Getting the adrenaline going: crystal structure of the adrenaline-synthesizing enzyme PNMT.

BACKGROUND: Adrenaline is localized to specific regions of the central nervous system (CNS), but its role therein is unclear because of a lack of suitable pharmacologic agents. Ideally, a chemical is required that crosses the blood-brain barrier, potently inhibits the adrenaline-synthesizing enzyme PNMT, and does not affect other catecholamine processes. Currently available PNMT inhibitors do not meet these criteria. We aim to produce potent, selective, and CNS-active PNMT inhibitors by structure-based design methods. The first step is the structure determination of PNMT. RESULTS: We have solved the crystal structure of human PNMT complexed with a cofactor product and a submicromolar inhibitor at a resolution of 2.4 A. The structure reveals a highly decorated methyltransferase fold, with an active site protected from solvent by an extensive cover formed from several discrete structural motifs. The structure of PNMT shows that the inhibitor interacts with the enzyme in a different mode from the (modeled) substrate noradrenaline. Specifically, the position and orientation of the amines is not equivalent. CONCLUSIONS: An unexpected finding is that the structure of PNMT provides independent evidence of both backward evolution and fold recruitment in the evolution of a complex enzyme from a simple fold. The proposed evolutionary pathway implies that adrenaline, the product of PNMT catalysis, is a relative newcomer in the catecholamine family. The PNMT structure reported here enables the design of potent and selective inhibitors with which to characterize the role of adrenaline in the CNS. Such chemical probes could potentially be useful as novel therapeutics.

Effects of a 3-alkyl-, 4-hydroxy- and/or 8-aromatic-substituent on the phenylethanolamine N-methyltransferase inhibitor potency and alpha2-adrenoceptor affinity of 2,3,4,5-tetrahydro-1H-2-benzazepines.

2,3,4,5-Tetrahydro-1H-2-benzazepine (THBA; 1) is nearly 100-fold more selective an inhibitor of phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) versus the alpha2-adrenoceptor than is 1,2,3,4-tetrahydroisoquinoline (THIQ; 2) (1: PNMT K(i)= 3.3 microM, alpha2-adrenoceptor K(i) = 11 microM, selectivity [alpha2 K(i)/PNMT K(i)] = 3.3; 2: PNMT K(i) = 9.7 microM, alpha2 K(i) = 0.35 microM, selectivity=0.036;). Since the PNMT inhibitory activity and selectivity of THIQ were enhanced by the introduction of a hydrophilic electron-withdrawing 7-substituent and a 3-alkyl-substituent, a similar study was conducted on THBA. 8-Nitro-THBA (3) was found to be as potent an inhibitor of PNMT as its THIQ analogue (21) and to be more selective due to its reduced alpha2-adrenoceptor affinity (3: PNMT K(i) = 0.39 microM, alpha2 K(i) = 66 microM, selectivity = 170; 21: PNMT K(i) = 0.41 microM, alpha2 K(i) = 4.3 microM, selectivity = 10). Introduction of a 3-alkyl substituent on the THBA nucleus decreased both the alpha2-adrenoceptor affinity and the PNMT inhibitory activity, suggesting an area of steric bulk intolerance at both sites. 4-Hydroxy-THBA (15), which can be considered a conformationally-restricted analogue of 3-hydroxymethyl-THIQ (30), exhibited poorer PNMT inhibitory activity and less selectivity than 30 (15: PNMT K(i) = 58 microM, alpha2 K(i) = 100 microM, selectivity = 1.7; 30: PNMT K(i) = 1.1 microM, alpha2 K(i) = 6.6 microM, selectivity = 6.0). While the addition of an 8-nitro group to 15 increased the selectivity of 16 as compared to its THIQ analogue (31), it was not as potent at PNMT nor as selective as 8-nitro-THBA (3) (16, PNMT K(i) = 5.3 microM, alpha2 K(i) = 680 microM, selectivity = 130; 31: PNMT K(i) = 0.29 microM, alpha2 K(i) = 19 microM, selectivity = 66). Compound 3 is the most selective (PNMT/alpha2) and one of the more potent at PNMT compounds yet reported in the benzazepine series, and should have sufficient lipophilicity to penetrate the blood-brain barrier (CLogP = 1.8).

