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Novel Machaeriol Analogues as Modulators of Cannabinoid Receptors: Structure–Activity Relationships of (+)-Hexahydrocannabinoids and Their Isoform Selectivities

By August 10, 2021August 18th, 2021No Comments

Journal List > ACS Omega > v.6(31); 2021 Aug 10 > PMC8359128

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 2021 Aug 10; 6(31): 20408–20421.
Published online 2021 Jul 28. doi: 10.1021/acsomega.1c02413
PMCID: PMC8359128
PMID: 34395989
Saqlain Haider,† Pankaj Pandey,† Chada Raji Reddy,‡ Janet A. Lambert,§ and Amar G. Chittiboyinacorresponding author*†
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Abstract

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Machaeriols are an important class of compounds that structurally resemble tetrahydrocannabinol (Δ9-THC), with the major differences being inverted stereochemistry at the ring junction as [6aR, 10aR] and an additional stereocenter at the C9 position of the A-ring due to saturation. A previous study reported that machaeriols did not show any cannabinoid receptor activity, even though these hexahydrodibenzopyran analogues mimic a privileged (+)-tetrahydrocannabinoid scaffold. To unravel structural requisites for modulation of cannabinoid receptors, a simple late-stage divergent approach was undertaken to functionalize the machaeriol scaffold using the Suzuki coupling reaction. Fourteen hexahydro analogues were synthesized and screened against both cannabinoid receptor isoforms, CB1 and CB2. Interestingly, many of the analogues showed a significant binding affinity for both receptors; however, two analogues, 11H and 11J, were identified as possessing CB2 receptor-selective functional activity in the GTPγS assay; they were found to be micromolar-range agonists, with EC50 values of 5.7 and 16 μM, respectively. Furthermore, molecular dynamics simulations between the CB2 receptor and two novel analogues resulted in unique interaction profiles by tightly occupying the active ligand-binding domain of the CB2 receptor and maintaining stable interactions with the critical residues Phe94, Phe281, and Ser285. For the first time, with the aid of structure–activity relationships of (+)-hexahydrocannabinoids, CB2 selective agonists were identified with late-stage diversification using palladium-mediated C–C bond formation. By simply switching to (R)-citronellal as a chiral precursor, enantiomerically pure (−)-hexahydrocannabinoids with better CB1/CB2 receptor isoform selectivity can be obtained using the current synthetic approach.

1. Introduction

More than 100 natural phytocannabinoids have been isolated and characterized from Cannabis sativa; tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) are the two best-studied phytoconstituents (Figure​Figure11). CB1R and CB2R, the cannabinoid receptors, are part of the endocannabinoid system (ECS), which comprises the endogenous ligands and their related enzymes and transporters. CB1 receptors are expressed in the central nervous system (CNS) and are also found in the body’s periphery, including the testes, eyes, vascular endothelium, and spleen, while CB2 receptors are found mostly in the immune and gastrointestinal systems. The expression of the CB2 receptor in the CNS is very low compared to that of the CB1 receptor, which makes it an attractive target to avoid possible CNS side effects. Some previously studied therapeutic benefits of CB2R agonists are analgesic and anti-inflammatory effects.− CB2R agonists have shown efficacy as potential therapeutic agents in peripheral diseases that involve inflammation, such as atherosclerosis, renal fibrosis, and liver cirrhosis. The ECS is involved in many human diseases and may provide potential drug development targets, including fatty acid amide hydrolase, monoacylglyceride lipase, and an anandamide transporter., Δ9–THC is a partial CB1 and CB2 receptor agonist, whereas CBD is a weak antagonist or a negative allosteric modulator (at the CB1R level) of the CB1/CB2 receptor.−

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Structural similarity between THC, CBD, and machaeriols.

The pharmacological activity of Δ9–THC is stereospecific, i.e., the (−)-trans-isomer (dronabinol, FDA approved) is 6–100 times more potent than the (+)-trans-isomer. Machaeriols are another important class of structurally similar compounds to THC and were first isolated by Muhammad et al. in 2001 from Machaerium multiflorum Spruce. Machaeriols have a hexahydrodibenzopyran scaffold (Figure​Figure11). The structural difference between THC and machaeriols is that the ring junction stereochemistry in machaeriols is inverted with an additional stereocenter at the C9 position in the A-ring; therefore, machaeriols are not tetrahydrocannabinoids but are instead hexahydrocannabinoids. Intrigued by the structural similarity between Δ9-THC and machaeriols and in continuation of our previous efforts, we report herewith the late-stage diversification of 14 novel derivatives of machaeriol-like analogues from a common precursor, hexahydrodibenzopyran. The cannabimimetic activities of these novel analogues were probed with CB1 and CB2 receptors in displacement assays, and their functional activity was confirmed with GTPγS assays. We further extended our study to evaluate the putative binding modes and interaction profiles of promising compounds 11J (against CB1R and CB2R) and 11H (against CB2R only), using molecular dynamics (MD) simulation and binding free-energy calculations.

2. Results and Discussion

2.1. Synthesis of Machaeriol Analogues

Continuing our previous synthetic work on the total synthesis of machaeriols A and B, lithiated methoxymethyl (MOM)-protected phloroglucinol was condensed with (S)-citronellal (Scheme 1). Mild acid-mediated deprotection of MOM groups induced the intramolecular hetero–Diels–Alder cycloaddition to produce hexahydrodibenzopyran (6) in 65% isolated yield with >98% diastereoselectivity. Selective triflation followed by MOM protection of the remaining phenol yielded a key intermediate, 8, amenable for the late-stage diversification. Palladium (0)-mediated Suzuki coupling of compound 8 with various boronic acids allowed the introduction of several aryl/alkyl moieties at the C3 position of the hexahydrochromane scaffold. Acid-mediated deprotection of the MOM group produced 14 diverse analogues, 11A–11N, with excellent yields (Scheme 1, Figure​Figure22).

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Structures of diverse compounds 11A–11N synthesized using Scheme 1.

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Reagents and Conditions

NaH, MOMCl, THF, 30 min, 95%; (b) n-BuLi, TMEDA, 0 °C, (S)-citronellal, 30 min, 85%; (c) 4% aqueous HCl in MeOH, rt, 12 h, 65%; (d) PhNTf2, Et3N, CH2Cl2, 3 h, 74%; (e) NaH, MOMCl, THF, 30 min, 97%; and (f) (1) boronic acid, Pd(PPh3)4, 2 M aq Na2CO3, MeOH, toluene, reflux, 2 h and (2) 1% aq HCl in MeOH, reflux, 30 min.

