Discovery of (3-Phenylcarbamoyl-3,4-dihydro-2 H-pyrrol-2-yl)phosphonates as Imidazoline I2 Receptor Ligands with Anti-Alzheimer and Analgesic Properties

Abstract

Imidazoline I₂ receptors (I₂-IRs) are altered in Alzheimer's disease (AD) patients and are associated with analgesia. I₂-IRs are not structurally described, and their pharmacological characterization relies on their modulation by highly affine ligands. Herein, we describe the synthesis of (3-phenylcarbamoyl-3,4-dihydro-2H-pyrrol-2-yl)phosphonates endowed with relevant affinities for I₂-IRs in human brain tissues. The optimal ADME and pharmacokinetic profile of a selected compound, 12d, secured its in vivo exploration in a senescence accelerated prone 8 mice revealing improvement in the cognitive impairment and unveiling the mechanism of action by analyzing specific AD biomarkers. The treatment of a capsaicin-induced mechanical hypersensitivity murine model with 12d revealed analgesic properties devoid of motor coordination issues. The target engagement of 12d was demonstrated by suppression of the analgesic effect by pretreatment with idazoxan. Overall, 12d is a putative candidate for advancing preclinical phases and supports the modulation of I₂-IRs as an innovative approach for therapeutics.

Figure 1

Structure of the I₂–IRs ligands: CR4056 (1), [¹¹C]BU99008 (2), LSL60101 (3), idazoxan (4), tracizoline (5), BU224 (6), and 2-BFI (7).

Figure 2

(A) Outline of the previous work and structure of compounds MCR5 (8), B06 (9), and BIN05 (10), and milestones achieved. (B) Outline of the present work.

Scheme 1. General Structures of the Starting Materials (11) and Final Products (12)

Reagents and conditions: (i) NaOH 0.05M, 2:1 THF/H₂O, r. t., 2.5 h.

Figure 3

X-ray structures of 12b, 12d and 12h.

Scheme 2. Putative Mechanism for the Formation of Compounds with the General Structure 12 from 11.

Reagents and conditions: (i) NaOH 0.05 M, 2:1 THF/H₂O, r. t., 2.5 h

Figure 4

(A1). Favorable var200: TIP-TP and favorable var507: N1-TIP of compound 12d (3D-QSAR I₂–IRs model); (A2) Favorable var200: TIP-TIP of compound B06 9; the steric hot spots (TIP) are presented in green and H-bond acceptor regions in blue. (B1) Unfavorable var291: DRY-N1 of compound 12h (3D-QSAR I₂–IRs model); (B2) Unfavorable var291: DRY-N1 of compound 12n (3D-QSAR I₂–IRs model); the hydrophobic regions (DRY) are labeled in yellow and H-bond acceptor regions in blue; (C1) Favorable var182: TIP-TIP and favorable var264: DRY-N1 of compound 12k (3D-QSAR I₂–IRs model); (C2) Favorable var182: TIP-TIP of compound 12j (3D-QSAR I₂–IRs model); the hydrophobic regions (DRY) are labeled in yellow, the steric hot spots (TIP) are presented in green and H-bond acceptor regions in blue. (D) Favorable var416: O-TIP, favorable var430: O-TIP and favorable var476: N1-TIP of compound 12d (3D-QSAR α₂-ARs model); the steric hot spots (TIP) are presented in green, H-bond acceptor regions in blue and H-bond donor regions in red.

Figure 5

Neuroprotective effect of compound 12d. SH-SY5Y cultures were exposed to 6-OHDA (35 μM) for at least 18 h. Previously, some cultures were pretreated 1h before with different concentrations of the compound 12d. CR4056 (1) (1 μM) was used as a reference. Basal cultures were treated with vehicle. Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT) assay, and nitrite production in the cell supernatant was quantified using the Griess reaction. The reported values represent the mean ± SD obtained from triplicate determinations repeated at least three times. Statistical analysis was performed: *p ≤ 0.05, **p ≤ 0.01 (versus 6-OHDA-treated cultures); ^###p ≤ 0.001 (compared to basal cultures).

