Binding of mazindol and analogs to the human serotonin and dopamine transporters.

Mazindol has been explored as a possible agent in cocaine addiction pharmacotherapy. The tetracyclic compound inhibits both the dopamine transporter and the serotonin transporter, and simple chemical modifications considerably alter target selectivity. Mazindol, therefore, is an attractive scaffold for both understanding the molecular determinants of serotonin/dopamine transporter selectivity and for the development of novel drug abuse treatments. Using molecular modeling and pharmacologic profiling of rationally chosen serotonin and dopamine transporter mutants with respect to a series of mazindol analogs has allowed us to determine the orientation of mazindol within the central binding site. We find that mazindol binds in the central substrate binding site, and that the transporter selectivity can be modulated through mutations of a few residues in the binding pocket. Mazindol is most likely to bind as the R-enantiomer. Tyrosines 95 and 175 in the human serotonin transporter and the corresponding phenylalanines 75 and 155 in the human dopamine transporter are the primary determinants of mazindol selectivity. Manipulating the interaction of substituents on the 7-position with the human serotonin transporter Tyr175 versus dopamine transporter Phe155 is found to be a strong tool in tuning the selectivity of mazindol analogs and may be used in future drug design of cocaine abuse pharmacotherapies.

Chiral pool synthesis of calystegine A3 from 2-deoxyglucose via a Brown allylation.

Calystegine A(3) is a naturally occurring nortropane iminosugar of which there previously have been three total syntheses. Inspired by our previous work we here report on a fourth approach using 17 steps from 2-deoxy-d-glucose applying a diastereoselective allylation protocol.

Synthesis and glycosidase inhibitory activity of noeurostegine-a new and potent inhibitor of beta-glucoside hydrolases.

A new, stable hemi-aminal nor-tropane christened noeurostegine was synthesised in 22 steps from levoglucosan and tested for inhibitory activity against glycoside hydrolases. Sweet almond and Thermotoga maritimabeta-glucosidases, coffee bean alpha-galactosidase, and Asp. oryzaebeta-galactosidase were inhibited in the low micromolar region but significant tightening of binding to K(i) 50 nM for almond beta-glucosidase was found to occur after pre-incubation. Yeast alpha-glucosidase and E. colibeta-galactosidase were not inhibited at 1 mM.

The two enantiomers of citalopram bind to the human serotonin transporter in reversed orientations.

The two enantiomers of the antidepressant citalopram inhibit the human serotonin transporter substantially differently. Previous studies revealed Tyr95 and Ile172 as important for citalopram binding, however, the overall orientation of the ligands in the binding site and the protein-ligand interaction points remain unknown. The binding of S- and R-citalopram to a human serotonin transporter homology model are herein examined via docking simulations including induced fit effects. For a better description of the formal charges of the ligand when bound inside the protein, polarization effects of the protein were included by additional quantum-polarized ligand docking calculations, where ligand charges are evaluated using QM/MM calculations. By this approach a much clearer picture emerged of the positions of the functional groups of citalopram. The two enantiomers are predicted to bind in the substrate binding pocket with opposite orientations of their aromatic groups. The predicted binding modes are experimentally validated using human wild type and 15 serotonin transporter mutants and 13 optically pure citalopram analogues. Important protein-ligand interaction points were identified validating one binding model for each enantiomer. In the validated model of the high affinity enantiomer, S-citalopram, the fluorine atom is located near Ala173 and Thr439 and the cyano group is in close proximity of Phe341; these contacts are found to be reversed for the R-enantiomer.

Binding and orientation of tricyclic antidepressants within the central substrate site of the human serotonin transporter.

