Article pubs.acs.org/jmc
Synthesis and Biological Evaluation of a Novel Series of Heterobivalent Muscarinic Ligands Based on Xanomeline and 1‑[3(4-Butylpiperidin-1-yl)propyl]-1,2,3,4-tetrahydroquinolin-2-one (77LH-28-1) Alessandro Bonifazi,† Hideaki Yano,‡ Fabio Del Bello,† Aniket Farande,† Wilma Quaglia,† Riccardo Petrelli,† Rosanna Matucci,§ Marta Nesi,§ Giulio Vistoli,∥ Sergi Ferré,‡ and Alessandro Piergentili*,† †
Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università di Camerino, Via S. Agostino 1, 62032 Camerino, Italy Integrative Neurobiology Section, Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse, Intramural Research Program, 333 Cassell Drive, Baltimore, Maryland 21224, United States § Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino (NEUROFARBA), Università di Firenze, Viale G. Pieraccini 6, 50139 Firenze, Italy ∥ Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy ‡
S Supporting Information *
ABSTRACT: Novel bitopic hybrids, based on the M1/M4 muscarinic acetylcholine receptor (mAChR) orthosteric agonist xanomeline (1) and the putative M1 mAChR allosteric agonist 1-[3-(4-butylpiperidin-1-yl)propyl]-1,2,3,4-tetrahydroquinolin-2-one (77-LH-28-1, 3) connected by an aliphatic linker of variable length, were prepared. The novel heterobivalent hybrids 4a−f along with the intermediate alcohols 5a−f were pharmacologically evaluated in radioligand binding assays and some of them for their functional efficacies in bioluminescence resonance energy transfer (BRET)-based assays to give an insight into the structure−activity relationships of bivalent and linkerattached compounds in mAChRs. The hybrid 4d exhibited high efficacy for β-arrestin2 engagement in M1 mAChR and alcohol 5c behaved much like 3 at M1 mAChR and showed full antagonism in both Gi activation and β-arrestin2 engagement at M4 mAChR. Moreover, docking simulations on the M1 mAChR model were performed to elucidate how the binding mode of the proposed compounds is influenced by the linker length.
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INTRODUCTION Muscarinic acetylcholine receptors (mAChRs) are members of the superfamily A guanine nucleotide-binding protein-coupled receptors (GPCRs) and mediate the action of the neurotransmitter acetylcholine (ACh) in both the peripheral and central nervous systems (CNS).1 Five mAChR subtypes (M1− M5) have been cloned and subdivided into two groups. M1, M3, and M5 mAChRs are insensitive to pertussis toxin and couple to Gq/11 to activate phospholipase C and increase phosphoinositol (PI) turnover, resulting in intracellular calcium release. M2 and M4 mAChRs are sensitive to pertussis toxin and mediate their signaling preferentially via Gi/o, whose activation leads to inhibition of cAMP production through inactivation of adenylate cyclase and influences subsequent downstream pathways.1 All five mAChR subtypes are expressed in the CNS, with the M1 mAChR being the most predominant as they are found in many brain regions including striatum, cortex, and hippocampus.2 M1 mAChR agonism has been suggested to have a role in the treatment of Alzheimer’s disease (AD)3,4 and cognitive impairment associated with schizophrenia.5 Xanomeline (1) (Chart 1), an M1/M4 mAChR preferring orthosteric © XXXX American Chemical Society
agonist and a potent agonist for 5-HT1A and 5-HT1B receptors as well as an antagonist for 5-HT2 receptors,6 had been implicated as a useful therapeutic agent for the treatment of AD7,8 and schizophrenia.9 However, adverse effects, such as eccessive salivation, lachrymation, gastrointestinal disturbances, and high incidence of syncope, were observed in an efficacious dose range that resulted in the removal of 1 for long-term clinical use.7,8 More recently, a next generation of M1 mAChRpreferring agonists, including 2 (AC-42)10 and 3 (77-LH-281)11 (Chart 1), has emerged. These compounds, originally classified as allosteric agonists, display mixed modes of orthosteric or allosteric pharmacology depending on the experimental assay conditions.12 It is worth noting here that 2 and 3 bear an alkyl chain in their structure as the orthosteric agonist 1, indicating perhaps a shared function of such chain structures in allosteric and orthosteric ligands. Allosteric modulators have classically been defined as ligands that bind at sites topographically distinct from the orthosteric Received: July 31, 2014
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dx.doi.org/10.1021/jm501173q | J. Med. Chem. XXXX, XXX, XXX−XXX
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Chart 1. Chemical Structures of Compounds 1−7
