Chem. Res. Toxicol. 1992,5, 625-633

625

Studies on 1,2,3,6-Tetrahydropyridine Derivatives as Potential Monoamine Oxidase Inactivators Larry Hall, Sam Murray, Kay Castagnoli, and Neal Castagnoli, Jr.* Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 Received March 2, 1992 The Parkinsonian-inducing neurotoxin l-methyl-Cpheny1-1,2,3,6-tetrahydropyridine (MPTP) and close structural analogs are the only known cyclic tertiary amines with good monoamine oxidase substrate properties. In addition t o its excellent substrate properties, M P T P is a mechanism-based inactivator of monoamine oxidase B (MAO-B). In an attempt to exploit the special interactions between this cyclic tertiary allylamine and MAO-B, we have initiated studies t o evaluate the enzymatic and biological properties of M P T P analogs bearing functional groups which are known t o mediate the metabolism-dependent inactivation of this enzyme. This paper describes the synthesis, enzyme substrate/inhibitor properties, and neurotoxic/neuroprotective properties of l-cyclopropyl-4-phenyl-l,2,3,6-tetrahydropyridine, the corresponding acyclic and 4-cyclopropyl-l-methyl-l,2,3,6secondary amine (E)-Ccyclopropyl-2-phenyl-2-butene, tetrahydropyridine. Scheme I. Proposed Pathway for the MAOCatalyzed Oxidation of Amines

Introduction Monoamine oxidase [EC 1.4.3.4 (MAO)'] is an FADcontaining enzyme that catalyzes the a-carbon oxidative deamination of acyclic amines including the biogenic amine neurotransmitters ( I , 2). Two isozymes, the A (3) and B ( 4 ) forms, have been identified, and the corresponding genes have been cloned (5, 6). The results of extensive studies by Silverman (7)and his colleagues on the inactivation of MAO-B by derivatives of cyclopropylamine (8,9),cyclobutylamine (IO),(aminomethy1)oxazolines(1I), and (aminomethy1)silanes (12) have led to the catalytic mechanism outlined in Scheme I. Initial electron transfer from the nitrogen lone pair of the substrate (1) to the oxidized flavin (FAD) generates an aminium radical (2) and the protonated flavin radical (FADH'). Proton loss from 2 yields the carbon-centered radical 3 which undergoes a second electron-transfer reaction to form the iminium product 4 and the fully reduced flavin (FADH2). Subsequent hydrolysis of 4 provides the deaminated product 5. In the case of strained cycloalkylamine derivatives such as N-benzylcyclopropylamine (61, the aminium radical 7 undergoes rapid ring opening to form the highly reactive primary carbon-centered radical 8, which is thought to mediate the inactivation of the enzyme (Scheme 11). Recent chemical studies on single electron transfer induced photochemical reactions of a model flavin with cyclopropylamines (13) and ESR studies on the interactions of cyclobutylamines with MAO-B (14)have provided convincing evidence for such radical intermediates. Although a cyclic tertiary amine, the Parkinsonianinducing nigrostriatal toxin l-methyl-4-phenyl-l,2,3,6-

* Address correspondence to this author at the Department of Chemistry, 107 Davidson Hall, Virginia Tech, Blacksburg, VA 24061. Abbreviations: DA, dopamine; DHBA, 3,4-dihydroxybenzylamine; ESR, electron spin resonance; FAD, flavin adenine dinucleotide; GC, gas chromatography; GC/MS, gas chromatography/mass spectrometry; HP, Hewlett Packard; HPLC, high-performance liquid chromatography; m-CBA, m-chlorobenzoic acid; m-CPBA, m-chloroperoxybenzoic acid; MAO, monoamine oxidase; MPDP+, l-methyl-4-phenyl-2,3-dihydropyridinium (10); MPTP, l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (9); TCA, trichloroacetic acid; TFAA, trifluoroacetic anhydride; TMS, tetramethylsilane. 0893-228~/92/ 2705-0625$03.00/0

H+ FAD FADH,

RCH,illh

H+

H+ FAOH, FADH,

H20 H ~ H ,

1L

RCH=I;H,URCHO

RCH,IJH,LRMH, 2

1

3

5

4

Scheme 11. The MAO-B-Catalyzed Oxidative Bioactivation of 6

VIA6

,HC,,& I

H 7

8

tetrahydropyridine (MPTP, 9, Chart I) is an excellent substrate (15)and weakinactivator (16)of MAO-B. Unlike acyclic amines, the dihydropyridinium species MPDP+ (10) generated by the MAO-B-catalyzed oxidation of MPTP is stable to hydrolysis and in vivo undergoes further oxidation to the pyridinium metabolite 11. Interest in the interactions of MPTP with MAO-B has been intense since inhibition of this biotransformation blocks the formation of 11, the putative ultimate nigrostriatal toxin (17,181,and protects susceptible animals against MPTP's neurotoxic properties ( 1 g 2 1 ) . The possibility that the etiology of idiopathic Parkinson's disease may involve MPTP-type endogenous (22) or environmental substance(s) (23) has prompted clinical trials with the MAOB-selective inactivator (S)-deprenyl (12) to evaluate the potential therapeutic utility of inhibiting this enzyme in the early stages of the disease. Preliminary results from this trial (24, 25) and related clinical studies with (S)deprenyl (26)have been encouraging. In an attempt to develop novel inactivators of MAO-A and/or MAO-B, we have initiated experiments designed to evaluate the substrate/inactivator and neurodegenerative/neuroprotective properties of MPTP analogs bearing substituents which may be activated by the electronjproton transfer sequence postulated for MA0 catalysis. In this paper we report our findings on 1-cyclopropyl-4-phenyl1,2,3,6-tetrahydropyidine (131, (E)-4-cyclopropyl-2-phen-

0 1992 American Chemical Society

626 Chem. Res. Toxicol., Vol. 5, No.5, 1992

Hall et al.

