{'fraction_non_alphanumeric': 0.03931406012254801, 'fraction_numerical': 0.047086400460583135, 'mean_word_length': 4.6396103896103895, 'pattern_counts': {'":': 0, '<': 3, '<?xml version=': 0, '>': 2, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Novel Machaeriol Analogues as Modulators of Cannabinoid Receptors: Structure–Activity Relationships of (+)-Hexahydrocannabinoids and Their Isoform Selectivities

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.


INTRODUCTION
More than 100 natural phytocannabinoids have been isolated and characterized from Cannabis sativa; tetrahydrocannabinol (Δ 9 -THC) and cannabidiol (CBD) are the two best-studied phytoconstituents ( Figure 1). 1 CB 1 R and CB 2 R, the cannabinoid receptors, are part of the endocannabinoid system (ECS), which comprises the endogenous ligands and their related enzymes and transporters. CB 1 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, 2 while CB 2 receptors are found mostly in the immune and gastrointestinal systems. 2 The expression of the CB 2 receptor in the CNS is very low compared to that of the CB 1 receptor, which makes it an attractive target to avoid possible CNS side effects. Some previously studied therapeutic benefits of CB 2 R agonists are analgesic and anti-inflammatory effects. 3−5 CB 2 R agonists have shown efficacy as potential therapeutic agents in peripheral diseases that involve inflammation, such as atherosclerosis, 6 renal fibrosis, 7 and liver cirrhosis. 8 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. 9,10 Δ 9 -THC is a partial CB 1 and CB 2 receptor agonist, whereas CBD is a weak antagonist or a negative allosteric modulator (at the CB 1 R level) of the CB 1 /CB 2 receptor. 11−13 The pharmacological activity of Δ 9 -THC is stereospecific, i.e., the (−)-trans-isomer (dronabinol, FDA approved) is 6−100 times more potent than the (+)-trans-isomer. 14 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. 15 Machaeriols have a hexahydrodibenzopyran scaffold (Figure 1). 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 Δ 9 -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 CB 1 and CB 2 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 CB 1 R and CB 2 R) and 11H (against CB 2 R only), using molecular dynamics (MD) simulation and binding free-energy calculations.

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). 16 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. Acidmediated deprotection of the MOM group produced 14 diverse analogues, 11A−11N, with excellent yields (Scheme 1, Figure  2).

