Design, Synthesis and Biological Evaluation of Novel Anthraniloyl-AMP Mimics as PQS Biosynthesis Inhibitors Against Pseudomonas aeruginosa Resistance

The Pseudomonas quinolone system (PQS) is one of the three major interconnected quorum sensing signaling systems in Pseudomonas aeruginosa. The virulence factors PQS and HHQ activate the transcription regulator PqsR (MvfR), which controls several activities in bacteria, including biofilm formation and upregulation of PQS biosynthesis. The enzyme anthraniloyl-CoA synthetase (PqsA) catalyzes the first and critical step in the biosynthesis of quinolones; therefore, it is an attractive target for the development of anti-virulence therapeutics against Pseudomonas resistance. Herein, we report the design and synthesis of novel triazole nucleoside-based anthraniloyl- adenosine monophosphate (AMP) mimics. These analogues had a major impact on the morphology of bacterial biofilms and caused significant reduction in bacterial aggregation and population density. However, the compounds showed only limited inhibition of PQS and did not exhibit any effect on pyocyanin production.

PqsA is an acyl-CoA synthetase enzyme belonging to the ANL superfamily of structurally related adenylating enzymes. PqsA catalyzes the first and key step in the biosynthesis of PQS, which involves transformation of anthranilic acid to anthranilate-CoA. Mechanistically, PqsA first catalyzes the ATP-dependent adenylation of anthranilic acid to form an anthranilate-AMP intermediate, which subsequently reacts with CoA-SH in a thioesterification reaction to yield anthranilate-CoA ( Figure 2). PqsA possesses some attractive features that validates it as a promising target for antimicrobial drug discovery. For example, PqsA-deficient mutant strains show reduced HHQ and PQS biosynthesis and poor biofilm formation in cell culture, as well as highly attenuated virulence in animal infection models. PqsA-deficient mutants also exhibit increased sensitivity towards antimicrobial therapy in vivo. PqsA inhibitors decrease PQS production and reduce mortality in a mice infection model [26]. Importantly, PqsA is exclusively present in bacterial cells, therefore, selective PqsA inhibitors are unlikely to affect host cell viability. Despite its essential role in the biosynthesis of PQS, only few attempts to inhibit the PqsA enzyme have been reported so far ( Figure 3). Halogenated anthranilic acids have been reported as competitive inhibitors of PqsA, however, they showed only weak inhibition, as millimolar concentrations are required to show any effect [23]. Recently, Ji et al. reported sulfonyl adenosine-based small molecule inhibitors mimicking the anthraniloyl-AMP intermediate for the PqsA is an acyl-CoA synthetase enzyme belonging to the ANL superfamily of structurally related adenylating enzymes. PqsA catalyzes the first and key step in the biosynthesis of PQS, which involves transformation of anthranilic acid to anthranilate-CoA. Mechanistically, PqsA first catalyzes the ATP-dependent adenylation of anthranilic acid to form an anthranilate-AMP intermediate, which subsequently reacts with CoA-SH in a thioesterification reaction to yield anthranilate-CoA ( Figure 2). PqsA possesses some attractive features that validates it as a promising target for antimicrobial drug discovery. For example, PqsA-deficient mutant strains show reduced HHQ and PQS biosynthesis and poor biofilm formation in cell culture, as well as highly attenuated virulence in animal infection models. PqsA-deficient mutants also exhibit increased sensitivity towards antimicrobial therapy in vivo. PqsA inhibitors decrease PQS production and reduce mortality in a mice infection model [26]. Importantly, PqsA is exclusively present in bacterial cells, therefore, selective PqsA inhibitors are unlikely to affect host cell viability. PqsA is an acyl-CoA synthetase enzyme belonging to the ANL superfamily of structurally related adenylating enzymes. PqsA catalyzes the first and key step in the biosynthesis of PQS, which involves transformation of anthranilic acid to anthranilate-CoA. Mechanistically, PqsA first catalyzes the ATP-dependent adenylation of anthranilic acid to form an anthranilate-AMP intermediate, which subsequently reacts with CoA-SH in a thioesterification reaction to yield anthranilate-CoA ( Figure 2). PqsA possesses some attractive features that validates it as a promising