Article pubs.acs.org/jmc
Synthesis, Pharmacology, and Biostructural Characterization of Novel α4β2 Nicotinic Acetylcholine Receptor Agonists Christine A. Ussing,†,⊥ Camilla P. Hansen,†,#,⊥ Jette G. Petersen,† Anders A. Jensen,† Line A. H. Rohde,†,‡ Philip K. Ahring,§ Elsebet Ø. Nielsen,§ Jette S. Kastrup,† Michael Gajhede,† Bente Frølund,† and Thomas Balle*,†,∥ †
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark ‡ NeuroSearch A/S, Pederstrupvej 93, DK-2750 Ballerup, Denmark § Aniona ApS, Pederstrupvej 93, DK-2750 Ballerup, Denmark ∥ Faculty of Pharmacy, Building A15, The University of Sydney, Sydney, NSW 2006, Australia S Supporting Information *
ABSTRACT: In our search for selective agonists for the α4β2 subtype of the nicotinic acetylcholine receptors (nAChRs), we have synthesized and characterized a series of novel heterocyclic analogues of 3-(dimethylamino)butyl dimethylcarbamate (DMABC, 4). All new heterocyclic analogues, especially N,N-dimethyl-4-(1-methyl-1H-imidazol-2-yloxy)butan-2-amine (7), showed an improved binding selectivity profile in favor of α4β2 over other nAChR subtypes, primarily due to impaired binding at β4 containing receptors. This observation can be rationalized based on cocrystal structures of (R)-4 and (R)-7 bound to acetylcholine binding protein from Lymnaea stagnalis. Functional characterization at both (α4)2(β2)3 and (α4)3(β2)2 receptors using two-electrode voltage clamp techniques in Xenopus laevis oocytes indicates that the investigated compounds interact differently with the two receptor stoichiometries. Compound 7 is an efficacious agonist at both α4-β2 and α4-α4 binding sites, while the close analogue N,N-dimethyl-4-(1,4-dimethyl-1H-imidazol-2-yloxy)butan-2-amine (9) primarily activates via α4-β2 binding sites. The results suggest that it may be possible to rationally design compounds with specific stoichiometry preferences.
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(α4)3(β2)2.11,12 The (α4)2(β2)3 receptors contain two identical binding sites with α4 as the principal and β2 as the complementary subunit (α4-β2) and display monophasic concentration−response relationships when challenged with acetylcholine (1, Figure 1) and other agonists. The (α4)3(β2)2
INTRODUCTION The α4β2 nicotinic acetylcholine receptor (nAChR) subtype is the most abundant nAChR in the central nervous system (CNS), and numerous studies have confirmed its significance in higher brain function and in relation to diseases and disorders of the nervous system.1,2 Animal models have indicated the potential of α4β2 receptors as drug targets in the treatment of schizophrenia3 and depression,4 and furthermore, the receptors are targeted by marketed smoking cessation aids including cytisine and varenicline.5−7 One of the major challenges when targeting nAChRs is the existence of many receptor subtypes. To date, 16 mammalian nAChR subunits have been identified, and when assembled into functional pentamers, they result in a plethora of receptor subtypes. Considerable efforts have gone into the synthesis of subtype selective nAChR ligands by exploiting differences in binding affinity between subtypes, but the concept of functional selectivity has also been pursued.8−10 The complexity of many subtypes is further complicated by subtypes assembling in different stoichiometric ratios. The α4β2 nAChR is known to assemble in two functional stoichiometries: (α4)2(β2)3 and © XXXX American Chemical Society
Figure 1. Chemical structures of reference compounds. Received: September 28, 2012
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dx.doi.org/10.1021/jm301409f | J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
Article
Scheme 1. Synthesis of Heterocyclic Analogues 5−11a
(a) NaH, 2-bromothiazole, THF, 0 °C, then reflux; (b) NaH, 2-chloropyrazine, THF, 0 °C, then reflux; (c) NaH, 2,4,5-tribromo-1-methyl-1Himidazole, THF, 0 °C, then reflux; (d) (1) n-BuLi, THF, −78 °C, (2) H2O; (e) (1) n-BuLi, Et2O, −78 °C, (2) H2O; (f) (1) n-BuLi, THF, −78 °C, (2) TMSCl, (3) n-BuLi, THF, (4) MeI, (5) Na, MeOH, reflux; (g) HBr/H2O, reflux; (h) NaH, 1H-pyrazol-1-ol, DMF, 0 °C, then rt; (i) NaH, pyridine-3-ol, DMF, 0 °C, then reflux. a
