3694
J. Med. Chem. 1996, 39, 3694-3700
Assessment of Structural Requirements for the Monoamine Oxidase-BCatalyzed Oxidation of 1,4-Disubstituted-1,2,3,6-tetrahydropyridine Derivatives Related to the Neurotoxin 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine Ste´phane Mabic and Neal Castagnoli, Jr.* Department of Chemistry, Virginia Tech, Blacksburg, Virginia 24061 Received May 31, 1996X
The monoamine oxidase B (MAO-B) substrate properties and distance measurements along the N1-C4 axis of 38 1,4-disubstituted-1,2,3,6-tetrahydropyridine derivatives, including seven newly synthesized MPTP analogs, were used to define the maximum size that can be accommodated by the MAO-B active site. Only those compounds measuring less than 12 Å displayed significant MAO-B substrate properties. The behavior of various 4-substituted-1cyclopropyltetrahydropyridine analogs also is discussed in terms of this N1-C4 distance parameter in an effort to understand factors which contribute to their substrate vs inactivator properties. We conclude that this distance parameter will predict the majority of substrates vs nonsubstrates with this class of compound. Introduction The flavoenzymes monoamine oxidase A and B (MAOA and MAO-B) catalyze the oxidative deamination of several primary and secondary amines, including the neurotransmitters serotonin and dopamine, and selected tertiary amines.1 Of special toxicological interest is a variety of 1,4-disubstituted-1,2,3,6-tetrahydropyridines, including the parkinsonian-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 1), which are good to excellent substrates or inactivators of MAO-A and/or MAO-B. The overall catalytic pathway (Scheme 1) involves the 2-electron C6 (allylic) R-carbon oxidation of the substrate molecule to yield the corresponding dihydropyridinium species 2, a reaction which is coupled to the reduction of oxidized flavin (FAD) to reduced flavin (FADH2). FADH2 subsequently is oxidized back to FAD by dioxygen (O2).2 Molecular modeling studies on MPTP analogs have identified lipophilicity as a contributing parameter to good MAO-A and MAO-B substrate behavior and also have provided some approximate estimates of the size and shape of molecules that display substrate properties.3,4 Two features of the active sites of these enzymes are pockets that accommodate the tetrahydropyridine (THP) ring and its C4 substituent. Both flexible (one or more sp3-hybridized atoms) and constrained (aryl) C4 substituents may be well tolerated by both MAO-A and MAO-B,5 although the MAO-A active site appears to accommodate bulkier groups at C4 better than does the active site of MAO-B.3,6 Only substitutions at the N1 and C4 positions of the THP ring are tolerated,7 and apparently, only small substituents can be accommodated at the N1 position. On the basis of results with a limited set of compounds, Efange suggested a maximum distance of 10.23 Å between the N-methyl carbon and the farthest carbon on the C4 substituent.4 The studies described in the present paper were undertaken in an effort to define more accurately the maximum distance along the N1-C4 axis [d (Å)max] of the THP ring that can be accommodated by the MAO-B active site.8 During the course of these studies, inconX
Abstract published in Advance ACS Abstracts, August 15, 1996.
S0022-2623(96)00388-3 CCC: $12.00
Scheme 1. MAO-B-Catalyzed Oxidation of MPTP (1) to Its Dihydropyridinium Metabolite (2)
sistencies in the MAO-B kinetic parameters reported for several compounds were identified.3,7,9 In order to resolve these conflicts and to add to the data base used to define [d (Å)max], we have synthesized and characterized the substrate properties of several p-phenylsubstituted MPTP derivatives. Results We have focused our analysis specifically on the estimated length of the N1-C4 axis which is defined as the distance between the two most distant atoms which fall on a line drawn through N1 and C4 (Figure 1). At the outset we encountered conflicting literature reports concerning the MAO-B substrate properties of the 4-(4methylphenyl) (3), 4-(4-chlorophenyl) (4), and 1-ethyl (5) analogs of MPTP. An early report10 concluded that the 4-methylphenyl analog 3 was not metabolized by MAOB. Even though the poor substrate properties of 3 were later confirmed,11 subsequent studies from the same laboratory reported a kcat/KM ) 345 min-1 mM-1 (over 60% the activity of MPTP) for this compound.12 Various authors have incorporated the poor7,13 or good3,4,14 substrate properties of 3 in the development of their structure-activity relationship (SAR) and quantitative SAR (QSAR) relationships. The 4-chlorophenyl derivative 4 also has been described as a poor (7%, 12% activity relative to MPTP)7,15-17 or moderate9 MAO-B substrate. Excellent substrate properties of 4 (kcat/KM ) 595 min-1 mM-1) were reported later12 and used to develop QSAR relationships.3 Finally, the N-ethyl derivative 5 was described as a poor substrate relative to MPTP (7%,10 13%,11 “poor substrate”18). Subse© 1996 American Chemical Society
Structural Requirements for MAO-Catalyzed Oxidation
Journal of Medicinal Chemistry, 1996, Vol. 39, No. 19 3695
Scheme 2. Synthesis of 4′-Substituted MPTP Analogs
Table 1. MAO-B Substrate Properties of N-R1-4′-R2-Substituted MPTP Derivatives
Figure 1. MM2 views of a few selected 1,4-disubstituted 1,2,3,6-tetrahydropyridine derivatives showing the distance [d (Å)].