obtained which, following crystallization from ethanol, provided 796 mg of white needles: mp 160.5-162 "C; lH NMR (DMSO-&) 6 7.38 (m, Ph H, 5 HI, 5.93 (td, J = 1.18 and 7.38 Hz, olefinic proton, 1H), 3.81 (d,J = 6.62 Hz, CHZN,2 H), 2.72 (bs,cyclopropyl methine, 1H), 2.10 (s, CH3,3 H), 0.97 (m, cyclopropyl ethylene protons trans to N, 2 H), 0.74 (m, cyclopropyl ethylene protons cis to N, 2 H); I3C NMR (DMSO-ds) 6 141.81 [Ph (Cl)], 140.90 [Ph (C4)1,128.34 [Ph (C3 and C5)1,127.63 (C2), 125.57 [Ph (C2 and C6)1,117.97 (C3), 45.29 (C4), 29.02 (Cl), 16.00 (cyclopropyl methine), 2.99 (cyclopropyl methylenes); GC/MS [isothermal at 100 "C for 1 min followed by a ramp of 25 "Cimin for 7 min (tR = 3.9 min)] m/z 187 (M+,4%), 172 (131,158 ( l l ) , 131 (loo), 115 (311, 91 (75). Anal. Calcd for C13H18NC1:C, 69.79; H, 8.11; N, 6.26. Found: C, 69.53; H, 8.04; N, 6.24. yl-2-butene (14), and 4-cyclopropyl-l-methyl-l,2,3,6(D)l-Cyclopropyl-4-phenyl-1,2,3,6-tetrahydropyridine tetrahydropyridine (15). Hydrochloride (13~HC1). The above oxazine 17 (2.0 g, 7.89 mmol) was heated in 20 mL of concentrated HC1 at 100 "C for 1 h. The solvent then was removed under reduced pressure to Materials and Methods yield 1.8 g of a white solid which was crystallized from 5% CH2CldCHCl3 to yield 1.36 g (73.2%) of fine white needles: mp 203Caution: l-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine (9) 204.5 "C; 'H NMR (DMSO-ds) 6 8.42 (bs, NH, 1HI, 7.36 (m, Ph is a known nigrostriatal neurotoxin and should be handled using H, 5 H), 6.18 (m, olefinic proton, 1 H), 3.95 (m, allylic CHzN, 2 disposable gloves in a hood. Detailed procedures for the safe H), 3.62 (bd, CHzN, 2 HI, 2.89 (m, CHzCHzNand cyclopropyl handling of MPTP have been reported (27). methine, 3 H), 1.15 (m, cyclopropyl ethylene protons trans to N, Chemistry. (A) General Methods. Synthetic reactions were 2 H), 0.82 (m, cyclopropyl ethylene protons cis to N, 2 H); l3C carried out under a nitrogen atmosphere. All chemicals (Aldrich, NMR (DMSO-ds) 6 138.31 [Ph (Cl)], 134.03 [Ph (C4)1, 128.45 Milwaukee, WI) were reagent or HPLC grade. Proton and 13C [Ph (C3 and C5)1, 127.83 (C4), 124.70 [Ph (C2 and C6)1,116.10 NMR spectra were recorded on a Bruker WP 270 or 200-MHz (C5), 50.33 (C6), 48.89 ((221, 37.92 (C3), 23.46 (cyclopropyl spectrometer. Chemical shifts (6) are reported in parts per million methine), 3.19 (cyclopropyl methylenes); GC/MS [isothermal at (ppm) relative to tetramethylsilane or sodium 3-(trimethylsilyl)100 "C for 1 min followed by a ramp of 20 "C/min for 8 min ( t ~ propionate-2,2,3,4-d4 (for DzO) as internal standard. Spin = 6.1 min)] m/z 199 (M+, 36), 184 (loo), 170 (61, 156 (12), 128 multiplicities are given as s (singlet), d (doublet), t (triplet), h (42), 115 (38), 91 (23). Anal. Calcd for Cl4Hl8NC1: C, 71.33; H, (heptuplet), or m (multiplet). Gas chromatography/mass spec7.70; N, 5.94. Found: C, 71.21; H, 7.72; N, 5.86. trometry (GC/MS) was performed under electron ionization (E)m-Chlorobenzoic Acid Salt of 1-Cyclopropyl-3,4conditions using a Hewlett Packard (HP) 5890 capillary GC epoxy-4-phenylpiperidine N-Oxide (21). A mixture of the equipped with an HP-1 capillary column (12-m X 200-pm X 0.33pm film thickness) coupled to a H P 5970 mass-selective detector. above tetrahydropyridine (707 mg, 3.6 mmol) and m-chloroData were acquired using a H P 5970 MS ChemStation. Norperoxybenzoic acid (m-CPBA, 1.14 g, 6.6 mmol) in 20 mL of malized peak heights are reported as percentage of the base peak. dichloromethane was stirred at room temperature for 24 h. The Both quantitative and qualitative HPLC analyses were performed solvent was removed, and the residue was chromatographed with on a Beckman 114M chromatograph employing an HP Model dichloromethane on 25 g of basic alumina to give a yellow oil that 1040A diode array detector. A Beckman Ultrasil 10-pm SCX upon crystallization from EtOH/H20 yielded 596 mg (81%)of cation-exchange column (25 cm X 4.6 mm) was utilized along 21 as its m-chlorobenzoate (m-CBA) salt: mp 193-195 "C; lH with a solvent system that was initially 80% acetate buffer (pH NMR (CDC13) 6 7.96 (t, J = 1.64 Hz, Ar H, 1 H), 7.88 (dt, J = 5) and 20% acetonitrile followed by a linear gradient for 8 min 1.53 and 7.46 Hz, Ar H, 1H), 7.60 (dq, J = 1.09 and 8.02 Hz,Ar to a final concentration of 55% acetate buffer and 45% H, 1H), 7.43 (m,Ar H a n d P h H , 6H),4.41 ( d , J = 2.08Hz,OCH, acetonitrile. Melting points were obtained on a Thomas-Hoover 1 H), 3.84 (dd, J = 2.15 and 12.31 Hz, NCHCH2, 1 H). 3.59 (m, melting point apparatus and are uncorrected. Microanalyses 4 H), 3.13 (dd, J = 2.13 and 14.55 Hz, NCHCHZ,1 H), 2.81 (h, were performed by Atlantic Microlab, Inc. (Norcross, GA). cyclopropyl methine, 1H), 1.46 (m, cyclopropyl ethylene protons (B) 3-Cyclopropyl-6-methyl-6-phenyltetrahydro-l,3-0~- trans to N, 2 H), 0.61 (m, cyclopropyl ethylene protons cis to N, 2 H); '3C NMR 6 164.65 (COz), 141.83 [m-CBA (C1)1, 135.78 azine (17). A 37% aqueous solution of formaldehyde (16.53 g, [m-CBA (C3)1, 134.45 [m-CBA (C2)1, 133.78 [Ph (C1)1, 131.41 204.2 mmol) was added to a solution of cyclopropylamine (5.71 [m-CBA (C6)], 130.44 [m-CBA (C4)1,129.35 [Ph (C2 and C6)], g, 100 mmol) in 10 mL of water (pH 6.0, adjusted with 129.02 [m-CBA (C5)1,128.92 [Ph (C4)1,126.99 [Ph(C3andC5)], concentrated HCl). The resulting mixture was heated to 65 "C 82.91 (c4), 73.38 (c3), 64.07 (c2), 63.02 (cs),51.37 (cs),23-29 for 10 min, and after cooling to room temperature, a-methyl(cyclopropyl methine), 1.04 (cyclopropyl methylene), 0.30 (cystyrene (5.85 g, 49.5 mmol) was added. The resulting mixture 242 nm. Anal. Calcd clopropyl methylene); UV (CHpC12) A, was heated to 65 "C for 2 h. After the addition of methanol (200 for CzlHzzN04C1: C, 65.03; H, 5.72; N, 3.61. Found: C, 65.28; mL) the mixture was left to stir overnight at room temperature. H, 5.87; N, 3.65. Removal of the solvent gave 10.61 g of a slightly yellow solid which, following crystallization from ethanol, yielded 7.16 g (F)Perchlorate Salt of 1-Cyclopropyl-4-phenylpyridin(61.4%) of product as fine white needles: mp 195-196.8 OC dec; ium (20). The above epoxy N-oxide salt (215 mg, 0.55 mmol) lH NMR (DzO) 6 7.51 (m, Ar H, 5 H),5.83 (d, OCHzN, 2 H), 3.0 in 20 mL of dichloromethane was treated dropwise with tri(m, CHZCH~N, 4 H), 1.82 (m, cyclopropyl methine, 1H),1.65 (d, fluoroacetic anhydride (TFAA, 1.05 g, 5.0 mmol) in 10 mL of CH3,3H),0.82 (m,cyclopropylethylene,4H); GCiMS [isothermal dichloromethane. After stirring 2 h at room temperature, the at 75 "C for 1 min followed by a ramp of 25 "Clmin for 7 min solvent was removed and the resulting yellow solid was dissolved (tR = 5.1 min)] mlz 217 (M+,6), 202 (5), 188 (461, 158 (191, 131 in 10 mL of 10% methanolic HC104. Upon cooling, the product (24), 117 (56), 97 (38), 91 (39), 82 (40), 77 (50), 70 (100). Anal. separated as small shiny plates (109 mg, 66.3% yield): mp 126Calcd for C14HzoNOC1J/3Hz0: C, 64.73; H, 8.02; N 5.39. Found: 127 "C; 'H NMR (DMSO-ds) 6 9.11 [d, J = 6.75 Hz, Py (C2 and C, 64.74; H, 7.81; N, 5.40. C6), 2 HI, 8.45 [d, J = 7.08 Hz, Py (C3 and C5), 2 HI, 8.10 [m, Ph (C2 and C6), 2 HI, 7.66 (m, Ph H, 3 H), 4.39 (h, cyclopropyl (C) (E)-l-(Cyclopropylamino)-3-phenyl-2-butene Hydromethine, 1H), 1.46 (m, cyclopropyl ethylene protons trans to N, chloride (14.HC1). Removal of the mother liquors from the 2 H), 1.30 (m, cyclopropyl ethylene protons cis to N, 2 H); 13C above crystallization gave 2.4 g of a yellow oil which dissolved NMR (CD30D) 6 146.50 (C2 and C6), 142.74 ((241, 133.38 [Ph in aqueous HC1. Upon concentration of this solution a solid was Chart I

