Novel, potent, orally bioavailable and selective mycobacterial ATP synthase inhibitors that demonstrated activity against both replicating and non-replicating M. tuberculosis

Abstract

The mycobacterial F₀F₁-ATP synthase (ATPase) is a validated target for the development of tuberculosis (TB) therapeutics. Therefore, a series of eighteen novel compounds has been designed, synthesized and evaluated against Mycobacterium smegmatis ATPase. The observed ATPase inhibitory activities (IC₅₀) of these compounds range between 0.36 and 5.45 μM. The lead compound 9d [N-(7-chloro-2-methylquinolin-4-yl)-N-(3-((diethylamino)methyl)-4-hydroxyphenyl)-2,3-dichlorobenzenesulfonamide] with null cytotoxicity (CC₅₀ >300 μg/mL) and excellent anti-mycobacterial activity and selectivity (mycobacterium ATPase IC₅₀ = 0.51 μM, mammalian ATPase IC₅₀ >100 μM, and selectivity >200) exhibited a complete growth inhibition of replicating Mycobacterium tuberculosis H37Rv at 3.12 μg/mL. In addition, it also exhibited bactericidal effect (approximately 2.4 log₁₀ reductions in CFU) in the hypoxic culture of non-replicating M. tuberculosis at 100 μg/mL (32-fold of its MIC) as compared to positive control isoniazid [approximately 0.2 log₁₀ reduction in CFU at 5 μg/mL (50-fold of its MIC)]. The pharmacokinetics of 9d after p.o. and IV administration in male Sprague–Dawley rats indicated its quick absorption, distribution and slow elimination. It exhibited a high volume of distribution (V_ss, 0.41 L/kg), moderate clearance (0.06 L/h/kg), long half-life (4.2 h) and low absolute bioavailability (1.72%). In the murine model system of chronic TB, 9d showed 2.12 log₁₀ reductions in CFU in both lung and spleen at 173 μmol/kg dose as compared to the growth of untreated control group of Balb/C male mice infected with replicating M. tuberculosis H37Rv. The in vivo efficacy of 9d is at least double of the control drug ethambutol. These results suggest 9d as a promising candidate molecule for further preclinical evaluation against resistant TB strains.

Introduction

Tuberculosis, primarily caused by Mycobacterium tuberculosis, is one of the globally leading infectious disease that continues as global epidemic with more than 9 million new cases and nearly 2 million deaths every year. ¹ In addition, over 2 billion people harbor latent TB infection (LTBI), thus representing an enormous reservoir of M. tuberculosis that can subsequently progress to active TB.¹ The main obstacles to the global TB control are: (i) the human immunodeficiency virus (HIV) epidemic proven to increase the risk of developing active TB dramatically, (ii) the increasing emergence of multi-drug resistant TB (MDR-TB), extensively drug-resistant TB (XDR-TB) and a recently identified TB state called total drug-resistant TB (TDR-TB).1, 2, 3, 4, 5 Current TB chemotherapy is mainly based on drugs that inhibit bacterial metabolism, and is characterized by efficient bactericidal and extremely weak sterilizing activities.⁶ The dormant or latent or metabolically inactive bacilli are tolerant to the anti-TB drugs that target cell division.7, 8 Although an extended chemotherapy may be partially successful in eradicating such dormant bacilli, drug(s) with efficient sterilizing activity are of current demand for properly tackling the dormant bacilli. In such scenario, targeting the particular enzyme that are essential for the survival of both actively growing and latent/dormant bacilli, is one of the most effective target-based drug discovery approach.²

