The importance of hydrogen bonding and aromatic stacking to the affinity and efficacy of cannabinoid receptor CB2 antagonist, 5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-N-[(1S,2S,4R)-1,3,3-trimethylbicyclo[2.2.1]hept-2-yl]-1H-pyrazole-3-carboxamide (SR144528)

Abstract

Despite the therapeutic promise of the subnanomolar affinity cannabinoid CB2 antagonist, 5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-N-[(1S,2S,4R)-1,3,3-trimethylbicyclo[2.2.1]hept-2-yl]-1H-pyrazole-3-carboxamide (SR144528, 1), little is known about its binding site interactions and no primary interaction site for 1 at CB2 has been identified. We report here the results of Glide docking studies in our cannabinoid CB2 inactive state model that were then tested via compound synthesis, binding, and functional assays. Our results show that the amide functional group of 1 is critical to its CB2 affinity and efficacy and that aromatic stacking interactions in the TMH5/6 aromatic cluster of CB2 are also important. Molecular modifications that increased the positive electrostatic potential in the region between the fenchyl and aromatic rings led to more efficacious compounds. This result is consistent with the EC-3 loop negatively charged amino acid, D275 (identified via Glide docking studies) acting as the primary interaction site for 1 and its analogues.

Figure 1

The molecular electrostatic potential maps of the docked conformations of compounds 1–6 are illustrated here. The electrostatic potential scale (in kJ/mol) is provided as a color scale. This scale is from blue (most electropositive) to red (most electronegative).

Figure 2

This figure illustrates the final Compound 1/CB₂ R complex. The view is from the lipid bilayer, with TMH6 and TMH7 closest to the viewer. The EC-3 loop residue, D2.75 (cyan) has been used as the primary interaction site for the most electropositive region of 1 (amide hydrogen). Residues displayed in orange are those for which the ligand interaction energy exceeded −3.50 kcal/mol (i.e., Int E<−3.50 kcal/mol). Additional interactions are present in the docked complex. Details of all interactions are provided in Tables S1 in Supporting Information.

Figure 3

This figure illustrates the final Compound 2/CB₂ R complex. In Compound 2, the amide group has been replaced with a trans-ethylene group to mimic the geometry of amide group in 1. The view is from the lipid bilayer, with TMH6 and TMH7 closest to the viewer. Glide docking studies reveal that 2 has highest electrostatic and Van der Waals interactions with K3.28(109) (magenta). Residues displayed in orange are those for which the ligand interaction energy exceeded −3.50 kcal/mol (i.e., Int E<−3.50 kcal/mol). Additional interactions are present in the docked complex. Details of all interactions are provided in Tables S2 in Supporting Information.

Figure 4

This figure illustrates the final Compound 3/CB₂ R complex. Compound 3 has a pyrrole central ring instead of a pyrazole so it has extra steric bulk where the N is substituted with CH moiety. The view is from the lipid bilayer, with TMH6 and TMH7 closest to the viewer. Here the amide group of 3 forms a hydrogen bond with D275 (orange). Residues displayed in orange are those for which the ligand interaction energy exceeded −3.50 kcal/mol (i.e., Int E<−3.50 kcal/mol). Additional interactions are present in the docked complex. Details of all interactions are provided in Tables S3 in Supporting Information.

Figure 5

This figure illustrates the final Compound 4/CB₂ R complex. The view is from the lipid bilayer, with TMH6 and TMH7 closest to the viewer. Compound 4 has no hydrogen bonding capability because it lacks both the amide group and has a pyrrole instead of a pyrazole central ring. However, 4 has all the aromatic stacking interactions found for 1, 2, 3 and 5. Residues displayed in orange are those for which the ligand interaction energy exceeded −3.50 kcal/mol (i.e., Int E<−3.50 kcal/mol). Additional interactions are present in the docked complex. Details of all interactions are provided in Tables S4 in Supporting Information.

Figure 6

This figure illustrates the final Compound 5/CB₂ R complex. The view is from the lipid bilayer, with TMH6 and TMH7 closest to the viewer. Compound 5 has a bornyl ring substituted for the fenchyl ring of 1. The main interaction energy contributor in the complex is D275 which forms a hydrogen bond with the amide group of 5. Residues displayed in orange are those for which the ligand interaction energy exceeded −3.50 kcal/mol (i.e., Int E<−3.50 kcal/mol). Additional interactions are present in the docked complex. Details of all interactions are provided in Tables S5 in Supporting Information.

Figure 7

This figure illustrates the final Compound 6/CB₂ R complex. The view is from the lipid bilayer, with TMH6 and TMH7 closest to the viewer. The lack of the methyl extension of the methyl phenyl ring causes a repositioning of 6 in the binding pocket such that the amide group of 6 is close enough to D275 to have favorable electrostatic interaction with it, but not close enough to form a hydrogen bond. Residues displayed in orange are those for which the ligand interaction energy exceeded −3.50 kcal/mol (i.e., Int E<−3.50 kcal/mol). Additional interactions are present in the docked complex. Details of all interactions are provided in Tables S6 in Supporting Information.

Chart 1

Chart 2

a) H₂NNH₂, HOAc ; b) NaH; c) LiAlH₄; d) PBr₃; e) Ph₃P; f) nBuLi; g) 15; h) Ph₂POCH₂OCH₃, LDA; i) Cl₃C-CO₂H, (85:15 endo:exo);

Chart 3

a) HCO₂H; b) POCl₃; c) NaH; d) ethyl acrylate, 21; e) 9; f) KOH, aq MeOH; g) SOCl₂; h) (IS)-endo-fenchylamine, 24

Chart 4

a) LiAlH₄, THF; b) CBr₄, Ph₃P; c) Dess-Martin periodinane, CH₂Cl₂, r.t.; d) G6, nBuLi, THF; e) MgBr₂•Et₂O, dioxane, 100 C; f) Ph₂POCH₂OCH₃, LDA; g) Cl₃C-CO₂H, (85:15 endo:exo); h) (MeO)₂P(O)H, LiHMDSi, THF, −78 C; i) S=C-im₂, THF, 50 C; j) Bu₃SnH, AIBN

Chart 5

a) KOH, aq MeOH; b) SOCl₂; c) 38; d) H₂NOH; e) Na⁰

Chart 6

a) Phenylhydrazine HCl, MeOH, b) NaOH, EtOH, c) SOCl₂; d) (IS)-endo-fenchylamine, 24