Design, Synthesis and Biological Evaluation of Chemical Probes Incorporating Aldehyde Dehydrogenase (ALDH) Recognition Motifs and Fluorescent Properties. An Investigation Towards the Development of ALDH-Affinic Fluorophores for Hypoxia Cell Tracking

The aldehyde dehydrogenase (ALDH) class of enzymes has become of great pharmacological interest. There is increasing evidence that these enzymes are highly expressed in several tumour types, with particular focus on their expression in cancer stem cells (CSCs). Diethylaminobenzaldehyde (DEAB) is a pan-ALDH inhibitor that is frequently used to explore ALDHs in in vitro. However, no specific inhibitor or probe has been developed that selectively targets any of the ALDH isoforms. Accordingly, this thesis has been focused on preparing chemical probes containing ALDH recognition motif (RM) with potential for attachment to an anthraquinone chromophore with fluorescent properties. As such the work has been focussed on three steps: (1) design of a suitable anthraquinone scaffold as a vehicle to probe cellular events associated with ALDH functional activity and/or hypoxia, (2) design of ALDH-affinic RMs using computational modelling and literature observations and (3) design of fluorescent chemical probes by combining information acquired in 1 and 2 to interrogate cancer tissues. Starting with the fluorophore, a series of probes were synthesised and investigated against U2-OS GFP cells to assess cellular and fluorescent activity, under 1% O2 (hypoxic) and 20% O2 (normoxic) conditions. Probe II-7 showed enhanced fluorescence (7-fold increase) in 1% O2 compared to 20% O2, but with profound effect on cell cycle only under hypoxia, and perhaps greater than AQ4N (used as a control). II-9 showed similar results (5-fold increase) in 1% O2 compared to 20% O2, but with much less perturbation to the cell cycle than AQ4N in 1% O2. II-13 showed enhanced fluorescence in A549 cell lines under 20% O2, however less than AQ4. Unfortunately, the N-oxide analogue of II-13 was not biologically evaluated due to poor solubility. II-24 and II-26 showed promising fluorescence ratios in hypoxia versus normoxia in A549 cell lines, and especially the latter perturbed the cell cycle to a lesser extent. II-33, II-36 and II-38 are N-oxide analogues that showed interesting fluorescent properties and await biological investigations. In parallel studies, initial computational screening of DEAB analogues was performed against ALDH7A1, and the docking results confirmed that the lipophilicity of the RM is vital in terms of binding affinity. Consequently, a series of DEAB analogues were prepared and evaluated in silico and in vitro. III-4 showed preferential activity against yeast ALDH enzyme, while compounds III- 9, III-14 and III-18 showed promising patterns when evaluated in human ALDH7A1-transfected lung cancer cells. Alongside, a virtual hit compound from a previous screen (called HAN) was biochemically assayed using ALDH7A1 lysates from H1299/ALDH7A1-transfected lung cancer cells. The results demonstrated that HAN, as the first reversible inhibitor, was able to reduce ALDH7A1 functional activity. Consequently, a new library of 19 analogues was prepared with the aid of computer simulation using dockingserver software. The computational docking results of the new analogues showed some selectivity for ALDH7A1 compared with ALDH1A1, 1A3, 2 and 3A1. As an extension of this research, chemical probes with ALDH RM tethered to anthraquinone chromophores were synthesised and biologically evaluated. Probe IV-10 showed an encouraging fluorescence profile at excitation/emission wavelength 485/680 nm. Probe IV-44 was found to emit almost no fluorescence at a wide wavelength range of excitation/emission pairs, while its hypoxia targeted N-oxide derivative IV-45 showed a relatively intense fluorescence at 485/538 nm wavelength. IV-50 and IV-51 showed relatively low fluorescence values in comparison to their potential metabolic products (IV-50-C and quinizarin, respectively), generated via enzymatic metabolism to potentially deliver DEAB, or any of its analogues into target cells including hypoxia-located CSCs.