Tricyclic antidepressants (TCAs) have been used for decades, but their orientation within and molecular interactions with their primary target is yet unsettled. The recent finding of a TCA binding site in the extracellular vestibule of the bacterial leucine transporter 11 A above the central site has prompted debate about whether this vestibular site in the bacterial transporter is applicable to binding of antidepressants to their relevant physiological target, the human serotonin transporter (hSERT). We present an experimentally validated structural model of imipramine and analogous TCAs in the central substrate binding site of hSERT. Two possible binding modes were observed from induced fit docking calculations. We experimentally validated a single binding mode by combining mutagenesis of hSERT with uptake inhibition studies of different TCA analogs according to the paired mutation ligand analog complementation paradigm. Using this experimental method, we identify a salt bridge between the tertiary aliphatic amine and Asp(98). Furthermore, the 7-position of the imipramine ring is found vicinal to Phe(335), and the pocket lined by Ala(173) and Thr(439) is utilized by 3-substituents. These protein-ligand contact points unambiguously orient the TCA within the central binding site and reveal differences between substrate binding and inhibitor binding, giving important clues to the inhibition mechanism. Consonant with the well established competitive inhibition of uptake by TCAs, the resulting binding site for TCAs in hSERT is fully overlapping with the serotonin binding site in hSERT and dissimilar to the low affinity noncompetitive TCA site reported in the leucine transporter (LeuT).

An extended study of dimeric phenyl tropanes.

A series of dimeric phenyl tropanes consisting of two molecules of 4-chloro, 4-iodo or 4-(3-thiopheno)-phenyl tropane tethered together at the carboxylic acid moiety by a diamine or diol linker were prepared. The diamines used were a variety of linear, cyclic and aromatic diamines, while the diol tethered compounds were prepared by 'click' chemistry and contained a triazole in the linker. The new compounds were tested for binding to hDAT, hSERT and hNET. Amide linked chlorophenyl tropanes with an aromatic linker was found to be potent and selective DAT inhibitors with the best K(i) value for hDAT being 6nM. The ester linked halophenyl tropanes were more potent but displayed little selectivity in inhibition of monoamine transporter binding. Among the studied compounds an ester linker of 10 atoms between the tropane moieties gave the highest affinity. One monomeric phenyl tropane was made for comparison and was found to be less potent than the dimeric counterparts towards SERT and NET but remain highly active against DAT. Dimeric thiophenophenyl tropanes were in general found to be comparatively poor monoamine transporter binders, but significant gains of affinity of up to 45-fold could be achieved with selected dimeric chlorophenyl tropanes compared to the parent monomer. This observation implies that a secondary binding site that has affinity for phenyl tropanes, most likely the putative S2 site, is located within 13A of the primary central S1 binding site.

Direct formation of beta-glycosides of N-acetyl glycosamines mediated by rare earth metal triflates.

A direct, mild and efficient protocol for the preparation of beta-glycosides of N-acetyl glucosamine (GlcNAc) and N-acetyl galactosamine (GalNAc) has been developed using peracetylated beta-GlcNAc and beta-GalNAc as donors. All rare Earth metal triflate promoters screened were found to promote glycosylation with Sc(OTf)(3) being superior in terms of reaction rate. Simple alcohol glycosylation was found to proceed smoothly in refluxing dichloromethane, whereas higher temperatures under microwave conditions were needed to attain acceptable yields with less reactive, carbohydrate based glycosyl acceptors. The protocol developed was applied to provide the first example of direct chemical formation of a disaccharide using both GlcNAc as a glycosyl donor and acceptor. The alpha-acetate donor was found to be significantly less reactive than the corresponding beta-anomer necessitating higher reaction temperatures under which glycoside anomerisation was found to occur. It was established, that the anomerisation only took place in the presence of both Sc(OTf)(3) and acetic acid.

Stereoelectronic substituent effects.

An investigation was carried out on the influence of the stereochemistry of substituents, particularly hydroxyl groups, on their electronic effects in piperidines, carbohydrates (pyranosides), and related compounds. Polar groups, such as OH, OR, and F, were found in the 3 and 4 position to be much more electron-withdrawing when positioned equatorially rather than axially. In contrast, little difference in electronic effects was observed from apolar groups as a result of epimerization. These observations were believed to be caused by differences in charge-dipole interactions and were used to explain why stereoisomeric glycosides hydrolyze with different rates. The conformational changes of hydroxylated piperidines and related compounds as a function of pH were likewise explained from the different substituent effects of axial and equatorial OH groups.

