15-PGDH 활성 억제를 위한 4-((2,4-dioxothiazolidin-5-ylidene) methyl)phenyl 2-phenylacetate 유도체 합성 및 구조-활성 상관관계 분석

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PGE 2, NAD +, 15-PGDH, inhibitors, TD, IC 50 values, halogen, wound healing
Background During the wound healing, Prostaglandin E2 (PGE2) as a lipid signaling molecule which is involved in pathological conditions such as neutrophils influx into the wound sites, angiogenesis, cell migration and proliferation synthesized by cyclooxygenase (COX-1/2) triggered by external stimulations. However it is rapidly metabolized to 15-keto prostaglandin E2 through chemical reaction of the NAD+ dependent 15-PGDH, which prevents specific EP-receptor coupling. This phenomenon delays wound healing in the inflammatory conditions with rapid in vivo degradation of PGE2. In this study, 15-PGDH was selected as the target protein to increase the PGE2 level which have short half-life in human body.
Experimental procedures In this study, 95 4-((2,4-dioxothiazolidin-5-ylidene)methyl) phenyl 2-phenylacetate derivatives, the series of Thiazolidiendione (TD) which was proved to have pharmacological effect through various research, were synthesized and analyzed for structure activity relationship (SAR). In order to analyze the inhibition effect of 15-PGDH, cDNA of human 15-PGDH was inserted into the pGEX-2T expression vector and transformed into E. coli BL-21 DE 3 and purified. Then, the optical density of NADH which is produced by inhibiting the enzyme was measured. The extracellular PGE2 levels was measured by enzyme-linked immunosorbent assay (ELISA) with sample from the A549 cell line. 4 lead compounds (10, 29, 31, 45, 66, 67, 69 and 86) were found by screening of the two data sets. These compounds were used in the MTT assay using HEK293 cell line and scratch-wound healing assay using human keratinocyte HaCaT cell line. The experimental data were expressed as mean ± standard deviation (SD) using IBM SPSS statistics version 20.0 (IBM inc., Armonk, NY, USA), and the average differences of the groups were tested with one-way ANOVA or general linear model (GLM - repeated measures) with Dunnet-t post-hoc test. A significance probability, P<0.05 was considered significant. Also IC50 values were calculated from the best-fit equation obtained by removing the outlier through the simple regression analysis. The curves were plotted and fitted using Sigmapolt for Windows. Ver. 10.0
Results The lead compounds showed IC 50 values ranging from 0.01 to 0.05 µM. They also increased levels of extracellular PGE2 in A549 cell. Especially, compounds 66, 67, 69 and 86 were significantly increased level of PGE2 approximately 3 - 4 fold higher than that of negative control. MTT assay were analyzed in confluent monolayers of human embryonic kidney cell line HEK293. Introduction of halogen at C-2 position of intermediate phenyl ring (66, 67, 69 and 86) showed higher IC 50 values which was represented lower cell cytotoxicity than the other substituents. They also showed significantly improved cell migration and proliferation with respect to negative control in the scratch - wound healing assay using confluent monolayers of human keratinacyte HaCaT cells.
Conclusion Among the lead compounds, introduction of halogen at both terminal phenyl ring and C-2 position of intermediate phenyl ring increased inhibitory potency of 15-PGDH. They also significantly increased extracellular PGE2 levels and improved wound closure rate with a lower toxicity. It was found that compound 69 [(Z)-2-bromo-4-((2,4 -dioxo-thiazolidin-5-ylidene)methyl)phenyl 2-(3-fluorophenyl)acetate] was the most potent inhibitor that was effective in the nanomolar range. Thus, it can be used for the tissue regeneration and treatment of diverse diseases caused by PGE2 deficiency.

Key words PGE 2, NAD +, 15-PGDH, inhibitors, TD, IC 50 values, halogen, wound healing
Alternative Title
Synthesis and structure-activity relationship(SAR) analysis of 4-((2,4-dioxothiazolidin-5-ylidene) methyl)phenyl 2-phenylacetate derivatives as 15-PGDH inhibitors
Alternative Author(s)
Park, So Hee
일반대학원 신재생에너지융합학과
Awarded Date
Table Of Contents
List of Tables ⅲ
List of Schemes ⅳ
List of Figures ⅴ
Abbreviations ⅵ
Abstract ⅷ
1. Introduction 1
2. Experimental procedures 14
2.1. Reagents and analysis apparatuses 14
2.2. Synthesis of 4-((2,4-dioxothiazolidin-5-ylidene)methyl)phenyl 2-phenylacetate
derivatives 16
2.2.1. General procedures for the synthesis of compounds a - e 16
2.2.2. General procedures for the synthesis of compounds 1 - 19 19
2.2.3. General procedures for the synthesis of compounds 20 - 38 27
2.2.4. General procedures for the synthesis of compounds 39 - 57 35
2.2.5. General procedures for the synthesis of compounds 58 - 76 44
2.2.6. General procedures for the synthesis of compounds 77 - 95 53
2.3. Expression and purification of 15-PGDH 62
2.4. Bradford protein assay 63
2.5. SDS-PAGE 63
2.6. 15-PGDH inhibition assay 65
2.7. Cell culture 66
2.8. Determination of extracellular PGE levels. 67
2.9. MTT assay 69
2.10. Scratch - wound healing assay 70
2.11. Statistical analysis 71
3. Results and Discussion 72
3.1. Chemical evaluation of 4-((2,4-dioxothiazolidin-5-ylidene) methyl) phenyl
2-phenylacetate derivatives 72
3.2. Biological evaluation 76
3.2.1. Inhibition effect of human 15-PGDH 76
3.2.2. Extracellular PGE2 levels 81
3.2.3. Cytotoxicity of the lead compounds for HEK293cell line 86
3.2.4. Determination of the wound healing effect 90
4. Conclusion 94
References 96
1H NMR Spectra 102
박소희. (2016). 15-PGDH 활성 억제를 위한 4-((2,4-dioxothiazolidin-5-ylidene) methyl)phenyl 2-phenylacetate 유도체 합성 및 구조-활성 상관관계 분석.
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