Motifs in Natural Products as Useful Scaffolds to Obtain Novel Benzo[d]imidazole-Based Cannabinoid Type 2 (CB2) Receptor Agonists

The endocannabinoid system (ECS) constitutes a broad-spectrum modulator of homeostasis in mammals, providing therapeutic opportunities for several pathologies. Its two main receptors, cannabinoid type 1 (CB1) and type 2 (CB2) receptors, mediate anti-inflammatory responses; however, their differing patterns of expression make the development of CB2-selective ligands therapeutically more attractive. The benzo[d]imidazole ring is considered to be a privileged scaffold in drug discovery and has demonstrated its versatility in the development of molecules with varied pharmacologic properties. On the other hand, the main psychoactive component of Cannabis sativa, delta-9-tetrahydrocannabinol (THC), can be structurally described as an aliphatic terpenoid motif fused to an aromatic polyphenolic (resorcinol) structure. Inspired by the structure of this phytocannabinoid, we combined different natural product motifs with a benzo[d]imidazole scaffold to obtain a new library of compounds targeting the CB2 receptor. Here, we synthesized 26 new compounds, out of which 15 presented CB2 binding and 3 showed potent agonist activity. SAR analysis indicated that the presence of bulky aliphatic or aromatic natural product motifs at position 2 of the benzo[d]imidazoles ring linked by an electronegative atom is essential for receptor recognition, while substituents with moderate bulkiness at position 1 of the heterocyclic core also participate in receptor recognition. Compounds 5, 6, and 16 were further characterized through in vitro cAMP functional assay, showing potent EC50 values between 20 and 3 nM, and compound 6 presented a significant difference between the EC50 of pharmacologic activity (3.36 nM) and IC50 of toxicity (30–38 µM).


Introduction
Natural products are known as a major source of chemical diversity, providing medicinal products throughout history [1,2]. Notable examples where natural products have become successful therapeutic agents include the analgesic morphine, the anticancer drug vincristine, and the antimalarial artemisinin [2].
Evidence, thus far, shows the ubiquitous presence of the components of the endocannabinoid system (ECS) across the human body, including both central and peripheral tissues. The widespread nature of the ECS highlights its role as a broad-spectrum modulator of homeostasis, bringing forth its therapeutic potential for the treatment of inflammation, and neurodegenerative disorders [3][4][5].
The ECS is a lipid signaling system, and the primary receptor proteins are the binoid receptors type I (CB1) and type II (CB2), which are members of the G-prote pled receptor (GPCR) family and signal through Gi-mediated mechanisms. Endo ligands that activate the cannabinoid receptors include 2-AG (2-arachidonoyl g and anandamide (AEA [N-arachidonoyl ethanolamine]), although other structur lated lipids have also been identified as endocannabinoids. Additionally, enzymes ated with the biosynthesis of endocannabinoids include NAPE-PLD (N-acylphosph ethanolamine-specific phospholipase D-like hydrolase) and DAGLα/β (Diacylgly pase α/β), which catalyze the biosynthesis of AEA and 2-AG, respectively, as well a responsible for endocannabinoid degradation, such as FAAH (fatty acid amide hyd and MAGL (monoacylglycerol lipase) [6,7].
Both the CB1 and CB2 receptors mediate anti-inflammatory responses but sh ferent patterns of expression [8]. While the CB1 receptor is mainly expressed wit CNS where it can be associated with the psychoactive property of marijuana, CB2 abundant in the immune tissues. Thus, the development of CB2-selective ligands an opportunity to regulate inflammatory responses while avoiding the psychoac fects associated with CB1 activation [9,10].
Within this context, our group has previously worked on the developm benzo [d]imidazole-based small molecules that target the cannabinoid receptors [ This heterocycle can be considered as a privileged scaffold in drug discovery a demonstrated its versatility as a framework to develop molecules with diverse p cologic properties [17][18][19][20][21]. On the other hand, tetrahydrocannabinol (THC) is th psychoactive component of Cannabis sativa, with affinity for both cannabinoid re ( Figure 1) [22,23]. This molecule can be structurally related to a terpenoid motif f an aromatic polyphenolic (resorcinol) structure. Inspired by the chemical structure phytocannabinoid and considering our previous experience with benzo [d]imidaz rivatives as an effective scaffold to develop cannabinoid ligands, we sought to c different natural product motifs with the heterocyclic core to access a new library pounds targeting the cannabinoid receptors. Various medicinal properties, such oxidant, antibacterial, and anti-inflammatory action, have been commonly associat natural products [24][25][26], with many of them being designated as GRAS (Genera ognized as Safe) substances by the FDA [27]. Therefore, the strategy to combine products with a synthetic scaffold can complement the chemical space of cannabin ands with interesting pharmacologic properties.
Herein, we report the synthesis and pharmacological characterization of a new of CB2 ligands based on natural product motifs conjugated to a benzo[d]imidazo compounds were evaluated for CB2 affinity using a radioligand binding assay and activity through cAMP accumulation assay.

