent NaBD4 the absorbance at 343 nm (the Amax of the resulting dihydropyReduction to 1-Cyclopropyl-4-(2-methylphenyl)-1,2,3,6- ridinium metabolite 2)every 3 s for 2 min. tetrahydropyridine-6.dl (30). A solution of m-chloroperoxybenzoic acid (0.56 g, 5596, 1.79 mmol) in CHzCl2 (10 mL) was Results and Discussion added dropwise t o 26d (0.39 g, 1.83 mmol) in CHzCln (10 mL) at 0 "C with stirring. After stirring for 3.5 h at this temperature, Chemistry. Due to the resistance of cyclopropyl the reaction mixture was chromatographed on basic alumina halides to nucleophilic attack, the preparation of the (30 g; eluent: 5% CH30H in CHzCl2) to afford the N-oxide 28 desired 4-substituted-l-cyclopropyl-1,2,3,6-tetrahydropy(0.40 g, 97.5%): 'H NMR (CDC13) d 7.10-7.21 (m, 4H, PhH), ridines had to be approached by a synthetic route other 5.53 (bs l H , NCHZCH).4.02 (m. 2H, NCHzCH), 3.49 (m, 2H, than the usual condensation reactions with appropriately NCH~CHZI. 3.08 (m, l H , NCH), 2.88 and 2.63 (m, 2H, NCHZCH~), 7.67 (m, 4H, PhH), 6.33 (bs, l H , NCHzCH), 3.60 (bs, 2H, NCHz2.62 (m, 2H, NCHzCHz), CH), 3.16 (t,J = 5.7, 2H, NCHZCH~), 2.30 (m, l H , NCH), 0.65 (m, 4H, NCHCH2). Anal. Calcd for CnHlsF3N04: C, 57.14; H, 5.08; N, 3.92. Found: C, 56.68; H, 5.20; N, 3.89.

Chem. Res. Toxicol., Vol. 8, No.5, 1995 707

Mechanistic Studies on MAO-B Catalysis

Scheme 4. Synthesis of 4-(Aryloxy)-and 4-Thiophenoxy- 1-cyclopropyl-1,2,3,6-tetrahydropyridinesa

-r"; iv

+/

a

x

X=O;Ar=C&j x = 0;Ar =8-CI-C& C: x = 0; Ar = 3,5-C&H3 d: X = o; Ar = 2,4-C1&& 8: X = 0; Ar = 4-N0&6H4 f: x = s; Ar = C & ,

8:

b:

19 20 21 22a-f 23a-f Reagents: (i) cyclopropylamine, HzO, 100 "C, 3 h; (ii) SOC12, reflux, 4 h; (iii) HXAr, NEts, CHsCN, 25 "C, 20 h; (iv) NaBH4, CHsOH,

1 h.

Scheme 5. Synthesis of 4-Aryl-1-cyclopropyl-1,2,3,6-tetrahydropyridinesa

QA 24

Scheme 6. Synthesis of l-Cyclopropyl-2,S-dihydro-4-pyridone (27)

6 6

- 0HO

ii

26a-e

Reagents: (i) BrAr, Mg, EtzO, 25 "C, 1 h, then 24,25 "C, 1 h; (ii) HC1-HOAc, reflux, 15-20 h.