CH,CECH

Potential

MA0 Inactivators

(Cl)], 130.86 [Ph (C2, C4, and C6)1, 125.76 [Ph (C3 and C5)1, 43.42 (cyclopropyl methine), 8.12 (cyclopropyl methylenes); UV (H2O) A, 305 nm (c = 10 500), (CH2C12) A, 295 nm. Anal. Calcd for C14H14NClO4: C, 56.86; H, 4.77; N, 4.74. Found: C, 56.73; H, 4.80; N, 4.70. (G)Oxalate Salt of 4-Cyclopropyl-l-methyl-4-piperidino1 (24). A solution of cyclopropyl bromide (4.5 mL, 55.8 mmol) in 75 mL of anhydrous tetrahydrofuran was added over a 20-min period with vigorous stirring to a mixture of Mg turnings (13.37 g, 550 mmol) which had been exposed previously to IZvapor. The warm reaction mixture was maintained under reflux for an additional 15 min, following which a solution of 1-methyl-4piperidone (23,4.53 mL, 36.8 mmol) in 75 mL of anhydrous tetrahydrofuran was added dropwise over a 15-minperiod. After heating under reflux for 1.5 h, the reaction mixture was cooled and the tetrahydrofurin was decanted from the Mg turnings, which were washed with additional tetrahydrofuran. The combined organic layers were extracted with 2 N Hi304 (4 X 25 mL), and the pH of the combined extracts was adjusted to 12 by the careful addition of 2 N NaOH. The product then was extracted with diethyl ether (5 X 30 mL), and the extract was dried over Na2S04,filtered, and evaporated to yield 4.27 g of a slightly yellow oil. This material was distilled under vacuum (57 "C/5 Torr) to yield a colorless oil (3.77 g, 66% yield): 'H NMR (CDC13) 6 4.74 (8, OH, 1H), 2.68 [dt, C(2)Hq and C(6)H,, 2 HI, 2.46 [dt, C(2), and C(6),, 2 HI, 2.37 (s, NCH3, 3 H), 1.78 [m, C(3)Hz and C(5)H2, 4 H), 1.07 (m, cyclopropyl methine, 1 H), 0.62 (m, cyclopropyl CH2CH2,4 H); GC/MS [isothermal at 80 "C for 1 min followed by a ramp of 20 W m i n for 8 min ( t =~ 3.9 min)] m/z 155 (M+, 43), 154 (22), 140 (5), 138 (8), 136 (91, 126 (191, 122 (loo), 108 (141, 98 (15), 70 (91),57 (29). The oxalate salt was prepared in and recrystallized from acetone: mp 92-94 "C; l3C NMR (CD30D) 6 166.45 (oxalic acid), 66.03 (C4), 51.83 (C2and C6), 43.96 (NCHs),34.97 (C3 and C5), 22.01 (cyclopropyl methine), 0.18 (cyclopropyl methylenes). Anal. Calcd for CllH1~N06.0.5H~O:C, 51.96; H, 7.87; N, 5.51. Found: 52.20; H, 7.88; N, 5.49. (H)Oxalate Salt of 4-Cyclopropyl-l-methyl-l,2,S,6-tetrahydropyridine. A solution of the above piperidinol (1.50 g, 9.67 mmol) and p-toluenesulfonic acid (2.02 g, 10.6 mmol) in 25 mL of benzene was heated under reflux, and the water collected by azeotropic distillation was measured with a Dean-Stark trap. After 5 h, 180r L of water (100%) had collected,and the reaction mixture was cooled and extracted with 1N NaOH (3 X 10 mL) followed by water (2 X 20 mL). The organic layer was dried over Na2SO4, filtered, and treated with a solution of oxalic acid (1.8 g, 20 mmol) in diethyl ether. The resulting white precipitate was recrystallized twice from hot acetone to provide 952 mg (53.3 %) of pure product: mp 110-111.8 "C; lH NMR (CDC13) 6 7.51 (bs, NH, 1 H), 5.35 (m, C-3 olefinic proton, 1 H), 3.55 and 3.40 (bs, allylicCH2N),3.04 (bs, NCH2,l H), 2.98 (s,NCH3,3H), 2.60 (bs, NCH2,l H), 2.22 (bs, CHZ,2 H), 1.45 (m, cyclopropyl methine, 1H), 0.75 (m, cyclopropyl ethylene protons trans to N, 2 H), 0.53 (m, cyclopropyl ethylene protons cis to N, 2 H); 13CNMR (CD3OD) 6 166.50 (oxalic acid), 139.47 (C4), 112.72 (C3), 53.18 (Cl), 51.78 (C6),42.75(C5),25.11 (NCHs),17.04 (cyclopropylmethine), 5.27 (cyclopropyl methylenes); GC/MS [isothermal at 60 OC for 1min followed by a ramp of 20 "C/min for 6 min ( t =~2.9 min)] m/z 137 (M+, 30), 136 (17), 134 (5), 122 (loo), 108 (181, 94 (19), 79 (34). Anal. Calcd for CllH17NOdJ/3H20: C, 56.64; H, 7.58; N, 6.00. Found C, 56.75; H, 7.38; N, 6.04. (I) 4-Cyclopropyl-1-methyl-4-piperidinol N-Oxide Hydrochloride (25-HC1). A solution of 4-cyclopropyl-1-methyl4-piperidinol (1.0 g, 6.5 mmol) and 50% m-CPBA (4.40 g, 12.7 mmol) in 25 mL of dichloromethane was stirred at room temperature for 2 h. The solvent was removed, and the oily residue was washed with diethyl ether to remove m-CBA. The ether-insoluble product was dissolved in 30 mL of ethanol containing 297 mg (8.13 mmol) of HCl. The addition of diethyl ether gave a precipitate that crystallized from ethyl acetate to provide 844 mg (62.4%) of analytically pure product: mp 169170 "C; 'H NMR (CD30D) 6 4.92 (8, OH, 2 H), 3.85 [dt, J = 1.26

