Synthesis and MAO-B substrate properties of 1-methyl-4-heteroaryl-1,2,3,6-tetrahydropyridines

The parkinsonian inducing drug 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is bioactivated in a reaction catalyzed by the flavoenzyme monoamine oxidase B (MAO-B) to form the corresponding dihydropyridinium and subsequently pyridinium metabolites. As part of our ongoing studies to characterize the structural features responsible for this unexpected biotransformation, we have examined the MAO-B substrate properties of a variety of MPTP analogues bearing various heteroaryl groups at the 4-position of the tetrahydropyridinyl ring. The newly synthesized analogues are 4-(1-methylimidazol-2-yl)-, 4-(3-methylfuran-2-yl)-, 4-(3-methylthien-2-yl)-, 4-(3,4-dimethylpyrrol-1-yl)-, 4-(3-methylpyrrol-2-yl)-, and 4-(1,3-dimethylpyrrol-2-yl)-1-methyl-1,2,3,6-tetrahydropyridine. Except for the 4-(1-methylimidazol-2-yl) analogue, all compounds displayed good to excellent substrate properties. The 1-methyl-4-(3-methylfuran-2-yl) analogue is the most active member of this series with a kcat/Km value greater than 8,500 min(-1)mM(-1). The results of these studies are discussed in terms of catalytic pathways proposed for MAO-B.     

Synthesis and in vitro biological evaluation of fluoro-substituted-4-phenyl-1,2,3,6-tetrahydropyridines as monoamine oxidase B substrates

The substrate properties of three beta-fluoro-4-phenyl-1,2,3,6-tetrahydropyridines related to the proneurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine have been examined in an effort to evaluate the contribution of electronic parameters to the MAO-B catalyzed allylic-alpha-carbon oxidation of the tetrahydropyridinyl system. The design, synthesis, and biological evaluation of these analogues are presented and correlations to amine ionization potentials versus substrate activity are discussed.     

Time-dependent slowly-reversible inhibition of monoamine oxidase A by N-substituted 1,2,3,6-tetrahydropyridines

A novel class of N-substituted tetrahydropyridine derivatives was found to have multiple kinetic mechanisms of monoamine oxidase A inhibition. Eleven structurally similar tetrahydropyridine derivatives were synthesized and evaluated as inhibitors of MAO-A and MAO-B. The most potent MAO-A inhibitor in the series, 2,4-dichlorophenoxypropyl analog 12, displayed time-dependent mixed noncompetitive inhibition. The inhibition was reversed by dialysis, indicating reversible enzyme inhibition. Evidence that the slow-binding inhibition of MAO-A with 12 involves a covalent bond was gained from stabilizing a covalent reversible intermediate product by reduction with sodium borohydride. The reduced enzyme complex was not reversible by dialysis. The results are consistent with slowly reversible, mechanism-based inhibition. Two tetrahydropyridine analogs that selectively inhibited MAO-A were characterized by kinetic mechanisms differing from the kinetic mechanism of 12. As reversible inhibitors of MAO-A, tetrahydropyridine analogs are at low risk of having an adverse effect of tyramine-induced hypertension.     

Photoinactivation of B-type monoamine oxidase by a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine metabolite

The reaction of the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) with monoamine oxidase from a variety of tissues including rat and monkey brain, bovine liver, and human placenta and platelets was found to yield, as a primary product, a reactive photosensitive substance with an absorbance maximum at 345 nm which is not the cation 1-methyl-4-phenylpyridinium ion previously reported as a monoamine oxidase-MPTP metabolite in vivo and in vitro. Our results suggest that the 1-methyl-4-phenyl-pyridinium ion is probably only generated in subsequent nonenzymatic transformations of this reactive monoamine oxidase metabolite. This substance was found to specifically inactivate the B-form of monoamine oxidase by a photo-induced mechanism and to react directly with NADPH and dopamine. Properties of the metabolite and potential significance of its reactions to MPTP neurotoxicity are discussed.     

