- Title
- Synthesis of bolinaquinone analogues as clathrin inhibitors and cytotoxic agents
- Creator
- Ghods, Azadeh
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2017
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Bolinaquinone (BLQ), a marine sesquiterpene quinone, was considered as a lead compound to develop potent clathrin inhibitors for investigation in two distinct clathrin’s functions in clathrin- mediated endocytosis and mitosis. Moreover, BLQ possessed cytotoxic property that can potentially be exploited as a lead compound for development of cytotoxic agents. The complex structure of BLQ was modified to simpler analogues through scaffold simplification strategy, enabling to synthesis variety of analogues for evaluation of clathrin inhibition and cytotoxicity. In regard to the development of BLQ analogues as clathrin inhibitors, the predicted binding site of BLQ within the clathrin terminal domain (TD) assisted to design three types of analogues (A, B and C). Given that the predicted interaction of BLQ - clathrin terminal domain (TD) was dominated by hydrogen bond donor/acceptor, BLQ structural simplification strategy led to design TYPE A (2-hydroxy-1,4-benzoquinone-5-substituted derivatives), TYPE B (2-arylmethyl-1,4-benzoquinones) and TYPE C (amino-1,4-naphthoquinones) analogues. The evaluation of clathrin inhibition by ELISA-binding protocol revealed no activity for TYPE A and B analogues. However, TYPE C (amino-1,4-naphthoquinones) analogues exhibited promising clathrin inhibition. In TYPE C analogues, 2-(3-hydroxy-4-methylphenylamino)naphthalene-1,4-dione (68), and 2-(3-hydroxyphenylamino)naphthalene-1,4-dione (69) were identified as clathrin inhibitors with moderate level of inhibitory activity (IC₅₀ ~ 24 μM). The structural modification through the combination of chlorine and m-aminophenol moieties on TYPE C analogue showed the highest level of clathrin inhibition for 2-(3-hyroxyphenylamino)-3-chloronaphthalene-1,4-dione (71) with an IC₅₀ value of 2.77± 0.9 μM. However, subsequent modification of the regions in TYPE C analogues did not result in the higher inhibitory activity and a moderate clathrin inhibition was identified for 2-(4-hydroxyphenylamino)-3-chloronaphthalene-1,4-dione (74) and 5-(2-chloro-1,4-dihydro-1,4-dioxonaphthalen-3-ylamino)-2-hydroxybenzoic acid (75). Thus, the evaluated clathrin inhibition for the modified BLQ analogues was indicative to the pivotal roles of chlorine and m-aminophenol moieties on TYPE C analogues for the optimum interaction with the clathrin TD. The cell permeability of the most potent clathrin inhibitor (71) was assessed through a CME assay, which indicated that this compound was highly cell permeable. The off-target effect of the compound 71 was examined using dynamin GTPase activity in a Malachite green assay. The evaluated dynamin inhibition showed a moderate inhibitory activity for compound 71. However, comparing the clathrin inhibitory activity of compound 71 with dynamin inhibition was indicative to a 10-fold higher inhibition towards clathrin. This compound was also revealed cytotoxic activity towards a selected panel of twelve cancer cell lines with preferential growth inhibition against ovarian (A2780, GI₅₀= 0.83 ± 0.1 μM) and spontaneous murine astrocytoma (SMA, GI₅₀= 0.90 ± 0.2 μM) cell lines. To assess the effect of the potent clathrin inhibitor (71) in mitosis and cell division, mitotic assays (trypan blue exclusion, immunofluorescence microscopy, multi-nucleation, mitotic index and abnormality in metaphase) were applied. This anti-mitotic evaluation revealed promising result with the multinucleated and star-shaped cells, showing the potential effect of compound 71 on the spindle assembly checkpoint during the mitotic division. Regarding the development of BLQ analogues as cytotoxic agents, three types of analogues (A, B and C) were evaluated through a phenotypic screening using a MTT assay. While TYPE A and B analogues did not display noteworthy cytotoxicity towards the panel of twelve cancer cell lines, TYPE C analogues, 2-(3-hyroxyphenylamino)-3-chloronaphthalene-1,4-dione (71) and 2-(3-hydroxy-4-methylphenylamino)-3-chloronaphthalene-1,4-dione (72) revealed the high level of cytotoxic activity towards two cancer cell lines. The highest cytotoxicity for the compounds 71 and 72 were found towards ovarian carcinoma (A2780) with GI₅₀ values of 0.83 and 0.85 μM and spontaneous murine astrocytoma (SMA) with GI₅₀ values of 0.90 and 0.79 μM respectively. Given that the promising cytotoxic property of BLQ analogues, a new scaffold simplification strategy was applied to design TYPE D-G analogues (aryldimethoxy-1,4-benzoquinones and aryldiamino-1,4-benzoquinones). Compound 107, a TYPE D analogue (5-dimethoxy-3-(naphthalene-1-yl)cyclohexa-2,5-diene-1,4-dione), showed moderate and preferential cytotoxic activity towards neuroblastoma cell line (BE2-C) with a GI₅₀ value of 8.0 ± 0.5 μM. However, 3-biphenyl-4-yl-2,5-dimethoxycyclohexa-2,5-diene-1,4-dione (109, TYPE D analogue) exhibited a broad and moderate cytotoxicity with an average GI₅₀ value of 4 μM across the six cancer cell lines. In TYPE E analogues (diaryldimethoxy-1,4-benzoquinones), an extension of the hydrophobic region on benzoquinone core resulted in the modest growth inhibition towards cancer cell lines. As a moderate and preferential cytotoxic activity towards breast cancer cell line (MCF-7) was found in 2,5-dimethoxy-3-(biaryl-2-yl)-6-(naphthalene-3-yl)cyclohexa-2,5-diene-1,4-dione (125) with a GI₅₀ value of 5.1 ± 0.5 μM. Of TYPE F and G analogues (monoaryldiamino-1,4-benzoquinones and diaryldiamino-1,4-benzoquinones), the introduction of an aliphatic amine (n-propyl amine) on the benzoquinone core, supported identification of the most potent cytotoxic agents. The evaluated cytotoxicity for 3-(biaryl-2-yl)-2,5-bis-(propylamino)cyclohexa-2,5-diene-1,4-dione (132) and 2-(naphthalene-1-yl)-5-naphthalen-3-yl)-3,6-bis(propylamino)cyclohexa-2,5-diene-1,4-dione (134) were indicative of a broad-spectrum of cytotoxicity and the highest level of inhibition across cancer cell lines with an average GI₅₀ values of 2.53 and 0.77 μM respectively. The cytotoxic property of TYPE F and G analogues suggested that n-propyl amine moiety can potentially participate in a hydrogen bond donor/acceptor interaction with a cellular target (e.g. protein or DNA), resulting in an enhanced cytotoxicity compared to TYPE D and E analogues.
- Subject
- bolinaquinone; quinone; clathrin inhibitors; cytotoxic agents
- Identifier
- http://hdl.handle.net/1959.13/1335861
- Identifier
- uon:27507
- Rights
- Copyright 2017 Azadeh Ghods
- Language
- eng
- Full Text
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