Discovery of AZD4831, a Mechanism-Based Irreversible Inhibitor of Myeloperoxidase, As a Potential Treatment for Heart Failure with Preserved Ejection Fraction

Abstract

Myeloperoxidase is a promising therapeutic target for treatment of patients suffering from heart failure with preserved ejection fraction (HFpEF). We aimed to discover a covalent myeloperoxidase inhibitor with high selectivity for myeloperoxidase over thyroid peroxidase, limited penetration of the blood-brain barrier, and pharmacokinetics suitable for once-daily oral administration at low dose. Structure-activity relationship, biophysical, and structural studies led to prioritization of four compounds for in-depth safety and pharmacokinetic studies in animal models. One compound (AZD4831) progressed to clinical studies on grounds of high potency (IC₅₀, 1.5 nM in vitro) and selectivity (>450-fold vs thyroid peroxidase in vitro), the mechanism of irreversible inhibition, and the safety profile. Following phase 1 studies in healthy volunteers and a phase 2a study in patients with HFpEF, a phase 2b/3 efficacy study of AZD4831 in patients with HFpEF started in 2021.

Figure 1

Enzymatic cycles of myeloperoxidase. MPO is activated by hydrogen peroxide and is converted into the highly reactive species “compound I”, through a two-electron oxidation. “Compound I” can react with halides (Cl^–, Br^–, I^–) and pseudohalides (SCN^–) to form the corresponding hypohalous acid and reduce MPO back to the native state. This is known as the halogenation cycle. “Compound I” can also act as a peroxidase by sequentially reacting with single-electron donors to form the redox intermediate “compound II”, which is in turn reduced back to the native state. This is known as the peroxidase cycle. Thioxanthines (TX) or deazathioxanthines (DTX) can react with “compound I” to form an inactive species of MPO.^,

Figure 2

Myeloperoxidase inhibitors evaluated in clinical trials.

Figure 3

Tight binding assay with human myeloperoxidase and inhibitors. Concentration–response curves obtained with (A) the reversible inhibitor 4-(5-fluoro-1H-indol-3-yl)butanamide; (B) compound 2, indicating partial reversibility; and (C) compound 16, indicating irreversible inhibition. ● represents inhibition in the presence of the inhibitor (before washing) and the △ represents inhibition without re-addition of inhibitor after repeated washing. Shaded areas represent 95% confidence intervals for the modeled inhibition. Data are mean and standard deviation of results from triplicate wells.

Figure 4

Native myeloperoxidase in complex with (A) compound 3 and (B) compound 9. Myeloperoxidase is shown in blue stick representation and compounds 3 and 9 are shown in orange and green, respectively. The coordinates have been deposited in the PDB with coordinates 7NI1 and 7NI3.

Figure 5

Covalent binding of compound [¹⁴C]-8 in human liver microsomes.

Scheme 1. Tentative Mechanism for Formation of the Cyanomethylene Amine (Q) from Compound 8

Figure 6

Whole-body autoradiography 24 h after an oral dose of [¹⁴C]-16 (AZD4831) in rats, before (left) and after (right) extraction.

Figure 7

Dose-dependent inhibition of peritoneal myeloperoxidase by compound 16 (AZD4831) in mice. ****p < 0.0001. *p < 0.05; ns, not significant. Data are expressed as photons per second (LPS), and each symbol represents one mouse. The statistical analysis was performed on log-transformed data by one-way analysis of variance followed by Dunnett’s multiple comparison test.

Figure 8

Predicted myeloperoxidase activity and plasma drug concentration versus time after once-daily oral dosing of 20 mg of compound 16 (AZD4831) in humans. Dark blue line shows the total inhibition of myeloperoxidase activity relative to the baseline, and the light blue line shows the contribution from irreversible inhibition. Red and orange lines show the predicted total and unbound plasma concentration of compound 16, respectively.

Scheme 2. Synthesis of Compound 16 (AZD4831)

(1) (S)-2-methylpropane-2-sulfinamide, Cs₂CO₃, and DCM, reflux. (2) MeMgBr and DCM, −45 °C to rt. (3) HCl and MeOH. (4) Boc₂O, TEA, and DCM. (5) Boc₂O, DMAP, and 2-MeTHF. (6) CO:H₂ (1:1, 5 bar), Pd(OAc)₂, cataCXium A, TMEDA, and toluene, 100 °C. (7) (i) 27, DIPEA, EtOH, (ii) HOAc, and (iii) NaBH₃CN. (8) (i) Benzoylsothiocyanate, MeOH, rt and (ii) C₂CO₃, 60 °C. (9) HCl and MeOH, 50 °C.