On the electronic effects of OH groups. Synthesis and investigation of tetrahydroxylated azabicycloheptanes.

Two stereoisomeric 2,3,5,6-tetrahydroxyazabicyclo[2.2.1]heptanes were synthesised and their base strengths determined. The 2,3,5,6-exo-isomer 1 and the 2,3-exo-5,6-endo-isomer 2 were prepared from the Diels-Alder adduct of Boc-pyrrole and tosylacetylene by a route involving osmium catalyzed dihydroxylation and protection, tosyl group reduction and repeated dihydroxylation. Deprotection gave 1, while 2 was obtained by conversion of the diol into the ditriflate, followed by nucleophilic inversion with KNO(2) and deprotection. Synthesis of the 2,3,5,6-endo-isomer by a similar strategy was attempted but failed. The pK(a) of 1 and 2 was determined to be 7.0 and 6.4 respectively. This means that the change in base strength as a result of stereoisomerism of an OH is smaller in the [2.2.1]-azabicyclic system than in the piperidines. This is explained by a difference in charge-dipole interactions in the two systems.

The disarming effect of the 4,6-acetal group on glycoside reactivity: torsional or electronic?

An evaluation of whether the well-known deactivating effect of a 4,6-acetal protection group on glycosyl transfer is caused by torsional or an electronic effect from fixation of the 6-OH in the tg conformation was made. Two conformationally locked probe molecules, 2,4-dinitrophenyl 4,8-anhydro-7-deoxy-2,3,6-tri-O-methyl-beta-D-glycero-D-gluco-octopyranoside (18R) and the L-glycero-D-gluco isomer (18S), were prepared, and their rate of hydrolysis was compared to that of the flexible 2,4-dinitrophenyl 2,3,4,6-tetra-O-methyl-beta-D-glucopyranoside (21) and the locked 2,4-dinitrophenyl 4,6-O-methylidene-2,3-di-O-methyl-beta-D-glucopyranoside (26). The rate of hydrolysis at pH 6.5 was 21 > 18R > 18S > 26, which showed that the deactivating effect of the 4,6-methylene group is partially torsional and partially electronic. A comparison of the rate of acidic hydrolysis of the corresponding methyl alpha-glycosides likewise showed that the probe molecules 17S and 17R hydrolyzed significantly slower than methyl tetra-O-methyl-glucoside 19, confirming a deactivating effect of locking the saccharide in the (4)C(1) conformation. The experiments showed that the hydroxymethyl rotamers deactivate the rate of glycoside hydrolysis in the order tg >> gt > gg.

Steric effects are not the cause of the rate difference in hydrolysis of stereoisomeric glycosides.

[structure: see text] A long-lived and plausible explanation as to why glycosides with axial substituents are more reactive than those with equatorial substituents was given in 1955 by Edward based on sterical hindrance being relieved in the transition state. Using model compounds 5, 6, 8, and 10, we here show conclusively that sterical hindrance is not the controlling factor in glycoside hydrolysis.

Stereoelectronic substituent effects in polyhydroxylated piperidines and hexahydropyridazines.

From the pK(a) values of the conjugate acids of a large series of hydroxylated piperidines and hexahydropyridazines, a consistent difference in basicity was found between stereoisomers having an axial or equatorial hydroxyl (OH) group either beta or gamma to the amine. Compounds with an equatorial OH group in the 3-position were 0.8 pH units more acidic than otherwise identical compounds with an axial OH group, whilst compounds with an equatorial OH group in the 4-position relative to the amine were 0.4 pH units more acidic than the corresponding compound with an axial OH. A similar effect was observed for the COOMe substituent. The difference in electron-withdrawing power of axial and equatorial substituents was explained by a difference in charge-dipole interactions in the two systems. Since this stereoelectronic substituent effect causes differences in basicity in different conformers, certain piperidines and hexahydropyridazines were found to change conformation upon protonation. A method for predicting the pK(a) of piperidines which takes stereochemistry into account is described.