Chem. Res. Toxicol., Vol. 5, No. 5, 1992 627 and 13.10 Hz, C(2)H, and C(6)H,, 2 HI, 3.63 [bd, J = 14.10 Hz, C(2)H, and C(6)H,, 2 HI, 3.55 (8, NCH3,3 H), 2.20 [dt,J 4.39 and 14.61 Hz, C(3)H, and C(5)H,, 2 HI, 1.72 [bd, J = 14.10 Hz, C(3)H, and C(5)H,, 2 HI, 0.94 (m, cyclopropyl methine, 1 H), 0.45 (m, cyclopropyl CHzCHz, 4 H); l13C NMR (CDaOD) 6 66.55 (C4), 62.75 (C2 and C6), 58.34 (C3 and C5), 32.33 (NCH,), 21.77 (cyclopropyl methine), 0.19 (cyclopropyl methylenes). Anal. Calcd for CgHlsN02CI: C, 52.05; H, 8.74; N, 6.74; C1, 17.07. Found C, 51.94; H, 8.73; N, 6.72; C1, 16.78. (J)Picrate Salt of 4-Cyclopropyl-l-methyl-2,3-dihydropyridinium (26). A solution of 4-cyclopropyl-1-methyl-4piperidinol N-oxide hydrochloride (283mg, 1.37 mmol) in lOmL of dichloromethane was treated dropwise with TFAA (1.58 g, 7.5 mmol) in 10 mL of dichloromethane. After stirring for 2 h at room temperature, the solvent was removed, and the UV, lH and 13C NMR spectra of the crude yellow solid were recorded: UV 318 nm (e = 6300); lH NMR (CDCl3) 6 8.10 (d, J = (H2O) A, 4.34 Hz, CH=N, 1H), 6.22 (d, J = 4.88 Hz, olefinic H, 1H), 3.81 (t,J = 9.42 Hz, allylic CH2N, 2 H), 3.61 (s, NCH3,3 H), 2.54 (t, J = 9.42 Hz, CH2,2 H), 1.86 (m, cyclopropyl methine, 1H), 1.32 (m, cyclopropyl ethylene protons trans to N, 2 H), 1.07 (m, cyclopropyl ethylene protons cis to N, 2 H); 13C NMR (CDC13) b 174.21 (Cs), 163.70 (Cs), 113.51 (Cd), 48.69 (NCHd, 46.81 (Cz), 24.68 (C3), 19.89 (cyclopropyl methine), 11.72 (cyclopropyl methylene). HPLC analysis (see General Methods) showed a single peak (tR = 7.33 min). The above residue in 20 mL of diethyl ether was added to a solution of picric acid (342 mg, 1.5 mmol) in acetone/EtzO (15 mL) which upon cooling gave 195 mg (39.1 %) of small orangish yellow needles: mp 103-104.4 "C; lH NMR (CD30D) 6 8.74 (8, Ar H, 2 H), 8.18 (dq, J = 1.12 and 4.84 Hz, CH-N, 1 H), 6.33 (d, J = 4.83 Hz, olefinic H, 1H), 3.83 (t, J = 9.13 Hz, allylic CHzN, 2 H), 3.60 (8, NCH3, 3 H), 2.57 (t,J = 9.37 Hz,CH2, 2 H), 1.95 (m, cyclopropyl methine, 1H), 1.22 (m, cyclopropyl ethylene protons trans to N, 2 H), 1.11 (m, cyclopropyl ethylene protons cis to N, 2 H); UV-vis (H2O) A,, 318 nm (C = 6300). Anal. Calcd for C15H16N407: C, 49.45; H, 4.43; N, 15.38. Found C, 49.36; H, 4.46; N, 15.29. (9) 4-Cyclopropyl-1-methylpyridiniumIodide (27). A tetrahydrofuran solution of cyclopropylmagnesium bromide, prepared as described above from magnesium turnings (970 mg, 40 mmol) and cyclopropyl bromide (2.42 g, 20 mmol), was added dropwise over a period of 15 min via a double needle to a solution of 4-bromopyridine (28),2.37g, 15 mmol) in 40 mL of anhydrous diethyl ether containingtetrakis(tripheny1phosphine)palladium(0) (58 mg, 0.05 mmol). The dark purple reaction mixture was heated under reflux for 24 h and then was passed through a column of neutral alumina (50 g), eluting first with diethyl ether (100 mL) followed by chloroform (100 mL) and finally 10% methylene/chloroform(240 mL). Iodomethane (10.6 g, 75 mmol) was added to the 10% methylene/chloroform eluent and the mixture was stirred for 18h. The residue obtained after removing the solvent was passed through a second column of neutral alumina (25 g) with 10% methylene/chloroform to yield 1.34 g of a yellow solid. Recrystallization from acetone yielded 1.05 g (51.9%)of small yellow needles: mp 137-138 "C; UV (H2O) A, 254 nm (c = 6900); 'H NMR (D20) 6 8.56 [d, J = 6.85 Hz, Py (C2 and C6), 2 HI, 7.69 [d, J = 6.85 Hz, Py (C3 and C5), 2 HI, 4.33 (8, NCH3,3 H), 2.29 (h, J = 4.73 Hz, cyclopropyl methine, 1H), 1.48 (m, cyclopropyl ethylene protons trans to N, 2 H), 1.16 (m, cyclopropyl ethylene protons cis to N, 2 HI; 13C NMR (D20, CH30Hused as external reference) 6 169.50 (C4), 146.46 (C2 and C6), 126.75 (C3 and C5), 50.10 (NCHa), 19.22 (cyclopropyl methine), 17.33 (cyclopropyl methylenes); GC/MS [isothermal at 50 "C for 1 min followed by a ramp of 25 "C/min for 8 min ( t =~ 2.9 min)] m/z 119 (M - 15,51),118 (loo), 117 (261,104 (6), 91 (37),78 (8),65(16),63 (12),51(19). Anal. Calcd for CgH12NI: C, 41.40; H, 4.63; N, 5.36. Found: C, 41.42; H, 4.58; N, 5.34. Enzymology. MAO-B was isolated from bovine liver mitochondria by the method of Salach (28),and traces of heme protein were removed by centrifugation through a sucrose gradient (29). The activity of the enzyme was determined spectrophotometrically at 250 nm (benzaldehyde) on a Beckman DU 50 spec-

628 Chem. Res. Toxicol., Vol. 5, No. 5, 1992 trophotometer using initial rate measurements (30-120 s) of the oxidation of 3.2 mM benzylamine. A unit of activity (equivalent to 3.7 nmol of protein) is defined as the amount of enzyme required to convert 1pmol of benzylamine to benzaldehyde in 1min. Stock solutions of the test compounds were prepared in phosphate buffer (100 mM, pH 7.4) and diluted with the same buffer to give concentrationsof0.05,0.067,0.10,0.20,1.0, and 2.5 mM. A0.5mL aliquot of each solution was added to a sample cuvette, which then was placed in a spectrophotometer that was maintained at 37 OC. After a 2-min equilibration period, MAO-B (0.185 nmol, 0.37 pM final concentration) was added and the absorbance at 320 nm (for compound 15) and that at 355 nm (for compounds 13 and 14) were monitored. MAO-B inhibition studies were conducted as follows: A 250 pM stock solution of the test compound in 100 mM sodium phosphate buffer (pH = 7.4) was diluted with buffer to obtain solutions of 10-200 pM. MAO-B (0.70 nmol, 1.85 pM final concentration) was added, and incubations were carried out with gentle agitation in a water bath incubator at 37 OC. A 50-pL aliquot was removed at 0, 5,10, 15, and 20 min and was added to a sample cuvette containing 5 mM MPTP2in phosphate buffer (450 pL, pH = 7.4). The rate of MPDP+ formation was determined by monitoring the absorbance at 345 nm every 3 s for 2 min. Similar substrate protection experiments were performed with benzylamine (2.5 mM) and the following concentrations of the cyclopropyl derivatives: 13 (25 pM); 14 (100 pM); 15 (1.0 mM). Each mixture (total volume = 400 pL) was agitated gently in a water bath incubator at 37 "C in the presence of MAO-B (1.88pM). Aliquots (25 pL) removed at 0, 5, 10, 15, 20, and 25 min were assayed for remaining enzyme activity using 5 mM MPTP (475 pL) as described above. Animal Toxicity Studies. Retired male breeder C57BL/6 mice (Harlan Sprague-Dawley, Inc., Madison, WI 53711) (22-30 g) were housed one per cage with free access to food and water under controlled temperature (19-22 OC),humidity, and lighting (12/12 h; lights on at 07:OO). Treated mice were administered the test compounds intraperitoneally in 0.1-0.2 mL of normal saline or, in the case of the poorly water-soluble compound 13, in Tween 80/normal saline (20230). The animals were sacrificed by cervical dislocation on day 14 following the first injection. The striata were dissected immediately (31),placed in a microcentrifuge tube, weighed, and homogenized in 10pLimgwet tissue weight using a solution of 5% trichloroacetic acid (TCA) containing 62.4 ng of 3,4-dihydroxybenzylamine(DHBA) as the internal standard. All samples were stored at -70 "C until analyzed. The frozen striata samples were brought to room temperature, and the supernatant obtained after centrifugation (14 000 rpm, 2 min, Eppendorf 5415 microcentrifuge) was analyzed for dopamine (DA) utilizing an HPLC system consisting of an Altex single piston pump, a Bioanalytical System LC-3 amperometric detector (potential set at +650 mV), a Rheodyne 7125 injector, a Kipp and Zonen single- or dual-pen strip chart recorder, and an Alltech EconosilC18, lO-pm, 25-cm X 4.6-mm column at room temperature. The mobile phase [90% aqueous (0.1 M sodium acetate, 1.98mM heptanesulfonic acid, 0.33 mM EDTA, adjusted to pH 3.9-4.1 with glacial acetic acid)/lO% methanol (v/v)l,was maintained at a flow rate of 1.0 mL/min. The quantitative estimations of DA were determined by using linear calibration curves derived from peak height ratios (DAIDHBA) obtained from standard stock solutions (5% TCA) containing DA at multiple levels and DHBA as the internal standard. 2 We have elected to monitor MAO-B activity with MPTP instead of benzylamine because of the ease with which one can estimate initial rates of formation of MPDP+ (10) (at 343 nm) and since the inhibition of the metabolism of MPTP may relate more directly to potential neurotoxic substrates of unknown structure that may be bioactivated by this enzyme. Studies were conducted with the N-cyclopropyl compound 13 (0.25-2.0 rM) and human placental MAO-A (0.93 pM) using kynuramine (5 mM) as substrate and monitoring rate of product formation at 314 nm every 3 s for 2 min (30).

