Optimization of physicochemical properties and safety profile of novel bacterial topoisomerase type II inhibitors (NBTIs) with activity against Pseudomonas aeruginosa

doi:10.1016/j.bmc.2014.07.040

Bioorganic & Medicinal Chemistry

Volume 22, Issue 19, 1 October 2014, Pages 5392-5409

https://doi.org/10.1016/j.bmc.2014.07.040 Get rights and content

Abstract

Type II bacterial topoisomerases are well validated targets for antimicrobial chemotherapy. Novel bacterial type II topoisomerase inhibitors (NBTIs) of these targets are of interest for the development of new antibacterial agents that are not impacted by target-mediated cross-resistance with fluoroquinolones. We now disclose the optimization of a class of NBTIs towards Gram-negative pathogens, especially against drug-resistant Pseudomonas aeruginosa. Physicochemical properties (pK_a and log D) were optimized for activity against P. aeruginosa and for reduced inhibition of the hERG channel. The optimized analogs 9g and 9i displayed potent antibacterial activity against P. aeruginosa, and a significantly improved hERG profile over previously reported analogs. Compound 9g showed an improved QT profile in in vivo models and lower clearance in rat over earlier compounds. The compounds show promise for the development of new antimicrobial agents against drug-resistant Pseudomonas aeruginosa.

Graphical abstract

Introduction

The emergence of multi-drug-resistant Gram-negative pathogens is an area of increasing concern in the medical community¹ and provides the impetus to discover new classes of antibacterial agents that are not cross resistant with existing drugs. Efforts aimed at addressing resistance have focused on both novel targets, as well as novel molecules that inhibit established targets through a different mode of action.

We were interested in bacterial type II topoisomerases (DNA gyrase and topoisomerase IV), which are clinically proven antibacterial targets for the fluoroquinolone class of drugs that target the GyrA and ParC subunits of DNA gyrase and topoisomerase IV, respectively. The clinical utility of fluoroquinolones is severely limited by mutations in the quinolone-resistance determining region (QRDR) of the targets.2, 3 The new class of NBTIs (novel bacterial type II topoisomerase inhibitors) engage with the GyrA/ParC subunits of bacterial type II topoisomerases through a different mode of inhibition that is not impacted by QRDR mutations.4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 Other classes of inhibitors of the GyrA/ParC subunits of bacterial type II topoisomerases that do not show cross resistance with fluoroquinolones have been described.19, 20, 21, 22, 23

The pharmacophore of the NBTIs suffers from a hERG liability and the mitigation of this risk has been the focus in the lead optimization phase of our program.8, 10 We recently reported on efforts to optimize physicochemical properties of NBTIs for selective Gram-positive activity, during which the pK_a of the compounds was reduced and compounds displayed an improved cardiac safety profile.¹⁰ The physicochemical property space for drugs targeting Gram-negative bacteria differs for compounds targeting Gram-positive bacteria, which provides guidelines for different strategies of optimization.²⁴ In particular, optimization of molecules targeting intracellular targets in Gram-negative pathogens requires consideration of drug permeability through porins of the outer membrane as well as permeability by passive diffusion through the inner membrane. In addition, many Gram-negative pathogens possess sophisticated efflux pump systems.²⁵ We report herein the optimization of NBTIs against serious Gram-negative pathogens, with focus on the problematic hospital pathogen Pseudomonas aeruginosa. In depth biochemical and microbiological characterization of an advanced lead from his series has been described elsewhere.¹⁸

Section snippets

Chemistry

Most final compounds were assembled by reductive aminations of chiral amines 5 with aldehydes 6²⁶ or 7²⁷ (Scheme 1).²⁸ The nosyl groups were removed using thiophenol, in most cases in good yields. With fluoro-substituted heterocyclic analogs, especially with naphthyridone 8j, care had to be taken to minimize substitution of the fluoro group by thiophenol.

