Design and Synthesis of Novel Indole Ethylamine Derivatives as a Lipid Metabolism Regulator Targeting PPARα/CPT1 in AML12 Cells

Abstract

Peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase 1 (CPT1) are important targets of lipid metabolism regulation for nonalcoholic fatty liver disease (NAFLD) therapy. In the present study, a set of novel indole ethylamine derivatives (4, 5, 8, 9) were designed and synthesized. The target product (compound 9) can effectively activate PPARα and CPT1a. Consistently, in vitro assays demonstrated its impact on the lipid accumulation of oleic acid (OA)-induced AML12 cells. Compared with AML12 cells treated only with OA, supplementation with 5, 10, and 20 μM of compound 9 reduced the levels of intracellular triglyceride (by 28.07%, 37.55%, and 51.33%) with greater inhibitory activity relative to the commercial PPARα agonist fenofibrate. Moreover, the compound 9 supplementations upregulated the expression of hormone-sensitive triglyceride lipase (HSL) and adipose triglyceride lipase (ATGL) and upregulated the phosphorylation of acetyl-CoA carboxylase (ACC) related to fatty acid oxidation and lipogenesis. This dual-target compound with lipid metabolism regulatory efficacy may represent a promising type of drug lead for NAFLD therapy.

Keywords: CPT1; NAFLD; PPARα; indole ethylamine derivatives.

Figure 1

Some representative leading compounds of indole-based PPAR agonists.

Scheme 1

Synthetic route of compound 9. Reagents and conditions: (a) (Boc)₂O, NEt₃, THF, rt, quant.; (b) DDQ, THF, H₂O, rt, overnight, 71% yield; (c) PMBBr, NaH, THF, 0 °C to rt, 64% yield; (d) DIBAL-H, −78 °C, 55% yield; (e) MeONH₂•HCl, NEt₃, toluene, 110 °C, 55% yield; (f) NaH, PMBBr, THF, 0 °C to rt, 34% yield; (g) methyl 3-chloro-3-oxopropanoate, AlCl₃, CH₂Cl₂, 0 °C to rt, 36% yield; (h) p-ABSA, NEt₃, CH₃CN, rt, 27% yield; (i) PhCF₃, 110 °C, 37% yield; and (j) TMSOTf, 2,6-lutidine, CH₂Cl₂, 0 °C to rt, 57% yield.

Figure 2

X-ray structure of compound 9.

Figure 3

Novel indole-derived structure 9 has comparable high binding affinity to PPARα. In silico docking of novel indole ethylamine derivative 9 with the homology model of human PPARα. The compound and important amino acids in the binding pockets are shown as sticks with the following colors according to atom type: hydrogen atoms in cyan, carbon atoms in white, nitrogen atoms in blue, and oxygen atoms in red. Hydrogen bonds are represented by yellow dotted lines. A surface representation and a ribbon diagram of the PPARα structure are shown.

Figure 4

Effects of novel indole ethylamine derivatives (4, 5, 8, 9) on PPARα agonist activity in hepatocytes. AML12 cells were treated with different concentrations of the compounds for 24 h: (a) PPARα levels in AML12 cells treated with all compounds (20 μM). Fenofibrate was used as the positive control; and (b) PPARα levels in AML12 cells treated with varying concentrations of 9 (5, 10, and 20 μM); n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. the negative control group.

Figure 5

Novel indole ethylamine derivatives (4, 5, 8, 9) prevent intracellular triglyceride (TG) accumulation in hepatocytes challenged with OA. AML12 cells were treated with different concentrations of the compounds for 2 h and then stimulated in the presence or absence of 500 μM OA for 24 h. (a) Hepatocyte TG levels in AML12 cells treated with all the compounds (20 μM) and/or OA. Fenofibrate was used as the positive control (n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the OA-treated control group). (b) Cell viability in AML12 cells treated with all the compounds (20 μM) (n = 4. * p < 0.05, ** p < 0.01, NS, not significant, p > 0.05 vs. the negative control group). (c) Hepatic hepatocyte TG levels in AML12 cells treated with varying concentrations of 9 (5, 10, and 20 μM) and/or OA (n = 3. **** p < 0.0001 vs. the OA-treated control group). (d) Cell viability in AML12 cells treated with varying concentrations of 9 (5, 10, and 20 μM) (n = 4. NS, not significant, p > 0.05 vs. the OA-treated negative control group).

Figure 6

Effects of 9 on the PPARα/CPT1 pathway in OA-induced AML12 cells. AML12 cells were treated with 9 (5, 10, and 20 μM) for 2 h and then stimulated in the presence or absence of OA (500 μM) for 24 h. (a) The mRNA expressions of PPARα and CPT1a were analyzed via real-time RT-PCR (n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the OA-treated control group). (b) The protein expressions of PPARα and CPT1a were extracted and analyzed via Western blotting. β-actin was used as an internal control. The relative intensity of the indicated proteins was quantified using NIH ImageJ 1.53a software (right panel) (n = 3. * p < 0.05, ** p < 0.01, NS, not significant, p > 0.05 vs. the OA-treated control group).

Figure 7

Effects of 9 on the lipid synthesis and metabolism of OA-induced AML12 cells. AML12 cells were treated with 9 (5, 10, and 20 μM) for 2 h and then stimulated in the presence or absence of OA (500 μM) for 24 h. (a) The mRNA expressions of HSL and ATGL were analyzed via real-time RT-PCR. n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, NS, not significant, p > 0.05 vs. the OA-treated control group. (b) The protein expressions of p-ACC/ACC and HSL were extracted and analyzed via Western blotting. β-actin was used as an internal control. The relative intensity of the indicated proteins was quantified using NIH ImageJ 1.53a software (right panel). (n = 3. * p < 0.05, ** p < 0.01, NS, not significant, p > 0.05 vs. the OA-treated control group).

Figure 8

Effects of 9 on the intracellular lipid accumulation in hepatocytes challenged with OA. AML12 cells were treated with different concentrations of 9 (5, 10, and 20 μM) and 20 µM fenofibrate for 2 h and then stimulated in the presence or absence of 500 μM OA for 24 h. Fenofibrate was used as the positive control. (a) Oil red O staining (200×). (b) MFI of oil red O staining was quantified using NIH ImageJ 1.53a software. (n = 3. *** p < 0.001, **** p < 0.0001 vs. the OA-treated control group). (c) BODIPY staining (200×). (d) MFI of BODIPY staining was quantified using NIH ImageJ 1.53a software. (n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the OA-treated control group).