Deuterium isotope effect measurements on the interactions of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine with monoamine oxidase B

Kinetic deuterium isotope effects for the noncompetitive, intermolecular monoamine oxidase B-catalyzed oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to the corresponding 1-methyl-4-phenyl-2,3-dihydropyridinium species MPDP+ were found to be 3.55 on Vmax and 8.01 on Vmax/Km with MPTP-6,6-d2 as the deuterated substrate. Similar values were obtained with MPTP-2,2,6-d4 and MPTP-CD3-2,2,6,6-d4. The deuterium isotope effect for the electrochemical oxidation of 1 mM MPTP-2,2,6,6-d4 was only 1.35. These results indicate that the monoamine oxidase B-catalyzed oxidation of this substrate may not proceed via a reaction pathway involving alpha-carbon deprotonation of an aminium radical intermediate. Isotope effect measurements also established that the rate of inactivation of monoamine oxidase B by MPTP is unaffected by replacement of the C-6 methylene protons with deuterons, but is retarded by replacement of the C-2 methylene protons (DKi = 1.9). The mechanism-based inactivation of monoamine oxidase B by MPTP, therefore, is likely to mediated by a species derived from the enzyme-generated 2,3-dihydropyridinium oxidation product.     

Neurotoxicity studies with the monoamine oxidase B substrate 1-methyl-3-phenyl-3-pyrroline

The neurotoxic properties of the parkinsonian inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are dependent on its metabolic activation in a reaction catalyzed by centrally located monoamine oxidase B (MAO-B). This reaction ultimately leads to the permanently charged 1-methyl-4-phenylpyridinium species MPP(+), a 4-electron oxidation product of MPTP and a potent mitochondrial toxin. The corresponding 5-membered analogue, 1-methyl-3-phenyl-3-pyrroline, is also a selective MAO-B substrate. Unlike MPTP, the MAO-B-catalyzed oxidation of 1-methyl-3-phenyl-3-pyrroline is a 2-electron process that leads to the neutral 1-methyl-3-phenylpyrrole. MPP(+) is thought to exert its toxic effects only after accumulating in the mitochondria, a process driven by the transmembrane electrochemical gradient. Since this energy-dependent accumulation of MPP(+) relies upon its permanent charge, 1-methyl-3-phenyl-3-pyrrolines and their pyrrolyl oxidation products should not be neurotoxic. We have tested this hypothesis by examining the neurotoxic potential of 1-methyl-3-phenyl-3-pyrroline and 1-methyl-3-(4-chlorophenyl)-3-pyrroline in the C57BL/6 mouse model. These pyrrolines did not deplete striatal dopamine while analogous treatment with MPTP resulted in 65-73% depletion. Kinetic studies revealed that both 1-methyl-3-phenyl-3-pyrroline and its pyrrolyl oxidation product were present in the brain in relatively high concentrations. Unlike MPP(+), however, 1-methyl-3-phenylpyrrole was cleared from the brain quickly. These results suggest that the brain MAO-B-catalyzed oxidation of xenobiotic amines is not, in itself, sufficient to account for the neurodegenerative properties of a compound like MPTP. The rapid clearance of 1-methyl-3-phenylpyrroles from the brain may contribute to their lack of neurotoxicity.     

Inactivation of monoamine oxidase by 3,3-dimethyl analogues of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenyl-2,3-dihydropyridinium ion. Dramatic effect of beta-mercaptoethanol on substrate turnover and enzyme inactivation

It was previously shown (Sayre, L. M., Arora, P. K., Feke, S. C., and Urbach, F. L. (1986) J. Am. Chem. Soc. 108, 2464-2466) that 1,3,3-trimethyl-4-phenyl-2,3-dihydropyridinium salt (the 3,3-dimethyl analogue of 1-methyl-4-phenyl-2,3-dihydropyridinium ion or MPDP+) is a good model for MPDP+ on the basis of its redox potential and was used to show that MPDP+ is unlikely to possess reactivity characteristics which could contribute to the neurotoxicity observed with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). 3,3-Dimethyl-MPTP and 3,3-dimethyl-MPDP+ are now shown to interact with monoamine oxidase similar to MPTP and MPDP+, but only in the presence of beta-mercaptoethanol (beta-ME). In the absence of beta-ME, mixed competitive-noncompetitive inhibition kinetics are observed for 3,3-dimethyl-MPTP and 3,3-dimethyl-MPDP+, whereas competitive inhibition kinetics are exhibited by MPTP. In the presence of beta-ME, however, 3,3-dimethyl-MPTP also is a competitive inhibitor. 3,3-Dimethyl-MPTP and 3,3-dimethyl-MPDP+ also are time-dependent inactivators of monoamine oxidase, having identical kinetic constants, as is the case with MPTP and MPDP+. In the presence of beta-ME, but not glutathione, the rate of inactivation increases dramatically. When [beta-ME] and [3,3-dimethyl-MPTP] or [3,3-dimethyl-MPDP+] are varied, there is an optimal concentration of 1.0 mM for all three at which maximal inactivation rates are obtained. Another dramatic effect of the beta-ME is to lower the partition ratio for inactivation from greater than 50 to about one. This suggests that the effect of the beta-ME toward inactivation may be to induce a conformational change in the enzyme, which reorients an active site nucleophile for attack on the activated species. Support for involvement of an active site nucleophile is the finding that inactivation does not lead to a flavin adduct. Three possible mechanisms for inactivation of monoamine oxidase by MPTP and MPDP+ are suggested.     