Design of Compounds
The general structure of the synthesized compounds is outlined in Table 1. Our previous studies on benzo [d]imidazole derivatives suggested that substitutions at position 2 of the heterocycle with bulky and hydrophobic groups were preferred for CB2 affinity, and the presence of electronegative substituents at the same position could be favorable [16]. Therefore, hydrocarbons, such as adamantane, terpenes, and polyphenols (resorcinol), which fulfill these characteristics, were chosen to be functionalized at position 2 of the benzo[d]imidazole scaffold using either an oxygen or sulfur linker. Additionally, to probe the steric requirements at position 1 of the heterocycle, both short-chain hydrocarbon and bulky aromatic groups were substituted at this position.

Design of Compounds
The general structure of the synthesized compounds is outlined in Table 1. Our previous studies on benzo[d]imidazole derivatives suggested that substitutions at position 2 of the heterocycle with bulky and hydrophobic groups were preferred for CB2 affinity, and the presence of electronegative substituents at the same position could be favorable [16]. Therefore, hydrocarbons, such as adamantane, terpenes, and polyphenols (resorcinol), which fulfill these characteristics, were chosen to be functionalized at position 2 of the benzo[d]imidazole scaffold using either an oxygen or sulfur linker. Additionally, to probe the steric requirements at position 1 of the heterocycle, both short-chain hydrocarbon and bulky aromatic groups were substituted at this position.

Design of Compounds
The general structure of the synthesized compounds is outlined in Table 1. Our previous studies on benzo[d]imidazole derivatives suggested that substitutions at position 2 of the heterocycle with bulky and hydrophobic groups were preferred for CB2 affinity, and the presence of electronegative substituents at the same position could be favorable [16]. Therefore, hydrocarbons, such as adamantane, terpenes, and polyphenols (resorcinol), which fulfill these characteristics, were chosen to be functionalized at position 2 of the benzo[d]imidazole scaffold using either an oxygen or sulfur linker. Additionally, to probe the steric requirements at position 1 of the heterocycle, both short-chain hydrocarbon and bulky aromatic groups were substituted at this position.

Design of Compounds
The general structure of the synthesized compounds is outlined in Table 1. Our previous studies on benzo[d]imidazole derivatives suggested that substitutions at position 2 of the heterocycle with bulky and hydrophobic groups were preferred for CB2 affinity, and the presence of electronegative substituents at the same position could be favorable [16]. Therefore, hydrocarbons, such as adamantane, terpenes, and polyphenols (resorcinol), which fulfill these characteristics, were chosen to be functionalized at position 2 of the benzo[d]imidazole scaffold using either an oxygen or sulfur linker. Additionally, to probe the steric requirements at position 1 of the heterocycle, both short-chain hydrocarbon and bulky aromatic groups were substituted at this position.

