Synthesis and antitubercular activity of novel 4-arylalkyl substituted thio-, oxy-and sulfoxy-quinoline analogues targeting the cytochrome bc 1 complex

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Introduction
Tuberculosis (TB) is an ancient infectious disease caused by Mycobacterium tuberculosis that remains a threat to global health.In 2021, TB killed 1.6 million people and an estimated 10.6 million people contracted this disease worldwide, although there was a significant reduction in reported cases (from 7.1 million to 5.8 million) between 2019 and 2020 due to disruptions caused by the COVID-19 pandemic on TB services including cases notifications [1].Notably, the TB incidence rate increased by 3.6% between 2020 and 2021 inverting the 2% yearly decline observed in the past two decades [1].Most cases were in the WHO regions of South-East Asia (43%) and Africa (25%), and Western Pacific (18%), with lower percentages in Europe (2.3%).Amongst the top 30 high TB burden countries, eight accounted for the total number of cases worldwide, e.g., India, China, Indonesia, the Philippines, Pakistan, Nigeria, Bangladesh and South Africa [1].However, TB is also a healthcare challenge in Europe, where 230,000 new cases with an estimated 20,000 deaths were reported in 2021.Further, an estimated 73,000 new cases of rifampicin-resistant and multidrug-resistant TB (RR/MDR TB) were reported in 2021 [1].This is about 16% of the 450,000 RR/MDR TB cases worldwide in 2021 and Europe has the highest MDR TB rates in the world (SURVEILLANCE REPORT.Tuberculosis surveillance and monitoring in Europe 2018 (europa.eu)).
During the COVID-19 pandemic in 2020, TB was the second leading cause of death by infectious disease after COVID-19, and the annual number of estimated global TB deaths increased by 7.45% for the first time since 2005 from a previous year (i.e., from 1.2 million in 2020 to 1.3 million in 2021) [1].This spike in TB deaths was linked to the restrictions put in place during the COVID-19 pandemic, which hamstrung the services required to fight TB due to lack of funding and focus, distribution of essential treatments and delays in initial diagnosis.Notwithstanding the COVID-19 pandemic, the progress made against the goals laid out in 2014 by the World Health Assembly has been very disappointing, as incident and mortality rates were expected to be reduced by a minimum of 20% and 35% respectively from the initial 2015 rates by 2021.However, both rates fell by only a fraction of the desired goal, with the rate of incident in 2021 falling only by 11% and mortality falling by 9.2% [2].
Q203 has recently completed phase II clinical trials [22] for TB treatment (https://clinicaltrials.gov/ct2/show/NCT03563599) and was found to be effective against MDR and XDR TB by targeting cytochrome bc 1 .Mutations T313I or T313A (e.g., mutation of T313 to isoleucine and alanine, T313I and T313A, respectively) within QcrB were found to be cause of resistance against Q203 [23], whereas a L176P mutation was responsible for the resistance to lansoprazole [13].
The remarkable anti-tubercular activity of previously reported 4substituted oxy-and thio-quinolines, such as 4-((4-(tert-butyl)benzyl) thio)-6-methoxy-2-methylquinoline (1, also referred to as 5a in this study), and their facile synthesis, prompted us to further investigate the mechanism of action of this class of compounds [8,24].Given the structural analogy of 4-thio-quinolines and their oxy-and sulfoxy-analogues with the QOA scaffolds, we postulated, along with other research groups, [8] that 4-substituted quinoline-compounds might exert their anti-tubercular activity by targeting the QcrB unit of the cytochrome bc 1 oxidoreductase complex.
Here, a library of 4-substituted sulfoxide-, oxy-and thio-quinoline analogues was synthesised and evaluated for their ability to inhibit the growth of M. tuberculosis wild-type and T313I mutant strains.The library included both previously reported oxy-and thio-quinolines [8,24] and novel derivatives containing an expanded range of aryl moieties, e.g., pyridine, quinoline, indole and meta-chloro substituted methoxybenzyl rings, attached to the quinoline unit of the final products.Moreover, sulfoxy-and oxy-quinoline derivatives were tested for antitubercular activity for the first time.In general, QcrB mutant strains were resistant to the 4-substituted thioquinoline compounds, which induced depletion of intracellular ATP, thus showing evidence of targeting the cytochrome bc 1 oxidase complex.On the other hand, 4-oxyquinoline and sulfoxy-quinoline derivatives were less active against M. tuberculosis H37Rv compared to the thio-quinoline class.However, two novel derivatives, 4i and 5 g, had greater growth inhibitory properties against the QcrB mutant strains, with the 4-quinolinyl-oxy-quinoline compound 4i showing 6.5-fold higher selectivity for the QcrB T313I mutant strain compared to the wild-type, indicating QcrB as the cellular target.

