Stimuli-driven heterocycle synthesis by quenching the (aza)quinone methide of self-immolative prodrugs
Edupuganti, Veera Venkat Shivaji Rama Rao
A prodrug is an inactive compound that is converted into therapeutically active drug by enzymatic or chemical transformation. The research in this field is constantly aiming to develop new prodrugs to improve the physicochemical and pharmacological properties of the drug cargo being delivered. A self-immolative linker can play an important role in prodrug design as it acts as a spacer between trigger and payload, and enables rapid release of the active payload (drug) upon activation of the trigger. However, the by-products of the linker generated after activation are often neglected, and is particularly problematic for a commonly used self-immolative linker, p-aminobenzyloxycarbonyl (PABC) and p-(hydroxyl)benzyloxycarbonyl (PHBC). With these two linkers there is potential toxicity associated with the in situ formation of a quinone or azaquinone methide. It is the electrophilic nature of the methide that poses many potential toxicity problems in vivo, such as alkylation of DNA and proteins, and liver toxicity associated with depletion of antioxidants (e.g., glutathione). The primary aim of this thesis was to develop a self-immolative linker that could capture the electrophilic azaquinone methide (by-product) before it can react with biological nucleophiles. The designed linkers incorporate a strategically placed nucleophile that can trap the electrophile as it is formed in situ, providing a detoxification mechanism (Chapters 2-5) or a method for in situ heterocycle synthesis (Chapter 6). To address the toxicity associated with azaquinone methide, model tripartite prodrugs consisting of a trigger, self-immolative linker (containing a built-in nucleophile) and a model drug or probe (leaving group) were developed. In Chapter 2 a general synthetic approach to the new methide-trapping self-immolative linkers was developed. A multistep procedure was designed, facilitating the nucleophilic handle (an amine) to be introduced onto the self-immolative linker part of the prodrug in close proximity to the electrophilic methide, such that the methide would be quenched by the proximal nucleophile via an intramolecular cyclisation. The cyclisation step involved a 1,6-addition to the azaquinone methide to generate an N-benzyl-protected tetrahydroisoquinoline (THIQ) 209. The synthesis was designed with a cyclic anhydride 175 as the key intermediate, that could be used to introduce a diverse range of nucleophiles by a simple ring-opening reaction. The versatile design in the synthesis also allowed for alternate stimuli-responsive prodrug trigger groups to be installed, including a nitro group (Chapter 2), amide group (Chapter 3), azide group (Chapter 4) and boronic ester group (Chapter 6). Following the successful synthesis of the cyclic anhydride 175 in Chapter 2, a series of nitro-functionalised self-immolative linkers conjugated to the model drugs via a carbamate, carbonate or ether bond were synthesised (177-183). Using a HPLC assay developed during this project, each of the prodrugs were activated using Zn/AcOH as a reductive stimulus of the nitro group, producing THIQ 209 after the in situ trapping of the azaquinone methide. THIQ 209 was also synthesised and isolated by silica gel column chromatography (69% yield) and its structure confirmed using 2D NMR experiments. HPLC and LC-MS-monitored activation studies of two prodrugs (177 and 182) were conducted in the presence of a competing nucleophile, glutathione (GSH), and the amount of THIQ 209 generated was quantified and compared to the experiment in the absence of the GSH nucleophile. The results demonstrate that the secondary amine nucleophile appended to the linker is able to trap the methide in preference to a thiol nucleophile (GSH), providing proof-of-concept for a reactive methide prodrug detoxification strategy. A limitation of this experiment was that, for Zn/AcOH-mediated reduction, the reaction mixture was more acidic than that expected in a biological system, leading to reduced nucleophilicity of the methide trap (an amine) and the competing thiol (GSH). In an attempt to mimic in vivo reduction, activation of prodrugs 177 and 182 were attempted with nitroreductase enzyme, but preliminary experiments suggested that the enzyme used was inactive and did not