2.2.
In Vitro Competitive Radioligand Displacement Assays for CB 1 and CB 2 Receptors. In preliminary probing, the synthesized compounds were assayed at a single concentration of 10 μM for their in vitro CB 1 and CB 2 percent displacement. The highly potent and nonselective CB agonist CP55,940 was used as a positive control. 17 The compounds that showed >50% displacement of the radioligand [ 3 H]-CP55,940 at the CB receptors were further assayed over a range of concentrations using a competitive radioligand binding assay to estimate binding affinities (K i values). Two compounds (11E and 11J) exhibited low micromolar CB 1 R displacement, with IC 50 values ≤1.0 μM ( Figure 3 and Table 1). Among the 14 compounds evaluated in the competitive radioligand binding assay (Table 1), compounds 11B, 11H, and 11J ( Figure 4) showed significant displacement at the CB 2 receptor, yielding binding affinities with IC 50 values in submicromolar/high nanomolar levels except for 11A and 11E ( Figure 4 and Table  1). Compound 11E, having an octenyl chain at the C3 position similar to CBD and Δ 9 -THC (pentyl chain), exhibited a higher CB 1 R 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 CB 2 R binding affinity in comparison with those having small aromatic rings (11A, 11C, 11D, and 11K).
2.3. In Vitro GTPγS Functional Assays for CB 1 and CB 2 Receptors. Using membrane preparations similar to the radioligand binding methods and GTPγ[ 35 S], the functional behavior (e.g., agonists, antagonists, or inverse agonists) of the most promising compounds was determined using GTPγS functional assays. 9 Compound 11J was tested using CB 1 and CB 2 functional assays, while 11H was tested using the CB 2 functional assay only. All were determined to act as agonists, with the most promising being 11H (EC 50 = 5730 ± 3289 nM) against the CB 2 receptor. Compound 11J showed an EC 50 value of 1471 ± 708 nM against the CB 1 receptor, while at the CB 2 receptor, it showed a moderate EC 50 value of 15 993 ± 8631 nM, confirming its preference toward the CB 1 receptor as an agonist ( Figures 5 and 6). Compound 11E was tested using the CB 1 functional assay and was identified as a CB 1 R agonist with an EC 50 value of 239 ± 68 nM.
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 CB 1 and CB 2 receptor protein crystal structures. Extra precision (XP) docking (Glide, Schrodinger) was used with flexible ligand sampling, keeping the receptor rigid. 18,19 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 7A,C). Similarly, the hexahydrochromane scaffold and the benzothiophene moiety of compound 11J exhibited strong π−π stacking interactions with Phe170, Phe268, and Trp279 ( Figure 7B,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 cocrystallized agonist in the active-state X-ray crystal structure (PDB ID: 5XRA) of the CB 1 receptors. 17 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 CB 1 receptor Ser383, 17 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 7. Furthermore, we compared the docked pose of Δ 9 -THC with 11E and 11J into the active site of the CB 1 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 Δ 9 -THC docked pose. Interestingly, upon close analysis, the ligand-binding orientation of hexahydrochromane 11E and 11J was significantly different from that of Δ 9 -THC ( Figure 7D). 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 CB 1 . . Binding displacement curves for the CB 1 receptor were obtained for compounds 11E and 11J with a radioligand binding assay. CP55,940 was used as a positive control. IC 50 and K i were determined by GraphPad Prism 9.1 and are listed in Table 1. The data represent mean ± SEM. Each compound was tested in triplicate.
We compared the docked pose of Δ 9 -THC with 11J and 11H against the CB 2 receptor and found that they overlaid well with Δ 9 -THC in the active site of the CB 2 receptor ( Figure S1). However, the substituted C3 moieties of 11H and 11J were vertically inverted compared to the C5 alkyl chain of Δ 9 -THC ( Figure 8). They also maintained the key interactions of Δ 9 -THC with the CB 2 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   Table  1. The data represent mean ± SEM. Each compound was tested in triplicate.  ligand within the protein active site. 18,21 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 CB 2 R−11H, CB 2 R− 11J, and CB 1 R−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 9 and 10. The RMSD of the protein Cα atoms of CB 2 R proteins in the complex of CB 2 −11H and CB 2 −11J varied between 1 and 1.5 Å during the whole simulation, which is an acceptable range for GPCR proteins. 19 Similarly, the RMSD of ligand heavy atoms of CB 2 R−11H and CB 2 R−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 CB 2 protein Cα atoms and ligand heavy atoms suggest that CB 2 R− 11H and CB 2 R−11J have strong predicted binding interactions. 2.5.1. CB 1 R−11J Complex. The RMSD of the ligand heavy atoms of CB 1 R−11J was very stable throughout the 200 ns simulation and suggested that CB 1 R−11J has strong binding interactions with the CB 1 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 CB 1 R for complexes with CB 1 R−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 11) and 2D-ligand contact map ( Figure 12) of 11J with the CB 1 receptor indicated Hbonding 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 CB 1 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 CB 1 R−11J complex (Table 2). In summary, 11J formed stable and strong interactions with the key residues of the CB 1 receptor.
2.5.2. CB 2 R−11J Complex. The RMSD of the protein Cα atoms of CB 2 R protein in the complex of CB 2 R−11J reached an equilibrium state just after 50 ns and remained stable in the rest of the simulation ( Figure 9A). Similarly, the RMSD of ligand's heavy atoms in the CB 2 R−11J complex was very stable throughout the 200 ns simulation ( Figure 9B). The lower RMSD values of CB 2 R protein Cα atoms and ligand heavy atoms suggest that CB 2 R−11J has strong binding interactions with the CB 2 receptor. The RMSF plot based on the Cα atoms of CB 2 R 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 13) and 2D ligand contact map (Figure 14) of 11J with the CB 2 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 CB 2 R−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 CB 2 receptor affirmed its complex stability (Table 2). Overall, 11J formed stable and strong interactions with the CB 2 receptor.
2.5.3. CB 2 R−11H Complex. The RMSF plot based on the Cα atoms of CB 2 R 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 15) and 2D ligand contact map (Figure 16) of 11H with the CB 2 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 CB 2 −11H complex (Table 2). In summary, strong H-bonding of 11H with Ser285 and multiple π−π stacking with CB 2 R residues resulted in a stable complex of CB 2 R−11H.

