Pharmacology & Toxicology 1991, 69, 3842.

Investigation of the Possible Dopaminergic Toxicity of l-Methyl-3-phenyl-1,2,3,6-tetrahydropyridine, an Isomer to

the Neurotoxin MPTP Kristina Nilsson', Anders Hallberg', Erik Pileblad2* and Anders h e k 3

'Department of Organic Pharmaceutical Chemistry, Uppsala Biomedical Center, Uppsala University, Box 574, S-75 1 23 Uppsala, *Department of Pharmacology, University of Goteborg, Box 33031, S-400 33 Goteborg, and 'Department of

Pharmacology 2, AB Draco, Box 34, S-22100 Lund, Sweden

(Received December 6, 1990; Accepted February 14, 1991)

Abstract: I-Methyl-3-phenyl- 1,2,3,6-tetrahydropyridine (M-3-PTP) is an analogue to the Parkinson-producing dopamin- ergic toxin I-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP). M-3-PTP, and simple analogues thereof, are versatile intermediates in organic synthesis. The present study was undertaken to investigate the possible dopaminergic toxicity of M-3-PTP. Male albino mice were injected with 50 mg/kg of either MPTP or M-3-PTP and dopamine (DA) and its metabolites were determined 2 hr and 7 days after the administration. Two hr after MPTP profound acute changes in brain DA metabolism were found, i.e. an approximately 50% reduction in the concentration of DA together with a 10- fold increase in the level of 3-methoxytyramine. Seven days after MPTP, DA and metabolites were markedly reduced which is consistent with a degeneration of the dopaminergic neurones. In contrast M-3-PTP produced no acute or long- term alterations in the concentrations of DA and its metabolites in mouse brain. Furthermore, in vitro experiments show that M-3-PTP does not inhibit monoamine oxidase B. Thus, the present data show that M-3-PTP is devoid of dopaminergic toxicity in mouse brain and is not likely to produce Parkinson's disease in humans. The lack of toxicity is probably explained by the low affinity of M-3-PTP for monoamino oxidase B.

1 -Methyl-Cphenyl- 1,2,3,6-tetrahydropyridine (MPTP) (fig. 1,l) is a neurotoxin known to destroy central dopaminergic neurones in several species, including humans (Davis et al. 1979; Langston et al. 1983), monkeys (Burns et al. 1983), and mice (Hallman et al. 1984; Heikkila et al. 1984), and to produce symptoms indistinguishable from those seen in Parkinson's disease (Langston et al. 1983). These obser- vations have gained a considerable interest and it is hoped that research regarding the mechanism of action of MPTP, and analogues thereof, would increase the understanding of the aetiology and pathogenesis of Parkinson's disease.

A number of MPTP analogues produce MPTP-like neurotoxicity in experimental animals following parenteral administration. These synthesized analogues are 4-aryl- (Johannessen et al. 1987; Youngster et al. 1987), 4-heteroar- yl- (Fuller et al. 1986; Fuller & Hemrick-Luecke 1987), or 4-cyclohexyl- (Johannessen et al. 1987; Youngster et al. 1987) tetrahydropyridine derivatives. It seems, however, un- likely that they would cause Parkinson's disease because they are not known to occur in foods or elsewhere in the environment. The Perry group studied 2-phenylpyridine and 3-phenylpyridine, the only compounds related to MPTP and known to be present in human diet (Perry et al. 1988). Both are present in tea (Vitzthum et al. 1975a), and 3- phenylpyridine is also present in peppermint, spearment oil (Sakurai et ~1.1983) and in roasted cocoa (Vitzthum et al. 1975b). The related N-methylated tetrahydro derivates of

* To whom correspondence should be directed.

2- and 3-phenylpyridine were also studied, and found to be neurotoxic in mice, producing neurological symptoms immediately after injection and causing death in some ani- mals. The 3-phenyl-tetrahydropyridine mixture studied (Perry et al. 1988), consisting of a 1:l ratio of l-methyl- 3-phenyl-l,2,5,6-tetrahydropyridine (fig. 1,2) and (R,S)-1- methyl-3-phenyl-l,2,3,6-tetrahydropyridine (M-3-PTP) (fig. 1,3), did however not lower the content of dopamine (DA) and its metabolites in the striatum of mice. Youngster et al. (1987) studied 2 (fig. 1) and found that, at relatively low doses, this compound lacked dopaminergic neurotoxicity. Interestingly, Rollema et al. (1990) recently found that the quarternary analogue of 3, i.e. M-3-PPf, is toxic to dopam- inergic neurones in rat brain.