length (4a−f; Chart 1). Because both lead compounds bear in their structure a lipophilic chain, the aliphatic spacer can be considered as an extension, or shortening, of the O-hexyl chain or butyl chain of 1 and 3, respectively (Chart 1). Moreover, the aliphatic chain might not only serve as a linker but also constitute an essential moiety contributing to the interaction of the ligand with the mAChRs. To investigate the role of the two pharmacophoric units of the heterobivalent hybrids 4a−f and the nature of the corresponding receptor sites, the homobivalent derivatives of 1 and 3 (618 and 7, respectively) (Chart 1) were also synthesized and evaluated. Although 3 has been reported to interact with a site different from the orthosteric site in mAChR, it also displaces [3H]Nmethylscopolamine ([3H]NMS), a classic orthosteric mAChR antagonist, from its binding site.21 Therefore, the novel heterobivalent hybrids 4a−f along with the intermediate alcohols 5a−f, which can be considered analogues of 3, were pharmacologically evaluated in radioligand binding assays at human M1−M5 (hM1−M5) mAChR subtypes expressed in Chinese hamster ovary (CHO) cell lines by competition assays using [3H]NMS as a radioligand, following a previously described protocol.22 Because 1 and 3 are an M1/M4 mAChR preferring orthosteric agonist and an M1 mAChR allosteric agonist, respectively, and both the M1 and M4 mAChRs are involved in schizophrenia and Alzheimer’s disease, based on their binding affinities, hybrids 4b−d and alcohols 5b−d were evaluated for their functional efficacies at M1 and M4 mAChR subtypes in bioluminescence resonance energy transfer (BRET)-based assays. BRET proximity assay is based on a resonance energy transfer principle that allows detection of close interactions between specific GPCRs and their signaling machinery, thus detecting their activation in intact cells.23 A series of parametric analyses using three modes of BRET assays (G protein engagement, G protein activation, and β-arrestin engagement) allowed us to evaluate effector coupling differences in two families of mAChR subtypes, namely M1-like Gq coupled (M1, M3, M5) and M2-like Gi coupled (M2, M4) mAChRs. The G
site and modulate orthosteric ligand affinity. However, some allosteric ligands can also induce conformational changes in the receptor that allow them to act as (allosteric) agonists in the absence of orthosteric ligands. These allosteric agonists have the potential advantage of having a greater selectivity due to the less conserved sequence homology of the allosteric sites across different receptor subtypes. Also, because the contact residues within a receptor are different from orthosteric compounds, allosteric ligands may set the receptor in a distinct conformation that can potentially lead to functional selectivity.13 Bivalent ligands are a promising new strategy in the quest for developing novel drug entities targeting GPCRs with greater affinity and/or subtype selectivities.14 Bivalent ligands are constituted by two pharmacophores joined by a linker and are classified as homo- or heterobivalent ligands depending on the pharmacophores being identical or different, respectively. Heterobivalent ligands that combine defined orthosteric and allosteric pharmacophores are known as bitopic ligands.13,15 It has been suggested that, compared to the parent orthosteric and allosteric ligands, a bitopic ligand may select a unique receptor conformation, which can be associated with functional selectivity.13,15 Muscarinic homobivalent agonists have been synthesized with different results: arecaidine propargyl ester derivatives displayed lower potency than their monovalent analogues,16 while those of 1 showed an interesting profile with higher affinity and efficacy.17,18 Among bitopic agonists that bind simultaneously to the orthosteric and allosteric sites of mAChRs, derivatives linking the nonselective muscarinic orthosteric agonist oxotremorine with a fragment of the selective M2 mAChR allosteric modulator W84 have been described.19 Hybrid molecules comprising moieties of the orthosteric M1 mAChR agonist 1 and the cholinesterase inhibitor and allosteric M1/M2 mAChR modulator tacrine have also been reported.20 In an attempt to obtain a novel series of bitopic ligands, we designed and synthesized hybrid molecules based on the M1/ M4 mAChR orthosteric agonist 1 and the putative M1 mAChR allosteric agonist 3 connected by an aliphatic linker of variable B
dx.doi.org/10.1021/jm501173q | J. Med. Chem. XXXX, XXX, XXX−XXX
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Scheme 1a
a
(a) n-BuLi, THF, −78 °C; (b) H2/PtO2, MeOH, 4 N HCl; (c) NaH, DMF; (d) TBAF, THF; (e) NaH, THF.