Catalytic biotransformation of physiologically active 1-methyl-4- aryl-1,2,3,6-tetrahydropyridines under the action of monoaminooxidase

Kinetics of monoamine oxidase (MAO) catalyzed dehydrogenation of neurotropic analogues of biogenic monoamines in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine series were studied. It is shown that methyl substitution in the phenyl ring increases significantly the enzyme-substrate affinity, but the substituent's effect on the catalytic stage largely depends upon its position in the ring. o- and m-Methyl derivatives were preferably oxidized by B type of MAO, whereas p-total derivative was oxidized by B type as well as by A type of the enzyme. In the course of the oxidation reactions MAO is irreversibly inhibited by the dihydropyridinium product of the reaction, particularly in case of methyl derivatives. The significant and structure-dependent inhibition of the enzyme might be responsible for the differences in neurotropic properties of the above substrate homologues.     

A novel and selective monoamine oxidase B substrate

Cyclic five- and six-membered tertiary allylamines constitute a unique class of monoamine oxidase substrates that undergo a net two-electron alpha-carbon oxidation to form the cyclic, conjugated eniminium metabolites. The corresponding saturated pyrrolidinyl and piperidinyl systems are not substrates for this flavoenzyme system. In an attempt to evaluate possible contributions that pi-orbital stabilization of the putative alpha-carbon radical intermediates may play in the catalytic pathway, we have examined the substrate properties of 3-methyl-6-phenyl-3-aza-bicyclo[4.1.0]heptane, the 3,4-cyclopropyl analog of the selective monoamine oxidase B substrate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The results, which document the first reported example of a saturated, cyclic tertiary amine with monoamine oxidase substrate properties, are consistent with alpha-carbon radical stabilization as a contributing factor in the catalytic pathway.     

Interactions of the neurotoxic amine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine with monoamine oxidases.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a thermal breakdown product of a meperidine-like narcotic used by drug abusers as a heroin substitute, produces Parkinsonian symptoms in humans and primates. The nigrostriatal toxicity is not due to MPTP itself but to one or more oxidation products resulting from the action of monoamine oxidase (MAO) on this tertiary allylamine. Both MAO A and B catalyse the oxidation of MPTP to the 1-methyl-4-phenyl-2,3-dihydropyridinium species (MPDP+), which undergoes further oxidation to the fully aromatic 1-methyl-4-phenylpyridinium species (MPP+). These bio-oxidations are blocked by selective inhibitors of MAO A and B. Additionally, MPTP, MPDP+ and MPP+ are competitive inhibitors of MAO A and B. The A form of the enzyme is particularly sensitive to this type of reversible inhibition. Both MAO A and B also are irreversibly inactivated by MPTP and MPDP+, but not by MPP+. This inactivation obeys the characteristics of a mechanism-based or 'suicide' process. The inactivation, which is accompanied by the incorporation of radioactivity from methyl-labelled MPTP, is likely to result from covalent modification of the enzyme.     

Comparative toxicity and antioxidant activity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and its monoamine oxidase B-generated metabolites in isolated hepatocytes and liver microsomes

MPTP (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is converted by monoamine oxidase B to its putative toxic metabolite MPP+ (1-methyl-4-phenylpyridinium ion) via MPDP+ (1-methyl-4-phenyl-2,3-dihydropyridinium ion). Both the parent compound and these two major metabolites were toxic to isolated rat hepatocytes with MPDP+ being the most toxic and MPP+ the least effective. MPP+ produced a slight increase in lipid peroxidation above control levels in hepatocytes, while both MPTP and MPDP+ showed antioxidant effects. The latter two compounds also protected against chemically and nonchemically induced lipid peroxidation in rat liver microsomes. MPDP+ was effective at much lower concentrations than MPTP. MPDP+ was also markedly more efficient when NADPH was used to induce microsomal lipid peroxidation. Lipid peroxidation as a consequence of oxygen radical generation is therefore unlikely to be involved in MPTP toxicity in vitro and the rationale of using chain-breaking antioxidants as protective agents in vivo needs a more careful evaluation.     

