Structure-activity relationships of uridine 5'-diphosphate analogues at the human P2Y6 receptor

Abstract

The structure-activity relationships and molecular modeling of the uracil nucleotide activated P2Y6 receptor have been studied. Uridine 5'-diphosphate (UDP) analogues bearing substitutions of the ribose moiety, the uracil ring, and the diphosphate group were synthesized and assayed for activity at the human P2Y6 receptor. The uracil ring was modified at the 4 position, with the synthesis of 4-substituted-thiouridine 5'-diphosphate analogues, as well as at positions 2, 3, and 5. The effect of modifications at the level of the phosphate chain was studied by preparing a cyclic 3',5'-diphosphate analogue, a 3'-diphosphate analogue, and several dinucleotide diphosphates. 5-Iodo-UDP 32 (EC50 = 0.15 microM) was equipotent to UDP, while substitutions of the 2'-hydroxyl (amino, azido) greatly reduce potency. The 2- and 4-thio analogues, 20 and 21, respectively, were also relatively potent in comparison to UDP. However, most other modifications greatly reduced potency. Molecular modeling indicates that the beta-phosphate of 5'-UDP and analogues is essential for the establishment of electrostatic interactions with two of the three conserved cationic residues of the receptor. Among 4-thioether derivatives, a 4-ethylthio analogue 23 displayed an EC50 of 0.28 microM, indicative of favorable interactions predicted for a small 4-alkylthio moiety with the aromatic ring of Y33 in TM1. The activity of analogue 19 in which the ribose was substituted with a 2-oxabicyclohexane ring in a rigid (S)-conformation (P = 126 degrees , 1'-exo) was consistent with molecular modeling. These results provide a better understanding of molecular recognition at the P2Y6 receptor and will be helpful in designing selective and potent P2Y6 receptor ligands.

Figure 1

Molecular models of the complexes of the human P2Y₆ receptor with UDP analogues 32, (a and b, 5-iodo), 6 (c, (S)-methanocarba), 31 (d, 3-methyl), 23 (e, 4-ethylthio), and 28 (f, 4-carboxyethylthio). In all cases, the diphosphate moiety of the nucleotide is coordinated by three cationic residues from TM3 (3.29), TM6 (6.55), and TM7 (7.39). The donation of an H-bond from the NH at position 3 of the uracil ring to Y33(1.39) seems to contribute to higher potency in the activation of the P2Y₆ receptor. To facilitate the comparison, in the six panels of this figure the receptor maintains the same orientation relative to the plane of the membrane. In the schematic representation of the P2Y₆ receptor complexed with Cpd. 32 (a), the tube represents the backbone of the receptor and is colored according to residue positions, with a spectrum of colors that ranges from red (N-terminus) to purple (Cterminus): TM1 is in orange, TM2 in ochre, TM3 in yellow, TM4 in green, TM5 in cyan, TM6 in blue, TM7 in purple.

Figure 2

Graphical representation of the pseudorotational cycle, which defines all of the possible ribose ring conformations. The phase angle of pseudorotation (P) describes the geometry of the ribose puckering. The (N)-methanocarba ring system adopts a C2′-exo conformation and the (S)-methanocarba ring system a C3′-exo conformation. The 2-OBH ring system maintains a C1′-exo conformation, in the (S) region.

Scheme 1

Preparation of ribose- and uracil-modified UDP analogues. Reagents and conditions: (i) (1) POCl₃, proton sponge, PO(OMe)₃, 0 °C; (2) (Bu₃NH⁺)₂PO₄H, Bu₃N, DMF, 0 °C; (ii) CF₃COOEt, DIEA, DMF, rt. (iii) H₂, Pd/C, MeOH, rt.

Scheme 2

Preparation of 2-OBH UDP analogue. Reagents and conditions: (i) (1) POCl₃, proton sponge, PO(OMe)₃, 0 °C; (2) (Bu₃NH⁺)₂PO₄H, Bu₃N, DMF, 0 °C.

Scheme 3

Preparation of 4-substituted-thio-UDP analogues. Procedure B. Reagents and conditions: (i) (1) 0.25 M NaOH, MeOH, rt; (2) RX, DMF, rt; (ii) 0.25 M NaOH, H₂O, rt.

Scheme 4

Preparation of 4-substituted-thio-UDP analogues. Procedure C. Reagents and conditions: (i) (1) 0.25 M NaOH, MeOH, rt; (2) R₁X, DMF, 90 °C; (ii) (1) 1,1’-carbonyldiimidazole, DMF, rt; (2) Et₃N 5% in H₂O/MeOH 1/1, rt; (3) (Bu₃NH⁺)₂PO₄H, Bu₃N, DMF, rt; (iii) 0.25 M NaOH, H₂O, rt.

Scheme 5

Preparation of 5-halo and phosphate- and ribose-modified UDP analogues. Reagents and conditions: (i) POCl₃, proton sponge, PO(OMe)₃, 0 °C; (ii) (1) 1,1’- carbonyldiimidazole, DMF, rt; (2) Et₃N 5% in MeOH, rt; (3) (Bu₃NH⁺)₂PO₄H^2-, Bu₃N, DMF, rt.

Chart 1

Structures of UDP and various analogues and the reported EC₅₀ values in stimulation of PLC through the recombinant P2Y₆ receptor (human, unless noted).^,,,, The EC₅₀ for INS48823 is similar to UDP.