The amine building blocks 5a–g were obtained by alkylation of heterocycles 2a–g8, 28, 29, 30, 31, 32, 33, 34 with the aziridine 328, 35

Results and discussion

Our lead for this effort was compound 1 (Fig. 1), which displays potent broad spectrum antibacterial activity.¹⁰ However, 1 is inhibiting the hERG cardiac channel (IC₅₀ = 35 μM, Table 1), and caused prolongation of the corrected QT (QT_c) interval in a dog toxicology study (Fig. 4). We aimed to reduce the changes in QT_c by reducing hERG inhibition, while retaining Gram-negative activity, especially against multi-drug resistant P. aeruginosa. We have previously reported that addressing the hERG

Conclusions

The NBTI lead 1 was optimized for reduced hERG channel activity and for activity against the Gram-negative organism P. aeruginosa by introduction of a second basic group in the linker that both reduces the log D of the compounds and allows formation of an intramolecular H-bond. The resulting optimized amino ethyl cyclohexyl NBTI 9g shows a significantly reduced inhibition of hERG relative to 1. The effects of compound 9g on the monophasic action potential (MAP) were studied in the guinea pig

Minimum inhibitory concentration testing

Minimum inhibitory concentrations (MICs) were determined by broth microdilution according to the Clinical and Laboratory Standards Institute guidelines.⁴⁹ Bacterial cultures were incubated at 35 °C on blood agar plates (Remel #01202) for 18–20 h prior to MIC determinations. Culture media for bacterial MIC determinations was Mueller Hinton Broth, Difco #275730 in 96 well microtiter plates. Compounds were dissolved in 100% DMSO and diluted to 2% DMSO (v/v) in culture medium in doubling dilutions

General chemical methods

All commercially available solvents and reagents were used without further purification. All moisture-sensitive reactions were carried out under a nitrogen atmosphere in commercially available anhydrous solvents. Column chromatography was performed on 230–400 mesh silica gel 60. Aluminium-backed sheets of silica gel 60 F254 (EM Science) were used for TLC. Melting points were obtained with a Mel-TempII melting point apparatus from Laboratory Devices, Inc and are uncorrected. ¹H NMR spectra were

General procedure for 4, by alkylation of 2

To a solution of 2 (2 mmol) in DMF (10 mL) was added sodium hydride (120 mg, 3.0 mmol), and the mixture was stirred at room temperature for 15 min under nitrogen. A solution of 3 (850 mg, 2.00 mmol) in DMF (6 mL) was added and the mixture was stirred at room temperature over night. The mixture was quenched with ice water and extracted with ethyl acetate (100 mL). The organic phase was washed with brine twice (100 mL) and dried over magnesium sulfate, then concentrated under reduced pressure.

General procedure for 5, by deprotection of 4

A solution of 4 (1 mmol) in dichloromethane (3 mL) was treated with trifluoroacetic acid (1 mL) at room temperature for 2 h. The solvent was removed under reduced pressure and the residue codistilled with methanol twice, to give the products as bis-trifluoroacetate salts.

General procedure for 8, by reductive amination of 5

A solution of 5 (0.20 mmol) and 1 equiv aldehyde, either 3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine-6-carbaldehyde 6²⁶ or 7-oxo-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazine-2-carbaldehyde 7⁵³ in dry DMF (2 mL) were heated over freshly activated 3 Å molecular sieves (pearled) at 60 °C for 3 h. The reaction mixture was cooled to 0 °C, and sodium triacetoxy borohydride (0.6 mmol) was added. The reaction mixture was stirred at room temperature for 30 min, then filtered. The filtrate was concentrated to

General procedure for 9 by deprotection of 8

To a solution of 8 (0.68 mmol) in anhydrous DMF (5 mL) was added anhydrous K₂CO₃ (467 mg, 3.38 mmol) and thiophenol (0.348 mL, 3.38 mmol). The mixture was stirred at room temperature for 2 h under a blanket of nitrogen. The volatile portion of the mixture was removed by rotary evaporation and saturated aqueous NaHCO₃ (20 mL) was added to the resulting residue. The residue was extracted with MeOH/CH₂Cl₂ (10%, twice with 100 mL). The organic layer was dried over Na₂SO₄ and concentrated under reduced

Acknowledgments

The authors thank Dr. Camil Joubran, Analytical Chemistry Department of AstraZeneca R&D Boston for performing nOe and HMBC NMR experiments, AstraZeneca R&D Susceptibility Testing Group for MIC testing, and Lena Grosser, Pharmacology Department, AstraZeneca R&D Boston for efficacy studies.