2.2. In Vitro Competitive Radioligand Displacement Assays for CB1 and CB2 Receptors

In preliminary probing, the synthesized compounds were assayed at a single concentration of 10 μM for their in vitro CB1 and CB2 percent displacement. The highly potent and nonselective CB agonist CP55,940 was used as a positive control. The compounds that showed >50% displacement of the radioligand [3H]-CP55,940 at the CB receptors were further assayed over a range of concentrations using a competitive radioligand binding assay to estimate binding affinities (Ki values). Two compounds (11E and 11J) exhibited low micromolar CB1R displacement, with IC50 values ≤1.0 μM (Figure​Figure33 and Table 1). Among the 14 compounds evaluated in the competitive radioligand binding assay (Table 1), compounds 11B, 11H, and 11J (Figure​Figure44) showed significant displacement at the CB2 receptor, yielding binding affinities with IC50 values in submicromolar/high nanomolar levels except for 11A and 11E (Figure​Figure44 and Table 1). Compound 11E, having an octenyl chain at the C3 position similar to CBD and Δ9-THC (pentyl chain), exhibited a higher CB1R binding affinity as compared to other compounds lacking the alkyl chain. The presence of a bulky aromatic substitution at the C3 position (11H and 11J) resulted in a superior CB2R binding affinity in comparison with those having small aromatic rings (11A, 11C, 11D, and 11K).

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Binding displacement curves for the CB1 receptor were obtained for compounds 11E and 11J with a radioligand binding assay. CP55,940 was used as a positive control. IC50 and Ki were determined by GraphPad Prism 9.1 and are listed in Table 1. The data represent mean ± SEM. Each compound was tested in triplicate.

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Binding displacement curves for the CB2 receptor were obtained for compounds 11A, 11B, 11E, 11H, and 11J with a radioligand binding assay. CP55,940 was used as a positive control. IC50 and Ki were determined by GraphPad Prism 9.1 and are listed in Table 1. The data represent mean ± SEM. Each compound was tested in triplicate.

Table 1

Percent (%) Displacement and Binding Affinity (Ki) of 11A–11N against CB1 and CB2 Receptors in Radioactive Competition Assaysa
% displacement at 10 μM

 Ki ± SEM (nM)

 IC50 ± SEM (nM)
compound CB1 CB2 CB1 CB2 CB1 CB2
11A 34.33 57.34 nd 1574 ± 836 nd 3148 ± 1672
11B 30.77 66.97 nd 117.2 ± 11.7 nd 2350 ± 23
11C –7.02 22.14 nd nd nd nd
11D 34.03 44.40 nd nd nd nd
11E 66.85 50.41 342.0 ± 95.8 572.8 ± 105.8 683.9 ± 325.9 1146 ± 212
11F 43.21 23.94 nd nd nd nd
11G 7.54 8.70 nd nd nd nd
11H 29.45 76.18 nd 63.68 ± 8.19 nd 127.4 ± 16.4
11I 34.37 16.31 nd nd
11Jb 56.36 70.40 >1000 40.18 ± 2.91 >2000 80.35 ± 5.82
11K –26.66 11.21 nd nd nd nd
11L nd nd nd nd nd nd
11M nd nd nd nd nd nd
11N nd nd nd nd nd nd
CP55,940 101.12 98.94 1.43 ± 0.24 1.07 ± 0.12 2.86 ± 0.46 2.15 ± 0.24
and, not determined. Each compound was tested in triplicate unless stated otherwise.
bDid not reach baseline.

2.3. In Vitro GTPγS Functional Assays for CB1 and CB2 Receptors

Using membrane preparations similar to the radioligand binding methods and GTPγ[35S], the functional behavior (e.g., agonists, antagonists, or inverse agonists) of the most promising compounds was determined using GTPγS functional assays. Compound 11J was tested using CB1 and CB2 functional assays, while 11H was tested using the CB2 functional assay only. All were determined to act as agonists, with the most promising being 11H (EC50 = 5730 ± 3289 nM) against the CB2 receptor. Compound 11J showed an EC50 value of 1471 ± 708 nM against the CB1 receptor, while at the CB2 receptor, it showed a moderate EC50 value of 15 993 ± 8631 nM, confirming its preference toward the CB1 receptor as an agonist (Figures​Figures55 and ​and6).6). Compound 11E was tested using the CB1 functional assay and was identified as a CB1R agonist with an EC50 value of 239 ± 68 nM.

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GTPγS functional curves for compounds 11E and 11J against the CB1 receptor. EC50 values were determined by GraphPad Prism 9.1. Each compound was tested in duplicate.

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GTPγS functional curves for compounds 11H and 11J against the CB2 receptor. EC50 values were determined by GraphPad Prism 9.1. Each compound was tested in duplicate.

2.4. Molecular Docking Studies

Molecular docking studies were performed to understand the binding pose and orientation of 11E, 11J, and 11H into the active sites of the CB1 and CB2 receptor protein crystal structures. Extra precision (XP) docking (Glide, Schrödinger) was used with flexible ligand sampling, keeping the receptor rigid., Compound 11E exhibited strong π–π stacking interactions, with Phe170 and Phe268, resulting in a GlideScore of −9.79 kcal/mol and a binding free energy (ΔG) of −68.02 kcal/mol. Furthermore, the octenyl chain at the C3 position of 11E showed strong hydrophobic interactions with an array of residues, Val196, Phe200, Ile267, Leu276, Trp279, Trp356, Leu359, Met363, and Cys386 (Figure​Figure77A,C). Similarly, the hexahydrochromane scaffold and the benzothiophene moiety of compound 11J exhibited strong π–π stacking interactions with Phe170, Phe268, and Trp279 (Figure​Figure77B,C), resulting in a GlideScore of −9.91 kcal/mol and a binding free energy (ΔG) of −66.39 kcal/mol. This double π–π interaction is reported with a co-crystallized agonist in the active-state X-ray crystal structure (PDB ID: 5XRA) of the CB1 receptors. The octenyl chain at the C3 position and the benzothiophene moiety of compounds 11E and 11J, respectively, were oriented toward the toggle switch residues Phe200 and Trp356. The benzothiophene moiety of 11J formed π–π stacking with Trp279. The oxygen atom of the benzopyran ring system of 11J was found to be at a distance of 3.5 Å from the key residue of the CB1 receptor Ser383, indicating that 11J can form hydrogen bonding with Ser383, if residue flexibility is permitted. In addition, 11J showed strong hydrophobic interactions with an array of hydrophobic residues, including Phe108, Phe174, Phe177, Leu193, Val196, Phe200, Ile267, Trp279, Trp356, Leu359, Phe379, Ala380, and Cys386, as shown in Figure​Figure77. Furthermore, we compared the docked pose of Δ9-THC with 11E and 11J into the active site of the CB1 receptor and found that they overlaid in a similar fashion and exhibited identical π–π stacking interactions with Phe170 and Phe268. However, 11E and 11J did not form H-bonding with Ser383, which was observed in the Δ9-THC docked pose. Interestingly, upon close analysis, the ligand-binding orientation of hexahydrochromane 11E and 11J was significantly different from that of Δ9-THC (Figure​Figure77D). The core scaffold of 11E and 11J was horizontally inverted by positioning the hydroxyl group away from the Ser383 residue, which showed the lack of direct H-bonding between 11J and CB1.

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2D interaction diagrams of 11E (A) and 11J (B) along with the 3D overlaid representation of 11E (carbon in orange) with 11J (carbon in plum) (C) and 11J (carbon in plum) with Δ9-THC (carbon in cyan) (D) against the CB1 receptor. The key residues are shown in the ball and stick model (carbon in gray), and transmembrane regions are shown as ribbons (green-colored).