target for antimicrobial drug discovery. For example, PqsA-deficient mutant strains show reduced HHQ and PQS biosynthesis and poor biofilm formation in cell culture, as well as highly attenuated virulence in animal infection models. PqsA-deficient mutants also exhibit increased sensitivity towards antimicrobial therapy in vivo. PqsA inhibitors decrease PQS production and reduce mortality in a mice infection model [26]. Importantly, PqsA is exclusively present in bacterial cells, therefore, selective PqsA inhibitors are unlikely to affect host cell viability. Despite its essential role in the biosynthesis of PQS, only few attempts to inhibit the PqsA enzyme have been reported so far ( Figure 3). Halogenated anthranilic acids have been reported as competitive inhibitors of PqsA, however, they showed only weak inhibition, as millimolar concentrations are required to show any effect [23]. Recently, Ji et al. reported sulfonyl adenosine-based small molecule inhibitors mimicking the anthraniloyl-AMP intermediate for the While these inhibitors showed high binding affinity (K i < 0.2 µM) for the PqsA enzyme, they did not exhibit satisfactory antibacterial potency in vitro (IC 50 > 300 µM). Compound accumulation studies showed poor bacterial cell permeability, which possibly limited their cellular activity [26]. Recently, the crystal structures of PqsA complexed with the ligand anthraniloyl-AMP with the inhibitor 6-fluoroanthraniloyl-AMP (6-FABA-AMP) were reported by Witzgall et al. [27]. The structures revealed that the N-terminal domain of PqsA contains the active site that recognizes and binds the natural ligand anthraniloyl-AMP as well as the competitor inhibitor, 6-FABA-AMP.
Compound accumulation studies showed poor bacterial cell permeability, which possibly limited their cellular activity [26]. Recently, the crystal structures of PqsA complexed with the ligand anthraniloyl-AMP with the inhibitor 6-fluoroanthraniloyl-AMP (6-FABA-AMP) were reported by Witzgall et al. [27]. The structures revealed that the N-terminal domain of PqsA contains the active site that recognizes and binds the natural ligand anthraniloyl-AMP as well as the competitor inhibitor, 6-FABA-AMP.

Design and Synthesis of Novel PqsA Inhibitors
Previously reported PqsA inhibitors based on anthranilate-AMP have shown high binding affinity and promising activity in enzymatic assays. However, due to their highly polar nature, these inhibitors cannot penetrate the bacterial cell membrane, and are therefore, devoid of any cellular activity in P. aeruginosa, which limits their further development as novel antimicrobial agents.
Nucleotide analogues can be generated by modifications of the phosphate backbone, ribose sugar or base [28]. To develop nonpolar nucleotide analogues of anthraniloyl-AMP, we first used the neutral triazole linker as an isostere for the charged phosphodiester linkage. One advantage of using triazole is that it is metabolically more stable than the phosphodiester linkage, because of its resistance to degradation by nuclease. The second modification was to replace the adenine base of anthraniloyl-AMP with pyrimidine bases, such as thymidine, 2′-deoxyuridine and 2′-deoxycytidine [29]. Finally, the ribose sugar of the natural ligand was replaced with 2′-deoxyribose ( Figure 4).

Design and Synthesis of Novel PqsA Inhibitors
Previously reported PqsA inhibitors based on anthranilate-AMP have shown high binding affinity and promising activity in enzymatic assays. However, due to their highly polar nature, these inhibitors cannot penetrate the bacterial cell membrane, and are therefore, devoid of any cellular activity in P. aeruginosa, which limits their further development as novel antimicrobial agents.
Nucleotide analogues can be generated by modifications of the phosphate backbone, ribose sugar or base [28]. To develop nonpolar nucleotide analogues of anthraniloyl-AMP, we first used the neutral triazole linker as an isostere for the charged phosphodiester linkage. One advantage of using triazole is that it is metabolically more stable than the phosphodiester linkage, because of its resistance to degradation by nuclease. The second modification was to replace the adenine base of anthraniloyl-AMP with pyrimidine bases, such as thymidine, 2 -deoxyuridine and 2 -deoxycytidine [29]. Finally, the ribose sugar of the natural ligand was replaced with 2 -deoxyribose ( Figure 4).
