設計與合成二酮酸生物等效性之藥效基團作為C肝毒NS5B聚合酶抑制劑及其生物活性評估

Design, Synthesis and Biological Evaluation of Heterocyclic Compounds with Diketo Acid Bioisosteric Pharmacophores as HCV NS5B Polymerase Inhibitors

迪歐(Ravindra Ramesh Deore)

指導教授 ： 陳基旺

國立臺灣大學/醫學院/藥學研究所/博士(2011年)

https://doi.org/10.6342/NTU.2011.00199

摘要

無資料

關鍵字

二酮酸生物等效性藥效基團 ； C型肝炎病毒 ； NS5B聚合酶抑制劑

並列摘要

Abstract

The chronic hepatitis C virus (HCV) infection progressively leads to major liver disorders such as cirrhosis, end stage liver disease and hepatocellular carcinoma. While almost 3% of world’s population is infected by HCV, the lone treatment option of ribavirin in combination with pegylated interferon is insufficient to address this global epidemic. Therefore, there is an unmet clinical need for more effective and safe anti-HCV drugs. NS5B polymerase is currently pursued as the most popular target to develop safer anti-HCV agents because it is not expressed in uninfected cells. In chapter 1 lead identification and structure activity relationship (SAR) studies of more than 50 scaffolds leading to the potent NS5B inhibitors is reviewed. Even though, allosteric site inhibitors have gained much attention due to their nanomolar potency in enzyme as well as replicon assays, emergence of resistant mutants and lack of specificity against all HCV subtypes have limited their transition to the clinic. Active site inhibitors of NS5B are noncompetitive with respect to both the RNA template and to the nucleotides. Existence of a strong sequence homology within the active site of NS5B from all the nine subtypes makes active site a potential target for safe drug development against all subtypes of HCV.
Scaffolds such as alpha, gamma-diketo acid (DKA) and its bioisosters such as monoethyl ester of meconic acid and dihydroxypyrimidine carboxylic acid are the only active site inhibitors of NS5B in the literature. Therefore, in chapter 2 a novel series of 2-hydroxy-1-oxo-1,2-dihydroisoquinoline-3-carboxylic acid derivatives with the built-in β-N-hydroxy-γ-keto-acid as DKA bioisoster was designed. Compounds 16a-p were synthesized via isocoumarin intermediates 18a-l. 2-Hydroxy-1-oxo-4-phenyl-1,2-dihydroisoquinoline-3-carboxylic acid (16c) was the most active compound in this series. Compound 16c inhibited NS5B polymerase in assays based on the inorganic pyrophosphate generation (IC50 = 9.5 µM) and NTP incorporation by NS5B enzyme (IC50 = 5.9 µM). Consequently, it demonstrated moderate antiviral activity (EC50 = 15.7 µM) and good selectivity in HCV genotype 1b replicon Ava5 cells. Job plot, NMR spectroscopy and docking studies showed that compound chelated to two Mg2+ ions and also deteriorated the interaction of NTP to NS5B polymerase in BIAcore assay.
Enhancement of druggability is a major challenge for active site inhibitors as they possess functional groups such as hydroxyl and carboxylic acid that add to their polar nature. Change in isoquinoline core to quinazoline and carboxylic acid to carboxamide generated β-N-hydroxy-γ-keto-carboxamide pharmacophore that would act as a DKA bioisoster. Thus, in chapter 3 a series of N-substituted-3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-carboxamides (8a-k) was synthesized. N-Benzyl-3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-carboxamide (8a) was defined as a minimum structure to inhibit NS5B polymerase. Predicted physicochemical properties such as MolLogP, cLogP, MolPSA, and number of hydrogen bond donors (HDB) and hydrogen bond acceptors (HBA) of N-hydroxyquinazolinone analog 8a were in acceptable range and possessed higher drug likeness score than the N-hydroxyisoquinolinones. Structure activity relationship studies have led to identification of more flexible and lipophilic N-hydroxyquinazolinone 8k (IC50 = 8.77 µM and EC50 = 15.7 µM) as the most potent analog of this series. Moreover, it was nontoxic to parent Huh7 cells rendering it a safe candidate for further development. Binding predictions by molecular docking suggested that compound 8k forms chelating interactions with two magnesium ions present in the active site of NS5B polymerase. In addition to metal chelation, backbone atom of Phe224 and Asp225 form hydrogen bonds with compound, while alkyl linker and aromatic ring also have a hydrophobic interaction with Val52 and Leu159 of NS5B polymerase.
A pharmacophore wherein 1,3-diketo moiety integrated with 2-N-hydroxyl group is proposed as DKA bioisoster in chapter 4. A small fragment like molecule N-hydroxysuccinimide weakly inhibited NS5B polymerase, and was subjected to structural change to generate an equipotent lead 2-hydroxyisoindole-1,3-dione (13a). A series of isoindole-1,3-diones (13a-n) was synthesized from respective phthalic anhydrides. 4-Nitro analog (13b) demonstrated moderate enzyme inhibition with an IC50 of 9.5 µM. Alternatively, a modified 1,3-diketo scaffold 2-aroylisoindoline-1,3-dione (14a-g) was developed, of which 2-benzoyl-isoindole-1,3-dione (14a) inhibited generation of pyrophosphate by NS5B polymerase with IC50 of 6.8 µM, which is more than 10 fold enhancement as compared to parent 2-hydroxyisoindole-1,3-dione. 5,6-Dichloro analog 14d was found to be selectively cytotoxic to Ava5 cells with an IC50 of 18.0 µM.
Chapter 5 describes the overall conclusion of this thesis and perspectives thereof. Literature reported NS5B inhibitors are classified. The summary of three different classes of NS5B polymerase inhibitors namely, isoquinolinone, quinazolinone and isoindoledione with hydroxamate inbuilt DKA bioisosteric pharmacophores have been discussed. Though investigated for their NS5B polymerase inhibitory activity and anti-HCV activity, these classes of DKA bioisosters offer an opportunity for medicinal chemists to provide lead structures for targeting other biological targets that are metal dependent.

並列關鍵字

diketoacid bioisoster ； hepatitis C virus ； NS5B polymerase ； inhibitor ； pharmacophore

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