We have a particular interest in M-3-PTP, since we handle large quantities of closely related derivatives (fig. 2,4). The pharmacological profile of these compounds are under evaluation and, besides, they are useful as precursors to various bioactive molecules, e.g. the widely investigated DA autoreceptor agonist 3-PPP (fig. 2,s) (e.g. Clark et al. 1985). Most of our precursors are substituted in the aromatic ring, but aromatic substitution of MPTP does not preclude toxicity, for example the tetrahydropyridine (fig. 2,6) in- duces MPTP-like neurotoxicity (Youngster et al. 1987). Ful- ly saturated phenylpiperidines are not substrates of mono- amine oxidase B (MAO-B) (Heikkila et al. 1985a & b) indicating that an allylic double bond is required fo activity.

We have undertaken an acute as well as a long term study on the effects of M-3-PTP (racemic) (fig. 1,3) on central dopaminergic metabolism.

NO DOPAMINERGIC TOXICITY OF M-3-PTP 39

A

I (MPTP) 2 3 (M-3-PTP)

Fig. 1. Structural formulas for I-methyl-4-phenyl-l,2,3,6-tetrahyd- ropyridine (MPTP) (I), I-methyl-3-phenyl- 1,2,5,6-tetrahydropyridi- ne (2) and (R,S)- I-methyl-3-phenyl- 1,2,3,6-tetrahydropyridine (M- 3-PTP) (3).

Materials and Methods

Chemistry. Materials. Palladium acetate (Johnson Matthey Chemicals), tri- o-tolylphosphine (Aldrich), silver nitrate (Labassco), iodobenzene (Janssen), and 1 -methyl- 1,2,3,6-tetrahydropyridine hydrochloride (Dr. Schuler & Lange) were used as received. Triethylamine was distilled from potassium hydroxide, and solvents were distilled and stored over appropriate molecular sieves before use.

Chemical analysis. 'H NMR spectra were recorded at 300 MHz in deuteriochloroform on a Varian XL 300 spectrometer. Mass spectra were obtained on a Finnigan 4021 (Data System Incos 2100) gas chromatograph-mass spectrometer at an ionization potential of 70 eV. Quantiative gas chromatographic analyses were performed on a Varian 3300 instrument equipped with a (2 m x 2 mm) glass column of 5 % OV 17 on Chromosorb W. GC-yields were deter- mined using naphthalene as internal standard. For flash chromato- graphy TLC-silica gel 60 H (15 pm, E. Merck, No. 11695) was used. Elemental analyses were obtained from Mikro AB, Uppsala, Sweden.

Experimental. I-Methyl-3-phenyl-I,2,3.6-tetrahydropyridine ( M - 3-PTP) (fig. 3,3). A mixture of 1.7 g (10 mmol) of silver nitrate, 0.35 g (2.7 mmol) of naphthalene, 0.1 1 g (0.50 mmol) of palladium acetate, 0.61 g (2.0 mmol) of tri-o-tolylphosphine, 4.0 g (40 mmol) of triethylamine, 2.0 g (10 mmol) of iodobenzene, 2.7 g (20 mmol) of I-methyl-l,2,3,6-tetrahydropyridine hydrochloride, and 20 mL of acetonitrile was magnetically stirred and heated at 100" for 3 hr, in a 50 ml heavy-walled and thin-necked Pyrex tube, sealed with a Screw cap fitted with a Teflon gasket. The reaction mixture was diluted in diethyl ether and the tarry mixture was filtered by suction. The ethereal solution was extracted with 0.1 M HCI, and the com- bined water phases were made alkaline (2 M NaOH) and extracted with diethyl ether. The organic phase was dried (Na,SO,) and evap- orated thoroughly. The resulting oil was subjected to flash chrom- atography, the crude product was dissolved in dichloromethane and evaporated on coarse gel before application to the column. Dichloromethane/methanol, 95/5, was used as eluent, 0.62 g (36%) of the title compound (3) was obtained as a yellow oil.