Synthesis and evaluation of 4-fluoro-8-substituted-2,3,4,5-tetrahydro-1H-2-benzazapines as selective inhibitors of phenylethanolamine N-methyltransferase versus the alpha(2)-adrenoceptor.

A small series of 4-fluoro-8-substituted-2,3,4,5-tetrahydro-1H-2-benzazapines (4-fluoro-THBAs; 12-15) were synthesized and evaluated as inhibitors of phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28) and as inhibitors of the binding of clonidine at the alpha(2)-adrenoceptor. 4-Fluoro-THBAs 13-15 displayed selectivity ratios (alpha(2) K(i)/PNMT K(i)) greater than 75 and 4-fluoro-8-nitro-THBA (13) was found to be one of the most selective inhibitors of PNMT known, with a selectivity ratio of greater than 900. These compounds are also quite lipophilic and according to previous results from this laboratory should be able to penetrate the blood-brain barrier. These 4-fluoro-THBAs represent important leads in the development of new, more selective, CNS-active inhibitors of PNMT.

Phenylethanolamine N-methyltransferase kinetics: bovine versus recombinant human enzyme.

Epinephrine (Epi) acts as a neurotransmitter in the brain, but its function therein is not well understood. Phenylethanolamine N-methyltransferase (PNMT) catalyzes the final step in the biosynthesis of Epi and is thus a pharmacological target to investigate the function of Epi in the central nervous system. The kinetic differences between bovine adrenal PNMT and human brain PNMT for a number of substrates and inhibitors are examined and the results reported.

Enantiospecific synthesis of 3-fluoromethyl-, 3-hydroxymethyl-, and 3-chloromethyl-1,2,3,4-tetrahydroisoquinolines as selective inhibitors of phenylethanolamine N-methyltransferase versus the alpha(2)-adrenoceptor.

An enantiospecific method was developed for the synthesis of 3-fluoromethyl-, 3-hydroxymethyl-, and 3-chloromethyl-7-substituted-1,2,3,4-tetrahydroisoquinolines (THIQs) from phenylalanine. Biochemical evaluation of the enantiomers of these compounds at both PNMT and the alpha(2)-adrenoceptor indicates that both sites display similar stereoselectivity. Overall the R-enantiomer was usually the more potent enantiomer at both PNMT and the alpha(2)-adrenoceptor for these 3-fluoromethyl-, 3-hydroxymethyl-, and 3-chloromethyl-THIQs. The one exception is 3-hydroxymethyl-7-nitro-THIQ (9), which was found to display the opposite stereoselectivity at the alpha(2)-adrenoceptor. A comparison of the PNMT inhibitory potency of the enantiomers of these 3-fluoromethyl-, 3-hydroxymethyl-, and 3-chloromethyl-THIQs indicates that all of the 3-substituted-THIQs displayed similar inhibitory potency for PNMT. However, the nature of the 3-substituent was found to have a major effect on the alpha(2)-adrenoceptor affinity of these compounds with the 3-hydroxymethyl- and 3-fluoromethyl-THIQs having the highest affinity and THIQs containing the 3-chloromethyl moiety the least. Compounds R-3-fluoromethyl-7-cyano-THIQ (R-12) and R-3-fluoromethyl-7-N-(4-chlorophenyl)aminosulfonyl-THIQ (R-13) and both enantiomers of 3-chloromethyl-7-nitro-THIQ (R- and S-30) are the most selective inhibitors in this study and display selectivities (alpha(2)-adrenoceptor K(i)/PNMT K(i)) greater than 200. These compounds give important insight into the steric and stereochemical preferences of both PNMT and the alpha(2)-adrenoceptor, which should assist in the development of new PNMT inhibitors.

