Synthesis of Novel Baicalein Amino Acid Derivatives and Biological Evaluation as Neuroprotective Agents

Abstract

Baicalein, a famously effective component of the traditional Chinese medicine Rhizoma Huang Qin (Scutellaria altissima L.), has been proved to have potent neuroprotection and anti-platelet aggregation effects with few side effects. Meanwhile, recent studies have revealed that the introduction of amino acid to baicalein could improve its neuroprotective activity. In the present study, a series of novel baicalein amino acid derivatives were designed, synthesized, and screened for their neuroprotective effect against tert-butyl, hydroperoxide-induced, SH-SY5Y neurotoxicity cells and toxicity on the normal H9C2 cell line by standard methylthiazol tetrazolium (MTT) assay. In addition, all of the newly synthesized compounds were characterized by ¹H-NMR, ¹³C-NMR, and high resolution mass spectrometry (HR-MS). The results showed that most of the compounds provided more potent neuroprotection than baicalein, and were equivalent to the positive drug edaravin. They showed no obvious cytotoxicity on normal H9C2 cells. Notably, the most active compound 8 displayed the highest protective effect (50% effective concentration (EC₅₀) = 4.31 μM) against tert-butyl, hydroperoxide-induced, SH-SY5Y neurotoxicity cells, which was much better than the baicalein (EC₅₀ = 24.77 μM) and edaravin (EC₅₀ = 5.62 μM). Further research on the chick chorioallantoic membrane (CAM) model indicated that compound 8 could significantly increase angiogenesis, which might promote neurovascular proliferation. The detection of apoptosis analysis showed that compound 8 could dramatically alleviate morphological manifestations of cell damage. Moreover, the benzyloxycarbonyl (cbz)-protected baicalein amino acid derivatives showed better neuroprotective activity than the t-Butyloxy carbonyl (boc)-protected derivatives.

Keywords: SH-SY5Y cell; amino acids derivatives; baicalein; chick chorioallantoic membrane; neuroprotective effect.

Scheme 1

Synthesis of the baicalein derivatives (1–15). Reagents and conditions: (a) boc-amino acids or cbz-amino acids, dichloromethane (DCM), 4-dimethylaminopyridine (DMAP), and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI); 25 °C; 12 h.

Scheme 1

Synthesis of the baicalein derivatives (1–15). Reagents and conditions: (a) boc-amino acids or cbz-amino acids, dichloromethane (DCM), 4-dimethylaminopyridine (DMAP), and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI); 25 °C; 12 h.

Figure 1

Structure–activity relationships of baicalein derivatives. The results show that cbz-protected baicalein amino acid derivatives show better neuroprotective activity than the boc-protected baicalein amino acid derivatives.

Figure 2

Morphological detection of apoptosis using Giemsa staining (200×) on injured SH-SY5Y cells treated with compound 8: (a) control group, (b) injury group, (c) 6.25 μM, (d) 12.5 μM, and (e) 25 μM.

Figure 3

Morphological detection of apoptosis using 4′,6-diamidino-2-phenylindole (DAPI) staining (200×) on injured SH-SY5Y cells treated with compound 8, (a) control group, (b) injury group, (c) 6.25 μM, (d) 12.5 μM, and (e) 25 μM.

Figure 4

Microvascular proliferation of compound 8 on chick chorioallantoic membrane (CAM) (×50). (a) Blank control group, (b) 1 mg/mL for compound 8 group, and (c) 4 mg/mL for compound 8 group.

Figure 5

Effect of compound 8 on angiogenesis promotion. * p < 0.05; large vessels: Inner diameter > 100 µm; medium vessels: 10 µm < inner diameter < 100 µm; small vessels: Inner diameter < 10 µm.