Design, Synthesis, and Characterization of Ogerin-Based Positive Allosteric Modulators for G Protein-Coupled Receptor 68 (GPR68)

Abstract

G protein-coupled receptor 68 (GPR68) is an understudied orphan G protein-coupled receptor (GPCR). It is expressed most abundantly in the brain, potentially playing important roles in learning and memory. Pharmacological studies with GPR68 have been hindered by lack of chemical tools that can selectively modulate its activity. We previously reported the first small-molecule positive allosteric modulator (PAM), ogerin (1), and showed that 1 can potentiate proton activity at the GPR68-G_s pathway. Here, we report the first comprehensive structure-activity relationship (SAR) study on the scaffold of 1. Our lead compound resulted from this study, MS48107 (71), displayed 33-fold increased allosteric activity compared to 1. Compound 71 demonstrated high selectivity over closely related proton GPCRs and 48 common drug targets, and was bioavailable and brain-penetrant in mice. Thus, our SAR study has resulted in an improved GPR68 PAM for investigating the physiological and pathophysiological roles of GPR68 in vitro and in vivo.

Figure 1.

Chemical structure of 1 and three highlighted regions for SAR study.

Figure 2.

Selectivity of the GPR68 PAM 71. Proton concentration-response curves in the absence and presence of increasing concentrations of 1 at human GPR68 (A), 71 at human GPR68 (B), GPR4 (C), and GPR65 (D). Proton-mediated G_s−cAMP production was measured in HEK293T cells transiently transfected with GPR68, GPR4, or GPR65, and GloSensor cAMP plasmids. Results were normalized to the proton activity in the absence of modulator and represented means ± SEM from a minimum of three independent assays, each in quadruplicate. Curves were analyzed in Prism using the built-in four parameter logistic functions. (E) Selectivity of 71 over 48 common GPCRs, ion channels, and transporters in radioligand binding assays. Values represent average binding affinity (pK_i) from a minimum of three independent assays, each in triplicate. Targets with less than 50% inhibition at 10 μM (in quadruplicate) are not shown. BZP for benzodiazepine receptor; DAT for dopamine transporter; DOR for delta (δ) opioid receptor; hERG for human ether-a-go-go-related potassium channel; KOR for kappa (κ) opioid receptor; MOR for mu (μ) opioid receptor; NET for noradrenaline transporter; PBR for peripheral benzodiazepine binding site; SERT for serotonin transporter. (F) Off-target agonist activity of 71 at human GPCRome. 71 was screened across the human GPCRome (318 GPCRs) for off-target agonist activity at 1 μM using the PRESTO-Tango screening platform. Results (fold of basal) represent means of four replicates. Dopamine receptor D₂ with 100 nM quinpirole served as an assay control. The dashed lines indicate an arbitrary cutoff line at 3× fold of basal.

Figure 3.

Allosteric potentiation of proton activity by Compound 71 at mouse GPR68. G_s GloSensor cAMP assays were carried out in HEK293T cells transiently transfected with mouse GPR68 as with human GPR68. Proton concentration responses were measured in the absence and presence of increasing concentrations of modulator. Results were normalized to proton activity in the absence of modulator and represented means ± SEM from a minimum of three independent assays, each in quadruplicate. Curves were analyzed in Prism. Allosteric parameters are summarized in Table 5.

Figure 4.

Mouse pharmacokinetic profile of Compound 71. Compound concentrations were quantified in both plasma (red curve) and brain (blue curve) at 0.5, 1.0, and 2.0 h after a single 25 mg/kg intraperitoneal (IP) administration. Compound concentrations in plasma and brain at each time point are average values from three test mice. The error bars represent ± SEM.

Scheme 1.. Synthesis of 5–7 and 10^a

^aReagents and conditions: (a) tetrahydrofuran (THF), 0 °C to room temperature (rt), 48–67%; (b) dioxane, reflux, 1 h, 56–96%; (c) Pd(PPh₃)₄, K₂CO₃, dioxane/H₂O = 5:3, 120 °C, microwave, 20 min, 42–98%.

Scheme 2.. Synthesis of 13–15, 21, 16, 18, 19, 22–29, 31, 32, 37, and 38^a

^aReagents and conditions: (a) dioxane, reflux, 1 h, 78–94%; (b) Pd(PPh₃)₄, K₂CO₃, dioxane/H₂O = 5:3, 120 °C, microwave, 20 min, 56–99%; (c) NaBH_4, MeOH, 0 °C to rt, 42%; (d) NH₃·H₂O or NH₂Me, MgSO₄, NaBH₄, THF, rt, 2 days, 45–67%. (e) Ethylene glycol, toluene, reflux, 4–8 h, 82–96%; (f) alkyl bromide, K₂CO₃, dimethylformamide, 85 °C, 34–78%; (g) i. n-BuLi, THF, −78 °C, 30 min; ii. B(OMe)₃, −78 °C, 1 h, then rt, overnight; iii. HCl (3 N), rt, 1 h, 26–57%; (h) 12, Pd(PPh₃)₄, K₂CO₃, dioxane/H₂O = 5:3, 120 °C, microwave, 20 min; (i) NaBH₄, THF, 0 °C to rt, 23–97%.

Scheme 3.. Synthesis of 41–54, 56–60, and 64–81^a

^aReagents and conditions: (a) dioxane, reflux, 1 h, 75–96 %; (b) Pd(PPh₃)₄, K₂CO₃, dioxane/H₂O = 5:3, 120 °C, microwave, 20 min, 16–99%.