The oxetane conformational lock of paclitaxel: structural analysis of D-secopaclitaxel.

Analysis of the 1H NMR data of paclitaxel in comparison with its oxetane ring-opened analogue D-secopaclitaxel suggests that the oxetane moiety (D-ring) of paclitaxel serves as a conformational lock for the diterpene moiety and the C13 side chain.

Synthesis and biochemical evaluation of 3-fluoromethyl-1,2,3, 4-tetrahydroisoquinolines as selective inhibitors of phenylethanolamine N-methyltransferase versus the alpha(2)-adrenoceptor.

A series of 3-fluoromethyl-1,2,3,4-tetrahydroisoquinolines (3-fluoromethyl-THIQs) was proposed, and their phenylethanolamine N-methyltransferase (PNMT) and alpha(2)-adrenoceptor affinities were predicted through the use of comparative molecular field analysis (CoMFA) models. These compounds were synthesized and evaluated for affinity at PNMT and the alpha(2)-adrenoceptor. It was discovered that these compounds are some of the most selective inhibitors of PNMT versus the alpha(2)-adrenoceptor known. To determine the ability of these compounds to penetrate the blood-brain barrier (BBB), a series of THIQs possessing a variety of calculated partition coefficients (Clog P) were assayed using an in vitro BBB model. This study found a good correlation between lipophilicity (Clog P) and BBB permeability, which indicated that THIQs possessing Clog P values of at least 0.13-0.57 should have some penetration into the brain. Two compounds [3-fluoromethyl-7-N-(4-chlorophenyl)aminosulfonyl-THIQ (18) and 3-fluoromethyl-7-cyano-THIQ (20)] possess calculated partition coefficients greater than 0.57 and display selectivities (alpha(2)-adrenoceptor K(i)/PNMT K(i)) greater than 200 and thus represent promising leads in the development of highly selective inhibitors of PNMT with the ability to penetrate the BBB.

Synthesis and evaluation of 3-trifluoromethyl-7-substituted-1,2,3, 4-tetrahydroisoquinolines as selective inhibitors of phenylethanolamine N-methyltransferase versus the alpha(2)-adrenoceptor.

A series of 3-trifluoromethyl-1,2,3,4-tetrahydroisoquinolines was synthesized and evaluated as inhibitors of phenylethanolamine N-methyltransferase (PNMT) and as inhibitors of the binding of clonidine at the alpha(2)-adrenoceptor. These compounds were found to be selective inhibitors of PNMT due to their decreased affinity for the alpha(2)-adrenoceptor, which was attributed to steric bulk intolerance around the 3-position of 1,2,3,4-tetrahydroisoquinoline (THIQ) at the alpha(2)-adrenoceptor and to the decreased pK(a) of the THIQ amine due to the 3-trifluoromethyl moiety. Overall, these compounds displayed less affinity for PNMT compared to previously studied THIQ-type inhibitors, except for 16 which was found to have good affinity for PNMT (PNMT K(i) = 0.52 microM). Compounds 14 and 16 proved to be the most selective inhibitors in this small series of compounds and are some of the most selective inhibitors of PNMT known (14, selectivity alpha(2) K(i)/PNMT K(i) = 700; 16, selectivity alpha(2) K(i)/PNMT K(i) > 1900). Compounds 14 and 16 are also quite lipophilic due to the 3-trifluoromethyl moiety and represent promising new leads for the development of new highly selective inhibitors of PNMT, which should be sufficiently lipophilic to penetrate the blood-brain barrier.

1,3-Dimethyl-7-substituted-1,2,3,4-tetrahydroisoquinolines as probes for the binding orientation of tetrahydroisoquinoline at the active site of phenylethanolamine N-methyltransferase.