Scheme 2a
a
(a) n-BuLi, THF, −78 °C; (b) H2/PtO2, MeOH, 4 N HCl; (c) NaH, DMF.
eliminates the possibility of signal contamination from other receptor activation.24 This is especially crucial because HEK 293T cells express M3 mAChR.25 To determine whether compounds displayed distinct efficacy bias toward Gq- and Giproteins of M1 and M4 mAChR, respectively, BRET-based assays, monitoring the interaction between M1 or M4 mAChR− renilla luciferase (RLuc) and Gα−Venus fusion proteins (Gq or Gi engagement, respectively) and the interaction between Gα− RLuc and Gγ−Venus fusion proteins (Gq or Gi activation, respectively), were performed. β-arrestin2 can act as a GPCR signal transducer, linking GPCRs to mitogen-activated protein kinase signaling pathways. Therefore, BRET assays were also performed using β-arrestin2 as a biosensor for M1 and M4 mAChR activation. This series of effector activation assays gave us an insight into the structure−activity relationships (SARs) of
protein engagement assay detects the drug-induced proximity change between the receptor fused with a bioluminescent donor and G protein fused with an acceptor. Therefore, a positive BRET change indicates the G protein engagement to the receptor. G protein activation assay, on the other hand, detects the drug-induced conformational changes within the heterotrimeric G protein complex (previously studied to correlate with G protein activation)24 and activation leads to a negative BRET change. In a same way as the G protein engagement assay, β-arrestin engagement assays detect conformational changes caused by β-arrestin recruitment to the receptor and positive drug-induced BRET indicates the engagement activity. Both types of engagement assays (i.e., G protein and β-arrestin) are particularly useful because the readout only detects specific receptor−effector coupling and C
dx.doi.org/10.1021/jm501173q | J. Med. Chem. XXXX, XXX, XXX−XXX
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Table 1. Affinity Constants, Expressed as pKi,a of Compounds 1, 3, 4a−f, 5a−f, 6, and 7 for Cloned hM1−M5 mAChRs, Expressed in CHO Cells
pKia compd 1 3 4a 4b 4c 4d 4e 4f 5a 5b 5c 5d 5e 5f 6 7 a
n
3 5 7 9 11 13 3 5 7 9 11 13
hM1 8.10 6.50 7.11 8.17 9.04 8.99 7.96 7.35 5.60 6.33 6.45 6.55 6.91 6.73 8.78 7.53
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.11 0.07 0.05 0.06 0.07 0.07 0.06 0.05 0.12 0.08 0.11 0.07 0.08 0.05 0.07 0.08
hM2 8.09 6.69 7.18 7.96 8.86 8.77 7.05 7.07 5.49 6.22 6.68 6.67 6.38 6.32 8.50 7.10
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
hM3
0.09 0.05 0.04 0.09 0.18 0.10 0.06 0.06 0.08 0.05 0.06 0.04 0.02 0.07 0.03 0.05
8.11 5.92 7.03 8.33 9.10 8.96 8.00 7.35 4.93 5.72 6.20 6.70 6.24 6.47 8.54 7.24
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.08 0.08 0.04 0.10 0.06 0.08 0.10 0.08 0.10 0.10 0.06 0.06 0.06 0.09 0.10 0.07
hM4 7.95 6.68 7.58 8.10 9.05 8.97 8.18 7.84 6.23 6.35 6.41 6.81 6.95 7.02 9.11 7.49
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.08 0.11 0.05 0.11 0.12 0.05 0.05 0.11 0.13 0.05 0.07 0.05 0.15 0.10 0.16 0.08
hM5 8.03 5.27 6.99 8.25 8.64 8.53 8.06 7.51 4.84 5.34 5.51 6.07 6.46 6.47 8.40 7.31
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.07 0.10 0.06 0.09 0.08 0.05 0.06 0.05 0.08 0.37 0.06 0.09 0.04 0.09 0.05 0.05
The values represent the arithmetic mean ± SEM of at least three experiments.
