CoMFA contour maps analysis was performed to visualize the important region in 3D molecules where the steric and electrostatic fields may affect the affinity and selectivity of the studied compounds in the cannabinoid CB1 and CB2 receptors. The weight of StDev*Coeff was used to calculate the field energies for all fields in CoMFA models. The highly active compound 90 was shown as the template ligand for all contour maps. CB1 and CB2 contour maps were generated from binding affinity of series of indole ligands evaluated at recombinant human CB1 and CB2 receptors. The steric and electrostatic contour maps of CoMFA are displayed in Figure 3. In order to systematize the analysis and obtain useful information on SAR, we had divided the molecule into three regions:

Region I. Close examination of the CoMFA maps allows us to see in the steric CB1 model a large green polyhedron around this region, which continues along the entire region III. At a greater distance from the green polyhedron in region I, we can see a yellow region. This suggests that bulky substituents at any position of the benzene will increase biological activity and selectivity for this receptor. However, as it was already mentioned, the yellow region restricts the size and length of the substitutions to some extent in the position 5 of the indole system. In the steric CB2 model, the big restrictive contour map near the positions 4 and 5 of the indole core limits drastically the possibility of substitute these positions. In fact, the most active compounds in the CB2 receptor (compounds 33, 39, 41, 66 and 90) bear a hydrogen atom in positions 4 and 5. On the other hand, in the electrostatic contour maps, the CB1 model indicates a red area contiguous to the positions 4 and 5 of the indole nucleus. According to this, electronegative substituents can be connected to the benzene or it is possible to replace the ring by an isostere with electronegative properties such as a thiophene or pyrrole. In the CB2 model the data implicate an inverse situation. In this case would be favorable the insertion of electropositive groups or the exchange for rings capable of protonation at physiological pH, such as pyridine, pyrimidine, piperidine and aniline, among others. Region I is, therefore, from the electronic standpoint a key area for selectivity.

Region II. The steric contour maps show that region II is inside a yellow polyhedron in CB1, while it is inside a green contour map surrounded by two yellow areas in CB2. This noteworthy difference suggests that a controlled increase of the size of the ring substituents or the replacement of this ring for bulkier systems like naphthoyl (for example in compounds 82 and 83), will raise the selectivity for the CB2 receptor. In fact, the comparative lower CB2 receptor Ki values for compounds 38–42 (Ki < 20 nM), are consistent with the existence of bulky rings in region II, and, as expected, they displayed less affinity for the CB1 receptor (Ki > 60 nM in all cases). On the other hand, there are not electrostatic surfaces around the region II in the CB1 model. The absence of information in this particular region is consequence of the similarity of the rings occupying that area which are electrically neutral: cyclopropyl, adamantyl, cyclohexyl, and phenyl. This, however, rather than being a limitation in the design, provides the ability to evaluate multiple electronic options. However, the CoMFA for CB2 shows the region II completely immersed in a red polyhedron. This suggests that when adding electronegative atoms or groups such as oxygen, halogens, sulfur, and no protonable nitrogens in this region, the CB2 affinity is boosted. As an example, compound 25, bearing an oxaadamantyl moiety in this region, and compound 36, with a 2-iodo-5-nitrophenyl, have 90 and 110 times greater affinity for the CB2 receptor than CB1, respectively.

Region III. As can be seen for compound 90, there is a green surface along the N-alkyl chain in the CB1 model but only a terminal green polyhedron in the CB2 model. This suggests that a branched chain would increase the affinity for the CB1 receptor, and the substitution with bulky groups at the end of it would be better for the CB2 affinity. Analogously to region II, in region III there are not any electrostatic contour maps in CB1 CoMFA model, while a blue region is observed in the CB2 receptor. This suggests that the introduction of protonable or electropositive groups would increase CB2 affinity. For example compounds 49, 72, 73 and 80 share the same structure but differ in the type of nitrogen at the end of the N-alkyl chain. Compounds 49 and 80 have the less basic nitrogen, whereas compounds 72 and 73 have strong basic sp3 nitrogen displaying 10 times higher CB2 affinity than compounds 49 and 80. This may suggests the presence of a hydrogen bond or ionic interaction in the stabilization of the complex ligand-receptor.