In order to determine the function of epinephrine (Epi) in the central nervous system, we have targeted the enzyme that catalyzes the final step in the biosynthesis of Epi, phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28). 1,2,3,4-Tetrahydroisoquinolines (THIQs) are inhibitors of this enzyme, but also display affinity for the alpha2-adrenoceptor. To gain further understanding about how THIQs bind at the PNMT active site and in an attempt to further increase the selectivity of THIQ-type inhibitors versus the alpha2-adrenoceptor, a series of cis- and trans-1,3-dimethyl-7-substituted-THIQs were synthesized. Evaluation of these compounds suggests that THIQs bind in two different orientations at the PNMT active site, based on the lipophilicity of the 7-substituent. However, no significant increases in selectivity versus the alpha2-adrenoceptor were observed for these compounds.

3,7-Disubstituted-1,2,3,4-tetrahydroisoquinolines display remarkable potency and selectivity as inhibitors of phenylethanolamine N-methyltransferase versus the alpha2-adrenoceptor.

3-Hydroxymethyl-1,2,3,4-tetrahydroisoquinoline (4) is a more selective inhibitor (PNMT Ki = 1.1 microM, alpha2 Ki = 6.6 microM, selectivity (alpha2 Ki/PNMT Ki) = 6.0) of phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28), with respect to its alpha2-adrenoceptor affinity, than is 3-methyl-1,2,3, 4-tetrahydroisoquinoline (2; PNMT Ki = 2.1 microM, alpha2 Ki = 0.76 microM, selectivity = 0.36) or 1,2,3,4-tetrahydroisoquinoline (1, THIQ; PNMT Ki = 9.7 microM, alpha2 Ki = 0.35 microM, selectivity = 0. 036). Evaluation of the O-methyl ether derivative of 4 suggested that the 3-hydroxymethyl substituent might be involved in a hydrogen-bond donor-type of interaction at a sterically compact region in the PNMT active site. The directionality of the steric bulk tolerance at both the PNMT active site and the alpha2-adrenoceptor appears to be the same. Since the presence of a hydrophilic electron-withdrawing substituent (such as NO2, SO2CH3, or SO2NH2) at the 7-position of THIQ reduced the binding affinity toward the alpha2-adrenoceptor, we investigated the combination of both a hydrophilic electron-withdrawing 7-substituent and a 3-alkyl substituent on a THIQ nucleus. A synergistic effect in increasing the PNMT-inhibitory potency of the THIQ nucleus and reducing the affinity toward the alpha2-adrenoceptor was observed with this 3, 7-disubstitution. Remarkably, 7-aminosulfonyl-3-hydroxymethyl-THIQ (12; PNMT Ki = 0.34 microM, alpha2 Ki = 1400 microM, selectivity = 4100) displayed a 23-680-fold enhanced selectivity over the parent compounds 27 (SK&F 29661; PNMT Ki = 0.55 microM, alpha2 Ki = 100 microM, selectivity = 180) and 4 (selectivity = 6.0) and is thus the most selective PNMT inhibitor yet reported.

Comparative molecular field analysis (CoMFA) models of phenylethanolamine N-methyltransferase (PNMT) and the alpha2-adrenoceptor: the development of new, highly selective inhibitors of PNMT.

As a guide to the development of new and more selective inhibitors of phenylethanolamine N-methyltransferase (PNMT) vs the alpha2-adrenoceptor, we have performed a comparative molecular field analysis (CoMFA) on a series of 80 benzylamine analogues. Using the models obtained, we have proposed a series of 3-trifluoromethyl-1,2,3,4-tetrahydroisoquinolines and predicted the activity of other analogues.

Synthesis, biochemical evaluation, and classical and three-dimensional quantitative structure-activity relationship studies of 7-substituted-1,2,3,4-tetrahydroisoquinolines and their relative affinities toward phenylethanolamine N-methyltransferase and the alpha2-adrenoceptor.