The mycobacterial F₀F₁-ATP synthase (F₀F₁-ATPase) is a validated anti-TB target, which is responsible for the production of adenosine 5′-triphosphate (ATP) for energy homeostasis in different mycobacterial species.⁹ Bedaquiline (TMC207, Sirturo™) is a member of the diarylquinoline class of anti-TB drugs targeting this new enzyme and has been approved by the U.S. Food and Drug Administration (US-FDA) specifically for the treatment of MDR-TB. This drug has shown promising efficacy against both the drug-sensitive and drug-resistant TB, and has remarkable potential for restricting the duration of TB treatment due to the fact that this drug is bactericidal to both the replicating as well as non-replicating (dormant) bacilli.9, 10, 11, 12 Bedaquiline (Sirturo™) has been reported by the World Health Organization (WHO) to disturb the function of the heart and liver in particular. Common observed side effects include nausea, joint and chest pain, and headache.¹³ Bedaquiline is proven to be a weak hERG (human Ether-à-go-go) potassium (K⁺) channel blocker (in vitro IC₅₀ = 0.2 μg/mL) that can cause prolongation of the heart QT interval (the time between the start of the Q wave and the end of the T wave in the heart’s electrical cycle), thus disturbing normal function of the heart.¹⁴ The observed efficacy of bedaquiline for the treatment of MDR-TB validates the target mycobacterium F₀F₁-ATPase, and the observed adverse events with this drug suggest further exploration of new chemical entities as selective F₀F₁-ATPase inhibitors lacking any potential adverse effects.

Structurally, the mycobacterial F₀F₁-ATPase is composed of a membrane-embedded F₀ portion comprising a₁b₂c_10–15 subunits and a hydrophilic F₁ portion consisting of α₃β₃δε.¹⁵ Proton (H⁺) migration through F₀ triggers the rotation of rotary ring formed by oligomeric subunits ‘c’ which is coupled to the rotation of the ‘γ’ subunit resided within the (αβ)₃ hexamer of F₁. This biochemical phenomenon thus drives the synthesis of ATP as a source of energy for mycobacterium species. A high-resolution crystal structure of mycobacterial ATP synthase or its subunits is lacking till date. Computational and biological studies have shown that TMC207 binds at the interface of subunit ‘a’, and rotary subunits ‘c’.15, 16, 17 This drug is biochemically shown to interfere with the rotary movement of subunit ‘c’ by mimicking a conserved basic residue Arg186 in the subunit ‘a’ needed for the proton transfer with Glu61 of the subunit ‘c’.16, 17 Through detailed computational studies, de Jonge and colleagues¹⁶ reported the chiral N,N-dimethylpropanol substructure of TMC207 to exhibit H-bond formation with the carboxylate side chain of Glu61, and a favorable location of quinoline substructure at the interface of subunits ‘a’ and ‘c’ that might lead to blockade of the rotation of rotary ring, and thus normal interfering with the proton transfer cascade process. Haagsma et al.¹⁵ have recently characterized the interaction between TMC207 and mycobacterial ATP synthase using biochemical assays and binding studies, where they have provided experimental support for ability of TMC207 to mimic a key residue Arg186 involved in the proton transfer cascade, and thus blocking rotary movement of subunits ‘c’ of the mycobacterial F₀F₁-ATP synthase.15, 16

In view of the success of mycobacterial F₀F₁-ATP synthase as validated target for TB chemotherapy, we utilized the state-of-the-art medicinal chemistry approach for the identification of novel inhibitors of mycobacterial F₀F₁-ATP synthase. This effort led to invention of quinoline class of aryl-sulfonamides as potent, orally bioavailable and selective mycobacterial ATP synthase inhibitors that we have patented recently.¹⁸ We have recently reported a short report on the biological screening of these compounds against Mycobacterium smegmatis ATP synthase along with in vitro testing against the dormant (non-replicating) M. tuberculosis H37Rv.¹² Herein, we report a full-length description of the rational design, synthesis and structure-activity relationship (SAR) studies of these novel compounds proven as low micromolar inhibitors of the mycobacterial F₀F₁-ATP synthase. Among the synthesized series of 18 compounds, two lead compounds were biologically explored in greater detail where one representative compound was proven as an orally bioavailable and selective mycobacterium ATP synthase inhibitor that demonstrated approximately 2.12 log₁₀ reductions in CFU in both lung and spleen at a dose of 173 μmol/kg as compared to the growth in the untreated control group of Balb/C male mice infected with M. tuberculosis H37Rv. Like the drug bedaquiline (Sirturo™, TMC207), this compound acts through a novel mechanism of action (ATP synthase), and is active against both the replicating as well as non-replicating M. tuberculosis H37Rv. This lead compound could be considered as candidate molecule for further preclinical evaluation that could lead to an effective TB therapeutics.