In a similar fashion, the docking and binding free-energy data revealed that compounds 11H (GlideScore = −10.80 kcal/mol; ΔG = −64.14 kcal/mol) and 11J (GlideScore = −10.07 kcal/mol; ΔG = −67.64 kcal/mol) bound more tightly and exhibited stronger interactions with the CB2 receptor. Compounds 11H and 11J were well docked into the active site of the CB2R cryo-EM structure (PDB ID: 6PT0) (Figure​Figure88A,B). The 3D overlaid representation of 11H and 11J against the CB2 receptor is shown in Figure​Figure88C. The hexahydrochromane moiety of compounds 11H and 11J was oriented toward the toggle-switch residues Phe117and Trp258. The benzofuran moiety of 11H formed strong π–π stacking interactions with Phe94 and His95. In addition, the hydroxyl group (C1) of compounds 11H and 11J showed H-bonding with Ser285, which is known to be a critical residue for CB2R activity. Furthermore, the benzothiophene and benzopyran rings of compound 11J exhibited π–π stacking interactions with Phe94 and Phe183, respectively. Both compounds 11H and 11J were surrounded by the hydrophobic residues of the CB2 receptor, including Tyr25, Ile27, Ile110, Phe117, Phe183, Tyr190, Leu191, Trp194, Ile198, Trp258, Val261, Leu262, and Phe281 (Figure​Figure88).

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2D interaction diagrams of 11H (A) and 11J (B) along with the 3D overlaid representation of 11H (carbon in yellow) and 11J (carbon in plum) (C) against the CB2 receptor. The key residues are shown in the ball and stick model (carbon in gray) and transmembrane regions are shown as ribbons (green-colored).

We compared the docked pose of Δ9-THC with 11J and 11H against the CB2 receptor and found that they overlaid well with Δ9-THC in the active site of the CB2 receptor (Figure S1). However, the substituted C3 moieties of 11H and 11J were vertically inverted compared to the C5 alkyl chain of Δ9-THC (Figure​Figure88). They also maintained the key interactions of Δ9-THC with the CB2 receptor, including Ser285 (H-bonding) and Phe183 (π–π interactions).

2.5. Molecular Dynamics Simulation Studies

Molecular docking represents a static snapshot of the protein–ligand complex and sometimes may not predict the exact pose of the ligand within the protein active site., Therefore, MD simulation is an excellent technique to further confirm the stability of the protein–ligand complex and study the interaction profiles as it evolves over time. To explore the conformation dynamics of the best-docked complexes of CB2R–11H, CB2R–11J, and CB1R–11J, 200 ns MD simulations were performed. The root-mean-square deviations (RMSDs) of the protein Cα atoms and ligand heavy atoms were calculated with reference to the starting structures (first frame at time 0 ns) and are shown in Figures​Figures99 and ​and10.10. The RMSD of the protein Cα atoms of CB2R proteins in the complex of CB2–11H and CB2–11J varied between 1 and 1.5 Å during the whole simulation, which is an acceptable range for GPCR proteins. Similarly, the RMSD of ligand heavy atoms of CB2R–11H and CB2R–11J was very stable throughout the 200 ns simulation, indicating that the starting conformation of the ligand did not change significantly throughout the simulation. The lower RMSD values of the CB2 protein Cα atoms and ligand heavy atoms suggest that CB2R–11H and CB2R–11J have strong predicted binding interactions.

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RMSD for the Cα atoms of (A) proteins and (B) ligand heavy-atom RMSD for MD simulations of complexes 11H and 11J with the CB2 receptor.

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Heavy-atom ligand RMSD for complex 11J with the CB1 receptor.

 

2.5.1. CB1R–11J Complex

The RMSD of the ligand heavy atoms of CB1R–11J was very stable throughout the 200 ns simulation and suggested that CB1R–11J has strong binding interactions with the CB1 receptor and 11J did not change its initial conformation during the 200 ns simulation. Furthermore, the root-mean-square fluctuation (RMSF) plot based on the Cα atoms of CB1R for complexes with CB1R–11J showed very low fluctuations for the residues that form the ligand-binding site. The overall fluctuation was observed to be <1.0 Å (Figure S2), supporting the stability of the complex.

The interaction histogram (Figure​Figure1111) and 2D-ligand contact map (Figure​Figure1212) of 11J with the CB1 receptor indicated H-bonding of phenolic hydroxyl with Ser383 (79% contribution), water-mediated H-bonding of the pyran oxygen of 11J with Ile267 (41% contribution), and π–π stacking with an array of hydrophobic residues such as Phe170, Phe174, Phe200, Phe268, and Trp279. The strong binding of 11J with the CB1 receptor is supported by the negative average binding free energy (ΔG = −82.95 ± 4.89 kcal/mol), calculated with Prime MM-GBSA for the entire trajectory of the CB1R–11J complex (Table 2). In summary, 11J formed stable and strong interactions with the key residues of the CB1 receptor.

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Simulation interaction diagram (SID) plot showing the protein–ligand interactions between the amino acid residues of the CB1 receptor binding site and 11J. Interaction-fraction values over 1.0 indicate that the residue has multiple contacts with the ligand.

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2D diagram of atomic-level interactions of the CB1R–11J complex with key CB1 residues during the 200 ns MD simulation.

Table 2

Prime MM-GBSA Binding Free Energies (Post-MD Simulations) for 11H (CB2) and 11J (CB1 and CB2) Receptorsa
ΔG average binding free energy (±SD) Coulomb (±SD) covalent (±SD) H-bond (±SD) Lipo (±SD) π-packing energy (±SD) SolvGB (±SD) vdW (±SD)
11H (CB2) –91.55 ± 4.90 –11.98 ± 2.14 3.66 ± 1.86 –0.48 ± 0.15 –39.56 ± 2.17 –5.74±1.02 22.85 ± 1.83 –60.29 ± 2.51
11J (CB1) –82.95 ± 4.89 –11.10 ± 2.80 2.31 ± 1.01 –0.42 ± 0.15 –33.96 ± 1.61 –4.77±0.79 23.25 ± 1.63 –58.25±1.83
11J (CB2) –81.42 ± 5.09 –12.74 ± 2.29 2.54 ± 1.09 –0.51± 0.10 –32.24 ± 1.80 –4.30±0.69 21.18 ± 1.95 –55.35±2.40
aCoulomb: Coulomb energy; covalent: covalent binding energy; vdW: van der Waals energy; Lipo: lipophilic energy; SolvGB: generalized Born electrostatic solvation energy; and H-bond: hydrogen-bonding energy.