7-Substituted-1,2,3,4-tetrahydroisoquinolines (7-substituted-THIQs) are potent inhibitors of phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28), the enzyme involved in the biosynthesis of epinephrine. Unfortunately, most of these compounds also exhibit strong affinity for the alpha2-adrenoceptor. To design a selective (PNMT vs alpha2-adrenoceptor affinity) inhibitor of PNMT, the steric and electrostatic factors responsible for PNMT inhibitory activity and alpha2-adrenoceptor affinity were investigated by evaluating a number of 7-substituted-THIQs. A classical quantitative structure-activity relationship (QSAR) study resulted in a three-parameter equation for PNMT (PNMT pKi = 0.599pi - 0.0725MR + 1. 55sigmam + 5.80; n = 27, r = 0.885, s = 0.573) and a three-parameter equation for the alpha2-adrenoceptor (alpha2 pKi = 0.599pi - 0. 0542MR - 0.951sigmam + 6.45; n = 27, r = 0.917, s = 0.397). These equations indicated that steric effects and lipophilicity play a similar role at either active site but that electronic effects play opposite roles at either active site. Two binding orientations for the THIQs were postulated such that lipophilic and hydrophilic 7-substituents would not occupy the same region of space at either binding site. Using these two binding orientations, based on the lipophilicity of the 7-substituent, comparative molecular field analysis (CoMFA) models were developed that showed that the steric and electrostatic interactions at both sites were similar to those previously elaborated in the QSAR analyses. Both the QSAR and the CoMFA analyses showed that the steric interactions are similar at the PNMT active site and at the alpha2-adrenoceptor and that the electrostatic interactions were different at the two sites. This difference in electrostatic interactions might be responsible for the selectivity of THIQs bearing a nonlipophilic electron-withdrawing group at the 7-position. These QSAR and CoMFA results will be useful in the design of potent and selective (PNMT vs alpha2-adrenoceptor affinity) inhibitors of PNMT.

Examination of the role of the acidic hydrogen in imparting selectivity of 7-(aminosulfonyl)-1,2,3,4-tetrahydroisoquinoline (SK&F 29661) toward inhibition of phenylethanolamine N-methyltransferase vs the alpha 2-adrenoceptor.

7-(Aminosulfonyl)-1,2,3,4-tetrahydroisoquinoline (SK&F 29661, 1) is a potent inhibitor of the enzyme phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28). In contrast to other inhibitors of PNMT, it is also highly selective toward PNMT in comparison with its affinity toward the alpha 2-adrenoceptor (PNMT Ki = 0.55 microM, alpha 2 Ki = 100 microM, selectivity [alpha 2 Ki/PNMT Ki] = 180). A diverse set of compounds was synthesized and evaluated to probe the role of the acidic hydrogen of the aminosulfonyl group of 1 in imparting this selectivity. Compounds were designed to investigate the effect on selectivity of the acidity of the NH group [the 7-N-methyl (compound 5) and 7-N-(p-chlorophenyl) (compound 4) derivatives of 1], the relative spatial position of the acidic hydrogen [7-(N-(methylsulfonyl)amino)-1,2,3,4-tetrahydroisoquinoline (6) and 7-((N-(methylsulfonyl)amino)methyl)-1,2,3,4-tetrahydroisoquinoline (8)], or the effect of the substitution of an acidic phenolic group for the aminosulfonyl moiety [1-(aminomethyl)-6-hydroxynaphthalene (23) and 8-hydroxy-1,2,3,4-tetrahydrobenz[h]isoquinoline (9)]. All of the compounds studied displayed lower affinity for PNMT than 1, and nine of the eleven compounds studied showed increased, rather than the desired decreased, affinity for the alpha 2-adrenoceptor. Specifically, compound 4, in which the aminosulfonyl NH group is more acidic than that in 1, showed greatly reduced selectivity on account of increased alpha 2-adrenoceptor affinity as compared to 1 (PNMT Ki = 2.6 microM, alpha 2 Ki = 6.3 microM, selectivity = 2.4). Compound 8, in which the acidic NH group is in the same region of space as that in 1, although the aminosulfonyl group is reversed with respect to the aromatic ring, showed poor PNMT affinity and modest alpha 2-adrenoceptor affinity (PNMT Ki = 330 microM, alpha 2 Ki = 18 microM, selectivity = 0.055). Compound 9, in which a phenolic group is in the same region of space as the acidic NH of 1, exhibited the best alpha 2-adrenoceptor affinity of any of the compounds studied (PNMT Ki = 0.98 microM, alpha 2 Ki = 0.078 microM, selectivity = 0.080). Results from this study suggest that the selectivity of 1 is not solely due to the presence of an acidic hydrogen on the 7-aminosulfonyl group of 1 but is likely also dependent on some other property (e.g. electron-withdrawing character) of the aminosulfonyl group.