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 CB 1 and CB 2 receptors. Two of the compounds (11E and 11J) exhibited low micromolar CB 1 displacement with an IC 50 value of ≤2.0 μM. Compounds 11A, 11B, 11E, 11H, and 11J showed significant displacement at the CB 2 receptor yielding binding affinities with an IC 50 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 CB 2 R 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 CB 1 receptor. MD simulations and binding free-energy calculations confirmed the stability of these compounds with CB 1 and CB 2 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 CB 1 and CB 2 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 CB 1 /CB 2 receptor isoform selectivities.

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). 1 H NMR and 13 C NMR spectra were recorded in CDCl 3 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.
4.2.4. Cell Membrane Preparation. Cell plasma membranes were prepared from HEK293 cells with stable expression of CB 1 and CB 2 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 MgCl 2 , 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. 17 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 (IC 50 , K i , and EC 50 ) were analyzed using a nonlinear regression curve fit model using GraphPad Prism 9.1 software (GraphPad Software, Inc., San Diego, CA, USA), and the K d value was calculated. Each compound was tested in triplicate unless stated otherwise.
4.2.6. GTPγS Binding Assay. The method for measuring agonist-stimulated [ 35 S]-GTPγS binding to the human CB 1 and CB 2 receptors was used as described previously. 24 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 [ 35 S]-GTPγS, 30 μM GDP, and compounds in assay buffer (50 mM Tris-HCl, 150 mM NaCl, 9 mM MgCl 2 , 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) 25 and the Cryo-EM structure of CB 2 (PDB ID: 6PT0) 26 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 Schrodinger software.
4.3.2. Ligand Preparation. The 2D structures of 11E, 11H, and 11J were drawn using the 2D-Sketcher module implemented in the Schrodinger software 27 and prepared using the LigPrep 28 module of the Schrodinger software, 27 using the OPLS3e force field 29 and a pH range of 7.0 ± 2.0 using Epik. 30 4.3.3. Ligand Docking within the Orthosteric Binding Site. The centroid of the orthosteric ligand co-crystallized with CB 1 R (PDB ID: 5XRA), and CB 2 R (PDB ID: 6PT0) were used to define the center of the receptor grid for docking. XP docking (Glide, Schrodinger) was used with flexible ligand sampling, keeping the receptor rigid. 31,32 The best-docked pose of the ligand was selected based on the Glide Emodel scores.
4.3.4. MD Simulations for CB 1 −11J, CB 2 −11J, and CB 2 − 11H Complexes. MD simulations were performed to further assess the stabilities and interaction profiles of the best complexes of CB 1 −11J, CB 2 −11J, and CB 2 −11H obtained after the docking study. A similar MD simulation protocol was applied to that described previously. 33,34 In brief, the complex was embedded in a POPC (1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine) bilayer and solvated with 11 Å TIP3P water buffer using the OPLS3e (optimized potentials for liquid simulations 3) force field in Desmond, 35 Schrodinger. 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.