Design, Synthesis, and Antimicrobial Activity of Novel Cyt-bd Inhibitors Targeting Energy Compensation in Mycobacterium tuberculosis

Abstract

Drug-resistant Mycobacterium tuberculosis (Mtb) poses a growing threat, necessitating new therapeutic strategies. The cytochrome bcc-aa ₃ oxidase (Cyt-bcc) is an attractive target, yet single-agent inhibition fails owing to compensation by the alternative cytochrome bd terminal oxidase (Cyt-bd). Herein, we describe a structure–activity relationship (SAR) study starting from the previously identified Cyt-bd inhibitor 12, leading to the discovery of 17aa (YH-685). YH-685 potently inhibits Cyt-bcc deficient Mycobacterium smegmatis (Δbcc) with a MIC of 0.8 μM and suppresses Cyt-bd dependent oxygen consumption in inverted membrane vesicles with an IC₅₀ of 0.45 μM. When combined with the Cyt-bcc inhibitor TB47, YH-685 enhanced inhibition of virulent Mtb H37Rv, reducing both planktonic growth and intracellular bacterial burdens in human macrophages, as assessed by high-content imaging. Under the tested in vitro conditions, this combination achieved growth inhibition comparable to standard regimens rifampicin–isoniazid and bedaquiline. Our study supports co-targeting Cyt-bd and Cyt-bcc as a therapeutic strategy for drug-resistant tuberculosis.

Tuberculosis (TB), a highly contagious infectious disease caused by Mycobacterium tuberculosis (Mtb), remains a major global health threat. Each year, over 10 million people develop active TB, and the incidence of multidrug-resistant TB (MDR-TB) continues to increase. Despite recent advances in MDR-TB treatment, drug resistance has reached alarming levels in several regions. − Current individualized MDR-TB regimens still require prolonged, toxic multidrug therapies lasting up to two years, achieving cure rates of only 40–60%. , The escalating challenge of drug-resistant TB underscores the urgent need for novel therapeutic strategies that can overcome resistance mechanisms and shorten the treatment duration.

In recent decades, bedaquiline (1) has received FDA approval, while pretomanid (2) is approved only as part of specified combination regimens, including the bedaquiline-pretomanid-linezolid regimen for highly drug-resistant or treatment-intolerant tuberculosis. Delamanid (3) has been authorized in other regions such as the European Union (Figure ). The discovery of these drugs highlights the importance of innovative approaches and new targets in antituberculosis drug research. While conventional anti-TB drugs primarily target cell wall synthesis, bedaquiline uniquely targets the F₁F₀-ATP synthase, disrupting Mtb energy metabolism by blocking the electron transport chain. , This has established respiratory chain inhibition as a promising strategy for inhibition of Mtb. Beyond the ATP synthase, the cytochrome oxidase system, comprising the cytochrome bcc-aa3 complex (Cyt-bcc) and cytochrome bd (Cyt-bd) oxidase, plays a crucial role in Mtb respiration and ATP production (Figure ). − The Cyt-bcc inhibitor Q203 (telacebec) , has completed a phase IIa study, whereas TB47 (TBA) is undergoing phase I evaluation in China (CTR20262349).

1.

Chemical structures of the newly approved anti-TB drugs.

2.

Schematic of the Mtb electron transport chain and energy compensation pathways. Under hypoxic or Cyt-bcc inhibited conditions, Cyt-bd serves as a compensatory terminal oxidase.

However, studies have demonstrated that the Cyt-bd oxidase serves as a compensatory respiratory bypass, sustaining bacterial viability even when Cyt-bcc is inhibited (Figure ). ,− This redundancy allows Mtb to persist under hypoxic and other stress conditions, wherein Cyt-bd plays a critical role in Mtb survival. Notably, simultaneous inhibition or genetic disruption of both Cyt-bcc and Cyt-bd completely collapses energy metabolism and leads to rapid mycobacterial death. ,, These findings highlight that the combined targeting of Cyt-bcc and Cyt-bd offers a compelling strategy for respiratory chain disruption in Mtb and warrants further investigation as a potential strategy for drug-resistant tuberculosis.