For analytical purpose the product was converted to the oxalate

salt and recrystalized from methanol/diethyl ether [mp 147-149" (uncorrected)].

'H NMR (free amine) 6 2.20 (dd, 1 H, NCH,CHPh, J = 1 1.2 and 8.8 Hz), 2.35 (s, 3 H), 2.84 (m, 1 H, NCH,CH=), 2.94 (dd, 1 H, NCH,CHPh, J = 11.2 and 5.5 Hz), 3.18 (m, 1 H, NCH,CH=), 3.66 (m, 1 H, CHPh), 5.80 (m. 1 H, NCH,CH=CH), 5.89 (m. 1 H, NCH,CH=), 7.2g7.34 (m, 5 H). The structure was further con- firmed by COSY and decoupling experiments, in which the vinylic hydrogens and the methine hydrogen were irradiated. MS m/z (rela- tive intensity) 173 (M+, 8), 130 (IOO), 115 (38), 104 (16), 91 (15), 77 (16).

Anal. calculated for C14H,,N0,: C, 63.87; H, 6.51; N, 5.32. Found: C, 63.5; H, 6.3; N, 5.2.

The GC-yields of the reaction mixture before work up were 53% of M-3-PTP (fig. 1,3) and approximately 35% of five unidentified diphenylated I-methyl-l,2,3,6-tetrahydropyridine.

Pharmacology. In vivo experiments. Male albino mice (2g30 g) of the NMRI strain were used. I-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine hydro- chloride (MPTP; Research Biochemical Inc., Wayland, MA, U.S.A.) was dissolved in 0.9% NaCl and I-methyl-3-phenyl- 1,2,3,6-tetrahy- dropyridine (M-3-PTP) was dissolved in a 2% ethanol/saline solu- tion. Both drugs were injected subcutaneously in volumes of 0.5 ml/ 25 g body weight. Control mice were given the same amount of vehicle.

The animals were killed by decapitation, their brains rapidly taken out, put on an ice-chilled petri dish and dissected into corpus striatum and the limbic forebrain (containing i.a. the nucleus accum- bens and the olfactory tubercles) as described by Carlsson & Lindqvist (1973). The brain parts were stored at -70" until further analysis.

Biochemical analysis. The brain parts were homogenized with 0.1 M HCIO, containing Na,-EDTA (4.3 mM) and reduced glutathione (1.6 mM). After centrifugation (lO,OOOxg, 0") for 10 min. the supernatant was taken for analysis of catechols (dopamine, DA; 3,4-dihydroxyphenylacetic acid, DOPAC) and for independent analysis of the 0-methylated compounds (3-methoxytyramine, 3- MT; homovanillic acid, HVA) by means of liquid chromatography with electrochemical detection (LCEC; Felice et al. 1978; Magnus- son et al. 1980; modified by Svensson et al. 1986).

For catechols 20 mg of acid-washed A1,03 was added to 0.7 ml supernatant. Then, 100 ng of alpha-methyl-DOPA (internal stan- dard) and, under vigorous stirring, 0.5 ml 3.0 M Tris buffer @H 8.6) were added. After mixing for 10 min. samples were washed twice with distilled water and finally eluted with 200 pl of a solution containing boric acid (0.25 M) and citric acid (0.125 M). For 3-MT and HVA 25 ng of N-methyl-5-HT (internal standard) was added to 0.25 ml supernatant.

The catechols were chromatographed using a mobile phase con- sisting of 0.017 M K,HP04, 0.033 M citric acid (pH 2.7-2.9), Na- octyl-sulphate (0.25-0.30 mM), Na,-EDTA (0.054 mM) and 8% v/ v methanol. 3-MT and HVA were chromatographed using a mobile phase consisting of 0.05 M citrate buffer (pH 4.2), Na-octyl-sulphate (0.25-0.30 mM), Na,-EDTA (0.054 mM) and 10% v/v methanol.

