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研究生:陳華鑫
研究生(外文):Hua-Sin Chen
論文名稱:4-(1-苄基-1H-吲唑-3-基)苯甲酸乙酯(YD-3)衍生物之合成與生物活性
論文名稱(外文):Synthesis and biological activities of ethyl 4-(1-benzyl-1H-indazol-3-yl)benzoate (YD-3) derivative
指導教授:郭盛助郭盛助引用關係黃麗嬌黃麗嬌引用關係
學位類別:博士
校院名稱:中國醫藥大學
系所名稱:藥物化學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:485
中文關鍵詞:血小板選凝素微波
外文關鍵詞:plateletselectinmicrowave
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在先前的研究中,4-(1-苄基-1H-吲唑-3-基)苯甲酸乙酯[ethyl 4-(1-benzyl-1H-
indazol-3-yl)benzoate;YD-3]被確認為第一個具有選擇性的非胜肽類蛋白酶活化受體4拮抗劑。
為了發展新型的PAR4拮抗劑,YD-3被選擇作為先導化合物。兩系列的N1-取代基芳香基甲基-3-(4-乙氧基羰基苯基)吲唑衍生物與N2-取代基芳香基甲基-3-(4-乙氧基羰基苯基)吲唑衍生物被合成出來。根據已知的方法來製備關鍵的中間物,4-(1H-吲唑-3-基)苯甲酸乙酯(8),然後將化合物8藉由直接的取代反應或Mitsunobu反應進行苄基化反應,可獲得相對應的主產物為N1-取代基芳香基甲基衍生物。然而,以微波輔助合成技術可以成功地促進主產物為N2-取代基芳香基甲基衍生物。
將所合成的化合物進行選擇性抗蛋白酶受體4活性之評估。經由構效活性關係(SAR)的研究,確認貢獻於抗蛋白酶受體4活性的重要官能基。在這些衍生物之中,4-[1-(3-氯苄基-1H-吲唑-3-基]苯甲酸乙酯(71)在蛋白酶受體4調控的血小板凝集顯示出最強效的抑制活性,化合物71值得更進一步的研究。
除此之外,也測試這些合成的化合物的抗發炎、抗過敏、細胞致毒與抗菌活性。在這些測試的化合物之中,4-(1H-吲唑-3-基)苯甲酸甲酯(6)與4-(1-苄基-1H-吲唑-3-基)-N-羥基苯醯胺(23)對於以LPS誘導RAW 264.7 cells生成TNF-α之抑制活性較SB-203580為強,其IC50值分別為4.8?b2.4與2.1?b0.2 μM。化合物8、4-[1-(2-氟苄基)-1H-吲唑-3-基]-N-羥基苯醯胺(117)、4-[1-(3-氟苄基)-1H-吲唑-3-基]-N-羥基苯醯胺(118)、4-[1-(2-甲氧基苄基)-1H-吲唑-3-基]-N-羥基苯醯胺(120)、4-[1-(3-甲氧基苄基)-1H-吲唑-3-基]-N-羥基苯醯胺(121)與4-[1-(4-甲氧基苄基)-1H-吲唑-3-基]-N-羥基苯醯胺(122)對於以LPS誘導RAW 264.7 cells生成TNF-α之抑制活性較genistein為強,其IC50值分別為1.7?b0.8、2.4?b0.7、1.7?b0.2、3.5?b0.5、3.5?b0.4與2.9?b1.3 μM。4-[1-(3-氯苄基)-1H-吲唑-3-基]-N-羥基苯醯胺(115)對於以fMLP/cytochalasin B誘導嗜中性白血球超氧自由基生成之抑制活性較DPI為強,其IC50值為0.8±0.2 μM。藉由β-glucuronidase之釋出偵測4-(1-苄基-1H-吲唑-3-基)苯醯胺(17)與23對於以fMLP/cytochalasin B誘導嗜中性白血球去顆粒化之抑制活性較genistein為強,其IC50值分別為6.4?b1.1與1.6?b0.5 μM。藉由lysozyme之釋出偵測4-(4,5,6,7-四氫-1H-吲唑-3-基)苯甲酸甲酯(5)、6、4-(2-benzyl-2H-吲唑-3-基)苯甲酸乙酯(11)、4-(1-苄基-1H-吲唑-3-基)苯甲酸(12)、17、23、135與4-(2-benzyl-2H-吲唑-3-基)苯甲酸甲酯(137)對於以fMLP/cytochalasin B誘導嗜中性白血球去顆粒化之抑制活性較genistein為強,其IC50值分別為8.6?b0.6、8.9?b0.6、8.7?b1.4、15.0?b0.6、4.7?b1.2、4.0?b0.4、12.0?b1.3、11.7?b1.2、10.5?b2.1與9.1?b1.1 μM。藉由β-glucuronidase之釋出偵測化合物8、117與[4-(1-苄基-6-甲氧基-1H-吲唑-3-基)苯基]甲醇(134)對於以fMLP/