Design of Compounds
The general structure of the synthesized compounds is outlined in Table 1. Our previous studies on benzo[d]imidazole derivatives suggested that substitutions at position 2 of the heterocycle with bulky and hydrophobic groups were preferred for CB2 affinity, and the presence of electronegative substituents at the same position could be favorable [16]. Therefore, hydrocarbons, such as adamantane, terpenes, and polyphenols (resorcinol), which fulfill these characteristics, were chosen to be functionalized at position 2 of the benzo[d]imidazole scaffold using either an oxygen or sulfur linker. Additionally, to probe the steric requirements at position 1 of the heterocycle, both short-chain hydrocarbon and bulky aromatic groups were substituted at this position.

Design of Compounds
The general structure of the synthesized compounds is outlined in Table 1. Our previous studies on benzo[d]imidazole derivatives suggested that substitutions at position 2 of the heterocycle with bulky and hydrophobic groups were preferred for CB2 affinity, and the presence of electronegative substituents at the same position could be favorable [16]. Therefore, hydrocarbons, such as adamantane, terpenes, and polyphenols (resorcinol), which fulfill these characteristics, were chosen to be functionalized at position 2 of the benzo[d]imidazole scaffold using either an oxygen or sulfur linker. Additionally, to probe the steric requirements at position 1 of the heterocycle, both short-chain hydrocarbon and bulky aromatic groups were substituted at this position.

Design of Compounds
The general structure of the synthesized compounds is outlined in Table 1. Our previous studies on benzo[d]imidazole derivatives suggested that substitutions at position 2 of the heterocycle with bulky and hydrophobic groups were preferred for CB2 affinity, and the presence of electronegative substituents at the same position could be favorable [16]. Therefore, hydrocarbons, such as adamantane, terpenes, and polyphenols (resorcinol), which fulfill these characteristics, were chosen to be functionalized at position 2 of the benzo[d]imidazole scaffold using either an oxygen or sulfur linker. Additionally, to probe the steric requirements at position 1 of the heterocycle, both short-chain hydrocarbon and bulky aromatic groups were substituted at this position.

Design of Compounds
The general structure of the synthesized compounds is outlined in Table 1. Our previous studies on benzo[d]imidazole derivatives suggested that substitutions at position 2 of the heterocycle with bulky and hydrophobic groups were preferred for CB2 affinity, and the presence of electronegative substituents at the same position could be favorable [16]. Therefore, hydrocarbons, such as adamantane, terpenes, and polyphenols (resorcinol), which fulfill these characteristics, were chosen to be functionalized at position 2 of the benzo[d]imidazole scaffold using either an oxygen or sulfur linker. Additionally, to probe the steric requirements at position 1 of the heterocycle, both short-chain hydrocarbon and bulky aromatic groups were substituted at this position.

2-alkoxybenzo[d]
imidazoles were synthesized by reacting the corresponding alcohol reagent in an aromatic nucleophilic substitution reaction. For aliphatic alcohol derivatives (Scheme 1), 2-chlorobenzo[d]imidazole was first alkylated at position 1 with the corresponding alkyl halides to obtain 1-alkyl-2-chlorobenzo[d]imidazoles I-III. Then, alcohols l-menthol, 1-adamantanol, and geraniol were reacted with I-III in the presence of NaH through nucleophilic aromatic substitution to yield products 4-6, 10-12, and 16-17. Unfortunately, naphthyl derivative of 1-adamantanol could not be obtained through this procedure, possibly due to steric hindrance of the bulky napthyl substituent, which impedes the substitution. Additionally, when the same synthetic methodology was carried out using anisyl alcohol, the alcoxy-substituted product could not be identified, and only a side product presumed to be 1-alkyl-2-benzo[d]imidazolone (analyzed by NMR) was identified.