 

2.5.2. CB2R–11J Complex

The RMSD of the protein Cα atoms of CB2R protein in the complex of CB2R–11J reached an equilibrium state just after 50 ns and remained stable in the rest of the simulation (Figure​Figure99A). Similarly, the RMSD of ligand’s heavy atoms in the CB2R–11J complex was very stable throughout the 200 ns simulation (Figure​Figure99B). The lower RMSD values of CB2R protein Cα atoms and ligand heavy atoms suggest that CB2R–11J has strong binding interactions with the CB2 receptor. The RMSF plot based on the Cα atoms of CB2R for complex with 11J showed very low fluctuations for the residues that form the ligand-binding site. The overall fluctuation was observed to be <1.4 Å (Figure S3), also supporting the stability of the complex. The interaction histogram (Figure​Figure1313) and 2D ligand contact map (Figure​Figure1414) of 11J with the CB2 receptor indicate a strong H-bonding of the OH of 11J with Ser285 (93% contribution) and π–π stacking with Phe183 (71% contribution), Phe87 (40% contribution), Phe91 (48% contribution), and Phe94 (22% contribution). Interestingly, no water-mediated interaction was observed in the entire simulation of the CB2R–11J complex. It also shows an array of hydrophobic interactions with the Ile27, Val113, Leu182, Pro184, Trp194, Val261, Phe281, and Ala282. The higher negative average binding free energy (ΔG = −81.42 ± 5.09 kcal/mol) after the post-MD of 11J with the CB2 receptor affirmed its complex stability (Table 2). Overall, 11J formed stable and strong interactions with the CB2 receptor.

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SID plot showing the protein–ligand interactions between the amino acid residues of the CB2 receptor binding site and 11J.

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2D diagram of atomic-level interaction of the CB2–11J complex with key CB2R residues during the 200 ns MD simulation.

 

2.5.3. CB2R–11H Complex

The RMSF plot based on the Cα atoms of CB2R for complexes with 11H showed very low fluctuations for the residues that form the ligand-binding site. The overall fluctuation was observed to be <1.3 Å (Figure S4), supporting the stability of the complex. The interaction histogram (Figure​Figure1515) and 2D ligand contact map (Figure​Figure1616) of 11H with the CB2 receptor indicate a strong H-bonding of the OH of 11H with Ser285 (85% contribution) and π–π stacking with Phe87 (75% contribution), Phe91 (47% contribution), Phe94 (39% contribution), and Phe183 (73% contribution), Interestingly, similar to 11J, no water-mediated interaction was observed during the entire 200 ns simulation. 11H also exhibited an array of hydrophobic interactions with Ile27, Val113, Leu182, Pro184, Trp194, Val261, Phe281, and Ala282. The negative average binding free energy (ΔG = −91.55 ± 4.90 kcal/mol) of 11H after post-MD simulation confirmed the stability of the CB2–11H complex (Table 2). In summary, strong H-bonding of 11H with Ser285 and multiple π–π stacking with CB2R residues resulted in a stable complex of CB2R–11H.

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SID plot showing the protein–ligand interactions between the amino acid residues of the CB2 receptor binding site and 11H.

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2D diagram of atomic-level interaction of the CB2–11H complex with key CB2R residues during the 200 ns MD simulation.

The most negative average binding free energy (ΔG = −91.55 ± 4.90 kcal/mol) for 11H (CB2) was contributed by the van der Waals interactions (vdW) (−60.29 ± 2.51 kcal/mol), along with other significant contributions from the Lipo term (a measure of hydrophobic interactions with water) (−39.56 ± 2.17 kcal/mol), π–π stacking interaction (−5.74±1.02 kcal/mol), and Coulombic term (Coulomb) or electrostatic interactions (−11.98 ± 2.14 kcal/mol). Similar trends were observed for 11J (CB1 and CB2 receptors). Binding free-energy data of 11J against CB1 and CB2 receptors showed correlation with experimental functional data in terms of EC50; however, the receptor binding affinity does not corroborate.

3. Conclusions

To probe the cannabimimetic activity of (+)-hexahydrocannabinoids, a small set of 14 novel analogues were synthesized readily from (S)-citronellal using a late-stage diversification approach. These analogues were screened against CB1 and CB2 receptors. Two of the compounds (11E and 11J) exhibited low micromolar CB1 displacement with an IC50 value of ≤2.0 μM. Compounds 11A, 11B, 11E, 11H, and 11J showed significant displacement at the CB2 receptor yielding binding affinities with an IC50 value of ≤3.20 μM. Two of the most promising compounds (11H and 11J) were further tested for functional activity and were found to be CB2R agonists. The XP Glide docking did not produce any pose for 11H, which is in accordance with the experimental low binding affinity of 11H (29.45% displacement) toward the CB1 receptor. MD simulations and binding free-energy calculations confirmed the stability of these compounds with CB1 and CB2 receptors. The MD study revealed that Ser173 and Ser285 are the two critical amino acids involved in the H-bonding interactions with these analogues for CB1 and CB2 receptors. In future, by simply switching to (R)-citronellal as a chiral precursor, enantiomerically pure (−)-hexahydrocannabinoids could be achievable to develop novel analogues with better CB1/CB2 receptor isoform selectivities.

4. Materials and Methods

4.1. Chemistry

All reactions were carried out under an argon atmosphere unless otherwise stated. Thin-layer chromatography was performed on precoated silica gel G and GP Uniplates. The plates were visualized with a 254 nm UV light, an iodine chamber, or charring with acid. Flash chromatography was carried out on silica gel 60 (particle size 32–63 μm, pore size 60 Å). 1H NMR and 13C NMR spectra were recorded in CDCl3 at 400 and 100 MHz or 500 and 125 MHz. The chemical shifts are reported in parts per million (ppm) downfield from tetramethylsilane, and J values are in Hz. The high-resolution mass spectra (HRMS) were recorded on a Waters Q-Tof Micro mass spectrometer with an ESI lock spray source. Dry dichloromethane was prepared by distilling it over calcium hydride.

 

4.1.1. General Procedure for the Preparation of Compounds (11A–11N)

To a solution of triflate 8 (80 mg, 0.18 mmol) in toluene/MeOH (9:1, v/v, 10 mL), boronic acid (0.27 mmol), 2 M aq Na2CO3 (100 μL), and tetrakis(triphenylphosphine)-palladium(0) (3 mg) were added and the reaction mixture was refluxed overnight. The reaction mixture was cooled, water was added, and the reaction mixture was extracted with ether. Combined organic layers were dried over MgSO4, concentrated under vacuum, and purified by column chromatography using ethyl acetate in hexanes. The purified product was dissolved in 1% aq HCl in MeOH, heated to reflux, and stirred for 30 min. MeOH was evaporated, and the crude products were purified by column chromatography to afford compounds 11A–11N. Triflate 8 was synthesized according to the procedure reported in our earlier work.

