Design, Synthesis, and Pharmacological Characterization of a Potent Soluble Epoxide Hydrolase Inhibitor for the Treatment of Acute Pancreatitis

Abstract

Acute pancreatitis (AP) is a potentially life-threatening illness characterized by an exacerbated inflammatory response with limited options for pharmacological treatment. Here, we describe the rational development of a library of soluble epoxide hydrolase (sEH) inhibitors for the treatment of AP. Synthesized compounds were screened in vitro for their sEH inhibitory potency and selectivity, and the results were rationalized by means of molecular modeling studies. The most potent compounds were studied in vitro for their pharmacokinetic profile, where compound 28 emerged as a promising lead. In fact, compound 28 demonstrated a remarkable in vivo efficacy in reducing the inflammatory damage in cerulein-induced AP in mice. Targeted metabololipidomic analysis further substantiated sEH inhibition as a molecular mechanism of the compound underlying anti-AP activity in vivo. Finally, pharmacokinetic assessment demonstrated a suitable profile of 28 in vivo. Collectively, compound 28 displays strong effectiveness as sEH inhibitor with potential for pharmacological AP treatment.

Figure 1

Rational design for sEH selective inhibitors.⁻

Scheme 1. Synthesis of Indoline Derivatives 3–8, 10, and 14

Scheme 2. Synthesis of Indole Derivatives 27–33 and 36–39

Scheme 3. Synthesis of Indole Derivatives 42, 45, and Carbazole Compound 48

Figure 2

Schematic representation of the accommodation into the 5-LOX (PDB ID: 3O8Y) catalytic site of indoline (A) and indole (B). The protein is depicted in tube, while ligands in wire. The green and red dashed lines represent the π–cation interaction and steric clashes, respectively.

Figure 3

Three-dimensional model of the interactions given by 6 (A), 3 (B), 5 (C), 7 (D), 4 (E), and 8 (F) with 5-LOX (PDB ID: 3O8Y). The protein is depicted by tube (colored: C, gray; polar H, white; N, dark blue; O, red). The small molecules are represented by sticks (black for 6, orange for 3, faded azure for 5, violet for 7, magenta for 4, faded yellow for 8) and balls (colored: C, as for the sticks; polar H, white; N, dark blue; O, red; S, yellow). The dashed black lines indicate the hydrogen bonds between the ligand and protein.

Figure 4

Three-dimensional model of the interactions given by 6 (A), 30 (B), 28 (C), 27 (D), 36 (E), and 45 (F) with sEH (PDB ID: 3I28). The protein is depicted by tube (colored: C, green; polar H, white; N, dark blue; O, red). The small molecules are represented by sticks (black for 6, faded yellow-green for 30, gold for 28, cyan for 27, kiwi for 36; light gray for 45) and balls (colored: C, as for the sticks; polar H, white; N, dark blue; O, red; S, yellow). The dashed black lines indicate the hydrogen bonds between the ligand and protein.

Figure 5

Plasma concentration–time curve after a single dose of compound 28 injected i.p. in mice. Values are represented as mean ± standard deviation (SD) (n = 6).

Figure 6

Effect of compound 28 in a murine model of acute pancreatitis. (A) Timescale for cerulein-induced murine pancreatitis. Pancreatitis was induced in mice by i.p. injections of cerulein (50 μg/kg) hourly (5 times). Mice received compound 28 (10 mg/kg, i.p.) or AUDA (10 mg/Kg, i.p.) 0.5 and 2.5 h after the first cerulein injection and were killed 6 h after the first cerulein injection. (B) Pancreas slices were stained for H&E. (C) Evaluation of pancreatic edema and inflammatory infiltration was performed by three-point scoring system. Edema: 0 absent or rare; 1, in the interlobular space; 2, in the intralobular space; 3, the isolated-island shape of pancreatic acinus. Inflammation: 0, absent; 1, mild (infiltration in ducts); 2, moderate (infiltration in parenchyma < 50%); 3, severe (infiltration in parenchyma > 50%). Asterisk = interlobular edema, hash sign = edema, arrows = inflammatory cells. (D) Immunohistochemical analysis of neutrophils in pancreas sections. (E) Semiquantitative determination of neutrophil expression was obtained with ImageJ/Fiji software. Values represent mean ± standard error of the mean (SEM); n = 5 mice for each group. Data were analyzed by one-way analysis of variance (ANOVA) plus Bonferroni. Statistical significance is reported as follows: °° P < 0.01 and °°° P < 0.001 vs Sham; * P < 0.05 and *** P < 0.001 vs Vehicle.

Figure 7

Effects of compound 28 over AP induced lung injury. Pancreatitis was induced in mice by i.p. injections of cerulein (50 μg/kg) hourly (5 times). Mice received compound 28 (10 mg/kg, i.p.) 0.5 and 2.5 h after the first cerulein injection and were killed 6 h after the first cerulein injection. (A) H&E staining of lung sections. (B) Evaluation of lung edema and inflammatory infiltration was performed with three-point scoring system: 0, absent; 1, mild; 2, moderate; 3, severe. Asterisk = edema, arrows = inflammatory cells. Analysis was performed in a blinded manner. (C) Immunohistochemical analysis of neutrophils in lung slices. (D) Semiquantitative determination of neutrophil expression was obtained with ImageJ/Fiji software. Plasma levels of AST (J) and ALT (K). Values represent mean ± SEM; n = 5 mice for each group. Data were analyzed by one-way ANOVA plus Bonferroni. Statistical significance is reported as follows: °° P < 0.01 and °°° P < 0.001 vs Sham; * P < 0.05 vs Vehicle.

Figure 8

Levels of EETs (A), DHETs (B), and HETEs (C) in pancreas homogenates; graph bar color: blue sham (n = 6), gray vehicle (n = 6) red compound 28 (n = 6); data are expressed as ng/mg of tissue plus standard error of the mean (SEM).