4-Aryl-substituted-thioquinolines disrupt energy metabolism in M. Tuberculosis
To test our hypothesis that aryl thiol ether quinolines target the M. tuberculosis H37Rv bc 1 complex, we synthesised previously reported anti-tubercular agent 1 (5a) [24].Compound 1 (5a) was evaluated for cytotoxicity in HepG2 cells and growth inhibitory activity against mutant strains expressing QcrB T313I or QcrB M342T .Analogue 1 (5a) was Complex II (coenzyme Q, or CoQ) receives electrons from Complex I, using FADH 2 as the electron source, and passes them onto Complex III, which contains cytochrome bc 1 .Cytochrome bc 1 , which includes the QcrB peptide, undergoes oxidation as the electrons are passed down the chain.For every two electrons transferred, four protons are translocated across the membrane.Complex IV (cytochrome aa 3 ) undergoes the same oxidization and reduction processes, as the electrons continue to be transferred through the chain.The electron transport chain is linked to ATP regeneration from ADP and the translocation of protons throughout the process creates a disparity in proton concentration between the cytoplasm and the intermembrane space.This imbalance creates a proton motive force that causes a flow of protons from the intermembrane space into the cytoplasm via ATP synthase.also tested for its ability to deplete intracellular ATP levels under aerobic conditions in M. tuberculosis (Fig. 3).In this study, we used previously reported resistant strains T313I and M342T [18,20] that were derived from the parental strain M. tuberculosis H37Rv-LP (ATCC 25618) carrying single-nucleotide polymorphisms of the qcrB gene, which encodes a subunit of the menaquinol cytochrome c oxidoreductase (bc 1 ) complex.
Compounds that inhibit cytochrome bc 1 oxidase have previously been shown to deplete intracellular ATP levels in aerobic cells [11].As can be noted in the graph of Fig. 3, there was a clear depletion of ATP in M. tuberculosis cells in response to compound 1 (5a) treatment.
Thioquinoline 1 (5a) was able to reduce ATP levels within 24 h of exposure under aerobic conditions.Q203, a known cytochrome bc 1 inhibitor, was used as the control.
After establishing that 4-substituted thio-quinolines targeted respiratory processes in M. tuberculosis, we set out to prepare a library of compound 1 analogues to gather structural activity relationship data.In addition to novel quinoline-based probes, the library included a number of previously reported derivatives, which were synthesised and screened to gather further information about the ability of variously substituted quinoline-including compounds to target respiratory processes in M. tuberculosis strains.The probes were tested against strains carrying the QcrB mutation at T313I.This is one the most common mutations that causes resistance to inhibitors [21].
Generally, oxyquinoline derivatives exhibited moderate activity against the H37Rv wild-type strain.For example, 4a was found to be the most potent of this series (MIC 90 = 1.7 µM), whereas 4d was > 100-fold less active than its thio-quinoline counterpart 5d (Table S2).
The presence of the sulfoxide group in the quinoline scaffold structure restored the anti-tubercular activity of the compounds.Novel sulfoxide containing compounds 6b, 6d and 6e effectively inhibited the growth of M. tuberculosis H37Rv with IC 90 values ranging between 0.2 and 0.83 µM and IC 50 .valuesranging from 0.043 to 0.26 µM.
IC screening of the compounds against QcrB resistant mutant T313I revealed a shift in activity for selected 4-arylalkyl substituted thio-and oxy-quinolines, thus confirming our hypothesis that this class of compounds targeted mitochondrial respiration enzymes in M. tuberculosis.It was noted that there were differences between IC 50 and IC 90 values in the growth inhibitory curves of QcrB mutants.This can be partially attributed to the fact that the T313I strain grows more slowly than the wild-type M. tuberculosis strain.As a result, IC 50 instead of IC 90 values were mainly used to assess the compounds activity against QcrB mutants.A decrease in growth inhibitory activity against the mutant strain compared to wild-type H37Rv was observed for both thioquinoline, i.e., 5d (42-fold) and 5b (3-fold), and to a lesser extent for oxyquinoline, i.e., 4a (8-fold), 4b (10-fold) and 4d (5.9-fold), analogues (Table S2).The diminished activity of 5b and 5d in the QcrB mutants might be ascribable to reduced interactions between the p-chlorophenyl group of 5d or trifluoromethyl group of 5b with the mutated aminoacid residue at position 313 of the QcrB unit.
Interestingly, quinolin-2-yl-methyl-oxyquinoline 4i and (1H-indol-3-yl)ethyl)-thioquinoline 5 g were the only library members tested that exhibited 1.7-fold and 2.2-fold higher activity against QcrB T313I (IC 50 QcrB T313I = 4.7 and 5.1 µM, respectively) compared to the wild-type strain (IC 50 H37Rv = 7.3 and 11 µM, respectively), thus indicating affinity toward the target within the electron transport chain of M. tuberculosis mitochondria.If the IC 90 values of 4i were used to compare the activity in wild-type vs mutated strains, the shift would be even more pronounced with a 6.5-fold decrease in activity against the wild-type (IC 90 = 47 µM) compared to T313I mutant strain (IC 90 = 7.3 µM).
Selected derivatives were screened for cytotoxicity in HepG2 cells and showed moderate to low toxicity.The new thioquinolines 5i and 5j were the least cytotoxic of the tested compounds with IC 50 values of 33 and 72 μM, respectively, and favourable selectivity index (SI = ratio between GIC 50 HepG2 and the IC 50 H37Rv ) values of 786 and 120, respectively.Novel sulfoxy-quinoline analogue 6b, which contained a trifluoromethyl-phenyl residue, showed an IC 50 HepG2 value of 6.8 μM and had a SI of 49, whereas 6e exhibited an IC 50 value of 0.093 against H37Rv and a SI of 30.

