"設計和合成2-胺基3,4-二氫喹唑啉衍生物作為潛在β位澱粉樣前體蛋白切割酵素抑制劑"

Abstract

本論文主旨在於設計與合成喹唑啉(quinazoline)衍生物作為潛能的β位澱粉樣潛體蛋白切割酵素(β-site amyloid precursor protein cleaving enzyme 1; BACE 1)的抑制劑，內容主要合成以喹唑啉為核心結構的十個化合物8, 60c-f, 61b-d, 80a-b，以及以喹唑啉酮(quinazolinone)為核心結構的四個化合物70-73。在方法開發上面總共分成四種途徑進行實驗設計，分別為兩種胺基的還原反應(reductive amination)及兩種親核性取代反應(nucleophilic substitution)，其中嘗試出途徑D (Approach D)為最佳合成方法。 根據楊森製藥公司所得到的化合物6a作為先導化合物，進行scaffold hopping計算及假設後，篩選出最具有潛力的化合物進行合成設計，根據折疊結果說明化合物8和化合物6a有一模一樣的折疊模式，且化合物8顯示良好抑制活性(IC50 = 0.75 µM)，間接說明化合物8在BACE 1空間中與化合物6a也有相同分布情形。從化合物8, 60c及61b-c的結果中發現4號位是飽和碳鏈取代時，活性會隨著碳鏈增長而下降；化合物60f在碳鏈末端增加羥基企圖增加親水性，而活性依然下降，因此說明碳鏈的長度越短、取代基親脂性越高會讓抑制BACE 1的活性越好。根據化合物9及60d-e的結果顯示取代基是甲基環己烷時，延長了環己烷的位置，因此能夠有效地折疊進入袋中，進而使得結構上的胺基與Asp228及Asp32間的距離變短，更能產生較佳活性。根據化合物61b-d結果我們推測pyrazolyl基團的存在，會推擠BACE 1結構中的S1口袋，因此4號位的取代基需要越小的基團才能夠讓胺基形成氫鍵的距離變短，提供較佳的抑制活性。依據上述推論，我們間接證明出S1口袋及S1’口袋與目標化合物的相對位置。甚至藉由化合物70-73的結果證明喹唑啉結構中的一級胺為提供BACE 1抑制活性主要官能基。 綜合上述的活性結果及物化性質預測結果，我們選出化合物60d (IC50 = 0.63 µM)及化合物61e (IC50 = 0.38 µM)作為未來最有機會發展的BACE 1抑制劑。

The aim of this thesis is to design and synthesize quinazoline and quinazolinone derivatives as potential inhibitors of β-site amyloid precursor protein cleaving enzyme 1 (BACE 1). The (S)-4-(2-amino-6-phenoxyquinazolin-3(4H)-yl)-N,4-dicyclo -hexyl-N-methylbutanamide (6a), invented by Johnson and Johnson as a new chemotype with BACE 1 inhibitory activity was selected as the lead molecule. Scaffold hopping strategy was applied to design two novel series of compounds bearing: (1) quinazoline core (8, 60c-f, 61b-d and 80a-b), and (2) quinazolinone core (70-73) as potential BACE 1 inhibitors. For synthesis of target molecules, in a key reaction step of coupling side chain with quinazoline or quinazolinone core four synthetic approaches (A-D) were considered. Two of these are based on reductive amination and other two are based on nucleophilic substitution reaction. Apporach D in which the side chain amino group undergoes necleophilic substitution reaction with quinazoline or quinazolinone core bearing suitable leaving group was found to be the most suitable approach for the synthesis of target molecules. The initial computational studies supporting the design hypothesis, indicated that compound 8 will mimic the bioactive topology of lead compound 6a by forming hairpin turn of the side chain and anchor itself with the BACE 1 active site by the virtue of an extensive hydrogen bonding by array of the two catalytic aspartic acids with the exocyclic amino group. This has been evident from its inhibition of BACE 1 enzyme activity (IC50 = 0.75 µM). Structure-activity-relationship studies revealed that introduction of methyl (8), cyclohexylmethyl (60c) and benzyl (60d) at C4 of quinazoline core, can retain the BACE 1 inhibitory activity. Based on simulation studies, evident by results of biochemical assay it can be postulated that these substituents can maintain the hairpin topology of sidechain similar to 6a and occupy the S1′ pocket of the BACE 1 enzyme. Interestingly, comparison of 9 (R1 = cyclohexyl, IC50 = 1.44 µM) and 60d (R1 = cyclohexylmethyl, IC50 = 0.63 µM cyclohexylmethyl) suggested that extra methyl group will extend the cyclohexane towards the S1′ pocket. Comparison of BACE 1 inhibition profile of compounds with quinazoline core (8-10, 60c-f and 61b-d) and quinazolinone (70-73) indicated that the exocyclic amino group is crucial for the hydrogen bonding with catalytic aspartate of enzyme active site. It can be hypothesized that N-pyrazolyl substituent on side chain will occupy the critical S1 binding pocket, however it is not compatible with larger substitution at C4 of quinazoline core (compare 61b, IC50 = 0.38 µM; 61c, IC50 = 2.41 µM; and 61d, IC50 = 2.00 µM). Finally, it was found that attempt to reduce lipophilicty of molecule by introduction of hydroxyl group at C4 of quinazoline core exemplified by 60f was not tolerated for BACE 1 inhibitory activity. Based on the results of biochemical assay and the computational calculation of druglike score, compound 60d (IC50 = 0.63 µM) and 61b (IC50 = 0.38 µM) were selected as potential leads for the further development as BACE 1 inhibitors.