Discovery of Pre-Clinical Candidate VU6008055/ AF98943: A Highly Selective, Orally Bioavailable, and Structurally Distinct Tricyclic M4 Muscarinic Acetylcholine Receptor Positive Allosteric Modulator (PAM) with Robust In Vivo Efficacy

Abstract

Herein, we report the structure-activity relationship to develop novel tricyclic M₄ positive allosteric modulator scaffolds with improved pharmacological properties. This endeavor involved modifying a 5-amino-3,4-dimethylthieno[2,3-c]pyridazine-6-carboxamide core via a "tie-back" strategy to discover a novel tricyclic 3,4-dimethylpyrimido[4',5':4,5]thieno[2,3-c]pyridazine core. From this exercise, VU6008055/AF98943 was identified as a preclinical candidate, which displays low nanomolar potency against both human and rat M₄. Moreover, VU6008055 is highly brain penetrant, has an overall superior pharmacological and DMPK profile to previously reported M₄ PAMs, and demonstrates efficacy in preclinical models of antipsychotic-like activity.

Keywords: Alzheimer’s disease; M4; Parkinson’s disease; VU6008055; muscarinic acetylcholine receptor (mAChR); positive allosteric modulator (PAM); schizophrenia; structure–activity relationship (SAR).

1

Discovery of novel, structurally distinct tricyclic cores as M₄ PAMs. Utilization of a “tie-back” strategy to mask the β-amino carboxamide moiety (dotted circle) of VU0467485 (4) revealed three unique M₄ PAM tricyclic chemotypes: 3,4-dimethylpyrimido­[4′,5′:4,5]­thieno­[2,3-c]­pyridazine core 5, 3,4-dimethylpyrido­[4′,3′:4,5]­thieno­[2,3-c]­pyridazine core 6, and 3,4-dimethylthieno­[2,3-c:4,5-d′]­dipyridazine core 7.

2

Structures of clinically advanced M₄-targeting therapeutics.

1. Synthesis of M₄ Analogs 13, 16, 18, 19, and 20

2. Synthesis of M₄ Analogs 26, 33, 38, and 41

3. Synthesis of Analogs 45, 50, 54, 55, and 57–59

3

Dose response curves of VU6008055 showing (A) selectivity with human M₁–M₅ and rat M₄ and (B) competition binding against [³H]­MK-6884 at human (n = 3) and rat (n = 2) M₄.

4

Systemic PO administration of VU6008055 blocked amphetamine-induced hyperlocomotion in male Sprague–Dawley rats. (A) Time course of locomotor activity and (B) total locomotor activity during the 55 min period following amphetamine administration. Data are means ± SEM of 6–8 animals per group. *p < 0.05, **p < 0.01, ***p < 0.001 vs vehicle + amphetamine. Vehicle = 10% Tween 80 in H₂O.

5

Systemic PO administration of VU6008055 attenuated MK-801-induced hyperlocomotion in male Sprague–Dawley rats. (A) Time course of locomotor activity and (B) total locomotor activity during the 55 min period following MK-801 administration. Data are means ± SEM of 10–12 animals per group. ***p < 0.001 vs vehicle + MK-801. Vehicle = 10% Tween 80 in H₂O.

6

Seven day, twice daily repeated PO dosing of VU6008055 does not alter its ability to block amphetamine-induced hyperlocomotion in male Sprague–Dawley rats. (A) Time course of locomotor activity and (B) total locomotor activity during the 55 min period following amphetamine administration. Rats were pretreated acutely or repeatedly (7 days, twice daily) by oral gavage with vehicle or VU6008055 (3 mg/kg). On day 8, vehicle or VU6008055 (3 mg/kg) was administered orally after 30 min by subcutaneous administration of amphetamine (0.75 mg/kg). Data are means ± SEM of 6 (blue curves, acute dosing) or 12 (red curves; repeated dosing) animals per group. **p < 0.01 vs vehicle + amphetamine (acute dosing), ^### p < 0.001 vs vehicle + amphetamine (7 day repeated dosing). Vehicle = 10% Tween 80 in H₂O.

7

Effects of VU6008055 in the CAR task in male Wistar-Han rats. (A) Effect of VU6008055 0.3–30 mg/kg in the CAR task in male Wistar–Han rats. A fully effective 0.64 mg/kg dose of risperidone used as a positive control for the assay. (B) Effect of VU6008055 0.03–30 mg/kg in the CAR task in male Wistar-Han rats in the presence of a threshold dose of 0.2 mg/kg risperidone. Data are expressed as means +SEM for groups of 8–16 rats. Comparisons by Brown–Forsythe and Welch test. Significant differences from the vehicle-treated controls are denoted by *p < 0.01, **p < 0.01, ***p < 0.001. Significant differences from Risperidone alone are denoted by #p < 0.05, ##p < 0.01, ###p < 0.001. Risperidone was dosed 30 min prior to testing. Other groups were dosed 60 min prior to testing.

8

Structures of most prominent metabolites of VU6008055 from multispecies hepatocyte MetID. Potency was determined for the most abundant, nonglucuronide human metabolite (MT4) using a calcium mobilization assay.

9

Systemic PO administration of VU6008055 attenuates amphetamine-induced increases in CBV in male Sprague–Dawley rats as measured by phMRI. Group-averaged CBV maps illustrate the dose-related blockade of amphetamine (1 mg/kg s.c.)-induced changes in CBV by 1 and 10 mg/kg p.o. of VU6008055. Group-averaged CBV maps were generated by colorizing mean percent change in CBV for minutes 31–40 onto corresponding voxels of the template brain. Data are means ± SEM of 8–10 rats per group.

10

Time course of regional CBV changes following administration of VU6008055 alone or in combination with amphetamine in male Sprague–Dawley rats. (A) Experimental timeline, structural MRI template, and identified brain regions assessed for CBV changes. Time courses of percent CBV changes for each region-of-interest (average of left and right hemispheres) following (B) administration of vehicle + amphetamine (red trace), VU6008055 1 mg/kg + amphetamine (black trace), and VU60080555 10 mg/kg + amphetamine or (C) vehicle + vehicle (black trace) and VU6008055 + vehicle (blue trace). Amphetamine was injected at time point 10 min. Statistical comparisons of the 31–40 min bin were made using one-way ANOVA and Dunnett’s post hoc test (B) or one-tailed Mann–Whitney U test (GraphPad Prism 8.0). Data are presented as mean ± SEM of 8–10 animals per group.

11

Systemic PO administration of VU6008055 increases arousal in male Sprague–Dawley rats. The changes in relative spectral power in the frontal cortex (% change from baseline, BL) during waking epochs within the 1–2 h period following dosing with VU6008055 are shown. Relative power was summed in 1 Hz bins (0.5–100 Hz) from all 10 s waking epochs and expressed as a percent change (±SEM) from respective power within the same frequency bin during waking epochs from the 1 h BL period prior to dosing. Gray/tan vertical bars represented frequency bands (Δ, delta 0.5–4 Hz; θ theta 4–8 Hz; α alpha, 8–13 Hz; β beta, 13–30 Hz; γ gamma 30–100 Hz). Corresponding colored horizontal dots/lines represented the frequencies at each dose that were statistically different from vehicle-treated rats. Vehicle = 10% Tween 80 in H₂O.