(6aS,9S,10aS)-6,6,9-Trimethyl-3-phenyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (11A): [α]D25 = +137.0 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz): δ 7.55 (d, J = 7.5 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.33 (t, J = 7.3 Hz, 1H), 6.71 (d, J = 1.5 Hz, 1H), 6.52 (d, J = 1.5 Hz, 1H), 4.93 (s, 1H), 3.11 (bd, J = 12.5 Hz, 1H), 2.56 (ddd, J = 2.5, 11.0, 13.5 Hz, 1H), 1.90 (m, 2H), 1.69 (m, 1H), 1.54 (t, J = 11.0 Hz, 1H), 1.44 (s, 3H), 1.17 (m, 2H), 1.14 (s, 3H), 1.0 (d, J = 6.5 Hz, 3H), 0.86 (dd, J = 11.5, 24.0, 1H). 13C NMR (CDCl3, 125 MHz): δ 155.3, 155.0, 140.3, 140.2, 128.5(2C), 127.2, 126.7(2C), 112.2, 109.0, 106.1, 77.5, 49.4, 39.2, 35.8(2C), 33.2, 28.4, 28.1, 23.0, 19.5. HRMS (ESI+): calcd for C22H27O2, 323.2011 (M + H)+, found 323.2008.

(6aS,9S,10aS)-3-(Benzo[d][1,3]dioxol-5-yl)-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (11B): [α]D25 = +92.0 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz): δ 7.03 (s, 1H), 7.02 (d, J = 9.0 Hz, 1H), 6.85 (d, J = 9.0 Hz, 1H), 6.62 (d, J = 1.5 Hz, 1H), 6.43 (d, J = 1.4 Hz, 1H), 6.0 (s, 2H), 4.91 (bs, 1H), 3.09 (bd, J = 13.0 Hz, 1H), 2.54 (ddd, J = 2.5, 11.0, 13.5 Hz, 1H), 1.90 (m, 2H), 1.68 (m, 1H), 1.52 (t, J = 11.2 Hz, 1H), 1.43 (s, 3H), 1.16 (m, 2H), 1.13 (s, 3H), 0.98 (d, J = 6.5 Hz, 3H), 0.84 (dd, J = 12.0, 24.0, 1H); 13C NMR (CDCl3, 125 MHz): δ 155.3, 155.0, 147.8, 146.8, 140.0, 134.7, 120.2, 111.9, 108.7, 108.4, 107.3, 105.9, 101.0, 77.5, 49.4, 39.2, 35.7(2C), 33.1, 28.3, 28.0, 22.9, 19.4. HRMS (ESI+): calcd for C23H27O4, 367.1909 (M + H)+, found 367.1891.

4-((6aS,9S,10aS)-1-Hydroxy-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl)benzonitrile (11C): [α]D25 = +103.0 (c 0.1, CHCl3); 1H NMR (MeOH-d4 + CDCl3, 500 MHz): δ 7.70 (dd, J = 8.5, 11.5 Hz, 4H), 6.60 (d, J = 1.5 Hz, 1H), 6.56 (d, J = 1.6 Hz, 1H), 3.23 (bd, J = 13.0 Hz, 1H), 2.51 (ddd, J = 2.5, 11.5, 13.5 Hz, 1H), 1.87 (m, 2H), 1.66 (m, 1H), 1.47 (t, J = 10.5 Hz, 1H), 1.38 (s, 3H), 1.16 (m, 2H), 1.09 (s, 3H), 0.96 (d, J = 6.5 Hz, 3H), 0.7 (dd, J = 11.5, 24.0 Hz, 1H); 13C NMR (MeOH-d4 + CDCl3, 125 MHz): δ 157.0, 155.2, 145.5, 137.7, 132.1(2C), 127.1(2C), 118.7, 113.8, 110.0, 107.4, 105.6, 77.2, 49.5, 38.8, 36.0, 35.8, 33.1, 28.2, 27.4, 22.3, 18.7. HRMS (ESI+): calcd for C23H26NO2, 348.1964 (M + H)+, found 348.1968.

(6aS,9S,10aS)-3-(Furan-3-yl)-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (11D): [α]D25 = +114.0 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz): δ 7.65 (s, 1H), 7.44 (s, 1H), 6.62 (d, J = 1.4 Hz, 1H), 6.60 (d, J = 1.6 Hz, 1H), 6.41 (d, J = 1.5 Hz, 1H), 4.94 (s, 1H), 3.07 (bd, J = 13.0 Hz, 1H), 2.52 (ddd, J = 2.5, 11.5, 13.5 Hz, 1H), 1.89 (m, 2H), 1.67 (m, 1H), 1.51 (t, J = 11.0 Hz, 1H), 1.42 (s, 3H), 1.16 (m, 2H), 1.12 (s, 3H), 0.98 (d, J = 6.5 Hz, 3H), 0.84 (dd, J = 11.5, 24.0 Hz, 1H); 13C NMR (CDCl3, 125 MHz): δ 155.3, 155.0, 143.3, 138.3, 131.5, 125.8, 112.1, 108.8, 107.8, 105.1, 77.5, 49.4, 39.2, 35.8, 33.2, 28.4, 28.1, 23.0, 19.4. HRMS (ESI+): calcd for C20H25O3, 313.1804 (M + H)+, found 313.1819.

(6aS,9S,10aS)-6,6,9-Trimethyl-3-((E)-oct-1-en-1-yl)-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (11E): [α]D25 = +126.0 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz): δ 6.45 (d, J = 1.5 Hz, 1H), 6.27 (d, J = 1.5 Hz, 1H), 6.15 (m, 2H), 4.86 (bs, 1H), 3.06 (bd, J = 12.5 Hz, 1H), 2.49 (ddd, J = 2.0, 10.5, 13.0 Hz, 1H), 2.18 (dd, J = 7.0, 14.0, 2H), 1.87 (m, 2H), 1.66 (m, 1H), 1.46 (m, 4H), 1.40 (s, 3H), 1.34 (m, 5H), 1.15 (m, 2H), 1.09 (s, 3H), 0.97 (d, J = 6.4 Hz, 3H) 0.92 (t, J = 6.5 Hz, 3H), 0.82 (dd, J = 12, 24 Hz, 1H); 13C NMR (CDCl3, 125 MHz): δ 155.0, 154.8, 137.0, 131.0, 128.9, 111.9, 107.9, 105.1, 77.3, 49.4, 39.2, 35.9, 35.8, 33.2(2C), 32.1, 29.7, 29.2, 28.4, 28.1, 23.0, 19.4, 14.5. HRMS (ESI+): calcd for C24H37O2, 357.2794 (M + H)+, found 357.2793.

(6aS,9S,10aS)-6,6,9-Trimethyl-3-((E)-4-(trifluoromethyl)styryl)-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (11F): [α]D25 = +106.0 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz): δ 7.6 (d, J = 8.5 Hz, 2H), 7.55 (d, J = 8.5 Hz, 2H), 7.02 (dd, J = 6.5 Hz, 2H), 6.64 (d, J = 1.0 Hz, 1H), 6.45 (d, J = 1.5 Hz, 1H), 4.91 (s, 1H), 3.07 (bd, J = 13.0 Hz, 1H), 2.53 (ddd, J = 2.5, 11.0, 13.0 Hz, 1H), 1.9 (m, 2H), 1.68 (m, 1H), 1.52 (t, J =11.0 Hz, 1H), 1.43 (s, 3H), 1.16 (m, 2H), 1.12 (s, 3H), 0.99 (d, J = 6.3 Hz, 3H), 0.83 (dd, J = 11.0, 23.0 Hz, 1H); 13C NMR (CDCl3, 125 MHz): δ 155.3, 155.0, 140.6, 135.9, 130.5, 126.8, 126.4(4C), 125.5(2C), 113.7, 108.8, 105.8, 77.5, 49.3, 39.1, 36.0, 35.8, 33.2, 28.4, 28.0, 22.9, 19.4. HRMS (ESI+): calcd for C25H28O2F3, 417.2041 (M + H)+, found 417.2040.

