뇌질환 치료제로써 피루베이트 유사체의 합성, 분석 및 약리효과 연구
Synthesis, analysis and biological evaluation of the pyruvate analogues as a neuroprotective agent
- 발행기관 아주대학교
- 지도교수 윤성화
- 발행년도 2011
- 학위수여년월 2011. 8
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000011858
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Part 1. Synthesis and biological evaluation of the pyruvoylsalicylate derivatives for the treatment of ischemic stroke It has been found that the co-treatment of ethyl pyruvate and aspirin enhanced neuroprotective effect compared to individual treatment of ethyl pyruvate (EP) or aspirin in the postischemic brain, and that pyruvoylsalicylate, the hybrid compound of a salicylic acid and pyruvate, possess excellent potency and remarkable reduction of an infarct volume of MCAO in a rat model for ischemia/reperfusion. In a continuous effort to find more potent neuroprotective drugs, twenty one pyruvoylsalicylate derivatives composed of pyruvate group and salicylate moiety were synthesized and evaluated for in vitro biological activities. Regarding the metal chelating activity of pyruvoylsalicylate derivative with iron, 1a-3b, which has the 2-oxopropanoyloxy benzoic acid group, showed higher chelating activity (EC50 = 0.05 - 2.1 μg/mL) with iron metal than edaravone (EC50 = 34.9 μg/mL) and NEU2000 (EC50 = 9.7 μg/mL). The EC50 value of derivatives, 1e, 1h, 1j, 1k, 2, 5b, and 5c on superoxide radical scavenging assay were found to be 4.6, 5.5, 3.2, 3.5, 2.8, 2.2, and 5.4 ng/mL compared with 4.5 and 3.1 ng/mL those of edaravone and NEU2000, respectively. The hydrogen peroxide scavenging activities of 1e, 1j, 3a, 5a, and 5b was found to be 69.3, 93.9, 74.9, 74.9, and 97.0% compared with 2.0 and 51.3% those of edaravone and NEU2000, respectively. The administration of 1a, 1b, 1e and 1h reduced infarct volumes against focal cerebral ischemia by 57.3, 62.4, 29.0 and 65.6%, respectively, while Edaravone, NEU2000, and EP reduced the infarct volume to 8.3, 42.8, 66.4%, respectively. Based on the results of antioxidant activities, a metal chelating activity and a reduction of infarct volume on ischemic stroke, 1e showed the most potent neuroprotective effect. To determine the neuroprotective effect in the severe condition which transient focal ischemia was induced in 90 min MCAO rat ischemia model, 1e reduced infarct volumes significantly by 50.0%, which is higher protective activity than three known drugs such as edaravone, NEU2000, and EP with 88.3, 65.1 and 64.8% reduction of infarct volumes, respectively. To know the antioxidant and anti-inflammatory activity in the neuron cell, the protective effects of 1e against LPS treatment, NMDA treatment, oxidative stress induced Fe2+, and H2O2 damaged cells were evaluated via cell viability and LDH release, and the effects were compared with triflusal and pyruvate which were metabolites of 1e. In the neuron cell damaged from oxidative stress induced by Fe2+ and H2O2, 1e and pyruvate suppressed the LDH release. Neuroprotective effects of 1e and triflusal were observed in NMDA and LPS treat primary cortical cultures. Neuron cell tests showed that the neuroprotective activity of 1e resulted from an intrinsic activity of 1e and not from activities from its metabolites. Toxicities of 1e were evaluated in AMES test and high single dose test (100, 200, 400 mg/kg in rats) for further study to develop as a candidate drug for the treatment of ischemic stroke. These tests showed that 1e had no genetic toxicities and no inhibition on any of the cytochoromeP450 isozymes tested except CYP3A4. The chemical and plasma stabilities of 1e were in an acceptable range and the clearance in hepatocytes of 1e were low, suggesting that it was not rapidly metabolized in vivo. The 3D structure of 1e by a single crystal X-ray analysis and conformation of 1e indicated that a reciprocal arrangement of pyruvate and carboxylate groups made easily a bond with metal ion. Since 1e