Hall et al.

Scheme 111. Synthetic Pathway to the NCyclopropyl Compounds 13 and 14

14

/ \

U

17

Scheme IV. Synthetic Pathway to the 1Cyclopropyl-4-phenylpyridiniumSpecies 20

13-

18

22 R = O H 19 R = H

20

Results and Discussion Chemistry. Reaction of a-methylstyrene (16) with formaldehyde and cyclopropylamine (32)gave the 1,3oxazine 17which, upon further treatment with acid, yielded the desired 1,2,3,6-tetrahydropyridine13 (Scheme 111). A byproduct isolated from this reaction mixture has been assigned the structure (E)-l-(cyclopropylamino)-3-phenyl2-butene (14). lH NMR, HPLC, TLC, and GC/MS analyses (see Materials and Methods) indicated a single species with spectral properties expected for 14. Since 14 was formed under acidic conditions, we have assumed that the geometry about the double bond is E. This allylamine was included in our enzyme inactivation studies as an openchain analog of 13. Synthesis of the postulated MAO-B-catalyzedoxidation products of 13,the dihydropyridinium (19)and pyridinium (20) species, was approached by oxidation of 13 with m-CPBA in an attempt to generate the N-oxide intermediate 18 (Scheme IV). The anticipated decreased reactivity of the cyclopropylamino group relative to simple aliphatic amines due to its decreased basicity (33) led us to run the m-CPBA reaction at room temperature (rather than at 0 "C) and in the presence of an excess of the oxidizing agent. Treatment of the crude isolate from this reaction with TFAA produced an unstable intermediate (Amm 340 nm) which within minutes underwent conversion to a stable product (Amm 295 nm). This product proved to be the pyridinium species 20 which was characterized fully at its perchlorate salt (see Materials and Methods). These results argued against the N-oxide structure 18since, although unstable (34), dihydropyridinium derivatives such as 19 would not be expected to autoxidize so readily. Spectral and elemental analyses (see Materials and Methods) led us to assign the epoxy N-oxide structure 21 to this product. The stereochemical features of 21 remain to be established. The 340-nm-absorbing intermediate observed followingtreatment of 21 with TFAA presumably corresponds to a hydroxydihydropyridiniumspecies such as 22. The preparation of the 4-cyclopropyl analog 15 of MPTP was readily accomplished by treatment of l-methyl-4piperidone (23)with cyclopropylmagnesium bromide to yield the corresponding piperidinol24 which underwent acid-catalyzed dehydration t o give the desired tetrahy-

Chem. Res. Toxicol., Vol. 5, No. 5, 1992 629

Potential MA0 Znactivators

Scheme V. Synthetic Pathway to 15,26, and 27 15

23

24

25

V

Br I

28

29

CH3 127

dropyridine product 15 (Scheme V). Oxidation of 24 with m-CPBA yielded the corresponding N-oxide 25, which was purified as its hydrochloride salt since the free base isolated by alumina chromatography was contaminated with an inorganic substance, presumably alumina. Treatment of 25 with TFAA yielded the expected dihydropyridinium product 26, which was purified as its picrate salt. Compound 26 proved to be stable to air oxidation even in the presence of Pd/C, which is known to catalyze the air oxidation of dihydropyridinium compounds to the corresponding pyridinium products (35). The underlying physicochemical properties of 26 that lead to this stability are not apparent. An alternative synthetic approach to the pyridinium compound 27 (Scheme V) took advantage of the vinylic character of the cyclopropyl group (36) and the reactivity of the corresponding Grignard reagent in a tetrakis(triphenylphosphine)palladium(O)catalyzed cross-coupling reaction (37) with 4-bromopyridine (28). The resulting volatile 4-cyclopropylpyridine (29)was converted to the corresponding methiodide prior to purification to yield the desired pyridinium product 27 in 51.9% overall yield. To our knowledge, this is the first example of a palladium-catalyzed cross-coupling reaction using cyclopropylmagnesium bromide. Enzymology. Spectral analysis of incubation mixtures containing MAO-B (0.37 pM) and 13 (up to 2.5 mM) gave no evidence of substrate turnover. The poor substrate properties of 13 may be due to the electronic features of the cyclopropyl moiety but also are consistent with literature reports describing the lack of significant turnover of MPTP derivatives bearing an N-substituent larger than methyl (38,391. Similar steric considerations may apply to the acyclic allylamine 14 since no metabolite was detected spectrophotometrically in incubation mixtures of 14 and MAO-B even though cinnamylamine (30,Chart I) (40) and Nfl-dimethylcinnamylamine (31) (41) are excellent MAO-B substrates. Despite their limited substrate properties, several tertiary amines (421, including the clinically used propargylamine (Sbdeprenyl (12, Chart I), are excellent inhibitors of the A and/or B forms of the enzyme (43). In contrast to the weak inhibitor properties of MPTP, the N-cyclopropyl analog 13 of MPTP proved to be a good inhibitor of this enzyme. The extent of inactivation was time and inhibitor concentration dependent as expected for a mechanism-based inactivator. A plot of 1/[131 vs l/k&,, (obtained from the linear semilog plots of remaining enzyme activity vs time over an inhibitor concentration range of 0-100 pM 13) gave a straight line (Figure la)

from which kinact (0.7 min-') and KI (182 pM) were calculated. As shown in the inactivation plot (Figure lb), the preferred substrate benzylamine (BnNH2, 2.5 mM) protected MAO-B (1.85 pM) against inactivation by 13 (250 pM). Compound 13 also proved to be a good time- and concentration-dependent inhibitor of human placental MAO-A. The double-reciprocal plot (Figure 2) provided values for kinact and KI of 0.04 min-' and 0.93 pM, respectively. Thus, although the maximum rate of inactivation was almost 20 times slower for the A form than the B form of the enzyme, the affinity of 13 for the active site of MAO-A is almost 200 times greater than for MAOB. The limited amount of enzyme available prevented additional studies with MAO-A. The evidence that 13 is a mechanism-based inactivator of MA0 implies that the enzyme does process the compound as a substrate but that an intermediate formed in the catalytic process inactivates the enzyme prior to escaping the active site (44). In an additional attempt to detect product formation, we examined an incubation mixture consisting of 3.7 nmol (1 unit) of MAO-B (7.4 pM) and 12.5 nmol of 13 (25 pM). Under these conditions the formation of an unstable species absorbing a t 355 nm for the dihydropy(corresponding to the expected ,A, ridinium intermediate 19) was observed and reached a maximum by 10 min. By 90 min the UV maximum had corresponding to shifted completely to 305 nm, the ,A, the pyridinium product 20. The maximum conversion of 13 to 20 was estimated to be 3.3 nmol. Since 3.7 nmol of enzyme was inactivated during the process, the total amount of substrate processed by the enzyme must be 3.3 nmol + 3.7 nmol or 7.0 nmol, giving a partition ratio of 1.90. The efficiency with which compound 13 inactivates MAO-B and MAO-A argues that the N-cyclopropyl group must play an important role in the inactivation process. I t seems reasonable to postulate a radical reaction pathway (Scheme VI) analogous to that proposed by Silverman for N-benzylcyclopropylamine (Scheme 11) (7). Initial oneelectron transfer from 13 to FAD would generate the aminium radical 32. This intermediate would have the option to ring open to the reactive primary radical 33 (pathway a leading to enzyme inactivation) or to lose an a-proton to give the secondary carbon-centered radical 34, which subsequently undergoes a second one-electron loss to generate the dihydropyridinium intermediate 19 (pathway b) . The acyclic derivative 14 also inactivated MAO-B. The double-reciprocal plot (Figure 3) constructed from the linear kinetic data of inhibitor concentration vs rate of enzyme inactivation gave values for kinact and KI of 0.38 min-l and 197 pM, respectively. Excellent protection against the inactivation MAO-B (1.85 pM) by 14 (100pM) was obtained with 2.5 mM benzylamine (data not shown). Silverman has reported that cinnamylamine (30) exhibits no inactivation properties with MAO-B while N,Ndimethylcinnamylamine (31) is only a weak inhibitor: K I = 1330 pM; kinact = 0.21 min-' (39). Consequently, the importance of the cyclopropyl moiety in the inactivation process is reinforced by these resulta and is consistent with the large number of primary and secondary N-cyclopropyl-bearing amines which are effective inhibitors of MAO-B (8, 9, 45, 46).