Interactions of 1-methyl-3-phenylpyrrolidine and 3-methyl-1-phenyl-3-azabicyclo[3.1.0]hexane with monoamine oxidase B

The parkinsonian inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its corresponding five-membered ring analogue 1-methyl-3-phenyl-3-pyrroline are cyclic tertiary allylamines and good substrates of monoamine oxidase B (MAO-B). The MAO-B catalyzed 2-electron α-carbon oxidation of this class of substrates appears to be dependent on the presence of the allylic π-bond since the corresponding saturated piperidinyl analogue of MPTP is reported not to be an MAO-B substrate. The only saturated cyclic tertiary amine known to act as an MAO-B substrate is the 3,4-cyclopropyl analogue of MPTP, 3-methyl-6-phenyl-3-azabicyclo[4.1.0]heptane. As part of our ongoing studies we have examined the MAO-B substrate properties of the corresponding pyrrolidinyl analogue, 1-methyl-3-phenylpyrrolidine, and the 3,4-cyclopropyl analogue, 3-methyl-1-phenyl-3-azabicyclo[3.1.0]hexane. The results document that both the pyrrolidinyl analogue [K_m = 234 μM; V_max = 8.37 nmol/(min-mg mitochondrial protein)] and the 3,4-cyclopropyl analogue [K_m = 148 μM; V_max = 16.9 nmol/(min-mg mitochondrial protein)] are substrates of baboon liver mitochondrial MAO-B. We also have compared the neurotoxic potential of these compounds in the C57BL/6 mouse. The results led us to conclude that these compounds are not MPTP-type neurotoxins.     

Influence of selective, reversible inhibitors of monoamine oxidase on the prolonged depletion of striatal dopamine by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) hydrochloride injected s.c. at 20 mg/kg once daily for four days resulted in marked depletion of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in mouse striatum one week after the last dose. Pretreatment with MD 240928, (R)-[4-((3-chlorophenyl)-methoxy)phenyl]-5-[(methylamino)methyl]-2- oxazolidinone methanesulfonate, prevented the depletion of striatal dopamine, DOPAC and HVA, whereas pretreatment with harmaline did not. MD 240928 selectively inhibited type B not type A monoamine oxidase (MAO), whereas harmaline selectively inhibited type A MAO in mouse striatum. Acutely after injection of harmaline, DOPAC and HVA concentrations were decreased in mouse striatum; these changes were not produced by MD 240928. The acute changes in dopamine metabolites reveal that MAO-A not MAO-B is responsible for the oxidation of dopamine in mouse striatum. Protection against the neurotoxic effects of MPTP by MD 240928 but not by harmaline indicates that prevention of dopamine oxidation is not the mechanism of the protective effect; instead the protection probably is due to prevention of MPTP metabolism by MAO-B, this metabolism having been shown to occur by other workers. The results with these reversible, competitive inhibitors of the two types of MAO are in agreement with previously reported results from studies using irreversible inhibitors of MAO.     

Phenylethanolamine is a specific substrate for type B monoamine oxidase

The characteristics of phenylethanolamine as both a competitive inhibitor and as a substrate for monoamine oxidase (MAO) were studied using rat brain and liver homogenates. Although phenylethanolamine, even at high concentrations (1 mM), produced minimal inhibition of MAO when serotonin (a substrate for type A MAO) was used as the substrate, it was a potent competitive inhibitor (K_i=11 μM) of the deamination of phenylethylamine (a substrate for type B MAO). When phenylethanolamine was used as a substrate, deprenyl, a selective inhibitor of type B MAO, was found to produce a single sigmoid inhibition curve at low concentrations of the inhibitor (pI₅₀=7.5). These results indicate that phenylethanolamine is a specific substrate for type B MAO. Identification of the products formed under the assay conditions show that phenylethanolamine is converted to both mandelic acid and phenylethylene glycol by liver homogenates but only to the latter, neutral metabolite by brain homogenates.     