2-alkoxybenzo[d]
imidazoles were synthesized by reacting the corresponding alcohol reagent in an aromatic nucleophilic substitution reaction. For aliphatic alcohol derivatives (Scheme 1), 2-chlorobenzo[d]imidazole was first alkylated at position 1 with the corresponding alkyl halides to obtain 1-alkyl-2-chlorobenzo[d]imidazoles I-III. Then, alcohols l-menthol, 1-adamantanol, and geraniol were reacted with I-III in the presence of NaH through nucleophilic aromatic substitution to yield products 4-6, 10-12, and 16-17. Unfortunately, naphthyl derivative of 1-adamantanol could not be obtained through this procedure, possibly due to steric hindrance of the bulky napthyl substituent, which impedes the substitution. Additionally, when the same synthetic methodology was carried out using anisyl alcohol, the alcoxy-substituted product could not be identified, and only a side product presumed to be 1-alkyl-2-benzo[d]imidazolone (analyzed by NMR) was identified.

2-alkoxybenzo[d]
imidazoles were synthesized by reacting the corresponding alcohol reagent in an aromatic nucleophilic substitution reaction. For aliphatic alcohol derivatives (Scheme 1), 2-chlorobenzo[d]imidazole was first alkylated at position 1 with the corresponding alkyl halides to obtain 1-alkyl-2-chlorobenzo[d]imidazoles I-III. Then, alcohols l-menthol, 1-adamantanol, and geraniol were reacted with I-III in the presence of NaH through nucleophilic aromatic substitution to yield products 4-6, 10-12, and 16-17. Unfortunately, naphthyl derivative of 1-adamantanol could not be obtained through this procedure, possibly due to steric hindrance of the bulky napthyl substituent, which impedes the substitution. Additionally, when the same synthetic methodology was carried out using anisyl alcohol, the alcoxy-substituted product could not be identified, and only a side product presumed to be 1-alkyl-2-benzo[d]imidazolone (analyzed by NMR) was identified.

4-6 10-12 16-17
For aryl alcohol derivatives, 1-alkyl-2-benzylsulphonylbenzimidazoles IV-VI were first synthesized, as described in Scheme 2. 2-(benzylthio)-1H-benzo[d]imidazole was first alkylated at position 1 with the corresponding alkyl halide, and the resulting dialkylated thioether was oxidized to the corresponding sulphone derivative using m-CPBA. Lastly, sulphones VII-IX were reacted with resorcinol and 1,1-dimethylheptylresorcinol via nucleophilic aromatic substitution reaction to yield products 21-26. The synthesis of 2-thioxybenzo[d]imidazole derivatives is described in Schemes 3 and 4. Compounds 1-3 were obtained by first tosylating l-menthol and geraniol using the procedure described by Hartung et al. [28]. The obtained products were then employed in the alkylation of 2-mercaptobenzo[d]imidazole to yield compounds XII-XIII, which were alkylated with the corresponding alkyl halide using the same procedure described before to obtain compounds 1-3 and 7-9. The synthesis of 2-thioxybenzo[d]imidazole derivatives is described in Schemes 3 and 4. Compounds 1-3 were obtained by first tosylating l-menthol and geraniol using the procedure described by Hartung et al. [28]. The obtained products were then employed in the alkylation of 2-mercaptobenzo[d]imidazole to yield compounds XII-XIII, which were alkylated with the corresponding alkyl halide using the same procedure described before to obtain compounds 1-3 and 7-9. For compounds 13-15, 1-adamantanol was reacted with 2-mercaptobenzo[d]imidazole via SN1 conditions using CF3COOH as a solvent, and the obtained thioxybenzo[d]imidazole XIV was alkylated with the corresponding naphthyl, benzyl, and ethyl halides at position 1, as described above. For compounds 18-20, 2-mercaptobenzo[d]imidazole was selectively monoalkylated at position 2 using an equivalent of anisyl chloride in the presence of TBAB and a base to give compound XV, which was further alkylated at position 1 using the same alkyl halides mentioned above.