\ OMe

Pd(OAc1,

/

I I I 3 (M-3-PTP) \ \ 3 (M-3-PTP)

4 5 6 Fig.3. The preparation of 1 -methyl-3-phenyl- I ,2,3,6-tetrahydropyri- dine (M-3-PTP) (3), starting from commercially available I-methyl- 1,2,3,6-tetrahydropyridine hydrochloride and iodobenzene in the presence of silver nitrate, and with palladium acetate and o-tolyl-

Fig. 2. Structural formulas for (R,S)-l-propyl-3-aryl-l,2,3,6-tetra- hydropyridine (4), (S)-l-propyl-3-(3-hydroxyphenyl)-piperidine (3- PPP) ( 5 ) and 1-methyl-4-(3-methoxyphenyl)-1,2,3,6-tetrahydropyri- dine (6). phosphine as catalyst.

40 KRISTINA NILSSON ET AL.

The LCEC-system consisted of a LDC-minipump (Laboratory Data Control, LDC, Riviera Beach, FL, U.S.A.) a stainless steel column (0.45 x 15.0 cm) packed with Nucleosil, RP-18,5 pm (Mach- erey-Nagel, Duren, FRG). Detection was achieved by means of a thin layer cell, TL-3 (Bioanalytical Systems, BAS, West Lafayette, IN, U.S.A.) and an amperometric detector (Lc-3, BAS). The detec- tor was operated at +0.65 V (catechols) and +0.85 V (3-MT, HVA). The current produced was monitored using an integrator model SP 4270 (Spectra-Physics, San JosC, CA, U.S.A.).

In vitro experiments. Preparation of mitochondria. Mitochondria were prepared from rat liver as described by Ask (1984). The liver was taken from a male Sprague-Dawley rat weighing 300 g. After anaesthesia with mebum- al, the liver was perfused in situ with saline, and then dissected out. The liver was homogenized in ice-cold 0.32 M sucrose (5 ml/g tissue) using a glass-tephlon Potter-Elvehjem homogenizer. The homogena- te was centrifuged at 4” at 800 x g for 10 min. in a Hereus Omnifuge 2.0 RS. The pellet was suspended in 0.32 M sucrose and recentri- fuged under the same conditions. The pellet from the second centri- fugation was suspended in the sucrose solution (6 ml/g liver) by homogenization, and suspension then filtered through gauze. The filtrate is called the “mitochondrial fraction” and was stored at - 20“.

Enzyme assay. Monoamine oxidase B was assayed as described by Salach (1979) with benzylamine HC1 as substrate. The reaction mixture contained 400 p1 mitochondrial fraction, 6 mg Triton X- 100, and 10 pmoles benzylamine HCI in a total volume of 3 ml. The buffer was 0.1 M potassium phosphate, pH 7.5, containing 0.05% EDTA. The reaction was started by addition of the substrate. The inhibitors were dissolved in water and added to the incubation mixtures 1 min prior to the substrate. The experiments were carried out at 37”. The formation of benzaldehyde was monitored at 250 nm using a Varian DMS 100 Spectrophotometer. The molar extinc- tion of benzaldehyde was followed for 8 min. during which time the reaction was relatively linear.

Results

Chemistry. Sodium borohydride reduction of the N-methyl pyridinium salt, obtained after methylation of 3-phenylpyridine, furnish a 1:l mixture of 2 (fig. 1) and M-3-PTP (3) (fig. 1) (no physical data were reported) (Perry et al. 1988).

We have synthesized M-3-PTP by the procedure that we have developed for the preparation of 4 (fig. 2). M-3-PTP is formed in a one pot synthesis by reaction of iodobenzene with commercially available N-methyl-l,2,3,6-tetrahydro- pyridine hydrochloride in the presence of silver nitrate, and with palladium acetate as catalyst (fig. 3). The undesired side products, easily separate from M-3-PTP by chromato- graphy, are derived from diarylation of the starting material, N-methyl-l,2,3,6-tetrahydropyridine. Scope and limitations of the reaction, chelation controlled regioselectivity, and the role of silver ion on double bond isomerization is discussed elsewhere (Nilsson & Hallberg, unpublished results).