LiAIH4

A

A

25a-e

I-

I

Ar

i

A

20

27

a

substituted pyridine derivatives (25). Syntheses of the aryloxy derivatives 23a-e and the thiophenoxy derivative 23f started with the preparation of y-pyrone (19)(26) which was obtained from the thermal decarboxylation of chelidonic acid (27). Condensation of 19 with cyclopropylamine gave the pyridone 20 which, upon reaction with thionyl chloride, generated 4-chloro-1-cyclopropylpyridinium chloride (21). Subsequent condensation of 21 with the appropriate arenol in the presence of triethylamine generated the corresponding 4-(aryloxy)-l-cyclopropylpyridinium compounds (22a-f). Due to their hygroscopicity and susceptibility to hydrolysis, these pyridinium intermediates were not isolated but rather were reduced directly with sodium borohydride in methanol to yield the requisite tetrahydropyridine derivatives (23a-f) (Scheme 4). The 4-aryl-1-cyclopropyltetrahydropyridines26a-e were synthesized by the reaction of l-cyclopropyl-4piperidone (24) (22)with the appropriate aryl Grignard reagent to give the corresponding piperidinols 25. The crude tertiary alcohols were subjected to acid catalyzed dehydration with HCYHOAc to generate the desired tetrahydropyridines 26a-e, which were characterized as their oxalate salts (Scheme 5). We anticipated that those 4-(aryloxy)-l-cyclopropyl analogs displaying MAO-B substrate properties would yield dihydropyridinium metabolites that, like the corresponding 1-methyl analogs (7,9),would undergo rapid hydrolysis to form the arenol and 1-cyclopropyl-2,3dihydro-Cpyridone (27). An authentic sample of 27, synthesized by the controlled reduction of pyridone 20 with LiAlH4 (Scheme 6) (281, was available to help identify this aminoenone in incubation mixtures of the 4-(arylo~y)-l -cyclopropyl-1,2,3,6-tetrahydropyridines. An additional synthetic effort was required to characterize the dihydropyridinium metabolite 29 generated from the MAO-B catalyzed oxidation of l-cyclopropyl-4(2-methylphenyl)-l,2,3,6-tetrahydropyridine (26d). Compound 29 was synthesized in a two-step reaction sequence starting with the m-chloroperoxybenzoic acid oxidation of 26d, which yielded the N-oxide 28. Treatment of 28 with trifluoroacetic anhydride at room tem-

Scheme 7. Synthesis of the 1-Cyclopropyl4-(2-methylphenyl)-2,3-dihydropyridinium Species 29 and Its in Situ Reduction to 30"

6 6 i

__t

+

ii

__c

1 d"o

(j-& +/

x

iii

H

XD

30 26d 28 29 Reagents: (i) m-CPBA, CHzClz, 25 "C, 3.5 h; (ii) (CF3C0)20, CHC13, 25 "C, 20 min; (iii) NaBD4, CHsOH, 25 "C, 20 min.

perature under carefully controlled conditions gave rise to the unstable dihydropyridinium species 29 which displayed all of the expected 'H NMR signals (see Experimental Section). The reaction was complete within 15 min. Because of its instability, the characterization of this dihydropyridinium product was achieved via the monodeuterated tetrahydropyridine derivative 30 which could be obtained in pure form by treatment of crude 29 in methanol with NaBD4 (Scheme 7). Enzymology. The unexpected substrate properties of the MPTP analog 4-benzyl-l-cyclopropyl-1,2,3,6-tetrahydropyridine (14) have prompted us to consider catalytic pathways other than the SET pathway for the MAO-B catalyzed oxidation of these cyclic tertiary allylamines. In particular, we have raised the question of whether or not the putative aminyl radical cation generated by the SET step is an obligatory intermediate. If this is the case, then the general expectation that the relative rates of cyclopropylaminyl radical cation ring opening will be considerably faster than a-proton loss may be in question. A direct loss of H from the substrate (see Scheme 3) that would bypass the radical cation intermediate, however, would be consistent with the substrate behavior of 14. In an effort to provide additional information on the potential substrate properties of l-cyclopropyltetrahydropyridines, we synthesized a series of 4-substituted analogs with the intention of examining both the steric and stereoelectronic factors at C-4 which may contribute

Rimoldi et al.