Mutagenic activity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is a neurotoxin, which can damage dopaminergic neurons. It causes symptoms resembling those observed in patients suffering from Parkinson's disease, and hence this toxin is widely used in studies on animal models of this disorder. Mutagenicity of MPTP was also reported by some authors, but results obtained by others suggested that this compound is not mutagenic. Interestingly, those contrasting results were based on the same assay (the Ames test). Therefore, we aimed to test MPTP mutagenicity by employing a recently developed Vibrio harveyi assay, which was demonstrated previously to be more sensitive than the Ames test, at least for some mutagens. We found that MPTP showed a significant mutagenic activity. Moreover, MPTP mutagenicity was attenuated by methylxanthines, compounds that are known to form complexes with aromatic mutagens.     

Reversible inhibition and mechanism-based irreversible inactivation of monoamine oxidases by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

It has been suggested (Chiba et al., Biochem. Biophys. Res. Communs. (1984) 120, 574) that the neurotoxic effects of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), which causes Parkinsonian symptoms in humans and other primates, are due to compounds resulting from the oxidation of MPTP by monoamine oxidase B in the brain. We reported recently that both monoamine oxidase A and B oxidize MPTP to MPDP+, the 2,3-dihydropyridinium form and that the reaction is accompanied by time-dependent, irreversible inactivation of the enzymes. Of the two forms of monoamine oxidase, the B enzyme oxidizes MPTP more rapidly and is also more sensitive to inactivation. We now wish to report that MPTP, as well as its oxidation products, MPDP+ and MPP+, the 4-phenylpyridinium form, are also potent reversible, competitive inhibitors of both monoamine oxidase A and B, particularly the former, and that the order of inhibition for the A enzyme is MPDP+ greater than MPP+ greater than MPTP, while for the B enzyme MPTP greater than MPDP+ greater than MPP+. We further report on the spectral changes and isotope incorporation accompanying the irreversible inactivation.     

1,4-Methylhistamine is a specific substrate for type B monoamine oxidase

1,4-Methylhistamine was characterized as substrate for monoamine oxidase (MAO) in rat liver mitochondria. The $\text{K}$_m and $\text{V}$_max values were 38.8 μM and 6.33 nmoles/mg protein/60 min, respectively. The inhibition experiments with clorgyline and deprenyl, the selective inhibitors for type A and type B MAO, showed that 1,4-methylhistamine was specific for type B MAO.     

Oxidation and enzyme-activated irreversible inhibition of rat liver monoamine oxidase-B by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

The compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces symptoms resembling Parkinson's disease in humans, acts both as a substrate and an enzyme-activated irreversible inhibitor of the B-form of monoamine oxidase from rat liver. Analysis of the inhibitory process showed the compound to be considerably more efficient as a substrate than as an irreversible inhibitor, with about 17000 mol of product being formed per mol of enzyme inactivated. The half-time of the inhibitory process was about 22 min. With the A-form of the enzyme, the compound had a lower Km value and a considerably lower maximum velocity than the corresponding values obtained with the B-form. Under the conditions used in the present work the inhibition of the A-form of the enzyme was largely reversible.     

Interaction of flexible analogs of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and of N-methyl-4-phenylpyridinium with highly purified monoamine oxidase A and B.

Sixteen analogs of N-methyl-1,2,3,6-tetrahydropyridine (MPTP) of varying degrees of flexibility have been studied as substrates of highly purified monoamine oxidases (MAO) A and B. The relative effectiveness of the various tetrahydropyridines as substrates of MAO A and B were evaluated in terms of the function turnover number/Km, as determined by initial rate measurements. The insertion of a methylene bridge between the phenyl and tetrahydropyridine moieties of MPTP to yield N-methyl-4-benzyl-1,2,3,6-tetrahydropyridine, rendering the molecule more flexible, greatly enhances reactivity with MAO B, but not with MAO A, as compared with MPTP itself, in accord with data in the literature (Youngster et al., 1989a). The ethylene-bridged MPTP analog, on the other hand, is a far better substrate of both forms of MAO than is MPTP itself. The effect of molecular flexibility on the rate of oxidation of these compounds is obscured by substituents on the aromatic ring. Branching and rigidity were detrimental to the activity as substrates of both forms of MAO. Those analogs of 1 which contain small electron-withdrawing substituents in the phenyl ring were found to be more selective for MAO B, while those substituted with bulky groups were selectively oxidized by MAO A. The substrate binding site of MAO A probably contains a lipophilic pocket larger than that found in a similar site in MAO B.