Hall et al.

630 Chem. Res. Toxicol., Vol. 5, No. 5, 1992

301 -0.020.00

' I

1 : 0

0.02 0.04 0.06 0.08 0 . 1 0 0.12

b)

10

20

30

Time (min)

Figure 1. Kinetic analysis of the inactivation of MAO-B by 13: (a) l / k & vs 1/[13] plot constructed from linear semilog inactivation plots; (b) plot showing protection by BnNHz of the MAO-B inactivation by 13. 140120-

I

-1.5

-0.5

0.5

1.5

2.5

3.5

4.5

1 / [ 1 3 (pM)1

I

Figure 2. Double-reciprocal plot of data obtained from the concentration- and time-dependent inhibition of MAO-A by 13. Scheme VI. Proposed Mechanism of M A 0 Inactivation by Compound 13

32

33

I

34

Inactivated Enzyme

The 4-cyclopropylanalog 15of MPTP also was examined for its potential MAO-B inactivator properties. The rationale for this effort was based in part on earlier results which led us to postulate that the inactivation of MAO-B be MPTP involves a radical species derived from MPDP+ (10, Chart I), the two-electron oxidation product of the reaction (47).We had observed that the rate of inactivation of MAO-B in the presence of the 6,6-dideuterio-MPTP analog 9 - 4 (Chart I) was the same as that observed with MPTP even though the D(V,,/K,) value for the conversion of MPTP to MPDP+ was 8.01. On the other hand, a kinetic isotope effect of 1.9 was observed for the inactivation of MAO-B by MPTP-2,2,6,6-d4 ( 9 - 4 ) . On the basis of these data, we postulated that the dihydropyridine free base 35 derived from MPDP+ is converted to the corresponding resonance-stabilized carbon radical 36 which, being benzylic and bisallylic, might possess the appropriate stability/reactivity characteristics to alkylate and inactivate the enzyme (Scheme VII). The reported

metabolism-dependent covalent binding of tritium-labeled MPTP to MAO-B (16) is consistent with this proposal as is the report that MPDP+ inactivates MAO-B at a faster rate than does MPTP (48). An analogous reaction sequence with the 4-cyclopropyl derivative 15 would lead to the dihydropyridine free base 37 and thence to the radical 38. Ring opening of this cyclopropyl carbinyl radical intermediate would give the primary carboncentered radical 39, the putative inactivating species. This pathway, however, is not consistent with a recent report showing that the 3,3-dimethyl analog 40 (Chart I) of MPDP+ is as effective an inactivator of MAO-B as is MPDP+ (49). The 4-cyclopropyl compound 15 proved to be a timeand concentration-dependent inhibitor of MAO-B. Furthermore, benzylamine (2.5 mM) protected against enzyme (1.85pM) inactivation by 1mM 15(data not shown).Values for kinactand K I ,however, could not be obtained because, at lower substrate concentrations, no loss of enzyme activity was detected. Under saturating conditions (1.0 mM 15), the rate constant for inactivation was approximately 0.1 min-l. The possibility that the weak inactivator properties of 15 were due to its inefficient initial oxidation to the dihydropyridinium intermediate 26 could be ruled out. As has been reported with many l-methyl-4-substituted1,2,3,64etrahydropyridinederivatives (151, the 4-cyclopropyl analog 15 of MPTP proved to be an excellent MAO-B substrate. The following evidence established the structure of the enzyme-catalyzed product as the dihydropyridinium species 26: (1)The chromophore (Amm 320 nm) of the enzyme-generated product corresponded to that of synthetic26; (2) treatment of the postincubation 320 nm) with sodium borohydride (to reduce mixture (A, the azomethine double bond) or sodium cyanide [to add across the azomethine double bond (50)] led to the immediate loss of the 320-nm chromophore; and (3) the HPLC/diode array tracing of the supernatant fraction obtained following centrifugation of the trifluoroacetic acid-treated postincubation mixture was identical with that of the synthetic dihydropyridinium trifluoroacetate standard. Kinetic studies provided linear initial velocity vs concentration plots from which the Lineweaver-Burk plot shown in Figure 4 was constructed. The respective Vmaxand K , values for 15,187 min-l and 192 pM, and for MPTP, 204 min-1 and 390 pM (15), are comparable. No evidence of further oxidation of the dihydropyridinium metabolite was observed during the course of the experiment, suggesting that 26 is likely to be a weak inactivator

Chem. Res. Toxicol., Vol. 5, No. 5, 1992 631

Potential MA0 Znactivators

Table I. Summary of Neurotoxic/NeuroprotectiveProperties of the N-Cyclopropyl Derivatives 13, 14, and 15 in the C57BL/6 Mouse Model compound none

treatment (administered ip)

MPTP

191 pmollkg 191 pmol/kg 288 pmol X 2, hourly (total dose: 576 pmol)

13 15 none 13 13 none 13 none 13 14 deprenyl

191 pmollkg X 2, hourly (total dose: 382 pmollkg) 191 pmollkg X 3, hourly (total dose: 573 pmollkg) 191 pmollkg X 2, hourly (total dose: 382 pmollkg) 96 pmol/kg followed by 191 pmollkg MPTP 6 h later 96 pmollkg followed by 191 pmollkg MPTP 6 h later 48 pmol/kg followed by 40 mglkg MPTP 6 h later 30

1

m

20-

Y

.

a

10-

I

/ o

'

.io

1

'

io

1

'

1

'

1

40

30

20

Figure 3. Double-reciprocal plot for the inactivation of MAO-B by 14. 5.4

-

-6

10

0

20

Figure 4. Double-reciprocal plot of the MAO-B-catalyzed oxidation of 15.

Scheme VII. Proposed Pathway for the Inactivation of MAO-Bby Tetrahydropyridines R

I 37: R

I

I

CH3 35: R = C,H, C3H5

CH3 CH3 36: R = C,H,

38: R

I CH,

39

C3H5

of MAO-B at best. According to the mechanism proposed in Scheme VII, the lack of enzyme inhibitor properties of 15 (and 26) may be a reflection of the stability of the dihydropyridinium metabolite 26 with respect to its conversion to the putative reactive intermediate 39. Potential Neurotoxic/NeuroprotectiveProperties. The potential MPTP-type nigrostriatal degenerative properties of the N-cyclopropyl compound 13 were compared with those of MPTP in the C57BL/6 mouse model developed by Heikkila (51). A very modest reduction of

dopamine levels (pg/g of tissue) 21.8 f 4.0 (n = 11) 2.9 f 0.8 (n = 3) 16.6 f 5.1 ( n = 5) 22.0 f 1.1 (n = 5) 16.7 f 5.7 (n = 6) 14.1 f 5.6 (n = 10) 12.4 f 5.6 (n = 6) 19.4 f 6.8 (n = 10) 17.8 f 4.8 (n = 6) 22.95 f 0.96 ( n = 6) 12.90 f 4.00 ( n = 6) 11.36 f 4.43 (n = 6) 23.67 f 1.30 (n = 6)

