23 1.12. Conclusions
Analogues of HHQ were synthesised with substitutions at the C-5 to C-8 positions (10–21). Two of the quinolone compounds were methylated to give the N-Me quinolone and O-Me quinoline isomeric products (Scheme 8). A C-3 substituted quinazolinone analogue (28) was prepared, and a HHQ analogue with a terminal alkene on the heptyl chain at C-2 was also synthesised (31). An improved route to access 6-NO2 HHQ 3 via regioselective nitration of HHQ 1 was developed, and the secondary (33) and tertiary (34) methylated analogues of Hartmann’s antagonist 5 were prepared.
All of the HHQ analogues prepared were sent to Dr Reen at the BIOMERIT Research Centre at UCC to be tested in various biological studies. The experimental details of these tests are outside the scope of this thesis. However, due to the fact that the results of the biological analysis often guide the design of structural analogues of lead compounds, the results of the studies are summarised in the next section. Full details of the biological experiments can be found in the various publications, and are included in Appendix I.
24 1.13. Biological Results
A Structure Activity-Relationship Study of Bacterial Signal Molecule HHQ Reveals Swarming Motility Inhibition in Bacillus atrophaeus (Organic & Biomolecular Chemistry, 2015, 13, 5537–5541)
Bacillus atrophaeus is a Gram-positive species of bacteria that co-inhabits with P.
aeruginosa in soil. P. aeruginosa QS molecules have been shown to influence behaviour in B. atrophaeus.[28, 54] B. atrophaeus also exhibits strong swarming and biofilm phenotypes that are characteristic of the multicellular behaviour underpinning virulence in many pathogens.[9] Swarming motility is considered a key virulence phenotype in many organisms controlled by QS.[55]
The aim of the SAR in this study was to learn more about the influence of AHQs on Gram-positive bacteria and provide a platform for therapeutic developments. To achieve this, a diverse range of novel aryl-substituted HHQ analogues were tested on B. atrophaeus. A previous report from Reen et al. showed that swarming motility of B. atrophaeus was abolished in the presence of PQS, while use of HHQ showed little reduction.[28] Interestingly, some of the aryl-substituted HHQ analogues showed significant alteration in activity relative to the parent HHQ, with some compounds exhibiting inhibition comparable to PQS. In particular, RC5 and RC6 completely inhibited swarming, despite differing in the alkyl chain length at C-2. It appears that for compounds with a C-2 heptyl chain, an electron-withdrawing substituent at C-6 is required for anti-swarming activity, while for compounds with a C-2 nonyl chain, an electron-donating substituent at C-6 is required.
As mentioned, 4-quinolones such as HHQ exist as two interconverting tautomeric forms: 4-quinolone and 4-hydroxyquinoline. It would be interesting to determine if either structure maintained anti-swarming activity if ‘locked’ in these tautomeric forms by methylation at the oxygen or nitrogen atom. Indeed, when anti-swarming molecule RC6 was methylated, both the quinolone form 24 and the quinoline form 25 lost this activity (Figure 6). Compound 12 was also methylated and both isomers tested for their impact on swarming motility in B. atrophaeus. Again, both methylated derivatives 22 and 23 exhibited reduced activity relative to the parent molecule. In fact, the quinoline analogue 23 appeared to promote swarming.
25
12 RC5 RC6
N-Me Compound 22 O-Me Compound 23
N-Me Compound 24 O-Me Compound 25 Figure 6 Impact of HHQ analogues on swarming motility of B. atrophaeus. The smaller the swarming diameter, the greater the anti-swarming activity of the compound.[56]
Modification of the quinolone at C-6 resulted in compounds possessing anti-swarming activity similar to that of PQS. These results suggest that the C-3 hydroxyl group of PQS is not essential for anti-swarming activity of AHQs towards B. atrophaeus.