from its binding site. Compared to 3, only alcohol 5a, bearing the shortest aliphatic chain (n = 3), exhibits lower affinity values at all mAChR subtypes, while its higher homologues 5b−f (n = 5, 7, 9, 11, and 13, respectively) show similar binding profiles. Among these last derivatives, analogous to 3, 5b (n = 5) displays a preference for M1, M2, and M4 compared to M3 and M5 mAChR subtypes. The chain elongation is not generally accompanied by a decrease in affinity, suggesting that the butyl chain of 3 interacts with an accessory site of the allosteric hydrophobic pocket of mAChRs that can also accommodate carbon chains longer than four methylene moieties, owing to their flexibility. The hydroxyl function seems to be a disturbing element in the interaction only when there is a three-carbon chain, as demonstrated by the decrease of affinity observed for 5a with respect to 5b−f and 3. Indeed, the elongation of the aliphatic chain (5b−f), leading to affinity values similar to those of lead 3, seems to negate this effect demonstrating that at least four methylene moieties are necessary for the interaction independent from the presence of a hydroxyl function. Interesting SARs were obtained for the heterobivalent compounds 4a−f which are significantly more affine than 3, whereas compared to 1 the increase of affinity for all mAChR subtypes depends on the length of the linker between the two pharmacophores as graphically shown in Figure 1. The highest affinity values are associated with a seven- or nine-carbon chain (compounds 4c and 4d, respectively), although compounds 4b and 4e, bearing 5 and 11 methylene groups, respectively, retain high affinities not significantly different from those of 1. Further shortening (4a, n = 3) or lengthening (4f, n = 13) of the chain causes a decrease in affinity at all mAChR subtypes. Analogous to 1, no subtype selectivity was observed. The above results indicate that the two pharmacophores probably interact with two distinct sites located at a distance of 7 to 9 methylene moieties. To investigate the role of the two pharmacophoric
bivalent and linker-attached compounds in mAChRs. Finally, docking simulations on the M1 mAChR model were performed to elucidate how the binding mode of the proposed compounds is influenced by the linker length.
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RESULTS AND DISCUSSION The novel compounds 4a−f, 5a−f, and 7 were prepared according to the procedures reported in Schemes 1 and 2. To prepare the intermediate aminoalcohols 5a−f, 4-methylpyridine was reacted with the appropriate bromoderivatives 8a−f26 in the presence of n-butyllithium in THF to yield the pyridine derivatives 9a−f, whose hydrogenation over Adams’s catalyst (PtO2) afforded piperidines 10a−f (Scheme 1). The reaction of 4-alkylpiperidines 10a−f with 1127 in the presence of sodium hydride in DMF yielded derivatives 12a−f, whose treatment with tetra-n-butylammonium fluoride (TBAF) in DMF gave 5a−f, which were reacted with 1328 in the presence of sodium hydride in THF to obtain the final compounds 4a−f (Scheme 1). The homobivalent ligand of 1 (6) was prepared as reported in the literature.18 The reaction of 4-methylpyridine with 1,7dibromoheptane in the presence of n-butyllithium in THF gave compound 14, whose reduction with PtO2 followed by alkylation with 1127 yielded 7 (Scheme 2). The affinity values, expressed as pKi, of compounds 4a−f, 5a−f, 6, and 7 are reported in Table 1 along with those of 1 and 3 included for comparison. An analysis of the hM1−M5 mAChR binding data reported in Table 1 shows that 1 binds with higher affinity than 3 across subtypes. However, while 1 has similar affinity values (7.95−8.11) at all five mAChR subtypes, 3 exhibits a preference for M1 (6.50), M2 (6.69), and M4 (6.68) compared to M3 (5.92) and M5 (5.27) mAChR subtypes. Analogous to 3, all the alcohols 5a−f bind with significantly lower affinities than 1, but they are able to recognize the five mAChR subtypes and displace [3H]NMS D
dx.doi.org/10.1021/jm501173q | J. Med. Chem. XXXX, XXX, XXX−XXX
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