Figure 6

Role of compound 12d in an in vitro neuroinflammation model. Glial cells, astrocytes (A) and microglial cells (B) were isolated from murine cortex and stimulated with bacterial LPS (10 μg/mL) in the presence of the compound 12d (at different doses). CR4056 1 (1 μM) was used as a reference and basal cultures were treated with vehicle. After 24h in culture, nitrite production was determined in the supernatant of glial cells using the Griess reaction. Values presented represent the mean ± SD from triplicate determinations, repeated at least three times. Statistical analysis revealed significant differences: *p ≤ 0.05, **p ≤ 0.01 compared to LPS-treated cultures; ^###p ≤ 0.001 compared to basal cultures. Immunocytochemical analysis of the expression of pro-inflammatory factors in primary glial cultures is shown, cyclooxygenase 2 (COX-2, green) and tumor necrosis factor alpha (TNFα, red). Cell nuclei were stained with DAPI. Scale bar, 20 μM.

Figure 7

Acute and repeated effects of compound 12d at core body temperature in mice. (A) The hypothermic effect was induced by acute treatment with 12d (20 mg/kg, i.p., 1 h). Columns are means ± SEM of the difference in body temperature (°C) (Δ, 1 h minus basal value) for 12d-treated mice (n = 12) and vehicle-treated control mice (n = 13). Symbols represent individual values for each mouse. ***p < 0.001 vs control group (Student’s t test, t = 5.588, df = 23). (B) Hypothermic effects observed during the repeated treatment with 12d (20 mg/kg, i.p., 5 days). Columns are means ± SEM of the daily difference in body temperature (°C) (Δ, 1 h minus basal value) for 12d-treated mice (n = 6) and vehicle-treated control mice (n = 6). **p < 0.01 or ***p < 0.001 vs control group (two-way repeated measures ANOVA followed by Sidak’s multiple comparisons test).

Figure 8

Acute and repeated effects of compound 12d on the content of hippocampal neurochemical markers (FADD, Cdk5, pERK/ERK) in mice. (A) Acute (20 mg/kg, i.p., 1 h, n = 6) and repeated (20 mg/kg, i.p., 5 days, n = 6) effects induced by 12d. Columns are means ± SEM of the target protein (% Control (C), n = 7–6). Symbols represent individual values for each mouse. *p = 0.0025 vs respective C group (Student’s t-test, t = 2.644, df = 10). (B) Representative immunoblots depicting the labeling of FADD, Cdk5 and pERK/ERK for each treatment group.

Figure 9

Results obtained from NORT, both short- (A) and long-term memory (B), and OLT (C) evaluations in SAMP8. Control mice group and SAMP8 treated with the compound 12d mice group represented as the summary of DI. Values represented are mean ± Standard error of the mean (SEM); (n = 8 per group); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 10

Representative Western blots and quantifications for PDE1B, HPCA, SYT7, and CAMKII (A – E). Values in bar graphs are adjusted to 100% for protein levels of the SAMP8 Control group. Representative gene expression for TrkA, Ngf, TrkB, and Bdnf (F). Gene expression levels were determined by real-time PCR. Values represented are mean ± Standard error of the mean (SEM); (n = 3–5 per group); *p < 0.05; **p < 0.01.

Figure 11

Reduction of capsaicin-induced mechanical hypersensitivity by the administration of 12d, clonidine and gabapentin in mice. (A) Dose dependency of the antinociceptive effects induced by the subcutaneous (s.c.) administration of 12d, clonidine and gabapentin. (B) Effects of 12d, clonidine or gabapentin alone and associated with the administration of the I₂–IRs antagonist idazoxan (4) or its methoxy analog RX 821002 (both at 3 mg/kg, s.c.). Values are the mean ± SEM obtained from 7–9 animals per group: **p < 0.01 vs nonsensitized animals treated with the vehicle of the drugs tested; ## p < 0.01 vs capsaicin-injected mice treated with the vehicle of the drugs tested; †† p < 0.01 12d-treated animals associated with idazoxan (4) or its solvent, and clonidine-treated animals associated with RX 821002 or its solvent (one-way ANOVA followed by Bonferroni test).

Figure 12

Effect of 12d and gabapentin on motor coordination. The latency to fall-down from the rotarod was recorded in each mouse immediately before (time 0) and at several times after the subcutaneous (s.c.) administration of 12d (20 mg/kg), gabapentin (40 mg/kg) or their vehicle. Values are the mean ± SEM from 8 animals: ∗p < 0.05, ∗∗p < 0.01 between the values at time 0 and after drug administration; ##p < 0.01 between gabapentin- and vehicle-treated groups on the same time after treatment (2-way repeated measures ANOVA followed by Bonferroni test).