showed a potent neuroprotective activity via antioxidant and anti-inflammatory effects without much toxicity, it can be developed as a candidate of neuroprotective drug for the treatment of an ischemic stroke. Part II. Synthesis, analysis and biological evaluation of bioisosters of ethyl pyruvate As a scavenger of reactive oxygen species (ROS), ethyl pyruvate (EP) exhibited protective effects against oxidative stress and developed a relationship with various ROS-mediated diseases. The objective of this study is to investigate the anti-oxidant activity of EP's bioisosters and evaluate their neuroprotective effect on focal brain ischemia injury in rats. Three EP's bioisosters were synthesized and evaluated for the neuroprotective activity with in vivo MCAO rat model as well as antioxidant activity against in vitro 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH•), 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+), hydroxyl radical (OH•), superoxide radical (O2•-) and hydrogen peroxide (H2O2) and iron chelation. According to in vivo study, the reduction of infarct volume by 1 and 2 were observed with 34.8% and 27.5% reduction, respectively, while 3 and 4 were less effective with 49.5% and 47.2% reduction when 5 mg/kg of each compound was administrated after 6 hours MCAO/reperfusion. This data is comparable with 33.6% reduction by EP. Although EP showed a high antioxidant capacity with iron chelation and scavenging activity of H2O2 and hydroxyl radical, EP bioisosters revealed different antioxidant and iron chelation effects. Accordingly, 1 showed a high antioxidant capacity with iron chelation and superoxide radical (O2•-) scavenging activity while 2 showed a high antioxidant capacity with iron chelation and scavenging activity of H2O2 and DPPH•. In contrast, 3 showed a high antioxidant capacity with ABTS•+, superoxide radical (O2•-) and H2O2 scavenging activity while 4 showed a high antioxidant capacity with DPPH•, ABTS•+ superoxide radical (O2•-) and hydroxyl radical scavenging activity. From in vivo and in vitro results such as metal chelating activity with iron, hydroxyl radical scavenging activity, and hydrogen peroxide scavenging activity, EP bioisosters generally revealed a better neuroprotective effect than EP on ischemic stroke. Despite interesting biological effects of EP, surprisingly, because there is no report regarding the quantification of EP in biological media, we need to develop and validate an assay for quantifying EP in biological media. EP in rat plasma was determined by the non-covalent bonded with alkali metal and solvent molecules and there were 7 species of EP-alkali metal cation complex. The relative abundance (%) of m/z 255 is most predominated at acetonitrile/methanol (v/v, 1/9). By using LC/MS method to measure the relative area of the peak (m/z 255), EP concentration in rat plasma was accurately quantified. This new method was shown to be simple, highly sensitive, with a lower LOQ of 0.5 μg/mL, and linear over a wide range of 0.5 - 60 μg/mL. After administration of a single dose of 50 mg/kg EP in rats, the maximum concentration and time at maximum concentration were 25.01 μg/mL and 5 min respectively.
more목차
Acknoledgement (in Korean)
Abstract
List of Figures
List of Tables
Abbreviation
Part 1. Synthesis and biological evaluation of the pyruvoylsalicylate derivatives for ischemic stroke
I. Introduction 1
II. Drug design 22
III. Results and discussion
1. Chemistry 28
2. Biological evaluation 37
3. X-ray structure of 1e 61
4. Discussion 63
IV. Experimental section 78
V. Conclusions 103
VI. References 104
Part II. Synthesis, analysis and biological evaluation of bioisosters of ethyl pyruvate
I. Introduction 117
II. Results and discussion
1. Neuroprotective and antioxidant activities of EP bioisosters 123
2. Determination of EP in biological media by HPLC-ESI/MS 141
III. Experimental section 152
IV. Conclusion 166
V. References 168
Appendix 182