(6aS,9S,10aS)-3-((E)-4-Chlorostyryl)-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (11G): [α]D25 = +112.0 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz): δ 7.39 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 8.5 Hz, 2H), 6.91 (dd, J = 16.0 Hz, 2H), 6.61 (d, J = 1.4 Hz, 1H), 6.42 (d, J = 1.5 Hz, 1H), 4.96 (s, 1H); 3.07 (bd, J = 12.5 Hz, 1H), 2.52 (ddd, J = 2.5, 11.0, 13.5 Hz, 1H), 1.89 (m, 2H), 1.67 (m, 1H), 1.51 (t, J = 11.0 Hz, 1H), 1.43 (s, 3H), 1.17 (m, 2H), 1.11 (s, 3H), 0.98 (d, J = 6.4 Hz, 3H), 0.82 (dd, J = 11.5, 24.0 Hz, 1H); 13C NMR (CDCl3, 125 MHz): δ155.2, 154.9, 136.3, 135.7, 132.9, 128.7(2C), 128.6, 127.5, 127.1(2C), 113.3, 108.6, 105.6, 77.5, 49.2, 39.1, 36.0, 35.9, 35.7, 33.2, 28.3, 28.0, 22.9, 19.4. HRMS (ESI+): calcd for C24H28O2Cl, 383.1778 (M + H)+, found 383.1768.

(6aS,9S,10aS)-3-(Dibenzo[b,d]furan-4-yl)-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (11H): [α]D25 = +142.0 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz): δ 7.98 (d, J = 7.5 Hz, 1H), 7.92 (dd, J = 1.0, 7.5 Hz, 1H), 7.60 (dd, J = 7.0, 8.0 Hz, 2H), 7.48 (dt, J = 1.0, 8.0 Hz, 1H), 7.38 (d, J =7.5 Hz, 2H), 7.02 (d, J =1.5 Hz, 1H), 6.94 (d, J = 1.6 Hz, 1H), 5.12 (s, 1H), 3.17 (bd, J = 13.0 Hz, 1H), 2.61 (ddd, J = 3.0, 11.5, 14.0 Hz, 1H), 1.91 (bd, 2H), 1.71 (m, 1H), 1.59 (t, J =11.3 Hz, 1H), 1.47 (s, 3H), 1.17 (s, 3H), 1.15 (m, 2H), 1.01 (d, J = 6.7 Hz, 3H), 0.88 (dd, J = 11.5, 23.5 Hz, 1H); 13C NMR (CDCl3, 125 MHz): δ 155.9, 155.3, 154.8, 153.0, 135.4, 127.1, 126.5, 125.1, 124.8, 124.1, 123.0, 122.6, 120.5, 119.5, 112.9, 111.9, 110.6, 107.9, 77.5, 49.4, 39.2, 36.0, 35.9, 33.3, 28.5, 28.2, 23.0, 19.5. HRMS (ESI+): calcd for C28H28O3, 413.2038 (M + H)+, found 413.2105.

(6aS,9S,10aS)-6,6,9-Trimethyl-3-(4-phenoxyphenyl)-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (11I): [α]D25 = +75.0 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz): δ 7.48 (d, J = 8.5 Hz, 2H), 7.38 (t, J = 8.0 Hz, 2H), 7.15 (t, J = 7.5 Hz, 1H), 7.07 (d, J = 8.0 Hz, 2H), 7.03 (d, J = 8.5 Hz, 2H), 6.89 (d, J = 1.5 Hz, 1H), 6.49 (d, J = 1.4 Hz, 1H), 5.33 (bs, 1H), 3.14 (bd, J = 13.0 Hz, 1H), 2.57 (ddd, J = 2.5, 11.0, 13.5 Hz, 1H), 1.90 (m, 2H), 1.69 (m, 1H), 1.55 (t, J =11.3 Hz, 1H), 1.45 (s, 3H), 1.16 (m, 2H), 1.14 (s, 3H), 0.99 (d, J = 6.4 Hz, 3H), 0.85 (dd, J = 11.5, 23.5 Hz, 1H); 13C NMR (CDCl3, 125 MHz): δ 156.9, 156.5, 155.3, 155.1, 139.6, 135.3, 129.7(2C), 127.9(2C), 123.3, 119.0(2C), 118.8(2C), 112.1, 108.6, 106.0, 77.6, 49.4, 39.2, 35.8(2C), 33.2, 28.4, 28.1, 23.0, 19.5. HRMS (ESI+): calcd for C28H31O3, 415.2273 (M + H)+, found 415.2284.

(6aS,9S,10aS)-3-(Benzo[b]thiophen-3-yl)-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (11J): [α]D25 = +95.0 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz): δ 8.0 (m, 1H); 7.91 (m, 1H); 7.37 (m, 2H); 7.33 (s, 1H); 6.72 (d, J = 1.4 Hz, 1H), 6.50 (d, J = 1.5 Hz, 1H), 5.37 (bs, 1H), 3.16 (bd, J = 13.0 Hz, 1H), 2.58 (ddd, J = 2.5, 11.0, 13.0 Hz, 1H), 1.91 (m, 2H), 1.70 (m, 1H), 1.57 (t, J = 11.3 Hz, 1H),1.46 (s, 3H), 1.18 (m, 2H), 1.17 (s, 3H), 1.0 (d, J = 6.3 Hz, 3H), 0.86 (dd, J = 11.5, 23.5 Hz, 1H); 13C NMR (CDCl3, 125 MHz): δ 155.2, 155.0, 140.5, 137.6, 137.3, 135.0, 124.3, 124.1, 123.2, 123.0, 122.8, 112.6, 110.5, 107.7, 77.6, 49.4, 39.1, 35.9(2C), 33.2, 28.4, 28.1, 23.0, 19.5. HRMS (ESI+): calcd for C24H27O2S, 379.1732 (M + H)+, found 379.1735.

(6aS,9S,10aS)-3-(3,5-Bis(trifluoromethyl)phenyl)-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (11K): [α]D25 = +135.0 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz): δ 7.98 (s, 2H), 7.83 (s, 1H), 6.72 (d, J = 1.6 Hz, 1H), 6.55 (d, J = 1.5 Hz, 1H), 5.40 (bs, 1H), 3.11 (bd, J = 13.0 Hz, 1H), 2.57 (ddd, J = 2.5, 11.0, 13.0 Hz, 1H), 1.91 (m, 2H), 1.69 (m, 1H), 1.54 (t, J = 11.0 Hz, 1H), 1.45 (s, 3H), 1.17 (m, 2H), 1.14 (s, 3H), 0.99 (d, J = 6.5 Hz, 3H), 0.86 (dd, J = 11.5, 24.0 Hz, 1H); 13C NMR (CDCl3, 125 MHz): δ 155.7, 155.6, 142.4, 137.1, 131.9, 126.7, 124.4, 122.2, 120.7, 114.0, 109.0(2C), 105.9(2C), 77.8, 49.3, 39.0, 35.8, 35.7, 33.2, 28.4, 28.0, 22.9, 19.4. HRMS (ESI+): calcd for C24H25O2F6, 459.1759 (M + H)+, found 459.1739.

