Research paper
Identification of novel benzothiopyranone compounds against Mycobacterium tuberculosis through scaffold morphing from benzothiazinones

https://doi.org/10.1016/j.ejmech.2018.09.042Get rights and content

Highlights

  • The benzothiopyranones displayed excellent anti-TB activity and low cytotoxicity.

  • Compound 6b exhibited good inhibitory activity against DprE1.

  • Compound 6b exhibited potent in vitro activity against both DS-TB and DR-TB.

  • Compound 6b displayed superior ADME/T and PK properties.

  • Compound 6b demonstrated potent in vivo efficacy in an acute mouse model of TB.

Abstract

In this study, three novel series of benzoxazinone, benzothiopyranone and benzopyranone derivatives were designed through scaffold morphing from benzothiazinones to target DprE1. All compounds were evaluated for their in vitro activities against Mycobacterium tuberculosis and cytotoxicity against Vero cell line. Among these three series, the benzothiopyranone series displayed excellent antimycobacterial activity and low cytotoxicity. In particular, compound 6b exhibited potent in vitro activity against both drug-susceptible and drug-resistant tuberculosis clinical strains with MICs <0.016 μg/mL. In addition, compound 6b demonstrated excellent ADME/T and PK properties and potent in vivo efficacy with bactericidal activity in an acute mouse model of tuberculosis. The antituberculosis effect of compound 6b is most likely attributed to its excellent anti-DprE1 activity. As such, compound 6b is under evaluation as a potential clinical candidate for treatment of tuberculosis. The current study provided new insight into the structural and pharmacological requirements for DprE1 inhibitors as potent antitubercular agents.

Introduction

Tuberculosis (TB) continues to be a major global health problem with millions of new cases every year. In the 2017 global TB report, TB was listed as the ninth leading cause of death worldwide, and had been the leading cause of death as a single infectious pathogen from 2012 to 2016, ranking above HIV/AIDS [1]. The prevalence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) has created an urgent demand for the discovery and development of novel TB drugs to improve the treatment outcomes. Due to the high attrition rate in new drug development, the unmet medical need for safer and more effective agents with novel mechanisms of action for the treatment of TB cannot be overemphasized [2].

The evaluation of novel chemical scaffolds possessing desirable pharmacological profiles against Mycobacterium tuberculosis (M. tuberculosis) and the identification of novel drug targets of M. tuberculosis are both at the forefront of new TB drug discovery and development. Re-positioning the existing antibacterial drug classes and structural modifications based on natural products with antibacterial activity are undoubtedly effective approaches to discover new antitubercular agents [[3], [4], [5]].

Cell wall is a functional and protective interface between external and internal environment for every living cell. Targeting the cell wall biosynthesis has been a successful strategy in TB drug discovery. For example, indolcarboxamides are a novel series of antitubercular agents identified recently. This series targets Mycobacterial membrane protein large 3 (MmpL3), a transporter of trehalose monomycolate that is essential for mycobacterial cell wall biosynthesis [6]. Two other cell wall biosynthesis drug targets, decaprenylphosphoryl-β-d-ribose 2′-epimerase (DprE1) and decaprenylphosphoryl-d-2-ketoerythropentose reductase (DprE2) were also identified recently, and they catalyze the epimerization of decaprenylphosphorylribose (DPR) to decaprenylphosphorylarabinose (DPA), a unique precursor for the synthesis of cell-wall arabinans [7]. DprE1, a vulnerable tuberculosis drug target due to its cell wall localization and specificity to mycobacteria and actinomycetes, has become an attractive target for discovering more effective and safer medicines for treatment of drug-sensitive TB as well as MDR/XDR-TB [[8], [9], [10]]. The first DprE1 inhibitor is nitrobenzothiazinone 1 (BTZ043) (Fig. 1) [11]. Phase I clinical trials for its next generation analogue 2 (PBTZ169) with improved efficacy and safety (Fig. 1) have been completed [1,12]. Compound 1 serves as a suicide substrate for the reduced form of DprE1. This compound irreversibly inactivates DprE1 through the reduction of nitro group to a nitroso species, which specifically forms a covalent adduct by reacting with the thiol side chain of the active site cysteine residue Cys387 [12,13]. The discovery of 1,3-benzothiazin-4-ones (BTZs), specifically 1 and its analogue 2, prompted further research related to the identification of potential antitubercular agents based on electron-deficient nitroaromatic structures. A series of potential DprE1 covalent inhibitors were reported with modification on the 2 position of BTZs, as represented by compounds 1a and 2a (Fig. 1) [[14], [15], [16], [17], [18], [19], [20]]. Interestingly, two compounds with relatively minor modifications to the BTZs core, benzothiazinethione (SKLB-TB1001, 3) and sulfoxide of 1 (BTZ-SO, 4), maintain potent antimycobacterial activities (Fig. 1) [21,22].