of a series of five α4β2 agonists with varying degrees of efficacy the data rather suggested that an intersubunit bridge formation governs efficacy.18 Over the past decade we have explored the SARs of several homologues of 2 through a classical medicinal chemistry approach with the specific aim of obtaining agonists with high affinity toward the α4β2 subtype.22−26 Of these, the ligand 3(dimethylamino)butyl dimethylcarbamate (DMABC, 4) (Figure 1) is suitable as lead compound for further investigation. Compound 4 has low muscarinic receptor affinity23 and captures the essence of previous SAR studies,22−25 which is in brief an essential tertiary amine, a methyl group in the C-3 position of the carbon chain, and a carbamate moiety bearing only small substituents. In the present study we have explored scaffold 4 further by replacing the aminocarbonyl part of the essential carbamate moiety with five- and six-membered heteroaromatic rings (compounds 5−11). On the basis of the structure of Ls-AChBP with 2 bound,16 we hypothesized that the tertiary amine of these compounds would form contacts to the principal subunit (α-subunit in α4-β2 interface), while the moiety corresponding to the carbamate would have contacts to the complementary subunit (β-subunit in α4-β2 interface). Hence, substitution of the carbamate moiety alters the β2 versus β4 preference and potentially leads to compounds with improved subtype selectivity and could help to clearly distinguish between α4β2 and α3β4 subtypes which are the more abundant and widespread subtypes in the CNS and PNS.27 Here, we present the synthesis and pharmacological characterization of a series of novel heterocyclic analogues of 4, all of which show improved selectivity toward the α4β2 nAChRs in radioligand binding experiments. Further, we present X-ray crystallographic data of Ls-AChBP in complexes with the parent compound 4 and one
receptors, on the other hand, display an inherent biphasic concentration−response relationship to 1 and many other agonists. As we recently demonstrated, this biphasic response can be decomposed into activation by α4-β2 and α4-α4 interface components with the latter being unique to the (α4)3(β2)2 receptor.13 The presence of distinct receptor subpopulations represents yet another challenge complicating rational nAChR drug discovery. However, it may also serve as an intriguing opportunity to obtain functional selectivity by selectively targeting specific receptor stoichiometries. For instance, a positive allosteric modulator, only enhancing the agonist response at (α4)3(β2)2 receptors, has been reported recently.14 In nAChR drug discovery, X-ray crystallographic studies of acetylcholine binding proteins (AChBPs) isolated from mollusks have played an important role in elucidating the molecular details of ligand binding and understanding SARs.15−18 AChBPs are soluble structural homologues of the extracellular domain (ECD) of nAChRs. Despite a relatively low overall sequence identity of 20−24% between AChBPs and the ECDs of the nAChRs, the sequence identity in the orthosteric binding pocket is high and AChBP has been shown to bind a wide range of orthosteric nAChR ligands including 1, carbamoylcholine (2), and (S)-nicotine (3).16,19 In contrast to the binding event, the mechanism by which the receptor is activated by agonists is under debate. Structural studies have revealed a high degree of flexibility of the so-called loop C. The loop caps the ligand upon binding, and the capping motion has been suggested to couple the binding event with channel activation.16,17,20 Some also suggest that the degree of loop closure correlates to efficacy.20,21 However, in a recent study using AChBP from the mollusk Lymnaea stagnalis (Ls-AChBP) as a surrogate for the ECD of the α4β2 receptor we found no correlation between loop C closure and efficacy.18 In the study B
dx.doi.org/10.1021/jm301409f | J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
Article
Table 1. Binding Characteristics of Compounds 1, 3, and 4−11 at Rat α4β2, α3β4, and α4β4 nAChRs and at the α7/5-HT3A and Ls-AChBP/5-HT3A Chimeraa competition binding,b Ki (μM)
selectivity ratio
compd
α4β2
α3β4
α4β4
α7/5-HT3A
Ls-AChBP/5-HT3A
α3β4/α4β2
α4β4/α4β2
1c 3 4e 5 6 7 8 9 10 11
0.033 [7.48 ± 0.11] 0.0076 [8.12 ± 0.05] 0.020 [7.70 ± 0.04] 0.013 [7.89 ± 0.03] 0.24 [6.62 ± 0.02] 0.014 [7.89 ± 0.06] 0.12 [6.92 ± 0.03] 0.28 [6.55 ± 0.05] 0.018 [7.74 ± 0.04] 0.030 [7.52 ± 0.06]
0.62 [6.21 ± 0.08] 0.25 [6.60 ± 0.03] 0.42 [6.38 ± 0.06] 4.7 [5.33 ± 0.06] 42 [4.38 ± 0.05] 7.2 [5.14 ± 0.04] ∼300 [∼3.5] 24 [4.62 ± 0.04] 8.1 [5.09 ± 0.03] 4.9 [5.31 ± 0.02]
0.058 [7.24 ± 0.12] 0.14 [6.85 ± 0.04] 0.15 [6.82 ± 0.04] 1.8 [5.74 ± 0.03] 8.3 [5.08 ± 0.06] 6.2 [5.21 ± 0.04] ∼50 [∼4.3] ∼50 [∼4.3] 11 [4.96 ± 0.05] 8.3 [5.08 ± 0.06]
170 [3.77 ± 0.08] nd >1000 [1000 [