4-((6aS,9S,10aS)-1-Hydroxy-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl)-N,N-dimethylbenzamide (11L): [α]D25 = +107.0 (c 0.1, CHCl3); 1H NMR (CDCl3, 400 MHz): δ 7.39 (dd, J = 8, 17.2 Hz, 1H), 6.51 (d, J = 1.2 Hz, 1H), 6.19 (d, J = 1.6 Hz, 1H), 3.27 (br d, J = 13.2 Hz, 1H), 3.19 (s, 3H), 3.05 (s, 3H), 2.53 (ddd, J = 2.0, 10.8, 13.2 Hz, 1H), 1.88–1.84 (m, 2H), 1.68–1.67 (m, 1H), 1.48 (t, J = 11.2 Hz, 1H), 1.39 (s, 3H), 1.19–1.13 (m, 2H), 1.08 (s, 3H), 0.97 (d, J = 6.8 Hz, 3H), 0.77 (dd, J = 11.6, 23.6 Hz, 1H); 13C NMR (CDCl3, 100 MHz): δ 172.35, 156.7, 155.2, 142.3, 138.6, 133.8, 127.2 (2C), 126.9(2C), 112.9, 107.5, 106.0, 77.2, 77.1, 49.2, 39.6, 38.6, 35.6, 32.9, 31.1, 28.1, 27.7, 22.6, 19.1; HRMS (ESI+): calcd for C25H32NO3, 394.2382 (M + H)+, found 394.2375.

(6aS,9S,10aS)-6,6,9-Trimethyl-3-(pyridin-3-yl)-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (11M): [α]D25 = +123.0 (c 0.1, CHCl3); 1H NMR (CDCl3, 400 MHz): δ 9.13 (d, J = 1.6 Hz, 1H), 8.6 (dd, J = 1.2, 4.8 Hz, 1H), 7.96 (dt, J = 1.6, 3.6, 8.0 Hz, 1H), 7.43 (dd, J = 4.8, 8.0 Hz, 1H), 6.87 (d, J = 1.6 Hz, 1H), 6.62 (d, J = 1.6 Hz, 1H), 3.37 (d, J = 12.8 Hz, 1H), 2.61 (ddd, J = 2.4, 11.2, 13.6 Hz, 1H), 1.91–1.89 (m, 2H), 1.75–1.73 (m, 1H), 1.55 (t, J = 11.2 Hz, 1H), 1.44 (s, 3H), 1.20–1.16 (m, 2H), 1.15 (s, 3H), 1.01 (d, J = 6.8 Hz, 3H), 0.83 (dd, J = 11.6, 24.0 Hz, 1H); 13C NMR (CDCl3, 100 MHz): δ 157.4, 155.7, 146.8, 146.6, 137.4, 135.8, 135.1, 124.1, 113.9, 107.4, 106.3, 77.5, 49.4, 38.8, 36.1, 35.9, 33.2, 28.4, 28.1, 23.0, 19.5; HRMS (ESI+): calcd for C21H26NO2, 324.1964 (M + H)+, found 324.1966.

(6aS,9S,10aS)-3-(4-(Dimethylamino)phenyl)-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (11N): [α]D25 = +118.0 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz): δ 7.45 (d, J = 8.5 Hz, 2H), 6.78 (d, J = 8.5 Hz, 2H), 6.66 (d, J = 1.5 Hz, 1H), 6.46 (d, J = 1.5 Hz, 1H), 4.97 (s, 1H), 3.11 (br d, J = 12.5 Hz, 1H), 2.99 (s, 6H), 2.54 (ddd, J = 2.5, 11.0, 13.5 Hz, 1H), 1.91–1.88 (m, 2H), 1.7–1.68 (m, 1H), 1.53 (t, J = 11 Hz, 1H), 1.42 (s, 3H), 1.19–1.15 (m, 2H), 1.13 (s, 3H), 0.99 (d, J = 6.5 Hz, 3H), 0.85 (dd, J = 11.5, 24.0 Hz, 1H); 13C NMR (CDCl3, 125 MHz): δ 155.2, 154.9, 149.4, 140.1, 134.6, 127.2(4C), 112.8, 107.9, 105.4, 77.2, 49.4, 40.9 (2C), 39.3, 35.9, 35.8, 33.2, 28.4, 28.1, 22.9, 19.5; HRMS (ESI+): calcd for C24H32NO2, 366.2433 (M + H)+, found 366.2426.

(6aS,9S,10aS)-1-Hydroxy-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl trifluoromethanesulfonate (7): 1H NMR (CDCl3, 500 MHz): δ 6.35 (d, 1H, J = 2.5 Hz); 6.25 (d, 1H, J = 2.5 Hz); 5.42 (bs, 1H); 3.00 (bd, 1H, J = 12.5 Hz); 2.46 (ddd, 1H, J = 2.5, 11.5, 14.0 Hz); 1.88 (m, 2H); 1.62 (m, 1H); 1.46 (m, 1H); 1.39 (s, 3H); 1.17 (m, 2H); 1.08 (s, 3H); 0.97 (d, 3H, J = 6.5 Hz); 0.78 (dd, 1H, J = 11.5, 24.5 Hz). 13C NMR (CDCl3, 125 MHz): δ 156.0, 155.8, 120.3, 117.0, 113.5, 103.2, 100.6, 78.3, 49.5, 48.7, 38.4, 35.3, 32.8, 29.3, 27.9, 27.5, 22.5, 19.0. HRMS (ESI+): calcd for C17H22F3O5S, 395.1140 (M + H)+, found 395.1146.

4.2. Biological Evaluation

 

4.2.1. Materials

CP55,940 was purchased from Tocris (Bristol, U.K.), and BSA, Trizma hydrochloride, l-glutamine, penicillin, and streptomycin, nonenzymatic cell dissociation solution, and guanosine 50-diphosphate (GDP) were obtained from Sigma-Aldrich (St. Louis, MO, USA). G418 (geneticin) sulfate was purchased from Gibco (Paisley, U.K.). [3H]-CP55,940 was obtained from AP Biotech (Little Chalfont, U.K.) or PerkinElmer (Boston, MA, USA), and [35S]-GTPγS was obtained from PerkinElmer (Boston, MA, USA). GTPγS adenosine deaminase and hygromycin B were obtained from Roche Diagnostic (Indianapolis, IN, USA). Expression clones containing CB1R and CB2R full-length cDNA were purchased OriGene (Rockville, MD, USA).

