Novel Neuroprotective Lead Compound Ligustrazine Derivative Mass Spectrometry Fragmentation Rule and Metabolites in Rats by LC/LTQ-Orbitrap MS.

Xinyu Zhang,Kang Fang,Penglong Wang,Tao Ma,Wenxi Zhang,Rui Zhao,Meng Chen,Hui Wang,Nannan Xue,Gaorong Wu,Haimin Lei,Guoliang Li

doi:10.3390/molecules23051154

Abstract

The neuroprotective evaluation of ligustrazine derivatives has become a research focus all over the world. A novel ligustrazine derivative, (3,5,6-Trimethylpyrazin-2-yl)methyl(E)-3-(4-((3,5,6-trimethylpyrazin-2-l)methoxy)phenyl)acrylate (T-CA), has shown protective effects against CoCl2-induced neurotoxicity in a differentiated PC12 cell model and middle cerebral artery occlusion (MCAO) model in our previous studies. However, nearly none of the parent drugs existed after rapid metabolism due to uncertain reasons. Thus, the fragmentation regularities of mass spectra, and metabolites, of T-CA in rats were examined using liquid chromatography-electrospray ionizationion trap mass spectrometry (LC/LTQ-Orbitrap MS) in this research. The main fragment ion, mass spectrum characteristics, and the structural information were elucidated. When compared with a blank sample, we identified five kinds of T-CA metabolites, including three phase I metabolites and two phase II metabolites. The results showed that the metabolic pathways of T-CA in rats via oral administration were hydrolysis (ether bond rupture, ester bond rupture), oxidation, reduction, glucose aldehyde acidification, etc. In addition, three main metabolites were synthesized and their structures were identified by superconducting high-resolution NMR and high-resolution mass spectroscopy (HR-MS). The neuroprotective activity of these metabolites was validated in a PC12 cell model. One of the metabolites (M2) showed significant activity (EC50 = 9.67 μM), which was comparable to the prototype drug T-CA (EC50 = 7.97 μM). The current study provides important information for ligustrazine derivatives, pertaining to the biological conversion process in vivo.

Highlights

It is important to evaluate the effects and toxicity of new drugs from metabolites, such as desloratadine, which showed a better anti-allergic activity than its parent drug loratadine [1]; amoxapine and its metabolites have the potential to alleviate irinotecan-induced diarrhea; and many microbial metabolites have the possibility to become therapeutic agents [2,3]
T-CA was evaluated in CoCl2-induced neurotoxic PC12 cell model, differentiated by nerve growth factor (NGF) in vitro, and middle cerebral artery occlusion (MCAO) rats model in vivo [11,14,16]
The LC-MSn detection method was established by analyzing T-CA and its metabolites in vivo

Summary

Introduction

It is important to evaluate the effects and toxicity of new drugs from metabolites, such as desloratadine, which showed a better anti-allergic activity than its parent drug loratadine [1]; amoxapine and its metabolites have the potential to alleviate irinotecan-induced diarrhea; and many microbial metabolites have the possibility to become therapeutic agents [2,3]. Tetramethylpyrazine was combined with small molecules of phenolic compounds, based on the traditional Chinese medicine combination principles [9,10,11,12,13,14,15]. According to different conjunctive positions and numbers of hydroxyls, over 100 novel ligustrazine-phenolic acid derivatives were synthesized and were proved to possess neuroprotective effects, both in vivo and in vitro [9,10,11,12,13]. The ligustrazine derivative (3,5,6-Trimethylpyrazin-2-yl)methyl(E)-3-(4-((3,5,6-trimethylpyrazin-2-l)methoxy)phenyl)acrylate (T-CA) was the best one among these tetramethylpyrazine derivatives because it showed an excellent neuroprotective activity. The internal absorption experiment indicated that the prototype compound was metabolized rapidly and few of it remained in plasma

Methods

Results

Conclusion