Discovery of VU6007496: Challenges in the Development of an M1 Positive Allosteric Modulator Backup Candidate

Abstract

Herein we report progress toward a backup clinical candidate to the M₁ positive allosteric modulator (PAM) VU319/ACP-319. Scaffold-hopping from the pyrrolo[2,3-b]pyridine-based M₁ PAM VU6007477 to isomeric pyrrolo[3,2-b]pyridine and thieno[3,2-b]pyridine congeners identified several backup contenders. Ultimately, VU6007496, a pyrrolo[3,2-b]pyridine, advanced into late stage profiling, only to be plagued with unanticipated, species-specific metabolism and active/toxic metabolites which were identified in our phenotypic seizure liability in vivo screen, preventing further development. However, VU6007496 proved to be a highly selective and CNS penetrant M₁ PAM, with minimal agonism, that displayed excellent multispecies IV/PO pharmacokinetics (PK), CNS penetration, no induction of long-term depression (or cholinergic toxicity) and robust efficacy in novel object recognition (minimum effective dose = 3 mg/kg p.o.). Thus, VU6007496 can serve as another valuable in vivo tool compound in rats and nonhuman primates, but not mouse, to study selective M₁ activation.

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

Figure 1

Structures of xanomeline (1), representative M₁ ago-PAMs with cholinergic AEs 2–5, representative “pure” PAMs devoid of M₁ agonism 6–8, and the low cooperative M₁ ago-PAM TAK-071 (9).

Figure 2

Initial scaffold-hopping exercise from 7 that led to 8, a novel M₁ PAM devoid of cholinergic toxicities and AEs.

Figure 3

Scaffold-hopping from 8 to regioisomeric cores 9 and 10, which led to the discovery of VU6007496 (11) and VU6006874 (12) that were advanced into further profiling.

Scheme 1. Synthesis of 7-Chloro-1-methyl-1H-pyrrolo[3,2-b]pyridine-5-carbonitrile 13 and a 7-Chlorothienyl[3,2-b]pyrdine-5-carbonitrile 14

Reagents and conditions: (a) m-CPBA, n-BuOAc:heptane (3:5), 0 °C—rt, 83%; (b) (MeO)₂SO₂, n-BuOAc, 75 °C, 16 h; (c) KCN, aq. NH₄Cl, 50 °C, 2 h; quantitative yield (2 steps); (d) MeI, NaH, DMF, 0 °C—rt, 98%; (e) m-CPBA, DCM, 0 °C—rt, 54%; (f) TMSCN, (CH₃)₂NCOCl, DCM, rt, 16 h, 98%.

Scheme 2. Synthesis of VU6007496 (11)

Reagents and conditions: (a) bis(pinacolato)diboron, KOAc, Pd(dppf)Cl₂, 1,4-dioxane, 100 °C, 16 h; (b) benzyl chloride 20, Cs₂CO₃, Pd(dppf)Cl₂, THF/H₂O, 90 °C, 16 h; 56% (2 steps); (c) conc. HCl, reflux, 2 h; (d) tetrahydro-2H-pyran-4-amine, HATU, DIEA, DMF, rt, 20 min, 71% (2 steps).

Scheme 3. Synthesis of VU6006874 (12)

Reagents and conditions: (a) bis(pinacolato)diboron, KOAc, Pd(dppf)Cl₂, 1,4-dioxane, 100 °C, 16 h, 95%; (b) benzyl chloride 20, Cs₂CO₃, Pd(dppf)Cl₂, THF/H₂O, 90 °C, 16 h, 99%; (c) conc. HCl, 100 °C, 3 h; (d) tetrahydro-2H-pyran-4-amine, HATU, DIEA, DMF, rt, 1 h, 61% (2 steps).

Scheme 4. Synthesis of Metabolite VU6036463 (24)

Reagents and conditions: (a) benzyl chloride 28, Cs₂CO₃, Pd(dppf)Cl₂, THF/H₂O, 90 °C, 16 h; 58%; (b) conc. HCl, reflux, 2 h; (c) tetrahydro-2H-pyran-4-amine, HATU, DIEA, DMF, rt, 20 min, 81% (2 steps).

Figure 4

(A) Modified Racine Score test in mice with M₁ PAMs. Pretreatment with M₁ PAMs (100 mg/kg, i.p., 10 mL/kg, 180 min) BQCA (2), MK-7622 (3), PF-0674427 (4), resulted in robust behavioral convulsions at 3 h post administration, while VU6007496 (11) and VU6006874 (12) did not cause any observed adverse effects. N = 3/group of male C57Bl/6 mice. ANOVA p < 0.0001; ****p < 0.0001 as compared to vehicle control. (B) Time course graph showing that bath application of 3 μM VU6007496 (11) for 20 min led to no significant change in fEPSP slope. N = 8 brain slices from 3 different male C57Bl/6 mice.

Figure 5

Novel object recognition (NOR) test in rats with VU6007496 (11). PAM 11 dose-dependently enhanced recognition memory in rats. Pretreatment with 0.1, 0.3, 1, and 3 mg/kg VU6007496 (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 = 15–18/group of male Sprague–Dawley rats. ANOVA p = 0.0283; **p < 0.01.

Figure 6

Metabolism of VU6007496 (11) in rat and human liver S9, with the major metabolite, VU6036463 (24), an N-demethylation product, exemplified.

Figure 7

Rat dose escalation study with VU6007496 (11).

Figure 8

Metabolism of VU6007496 (11) in rat, dog, primate and human hepatocytes, with the major metabolite, VU6036463 (24), an N-demethylation product. Unexpectedly, no parent PAM 11 remained in human after the 4 h incubation.

Figure 9

Extensive metabolism (95.4%) of VU6007496 (11) in mouse hepatocytes, with metabolite F (25) produced in high abundance.

Figure 10

In vivo MET ID and metabolic pathways of VU6007496 (11) in mouse rat plasma at 100 mg/kg and 300 mg/kg p.o. at 5–30 min and 160–240 min at each dose. Three metabolites are produced in vivo: the N-demethylated 24, and two oxidative metabolites, a dioxygenated species M461 (26) and a mono-oxygenated species M445 (27) on the tetrahydropyranyl moiety.

Figure 11

Structure and human M₁ pharmacology of 30, an extremely potent M₁ ago-PAM with CNS penetration in rat.