Structure-Guided Modification of Heterocyclic Antagonists of the P2Y14 Receptor

Abstract

The P2Y₁₄ receptor (P2Y₁₄R) mediates inflammatory activity by activating neutrophil motility, but few classes of antagonists are known. We have explored the structure-activity relationship of a 3-(4-phenyl-1 H-1,2,3-triazol-1-yl)-5-(aryl)benzoic acid antagonist scaffold, assisted by docking and molecular dynamics (MD) simulation at a P2Y₁₄R homology model. A computational pipeline using the High Throughput MD Python environment guided the analogue design. Selection of candidates was based upon ligand-protein shape and complementarity and the persistence of ligand-protein interactions over time. Predictions of a favorable substitution of a 5-phenyl group with thiophene and an insertion of a three-methylene spacer between the 5-aromatic and alkyl amino moieties were largely consistent with empirical results. The substitution of a key carboxylate group on the core phenyl ring with tetrazole or truncation of the 5-aryl group reduced affinity. The most potent antagonists, using a fluorescent assay, were a primary 3-aminopropyl congener 20 (MRS4458) and phenyl p-carboxamide 30 (MRS4478).

Figure 1.

Computational pipeline applied for the design of new derivatives as hP2Y₁₄R antagonists. A library of compounds was designed according to the general scheme reported in Chart 2. The compounds were docked at a hP2Y₁₄R homology model (Glide, SP scoring function). Hit selection was performed by visual inspection followed, for a few selected candidates, by membrane molecular dynamics (MD) refinement (30 ns, run in triplicate, CHARMM36/CGenFF, POPC lipid model). The MD refinement phase was sped up using High Throughput MD (HTMD).

Figure 2.

Upper panels: Selection of the aromatic ring. Docking pose of (A) thiophene derivative 13 (cyan carbon atoms) and (B) benzene derivative 16 (salmon carbon atoms) at the hP2Y₁₄R. Both the thiophene and the benzene rings establish an additional π-cation interaction with Arg274^7. (dark green dashed lines). Lower panel: Selection of the optimal spacer between the aromatic and amine functions. Docking poses of derivatives bearing a spacer of three methylene groups and a (C) terminal primary amine (compound 7, light blue carbon atoms) or (D) a hydroxyl group (dark green carbon atoms) at the hP2Y₁₄R. Side chains of residues important for ligand recognition are reported as sticks (grey carbon atoms). H-bonds, salt bridges, and π-π stacking interactions are pictured as orange, magenta, and cyan dashed lines, respectively. Nonpolar hydrogen atoms are omitted.

Figure 3.

Selection of the optimal linker geometry: alkyl sulfonamide vs amide. Upper panel: docking analysis. (A) Superimposition of docking poses of sulfonamide derivative 7 (light blue carbon atoms) and amide derivative 20 (purple carbon atom) representing the starting geometry for subsequent MD simulations. (B) Detail of the shape (highlighted with a solid yellow line) of the narrow pocket surrounding the thiophene ring delimited by the conserved disulfide bridge and the EC tip of TM2 of the hP2Y₁₄R. Lower panel: lowest interaction energy (IE) structure extracted from the selected trajectory of 30 ns of MD simulation of the (C) 7-hP2Y₁₄R and (D) 20-hP2Y₁₄R complexes. Side chains of residues important for ligand recognition are reported as sticks (grey carbon atoms). H-bonds and salt bridges are pictured as orange and magenta dashed lines, respectively. Nonpolar hydrogen atoms and π-π stacking interactions are omitted. The binding site surface is color-coded by residue property (blue: positively charged, red: negatively charged, green: hydrophobic, cyan: polar). The interaction pattern around the carboxylate moiety is highlighted with a yellow circle.

Figure 4.

Fluorescent assay of specific hP2Y₁₄R binding of selected 3-(4-trifluorophenyl-1H-1,2,3- triazol-1-yl)-5-(aryl) derivatives. AlexaFluor488-labeled tracer 3a binding was determined by flow cytometry using P2Y₁₄R-expressing CHO cells. (A) 5-Thienyl 12 and 20, 5-furyl 24 and primary 4- carboxamidophenyl 30 derivatives. (B) Primary 4-substituted carboxamides with 5-pyridyl 27 and 28 and 5-pyrimidyl 29 aromatic groups. (C) N-alkyl elaboration of a potent 4-carboxamidophenyl congener 30: Me, 31; Et, 32; n-Pr, 33; tert-Bu, 34. The IC₅₀ values are given in Table 1.

Chart 1.

