To synthesize the target azaindole-2-carboxamides, it requires corresponding azaindole-2-carboxylic acids (i.e., 1b and 8) as the key building blocks (Fig. 2). To access these azaindole-2-carboxylic acids, we used the Hemetsberger–Knittel indole synthesis, which involves Knoevenagel condensation between a pyridinecarbaldehyde and the azido acetate 5.²⁰ However, the condensation between 5-chloro-pyridine-3-carbaldehyde and the azido acetate 5 failed to provide the desired Knoevenagel product. In this investigation, the 6-azaindole-2-carboxylic acid 1b was obtained from a commercial source (Achemtek, Worcester, MA). In contrast, the condensation between 2-chloro-pyridine-4-carbaldehyde (4) and the azido acetate 5 successfully produced the corresponding Knoevenagel product 6, from which the required 7-azaindole-2-carboxylic acid (8) can be easily obtained. Unlike the indole-2-caboxylic acids, the coupling of the azaindole-2-carboxylic acids 1b and 8 with corresponding amines under the catalysis of HOBT or BOP and Hünig's base leads to low yields of the amide 3c-d and 9a-b. In contrast, catalyzing the coupling reaction by DMTMM and NMM generally provided the corresponding amides in good yields. The synthesized compounds were assessed by equilibrium binding assay,^25,26 which identifies two important parameters for initial characterization of allostery: K_B, the equilibrium dissociation constant that defines the affinity of an allosteric modulator for its receptor; and α, the cooperativity factor, which defines the magnitude and direction of impact that the allosteric modulator and orthosteric ligand have on each other when both occupy the receptor. At the receptor binding level, when α is >1, the ligand is a positive allosteric modulator, whereas when α is <1, the ligand is a negative allosteric modulator. Accordingly, when α is equal to 1, it indicates no allosteric modulation on orthosteric ligand binding. The K_B and α values of the synthesized compounds are presented in Table 1. In comparison with their indole-2-carboxamide counterparts 3a and 3b, the 7-azaindole-2-carboxamides 9a and 9b completely lost their binding affinity to the CB₁ receptor, likely influenced by the poor solubility of these compounds. In contrast, the 6-azaindole-2-carboxamides 3c and 3d exhibited modest binding affinity to the CB₁ receptor. Interestingly, 6-azaindole-2-carboxamide 3d showed comparable allosteric modulation on the binding of orthosteric agonist CP55,940, while its binding to the CB₁ receptor was reduced by about 25-fold in comparison with its indole-2-carboxamide counterpart 3b. These results from 9a and 9b suggested that the 7-azaindole is not an optimal bioisostere for replacing the indole ring in the class of CB₁ allosteric modulators, although it has been successfully used as an effective bioisostere in other indole-containing bioactive molecules.¹⁴

To investigate G-protein coupling induced by the allosteric modulators, the 6-azaindole-2-carboxamides 3c and 3d were tested for their impact on GTPγS binding. These results, plotted as percent of basal levels of CB₁ activity, are shown in Figure 3. Both 3c and 3d showed an inhibition of GTPγS binding at 5 and 10 μM in the presence of 0.1 μM CP55,940, compared to membranes treated with 0.1 μM of CP55,940 alone. These data suggest that although the allosteric modulators apparently enhanced the binding of CP55,940, the G-protein coupling induced by CP55,940 was inhibited. In addition, inhibition of G-protein coupling was also seen when CB₁ was treated with 3c and 3d alone. The level of G-protein coupling inhibition achievable is comparable to that observed with the inverse agonist SR141716A, although the orthosteric compound SR141716A and the allosteric modulators, 3c and 3d, likely inhibit G-protein coupling via different mechanisms.

In the solubility tests, we selected compounds 3c, 3d, and 9a because they represented the indole, 6-azaindole and 7-azaindole scaffold, respectively. We found that compound 9a has exceptionally low solubility in common organic solvents suitable for HPLC sample preparation such as methanol, acetone, and their mixtures with different stoichiometry. We tried the sampling method reported previously to prepare stock solution of 9d into a 2%–5% DMSO in Acetonitrile solution.²² Addition of aqueous PBS media led to precipitation of the compound. This made us unable to determine the standard curve for analysis of 9d. Hence, only compounds 3c and 3d were analyzed. The individual solubility of 3c, 3d, and 9a was also simulated using a program (Chemicalize). The simulated aqueous solubility of these three compounds was reported along with the experimental solubility of 3c, 3d, and 9a in Table 2.

The results from solubility studies showed that using 6- and 7-azaindole in lieu of the indole moiety led to enhancement of aqueous solubility compared to the indole counterpart although their solubility are still poor and far below optimal solubility. This finding is in agreement with the results in an early investigation, of which 6- and 7-azaindole analogs showed solubility enhancement in comparison with indole counterpart.¹⁶ The binding parameters of the synthesized compounds suggested that the 6-azaindole scaffold is worth further exploration, whereas the 7-azaindole ring is not a viable bioisostere of the indole ring in the Org27569 class of CB₁ allosteric modulators.

Although their binding affinity for CB₁ was less than their indole-2-carboxamide counterparts, they showed similar coupling characteristics as Org27569, in which they decreased G-protein coupling when compared with treatment with CP55,940 alone, indicating that they may induce signaling in a way similar to Org27569 and its analogs. Further research is needed to fully elucidate the signaling pathways of these compounds; however, it might involve beta-arrestin coupling and signaling such as Org27569 and its previously tested analogs.^20,27,28 We postulated that the reduced pharmacologic effects of the 6-azaindole-2-carboxamides in comparison with their indole counterparts are likely due to the impact of its electron-deficient pyridine ring on the aromatic π-π stacking during interaction with the allosteric binding site. Therefore, hypothetically, introduction of strong electron-donating groups such as alkoxyl, amino and alkylamino groups on the 6-azaindole moiety may help to improve the pharmacologic effects and aqueous solubility of future compounds bearing the 6-azaindole moiety.