Several Cyt-bd inhibitor chemotypes have been reported (Figure ). Aurachin D (4) inhibits Mtb Cyt-bd in IMV assays, although its cell activity varies among experimental systems. , ND-011992 (5) and related analogues show potent activity against Mtb, with evidence of Cyt-bd selectivity. , Additional chemotypes include HM2-16F (6), CK-2-63 (7), thieno­[3,2-d]­pyrimidin-4-amine (8), benzothiazole amides (9), ETX1975-3 (10), and pyrroloquinolone inhibitor (11). In the previous work, compound 12 was identified as a moderate inhibitor of the growth of a Cyt-bcc-deficient mycobacterial strain.

3.

Chemical structures of reported Cyt-bd inhibitors.

In this study, systematic optimization of compound 12 focused on three structural regions: the benzyl substituent (17a–17k), the side-chain linker (17l–17o), and the quinolinone core (17p–17aa) (Figure ). These efforts led to the identification of 17aa (YH-685), which inhibited Cyt-bcc-deficient M. smegmatis with an MIC of 0.8 μM and enhanced TB47-mediated inhibition of planktonic and intracellular Mtb.

4.

Optimization of Cyt-bd inhibitors based on the lead compound 12.

The synthesis of compounds 17a–17k, 17m, 17p–17aa is described in Scheme . Briefly, nucleophilic substitution of various commercially available benzyl bromides 13a–13k, 13m, 13p–13aa with ethyl acetoacetate (14) gave the intermediates 15a–15k, 15m, 15p–15aa. These intermediates were then converted into 4-hydroxyquinoline derivatives 16a–16k, 16m through the Conrad–Limpach reaction. Similarly, intermediates 15p–15aa were cyclized with various anilines under the same conditions to afford compounds 16p–16aa. Subsequent steps involved protecting the hydroxyl groups with propionyl chloride, oxidizing with m-CPBA, and finally removing the protecting groups to yield target compounds 17a–17aa. Additionally, compound 17h was demethylated to yield compound 17i.

1. Synthetic Route of Compounds 17a–17aa .

^a Reagents and conditions: (a) NaH, dry THF, 0°C, 1h, 72–89%; (b) (i) aniline, p-TsOH, benzene, 120 °C, overnight; (ii) Ph₂O, 230 °C, 2 h, 10–36% (two steps); (c) (i) propyl chloroformate, NaH, dry THF, rt, 3 h; (ii) m-CPBA, DCM, rt, 3 h; (iii) 5M KOH, EtOH, rt, 1 h, 55–80% (three steps).

The synthesis of compounds 17l, 17n, and 17o is described in Scheme . Bromination of compound 18 using N-bromosuccinimide (NBS) afforded 19, which was then treated with phosphorus oxychloride to generate intermediate 22a. Further Suzuki coupling between 22a and 4,4,5,5-tetramethyl-2-(4-(trifluoromethoxy)­phenyl)-1,3,2-dioxaborolane produced intermediate 23a. Subsequent oxidation with m-CPBA, followed by hydrolysis in acetic acid, furnished target compound 17l. For compound 17n, commercially available 22b was first converted to intermediate 23b via an amination reaction. Using similar oxidation and hydrolysis conditions of 23a as above, 23b was transformed into the title compound 17n. The synthesis of 17o commenced with the cyclization of commercially available compounds 20 and 21 to afford the intermediate 22c. A subsequent condensation reaction converted 22c to the quinoline derivative 23c. Finally, 23c underwent hydroxyl protection, oxidation, and hydrolysis to deliver target compound 17o.

2. Synthesis Route of Compounds 17l, 17n, and 17o .

^a Reagents and conditions: (a) NBS, AcOH, 60°C, 15 min, 67%; (b) POCl₃, 120°C, 4 h, 62%; (c) (i) Ph₂O, 230 °C, 2 h; (ii) NaOH, EtOH, H₂O, 70°C, 4 h, 25% (two steps); (d) 4,4,5,5-tetramethyl-2-(4-(trifluoromethoxy)­phenyl)-1,3,2-dioxaborolane, Pd­(dppf)₂Cl₂, K₂CO₃, dioxane, 100°C, overnight, 52% for 23a; 4-(trifluoromethoxy)­aniline, Ph₂O, 230 °C, 2 h, 64% for 23b; HATU, Et₃N, DMF, 80°C, overnight, 42% for 23c. (e) (i) m-CPBA, DCM, rt, 3 h; (ii) AcOH, H₂O, 100°C, 3 d, 10% (two steps) for 17l; (i) propyl chloroformate, NaH, dry THF, rt, 3 h; (ii) m-CPBA, DCM, rt, 3 h; (iii) 5M KOH, EtOH, rt, 1 h, 72% (three steps) for 17n and 17o.