References and notes (54)

D.M. Livermore
Int. J. Antimicrob. Agents
(2012)
L. Gomez et al.
Bioorg. Med. Chem. Lett.
(2007)
J.J.M. Wiener et al.
Bioorg. Med. Chem. Lett.
(2007)
B. Geng et al.
Bioorg. Med. Chem. Lett.
(2011)
T.J. Miles et al.
Bioorg. Med. Chem. Lett.
(2011)
T.J. Miles et al.
Bioorg. Med. Chem. Lett.
(2011)
M.J. Mitton-Fry et al.
Bioorg. Med. Chem. Lett.
(2013)
T.J. Miles et al.
Bioorg. Med. Chem. Lett.
(2013)
G. Brooks et al.
Tetrahedron Lett.
(2010)
P. Chen et al.
Tetrahedron Lett.
(2001)

A. Titz et al.

Tetrahedron Lett.

(2006)

H. Nikaido et al.

Outer Membrane Permeability of Pseudomonas aeruginosa

M.H. Bridgland-Taylor et al.

J. Pharmacol. Toxicol. Methods

(2006)

C.L. Andrew et al.

Biochim. Biophys. Acta

(1997)

W.A. Craig

Infect. Dis. Clin. North Am.

(2003)

O. Cuevas et al.

J. Antimicrob. Chemother.

(2011)

Pasca M. Rosalia et al.

Microb. Drug Resist.

(2012)

B.D. Bax et al.

Nature

(2010)

M.T. Black et al.

Antimicrob. Agents Chemother.

(2008)

F. Reck et al.

J. Med. Chem.

(2011)

F. Reck et al.

J. Med. Chem.

(2012)

J. Surivet et al.

J. Med. Chem.

(2013)

S.B. Singh et al.

ACS Med. Chem. Lett.

(2014)

C. Mayer et al.

Chem. Rev. (Washington, DC, U.S.)

(2014)

T.J. Dougherty et al.

Antimicrob. Agents Chemother.

(2014)

A.A. Miller et al.

Antimicrob. Agents Chemother.

(2008)

Barvian, K.; Basarab, G. S.; Gowravaram, M. R.; Hauck, S. I.; Zhou, F. WO2010043893,...

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Optimization of physicochemical properties and safety profile of novel bacterial topoisomerase type II inhibitors (NBTIs) with activity against Pseudomonas aeruginosa

Abstract

Graphical abstract

Introduction

Section snippets

Chemistry

Results and discussion

Conclusions

Minimum inhibitory concentration testing

General chemical methods

General procedure for 4, by alkylation of 2

General procedure for 5, by deprotection of 4

General procedure for 8, by reductive amination of 5

General procedure for 9 by deprotection of 8

Acknowledgments

Int. J. Antimicrob. Agents

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem. Lett.

Tetrahedron Lett.

Tetrahedron Lett.

Tetrahedron Lett.

J. Pharmacol. Toxicol. Methods

Biochim. Biophys. Acta

Infect. Dis. Clin. North Am.

J. Antimicrob. Chemother.

Microb. Drug Resist.

Nature

Antimicrob. Agents Chemother.

J. Med. Chem.

J. Med. Chem.

J. Med. Chem.

ACS Med. Chem. Lett.

Chem. Rev. (Washington, DC, U.S.)

Antimicrob. Agents Chemother.

Antimicrob. Agents Chemother.