 

4.2.2. Cell Lines and Culture

Human embryonic kidney (HEK) 293 cells were purchased from the American Type Culture Collection. The cells were grown in 150 cm2 Corning cell culture dishes with Dulbecco’s modified Eagle’s medium/Ham’s F12 medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, penicillin (100 U/mL), and streptomycin (100 μg/mL) in an atmosphere of 5% CO2.

 

4.2.3. Transfection and Stable Expression of CB1 and CB2 Receptors in Mammalian Cell Lines

HEK293 cells were collected and transiently transfected with the human CB1 and CB2 receptors. cDNA containing expression clones were used to generate separate cell lines expressing either the CB1 or the CB2 receptors (50 μg/mL) using electroporation (70 ms, single pulse, 150 V). The transfected cells were grown in a 150 cm2 cell culture Petri dish. For selection, G418 antibiotic solution (800 μg/mL) was used. After selection, the HEK293 cells were further cultured until single colonies were obtained. The colonies with a binding ratio (%) over 50% were chosen for binding and functional assays.

 

4.2.4. Cell Membrane Preparation

Cell plasma membranes were prepared from HEK293 cells with stable expression of CB1 and CB2 receptors. Cells grown to confluency were collected by scraping and spun at 2000g for 10 min at 4 °C. Crude membranes were prepared by homogenization of the cells in 50 mM Tris-HCl (pH 7.5) and centrifugation at 1000g for 5 min. The supernatant was centrifuged at 40,000g for 40 min at 4 °C, and the pellet was resuspended in a buffer consisting of 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, and 1 mM EDTA and stored at −80 °C until use.

 

4.2.5. Competitive Receptor Binding Assay

Competitive binding assays were performed with a recently modified rapid filtration assay referred to the methods described earlier.,, Briefly, cell membranes (5 μg of CB1R or 1 μg of CB2R) were incubated with 1.079 nM [3H]-CP55,940 (CB1R) or 1.002 nM [3H]-CP55,940 (CB2R) and test compounds in 50 mM Tris-EDTA buffer (50 mM Tris, pH 7.4, 20 mM disodium EDTA, 154 mM NaCl, and 0.2% bovine serum albumin) for 1.5 h at 37 °C with gentle shaking (total volume 200 μL). The reaction was terminated by rapid vacuum filtration onto a PerkinElmer Unifilter GF/C-96 filter plate and washed 10 times with ice-cold 50 mM Tris-EDTA containing 0.2% BSA (pH 7.4); bound radioactivity was quantified by the Packard TopCount Scintillation Counter. Specific binding was defined as the difference between the binding that occurred in the presence and the absence of 1 μM unlabeled CP55,940. All of the experimental data (IC50, Ki, and EC50) were analyzed using a nonlinear regression curve fit model using GraphPad Prism 9.1 software (GraphPad Software, Inc., San Diego, CA, USA), and the Kd value was calculated. Each compound was tested in triplicate unless stated otherwise.

 

4.2.6. GTPγS Binding Assay

The method for measuring agonist-stimulated [35S]-GTPγS binding to the human CB1 and CB2 receptors was used as described previously. In brief, binding reactions were carried out in 96-well microplates in a final volume of 500 μL. Cell membranes (20 μg) were incubated with 0.5 nM [35S]-GTPγS, 30 μM GDP, and compounds in assay buffer (50 mM Tris-HCl, 150 mM NaCl, 9 mM MgCl2, 0.2 mM EGTA, and 1.4 mg/mL BSA, pH 7.4) for 2 h at 37 °C with gentle shaking. The nonspecific binding (NSB) was determined using 40 mM nonradiolabeled guanosine 5′-(γ-thio) triphosphate (GTPγS) (PerkinElmer, Waltham, MA). The positive control was attained by utilizing 10 μM unlabeled CP55,940 for the test compound. The reaction was terminated by rapid vacuum filtration, and the membranes were harvested onto a PerkinElmer Unifilter GF/B-96 filter plate and washed three times with ice-cold washing buffer (10 mM Tris-HCl, pH 7.4), and the bound radioactivity was quantified by a Packard TopCount Scintillation Counter.

4.3. Computational Methods

 

4.3.1. Protein Preparation and Receptor Grid Generation

The X-ray crystal structure of cannabinoid receptors 1 (PDB ID: 5XRA) and the Cryo-EM structure of CB2 (PDB ID: 6PT0) were downloaded from the RCSB Protein Data Bank (PDB). These structures were prepared by adding hydrogen atoms, bond orders, and missing side chains and by proper ionization at physiological pH 7.4 using the Protein Preparation wizard module implemented in the Schrödinger software.

 

4.3.2. Ligand Preparation

The 2D structures of 11E, 11H, and 11J were drawn using the 2D-Sketcher module implemented in the Schrödinger softwareand prepared using the LigPrep module of the Schrödinger software, using the OPLS3e force field and a pH range of 7.0 ± 2.0 using Epik.

 

4.3.3. Ligand Docking within the Orthosteric Binding Site

The centroid of the orthosteric ligand co-crystallized with CB1R (PDB ID: 5XRA), and CB2R (PDB ID: 6PT0) were used to define the center of the receptor grid for docking. XP docking (Glide, Schrödinger) was used with flexible ligand sampling, keeping the receptor rigid., The best-docked pose of the ligand was selected based on the Glide Emodel scores.

 

4.3.4. MD Simulations for CB1–11J, CB2–11J, and CB2–11H Complexes

MD simulations were performed to further assess the stabilities and interaction profiles of the best complexes of CB1–11J, CB2–11J, and CB2–11H obtained after the docking study. A similar MD simulation protocol was applied to that described previously., In brief, the complex was embedded in a POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) bilayer and solvated with 11 Å TIP3P water buffer using the OPLS3e (optimized potentials for liquid simulations 3) force field in Desmond, Schrödinger. The system was neutralized, and 0.15 M NaCl was added to the system. The system was equilibrated using the following protocol. First, the system was simulated for 100 ps using Brownian dynamics in the NVT ensemble at 10 K with the restraint of 50 kcal/mol on solute heavy atoms. Second, a 500 ps simulation was run in the NVT ensemble using the Berendsen thermostat (10 K) while retaining the restraint on solute heavy atoms. Third, a 300 ps simulation was run in the NPT ensemble using the Berendsen thermostat (10 K) and barostat (1 atm) while restraints were retained. The system was gradually heated to 300 K over the next 500 ps. A final 500 ps simulation was performed in which all restraints were removed before the production run. The final production run (200 ns) was performed in the NPT ensemble using a timestep of 2 fs. The Langevin thermostat and Langevin were used for the production runs.

Acknowledgments

This research is in part supported by “Discovery & Development of Natural Products for Pharmaceutical & Agrichemical Applications” funded by the United States Department of Agriculture, Agricultural Research Service, Specific Cooperative Agreement No. 58-6060-6-015.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c02413.

  • 3D overlaid representation of 11H, 11J, and Δ9-THC against the CB2 receptor; RMSF plots for 11J and 11H; NMR and HR-MS spectral data for compounds 11A–11K (PDF)

 

Notes

The authors declare no competing financial interest.

Supplementary Material

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