Centrally active allosteric potentiators of the M4 muscarinic acetylcholine receptor reverse amphetamine-induced hyperlocomotor activity in rats

Abstract

Previous clinical and animal studies suggest that selective activators of M(1) and/or M(4) muscarinic acetylcholine receptors (mAChRs) have potential as novel therapeutic agents for treatment of schizophrenia and Alzheimer's disease. However, highly selective centrally penetrant activators of either M(1) or M(4) have not been available, making it impossible to determine the in vivo effects of selective activation of these receptors. We previously identified VU10010 [3-amino-N-(4-chlorobenzyl)-4, 6-dimethylthieno[2,3-b]pyridine-2-carboxamide] as a potent and selective allosteric potentiator of M(4) mAChRs. However, unfavorable physiochemical properties prevented use of this compound for in vivo studies. We now report that chemical optimization of VU10010 has afforded two centrally penetrant analogs, VU0152099 [3-amino-N-(benzo[d][1,3]dioxol-5-ylmethyl)-4,6-dimethylthieno[2,3-b]pyridine carboxamide] and VU0152100 [3-amino-N-(4-methoxybenzyl)-4,6-dimethylthieno[2,3-b]pyridine carboxamide], that are potent and selective positive allosteric modulators of M(4). VU0152099 and VU0152100 had no agonist activity but potentiated responses of M(4) to acetylcholine. Both compounds were devoid of activity at other mAChR subtypes or at a panel of other GPCRs. The improved physiochemical properties of VU0152099 and VU0152100 allowed in vivo dosing and evaluation of behavioral effects in rats. Interestingly, these selective allosteric potentiators of M(4) reverse amphetamine-induced hyperlocomotion in rats, a model that is sensitive to known antipsychotic agents and to nonselective mAChR agonists. This is consistent with the hypothesis that M(4) plays an important role in regulating midbrain dopaminergic activity and raises the possibility that positive allosteric modulation of M(4) may mimic some of the antipsychotic-like effects of less selective mAChR agonists.

Fig. 1

Chemical structures of xanomeline [3-[3-hexyloxy-1,2,5-thiadiazo-4-yl]-1,2,5,6-tetrahydro-1-methylpyridine] (1) and VU10010 (2).

Fig. 2

Chemical optimization of VU10010 using a diversity-oriented approach to achieve soluble, centrally penetrant M₄ positive allosteric modulators. A, β-aminoamide as a potential P-gp liability in series 3 and cyclization strategy to diminish this liability in series 4. B, solution-phase parallel synthesis of libraries of VU10010 analogs. Commercial heterocyclic carboxylic acids 5 (X, Y = C or N) were coupled to 12 different amines (HNR₁R₂) to afford focused libraries of VU10010 analogs 7 to 15 in yields ranging from 15 to 99%. C, generic structures of analogs of VU10010 evaluated in the chemical lead optimization program in an effort to develop soluble, brain penetrant M₄ positive allosteric modulators.

Fig. 3

Screening paradigm for analog libraries 2 to 12 allowing for the rapid triage of inactive analogs. A representative library of 61 analogs (scaffold 7) were tested at a single concentration (10 µM) for their ability to potentiate an EC₂₀ concentration of ACh in CHO K1 cells stably coexpressing the rat M₄ mAChR and the chimeric G protein, G_qi5. Calcium mobilization was measured using a FLEXstation II, as described under Materials and Methods. Of those tested, 16 compounds (denoted by an asterisk) were selected for further evaluation. The response to an EC₂₀ concentration of ACh alone is shown in the bar on the far left, and this level of activity is indicated by the solid line spanning the panel. Thus, test compounds increasing the % Max ACh response above this level are considered potentiators of the M₄ mAChR. VU10010 was included as a positive control. Bars represent the mean ± S.E.M. of three or more determinations, each performed in duplicate.

Fig. 4

VU0152099 and VU0152100 are potent positive allosteric modulators of rat M₄ in a functional calcium mobilization assay. A and D, chemical structure of VU0152099 (7o) (A) and VU0152100 (7p) (D). B and E, potency of VU0152099 (403 ± 117 nM) (B) and VU0152100 (380 ± 93 nM) (E) was evaluated at the rM₄ receptor by measuring calcium mobilization in CHO cells stably expressing rM₄ and the chimeric G protein G_qi5. A range of concentrations of test compound was added to cells, followed 1.5 min later by addition of an EC₂₀ concentration of ACh (ACh EC₂₀). In the absence of an EC₂₀ concentration of ACh (Vehicle), neither test compound elicited a response. Data were normalized as a percentage of the maximal response to 10 µM ACh and represent the mean ± S.E.M. of three independent experiments. C and F, VU0152099 (C) and VU0152100 (F) potentiate the response of rM₄ to ACh, as manifest by a dose-dependent leftward shift in the ACh CRC. At the highest concentrations tested, VU0152099 (30 µM) induced a 30-fold shift, and VU0152100 (10 µM) induced a 70-fold shift in the ACh CRC. Data were normalized as a percentage of the maximal response to 10 µM ACh and represent the mean ± S.E.M. of three to five independent experiments.