The synthetic route for the synthesis of the novel triazole-linked anthranilamide deoxynucleoside compounds began with the ring-opening of 5-substituted isatoic anhydrides (1a-e) using propargylamine (2) under mild reaction conditions. Different terminal alkyne-containing anthranilamide intermediate compounds (3a-e) were synthesized in excellent yields (Scheme 1).
Next, we synthesized nucleoside azides following a reported protocol [30]. Selective tosylation of the primary hydroxy group of thymidine nucleoside (4) using pyridine and tosyl chloride at 0 • C generated tosylate (5), which was further converted to the corresponding azide (6) by a nucleophilic substitution reaction with sodium azide in DMF at 70 • C (Scheme 2).
Furthermore, 2 -deoxyuridine (8) was converted to the corresponding triazole nucleoside analogues (9a-c) and (9e) by using the same three-step reaction sequence in moderate to good isolated yields (Scheme 4). The synthetic route for the synthesis of the novel triazole-linked anthranilamide deoxynucleoside compounds began with the ring-opening of 5-substituted isatoic anhydrides (1a-e) using propargylamine (2) under mild reaction conditions. Different terminal alkyne-containing anthranilamide intermediate compounds (3a-e) were synthesized in excellent yields (Scheme 1).
Next, we synthesized nucleoside azides following a reported protocol [30]. Selective tosylation of the primary hydroxy group of thymidine nucleoside (4) using pyridine and tosyl chloride at 0 °C generated tosylate (5), which was further converted to the corresponding azide (6) by a nucleophilic substitution reaction with sodium azide in DMF at 70 °C (Scheme 2).   The synthetic route for the synthesis of the novel triazole-linked anthranilamide deoxynucleoside compounds began with the ring-opening of 5-substituted isatoic anhydrides (1a-e) using propargylamine (2) under mild reaction conditions. Different terminal alkyne-containing anthranilamide intermediate compounds (3a-e) were synthesized in excellent yields (Scheme 1).
Next, we synthesized nucleoside azides following a reported protocol [30]. Selective tosylation of the primary hydroxy group of thymidine nucleoside (4) using pyridine and tosyl chloride at 0 °C generated tosylate (5), which was further converted to the corresponding azide (6) by a nucleophilic substitution reaction with sodium azide in DMF at 70 °C (Scheme 2).  The synthetic route for the synthesis of the novel triazole-linked anthranilamide deoxynucleoside compounds began with the ring-opening of 5-substituted isatoic anhydrides (1a-e) using propargylamine (2) under mild reaction conditions. Different terminal alkyne-containing anthranilamide intermediate compounds (3a-e) were synthesized in excellent yields (Scheme 1).
Next, we synthesized nucleoside azides following a reported protocol [30]. Selective tosylation of the primary hydroxy group of thymidine nucleoside (4) using pyridine and tosyl chloride at 0 °C generated tosylate (5), which was further converted to the corresponding azide (6) by a nucleophilic substitution reaction with sodium azide in DMF at 70 °C (Scheme 2). Scheme 2. Synthesis of thymidine azide intermediate 6.
