Design and synthesis of 1,4,8-triazaspiro[4.5]decan-2-one derivatives as novel mitochondrial permeability transition pore inhibitors

Abstract

Ischaemia/reperfusion injury (IRI) is a condition that occurs when tissues from different organs undergo reperfusion following an ischaemic event. The mitochondrial permeability transition pore (mPTP), a multiprotein platform including structural components of ATP synthase with putative gate function, is an emerging pharmacological target that could be modulated to facilitate the restoration of organ function after a hypoxic insult. Herein, we reported the synthesis and biological characterisation of new molecules with a 1,4,8-triaza-spiro[4.5]decan-2-one framework of potential interest for the treatment of IRI able to inhibit the opening of mPTP in a cardiac model in vitro. Modelling studies were useful to rationalise the observed structure-activity relationship detecting a binding site for the investigated molecules at the interface between the c₈-ring and subunit a of ATP synthase. Compound 14e was shown to display high potency as mPTP inhibitor combined with the capability to counteract cardiomyocytes death in an in vitro model of hypoxia/reoxygenation.

Keywords: Permeability transition pore; cardiac ischaemia/reperfusion injury; cytoprotection; mPTP inhibitors; mitochondria.

Graphical abstract

Figure 1.

From 1,3,8-triazaspiro[4.5]decane to 1,4,8-triaza-spiro[4.5]decane derivatives as mPTP inhibitors.

Scheme 1.

Reagents and conditions: i. 40% piperidine in DMF; ii DIC, HOBt, DMF; iii. N-benzyl-4-piperidone, p-TsOH, toluene, 80 °C, overnight (for 11a-f) or N-benzyl-4-piperidone, p-TSOH, 2,2-dimethoxypropane, 80 °C, 5h (for 12a-f); iv. TFA/H₂O/Et₃SiH (95:2.5:2.5 v/v), rt, 3h.

Figure 2.

In vitro evaluation of H/R damage and 14e-dependent cytoprotection. A) mPTP activity measured in 14e treated cells and compared to untreated conditions; representative kinetics and images from confocal microscopy are reported on the right; B) Cytometer-dependent quantification of living cells in percentage. Each value is the mean of 3 biological and 3 technical replicates. C) Quantification of AC16 cells positive to Annexin V staining following H/R. Each value is the mean of 3 biological and 3 technical replicates. D) Immunoblot detection of the main markers of apoptosis and necrosis such as Cleaved PARP, Cleaved Caspase 3 and Cleaved RIP1. This is representative of 3 biological replicates. E) Quantification of the MitoSOX probe intensity in cells following H/R. Each value is the mean of at least 3 biological and 3 technical replicates. F) Quantification of mitochondrial membrane potential and representative kinetics using the TMRM probe, reported as the ratio between the initial intensity and that one reached following 10 nM FCCP administration. Each value is the mean of at least 3 biological and 3 technical replicates. (***) p values < 0.001; (****) p values < 0.0001 (see Figure S1 for raw data).

Figure 3.

Molecular modelling. A) Location of the detected binding site at the interface between the c₈-ring (light blue cartoon) and helices ɑ5 and ɑ6 of subunit a (magenta cartoon) with respect to the overall human ATP synthase architecture. The site is represented as surface colour coded by lipophilicity with green, grey, and purple representing hydrophobic, neutral and hydrophilic regions, respectively. B) Representative docking pose of compound 14f (sphere representation, cyan carbon atoms) in the identified binding site. C) Details of established ligand-protein interactions in the predicted binding mode of compound 14f. Hydrophobic, salt-bridge and H-bond interactions are represented in dashed grey, dashed yellow, and solid blue lines, respectively. The ligand and interaction residues are represented in sticks with ligand carbon atoms in cyan and protein residue carbon atoms colour coded according to the region they belong to as follows: c₈-ring, blue; subunit a, magenta. The grey surface represents the contact surface area between the ligand aromatic R group and protein Tyr221 and Glu58 side chains. Figure in panel A was rendered with MOE, whereas figures in panel B and C were created with PyMOL open source v2.5.0.

Figure 4.

In vitro evaluation of the key parameters of mitochondrial respiration. A) Oxygen consumption rate (OCR) kinetics reporting mean values with SEM. B) Statistics for the calculation of basal respiration, C) Statistics for the calculation of ATP-linked respiration. D) Statistics for the calculation of Maximal respiratory capacity. Each value is the mean of at least 3 biological and 3 technical replicates.