26 Exploiting Interkingdom Interactions for Development of Small Molecule Inhibitors of Candida albicans Biofilm Formation (Antimicrobial Agents and Chemotherapy, 2016, 60, 5894–5905)
Candida albicans causes a variety of complications ranging from mucosal disease to deep-seated mycoses, particularly in immunocompromised individuals.[57-58] Along with other bacterial, fungal and yeast pathogens, C. albicans is known to form biofilms on medical devices, leading to recurring infections and in some cases death.[59-60] Once established in the biofilm phase, C. albicans presents a significant clinical problem, as biofilms themselves are considered a breeding ground for the emergence of antibiotic-resistant strains.[61]
C. albicans is known to coexist with P. aeruginosa in the cystic fibrosis lung, and interkingdom communication between the two organisms has previously been reported.[62-63] Previously, Reen et al. have shown that HHQ, but not PQS, suppresses biofilm formation in C. albicans (Figure 7).[28] In order to visualize the biofilm structures, attached cells were treated with specific intracellular stains to assess impact on key cellular components. These included cellulose and chitin with Calcofluor;
lectin (carbohydrate) structures with Concanavalin A; and live cellular vacuolar stain FUN-1 which functions as a live/dead stain. As both PQS and HHQ promote virulence and pathogenicity in P. aeruginosa,[24, 64] these compounds fall short of being a viable antifungal treatment. However, chemical modification of these molecules provides an opportunity to develop compounds with greater specificity of function.
Figure 7 C. albicans biofilms grown in the presence of PQS and HHQ.[65]
27 Analysis of the suite of analogues based on the core HHQ quinolone framework led to the identification of compounds that retain anti-biofilm activity toward C. albicans.
Several analogues displayed anti-biofilm activity similar to HHQ, including the C-3 substituted quinazolinone analogue 28. Overall, electron-withdrawing substituents (e.g. compounds RC4, RC5) promoted anti-biofilm activity in a similar manner to the HHQ parent, while electron-donating substituents (e.g. compounds 12, 13, RC3) resulted in some loss of activity. Extension of C-2 chain (20) did not seem to alter activity. Microscopic staining revealed that, like HHQ, the active compounds caused structural changes in the biofilm, causing atypical morphologies.
Importantly, the new compounds exhibited significantly reduced cytotoxicity toward IB3-1 airway epithelial cells compared with the parent HHQ molecule. Evaluating the cytotoxicity of synthetic compounds is crucial in the context of developing targeted therapeutics that are benign to human cellular physiology and ideal for use in a clinical environment. Furthermore, unlike HHQ, the lead compounds were inactive toward the P. aeruginosa PqsR receptor system, a critical requirement for their potential future development as anti-biofilm therapeutics.
28 Harnessing Bacterial Signals for Suppression of Biofilm Formation in the Nosocomial Fungal Pathogen Aspergillus fumigatus (Frontiers in Microbiology, 2016, 7, 2074)
Aspergillus fumigatus is a pathogenic fungus associated with hospital-acquired infections. A. fumigatus infections contribute to morbidity and mortality in people with respiratory diseases such as cystic fibrosis, as well as infecting open skin wounds in burn patients and areas surrounding medical implants.[66-67] However, as with its bacterial counterparts, the defensive biofilm of A. fumigatus has led to conventional antifungal therapies rapidly becoming redundant.[68] Additionally, the co-existence and mutual interaction of bacteria and fungi at the site of infection contributes to the pathogenesis of disease. In the case of CF, A. fumigatus has been isolated in up to 60%
of patients with P. aeruginosa infection, suggesting a close relationship between colonisation by P. aeruginosa and chronic infection by A. fumigatus.[69-70]
In this study, the anti-biofilm activity of P. aeruginosa AHQs against A. fumigatus was investigated. Biofilm biomass and structure was significantly altered in the presence of both HHQ and PQS. Confocal microscopic analysis revealed that cells treated with either HHQ or PQS appeared locked in a spore form, with minimal evidence for formation of hyphal structures, characteristic of A. fumigatus biofilms.
Next, the suite of analogues was tested and lead compounds were identified that retained activity against A. fumigatus (11, 12, 13, 20, 28). Of these, compound 12 (n-hexyl group at C-6) and compound 13 (methoxy group at C-7) gave the greatest reduction in biofilm formation. Compound 20, in which the alkyl chain length was extended relative to the parent molecule HHQ, retained its anti-biofilm activity.