Pharmacology. In vivo. Two hr after injection of MPTP the striatal DA concentration had decreased to 55% of controls (table 1). An insignificant 34% reduction in DA was also observed in the limbic region. In addition, MPTP produced a more than 10-fold increase in the concentration of the DA meta- bolite 3-methoxytyramine (3-MT) in both brain regions. 3,4-

Table 1. Acute effects of 1 -methyl-Cphenyl- 1,2,3,6-tetrahydropyridine (MPTP) ( I ) and its analogue l-methyl-3-phenyl-l,2,3,6-tetrahydro- pyridine (M-3-PTP) (3) on catecholamine metabolism in mouse brain. MPTP (50 mg/kg) and M-3-PTP (50 mg/kg) were adminis- tered subcutaneously two hours before death. Data are meansf S.E.M. in ng/g wet tissue for 5 mice. Statistics: Student’s t-test; ** P<O.OOl, * P<O.Ol.

DA 3-MT DOPAC HVA

Striatum Controls 7731 f 585 MPTP 4261 f492* Controls 6535 f 530

Limbic region Controls 2207 f 242 MPTP 1465f72 Controls 1626 f 248

M-3-PTP 6699f412

M-3-PTP 1659f 170

244 f 40

331 f 3 7 230f21

57 f3 601 f 54** 48f7 45f15

25 13 f 300** 549 f 57 957*73* 684 f 64 560 f 28

246f 10 220k19 270f 14 214f 10

572 f 58 1118f214 697f44 674f21

255 f 2 2 374 f 36 210f21 215k 14

Dihydroxyphenylacetic acid (DOPAC) increased slightly in the striatum but not in the limbic region. Homovanillic acid (HVA) appeared to increase in both brain regions, but this effect failed to reach statistical significance. In contrast, the MPTP-analogue M-3-PTP did not produce any acute changes in the DA metabolism in the striatum or in the limbic region (table 1).

One week after injection of MPTP the striatal and limbic levels of DA and its metabolites were markedly reduced (table 2). In contrast, M-3-PTP did not produce any long- term reduction in DA and metabolites (table 2).

In vitro. The rate of benzaldehyde formation at the con- ditions described above was 3.37 k0.14 nmol/min./ml (n= 5). M-3-PTP up to 0.5 mM did not inhibit the reaction, while MPTP inhibited the reaction in a concentration dependent fashion. At a concentration of 0.2 mM the inhibition was around 50%. The effects by MPTP are in agreement with data reported by Son et al. (1990). Higher concentrations

Table 2. Long term effects of 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP) (1) and its analogue l-methyl-3-phenyl-l,2,3,6-tetrahydro- pyridine (M-3-PTP) (3) on catecholamine metabolism in mouse brain. MPTP (50 mg/kg) and M-3-PTP (50 mg/kg) were adminis- tered subcutaneously 7 days before death. Data are means f S.E.M. in ng/g wet tissue for 4-6 mice. Statistics: Student’s t-test; ** P<O.OOI, * P<O.OI.

DA

Striatum Controls 6784 f 434 MPTP 2562 f 285** Controls 6896 f 187

Limbic region Controls 1871 f 171 MPTP 879+15** Controls 2257f 194

M-3-PTP 7745 f 173

M-3-PTP 2465 f 155

3-MT

479 f 29 291 f20** 485 f 28 417f30

74+8 58+3*

122k28 73k8

DOPAC HVA

776+47 1131 f 5 6 325 f 27** 653 +42** 895f57 1051f47 884f84 1031 f 6 0

294 & 19 248 & 26 137+8** 129+4** 443f67 431_+46 409k23 378522

NO DOPAMINERGIC TOXICITY OF M-3-PTP 41

of MPTP were not tested since this compound itself inter- feres with the assay at the wavelength used.