ATP depletion
Measurement of ATP production in mycobacteria can provide a Inhibitory concentrations (ICs) were determined after 5-day incubation period.IC 90 and IC 50 are the compound concentrations that would inhibit the growth of 90% and 50%, respectively, of the tested bacterial isolates.The parental strain is M. tuberculosis H37Rv-LP (ATCC 25618).b IC 50 is the concentration required to reduce HepG2 cell number by 50% and was determined after 3 days of exposure to compounds c SI is the ratio between GIC 50 and the IC 90 H37Rv .ND = Not Determined.d Compounds' structures and antimycobacterial activity previously reported [8,24].

Table 2
Docking scores of selected 4-substituted thio-, oxy-and sulfoxy-quinolines against M. tuberculosis wild type (WT) and mutant (T313I) QcrB protein as targets.The average docking scores obtained by Autodock Vina were calculated from the top scores obtained for representative structures of five most populated clusters in each trajectory.The Plants CHEMPLP docking scores [25] were obtained by rescoring all poses obtained against the second clusters for of QcrB and difference was calculated between corresponding T313I and H37Rv WT poses.additional information on compounds' ability to inhibit aerobic respiration processes (e.g., electron transport chain and ATP synthase).Inhibition of cytochrome bc 1 leads to reduced movement of protons across the membrane, thus resulting in less build-up of proton ions outside of the mitochondrial membrane.A low proton gradient results in a decrease of ATP production, as there are less proton ions passing through the ATP synthase, which in turn cannot catalyse the conversion of ADP to ATP.Compounds 4a, 4b, 4d, 4i and 5a were incubated with M. tuberculosis for 24 h and ATP levels were determined using the BacTiter-Glo assay kit (Promega).Growth was measured by optical density at 590 nm (OD 590 ).All of the tested compounds, including positive control Q203, were able to deplete ATP production in a dosedependent manner at concentrations that did not inhibit M. tuberculosis growth (i.e., lower than IC 50 growth inhibition values) (Fig. 3).Kanamycin, which does not target electron chain transport mechanisms, did not cause ATP depletion.This constitutes further evidence that thioquinoline and oxyquinoline probes mode of action may reside in their ability to target cytochrome bc 1 complex and disrupt electron transfer in the respiratory chain of tubercle bacilli mitochondria.

Molecular modelling
The molecular docking results reported in Table 2 revealed that on average ligands were predicted to bind tighter into the binding pocket of the mutant QcrB protein compared to that of the WT protein.Although the analogues are closer aligned with the inactive conformation of Q203 in the binding site of the QcrB T313I M. tuberculosis mutant, they have different binding modes.It can also be noted that quinolin-2yl-methyl-containing derivatives 4i (oxy-quinoline series), 5i (thioquinoline series) and 6i (sulfoxiquinoline series) were predicted to bind to the T313I mutated protein with higher affinity compared to other library members.These results might in part explain the 6.5-fold higher antitubercular activity of 4i against QcrB T313I compared to the wildtype strain.As can be seen in Fig. 4, 4i has an increased number of interactions with amino acid residues at the QcrB T313I binding site.These include contacts of the 4i oxyquinoline core with mutated isoleucine 313, hydrogen bonding of tyrosine 161 with the oxygen atom of the compound's methyl-oxy-bridge, van der Waals interactions between the phenylalanine 346 benzyl group and the quinolin-2-yl northern aryl moiety of the ligand.The full atomistic molecular dynamics simulation of mutated QcrB -4i complex has confirmed that the key interactions between the ligand and hydrophobic residues (A179, I183 and I313) were preserved throughout the 100 ns trajectory.This suggests that the hydrophobic pocket of T313I mutant (Fig. S1) could be the target site for these novel analogues and will be subject of further studies.