Quinazolinone compound 28 also displayed good anti-biofilm activity.
Importantly, anti-biofilm activity was retained against clinical isolates from paediatric samples. It has been shown that the genomes of clinical isolates can vary markedly from typed environmental strains, with niche-specific selective pressures manifesting with considerable specificity, even within species.[71] Both HHQ and PQS were capable of suppressing biofilm formation in each of the clinical isolates. Lead compounds 11 and 28 were also found to be effective against biofilm formation in clinical strains, underpinning their suitability for further therapeutic development (Figure 8). As expected, anti-biofilm compounds disrupted the formation of hyphal
29 structures, and appeared to lock the fungal cells in the spore state. Notably, however, the suppression of biofilm formation was not to the same extent as that seen against the lab strain Af293, suggesting some degree of tolerance or adaptation in the clinical isolates.
28 11
Figure 8 Confocal laser scanning microscopy analysis of biofilm formation in model strain (Af293) and clinical isolates (CFBRC1, CFBRC2, CFBRC3) in the presence of HHQ, PQS and lead compounds 28 and 11.[72]
This study identified compounds with anti-biofilm activity comparable to the parent HHQ molecule, providing important insights into the SAR underpinning the suppression of fungal biofilms.
28 11
30 The Requirements at the C-3 Position of Alkylquinolones for Signalling in Pseudomonas aeruginosa (Organic & Biomolecular Chemistry, 2017, 15, 306–310)
As described in the introduction, Hartmann and co-workers reported a potent PqsR antagonist could be accessed by placing a nitro group at the C-6 position of HHQ (Scheme 1).[41-42, 52] In order to suppress functional inversion by in vivo hydroxylation of the C-3 position, the C-3 position was ‘blocked’ with a primary amide, leading to the development of a highly active antagonist 5. Previous work form the McGlacken group and collaborators showed that altering the C-3 position of HHQ prevented restoration of phenazine production in P. aeruginosa mutants, and also resulted in loss of anti-biofilm activity displayed by HHQ towards B. subtilis.[37] However, since then, the observations made by Hartmann compel further investigation of the role played by the C-3 position of AHQs.
As previously stated, the P. aeruginosa QS system is known to be dependent on the PqsABCDE biosynthetic operon, which is positively regulated by PqsR. As PqsR controls the expression of PqsA, the monitoring of PqsA promoter activity can be used to determine PqsR agonism and antagonism. Thus all of the C-3 substituted 6-NO2
HHQ analogues were tested using promotor fusion analysis of the PqsR-regulated PqsABCDE operon. Two genotypes of P. aeruginosa were chosen to be tested with the prepared molecules: a wild-type P. aeruginosa (PAO1) which would appropriately reflect the natural biochemistry of the species, and an isogenic PqsA mutant (PAO1pqs−) in which the ability to produce HHQ and PQS has been abolished (Figure 9). Overall, while all of the compounds showed some decrease in PqsR activity, none proved to be as potent as Hartmann’s amide 5, particularly when tested on the wild-type strain PAO1.
Substituted amide compounds 33 and 34 showed minimal inhibition and were less potent than compound 5 (Figure 9). Inhibitory effects decreased with increasing substitution of the amide, indicating that the amide group is not acting solely as a blocking group. Furthermore, this result suggests that the H-bond donor capability of the amide N-H is important. This is in agreement with an extensive study on AHQ receptor interactions by Williams and co-workers,[43] which highlighted the requirement for an -NH2 group at the C-3 position of HHQ derivatives for competitive antagonism of PqsR. They suggested that strengthened electrostatic interactions
31 between the -NH2 group and carbonyl groups in the Leu207 backbone within the receptor pocket results in stronger binding than with native ligands such as PQS.
3 5
33 34
Figure 9 PqsR activation in the presence of 6-NO2 HHQ analogues.[53]
From this study, it is clear that the substituent at C-3 has a more complex role beyond acting as a blocking group to prevent hydroxylation by PqsH. Overall, it appears that antagonistic behaviour seems to be strongly dependent on the ability of the molecule to engage in hydrogen-bonding interactions at the C-3 position.
Experimental