The development of flow chemistry methodologies for the synthesis of potential anti-cancer agents

Research Doctorate - Doctor of Philosophy (PhD)The discovery and development of more specific and targeted small molecule anti-cancer drugs over the last decade, has led to a movement away from the ‘one-size-fits-all’ approach for cancer treatment. The synthesis of such molecules requires control over both chemical reactivity and reaction conditions. Advances in the control over reaction parameters, such as temperature and pressure, through the emergence of technology such as flow reactors and automated synthesisers has accelerated method development and discovery. The utilisation of flow chemistry throughout this research has enabled the optimization and development of a variety of synthetic methodologies, some of which were not previously achievable by batch synthesis. These protocols have enabled access to numerous potential anti-cancer agents. Through examination of acrylonitrile-based small molecules, a number of libraries were tested for their cytotoxicity. Access to the quinolone-2-(1<i>H</i>)-one library was achieved by a sequential four component Ugi-Knoevenagel condensation to afford 11 analogues in moderate to good yields (49–71%). A variation of the Ugi reaction was also carried out, in which three component compounds were generated to afford an α-amino amide library of 12 analogues in good yields (51-70%) (Chapter 2). Two libraries of highly decorated norcantharidin analogues were accessed <i>via</i> a number of chemoselective flow hydrogenation reactions to afford an isoindole-7-carboxylic acid library (14 analogues) and 7-oxa-bicyclic[2.2.1]heptane library (20 analogues) in moderate to good yields (21–75%), Whilst no analogues in the isoindole-7-carboxylic acid library showed any significant biological activity, four compounds from the 7-oxa-bicyclic[2.2.1]heptane library were found to exhibit a >85% growth inhibition against one or more cancer cell lines with a concentration of 100 μM (Chapter 3). Hydrodehalogenation proved to be a significant challenge whilst conducting hydrogenation reactions using flow chemistry conditions. In an attempt to circumvent this problem, a series of catalysts were screened for their ability to effect reductions in the presence of halogens. It was identified the 5% Pt/C (sulfided) catalyst was effective in retaining halogen atoms for reductive aminations, nitro reductions and alkene reductions (Chapter 5). Flow chemistry protocols were developed for the Suzuki-Miyaura cross coupling of 5-formyl-2-furanly boronic acid and a range of aryl bromides to synthesise a small library of 13 furan-based biaryl analogues. Such compounds will be used in the future as building blocks towards the development of potential inhibitors of the Hedgehog signalling pathway, a promising new class of potential anti-cancer agents. (Chapter 6)

Synthesis and cytotoxicity of octahydroepoxyisoindole-7-carboxylic acids and norcantharidin-amide hybrids as norcantharidin analogues

Octahydroepoxyisoindole analogues of norcantharidin were accessed through a Diels–Alder reaction of an amine-substituted furan with maleic anhydride and subsequent reduction of the bicyclo[2.2.1]heptene olefin. Despite retention of the carboxylate and the ether bridgehead known to impart cytotoxic activity to norcantharidin, none of these analogues displayed notable cytotoxicity against the 11 cell lines examined: HT29 (colon), MCF-7 (breast), A2780 (ovarian), H460 (lung), A431 (skin), Du145 (prostate), BE2-C (neuroblastoma), SJ-G2 and U87 (glioblastoma), MIA (pancreatic), and SMA (spontaneous murine astrocytoma). The incorporation of an amino-substituted system post-synthesis of norcantharidin afforded facile access to 14 acid/amide-substituted norcantharidin analogues. Of these, only four displayed sufficient activity at the initial 25 μm compound screening dose to warrant full evaluation of growth inhibition. Common to these analogues was the presence of a 4-biphenyl moiety, and in particular 3-(2-(furan-2-ylmethyl)-3-(4-biphenylamino)-3-oxopropylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (13 c) and 3-(2-(pyrrole-2-ylmethyl)-3-(4-biphenylamino)-3-oxopropylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid (24) displayed high levels of cytotoxicity, returning GI50 values of 15 nm (HT29) to 2.9 μm (U87) and 17 nm (SMA) to 2.8 μm (U87), respectively. These are the most cytotoxic norcantharidin analogues reported to date

The expanding utility of continuous flow hydrogenation

There has been an increasing body of evidence that flow hydrogenation enhances reduction outcomes across a wide range of synthetic transformations. Moreover flow reactors enhance laboratory safety with pyrophoric catalysts contained in sealed cartridges and hydrogen generated in situ from water. This mini-review focuses on recent applications of flow chemistry to mediate nitro, imine, nitrile, amide, azide, and azo reductions. Methodologies to effect de-aromatisation, hydrodehalogenation, in addition to olefin, alkyne, carbonyl, and benzyl reductions are also examined. Further, protocols to effect chemoselective reductions and enantioselective reductions are highlighted. Together these applications demonstrate the numerous advantages of performing hydrogenation under flow conditions which include enhanced reaction throughput, yields, simplified workup, and the potential applicability to multistep and cascade synthetic protocols

Investigation of the one-pot synthesis of quinolin-2-(1H)-ones and the discovery of a variation of the three-component Ugi reaction