Molecules 2020, 25, x FOR PEER REVIEW 6 of 17 The final step involved an azide-alkyne cycloaddition (click chemistry) reaction between alkynes (3a-e) and thymidine azide (6). The reaction was performed in the presence of CuSO4 catalyst and sodium ascorbate as reducing agents in t-BuOH:H2O (1:1) medium [31]. The desired triazolo nucleosides (7a-e) were synthesized between 59-74% yields (Scheme 3). Furthermore, 2′-deoxyuridine (8) was converted to the corresponding triazole nucleoside analogues (9a-c) and (9e) by using the same three-step reaction sequence in moderate to good isolated yields (Scheme 4). Furthermore, 2′-deoxyuridine (8) was converted to the corresponding triazole nucleoside analogues (9a-c) and (9e) by using the same three-step reaction sequence in moderate to good isolated yields (Scheme 4). In the case of deoxycytidine (10), selective tosylation of the primary alcohol did not give the desired product, most likely due to presence of the free amino group. We attempted to protect the amino group via acetyl and trifluoroacetyl protecting groups, however, both were found to be unstable and did not survive the subsequent tosylation reaction. After conducting a literature search, we found that a benzoyl group could be used for the protection of the amino group of deoxycytidine [32]. Therefore, selective benzoyl protection of deoxycytidine (10) was achieved by using TMSCl and benzoyl chloride in pyridine at 0 °C, giving N-benzoylated deoxycytidine (11) in excellent yields (Scheme 5). Next, compound (11) was converted to the benzoyl-protected deoxycytidine triazole analogues (12a-e) following the same reaction pathway as above. Finally, the benzoyl group was deprotected by mild hydrolysis using aqueous NH3(28%):MeOH (1:1) at room temperature to give the desired deoxycytidine analogues (13a-e) in excellent yields, from 81-92%. In the case of deoxycytidine (10), selective tosylation of the primary alcohol did not give the desired product, most likely due to presence of the free amino group. We attempted to protect the amino group via acetyl and trifluoroacetyl protecting groups, however, both were found to be unstable and did not survive the subsequent tosylation reaction. After conducting a literature search, we found that a benzoyl group could be used for the protection of the amino group of deoxycytidine [32]. Therefore, selective benzoyl protection of deoxycytidine (10) was achieved by using TMSCl and benzoyl chloride in pyridine at 0 • C, giving N-benzoylated deoxycytidine (11) in excellent yields (Scheme 5). Next, compound (11) was converted to the benzoyl-protected deoxycytidine triazole analogues (12a-e) following the same reaction pathway as above. Finally, the benzoyl group was deprotected by mild hydrolysis using aqueous NH 3 (28%):MeOH (1:1) at room temperature to give the desired deoxycytidine analogues (13a-e) in excellent yields, from 81-92%. 2D NMR experiments including COSY, HSQC and HMBC were performed to confirm the regioselectivity of the compounds, particularly the connectivity between the anthranilamide unit and the pyrimidine nucleoside via the 1,4-disubstituted triazole ring. In the COSY spectrum of compound 13a, the CH2 protons at C-17 (5′) of the sugar showed correlation with the proton at C-18 (4′) of the sugar ( Figure 5). Meanwhile, in the HMBC spectrum of 13a, the C-17 protons showed correlation with C-18 and C-19 (3′) carbons of the sugar, and with C-13 of the triazole ring. Similarly, the CH2 protons at C-11 showed a COSY correlation with the N-H proton of the anthranilamide and HMBC correlations with C-1, C-12, and C-13 carbons ( Figure 5). 2D NMR experiments including COSY, HSQC and HMBC were performed to confirm the regioselectivity of the compounds, particularly the connectivity between the anthranilamide unit and the pyrimidine nucleoside via the 1,4-disubstituted triazole ring. In the COSY spectrum of compound 13a, the CH 2 protons at C-17 (5 ) of the sugar showed correlation with the proton at C-18 (4 ) of the sugar ( Figure 5). Meanwhile, in the HMBC spectrum of 13a, the C-17 protons showed correlation with C-18 and C-19 (3 ) carbons of the sugar, and with C-13 of the triazole ring. Similarly, the CH 2 protons at C-11 showed a COSY correlation with the N-H proton of the anthranilamide and HMBC correlations with C-1, C-12, and C-13 carbons ( Figure 5). and the pyrimidine nucleoside via the 1,4-disubstituted triazole ring. In the COSY spectrum of compound 13a, the CH2 protons at C-17 (5′) of the sugar showed correlation with the proton at C-18 (4′) of the sugar ( Figure 5). Meanwhile, in the HMBC spectrum of 13a, the C-17 protons showed correlation with C-18 and C-19 (3′) carbons of the sugar, and with C-13 of the triazole ring. Similarly, the CH2 protons at C-11 showed a COSY correlation with the N-H proton of the anthranilamide and HMBC correlations with C-1, C-12, and C-13 carbons ( Figure 5).