Radioligand Displacement Assay
To assess ligand binding to CB2 receptors, radioligand displacement assay at a single dose (10 µM) was performed in membranes obtained from recombinant CHO cells expressing human CB2 receptors (Eurofins Cerep SA, France). The results are presented in Table 2 and Figure 2. Out of 26 compounds, more than 50% of the molecules presented >50% displacement, while 20% of the compounds (5, 6, 16, 19, 22) presented >80% displacement of radioligand binding at a 10 µM dose. Compounds 5, 6, 16, 19, and 22 were further tested for CB2 receptor activation (agonist activity, see below) and CB2/CB1 selectivity. For compounds 13-15, 1-adamantanol was reacted with 2-mercaptobenzo[d]imidazole via SN1 conditions using CF3COOH as a solvent, and the obtained thioxybenzo[d]imidazole XIV was alkylated with the corresponding naphthyl, benzyl, and ethyl halides at position 1, as described above. For compounds 18-20, 2-mercaptobenzo[d]imidazole was selectively monoalkylated at position 2 using an equivalent of anisyl chloride in the presence of TBAB and a base to give compound XV, which was further alkylated at position 1 using the same alkyl halides mentioned above.

Radioligand Displacement Assay
To assess ligand binding to CB2 receptors, radioligand displacement assay at a single dose (10 µM) was performed in membranes obtained from recombinant CHO cells expressing human CB2 receptors (Eurofins Cerep SA, France). The results are presented in Table 2 and Figure 2. Out of 26 compounds, more than 50% of the molecules presented >50% displacement, while 20% of the compounds (5, 6, 16, 19, 22) presented >80% displacement of radioligand binding at a 10 µM dose. Compounds 5, 6, 16, 19, and 22 were further tested for CB2 receptor activation (agonist activity, see below) and CB2/CB1 selectivity. For compounds 13-15, 1-adamantanol was reacted with 2-mercaptobenzo[d]imidazole via S N 1 conditions using CF 3 COOH as a solvent, and the obtained thioxybenzo[d]imidazole XIV was alkylated with the corresponding naphthyl, benzyl, and ethyl halides at position 1, as described above. For compounds 18-20, 2-mercaptobenzo[d]imidazole was selectively monoalkylated at position 2 using an equivalent of anisyl chloride in the presence of TBAB and a base to give compound XV, which was further alkylated at position 1 using the same alkyl halides mentioned above.

cAMP Accumulation Assay
Compounds were further characterized through in vitro functional assays by me uring the variation in forskolin-induced cAMP accumulation (Eurofins Cerep servic The tested compounds diminished the accumulation of cyclic AMP, indicating activity agonists (Figure 3).

cAMP Accumulation Assay
Compounds were further characterized through in vitro functional assays by measuring the variation in forskolin-induced cAMP accumulation (Eurofins Cerep services). The tested compounds diminished the accumulation of cyclic AMP, indicating activity as agonists (Figure 3). As shown in Table 3, all the values of EC50 varied within the nanomolar range of Data for compounds 19 and 22 could not be determined. WIN55212−2 was assessed in parallel for all assays; data presented as normalized response to 100 nM WIN55212−2 maximum response. Table 3, all the values of EC50 varied within the nanomolar range of activity, with compound 16 presenting an EC50 value of 20 nM, compound 5 14 nM, and the most potent, compound 6 3.36 nM.

CB1/CB2 Receptor Selectivity
To assess the compound selectivity between CB1 and CB2 receptors, binding constants were determined through a radioligand displacement assay by testing concentrationresponse curves for compounds 5, 6, 16, 19, and 22. As shown in Figure 4 and summarized in Table 4, three of the tested compounds (5, 19, and 22) presented moderate binding affinity in a low micromolar range to both CB1 and CB2 receptors. However, compounds 6 and 16 showed improved selectivity profiles, with at least ten-fold higher affinity toward the CB2 receptor. Noteworthily, although the tested compounds showed moderate binding affinities within the micromolar range, agonist activity measured through functional assays presented nanomolar values, with compound 6 being the most potent agonist (EC50 = 3.36 nM).

Molecular Docking
Compounds 5, 6, and 16 were further studied through molecular docking to insight into their binding mode within the orthosteric pocket of the CB2 receptor (F

Molecular Docking
Compounds 5, 6, and 16 were further studied through molecular docking to gain insight into their binding mode within the orthosteric pocket of the CB2 receptor ( Figure 5A-D). Docking was performed using the available cryo-EM structure of the CB2 receptor bound to WIN55212-2 (PDB ID: 6PT0).

