SAR studies on truxillic acid mono esters as a new class of antinociceptive agents targeting fatty acid binding proteins

Abstract

Fatty acid binding proteins (FABPs) serve as critical modulators of endocannabinoid signaling by facilitating the intracellular transport of anandamide and whose inhibition potentiates anandamide signaling. Our previous work has identified a novel small-molecule FABP inhibitor, α-truxillic acid 1-naphthyl monoester (SB-FI-26, 3) that has shown efficacy as an antinociceptive and anti-inflammatory agent in rodent models. In the present work, we have performed an extensive SAR study on a series of 3-analogs as novel FABP inhibitors based on computer-aided inhibitor drug design and docking analysis, chemical synthesis and biological evaluations. The prediction of binding affinity of these analogs to target FABP3, 5 and 7 isoforms was performed using the AutoDock 4.2 program, using the recently determined co-crystal structures of 3 with FABP5 and FABP7. The compounds with high docking scores were synthesized and evaluated for their activities using a fluorescence displacement assay against FABP3, 5 and 7. During lead optimization, compound 3l emerged as a promising compound with the Ki value of 0.21 μM for FABP 5, 4-fold more potent than 3 (Ki, 0.81 μM). Nine compounds exhibit similar or better binding affinity than 3, including compounds 4b (Ki, 0.55 μM) and 4e (Ki, 0.68 μM). Twelve compounds are selective for FABP5 and 7 with >10 μM Ki values for FABP3, indicating a safe profile to avoid potential cardiotoxicity concerns. Compounds 4f, 4j and 4k showed excellent selectivity for FABP5 and would serve as other new lead compounds. Compound 3a possessed high affinity and high selectivity for FABP7. Compounds with moderate to high affinity for FABP5 displayed antinociceptive effects in mice while compounds with low FABP5 affinity lacked in vivo efficacy. In vivo pain model studies in mice revealed that exceeding hydrophobicity significantly affects the efficacy. Thus, among the compounds with high affinity to FABP5 in vitro, the compounds with moderate hydrophobicity were identified as promising new lead compounds for the next round of optimization, including compounds 4b and 4j. For select cases, computational analysis of the observed SAR, especially the selectivity of new inhibitors to particular FABP isoforms, by comparing docking poses, interaction map, and docking energy scores has provided useful insights.

Keywords: Anti-inflammatory agent; Anti-nociceptive agent; Computer-aided design; FABP inhibitor; Fatty acid binding protein; Molecular docking; SAR study.

Fig. 1

Structures of (-)-incarvillateine and 3

Fig. 2

Enantiopure amines examined for optical resolution of 3

Fig. 3

X-ray crystal structure of (R,R,R,R)-3 (3-A)

Fig. 4

Structures of FABP5 and FABP7 in complex with (S,S,S,S)-3. (A) (S,S,S,S)-3 bound at the canonical site of human FABP5. (B) (S,S,S,S)-3 bound at the canonical site of human FABP7.

Fig. 5

Overlay of representative FABP5 inhibitors designed (Table S7)

Fig. 6

Docking analysis of 4d in FABP5. (A) Binding pose of 4d (gray) through the local search with AutoDock 4.2 in comparison with 3 (blue). (B) Steric clash of 4-hydroxyphenyl group with Lys61 visualized by AutoDock Tool. (C) Docking pose of 4d (gray) by AutoDock 4.2 in comparison with 3. The binding pose of 3 is from the co-crystal structure.¹³

Fig. 7

Docking poses of 4k (gray) in FABPs as compared to those of 3 (blue). (A) FABP3, (B) FABP5, (C) FABP7. The binding poses of 3 in FABP5 and FABP7 are from co-crystal structures,¹³ while that in FABP3 is predicted by the AutoDock 4.2.

Fig. 8

Docking poses of 4b in FABPs in comparison with 3 (blue). (A) FABP3; (B) FABP5; (C) FABP7. The binding poses of 3 in FABP5 and FABP7 are from co-crystal structures,¹³ while that in FABP3 is predicted by the AutoDock 4.2.

Fig. 9

In Vivo Efficacy of Select Compounds. (A) Hyperalgesia was induced by intraplantar injection of CFA and thermal latencies were measured in mice following inhibitor administration (20 mg/kg, i.p.). n = 6-9 per group. **p < 0.01, ***p < 0.001 versus vehicle controls by one-way ANOVA with Dunnett post-hoc test. (B) Thermal latencies of wild-type (WT) and FABP5 KO mice following administration of 3 (40 mg/kg, i.p.). n = 6. ns indicates no significant difference, **p < 0.01 by two-ANOVA with Bonferonni post-hoc test. All data are presented as mean ± standard error.

Scheme 1

Synthesis of α-truxillic acids 1a~g via solid-state [2+2]

Scheme 2

Synthesis of 1h, 1i and 1j

Scheme 3

Synthesis of α-truxillic acid monoesters 3x and 4x, as well as diesters 5x (for the R group, see Table S7)

Scheme 4

Synthesis of (-)-incarvillateine-mimicked 3-analogs, 4a and 4d

Scheme 5

Synthesis of compound 3j.

Scheme 6

Synthesis of 3s.

Scheme 7

Synthesis of optically pure 3l isomers.

Scheme 8

Synthesis of α-truxillic acid monoamides 6a and 6b

Scheme 9

Synthesis of 3o-γ and amide 6a-γ