Discussion

The MPTP-produced neurotoxic process leading to de- generation of brain DA neurones involves several steps. MPTP is first oxidized by MAO-B (Chiba et al. 1984), which catalyzes the two-electron oxidation to the corresponding 1,2-dihydropyridinium species. This reaction is thought to involve the formation of a stabilized a carbon-centered rad- ical intermediate (Silverman & Zieske 1986). Subsequent transformation yields 1-methyl-4-phenyl pyridinium ion (MPP+) (fig. 4,7). MPP+ is then taken up and accumulated within the dopaminergic neurones due to its affinity for the dopamine re-uptake carrier (Javitch & Snyder 1985). In line with this, re-uptake inhibitors like GBR 13098 are known to effectively protect the DA neurones from MPTP toxitity (Pileblad & Carlsson 1985). MPPf accumulates thereafter in the mitochondria via a passive transport mechanism in response to the transmembrane electrochemical potential gradient (Hoppel et al. 1987; Sayre et al. 1989). In the mitochondria, MPP+ interrupts the electron-transport chain by inhibiting the NADH-dehydrogenase; as a conse- quence the level of intracellular ATP drops which in turn may lead to a destruction of the DA neuron (Ramsay & Singer 1986; Niclas et al. 1987).

In the present study the acute effects of MPTP (fig. 1,l) and M-3-PTP (fig. 1,3) on catecholamine metabolism in mouse brain were compared. MPTP produced a marked acute reduction of DA together with a profound increase in the DA metabolite 3-MT (table 1). These changes are indicative of a marked release of DA from the nerve ter- minals into the extraneuronal space (Pileblad et al. 1984 & 1985; Pileblad & Carlsson 1988). Energy is required to keep DA within the synaptic vesicles and also within the neurones. Thus, the outflow of DA induced by MPTP is probably secondary to an acute drop in ATP (see Pileblad & Carlsson 1988). In contrast to these rather dramatic effects of MPTP, the analogue M-3-PTP did not produce any acute changes in DA metabolism in the striatum or in the limbic region.

In the long-term experiments, both MPTP and M-3-PTP were injected 7 days before death (table 2). A marked re- duction in brain DA and its metabolites were observed one week after MPTP, a change which is consistent with a destruction of the dopaminergic nerve terminals in the stria-

@ \+

I 7 (MPP*) 8 (M-3-PP’)

Fig.4. Structural formulas for I-methyl-4-phenyl pyridinium ion (MPP+) (7) and I-methyl-3-phenyl pyridinium ion (M-3-PP+) (8).

tum and in the limbic region (see Hallman et al. 1984). In contrast, M-3-PTP did not produce any long-term depletion of DA and metabolites, indicating that the compound is devoid of dopaminergic neurotoxicity.

The in vivo dopaminergic neurotoxic properties of MPPf, M-3-PPf (fig. 4,8), and other MPP+ analogues were examined by an intrastriatal microdialysis assay in conscious rats (Rollema et al. 1990). MPP+-like toxicity, as evidenced by the irreversible effects on DA release and enhancement of lactate formation, was observed with a variety of structural types although no compound was more toxic than MPP+. M-3-PP+, prepared by N-methylation of 3-phenylpyridine, exhibited a comparable toxicity to MPP+ following a 60 min. perfusion. Thus, M-3-PP+ appears to be toxic to the DA neurones whereas the present data suggest that M-3-PTP is not. The most likely explanation for this is that M-3-PTP has very low affinity for MA0 and that M-3-PP+ is not formed to any great extent in the brain following systemic administration of M-3-PTP. The present in vitro experiments showing that M-3-PTP, in contrast to MPTP, had no effect on the activity of MAO-B clearly support this assumption. The lack of alterations in DA metabolism following a rather high dose of M-3-PTP (table 1) would also be in line with such a notion.

In summary, M-3-PTP seems to lack toxicity to dopam- inergic neurones in mouse brain and thus it appears unlikely that the compound would produce Parkinson’s disease in humans. The lack of toxicity appears to be related to low affinity of M-3-PTP for MAO. We believe that structurally related propyl amines 4 (fig. 2) are also devoid of dopamin- ergic toxicity and we hope that these compounds would be recognized as versatile reaction intermediates and starting materials in organic synthesis.

Acknowledgements We thank the Swedish Natural Science Research Council,

the Swedish Medical Research Council (no X14-09076) Sig- urd and Elsa Golje’s Foundation and 8 k e Wiberg’s Founda- tion for financial support.

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37, 707-712.

213-222.

2221-2230.

128-137.

1990, 1845- 1848.

809-8 18.

341-346.