Investigating the Structure-Activity Relationship of 1,2,4-Triazine G-Protein-Coupled Receptor 84 (GPR84) Antagonists

Abstract

G-protein-coupled receptor 84 (GPR84) is a proinflammatory orphan G-protein-coupled receptor implicated in several inflammatory and fibrotic diseases. Several agonist and antagonist ligands have been developed that target GPR84; however, a noncompetitive receptor blocker that was progressed to phase II clinical trials failed to demonstrate efficacy. New high-quality antagonists are required to investigate the pathophysiological role of GPR84 and to validate GPR84 as a therapeutic target. We previously reported the discovery of a novel triazine GPR84 competitive antagonist 1. Here, we describe an extensive structure-activity relationship (SAR) of antagonist 1 and also present in silico docking with supporting mutagenesis studies that reveals a potential binding pose for this type of orthosteric antagonist. Lead compound 42 is a potent GPR84 antagonist with a favorable pharmacokinetic (PK) profile suitable for further drug development.

Figure 1

Chemical structures of known GPR84 activators.

Figure 2

Chemical structures of selected GPR84 antagonists.

Figure 3

Chemical structure, calculated physicochemical properties, and bioactivity of hit 1,2,4-triazine compound 1.

Scheme 1. Synthesis of 3,5,6-Trisubstituted 1,2,4-Triazines 4-72 via Condensation of 1,2-Diketones

Figure 4

X-ray crystal structures of (A) 37 and (B) 52 (carbon = gray, hydrogen = white, oxygen = red, nitrogen = blue).

Scheme 2. Late-Stage Structural Diversification of (A) 52 to Yield 73–81 (B) 51 to Yield Amides 82–90 (R₁ and R₂ Defined in Table 8).

Figure 5

Proposed binding mode of compound 1 at the human GPR84 receptor Alphafold (AF-Q9NQS5-F1). The overall view of the ligand–receptor complex and the zoomed ligand binding site are on the left and right. The ligand and the amino acids are shown by orange and blue sticks, respectively. Hydrogen bonds, π–π, and cation−π interactions are in pink, cyan, and green dashed lines, respectively. A section of helix 7 was hidden for a clear view in the zoomed image. The 1,2,4-triazine ring is numbered.

Figure 6

[³⁵S]GTPγS binding was performed on membrane preparations from Flp-In T-REx 293 cells expressing the indicated GPR84-Gα_i2 fusion proteins. Conversion of each of Phe101, Phe335, and Trp360 to Ala resulted in a substantial loss of affinity of compound 1 to block effects of the allosteric agonist PSB-16671. PSB-16671 showed pEC50 = 6.70 ± 0.05 at wild type, 6.97 ± 0.29 at Phe101Ala, 5.88 ± 0.02 at Phe335Ala, and 7.17 ± 0.08 at Trp360Ala.

Figure 7

[³⁵S]GTPγS binding was performed on membrane preparations from Flp-In T-REx 293 cells induced to express human FFAR2 (A), human FFAR3 (B), or a human GPR84-Gα_i2 fusion protein (C). Compounds 1, 4, 42, and 76 (at 10 μM) did not block effects of C3 at either FFAR2 or FFAR3.