The co-crystal structures of the DprE1 with 1 and DprE1 with 2 complexes as well as the structure-activity relationship (SAR) of this series indicated that the key constituents for potent activity are the sulfur atom and carbonyl group in the thiazinone ring, a strong electron-withdrawing group (CF3, CN, NO2, etc.) at the 6 position, and more importantly a nitro group at the 8 position [11,12]. However, we noticed that the nitrogen atom in the thiazinone ring has no direct interaction with the enzyme. Inspired by the above background information, we employed the bioisosteric replacement strategy by replacing the nitrogen atom at the 3 position with a carbon atom and replacing the sulfur atom at the 1 position with an oxygen atom. As such, we identified new DprE1 inhibitors with improved physicochemical properties and safety profiles. Herein, we report three novel structural series, benzoxazinones 5, benzothiopyranones 6 and benzopyranones 7, which maintain a CF3 group at the 6 position and a NO2 group at the 8 position (Fig. 2). After further evaluation, a benzothiopyranone compound 6b as a DprE1 inhibitor was identified as a promising preclinical candidate for treatment of drug-resistant tuberculosis.

Section snippets

Chemistry

The target compounds were synthesized following the procedure as outlined in Scheme 1. 2-Chloro-3-nitro-5-(trifluoromethyl)benzamide (8) [11] was treated with oxalyl chloride followed by an appropriate amine to afford intermediates 9a-r. Compounds 9a-r were heated in DMF in the presence of K2CO3 to give the benzoxazinones 5a-r after purification by column chromatography [23]. Benzothiopyranones 6a-r were prepared by a two-step process. The key intermediate 11 was first synthesized by treating

Identification of benzothiopyranones as a promising lead series

All target compounds were evaluated for their antimycobacterial activities against M. tuberculosis H37Rv using the microplate alamar blue assay (MABA) [29]. The minimum inhibitory concentration (MIC) was defined as the lowest concentration effecting a reduction in fluorescence of ≥90% relative to the mean of replicate bacterium-only controls. All target compounds were further tested for their cytotoxicity against mammalian cell line (Vero cells) as measured by the concentration required for

Conclusion

Antimycobacterial compounds targeting DprE1 and its inhibitor benzothiazinone series represent the new trend in TB drug discovery [[11], [12], [13]]. Benzothiopyranone compound 6b, obtained by scaffold morphing from benzothiazinones, has been identified as a promising TB drug candidate with potent activity, low toxicity and acceptable PK properties. Extensive SAR studies provide insight into the structural requirements for potent antimycobacterial activity and low cytotoxicity.

General experimental information

All the solvents and chemicals were obtained from commercial sources and used without further purification. TLC was performed on silica gel plates (GF254) with visualization of components by UV light (254 nm) or exposure to I2. Column chromatography was carried out on silica gel (300–400 mesh). The structural identities of the prepared compounds were confirmed by 1H NMR and 13C NMR spectroscopy and mass spectrometry. 1H NMR spectra were obtained on Varian Mercury-400 at 400 MHz. 13C NMR spectra

Notes

The authors declare no competing financial interest.

Acknowledgments

The research is supported in part by the National Science & Technology Major Project of China (Grant 2015ZX09102007-011) and the Fundamental Scientific Research Fund of Institute of Materia Medica (Grant 2013CHX10).

References (31)

  • D. Cappoen et al.

    1,2,3,4,8,9,10,11-Octahydrobenzo[j]phenanthridine-7,12-diones as new leads against Mycobacterium tuberculosis

    J. Med. Chem.

    (2014)
  • S.P.S. Rao et al.

    Indolcarboxamide is a preclinical candidate for treating multidrug-resistant tuberculosis

    Sci. Transl. Med.

    (2013)
  • J. Neres et al.

    Structural basis for benzothiazinone-mediated killing of Mycobacterium tuberculosis

    Sci. Transl. Med.

    (2012)
  • G. Riccardi et al.

    The DprE1 enzyme, one of the most vulnerable targets of Mycobacterium tuberculosis

    Appl. Microbiol. Biotechnol.

    (2013)
  • M. Brecik et al.

    DprE1 is a vulnerable tuberculosis drug target due to its cell wall localization

    ACS Chem. Biol.

    (2015)
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