Compounds 17a–17o were evaluated against Cyt-bcc-deficient M. smegmatis ΔqcrCAB (Δbcc), as a corresponding Mtb strain was unavailable. Deletion of qcrCAB removes the Cyt-bcc-aa3 branch, thereby redirecting respiratory electron flow through Cyt-bd and providing a genetically sensitized surrogate for evaluating activity under Cyt-bd-dependent conditions. Species-specific differences in compound uptake and metabolism, however, should be considered when extrapolating these results to Mtb. Initial SAR studies focused on systematic substitutions at the para-positions of the benzyl moiety. Introduction of small alkyl substituents (17a, 17b) resulted in moderate antimycobacterial activity against the Δbcc strain, with minimum inhibitory concentrations (MICs) of 50 μM for both compounds (Table ). In contrast, the tert-butyl-substituted analogue 17c showed no measurable inhibition at the highest concentration tested (MIC > 200 μM), whereas analogues bearing smaller para- substituents retained measurable activity.

1. Inhibitory Activity of Compounds with Benzyl Side Chain Substitutions against the Cyt-bcc Deficient M. smegmatis .

^aCyt-bcc deficient M. smegmatis (Δbcc).

We next examined additional para-substituted benzyl analogues. Among electron-withdrawing groups, the para-trifluoromethyl analogue 17f demonstrated the highest potency, with an MIC value of 25 μM against the Δbcc strain, whereas the cyano (17e) and fluoro (17g) analogues retained moderate activity (MIC = 50 μM). The para-nitro analogue 17d and the hydroxy analogue 17i both had MIC values of >200 μM, whereas the methoxy analogue 17h retained moderate activity (MIC = 50 μM).

To evaluate positional effects on the benzyl group, selected substituents were relocated from the para- to the meta-position. The meta-methyl analogue 17j exhibited equipotent activity to the lead compound 12 (MIC = 12.5 μM), whereas the meta-nitro analogue 17k displayed only weak activity (MIC = 100 μM). Although 17j matched the potency of compound 12, its inferior aqueous solubility prompted us to retain the para-trifluoromethoxy substituent for further optimization. Subsequent SAR efforts targeted the benzyl side-chain linker (compounds 17l–17o). All linker-modified analogues 17l–17o had MICs of >200 μM, indicating that the original linker was required for activity.

With the trifluoromethoxybenzyl group identified as the optimal substituent, we next examined the influence of modifications on the 1-hydroxy-2-methylquinolin-4­(1H)-one core, leading to the design and synthesis of compounds 17p–17aa (Table ). Fluorine substitution at different positions on the quinolinone scaffold had a pronounced impact on antimicrobial activity. Notably, the 6-fluoro analogue 17q was the most potent (MIC = 6.25 μM), representing a two-fold improvement over lead compound 12, while the 7-fluoro analogue 17r retained comparable activity (MIC = 12.5 μM). In contrast, the 5- and 8-fluoro analogues 17p and 17s both had MIC values of >200 μM, indicating a marked positional effect within this series.

2. MIC of Compounds 17p–17aa against Cyt-bcc Deficient M. smegmatis.

Compds	R2	Δbcc MIC (μM)	HepG2 IC50 (μM)
17p	5-F	>200	NT
17q	6-F	6.25	87.3
17r	7-F	12.5	97
17s	8-F	>200	NT
17t	6-OCH3	>200	NT
17u	6-CH3	>200	NT
17v	6-Br	>200	NT
17w	6-NO2	6.25	>100
17x	6-F,7-Cl	0.8	91.1
17y	6-F,5-Cl	>200	NT
17z	6-F,7- morpholine	>200	NT
17aa	6-F,7-F	0.8	61.7
12		12.5	>100
4		6.25	NT

^aCyt-bcc deficient M. smegmatis (Δbcc).

^bNT: not tested.