Conclusions
The facility to access the 4-substituted quinoline framework and the remarkable anti-tubercular activity of previously reported derivatives prompted us to further investigate this scaffold by expanding the array of aryl-group positioned at the C-4 position of the quinoline unit and evaluating the ability of thio-, oxy-and sulfoxide-derivatives to inhibit the cytochrome oxidase complex.It was found that the oxyquinoline series had moderate activity against both H37Rv and QcrB T313I mutant strains of M. tuberculosis used in this study, whereas novel sulfoxidebearing derivatives 6b, 6d and 6e, and thioquinolines 5f, 5i inhibited the growth of the Koch bacillus in the 42-260 nM range, with new compound 5i being active at low nanomolar levels (IC 50 H37Rv = 42 nM).
A shift in anti-tubercular activity against QcrB resistant mutant T313I for 4a, 4d, 5a, 5b and 5d confirmed that 4-arylalkyl substituted thio-and oxy-quinolines targeted mitochondrial respiration enzymes in M. tuberculosis.Measurement of depletion of intracellular ATP levels in M. tuberculosis further corroborated these results, as thio-and oxyquinolines induced ATP depletion.
Remarkably, novel quinolin-2-yl-methyl-oxyquinoline 4i showed higher activity against QcrB T313I compared to the wild-type strain.Further, 4i induced depletion of intracellular ATP levels and was predicted to bind to the T313I mutated protein with higher affinity compared to other analogues.Moreover, another novel thioquinoline compound, 5g, was also found to have higher activity against QcrB T313I.This is an interesting finding related to the potential ability of bulky aryl-moieties, i.e., quinolyl-and indolyl moieties, attached to the oxy-and thio-quinoline rings to modulate interactions with QcrB sites.It is also worth considering that bioisosteric exchange between oxygen and sulfur, coupled with insertions of bulky northern aryl unit in the quinoline frames, might maintain activity and interactions with the molecular target in the mutated M. tuberculosis strain.
Taken together, these findings suggests that quinoline derivatives might serve as a scaffold to develop new QcrB targeted probes and improve the level of efficacy previously observed for this class of compounds.Further, the novel quinolinic probes described here can be used as tool compounds to study oxidative phosphorylation processes in mycobacteria.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 1 .
Fig. 1.Schematic representation of the electron transport chain in M. tuberculosis.The chain includes Complexes I -IV and ATP synthase.Complex I (NADH dehydrogenase) oxidizes NADH to NAD+ , allowing protons to cross from the cytoplasm to the intermembrane space.Complex II (coenzyme Q, or CoQ) receives electrons from Complex I, using FADH 2 as the electron source, and passes them onto Complex III, which contains cytochrome bc 1 .Cytochrome bc 1 , which includes the QcrB peptide, undergoes oxidation as the electrons are passed down the chain.For every two electrons transferred, four protons are translocated across the membrane.Complex IV (cytochrome aa 3 ) undergoes the same oxidization and reduction processes, as the electrons continue to be transferred through the chain.The electron transport chain is linked to ATP regeneration from ADP and the translocation of protons throughout the process creates a disparity in proton concentration between the cytoplasm and the intermembrane space.This imbalance creates a proton motive force that causes a flow of protons from the intermembrane space into the cytoplasm via ATP synthase.
R.Murnane et al.

Fig. 4 .
Fig. 4. Overlay of selected active molecules and Q203 in the binding sites of the wt (left) and T313 mutant (right) QcrB.Q203 is shown in a thin stick representation and 5b is represented as a ball and stick, both having carbon atoms coloured in grey.The active analogues are shown in a thick stick representation with carbon atoms coloured as followed: 4i -green carbons; 5d -cyan carbons; 5i -pink carbons and 5b -purple carbons.The 2D protein ligand interactions plots are shown for 4i in the binding sites of the wt (left) and T313 mutant (right) strains.

Table 1
Inhibitory concentrations against wild-type and QcrB T313I M. tuberculosis strains a and HepG2 (IC 50 ) b toxicity of selected new 4-substituted quinoline analogues.