% of control 100 13 76 99 100 74 84 100 93 100 56 49 103

the neostriata1 level of DA was observed under conditions that led to an 87% depletion of DA by MPTP (Table I). Additional experiments were conducted with multiple doses of 13, but the depleting effects still were modest. Intracerebral microdialysis studies on a variety of pyridinium derivatives indicate that the N-cyclopropylpyridinium compound 20 should be a potent MPP+-type neurotoxin (52). Consequently, the marginal neurotoxic properties of 13 are likely to be due to its poor conversion in vivo to 20 as would be expected on the basis of its poor MAO-B substrate and good MAO-B inhibitor properties. The 4-cyclopropyl analog 15 displayed no MPTP-type neurotoxicity in the mouse at a total dose 3 times the toxic MPTP dose. This lack of toxicity was expected since, even though an excellent substrate of MAO-B,the stability of the resulting dihydropyridinium metabolite 26 precluded its conversion to the pyridinium product 27. The potential neuroprotective properties of the N-cyclopropyltetrahydropyridine derivatives 13 and 14 were compared with those of (SI-deprenyl by assessing the effects of drug pretreatment on the DA-depleting properties of a toxic dose of MPTP administered 6 h later. The results show moderate protection by these two compounds compared to (8)-deprenyl, which was considerably more effective at only 1/2 the dose. Since MPTP was administered 6 h after the animals had been treated with the test compounds, it is likely that the observed effects are a consequence of MAO-B inactivation. Summary The present studies represent an initial attempt to develop MAO-B inactivators that would exploit the special interactions observed between MPTP and MAO-B. On the basis of both theoretical considerations and published experimental results, the cyclopropyl group was selected as an attractive functionality to mediate the metabolismdependent inactivation of this enzyme. The results of these studies show that the C(4) cyclopropyl analog 15 is a poor inactivator of MAO-B possibly because the dihydropyridinium metabolite 26 resists subsequent oxidation to the radical 39, the species postulated to mediate enzyme inactivation. Studies on the MAO-B-catalyzed oxidation with the N-cyclopropyl compound 13 to the pyridinium species 20 confirmed its expected poor MAO-B substrate properties. This compound displayed moderately good time- and concentration-dependent inactivation properties but was without selectivity for the B form of the enzyme. The observed protection against the in vivo neurotoxicity of MPTP, while only partial, indicates that appropriately structured analogs may be able to block the in vivo

632 Chem. Res. Toxicol., Vol. 5, No. 5, 1992

bioactivation of MPTP. Future studies will focus on the mechanism of inactivation of MA0 by 13 and the evaluation of related tetrahydropyridine derivatives 8s potential enzyme inactivators.

Acknowledgment. We thank Dr. Creed Abell for the sample of human placental MAO-A. This work was supported by National Institute of Neurological and Communicative Disorders and Stroke Grant NS 28792 and the Harvey W. Peters Research Center for Parkinson’s Disease and Disorders of the Central Nervous System. References Singer,T. P.,Trevor, A. J.,and Castagnoli,N., Jr. (1987)Biochemistry of the neurotoxic action of MPTP-Or how a faulty batch of a ‘designer drug” led to Parkinsonism in drug abusers. Trends Biochem. Sci. 12, 266-271. Dostert, P. L., Strolin Benedetti, M., and Tipton, K. F. (1989) Interactions of monoamine oxidase with substrates and inhibitors. Med. Res. Reo. 9, 45-89. Johnston, J. P. (1968) Some observations upon a new inhibitor of monoamineoxidase in brain tissue. Biochem. Pharmacol. 17,12851297. Knoll, J., and Magyar, K. (1972) Some puzzling pharmacological effects of monoamine oxidase inhibitors. In Adu. Biochem. Psychopharmacol. (Costa, E., and Sandler, M., Eds.) Vol. V, pp 393408, Raven Press, New York. Bach, A. W. J., Lan, N. C., Johnson, D. L., Abell, C. W., Bembenek, M. E., Kwan, S.-W., Seeburg, P. H., and Shih, J. C. (1988) cDNA cloning of human liver monoamine oxidase A and B: Molecular basis of differences in enzymatic properties. Proc. Natl. Acad. Sci. U.S.A. 85, 4934-4938. Hsu, Y.-P. P., Powell, J. F., Sims, K. B., and Breakefield, X. 0. (1989) Molecular genetics of the monoamine oxidases. J. Neurochem. 53,12-18. Silverman, R. B. (1991) The use of mechanism-based inactivators to probe the mechanism of monoamine oxidase. Biochem. SOC. Trans. 19, 201-206. Silverman, R. B., and Yamasaki, R. B. (1984) Mechanism-based inactivation of mitochondrial monoamine oxidase by N-(1-methylcyclopropy1)benzylamine. Biochemistry 23, 1322-1332. Silverman, R. B., and Zieske, P. A. (1985)Mechanism of inactivation of monoamine oxidase by 1-phenylcyclopropylaine. Biochemistry 24, 2128-2138. Silverman,R. B., and Zieske, P. A. (1986) 1-Phenylcyclobutylamine, the first in a new class of monoamine oxidase inactivators. Further evidence for a radical intermediate. Biochemistry 25, 341-346. Gates, K. S., and Silverman, R. B. (1989) Model studies for the mechanism of inactivation of monoamine oxidase by 5-(aminomethyl)-3-aryl-2-oxazolidinones. J. Am. Chem. SOC.111,8891-8895. Silverman, R. B., and Banik, G. M. (1987) (Aminoalky1)trimethylsilanes. A new class of monoamine oxidase inactivators. J. Am. Chem. SOC.109, 2219-2220. Kim, J.-M., Bogdan, M. A,, and Mariano, P. S. (1991) SET photochemistry of flavin-cyclopropylamine systems. Models for proposed monoamine oxidase inhibition mechanisms. J.Am. Chem. SOC.113, 9251-9257. Yelekci, K., Lu, X., and Silverman, R. B. (1989) Electron spin resonance studies of monoamine oxidase B. First direct evidence for a substrate radical intermediate. J. Am. Chem. SOC.111,11381140. Youngster,S. K., McKeown, K. A., Jin,Y.-Z., Ramsay,R.R., Heikkila, R. E., and Singer, T. P. (1989) Oxidation of analogs of l-methyl4-phenyl-1,2,3,6-tetrahydropyridine by monoamine oxidases A and B and the inhibition of monoamine oxidases by the oxidation products. J. Neurochem. 53, 1837-1842. Singer, T. P., Salach, J. I., and Crabtree, D. (1985) Reversible inhibition and mechanism-based irreversible inactivation of monoam(MPine oxidases by l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine TP). Biochem. Biophys. Res. Commun. 127, 707-712. Kopin, I. J., and Markey, S. P. (1989)MPTP toxicity: Implications for research in Parkinson’s Disease. Annu. Reu. Neurosci. 11, 8196. Langston, J. W., Irwin, I., Langston, E. B., and Forno, L. S. (1984) 1-Methyl-4-phenylpyridiniumion (MPP+): Identification of a metabolite of MPTP, a toxin selective to the substantia nigra. Neurosci. Lett. 48, 87-92. Heikkila, R. E., Manzino, L., Cabbat, R. S., and Duvoisin, R. C. (1984) Protection against the dopaminergic neurotoxicity of l-methyl-l-phenyl-1,2,3,6-tetrahydropyridine by monoamine oxidase inhibitors. Nature 311, 467-469.