cytochalasin B誘導嗜中性白血球去顆粒化之抑制活性較TFP為強,其IC50值分別為8.4?b2.6、5.7?b1.4與9.4?b0.8 μM。藉由lysozyme之釋出偵測化合物8對於以fMLP/cytochalasin B誘導嗜中性白血球去顆粒化之抑制活性較TFP為強,其IC50值為 3.9?b2.3 μM。這些化合物被發現有顯著的抗發炎活性,而且可選為新的先導化合物。此外,化合物92、4-[1-(3-氯苄基)-1H-吲唑-3-基]苯甲酸(93)、4-[1-(3-氟苄基)-1H-吲唑-3-基]苯甲酸(96)、115與118對金黃色葡萄球菌ATCC 25923、金黃色葡萄球菌1與金黃色葡萄球菌2有抗菌活性。
In the previous study, ethyl 4-(1-benzyl-1H-indazol-3-yl)benzoate (YD-3) was identified as the first selective non-peptide protease-activated receptor 4 (PAR4) antagonist.
In order to develop noel PAR4 antagonist, YD-3 was selected as lead compound. Two series of N1-substituted arylmethyl-3-(4-ethoxycarbonylphenyl)indazole derivatives (I) and N2-substituted arylmethyl-3-(4-ethoxycarbonylphenyl)indazole derivatives (II) were synthesized. The key intermediate, ethyl 4-(1H-indazol-3-yl)-
benzoate (8) was prepared according to the know method. Then, compound 8 was subjected to alkylation with directly substitution or Mitsunobu reaction to yield corresponding N1-substituted arylmethyl derivatives (I) as major products. However, the microwave-assisted synthetic technique can be successfuly promote N2-
substituted arylmethyl derivatives (II) as major products.
The synthesized compounds were evaluated for their selective anti-protease-
activated receptor 4 (anti-PAR4) activity. Through structure-activity relationships (SAR) study the functional groups contributing to anti-PAR4 activity was identified. Several new compounds with anti-PAR4 activity comparable to YD-3 were found. Among them, ethyl 4-[1-(3-chlorobenzyl)-1H-indazol-3-yl]benzoate (71) showed the most potent inhibitory effect on PAR4-mediated platelet aggregation. Compound 71 is worthy of further investigation.