708 Chem. Res. Toxicol., Vol. 8, No. 5, 1995 Scheme 8. Metabolic Fate of Tetrahydropyridines 23a and 23f

3 6 x XPh

MAO-B

27

I

A

23a X = O 23f X = S

22f

31 32

to the substrate properties of these 1-cyclopropyl derivatives. The first target compound was the 4-phenoxy derivative 23a, an analog with steric features similar to those of the 4-benzyl analog. Metabolic screening of the phenoxy analog 23a clearly documented its substrate properties. The repeated W scans showed the formation of a chromophore with Am= 332 nm. The species responsible for this chromophore was tentatively identified by W analysis as l-cyclopropyl-2,3-dihydro-4-pyridone(27) by comparison with the synthetic sample (Amm 328 nm). GC-E1 mass spectral analysis of an ethyl acetate extract of an incubation mixture composed of 50 p L of the MAO-B preparation and 100 p L of 1 mM 23a incubated for 10 min at 37 "C displayed a peak with retention time 5.31 min and the following mass spectral characteristics: m / z 137 (M+), 109 (M' - CO), 108 (M+- CHO), 94,81,80,54. Identical GC-E1 mass spectral behavior observed with a synthetic sample confirmed the structure of the MAO-B generated product as 27. Based on these data, the metabolic fate of 23a (Scheme 8) is analogous to that observed with the corresponding N-methyl compound and the 4-benzyl-lcyclopropyl analog 14. Enzyme kinetic data of substrate turnover performed by monitoring the 332 nm chromophore established that this analog was an excellent MAO-B Substrate. Taking kcaJKMas an overall estimate of the efficiency of catalysis (Table 11, the substrate properties of 23a (1650 min-I mM-l) were found to be comparable to those of the benzyl derivative 14 (1540 min-I mM-'1. Unlike the benzyl compound, however, this phenoxy analog showed no enzyme inhibition properties even at high (1 mM) concentrations. Prompted by these results, several additional 4-(aryloxy) analogs were synthesized and screened for their MAO-B substrate properties. Incubation of the 4-thiophenoxy analog 23f with the enzyme led to the formation of a chromophore with Am= 365 nm which, unlike the phenoxydihydropyridinium species 31,did not shiR to 332 nm, the chromophore for aminoenone 27, but instead slowly shifted to Am= 305 nm. We suspected from the known behavior of the corresponding N-methylthiophenoxydihydropyridinium derivative (28) that, instead of undergoing hydrolysis, the relatively stable l-cyclopropyl4-thiophenoxydihydropyridiniummetabolite 32 had undergone slow oxidation to yield the corresponding pyridinium species 22f. The W spectrum of synthetic 22f and the final spectrum of the incubation mixture (Am= 305 nm) confirmed the identity of this product as 22f which presumably was formed by autoxidation andlor disproportionation (29) of the dihydropyridinium intermediate 32. Apparently, the weaker electronegativity of the sulfur vs the oxygen atom is responsible for the hydrolytic stability of this system. Like the other N cyclopropyl derivatives examined in this study, the

thiophenoxy analog 23f displayed no time- or concentration-dependent MAO-B inhibitor properties. The 4-(3-chlorophenoxy) analog 23b also proved to be an excellent MAO-B substrate while the 444-nitrophenoxy) analog 23e was a relatively weak substrate. Neither compound displayed detectable time-dependent inhibitor properties. The dichloro derivatives 23c and 23d proved to be neither substrates nor inactivators of MAO-B, a finding which was somewhat surprising in view of the excellent substrate properties we have observed for the related N-methyl analogs.2 Apparently, the combination of the sizes of the 1-cyclopropyl group and the disubstituted phenoxy moiety at C-4 leads to unfavorable enzyme-substrate steric interactions. We next turned our attention to substituted 4-aryl-lcyclopropyltetrahydropyridinederivatives which lack a methylene or heteroatom spacer group. Similar to the behavior of the 4-phenyl analog 8, the 4-(4-chloro-, 4-(4methoxy-, and 4-(3,5-dichlorophenyl)derivatives (26a,-c, and -e, respectively) all proved to be efficient time- and concentration-dependent inactivators of MAO-B. No evidence of substrate properties was found when 1 mM solutions of these 4-aryl-1-cyclopropyltetrahydropyridine derivatives were incubated with MAO-B. The 4-(trifluoromethyl) analog 26b could not be assayed due to its low solubility in buffer. The least expected result surfaced when l-cyclopropyl4-(2-methylphenyl)-l,2,3,6-tetrahydropyridine (26d)was incubated with MAO-B. This analog gave rise to a chromophore (Ama 338 nm) that was assigned to the dihydropyridinium species 29 by comparison with the W spectral properties of crude synthetic 29. Quantitative kinetic analyses3 established the Kcat (176 m i x ' ) and KM (0.28 mM) values for this oxidation. This compound, therefore, is a reasonably good MAO-B substrate (K,,J KM = 635 min-' mM-'). Equally surprising was the observation that 26d did not inhibit MAO-B. These results are in dramatic contrast to the corresponding characteristics observed with the 4-phenyl analog 8, which was not a substrate but instead was a good timeand concentration-dependent inactivator of MAO-B. The unexpected substrate properties of the N-cyclopropyltetrahydropyridines described in this study raise the possibility that cyclopropylaminyl radical cations may not be obligatory intermediates in MAO-B catalyzed oxidations. An alternative pathway follows the suggestion reported by Edmondson in which a hydrogen atom acceptor present in the enzyme abstracts the allylic hydrogen atom from the substrate molecule (33) to generate the corresponding stabilized allylic radical (35) directly. According to this suggestion, the parent amine would partition between an electron abstraction pathway, leading to enzyme inactivation, and a hydrogen atom abstraction pathway, leading to dihydropyridinium product formation (Scheme 9). Although the hydrogen atom abstraction pathway is attractive in that it obviates the need to invoke a kinetic preference for proton loss over ring opening of cyclopropylaminyl radical cations, the behavior of the 4-phenyl analog 8 vs the behavior of the o-methylphenyl analog 26d is difficult to rationalize in terms of this pathway. Unpublished results. Since we were unsuccessful in obtaining a pure sample of 29, a molar extinction coefficient (10000 M-l) was estimated on the basis of the corresponding value for the N-methyl compound (16 000 M-I) ( 8 ) and the reduced conjugation between the n-system of the aromatic group and the olefinic double bond due to the o-methyl group. 3