Hall et al. (20) Langston, J. W., Irwin, I., Langston, E. B., and Forno, L. S. (1984) Pargyline prevents MPTP-induced parkinsonism in primates. Science 225, 1480-1482. (21) Cohen, G., Pasik, P., Cohen, B., Leist, A. Mytilineou, C., and Yahr, M. D. (1984) Pargyline and deprenyl prevent the neurotoxicity of l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) in monkeys. Eur. J. Pharmacol. 106, 209-210. (22) Collins, M. A., and Neafsey, E. J. (1985) @-Carbolineanalogues of N-methyl-4-phenyl-l,2,5,6-tetrahydropyridine (MPTP): endogenous factors underlying idiopathic parkinsonism? Neurosci. Lett. 55, 179-184. (23) Tanner, C. M., and Langston, J. W. (1990)Do environmentaltoxins cause Parkinson’s disease?-A critical review. Neurology 40 (Suppl. 3), 17-30. (24) The Parkinson Study Group. (1989) Effect of deprenyl on the progression of disability in early Parkinson’s disease. N. Engl. J. Med. 321, 1364-1371. (25) Tetrud, J. W., and Langston, J. W. (1989) The effect of deprenyl (selegiline) on the natural history of Parkinson’s disease. Science 245, 519-522. (26) Birkmayer, W., Knoll, J., Reiderer, P., Youdim, M. B. H., Hars, V., and Marton, J. (1985) Increased life expectancy resulting from additionof L-deprenyl to Madopar treatment in Parkinson’sdisease:a long-term study. J. Neural Transm. 64, 113-127. (27) Pitts, S. M., Markey, S. P., Murphy, D. L., and Weisz, A. (1986) Recommended practices for the safe handling of MPTP. In MPTP: A Neurotoxin Producing a Parkinsonian Syndrome (Markey, S . P., Castagnoli, N., Jr., Trevor, A. J., and Kopin, I. J., Eds.) pp 703-716, Academic Press, New York. (28) Salach, J. I. (1979)Monoamine oxidase from beef liver mitochondria: simplified isolation procedure, properties, and determination of its cysteinyl flavin content. Arch. Biochem. Biophys. 192, 128-137. (29) Weyler, W., and Salach, J. I. (1981) Iron content and spectral properties of highly purified bovine liver monoamine oxidase. Arch. Biochem. Biophys. 212, 147-153. (30) Salach, J. I., and Weyler, W. (1987) Preparation of the flavincontaining aromatic amine oxidases of human placenta and beef liver. Methods Enzymol. 142, 627-637. (31) Ricaurte, G. A., Langston,J. W., Delanney, L. E., Irwin, I., Peroutka, S. J., and Forno, L. S. (1986) Fate of nigrostriatal neurons in young m a t u r e mice given l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine: A neurochemical and morphological reassessment. Brain Res. 376, 117-124. (32) Schmidle, C. J., and Mansfield, R. C. (1956) The aminomethylation of olefins. V. A new synthesis of 4-phenylpyridine and related compounds. J. Am. Chem. SOC.78, 1702-1705. (33) Hanzlik, R. P., Tullman, R. H., and Kishore, V. (1980)Inactivation of cytochromeP-450 enzymes by cyclopropylamines. InMicrosomes, Drug Oxidations and Chemical Carcinogenesis(Coon,M. J., Conney, A. H., Estabrook, W., Gelboin, H. V., Gillette, J. R., and O’Brien, P. J., Eds.) pp 327-330, Academic Press, New York. (34) Subramanyam, B., Woolf, T., and Castagnoli, N., Jr. (1991) Studies on the in vitro conversion of haloperidol to a potentially neurotoxic pyridinium metabolite. Chem. Res. Toxicol. 4, 123-128. (35) Gessner, W., and Brossi, A. (1985)Reduction of phenyl-substituted pyridinium methiodides with sodium borohydride. Formation of amine-borane complexes in water. Synth. Commun. 15,911-916. (36) Crisp, G. T., and Flynn, B. L. (1990) Palladium-catalyzed coupling ofuridinetriflatewithorganostannanes. TetrahedronLett. 31,13471350. (37) Pelter, A,, Rowlands, M., and Clements, G. (1987) Cross-coupling reactions for the preparation of 2-arylfurans, 2-benzylfuran and 2-cinnamylfuran. Synthesis, 51-53. (38) Heikkila, R. E., Manzino, L., Cabbat, F. S., and Duvoisin, R. C. (1985) Studies on the oxidation of the dopaminergic neurotoxin l-methyl-4-phenyl-l,2,5,6-tetrahydropyridine by monoamineoxidase B. J. Neurochem. 45, 1049-1054. (39) Brossi, A., Gessner, W. P., Fritz, R. R., Bembenek, M. E., and Abell, C. W. (1986) Interaction of monoamine oxidase B with analogues of l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine derived from prodinetype analgesics. J. Med. Chem. 29, 444-445. (40) Hiebert,C. K.,andSilverman,R. B. (1988) l-Methyl-4-phenyl-l,2,3,6tetrahydropyridine analogues. Inactivation of monoamine oxidase by conformationallyrigid analoguesof N,N-dimethylcinnamylamine. J. Med. Chem. 31, 1566-1570. (41) Fries, D. S., de Vries, J., Hazelhoff, B., and Horn, A. S. (1986) Synthesis and toxicity toward nigrostriatal dopamine neurons of l-methyl-l-phenyl-l,2,3,6-tetrahydropyridine (MPTP) analogues. J. Med. Chem. 29,424-427. (42) Barwell, C. J., Basma, A. N., Lafi, M. A. K., and Leake, L. D. (1989) Deamination of hordenine by monoamine oxidase and its action on vasa deferentia of the rat. J. Pharm. Pharmacol. 41, 421-423. (43) Fowler, C. J., and Ross, S. B. (1984) Selective inhibitors of monoamine oxidase A and B: biochemical, pharmacological, and clinical properties. Med. Res. Rev. 4, 323-358.

Potential MA0 Inactivators (44) Silverman, R. B., and Hoffman, S. J. (1984) The organic chemistry of mechanism-based enzyme inhibition: a chemical approach to drug design. Med. Rea. Reu. 4, 415-457. (45) Tipton, K. F., McCrodden, J. M., Kalir, A. S., and Youdim, M. B. H. (1982) Inhibition of rat liver monoamine oxidase by a-methyl andN-propargyl-amine derivatives. Biochem. Pharmacol. 31,12511255. (46) Stlverman, R. B., and Hoffman, S. J. (1981) N-(1-methyl)cyclopropylbenzylamine: a novel inactivator of mitochondrial monoamine oxidase. Biochem. Res. Commun. 101, 1396-1401. (47) Ottoboni, S., Caldera, P., Trevor, A., and Castagnoli, N., Jr. (1989) Deuterium isotope effect measurements on the interactions of the neurotoxin l-methyl-4-phenyl-l,2,3,6-tetrahydropyridme with monoamine oxidase B. J.Biol. Chem. 264, 13684-13688. (48) Singer,T.P.,andRamsay,R.R. (1991)Theinteractionofmonoamine oxidases and tertiary amines. Biochem. SOC. Trans. 19, 211-215. (49) Hiebert,C. K.,Sayre, L. M., and Silverman, R. B. (1989)Inactivation of monoamine oxidase by 3,3-dimethyl analogues of l-methyl-4phenyl-l,2,3,6-tetrahydropyridine and l-methyl-4-phenyl-2,3-dihydropyridinium ion. J. Biol. Chem. 264, 21516-21521. (50) Peterson, L. A., Caldera, P. S., Trevor, A., Chiba, K., and Castagnoli, N., Jr. (1985) Studies on the l-methyl-4-phenyl-2,3-dihydropyri-

Chem. Res. Toxicol., Vol. 5, No. 5, 1992 633 dinium species 2,3-MPDP+, the monoamine oxidase catalyzed oxidation product of the nigrostriatal toxin 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP). J.Med. Chem. 28,1432-1436. (51) Heikkila, R. E., Hess, A., and Duvoisin, R. C. (1984) Dopaminergic neurotoxicity of l-methyl-4-phenyl-l,2,5,6-tetrahydropyridine in mice. Science 224, 1451-1453. (52) Rollema, H., Johnson, E. A., Booth, R. G., Caldera, P., Lampen, P., Youngster, S. K., Trevor, A. J., Naiman, N. A., and Castagnoli, N., Jr. (1990)In vivo intracerebral microdialysisstudies in rata of MPP+ analogues and related charged species. J. Med. Chem. 33, 22212230.

Registry No. 13, 142798-22-9; 13-HC1, 142798-23-0; (E)-14, 142798-24-1; (E)-14sHCl, 142798-25-2; 15, 142798-26-3; 17.HC1, 142798-27-4;20+C104-,142798-29-6;21*3-C1CsHdCOzH,14279831-0; 23,1445-73-4; 24.oxalate, 142798-33-2; 25.HC1,142798-343; 26+.picrate-, 142798-36-5; 27+-1-, 142798-37-6; 28, 1120-87-2; HCHO, 50-00-0;cyclopropylamine, 765-30-0; cyclopropyl bromide, 4333-56-6; 4-cyclopropyl-1-methyl-4-piperidinol N-oxide, 142798-32-1; cyclopropylmagnesium bromide, 23719-80-4; monoamine oxidase, 9001-66-5; a-methylstyrene, 98-83-9.