At the 6-position, only the nitro analogue 17w retained activity (MIC = 6.25 μM), while analogues 17t–17v had MIC values of >200 μM. The 6,7-disubstituted analogues 17x and 17aa were the most potent compounds in the series (MIC = 0.8 μM), whereas 17y and 17z had MIC values of >200 μM. These results identify 6,7-disubstitution as the preferred substitution pattern within this series.

To assess mammalian-cell cytotoxicity, selected compounds were evaluated in HepG2 cells (Table ). The six compounds tested showed IC₅₀ values ranging from 61.7 to >100 μM. YH-685 (17aa) and compound 17x, the two most active compounds against the ΔqcrCAB surrogate strain (MIC = 0.8 μM), showed HepG2 IC₅₀ values of 61.7 and 91.1 μM, respectively. Within this limited compound set, no consistent relationship was observed between antibacterial potency and HepG2 cytotoxicity. Broader mammalian-cell profiling, including testing under galactose conditions, is needed to assess potential mitochondrial toxicity.

Molecular oxygen is the terminal electron acceptor in the mycobacterial respiratory chain, and oxygen consumption rate (OCR) therefore provides a functional readout of terminal respiratory activity. To assess the effects of selected compounds on Cyt-bd-dependent respiration, we measured OCR using inverted membrane vesicles (IMVs) prepared from the Δbcc strain overexpressing Mtb Cyt-bd, as previously described (Figure ). , At 10 μM, compound 17aa (YH-685) inhibited OCR to an extent similar to compound 12, whereas 17x was less active. Dose–response analysis gave OCR IC₅₀ values of 0.45 μM for YH-685 and 7.2 μM for 17x. Thus, although the two compounds had the same ΔqcrCAB MIC, YH-685 was more active in the OCR assay. TB47 did not inhibit OCR, consistent with the absence of the Cyt-bcc-aa3 branch. The OCR IC₅₀ is a functional potency value rather than a binding-affinity measurement, and the assay does not exclude effects on endogenous M. smegmatis Cyt-bd, upstream electron transfer, or membrane-associated respiratory processes.

5.

Effects of selected compounds on OCR in M. smegmatis ΔqcrCAB IMVs overexpressing Mtb Cyt-bd. YH-685 and 17x were selected because they were the two most active compounds in the ΔqcrCAB whole-cell assay (MIC = 0.8 μM). Compound 12, previously reported as 8d, served as an internal series reference; DMSO was the vehicle control. TB47 served as a branch-negative comparator because the Cyt-bcc-aa3 branch is absent in the ΔqcrCAB IMVs. (A) OCR after treatment with DMSO or 10 μM TB47, compound 12, 17x, or YH-685. (B) Concentration–response curves for 17x and YH-685.

The inhibitory effects of YH-685 in combination with TB47 against virulent Mtb H37Rv were further evaluated in planktonic cultures and inside human macrophages. To assessing intracellular Mtb growth, human macrophage infection model and high content imaging was used. In both experimental settings, co-treatment with YH-685 and TB47 produced a marked, dose-dependent enhancement of anti-Mtb activity (Figure A,B). Notably, comparable efficacy was observed with 100 nM versus 1000 nM TB47, suggesting no substantial additional benefit from the higher concentration. YH-685 also showed single-agent activity against wild-type H37Rv. Because Cyt-bd becomes conditionally essential when the Cyt-bcc-aa3 branch is inhibited, this activity cannot be explained solely by Cyt-bd inhibition and may reflect engagement of additional cellular targets, particularly at higher YH-685 concentrations.

6.

Combined anti-Mtb activity of YH-685 and TB47 against H37Rv. (A,B) Dose–response of YH-685 + TB47 on (A) planktonic and (B) intracellular H37Rv growth. (C,D) effects on (C) planktonic and (D) intracellular growth; Δ values indicate additional inhibition versus single agents (dotted lines). (E) Comparison with RIF+INH and BDQ in both settings. Antibiotic concentrations commonly reported in vitro as 1 or 10 μg/mL were converted to μM for direct comparison with the co-treatment. The average percentage growth inhibition of planktonic (grey bars) and intracellular (white bars) bacteria under the different conditions is indicated above each bar. (F) Representative fluorescence images of GFP-expressing H37Rv (green) in planktonic and intracellular conditions (20× magnification). Macrophages appear as light grey cells. The lower panel shows staining with a viability dye, demonstrating no detectable cytotoxicity compared with the untreated H37Rv control. DRAQ7-positive cells were few and are indicated by arrowheads where visible. All data were acquired using the IncuCyte system (days 3–7) and are shown as median and range from triplicate wells (n = 2 sub-cultures, n = 2 donors). Representative of three independent experiments. **p < 0.01, *p < 0.05.

