Discovery of β-Arrestin Biased, Orally Bioavailable, and CNS Penetrant Neurotensin Receptor 1 (NTR1) Allosteric Modulators

Abstract

Neurotensin receptor 1 (NTR1) is a G protein coupled receptor that is widely expressed throughout the central nervous system where it acts as a neuromodulator. Neurotensin receptors have been implicated in a wide variety of CNS disorders, but despite extensive efforts to develop small molecule ligands there are few reports of such compounds. Herein we describe the optimization of a quinazoline based lead to give 18 (SBI-553), a potent and brain penetrant NTR1 allosteric modulator.

Figure 1:

Selected previously reported NTR1 ligands

Figure 2:

Quinazoline NTR1 modulators

Figure 3.

Augmentation of [¹²⁵I]-NT binding (0.02 nM) to cell membranes in the presence of increasing doses of 18 (SBI-553). The EC₅₀ for SBI-553 at the human receptor for this experiment was 0.14 μM, with a maximum efficacy of 140% (Data were plotted and analyzed using GraphPad Prism V.7, n=2). Efficacy is relative to the maximal displacement of orthosteric ligands (i.e. −100%). Data point excluded from curve fitting is shown in red. NT peptide in these assays give K_ds of 0.082 nM (hNTR1), 0.59 nM (hNTR2) and 0.17 nM (mNTR) respectively.

Figure 4.

Dose response of NT peptide in the presence of 18 (SBI-553) in the β-Arrestin (A) and Ca²⁺ flux (B) assays. Data were plotted and analyzed using GraphPad Prism V.7 and are presented as an average ± SEM (n=4). The compound shows divergent behavior, with no effect on the EC₅₀ in the β-Arrestin assay but a strong rightward shift in the Ca²⁺ flux assay.

Figure 5:. 18 (SBI-553) attenuates basal hyperlocomotion in dopamine transporter knockout (DAT^−/−) mice.

Locomotor activity was evaluated using open field automated activity monitors (AccuScan Instruments, Columbus, OH). Male and female DAT^−/− mice were acclimated to the open field chamber for 30 min prior to administration of NTR1 ligands (indicated by arrow). After treatment, animals were immediately returned to chambers and horizontal locomotion was monitored over the next 2 hrs. (A) Animals received SBI-553 (12 mg/kg), PD149163 (1 mg/kg) or vehicle (4% DMSO in saline; 10 ml/kg) i.p. Beam breaks in 5 min intervals were recorded to generate horizontal distance traveled per time bin. *p<0.05-p<0.001 Vehicle vs. SBI-553; #p<0.05-p<0.001 Vehicle vs. PD149163; ~p<0.05-p<0.001 SBI-553 vs. PD149163, using a two-way, repeated measures ANOVA (F_Time(23, 1679)=13.5, p<0.0001; F_Treatment(2, 73)=45.1, p<0.0001; F_Interaction(46, 1679)=10.4, p<0.0001) followed by the post hoc Tukey test. (B) Time course data in (A) was summed to determine the total distance traveled in the 2 hr post-treatment. ***,###,~~~p<0.001, as indicated, using one-way ANOVA (F(2, 72)=46.6, p<0.0001) followed by the post hoc Tukey test. Data are represented as mean ± SEM of 18–29 animals per group. (C) Animals received SBI-553 10, 30 or 100 mg/kg or vehicle (distilled H₂O) p.o. Beam breaks in 5 min intervals were recorded to generate horizontal distance traveled per time bin. *p<0.05-p<0.001 Vehicle vs. 100 mg/kg SBI-553; #p<0.05 Vehicle vs. 30 mg/kg SBI-553; ~p<0.05 10 mg/kg SBI-553 vs. 100 mg/kg SBI-553, using a two-way, repeated measures ANOVA (F_Time(23, 989)=8.6, p<0.0001; F_Treatment(3, 43)=6.8, p<0.001; F_Interaction(69, 989)=1.1, p=0.2726) followed by the post hoc Tukey test. (D) Time course data in (C) was summed to determine the total distance traveled in the 2 hr post-treatment. **p<0.01, #p<0.05,~p<0.05, as indicated, using one-way ANOVA (F(3, 43)=6.8, p<0.001) followed by the post hoc Tukey test. Data are represented as mean ± SEM of 11–12 animals per group.

Scheme 1.

Representative synthesis of quinazoline compounds ^aReagents and conditions: (a) (COCl)₂, 35 °C, 1.5h; (b) Pyridine, DCM, rt, 2h 86% over 2 steps; (c) NaOH, H₂O₂, EtOH, reflux, 12 h, 66%; (d) K₃PO₄, CuI, proline, DMSO, 100 °C, 12h; (e) NaBH(OAc)₃, HCHO, MeOH, rt, 1h, 39% over 2 steps; (f) BOP, DBU, CH₃CN, rt, 12h, 87%