Chem. Res. Toxicol., Vol. 8, No. 5, 1995 709

Mechanistic Studies on MAO-B Catalysis

Table 1. Kinetic Parameters for the MAO-B Catalyzed OxidationLnhibiton of 4-Substituted l-Cyclopropyl-1,2,3,6-tetrahydropyridines at 37 "C 4-substituted l-cyclopropyl-1,2,3,6-tetrahydropyridine 4-phenyl ( 8 ) 4-benzyl (14) 4-phenoxy (23a) 4-(3-chlorophenoxy) (23b) 4-(3,5-dichlorophenoxy) ( 2 3 ~ ) ~ 4-(2,4-dichlorophenoxy) (23dY 4-(4-nitrophenoxy) (23e) 4-thiophenoxy (230 444-chlorophenyl) (26a) 4-[4-(trifluoromethyl)phenyll (26bIb 4-(4-methoxyphenyl) (26c) 4-(2-methylphenylj (26d) 4-(3,5-dichlorophenyl) (26e) a

KM(mM)

kcat (min-')

0.414 0.130 0.096

637 215 136

1420

1.06

152 228

150 1900

0.12

0.28

kcaJKM

KI (mM)

kinact

(min-l)

kinacJK1

0.18

0.70

3.85

0.46

1.07

2.45

0.53

0.24

0.46

0.09

0.03

0.33

1538 1650

176

630

Neither substrate or inactivator. Insoluble in 100 mM phosphate buffer.

Scheme 9. Possible Catalytic Pathways for the MAO-B Catalyzed Oxidation of 4-Substituted 1-Cyclopropyl-1,2,3,6-tetrahydropyridines R

R

A R

A

A

A\\ B. 34 I I

36

\

A

A

35

37

While inductive effects are consistent, the decreased resonance stabilization due to loss of coplanarity in the o-methylphenyl analog 26d is not consistent with this compound's good substrate properties relative to those observed with the 4-phenyl analog 8. Consequently, the geometries of these 4-substituted l-cyclopropyltetrahydropyridines may play an important role in determining the nature of their interactions with MAO-B, as has been suggested by Efange and co-workers (30). It also should be noted that the SET pathway cannot be ruled out since constraints in the active site may prohibit the appropriate geometry required for ring opening andor a-proton abstraction of the putative cyclopropylaminyl radical cation. Studies currently underway on a series of tetrahydropyridine derivatives bearing various heteroaromatic systems at C-4 as well as kinetic deuterium isotope effect studies on rates of substrate turnover and enzyme inactivation may shed additional light on the mechanism by which MAO-B processes these and related tetrahydropyridine derivatives. Acknowledgment. This work was supported by the National Institute of Neurological and Communicative Disorders and Stroke (NS 28792) and the Harvey W. Peters Research Center for Parkinson's Disease and Disorders of the Central Nervous System. We thank Mr. Kim Harich (Virginia Tech) for the high-resolution mass spectral data.

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