Co-treatment with 100 μM YH-685 and 0.1 μM TB47 produced significantly greater growth inhibition than either compound alone under the conditions tested (p < 0.05; Figure C,D). Relative to TB47 monotherapy, the combination produced an additional 29.7% inhibition in planktonic culture and 14.5% inhibition in infected human macrophages. Because checkerboard or diaMOND analyses were not performed, the greater inhibition observed with the combination cannot be classified as synergistic or additive. Some antimicrobial activity was also observed with YH-685 alone (Figure C,D), indicating a potential off-target effect that was not investigated further in this study. As expected, planktonic Mtb was more susceptible overall, whereas intracellular bacilli were comparatively more difficult to inhibit (Figure A–D).

Importantly, the inhibitory potency of the optimal YH-685 + TB47 demonstrated efficacy comparable to rifampicin plus isoniazid (RIF+INH) or bedaquiline (BDQ) (Figure E). Similar differences between extra- and intracellular efficacy were observed for these standard antibiotics, paralleling the pattern seen with YH-685 + TB47 (Figure E). Planktonic as well as intracellular growth of H37Rv inside human macrophages was visualized using green fluorescent protein (GFP)-expressing bacteria (Figure F). Imaging of Mtb-infected macrophages in the presence of a viability dye showed that neither compound exhibited any detectable cytotoxic effects on human cells as compared to the untreated bacterial control (Figure , lower panel).

In summary, optimization of compound 12 identified compound 17aa (YH-685), which showed improved activity against the Cyt-bd-dependent M. smegmatis ΔqcrCAB surrogate. YH-685 reduced oxygen consumption in ΔqcrCAB IMVs with an IC₅₀ of 0.45 μM, supporting inhibition of Cyt-bd-dependent respiration. In H37Rv assays, YH-685 enhanced the growth-inhibitory effect of TB47 in both planktonic cultures and infected human macrophages. However, the single-agent activity of YH-685 against wild-type H37Rv indicates that engagement of additional cellular targets cannot be excluded.

These findings support further evaluation of combined Cyt-bd/Cyt-bcc inhibition and identify YH-685 as an early stage chemical lead requiring further validation. Because formal checkerboard or diaMOND analyses were not performed, the interaction with TB47 cannot be classified as synergistic or additive. In addition, the absence of direct comparison with more advanced Cyt-bd inhibitors limits the assessment of the relative performance of YH-685. Further studies should establish direct target engagement and selectivity, improve whole-cell potency and pharmacokinetic exposure, and evaluate mitochondrial toxicity and in vivo efficacy.

Glossary

Abbreviations

Mtb

Mycobacterium tuberculosis

Cyt-bcc

cytochrome bcc-aa3 oxidase

Cyt-bd

cytochrome bd terminal oxidase

TB

tuberculosis

MDR-TB

multidrug-resistant tuberculosis

OCR

oxygen consumption rate

IMVs

inverted membrane vesicles

BDQ

bedaquiline

RIF

rifampicin

INH

isoniazid

GFP

green fluorescent protein

FCS

fetal calf serum

MOI

multiplicity of infection

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.6c00310.

• Experimental section detailing biological assays, compound synthesis along with analytical data and structural characterization data (¹H/¹³C NMR spectra, HRMS, and HPLC purity chromatograms) for key intermediates and all final compounds 17a–17aa (PDF)

The manuscript was written through contributions of all authors. X.L., G.M.C., M.M., S.B. and Y.Z. contributed to conceptualization. Q.Q., Z.L. and H.Y. performed the chemical synthesis of compounds. Z.L., X.L., G.M.C., M.M., S.B. and Y.Z. contributed to writing, review and editing. C.Y.C., M.M., J.X. contributed to the oxygen consumption assay in inverted membrane vesicles and MIC testing of M. smegmatis. S.K. and M.L. performed assessment of planktonic and intracellular growth of H37Rv using high-content imaging. All authors approved the submitted manuscript.

The authors declare no competing financial interest.

Safety Statement. No unexpected or unusually high safety hazards were encountered.