Biological Activity
For PQS inhibition assay, all the new triazole nucleotide analogues were screened for their ability to inhibit the pqs QS system using the PAO1 (a P. aeruginosa reporter strain) pqsA:gfp reporter assay. This assay measures the PqsR (MvfR) regulated expression of the pqsABCDE operon. It is expected that the PqsA inhibitors will block the biosynthesis of PQS and HHQ, which leads to lower activation of the PqsR (MvfR) receptor and ultimately, reduction in the expression of the pqsABCDE reporter. The expression of the reporter in the untreated control reached its maximum level after 6-8 h which then fell to basal expression levels at later time points. A known inhibitor of PQS was employed as a positive control which prevented this expression [33]. Preliminary assays revealed that concentrations of DMSO above 1.25% inhibited GFP expression of the reporter, even without the presence of any of the test compounds, although growth was unaffected. Therefore, the final

Biological Activity
For PQS inhibition assay, all the new triazole nucleotide analogues were screened for their ability to inhibit the pqs QS system using the PAO1 (a P. aeruginosa reporter strain) pqsA:gfp reporter assay. This assay measures the PqsR (MvfR) regulated expression of the pqsABCDE operon. It is expected that the PqsA inhibitors will block the biosynthesis of PQS and HHQ, which leads to lower activation of the PqsR (MvfR) receptor and ultimately, reduction in the expression of the pqsABCDE reporter. The expression of the reporter in the untreated control reached its maximum level after 6-8 h which then fell to basal expression levels at later time points. A known inhibitor of PQS was employed as a positive control which prevented this expression [33]. Preliminary assays revealed that concentrations of DMSO above 1.25% inhibited GFP expression of the reporter, even without the presence of any of the test compounds, although growth was unaffected. Therefore, the final DMSO concentration was kept at 1.25%. Based on the concentration of our compound stock solutions, this corresponded to 125 µg/mL as the highest concentration tested for each compound. Then, two-fold lower serial dilutions of the compound were tested whilst keeping the DMSO concentration constant. All compounds except one showed no or very slight inhibition of the pqsA reporter at all concentrations tested. Only 13e at 125 µg/mL (400 µM approx.) showed approximately 30% inhibition of the reporter when compared to the negative control ( Figure 6, Table 1). The LogP value of the compound plays a critical role in bacterial cell penetration and the cLogP value of compound 13e (−0.55) is highest among cytidine triazoles, which indicates that the compound 13e could be more permeable to bacterial cells compared to all other derivatives. Then, two-fold lower serial dilutions of the compound were tested whilst keeping the DMSO concentration constant. All compounds except one showed no or very slight inhibition of the pqsA reporter at all concentrations tested. Only 13e at 125 μg/mL (400 μM approx.) showed approximately 30% inhibition of the reporter when compared to the negative control ( Figure 6, Table 1). The LogP value of the compound plays a critical role in bacterial cell penetration and the cLogP value of compound 13e (−0.55) is highest among cytidine triazoles, which indicates that the compound 13e could be more permeable to bacterial cells compared to all other derivatives.  No inhibition - Figure 6. PQS inhibition by compound 13e using PAO1 pqsA:gfp reporter-based assay. For inhibition of biofilm formation, some of the representative compounds (7a, 7e, 9a, 9e, 13d and 13e) were tested for their potential ability to inhibit P. aeruginosa biofilm formation. PAO1 planktonic cultures were incubated with 400 µM of compounds and added to microtiter wells to initiate bacterial adhesion and biofilm growth. After 24 h, biofilms were visualized using phase contrast microscopy [34]. The compounds exerted a major impact on the morphology of bacterial biofilms. A drastic reduction in bacterial aggregation and population density was observed in the presence of compounds 13e and 13d as compared to both untreated and DMSO controls (Figure 7). Since the PQS system mediates bacterial aggregation and biofilm formation in P. aeruginosa, this result could indicate that the compounds interfere with PQS biosynthesis