Fig. 5

VU0152099 and VU0152100 potentiate GIRK-mediated thallium flux in response to ACh in HEK293 cells expressing human M₄. A, both VU0152099 (■) and VU0152100 (▲) potentiate hM₄-induced GIRK-mediated thallium flux in response to an EC₂₀ concentration of ACh with potencies of 1.2 ± 0.3 and 1.9 ± 0.2 µM, respectively. B, in the presence of 10 µM VU0152099 (▲) and VU0152100 (▼), the ACh CRC for induction of GIRK-mediated thallium flux was leftward shifted (≈30-fold) from 77 ± 1.2 (■, Veh) to 2.09 ± 0.3 nM (▲, VU0152099) and 2.35 ± 0.5 nM (▲, VU0152100). Data were normalized as a percentage of the maximal response to 10 µM ACh and represent the mean ± S.E.M. of three to four independent experiments performed in quadruplicate.

Fig. 6

VU0152099 and VU0152100 bind allosterically and increase ACh affinity at rM4. A, in competition binding studies, neither VU0152099 (■) nor VU0152100 (▲) displaced the orthosteric radioligand, [³H]NMS (0.1 nM), at concentrations up to 30 µM. However, the orthosteric antagonist, atropine (▲), potently inhibited [³H]NMS binding with a K_i of 0.54 ± 0.1 nM. B, in the presence of vehicle alone, an increasing concentration of ACh displaces [³H]NMS (0.1 nM) binding with a K_i of 252 ± 17.9 nM (■). In the presence of a fixed concentration (10 µM) of VU0152099 or VU0152100, the potency of ACh to displace [³H]NMS binding is shifted leftward, yielding K_i values of 10.4 ± 0.91 (▼, VU0152099) and 12.2 ± 0.49 nM (▲,VU0152100), which represent a 25- and 21-fold shift in ACh potency, respectively. Data represent the mean ± S.E.M. of three independent experiments performed in duplicate.

Fig. 7

VU0152099 (A) and VU0152100 (B) are functionally selective for the M₄ mAChR subtype. No shift in the ACh CRC was observed in the presence of 30 µM test compound at CHO K1 cells stably expressing M₁, M₂-G_qi5, M₃, or M₅ mAChRs. Calcium mobilization was measured in response to increasing concentrations of ACh following preincubation with either vehicle or test compound (30 µM), as described under Materials and Methods. Assay of M₁, M₃, and M₅ mAChRs took advantage of endogenous coupling to G_q proteins, whereas assay of M₂ activity required use of cells coexpressing the chimeric G protein, G_qi5, to allow coupling of this receptor to calcium mobilization. Points represent the mean ± S.E.M. of three independent experiments.

Fig. 8

Pharmacokinetic profiling of VU0152099 and VU0152100 in rats. Concentration-time profile of VU0152099 (A) and VU0152100 (B) in brain and plasma of male Sprague-Dawley rats following a 56.6 mg/kg i.p. administration of each compound. Blood and brain tissue were collected at 0.5, 1, 2, and 4 h after injection. Samples were extracted as described under Materials and Methods and analyzed by LC-MS-MS. Each time point represents the mean determination ± S.E.M. of three rats.

Fig. 9

VU0152099 and VU0152100 inhibit amphetamine-induced hyperlocomotor activity in rats without causing sedation. A, rats were pretreated for 30 min with vehicle or a 56.6 mg/kg dose of either VU0152099 or VU0152100 i.p. (data not shown). All rats received an injection of 1 mg/kg s.c. amphetamine, and locomotor activity was measured for an additional 60 min. Each point represents the mean of 8 to 16 rats. The error bars represent ± S.E.M. and are absent when less than the size of the point. Abscissa, dose of drug in milligrams per kilogram; ordinate, ambulations or total beam breaks per 5-min intervals; *, P < 0.05 versus veh + amphetamine control group, Dunnett’s test. B, lack of effect of VU0152100 on motor performance on the rotorod. After initial training trials, rats were pretreated for 30 min i.p. with vehicle or a dose of VU0152100, specifically 30, 56.6, or 100 mg/kg, and then the time each animal remained on the rotorod was recorded; animals not falling off the rotorod were given a maximal score of 85 s. Abscissa, dose of VU0152100 in milligrams per kilogram; ordinate, time spent on the rotorod in seconds. Each bar graph represents the mean of 8 to 10 rats. The error bars represent ± S.E.M.