Recombinant human phenylethanolamine N-methyltransferase: overproduction in Escherichia coli, purification, and characterization.

The gene encoding phenylethanolamine N-methyltransferase (PNMT) has been amplified from a human adrenal medulla cDNA library. Following ligation of the gene into a pET3a-derived expression vector and transformation into Escherichia coli BL21(DE3)pLysS, PNMT was expressed, yielding about 10% of the soluble protein. The enzyme was purified to homogeneity by ammonium sulfate fractionation followed by ion-exchange chromatography and gel filtration. The Km for phenylethanolamine and S-adenosyl-L-methionine were determined to be 130 and 16 microM, respectively. The enzyme could be inhibited by reagents expected to modify cysteine, arginine, tyrosine, and histidine residues, but not by methyl acetimidate, a reagent expected to modify lysine residues.

Effect of ring size or an additional heteroatom on the potency and selectivity of bicyclic benzylamine-type inhibitors of phenylethanolamine N-methyltransferase.

In the search for potent and selective inhibitors of the enzyme phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28), we examined the effect of ring size or an additional heteroatom in the conformationally-restricted benzylamine-type PNMT inhibitors. Based on semiempirical calculations (MNDO) and molecular modeling studies, PNMT-inhibitory activity of these compounds seemed to be dependent on (a) the torsion angle between the plane of the aromatic ring and the endo N atom lone pair ( tau 2 angle), with the optimal value of tau 2 being about - 75 degrees, and (b) the amount of steric bulk about the 3-position of 1,2,3,4-tetrahydroisoquinoline (5, THIQ). 2,3,4,5-Tetrahydro-1H-2-benzazepine (6) was found to have the highest selectivity (PNMT Ki = 3.34 microM, alpha 2 Ki = 11 microM, selectivity = 3.2) as compared to other homologues of THIQ (PNMT Ki = 9.67 microM, alpha 2 Ki = 0.35 microM, selectivity = 0.036). The higher PNMT-inhibitory activity of 6 was attributed to favorable steric interactions of the puckered methylene groups in the putative bioactive conformation of 6 at the PNMT active site, whereas unfavorable interactions of these puckered methylene groups at the alpha 2-adrenoceptor were thought to be the cause of reduced alpha 2 affinity of 6. No further enhancement of the selectivity of the benzazepine ring system could be obtained via introduction of a second heteroatom (N, O, S) at the 5-position in this ring system.

Structures of two conformationally defined phenylethanolamines: exo-1,4-epoxy-2-formamido-1,2,3,4-tetrahydro-8-trifluoromethylnaphthale ne and exo-1,4-epoxy-2-formamido-1,2,3,4-tetrahydro-6-trifluoromethylnaphthale ne.

