The Discovery and Development of Thienopyrimidines as Inhibitors of Helicobacter pylori That Act through Inhibition of the Respiratory Complex I

Abstract

The successful treatment of Helicobacter pylori infections is becoming increasingly difficult due to the rise of resistance against current broad spectrum triple therapy regimens. In the search for narrow-spectrum agents against H. pylori, a high-throughput screen identified two structurally related thienopyrimidine compounds that selectively inhibited H. pylori over commensal members of the gut microbiota. To develop the structure-activity relationship (SAR) of the thienopyrimidines against H. pylori, this study employed four series of modifications in which systematic substitution to the thienopyrimidine core was explored and ultimately side-chain elements optimized from the two original hits were merged into lead compounds. During the development of this series, the mode of action studies identified H. pylori's respiratory complex I subunit NuoD as the target for lead thienopyrimidines. As this enzyme complex is uniquely essential for ATP synthesis in H. pylori, a homology model of the H. pylori NuoB-NuoD binding interface was generated to help rationalize the SAR and guide further development of the series. From these studies, lead compounds emerged with increased potency against H. pylori, improved safety indices, and a good overall pharmacokinetic profile with the exception of high protein binding and poor solubility. Although lead compounds in the series demonstrated efficacy in an ex vivo infection model, the compounds had no efficacy in a mouse model of H. pylori infection. Additional optimization of pharmacological properties of the series to increase solubility and free-drug levels at the sequestered sites of H. pylori infection would potentially result in a gain of in vivo efficacy. The thienopyrimidine series developed in this study demonstrates that NuoB-NuoD of the respiratory complex I can be targeted for development of novel narrow spectrum agents against H. pylori and that thienopyrimines can serve as the basis for future advancement of these studies.

Keywords: Helicobacter pylori; NuoD; complex I inhibitor; thienopyridine.

Figure 1.

A) Structure of Hpi1, a selective anti-H.pylori compound that emerged as a positive hit from a high through-put screen against H. pylori. B, C) Structures of thienopyrimidines hits 1 and 2, that were identified using a similar screening approach with the H. pylori IC₅₀ of 1.55 μM and 1.72 μM respectively. D) Areas substituted in this study to improve potency, drug-like properties, and cytotoxicity.

Figure 2.

a. Homology model of the NuoD and NuoB subunits of complex I. b. Snapshot of lead compound 26 (gold) in the average conformation over a 250 ns molecular dynamic simulation in the wild type protein. The 250ns MD simulation shows that compound 26 is a hydrogen bond donor to the carbonyl oxygen at T395 and accepts a hydrogen bond from the hydroxyl side chain of T400 labeled with magenta dotted lines respectively. Compound 26 methoxybenzyl group pi-stacks with Y376 illustrated with blue dotted lines. c. A ligand interaction diagram between compound 26 and the protein. Pink lines show hydrogen bonds and the green lines show pi-pi interactions d. Analysis of compound 26 hydrogen bond interactions with respective residues over the entire 250 ns simulation.

Figure 3.

Sequence alignment of the wildtype nuoD and two H. pylori mutant NuoD. Resistant mutants were generated by exposing H. pylori to compound 1 or 28 on agar plates. Whole genome sequencing was performed, and single nucleotide polymorphism analysis identified two spontaneous mutants, which had non-synonymous point mutations in nuoD. The resulting amino acid changes were Threonine to Isoleucine (T400I) and Alanine to Proline (A402P). nuoD encodes for the subunit D of NADH:Quinone eductase also known as Complex I, a membrane protein that catalyzes the first step of oxidative phosphorylation. Complex I is the putative target of the thienopyrimidine.

Figure 4.

The effect of Compound 25 against H. pylori in an ex vivo infection model. Ten mice were infected with H. pylori strain SS1. After 3 days, stomachs were harvested and bisected laterally. One-half each stomach was placed in a 12-well plate containing either Brucella-FBS medium or Brucella-FBS medium with 0.6 μg/mL (1.6μM) of compound 25. Plates were incubated for 24 hours and tissues were washed, homogenized, diluted, and plated for colony counts.

Scheme 1.. Synthesis of Thienopyrimidine Derivatives.

Reagents and conditions: (i) Sulfur, Morpholine, Ethanol, 70˚C, Microwave, 20 min, 90%; (ii) Formamide, reflux, 18 h, 80%; (iii) POCl₃, N, N dimethylaniline, reflux, 14 h, 90%; (iv) Ethanol, 150˚C, Microwave, 1 h, 80-93%.

Scheme 2:. Synthesis of Amine Derivative 31.^a

^aReagents and conditions: (i) di-tert-butyl dicarbonate, Et₃N, DCM, RT, 2 h; (ii) phthalimide, DIAD, Ph₃P, DCM, RT, 4 h; (iii) 4 M HCl in dioxane, DCM, methanol, RT, 4 h; (iv) Et₃N, reflux, 6 h; (v) NH₂NH₂, methanol, RT, 6 h.

Scheme 3:. Synthesis of thienopyrimidine derivatives with modifications at position 5 and 6.^a

^aReagents and conditions: (i) Et₃N, 150˚C, Microwave, 1 h, 89-93%.