Rapid access to the quinolin-2-(1H)-one scaffold is afforded by a sequential 4 component Ugi-Knoevenagel condensation of an aminophenylketone, an aromatic aldehyde possessing electron donating moieties, cyanoacetic acid and an aliphatic isocyanide, in moderate to good yields (49-71%). Interestingly, when the reaction is performed using aromatic aldehydes bearing electron withdrawing moieties or isocyanides containing aromatic or ester units, a mixture of a quinolin-2-(1H)-one and an α-amino amide (Ugi three-component adduct) is afforded in varying ratios. Further when the reaction is performed utilizing a combination of an isocyanide-containing aromatic or carbonyl unit, and an aldehyde possessing an electron withdrawing functionality, the Ugi three-component adduct is exclusively afforded. In our hands this new variation of the Ugi 3CR proved to be efficient and robust affording analogues in good yields (51-70%)

Chemoselective flow hydrogenation approaches to diversify the cytotoxic tetrahydroepoxyisoindole carboxamide scaffold

An Intramolecular Diels-Alder cycloaddition reaction between a furan diene and an alkynic dienophile was performed within a flow hydrogenator fitted with an inert titanium column at 150°C, no H2, under 100bar pressure. A single column pass (tR=1.6min) afforded ≈55% conversion to the tetrahydroepoxyisoindole carboxamide scaffold with a product turnover of ≈0.035g/h, a 95% improvement over batch procedures. The cycloaddition protocol is performed in water and ethanol, and does not require catalysts or other additives. Quantitative hydrogenation of the resulting dual π-bonds within the oxabicyclo system was effected with either a 10% Pd/Al2O3 or 10% Pd/CaCO3 catalyst at 20°C, 20bar, with full H2 whereas utilisation of a Raney Ni catalyst under these conditions resulted in a quantitative mono-olefin reduction of the C4-C5 double-bond. With regard to di-olefin reduction, a degree of stereoselectivity was observed with the aforementioned palladium-based catalysts yielding exclusive formation of (R) configuration at C7 while utilisation of a 5% Pt/C (sulfided) catalyst at temperatures below 60°C promoted the formation of the (S)-isomer at C7. Hence this work further highlights that flow-hydrogenation provides unprecedented convenience for establishing robust protocols to effect chemoselective transformations

An efficient continuous flow approach to furnish furan-based biaryls

Suzuki cross-couplings of 5-formyl-2-furanylboronic acid with activated or neutral aryl bromides were performed under continuous flow conditions in the presence of (Bu)₄N⁺F⁻ and the immobilised <i>t</i>-butyl based palladium catalyst CatCart™ FC1032™. Deactivated aryl bromides and activated aryl chlorides were cross-coupled with 5-formyl-2-furanylboronic in the presence of (Bu)₄N⁺OAc⁻ using the bis-triphenylphosphine CatCart™ PdCl₂(PPh₃)₂-DVB. Initial evidence indicates the latter method may serve as a universal approach to conduct Suzuki cross-couplings with the protocol successfully employed in the synthesis of the current gold standard Hedgehog pathway inhibitor LDE225

Expanding the utility of flow hydrogenation - a robust protocol restricting hydrodehalogenation

A commonly observed limitation of conducting hydrogenations under flow chemistry conditions is hydrodehalogenation. In a bid to circumvent this limitation a series of hydrogenation catalysts were screened, with 5% Pt/C (sulfided) catalyst identified as an effective catalyst to selectively effect reductive aminations, nitro reduction, and alkene reductions in the presence of halogen atoms. Additionally the optimised protocol cleanly reduced imines in the presence of a furan moiety indicating potential amenability to other labile functionalities

Discovery of acrylonitrile-based small molecules active against Haemonchus contortus

We report the discovery of a series of acrylonitrile-containing molecules and α-amino amides which cause 99–100% lethality in H. contortus. Of the 22 acrylonitrile analogues investigated, the most active were 2-cyano-3-[1-(3-dimethylaminopropyl)-2-methyl-1H-indol-3-yl]-N-hexylacrylamide (13a), 2-cyano-3-[1(2-dimethylaminoethyl)-2-methyl-1H-indol-3-yl]-N-hexylacrylamide (13b), 2-cyano-3-{4-[3-(dimethylamino)propoxy]phenyl}-N-octylacrylamide (21), and 2-cyano-3-{1-[3-(dimethylamino)propyl]-1H-pyrrol-2-yl}-N-octylacrylamide (22) with each displaying LD₅₀ values <15 μM whilst the α-amino amide methyl-2-[2-(2-benzoylphenylamino)-2-(4-methoxyphenyl)acetamido]acetate (12a) had an LD₅₀ value of 10 μM. A cytotoxicity screen of the acrylonitrile analogues (13a, 13b, 21 and 22) against nine cancer cell lines indicated modest to high cytotoxicity. In contrast, the α-amino amide 12a displayed very low cytotoxicity, with a maximum of ∼30% cell death at 25 μM (A2780, an ovarian carcinoma derived cell line) and with a mean of 11% cell death across all cell lines evaluated. Thus, 12a is considered a promising lead candidate for the development of a new anthelmintic