(+/-)-exo-1,4-Epoxy-2-formamido-1,2,3,4-tetrahydro-8- trifluoromethylnaphthalene (1), C12H10F3NO2, Mr = 257.21, Pccn, a = 8.895 (1), b = 19.968 (5), c = 12.656 (3) A, V = 2247.9 (8) A3, Z = 8, Dx = 1.520 g cm-3, lambda(Mo K alpha) = 0.71069 A, mu = 1.49 cm-1, F(000) = 1056, T = 297 K, R = 0.0466 for 1481 independent reflections collected. The torsion angle for N(10)-C(2)-C(1)-C(8a) is 167.4 (2) degrees. (+/-)-exo-1,4-Epoxy-2-formamido-1,2,3,4-tetrahydro-6- trifluoromethylnaphthalene, (2), C12H10F3NO2, Mr = 257.21, P2(1)2(1)2(1), a = 8.52 (2), b = 26.15 (2), c = 5.06 (5) A, V = 1127 (11) A3, Z = 4, Dx = 1.516 g cm-3, lambda(Mo K alpha) = 0.71069 A, mu = 1.29 cm-1, F(000) = 528, T = 297 K, R = 0.108 for 932 independent reflections collected. The torsion angle N(10)-C(2)-C(1)-C(8a) is 172.5 (6) degrees; the formamido group in both (1) and (2) is exo. X-ray studies on (1) suggest a hydrogen bond between N(10) and O(12) and similarly for (2).

Sodium channel binding and anticonvulsant activities for the enantiomers of a bicyclic 2,4-oxazolidinedione and monocyclic models.

Racemic 7-phenyl-9,10-dioxo-1-aza-8-oxabicyclo[5.2.1]decane (1), a bicyclic 2,4-oxazolidinedione that we previously reported was a possible sodium channel anticonvulsant, was resolved into its enantiomeric forms, the absolute configurations were determined, and the stereoisomers were evaluated for relative sodium channel binding and whole animal anticonvulsant activities. Similar studies were carried out with two monocyclic models, 5-ethyl-5-phenyl-2,4-oxazolidinedione (2) and 5-ethyl-3-methyl-5-phenyl-2,4-oxazolidinedione (3). None of these isomers exhibited stereoselective effects in the sodium channel assay, and only modest enantioselectivities were observed for 2 and 3 in the anticonvulsant assays. (R)-(-)-1 was, however, 4 times more toxic than (S)-(+)-1 in the rotorod test, and due to its larger protective index, (S)-(+)-1 exhibited greater therapeutic potential than either (R)-(-)-1 or racemic 1.

Conformationally restricted and conformationally defined tyramine analogues as inhibitors of phenylethanolamine N-methyltransferase.

In a search for a selective inhibitor for the epinephrine synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28), phenolic 2-aminotetralins (12-15 as conformationally restricted analogues of tyramine) and phenolic benzobicyclo[3.2.1]octylamines (22-24 as conformationally defined analogues of tyramine) were used to gain information about the binding interactions of the catecholic hydroxyl groups in the natural substrate norepinephrine at the active site of PNMT. In addition, these analogues provided information about the effects of conformational flexibility on active-site interaction of the aminoethyl side chain in phenolic phenylethylamines that may aid in learning the manner in which norepinephrine binds at the active site of PNMT. Analogues 22-24 were synthesized by a nine-step sequence, in which a Friedel-Crafts type intramolecular cyclization was the key step in the construction of the benzobicyclo[3.2.1]octane skeleton. p-Tyramine (10, Ki = 294 microM) was more potent than phenylethylamine (1, Ki = 854 microM) but m-tyramine (9, Ki = 1250 microM) was less potent than phenylethylamine as an inhibitor of PNMT. Similarly, in the conformationally restricted and conformationally defined tyramine analogues (12-15 and 22-24, respectively), the analogues with the p-tyramine moiety (14, Ki = 4.7 microM; 23, Ki = 111 microM) bind to PNMT better than do the corresponding unsubstituted compounds (16, Ki = 6.8 microM; 25, Ki = 206 microM) while the analogues with the m-tyramine moiety (13, 15, 22, and 24) have a lower binding affinity than do 16 and 25. The greatly enhanced activity of the phenolic 2-aminotetralins (12-15) compared with m- and p-tyramine (9 and 10, respectively) is likely due to the restriction of the side-chain conformation. The conformationally defined analogues 22-24 were less active than the conformationally restricted ones, 12-15, although the low-energy half-chair conformation of 2-aminotetralin is defined in 22-24. The reduced activity of 22-24 compared with the activity of 12-15 is probably due to the steric hindrance from the extra bridging atoms in binding to PNMT. The interaction of the p-hydroxyl group of the tyramine moiety may involve hydrogen bonding since the corresponding methyl ethers show a greatly reduced affinity for the active site of PNMT (Ki = 34 and 389 microM for methoxy analogues 28 and 35, compared to Ki = 4.7 and 111 microM for the corresponding phenolic analogues 14 and 23).