Molecular Docking
Compounds 5, 6, and 16 were further studied through molecular docking to gain insight into their binding mode within the orthosteric pocket of the CB2 receptor ( Figure  5A-D). Docking was performed using the available cryo-EM structure of the CB2 receptor bound to WIN55212-2 (PDB ID: 6PT0).

Neutral Red Uptake Assay
Compound 6 was tested through a neutral red uptake assay, and cell viability was measured. Neutral red consists of a cationic dye that accumulates in lysosomes. Uptake of neutral red depends on a viable cell's capacity to maintain acidic pH in the interior of lysosomes [29]. Figure 6 and Table 5

Neutral Red Uptake Assay
Compound 6 was tested through a neutral red uptake assay, and cell viability was measured. Neutral red consists of a cationic dye that accumulates in lysosomes. Uptake of neutral red depends on a viable cell's capacity to maintain acidic pH in the interior of lysosomes [29]. Figure 6 and Table 5    Cell Line IC50 Viability (µM) Figure 6. Dose-response curve of neutral red uptake assay of compound 6 in two different cell lines: HEK-293 and MCF-7. Data presented as normalized response relative to control (DMSO). Experiments were performed in triplicate.

Discussion
The structural information obtained from Table 2 and Figure 2 showed that derivatives with either aliphatic or aromatic natural product motifs presented activities that spanned from limited to excellent, suggesting that the chemical nature of the motif has little impact on CB2 receptor recognition. Additionally, bulky groups such as adamantyl were well tolerated, but longer motifs in R2 seem to be detrimental for affinity, as geranyl and DMH derivatives presented lower percentages of radioligand displacement (compounds 10-12 and 24-26). Regarding the effect of the linker atom, by comparing compounds 1-3 (sulfur linker) with compounds 4-6 (oxygen linker), higher inhibition was observed for oxygen linker derivatives. This is also true when comparing compounds 14-15 (sulfur linker) and 16-17 (oxygen linker) with oxygen derivatives presenting equivalent or superior inhibition percentage.
Therefore, the data indicate that the presence of an oxygen linker is more favorable for affinity, in agreement with our previous QSAR study [16], which suggested that the presence of electronegative atoms at position 2 of benzo[d]imidazoles could increase the activity. Some exceptions are geranyl derivatives (compounds 7-12), where alkoxy derivatives present lower binding inhibition than their thioxy counterparts. Nevertheless, these geranyl derivatives can be considered as part of the "elongated" series of compounds, which were unfavorable for activity, as discussed before. Thus, maintaining the adequate size of the substituent at position 2 of benzo[d]imidazoles seems to be of greater importance than the presence of electronegative atoms at the same position. Additionally, the difference in atomic size between oxygen and sulphur linker atoms, which determines a change in the angle between the two substituent groups, could play a role in the proper orientation of the compounds within the binding site.
Regarding the effect of R1 substituents, again, the size of the introduced group affects binding affinity. The presence of an ethyl group at R1 yielded compounds with moderate to excellent affinity (compounds 3, 6, 9, 15, 17, 20, 23, and 26). Nevertheless, changing this group to a benzyl substituent maintained or even enhanced activity (compounds 2, 5, 8,  14, 16, 19, 22). However, the introduction of a bulkier naphthalen-1-ylmethyl substituent greatly diminished receptor recognition (compounds 4, 7, 13, 18 and 21) and, in some cases, was detrimental for binding (compound 1). Thus, the data suggest that the presence of lipophilic groups with moderate bulkiness is preferable on R1.