quently, Youngster et al. reported a kcat/KM of 200 min-1 mM-1 for 6,12 but the compound was later described as a “poor substrate” in a QSAR study.7 Unfortunately, the synthesis and chemical characteristics of many of the compounds for which enzyme kinetic data have been published12,14 have been reported in a thesis which does not include complete microanalytical and spectroscopic details.19 As the biological activities of several of these compounds were of particular relevance to the present study, we undertook the synthesis and full chemical characterization of the 4-chlorophenyl (4), 4-methylphenyl (3), and N-ethyl (5) derivatives. We also studied the following additional analogs: 4-(4-ethylphenyl) (6), 4-(4-bromophenyl) (7), 4-(4-hydroxyphenyl) (8), and 4-(4methoxyphenyl) (9), for which no definitive enzyme kinetic data could be found in the literature.20,21 Compounds 3, 4, and 6-9 were prepared by nucleophilic addition of the corresponding para-substituted phenyllithiated reagent to 1-methyl-4-piperidone (10) followed by dehydration of the resulting piperidinols 11-16 in acidic medium (Scheme 2).5,15,22 The 4-hydroxyphenyl analog 8 was prepared after protection of the 4-bromophenol as its tert-butyldimethylsilyl ether.23 The protecting group was cleaved during the acidic dehydration step.24 Compound 5 was obtained by reduction of the corresponding pyridinium species in protic solvent.25,26 All compounds were obtained as their crystalline oxalate salts which exhibited the expected elemental values and spectroscopic properties. The substrate properties of the synthesized compounds were evaluated with purified MAO-B at 30 °C (Table 1). The kcat/KM values of the 4-methylphenyl (345 min-1 mM-1) and 4-chlorophenyl (595 min-1 mM-1) analogs 3 and 4, respectively, compared favorably with the ‘excellent substrate’12,14 rather than the poor substrate10,11,15,16 properties reported for these two com-
R1
R2
kcata
KM
kcat/KM
source
1 3
Me Me
H Me
4
Me
Cl
5
Et
H
6 7 8 9 17 18 19 20
Me Me Me Me Me Me Me Me
Et Br OH OMe NH2 NO2 Ph F
204 175 169 97 119 74 34 85 71
0.39 0.50 0.49 0.18 0.20 1.09 0.17 1.89 0.14 inactive 0.94 b b inactive 0.22
523 349 345 551 595 68 200 45 493
12 c 12 c 12 c 12 c c c c 14 14 27 12
a
68 b b 93
73 54 16 423
Units: kcat in KM in mM, kcat/KM in min-1 mM. b Not reported. c Determined in this study. min-1,
pounds. On the other hand, the 1-ethyl-4-phenyl analog 5 was found to be a poor substrate (kcat/KM ) 68 min-1 mM-1), as reported in some papers10,11,15 and in contrast with the good substrate properties for this compound reported in other papers.12,14 The 4-bromophenyl analog 7 was an excellent substrate, while the 4-ethylphenyl (6) and 4-methoxyphenyl (9) analogs were poor MAO-B substrates. The 4-hydroxyphenyl analog 8 exhibited no substrate activity, as might be anticipated from results of earlier in vivo studies.20 For comparison purposes, the MAO-B substrate properties of the 11 MPTP analogs bearing a substituent at the para-position of the phenyl group and the 1-ethyl-4-phenyl analog 5 examined in this study are reported in Table 1. The values for the distance measurements of the N1C4 axis for the various tetrahydropyridine derivatives were estimated with the Mac Mimic-MM2 molecular modeling program (Version 91). With the purpose of accounting for the possible rotation of substituents such as C4′-ethyl and measuring the actual steric hindrance of such groups relative to a methyl group, a