Discovery of VU0467319: an M1 Positive Allosteric Modulator Candidate That Advanced into Clinical Trials

Abstract

Herein we detail the first disclosure of VU0467319 (VU319), an M₁ Positive Allosteric Modulator (PAM) clinical candidate that successfully completed a Phase I Single Ascending Dose (SAD) clinical trial. VU319 (16) is a moderately potent M₁ PAM (M₁ PAM EC₅₀ = 492 nM ± 2.9 nM, 71.3 ± 9.9% ACh Max), with minimal M₁ agonism (EC₅₀ > 30 μM), that displayed high CNS penetration (K_ps > 0.67 and K_p,uus > 0.9) and multispecies pharmacokinetics permissive of further development. Based on robust efficacy in multiple preclinical models of cognition, an ancillary pharmacology profile devoid of appreciable off-target activities, and a lack of cholinergic adverse effects (AEs) in rats, dogs and nonhuman primates, VU319 advanced into IND-enabling studies. After completing 4-week rat and dog GLP toxicology without AEs, including absence of cholinergic effects, the first in human Phase I SAD clinical trial of VU319 (NCT03220295) was performed at Vanderbilt, where a similar lack of adverse effects, including absence of cholinergic effects was noted. Moreover, signals of target engagement were seen at the highest dose tested. Thus, VU319 demonstrated the feasibility of achieving selective targeting of central M₁ muscarinic receptors without eliciting cholinergic AEs that have plagued other drugs targeting CNS cholinergic neurotransmission.

Keywords: cognition; metabolism; muscarinic acetylcholine receptor subtype 1 (M1); positive allosteric modulator (PAM).

Figure 1

Structures of the M₁/M₄-preferring xanomeline (1), representative M₁ PAMs with cholinergic AEs 2-5, and representative M₁ PAMs with lesser levels of M₁ agonism 6-10, that showed no cholinergic AEs.

Figure 2

Roadmap to the discovery of VU0467319 (VU319, 16). From an unselective, pan-G_q-mAChR (M₁, M₃, M₅) PAM 11, harboring an electrophilic and reactive isatin moiety, to a highly selective M₁ PAM clinical candidate, VU319 (16) with minimal agonism and no reactive functionality.

Scheme 1. Discovery Chemistry Route for the Synthesis of VU319 (16)

Reagents and conditions: (a) PdCl2(dppf)·DCM, Cs2CO3, THF:H2O, 40 °C, 12 h; (b) NH2OH·HCl, CH3COONa, EtOH, 2.5 h; (c) Zn, acetic acid, 2.5 h, 79% over three steps; (d) TCCA, DCM, rt, 24 h, 100%; (e) Hünig’s base, CH3CN, 80 °C, 12 h, 40%.

Scheme 2. CMC Process Route for the Synthesis of VU319 (16) on 3.3 kg Scale

Reagents and conditions: (a) Bis(pinacolato)diboron, PdCl₂(dppf)·DCM, 1,4-dioxane, KOAc, 85–90 °C, 10 h, 66%; (b) PdCl₂(dppf)·DCM, Cs₂CO₃THF:H₂O, 55–60 °C, 3 h, 95%; (c) NH₂OH·HCl, CH₃COONa, EtOH, 5 h, 95%; (d) Zn, acetic acid,2.5 h, 66%; (e) SeO₂, 1,4-dioxane, 135–140 °C, 2 h, 51%; (f) STAB, ZnCl₂, THF, 50–55 °C, 6 h, 73%.

Figure 3

(A) VU319 is a PAM of the human M₁ muscarinic receptor with minimal M₁ agonism. Data represent one experiment performed in triplicate. (B) VU319 is a PAM of the rat, mouse, and cynomolgus monkey M₁ muscarinic receptors. Representative data from one experiment for each species performed in triplicate.

Figure 4

VU319 is highly selective as an M₁mAChR PAM at human (A) and rat (B) M₁–M₅ receptors. Increasing concentrations of VU319 were applied approximately 2 min prior to an appropriate concentration of acetylcholine (ACh) designed to elicit an EC₂₀ response. Calcium mobilization responses were measured and normalized to a maximal ACh response elicited after activation of each receptor.

Figure 5

(A) In contrast to atropine, VU319 does not displace the binding of the orthosteric radioligand, [³H] NMS. (B) Fold shift of the ACh concentration–response curve in a calcium assay in cells expressing the rat M₁ receptor and exposed to increasing concentrations of VU319 prior to a full range of ACh concentrations. Data represent two experiments performed in duplicate or triplicate.

Figure 6

Racine Score test in mice. Pretreatment with M₁ PAMs (100 mg/kg, i.p., 10 mL/kg, 180 min) BQCA (2) resulted in robust behavioral convulsions at 30 min post administration, while VU319 did not cause any observed adverse effects out to 6 h post administration. N = 3/group of male C57Bl/6 mice. ANOVA p < 0.0001; ****p < 0.0001 as compared to vehicle control.

Figure 7

Effects of VU319 on the modified Irwin Neurological Test battery in mice. At doses of either 56.6 mg/kg or 100 mg/kg IP, and over a 6 h time course, no adverse cholinergic events (SLUDGE) were noted.

Figure 8

Effects of VU319 on Novel Object Recognition in rats. VU319 dose-dependently enhanced recognition memory in rats. Pretreatment with 0.3, 1, 3, and 5.6 mg/kg VU319 (p.o, 0.5% natrosol/0.015% Tween 80 in water, 30 min) prior to exposure to identical objects significantly enhanced recognition memory assessed 24 h later. N = 10–18/group of male Sprague–Dawley rats. ANOVA p = 0.0053, *p < 0.05.

Figure 9

VU319 (PO), in combination with an inactive dose of donepezil (0.3 mg/kg IP) produced enhanced recognition memory in the NOR task in rats. Dose-dependent enhancement of the recognition index during the NOR task (p = 0.0449, one-way ANOVA with a dunnett post hoc test * p < 0.05) (N = 11–12).

Figure 10

VU319 (PO) produced increases in high gamma (γ) power in rats in a dose-dependent manner. For all dose–response studies a two-way analysis of variance was applied to examine effects of dose and frequency for each of the frequency bands; significance was defined as p < 0.05.

Figure 11

Multispecies hepatocyte metabolite identification of VU319. Metabolite D (VU0481424, 26) is a major, inactive metabolite.

Scheme 3. Synthesis of Metabolite D (VU0481424, 26)

Reagents and conditions: (a) 19, Et₃N, DMF/MeCN, rt, 1 h, then HATU, 90%; (b) NaBH₄, DCM/MeOH, 24%.