Based on the results obtained from the radioligand displacement assay, compounds 5, 6, 16, 19, and 22 were selected and analyzed through a cAMP accumulation assay in recombinant cells expressing CB2 receptors, and agonist activity was confirmed (Table 3). Nevertheless, statistical analyses could not be performed for compounds 19 and 22 (EC50 not determined). In the case of compounds 5, 6, and 16, agonist activities in the nanomolar range were observed, with compound 6 (a menthol derivative) presenting the best profile, with an EC50 of 3.3 nM. Interestingly, although this compound presented potent agonist behavior over the CB2 receptor (as measured by cAMP accumulation), the affinity was near the micromolar range, indicating that compound 6 exerts a high pharmacological response at moderate affinity. Regarding the binding selectivity between CB1 and CB2 receptors (Table 4), the five tested compounds showed moderate selectivity indexes, but compound 6, the most potent identified compound, presented 20-fold higher recognition toward the CB2 receptor according to the Ki(CB1)/Ki(CB2) ratio, turning it as a potent and selective derivative for the CB2 receptor over CB1.
The analysis of the binding modes of compounds 5, 6, and 16 compared to that of WIN55212-2 (PDB ID: 6PT0) showed that all compounds adopt similar binding modes to the agonist WIN55212-2 ( Figure 5A), maintaining most of the described interactions in the orthosteric site. As the indole ring, the benzo[d]imidazole heterocycle acts as a central core directing the substituent groups at positions 1 and 2 towards TM2 and TM5, respectively. This binding mode produces a superposition of the terpenoid motif at position 2 with the naphthalene ring of WIN55212-2, while substituents at position 1 coincide with the morpholine moiety of the agonist. Figure 5B-D present the binding interactions established by compounds 5, 6, and 16 within the orthosteric pocket of the receptor. The predominance of hydrophobic interactions is seen in accordance with the highly lipophilic nature of the CB2 receptor binding pocket. Two hydrophobic pockets can be identified within the binding site. One of them extends into TM2 and the other one is composed of TM5 residues and capped with aromatic residues of ECL2. The obtained docking poses show that the natural motifs of compounds 5, 6, and 16 extend towards the pocket in TM2, engaging in hydrophobic interactions with Phe87, Phe91, and/or Phe94, while the second pocket harbors the substituents at position 1 also through hydrophobic and pi-stacking interactions with Ile110, Val113, Phe183, Ile186, and Trp194 ( Figure 5B-D). In this way, the heterocyclic core acts as a bridging scaffold between these two pockets and, at the same time, forms hydrophobic contacts with one of the toggleswitch residues Phe117, important for receptor activation. Additionally, comparison of the docking poses in Figure 5 shows that the most potent compound 6 can directly interact with residue Ser285, which has been described to play a role in ligand efficiency [30].
Lastly, the toxicity of compound 6 was evaluated in vitro through the neutral red uptake assay. Experiments were performed in both cancerous (MCF-7) and non-cancerous (HEK-293) cell line models, showing that compound 6 decreased cell viability to 50% at 38 µM and 30 µM, respectively. The results showed little difference in toxicity between cancerous and non-cancerous cell lines. Nevertheless, considering that compound 6 presents an EC50 of 3.3 nM in the cAMP accumulation assay, there is a difference by five orders of magnitude between pharmacologic activity and toxicity. Based on the obtained results, compound 6 represents a safe, potent, and selective derivative for CB2 receptor.
In summary, new potent and selective CB2 ligands based on natural product motifs linked to a benzo[d]imidazole core were obtained. SAR analysis suggested that the presence of bulky aliphatic or aromatic natural product motifs at position 2 of the benzo[d]imidazole ring is essential for receptor recognition, linked preferably by an electronegative atom. Furthermore, the presence of substituents with moderate bulkiness at position 1 of the heterocyclic scaffold is also important for receptor recognition, with a benzyl group being the optimal substituent. Functional evaluation identified five compounds with agonist activity for the CB2 receptor. Docking studies support a common binding mode for the analyzed compounds. The high potency to inhibit cAMP accumulation, albeit having moderate affinities over the CB2 receptor, highlights the importance of complementing both binding and functional data as well as showing that great affinity is not needed to perform a potent pharmacological response. Finally, the cell viability assay showed a low toxicity profile for the most potent compound. Future evaluation through different assays will be useful to further characterize the pharmacological profile of the new ligands.