cylindrical volume was defined around the N1-C4 axis. The diameter of this cylinder was defined as the carbon frame width of the tetrahydropyridine ring (C3-C5 distance). Atoms that did not fall in the cyclindrical volume were not included in the calculated distance d (Å). Virtual atoms were simulated on the N1-C4 axis, at the farthest position occupied by the substituents at both N1 and C4 (see Figure 1). van der Waals volumes also were not taken into account in these measurements. In addition to C4- and N1-substituted MPTP analogs, compounds representing the various classes of C4-
3696
Journal of Medicinal Chemistry, 1996, Vol. 39, No. 19
Mabic and Castagnoli
Table 2. Maximum Distance [d (Å)] Along the N1-C4 Axis and the Corresponding Kinetic Data [kcat/KM (min-1 µM-1)] of a Series of 1,4-Disubstituted-1,2,3,6-Tetrahydropyridine Derivativesa X 1 3 4 5 6 7 8 9 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
N1 substituent
C4 substituent
d (Å)
I1
Me
Ph
10.07
+
Me Me Et Me Me Me Me Me Me Me Me H allyl cyclopentyl cyclobutyl n-Pr H Me Me Me Me Me Me Me Me Me Me Me Me Me Me Me Me
4′-Me-Ph 4′-Cl-Ph Ph 4′-Et-Ph 4′-Br-Ph 4′-OH-Ph 4′-OMe-Ph 4′-NH2-Ph 4′-NO2-Ph 4′-Ph-Ph 4′-F-Ph 4′-Cl-Ph Ph Ph Ph Ph Ph acetylenyl Ph-acetylenyl vinyl trans-Ph-vinyl cis-Ph-vinyl benzyl phenoxy CO2Et OCONMe2 OCONMePh t-Bu N-Me-pyrrolyl thien-2-yl cyclohexyl phenylethyl cyclopropyl
10.86 10.80 11.30 12.09 10.84 10.66 11.87 10.78 10.95 14.27 10.28 9.86 12.32 12.84 12.15 12.15 9.25 8.40 12.68 7.96 12.16 7.96 6.72 6.60 8.08 7.93 7.93 7.82 9.09 9.37 10.02 7.82 7.90
+ + + + + + + + + + + + + + + + + + + + + + + + +
kcat/KM 523b 1431c 349b 551b 68b 45b 493b 4b 73b 54b 16b NSb 423b 382b NSc NSc NSc 4c 319b 790c NSc 2370c 19b 912b 3580c 4151c 433b 57b 67b 92b 347b 55b 688b 1130c 974c
I2 + + + + + + + + + + + + + + + + + + + + + + + +
ref 12 28 d d d d d d d 14 14 27 12 29 30 30 30 30 29 30 30 30 31 31 32 28 29 33 33 12 14 5 12 32 34
a I is set to + for compounds d e 12 Å that are predicted to be substrates and - for compounds d > 12 Å that are predicted to be 1 nonsubstrates. I2 corresponds to the experimentally determined substrate properties and was fixed to + for a substrate and - for a nonsubstrate (kcat/KM < 10%). b Measured at 30 °C. c Measured at 37 °C. d Determined in this study. NS: no MAO-B substrate activity demonstrated.
substituted tetrahydropyridine derivatives reported in the literature were included without exception. The following four classes of N-methyltetrahydropyridine derivatives were identified (Figure 1): 4-aryl, 4-ethynyl, 4-ethenyl and 4-aryloxy, and 4-arylmethyl, plus a fifth, miscellaneous group. The results (Table 2) compare the d (Å) values of 34 compounds with the literature (or newly estimated) kcat/KM values for those compounds which are substrates for MAO-B. Enzyme activity was based on kcat/KM values as used in similar SAR analyses.3,5 A comparison of the kcat/KM values with the d (Å) values of the 34 compounds listed in Table 1 leads us to propose a maximum d (Å) value [d (Å)max] of 12 Å for MAO-B substrates. In order to relate more easily the predicted vs the experimental values, an indicator (I1) was assigned to each compound to designate its predicted substrate (+) or nonsubstrate (-) behavior. A second indicator (I2) was assigned to each compound to correspond to the experimental result, and then I1 and I2 were compared. Those compounds with a kcat/KM value less than 10% of the kcat/KM value for MPTP (