Conformational and steric aspects of phenylethanolamine and phenylethylamine analogues as substrates or inhibitors of phenylethanolamine N-methyltransferase.

The conformational and steric aspects of binding to phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28) for phenylethanolamine substrates and phenylethylamine inhibitors were probed with three conformationally defined analogues (11, 12, and 13) of phenylethylamine (1) and phenylethanolamine (6) containing the benzobicyclo[3.2.1]octane skeleton. The 2-aminotetralin (2AT) moiety in conformationally defined analogues 11, 12, and 13 exists in a half-chair conformation with an equatorial amino group. Although conformationally restricted phenylethylamine analogue 2AT (3, Ki = 6.8 microM) and conformationally restricted phenylethanolamine analogues (cis)- and (trans)-2-amino-1-tetralol (9, Km = 22 microM; Vmax = 0.15; 100 X Vmax/Km = 0.68; 10, Ki = 9.4 microM) are good ligands for PNMT, none of the analogues 11, 12, and 13 showed activity as a substrate of PNMT. The fact that 11 (Ki = 206 microM) is more potent than analogues 4 (Ki = 1296 microM) and 5 (Ki = 479 microM), with a half-boat 2AT moiety, suggests that PNMT preferentially binds the half-chair conformation of 2AT at the active site. This is consistent with previous findings that a fully extended conformation for the aminoethyl side chain of phenylethylamine inhibitors is optimal for PNMT binding. The reduced activity of 11, 12 (Ki = 1246 microM), and 13 (Ki = 3000 microM), compared with 2AT and (cis)- and (trans)-2-amino-1-tetralol (9 and 10) is consistent with a negative steric interference from the extra ethano bridge in 11, 12, and 13. The results from 11, 12, and 13, combined with previous findings, suggest that PNMT interacts better with relatively planar ligands.

Inhibition of norepinephrine transport into synaptic vesicles by amphetamine analogs.

The abilities of several amphetamine analogs with restricted conformations to inhibit uptake of [3H]norepinephrine into synaptic vesicles isolated from rat brain cerebral cortex were compared. [3H]Norepinephrine was accumulated in the vesicles with a Km of 3.5 microM and a Vmax of 7.6 pmol/g of tissue per min. This uptake was inhibited by reserpine (IC50, 6.4 nM), amphetamine (IC50, 2.5 microM) and eight amphetamine analogs. 2-Aminotetralin, the most flexible of the analogs (capable of assuming both gauche and anticonformations), was the most potent (IC50, 22 microM). The side chain of amphetamine was held in one of its two low energy conformations [transantiperiplanar (extended) and gauche (folded)]. This was accomplished by using the benzobicyclo[2.2.1]heptane, benzobicylco[2.2.2]octane, or tetrahydroisoquinoline ring systems. The potencies of all of the conformationally defined analogs were reduced with IC50 values of 120 to 370 microM and the potency differences between anti- and gauche conformations were small. These results are in contrast to those obtained by us earlier for inhibition of neuronal reuptake and suggest that vesicular uptake may be more conformationally restrictive than neuronal reuptake. It is possible that: 1) the amphetamine pharmacophore must retain some conformational flexibility for vesicular uptake (hence activity for 2-aminotetralin but not for the rigid analogs); 2) there is another higher energy conformation of amphetamine not present in any of the rigid analogs evaluated that is required for optimal interaction with the vesicular uptake site; or 3) the extra steric bulk of the bridging atoms in the conformational analogs severely interferes with binding at the vesicular uptake site.