reduce the nitro group. In order to investigate the prodrug activation and nucleophilicity of the methide trap and the competing thiol under more neutral conditions (pH 7.4), amide-functionalised prodrugs 219-220 were synthesised and examined for enzymatic activation using penicillin G amidase (PGA) (Chapter 3). The prodrugs 219 and 220 were subjected to activation by enzymatic hydrolysis, and using a HPLC-monitored assay the amount of cargo released and THIQ 209 generated was measured in the absence and the presence of a highly nucleophilic thiol, N-acetyl cysteine (NAC). Without the thiol, the prodrugs displayed excellent 1,6-self-immolation, releasing the model drug 166 (⁓ 90% in 24 h) and generating THIQ 209 (⁓ 78% in 24 h). In the presence of the thiol, the amount of model drug released (⁓ 88% in 24 h) and THIQ 209 generated (⁓ 78% in 24 h) was relatively unaffected by the competing thiol, supporting the results from Chapter 2. To provide further confirmation for the in situ formation of 65 and to rule out the generation of any thiol or water (another potential competing nucleophile) adducts, LC-MS studies were executed. Only low levels of thiol and water adducts were identified by LC-MS (no detection by standard HPLC), demonstrating that the intramolecular cyclisation outcompetes the intermolecular reaction, even in the presence of 1 mM (2.5-fold excess) and 4 mM (10-fold excess) NAC. In Chapter 4 the synthesis of azide-functionalised prodrugs 247, 248, and 249 carrying benzyl amine (184), 7-hydroxycoumarin (166) and ciprofloxacin ester 238 as model drug cargo was carried out. The azide-prodrugs were triggered (reduced) with NaSH, a hydrogen sulfide donor, to mimic overexpression of H2S, commonly associated with inflammatory conditions. A 5-fold and 20-fold excess of NAC was used as a competing nucleophile for the azaquinone methide, and the formation of THIQ 209 and release of model drug 166 were quantified by HPLC assay and further analysed by LC-MS. The prodrugs showed promising percentage release of the model drug coumarin 166 and the generation of THIQ 209 in the absence of competing nucleophile, forming THIQ 209 (⁓ 75% in 24 h), and releasing model drug 166 (⁓ 87% in 24 h). In the presence of nucleophile, the amount of THIQ 209 (⁓ 73% in 24 h) generated and release of model drug 166 (⁓ 84% in 24 h) showed that the intramolecular reaction is more favoured, even in the presence of strong competing nucleophile. The prodrugs synthesised in Chapters 3 and 4 (221, 248, 249), drug cargo (166 and 238), and the THIQ 209 were investigated for cytotoxicity in vitro against an epithelial breast cancer cell line (4T1) and a normal cell line (Madin-Darby Canine kidney; MDCK). The concentrations tested ranged from 1.95-1000 µM (Chapter 5). The prodrug 248, released drug 166 and THIQ 209 did not display cytotoxicity towards the 4T1 cancer and MDCK normal cell lines. The prodrugs 221, 249 and ciprofloxacin ester 238 displayed moderate cytotoxicity (IC50 = 279 µM (221), IC50 = 274 µM (249), IC50 = 152 µM (238)), towards the cancer cell line, whereas prodrug 249 and ciprofloxacin ester 238 exhibited relatively high cytotoxicity against normal cell lines (IC50 = 158 µM at 48 h (249), IC50 = 62 µM at 48 h (238)). In Chapter 6 two acyclic prodrugs 288 and 317 that could lead to the in situ synthesis of THIQ bioactives with anti-tubulin activity and P-gp pump inhibition were identified. For the proof-of-concept, the boronic-ester-functionalised prodrug 288 was designed that could potentially release one drug following oxidation-mediated activation and produce a THIQ 277 that acts as a P-gp efflux pump inhibitor (co-drug type delivery). However, after successfully completing nine of the sixteen proposed synthetic steps, selective bromination at the 4-position of aryl acid 231 was unsuccessful. An alternate synthetic approach in which synthesis of anti-tubulin inhibitor (THIQ) 318 could be achieved was designed, however, due to time constraints, the synthesis was not executed as part of the current thesis.
Advisor: Gamble, Allan B.; Tyndall, Joel D.A.
Degree Name: Doctor of Philosophy
Degree Discipline: School of Pharmacy
Publisher: University of Otago
Keywords: Self-immolative linkers; prodrugs; tripartite; triggers; model drugs; nucleophilic handle; azaquinone methide; 1,6-elimination; Tetrahydroisoquinoline (THIQ); intramolecular cyclisation
Research Type: Thesis