General Procedure for the Synthesis of Compounds I-III
Here, 1 equivalent of 2-chlorobenzimidazol and 1.2 equivalent of NaH (as 60% oil disp.) were stirred at room temperature for 30 min under N 2 atmosphere and dry AcCN as solvent. Then, 1 equivalent of alkyl halide was added dropwise, and the reaction was heated in an oil bath at 40 • C overnight. Excess NaH was inactivated with MeOH, and the suspension was filtered and washed with DCM. The organic phase was distilled under vacuum, obtaining an oily residue that solidified over time.

General Procedure for the Synthesis of Compounds IV-VI
The synthetic procedure was adapted from Rao et al. [31]. In brief, 1 mmol of 2-(benzylthio)-1H-benzo[d]imidazole, 4 mmol of K 2 CO 3 , tetrabutylammonium bromide (TBAB), and 1 mmol of the corresponding alkyl halide were suspended in DMF, and the mixture was stirred overnight. The mixture was poured over water, and the aqueous phase was extracted with DCM and AcOEt. The combined organic phase was dried over Na 2 SO 4 and the solvent removed in vacuo. Products were purified using column chromatography. Here, 1 equivalent of compound IV-VI was dissolved in DCM, and the solution was cooled using an ice bath. Further, 2 equivalents of m-CPBA were carefully added to the agitating solution, and the mixture was gradually heated to room temperature and stirred overnight. The resulting suspension was filtered, and the organic layer concentrated, recovering a solid, which was resuspended in a saturated solution of NaHCO 3 and then filtered. For oily residues, the crude reaction was extracted with a solution of NaHCO 3 ; the organic layer was dried with Na 2 SO 4 and then distilled under vacuum.

General Procedure for the Synthesis of Derivatives XII-XIII and XV
The synthetic procedure was adapted from Rao et al. [31], where 1 mmol of 2-mercaptobenzimidazol, 4 mmol of K 2 CO 3 , tetrabutylammonium bromide (TBAB), and 1 mmol of the corresponding tosylate derivative were dissolved in DMF, and the mixture was heated overnight in an oil bath at 70 • C. The mixture was poured over water, and the aqueous phase was extracted with DCM. The organic phase was dried over Na 2 SO 4 and the solvent removed in vacuo. For compound XV, 4-methoxybenzyl chloride was used instead of a tosylate derivate. Products were purified via recrystallization.

Synthesis of Compound XIV
Here, 1 mmol of 2-mercaptobenzimidazol and 1 mmol of 1-adamatanol were dissolved in 1.33 mL of CF 3 COOH and heated in an oil bath at 80 • C for 1 h. Then, 5 mL of a solution of EtOH:H 2 O (1:1) was added, and the reaction was neutralized with NH 3 (ac). A precipitate formed, which was filtered and recrystallized with H 2 O:EtOH (1:9), obtaining white crystals. Here, 1 equivalent of 2-thioxybenzimidazol XII-XV, 4 equivalents of K 2 CO 3 , 0.05 equivalent of tetrabutylammonium bromide (TBAB), and 1 equivalent of the corresponding alkyl halide were dissolved in DMF, and the mixture was stirred overnight at room temperature. The mixture was poured over water, and the resulting precipitate was filtered and washed with water. When a filterable precipitate was not formed, the aqueous phase was extracted with DCM, the organic layer was dried over Na 2 SO 4, and the solvent removed in vacuo. Products were purified via column chromatography or preparative plate or recrystallization.
neutral red solution prepared in culture media was added to each well and incubated for 2 h at 37 • C in a humidified atmosphere containing 5% CO 2 . Then, media were aspirated, the plate was washed three times with PBS 1X, and 100 µL of neutral red distain solution (50:49:1, ethanol/water/glacial acetic acid) was added. The plate was placed for 15 min in a shaker, and fluorescence was measured using Cytation 5 apparatus (Biotek, Winooski, VT, USA) at 530/645 nm excitation/emission wavelengths.