Application of Deuterium in an M1 Positive Allosteric Modulator Back-Up Program: The Discovery of VU6045422

Abstract

Recently, we disclosed VU0467319, an M₁ positive allosteric modulator (PAM) clinical candidate that had successfully completed a phase I single ascending dose clinical trial. Pharmacokinetic assessment revealed that, in humans upon increasing dose, a circulating, inactive metabolite constituted a major portion of the total drug-related area under the curve (AUC). One approach the team employed to reduce inactive metabolite formation in the back-up program was the kinetic isotope effect, replacing the metabolically labile C-H bonds with shorter, more stable C-D bonds. The C-D dipole afforded VU6045422, a more potent M₁ PAM (human EC₅₀ = 192 nM, 80% ACh Max) than its proteocongener VU0467319 (human EC₅₀ = 492 nM, 71% ACh Max), and retained the desired profile of minimal M₁ agonism. Overall, the profile of VU6045422 supported advancement, as did greater in vitro metabolic stability in both microsomes and hepatocytes than did VU0467319. In both rat and dog in vivo, low doses proved to mirror the in vitro profile; however, at higher doses in 14-day exploratory toxicology studies, the amount of the same undesired metabolite derived from VU6045422 was equivalent to that produced from VU0467319. This unexpected IVIVC result, coupled with less than dose-proportional increases in exposure and no improvement in solubility, led to discontinuation of VU0467319/VU6045422 development.

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

Figure 1

Structures of VU0467319 1, and the corresponding oxidative metabolite 2, as well as the dideutero congener 3 (VU6030095) and the penta-deutero analogue 5 (VU6045422), with the putative oxidative metabolites 4 and 6, respectively.

Scheme 1. Synthesis of VU6030095 (3)

Reagents and conditions: (a) NaOD (40 wt % in D₂O), tetrahydrofuran (THF)/D₂O, 35 °C, 70%.

Scheme 2. Synthesis of VU6045422 (5)

Reagents and conditions: (a) ICD₃, Cs₂CO₃, MeCN, room temperature (rt), 33%; (b) bis-pinacolborane, PdCl₂(dppf)·CH₂Cl₂, KOAc, 1,4-dioxane, 100 °C, 72%; (c) 9, PdCl₂(dppf)·CH₂Cl₂, Cs₂CO₃, 1,4-dioxane/H₂O, 100 °C, 96%; (d) NH₂OH·HCl, NaOAc, EtOH, 98%; (e) Zn/HOAc, rt, 96%; (f) SeO₂, 1,4-dioxane, molecular weight (mw), 120 °C, 68%; (g) 13, sodium triacetoxyborohydride (STAB), dichloroethane (DCE), 89%; (h) NaOD (40 wt % in D₂O), THF/D₂O, 45 °C, 73%.

Scheme 3. Synthesis of VU0481424 (2)

Reagents and conditions: (a) (2,6-difluoro-4-(2-methyl-2H-indazol-4-yl)phenyl)amine, Et₃N, dimethylformamide (DMF)/MeCN, rt, 1 h, then 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), 90%; (b) NaBH₄, DCM/MeOH, 24%.

Scheme 4. Synthesis of VU6031240 (4) and VU6045587 (6)

Reagents and conditions: (a) NaBD₄, DCM/MeOD, 30–35%.

Figure 2

Multispecies hepatocyte metabolite identification of M₁ PAMs 1, 3, and 5 and quantification of oxidative metabolites 2, 4, and 6, respectively. In vitro hepatocyte assays clearly indicated greater stability toward oxidative metabolism for the deuterated congeners.

Figure 3

M₁ PAM 5 (VU6045422) did not induce a significant long-term depression of fEPSPs in the mouse layer 5 prefrontal cortex. (A) Normalized time course of the fEPSP slope during baseline, application of 10 μM VU6045422 and washout. (B) Bar graph summarizing the averaged fEPSP slope during the last 5 min of washout (gray bar in panel (A)) compared to the baseline period (ns, p > 0.05, two-tailed paired t test, n = 9).

Figure 4

Novel object recognition (NOR) test in rats with M₁ PAMs 3 and 5. (A) PAM 3 dose-dependently enhanced the recognition memory in rats. Pretreatment with 1, 3, and 10 mg/kg of 3 (PO 0.5% Natrasol/0.015% Tween 80 in water) 2 h prior to exposure to identical objects significantly enhanced recognition memory assessed 24 h later. Minimum effective dose (MED) is 10 mg/kg. (B) PAM 5 dose-dependently enhanced recognition memory in rats. Pretreatment with 0.3, 1, and 3 mg/kg of 5 (PO 10% Tween 80 in water) 1 h prior to exposure to identical objects significantly enhanced recognition memory assessed 24 h later. Minimum effective dose (MED) is 1 mg/kg. N = 13–18/group of male Sprague-Dawley rats. Analysis of variance (ANOVA) *p < 0.05; **p < 0.01 Dunnett posthoc test.

Figure 5

Rat oral dose escalation PK study at 3 (AUC last 8390 h·ng/mL; C_max = 1250 ng/mL), 10 (AUC last 23,900 h·ng/mL; C_max = 3380 ng/mL), 30 (AUC last 55,600 h·ng/mL; C_max = 6020 ng/mL), and 100 mg/kg (AUC last 87,800 h·ng/mL; C_max = 7850 ng/mL).