Progression of the structural modifications of naphthoic acid derivative 1 to the present set of derivatives 2b. The major focus was the introduction of diverse heteroaromatic rings in place of the bridging phenyl ring shown in green. The p-trifluoromethylphenyl moiety shown in red and the core benzene ring and adjacent triazole shown in blue were not modified in this study. Compound 3a is the fluorescent antagonist containing structure 1 as a pharmacophore that was used in the flow cytometric assay. Group X is a carboxylic acid or a bioisosteric replacement.

Chart 2.

Detailed general approach for the design of new derivatives. The expected orientation of the scaffold with respect to the hP2Y₁₄R is shown in a symbolic representation. Diverse 5- and 6- membered heteroaromatic rings linking the core (cyan) to the group facing the EC side of the receptor (yellow) were considered, although not all of these analogues were synthesized. Various linking groups or linkers (magenta), including amide, sulfonamide and methylene, were inserted to provide conformational diversity in the terminal alkylamino moiety. The terminal group on the spacer moiety was an H-bond donor/acceptor group, an amino, protected amino or a hydroxyl group. The spacer group consisted of either a straight chain alkyl moiety or a six-membered ring containing the secondary amine.

Scheme 1:

a) SOCl₂, CH₃OH, rt, 15 h, 98%; b) PTSA, NaNO₂ then NaN₃, H₂O, CH₃CN, rt, 15 h, 63%; c) 4-ethynyltrifluorotoluene, sodium ascorbate, CuSO₄, THF, H₂O, rt, 1 h, 84%; d) LiOH, MeOH, 60 °C, 2 h, 76%; e) B₂pin₂, KOAc, PdCl₂(dppf), dioxane, 70 °C, 15 h, 87%; f) i) 10% NaOH, H₂O₂, THF, 0 °C, 0.5 h; ii) KOH, MeOH, water, 50 °C, 5 h, 86%.

Scheme 2:

a) Boc₂O, DCM, 0 °C, 0.5 h, 70%; b) amine, Et₃N, DCM, rt, 78 – 99%; c) N-Boc piperazine, Et₃N, NaBH(OAc)₃, DCE, rt, 15 h, 90%; d) amine, HATU, DIPEA, DMF, rt, 14 – 88%; e) amine, Et₃N, CH₃CN, 0 °C, 73–99%.

Scheme 3:

a) PdCl₂(dppf), 2M Na₂CO₃, DME, 50 °C, 25 – 73%; b) Pd(PPh₃)4, Na₂CO₃ (KOAc for 79), DMF, H₂O, 11 – 43%; c) KOH, MeOH, H₂O, 50 – 70 °C, 30 – 99%; d) TFA, rt, 34 – 92%; e) AlCl₃, Me₂S, rt, 15 h, 29 – 61%. The methyl esters of 79 - 102 were hydrolyzed using KOH to give compound 4, 6, 8, 10, 11, 13, 14, 16, 17, 19, 21 and 27 - 34. The N-Boc groups of compounds 103,4,6, 8, 11, 14, 17, 19 and 21 were removed with TFA to give compound 2a, 5, 7, 9, 12, 15, 18, 20 and 22, respectively. Compound 23 and 25 - 26 were obtained using AlCL₃ and Me₂S.

Scheme 4:

a) NH₄Cl, HATU, DIPEA, DMF, rt, 1 h, 80–99%; b) (CF₃CO)₂O, Et₃N, DCM, 0 °C to rt, 1 h, 64–76%; c) TMSOTf, anisole, μW, 140 °C, 1 h, 80%; d) CuSO₄5H₂O, NaN₃, DMSO, 140 °C, 6 h, 69–99%; e) TFA, rt, 10 min; 44–74%.

Scheme 5:

a) Boc₂O, Et₃N, DCM, rt, 3 h, 80%; b) i) MsCl, Et₃N, DCM, 0 °C, 5 min; ii) NaN₃, NBu₄Br, H₂O, 100 °C, 15 h, 59%; c) KOH, MeOH, water, 50 °C, 10 h; d) i) Tf₂O, pyr, -78 °C to rt, 1 h; ii) B₂pin₂, PdCl₂(dppf), KOAc, dioxane, 85 °C, 4 h, 61%; e) tert-butyl 4-(4-bromophenyl)piperidine-1- carboxylate 55, Pd(PPh₃)₄, K₂CO₃, DMF, 80 °C, 5 h, 59%; f) TFA:THF=1:1, rt, 1 h, 93%; g) 6- bromohexyne-1, K₂CO₃, DMF, rt, 15 h, 70%; h) 117, CuSO₄5H₂O, sodium ascorbate, DCM:tBuOH:H₂O=1:1:1, rt, 15 h, 58%; i) LiOH, MeOH, 80 °C, 15 h, 76%; j) TFA, rt, 30 min, 78%; k) Ac₂O, pyr, rt, 1 h, 81%.