to reduce bacterial aggregation. However, given the weak PQS inhibitory activity of the compounds in the in vitro reporter assay, we do not exclude the possibility that the compounds might be acting through alternative pathways to inhibit biofilm formation. For the pyocyanin inhibition assay, to investigate the activity of these compounds against bacterial virulence factors, some of the derivatives (7a, 7e, 9a, 9e, 13d and 13e) were evaluated for their ability to inhibit the production of pyocyanin from P. aeruginosa PA01 at 400 μM concentration. However, out of six compounds tested, none of them showed any significant decrease in the production of pyocyanin. The findings in this study match with previously published data showing 20 µm For the pyocyanin inhibition assay, to investigate the activity of these compounds against bacterial virulence factors, some of the derivatives (7a, 7e, 9a, 9e, 13d and 13e) were evaluated for their ability to inhibit the production of pyocyanin from P. aeruginosa PA01 at 400 µM concentration. However, out of six compounds tested, none of them showed any significant decrease in the production of pyocyanin. The findings in this study match with previously published data showing compounds were effective in inhibiting PQS biosynthesis without any impact on pyocyanin production [26]. This is an indication of the complex relationship between the PQS biosynthesis and pyocyanin production.
Regarding effect on growth, none of the compounds had any effect on growth at concentrations up to 256 µg/mL except for 7a. For 7a, even the 32 µg/mL concentration was enough to affect growth and increase the lag phase. For all compounds, growth was inhibited at 512 µg/mL, although this could be due to the amount of DMSO present, since 5.12% DMSO alone affected growth (for details, see Supporting Information).

Synthesis
All the reagents and solvents were purchased from commercial sources (Combi-Blocks, Oakwood Chemicals, Sigma-Aldrich, Sydney, Australia) and used without further purification. Reactions were performed using oven-dried glassware under an atmosphere of nitrogen (if required). Room temperature refers to the ambient temperature (25 • C). Yields refer to compounds isolated after flash column chromatography unless otherwise stated. Progress of the reactions were monitored by thin layer chromatography (TLC) precoated with Merck silica gel 60 F254 plates and visualization using UV light (254 nm). Flash column chromatography was carried out using Grace Davison LC60A 40-63 µm silica gel as the stationary phase and solvent gradient (methanol in ethyl acetate) used as the mobile phase. High-resolution mass spectrometry (HRMS) was performed at Bioanalytical Mass Spectrometry Facility, UNSW by electrospray (ESI) ionization using a Thermo LTQ Orbitrap XL instrument (Thermo Scientific, Waltham, MA, USA). 1 H-and 13 C-NMR was recorded in deuterated solvent (DMSO-d6) using Bruker Avance 300, 400 or 600 MHz instruments (Bruker Pty Ltd., Preston, NSW, Australia) at 24 • C. Chemical shifts (δ) are reported as relative to the corresponding solvent peak, with tetramethylsilane as the internal standard and quoted in parts per million (ppm). Multiplicities in the NMR spectra are described as: s-singlet; d-doublet; t-triplet; q-quartet; m-multiplet; b-broad; coupling constants (J) are reported in hertz (Hz).

Conclusions
Inhibition of the PQS quorum sensing system is an attractive target for the development of alternative therapies against multidrug-resistant P. aeruginosa. Anthraniloyl-AMP mimics are competitive antagonists of the enzyme PqsA, and block production of virulence factors PQS and HHQ without affecting bacterial growth. In this study, we have designed and synthesized 14 novel anthraniloyl-AMP mimics containing a triazole linker as potential inhibitors of Pseudomonas quinolone biosynthesis. However, most of these analogues did not show PQS inhibitory activity against a P. aeruginosa reporter chain except for deoxycytidine analogue (13e), which inhibited PQS activity by 30% at 125 µM, possibly due to limited cell penetration. Interestingly, these compounds induced significant morphological changes in biofilms as observed by microscopy, suggesting that they could inhibit PQS or other signaling systems responsible for bacterial aggregation. Our future efforts will focus on modifying these analogues to increase bacterial cell penetration.