In addition, the synthesized compounds were examined for their anti-
inflammatory, anti-allergic, cytotoxic, and anti-bacterial activity. Among these tested compounds, methyl 4-(1H-indazol-3-yl)benzoate (6) and 4-(1-benzyl-1H-indazol-3-
yl)-N-hydroxybenzamide (23) showed the better inhibitory effect of LPS induced TNF-α formation than positive control SB-203580 (IC50≒4.8?b2.4 and 2.1?b0.2 μM, respectively). Compounds 8, 4-[1-(2-fluorobenzyl)-1H-indazol-3-yl]-N-hydroxy
benzamide (117), 4-[1-(3-fluorobenzyl)-1H-indazol-3-yl]-N-hydroxybenzamide (118), 4-[1-(2-methoxybenzyl)-1H-indazol-3-yl]-N-hydroxybenzamide (120), 4-[1-(3-
methoxybenzyl)-1H-indazol-3-yl]-N-hydroxybenzamide (121), and 4-[1-(4-methoxy
benzyl)-1H-indazol-3-yl]-N-hydroxybenzamide (122) showed the better inhibitory effect of LPS induced TNF-α formation than positive control genistein (IC50≒1.7?b0.8, 2.4?b0.7, 1.7?b0.2, 3.5?b0.5, 3.5?b0.4, and 2.9?b1.3 μM, respectively). 4-[1-(3-Chlorobenzyl)-1H-indazol-3-yl]-N-hydroxybenzamide (115) showed the better inhibitory effect of fMLP/cytochalasin B induced neutrophil superoxide formation than positive control DPI (IC50≒1.2?b1.1, 0.8±0.2, 1.3±0.6, and 2.0±0.6 μM, respectively). 4-(1-Benzyl-1H-indazol-3-yl)benzamide (17) and 4-(1-benzyl-1H-
indazol-3-yl)-N-hydroxybenzamide (23) showed the better inhibitory effect for β-glucuronidase expression of fMLP/cytochalasin B induced neutrophil degranulation than positive control genistein (IC50≒6.4?b1.1 and 1.6?b0.5 μM, respectively). Methyl 4-(4,5,6,7-tetrahydro-1H-indazol-3-yl)benzoate (5), 6, ethyl 4-(2-benzyl-2H-indazol-
3-yl)benzoate (11), 4-(1-benzyl-1H-indazol-3-yl)benzoic acid (12), 17, 23, 135, and methyl 4-(2-benzyl-2H-indazol-3-yl)benzoate (137) showed the better inhibitory effect for lysozyme expression of fMLP/cytochalasin B induced neutrophil degranulation than positive control genistein (IC50≒8.6?b0.6, 8.9?b0.6, 8.7?b1.4, 15.0?b0.6, 4.7?b1.2, 4.0?b0.4, 12.0?b1.3, 11.7?b1.2, 10.5?b2.1, and 9.1?b1.1 μM, respectively). Compounds 8, 117, and [4-(1-benzyl-6-methoxy-1H-indazol-3-
yl)phenyl]methanol (134) showed the better inhibitory effect for β-glucuronidase expression of fMLP/cytochalasin B induced neutrophil degranulation than positive control TFP (IC50≒8.4?b2.6, 5.7?b1.4, and 9.4?b0.8 μM, respectively). Compounds 8, showed the better inhibitory effect for lysozyme expression of fMLP/cytochalasin B induced neutrophil degranulation than positive control TFP (IC50≒3.9?b2.3 μM). They were found to exhibit significant anti-inflammatory activity and were selected as new lead compounds. Besides, compounds 92, 4-[1-(3-chlorobenzyl)-1H-indazol-
3-yl]benzoic acid (93), 4-[1-(3-fluorobenzyl)-1H-indazol-3-yl]benzoic acid (96), 115, and 118 were found to exhibit significant anti-bacterial activity against Staphylococcus aureus ATCC 25923, Staphylococcus aureus 1, and Staphylococcus aureus 2.
中文摘要
英文摘要
第一章 緒論…………………………………………………………..……………1
第一節 吲唑類化合物之合成方法…………….……………….………………….2
第二節 微波反應概述……………………………………………………………...6
第三節 血小板之生理和病理功能………………………………………………...8
第四節 凝血酶及凝血酶–受體拮抗劑…………………………………………...11
第五節 選凝素之生理功能……………………………………………………….14
第六節 肥大細胞與嗜中性白血球之生理與病理功能………………………….15
第七節 巨噬細胞之生理與病理功能…………………………………………….17
第八節 小神經膠質細胞之生理與病理功能…………………………………….19
第九節 YC-1與YD-3之研究概況………………………………………………20
第十節 研究動機與目的………………………………………………………….21
第二章 結果與討論……………………………………………………………..23
第一節 化學合成………………………………………………………………….23
第二節 生物活性試驗結果……………………………………………………….48
第三章 結論……………………………………………………………………..113
第四章 實驗部分……………………………………………………………….118
第一節 試藥與溶劑……………………………………………………………...118
第二節 重要儀器與實驗材料…………………………………….......................122
第三節 化合物之製備…………………………………………….......................124
第四節 藥理試驗方法…………………………………………….......................191
參考文獻…………………………………………………………….....................200
圖譜資料………………………………………………………………………….211
研究成果…………………………………………………………………...……..484
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