pubs.acs.org/jmc Article Design of Negative and Positive Allosteric Modulators of the Cannabinoid CB2 Receptor Derived from the Natural Product Cannabidiol Gemma Navarro, Angel Gonzalez, Adrià Sánchez-Morales, Nil Casajuana-Martin, Marc Gómez-Ventura, Arnau Cordomí, Félix Busqué, Ramon Alibés,* Leonardo Pardo,* and Rafael Franco* Cite This: J. Med. Chem. 2021, 64, 9354−9364 Read Online See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Cannabidiol (CBD), the second most abundant of the active compounds found in the Cannabis sativa plant, is of increasing interest because it is approved for human use and is neither euphorizing nor addictive. Here, we design and synthesize novel Downloaded via 83.50.3.167 on May 12, 2023 at 11:09:29 (UTC). compounds taking into account that CBD is both a partial agonist, when it binds to the orthosteric site, and a negative allosteric modulator, when it binds to the allosteric site of the cannabinoid CB2 receptor. Molecular dynamic simulations and site-directed mutagenesis studies have identified the allosteric site near the receptor entrance. This knowledge has permitted to perform structure-guided design of negative and positive allosteric modulators of the CB2 receptor with potential therapeutic utility. ■ INTRODUCTION Cannabidiol (CBD) is the second most abundant of the active legalize the use of Cannabis products for medical purposes.6 As a consequence, the CBD scaffold is of increasing interest for compounds found in the Cannabis sativa plant (more medicinal chemists due to its potential therapeutic utility.7 commonly known as marijuana) (Figure 1). However, in The actions of CBD were first assumed to be mediated through two members of the G-protein-coupled receptor (GPCR) family, the cannabinoid CB1 (CB1R) and CB2 (CB2R) receptors. However, there is evidence that CBD also modulates other molecular targets.8 These include serotonin, adenosine, opioid, and orphan GPCRs plus non-GPCR proteins.9 Within the endocannabinoid-related receptors, the pharmacology of CBD shows significant divergences as some authors support high potency as an antagonist of CB1R and Figure 1. Structures of CBD, (−)-trans-THC, and JWH-133. CB2R10 and others support a very low affinity as a CB1R agonist.11 On the other hand, recent results show that CBD may act as a negative allosteric modulator (NAM) of both contrast to (−)-trans-Δ9-tetrahydrocannabinol (THC), the CB1R12 and CB2R.13 In the case of CB2R, CBD would act at principal psychoactive constituent of Cannabis, CBD is micromolar concentration as an agonist14 and at nanomolar noneuphorizing and nonaddictive. In humans, CBD exhibits concentration as a NAM.15 a favorable safety profile.1 Although the exact medical Here, we have used the recently released structure of CB2R implications are currently being investigated, CBD is in its inactive16 and active,17,18 Gi-bound, conformations to generating considerable interest due to its beneficial neuro- identify the binding mode of CBD in the allosteric binding site protective, antiepileptic, anxiolytic, antipsychotic, and anti- inflammatory properties.2 Sativex, a 1:1 formulation of CBD and THC, is a cannabinoid medicine approved for the Received: March 28, 2021 treatment of spasticity due to multiple sclerosis.3 In addition, Published: June 23, 2021 an oral solution of CBD (Epidiolex) is the first and only US Food and Drug Administration-approved prescription that is used to treat refractory epilepsy due to Lennox−Gastaut or Dravet syndrome.4,5 Thus, there is a growing pressure to © 2021 The Authors. Published by American Chemical Society https://doi.org/10.1021/acs.jmedchem.1c00561 9354 J. Med. Chem. 2021, 64, 9354−9364 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Figure 2. Dose−response curves on forskolin-induced cAMP levels (top), on ERK1/2 phosphorylation (middle), and on DMR (bottom), upon the treatment of CB2R-expressing HEK-293T cells [wild-type CB2R (A) and Val1133.32Met (B), Val361.35Met (C), Ala2827.36Met (D), or Ser2857.39Leu (E) mutants]. Ligands used were CBD (black line), JWH-133 (green), and JWH-133 + CBD (red) ligands. Data for cAMP (n = 9, each in triplicates) are given in percentage (100% represents the forskolin effect), for ERK1/2 phosphorylation (n = 7, each in triplicates) are expressed as percentage with respect to basal levels, and for DMR tracings are representing the picometer (pm) shifts of reflected light wavelengths over time upon ligand treatment. Table 1. Functional Properties of JWH-133, CBD, and JWH-133 + CBD at Wild-Type and Mutant CB2R cAMP assays pERK1/2 assays DMR assays receptor ligand pEC50a Emaxb pEC50a Emaxc pEC50a Emaxd wild type CBD 7.3 ± 0.2 74.0 ± 3.0 7.9 ± 0.2 155.8 ± 4.5 6.3 ± 0.1 49.1 ± 2.4 JWH-133 7.5 ± 0.1 46.0 ± 3.5 8.1 ± 0.2 322.2 ± 14.6 6.2 ± 0.1 182.9 ± 8.1 JWH-133 + CBD 7.3 ± 0.2 57.8 ± 4.1 8.1 ± 0.2 250.3 ± 8.1 6.2 ± 0.1 117.0 ± 5.9 V1133.32M CBD 7.4 ± 0.3 89.1 ± 1.9 7.9 ± 0.2 186.1 ± 7.4 6.2 ± 1.1 5.3 ± 2.1 JWH-133 7.7 ± 0.1 72.5 ± 1.7 7.7 ± 0.2 275.8 ± 15.3 6.3 ± 0.1 79.9 ± 3.2 JWH-133 + CBD 7.6 ± 0.2 77.8 ± 2.2 7.4 ± 0.1 216.5 ± 5.2 6.4 ± 0.2 71.9 ± 6.2 V361.35M JWH-133 7.9 ± 0.1 56.7 ± 1.7 7.5 ± 0.2 363.3 ± 25.5 6.1 ± 0.1 193.2 ± 13.3 JWH-133 + CBD 7.8 ± 0.2 53.0 ± 3.4 7.4 ± 0.2 352.7 ± 18.8 6.2 ± 0.1 199.6 ± 13.8 A2827.36M JWH-133 7.9 ± 0.2 45.4 ± 4.2 7.7 ± 0.1 375.7 ± 6.7 6.2 ± 0.1 198.8 ± 12.3 JWH-133 + CBD 8.1 ± 0.2 48.8 ± 3.3 7.9 ± 0.5 325.2 ± 40.9 6.2 ± 0.1 188.6 ± 10.7 S2857.39L JWH-133 7.4 ± 0.2 49.3 ± 3.8 7.4 ± 0.3 373.5 ± 31.5 6.1 ± 0.1 222.6 ± 14.0 JWH-133 + CBD 7.4 ± 0.2 57.4 ± 4.7 7.5 ± 0.1 291.1 ± 8.1 6.1 ± 0.1 164.9 ± 11.0 a pEC50 (nM). bEmax (%), the maximum decrease of forskolin-stimulated cAMP levels (normalized to 100%). cEmax (%), the maximum increase of ERK1/2 phosphorylation expressed as a percentage of basal (normalized to 100%). dEmax, the maximum increase of picometer shifts of reflected light wavelengths expressed as a value above basal. by molecular dynamic (MD) simulations and site-directed induced signaling response of CBD with JWH-133, a potent mutagenesis studies. This knowledge has permitted to perform and selective CB2R agonist, in cAMP production, phosphor- structure-guided design of NAMs and positive allosteric ylation of signal-regulated kinases (pERK1/2), and label-free modulators (PAMs) of CB2R. The designed compounds are dynamic mass redistribution (DMR) assays that enable real- relevant because the combination of orthosteric agonists with time detection of integrated cellular responses in living cells22 PAMs could represent a therapeutic approach for neuro- (Figure 2A and Table 1). These correspond to different steps degenerative disorders and neuropathic pain.19 Until now, the of the signaling pathways. Cells stimulated with forskolin and only reported allosteric modulators of CB2R, in addition to treated with JWH-133 or CBD showed reduced cAMP CBD,15 are PAMs: the endogenous 12-residue peptide pepcan- production, as expected for Gi-coupled receptors. For the 1220 and a synthetic small molecule.21 ERK1/2 pathway, both JWH-133 and CBD increased ERK1/2 ■ RESULTS AND DISCUSSION CBD Is Both a Partial Agonist and a Negative phosphorylation. Finally, DMR assays also showed an increase of response by JWH-133 and CBD action. Thus, CBD is, relative to JWH-133, a partial agonist in all these assays Allosteric Modulator. We have first compared the agonist- because the decrease of cAMP or increase of ERK1/2 9355 https://doi.org/10.1021/acs.jmedchem.1c00561 J. Med. Chem. 2021, 64, 9354−9364 Journal of Medicinal Chemistry pubs.acs.org/jmc Article phosphorylation or increase of DMR response is of less magnitude. Figure S1 shows proposed computer models of JWH-133 and CBD bound to the orthosteric binding site of CB2R, superimposed to the crystal structure of CB2R in complex with the structurally similar AM12033 ligand.18 CBD and JWH-133 are similar in structure, so they both might elicit their agonist action by binding at the orthosteric binding site of CB2R in a similar manner. In order to validate this hypothesis, we mutated the side chain of Val1133.32, which is centrally located in the orthosteric cavity (Figure S1), to the much larger Met side chain (Figure 2B and Table 1). As expected, the Val1133.32Met mutation impairs the signaling of both CBD and JWH-133, indicating the binding at the orthosteric site. Figure 2A and Table 1 also show the effect of CBD on the signaling responses of JWH-133 (JWH-133 + CBD). Clearly, CBD blocks the decrease of forskolin-induced cAMP triggered Figure 3. Detailed view of the docking model (Figure S4) of CBD by JWH-133. Similar effects are observed in the other signaling (magenta sticks) into an allosteric binding site and JWH-133 pathways in which CBD blocks the increase of pERK1/2 and (transparent orange sticks) into the orthosteric binding site of CB2R (green ribbons). The stability of this model was evaluated by DMR responses. Thus, CBD is, in all these assays, a NAM that MD simulation (Figure S5). The Val361.35, Ala2827.36, and Ser2857.39 decreases the efficacy of the orthosteric JWH-133 agonist. residues mutated to verify the proposed binding mode of CBD are CBD Also Binds in an Allosteric Cavity near the shown in green spheres. Phe1173.36 and Trp2586.48, which have been Receptor Entrance. It was proposed that ligands binding a described as conformational toggle or trigger switches involved in the small cavity at the entrance of the orthosteric binding site initial agonist-induced receptor activation, are highlighted with red could act as allosteric modulators,23 as shown in the crystal transparent surfaces. structure of the muscarinic M2 receptor simultaneously bound to an orthosteric agonist and a PAM.24 However, in between Ser and one of the hydroxyl substituents of the meta- cannabinoid receptors, and in other GPCRs for lipid benzenediol moiety. This mutation only partly impairs the mediators, the extracellular N-terminus and ECL2 fold over NAM effect of CBD (Figure 2E and Table 1). the ligand-binding pocket blocking the access to the The pentyl chain of CBD expands toward an intracellular orthosteric binding cavity from the extracellular environ- hydrophobic cavity formed by Phe87 2.57 , Cys288 7.42 , ment.25 Previous ligand-binding pathway simulations have Leu2897.43, and, importantly, Phe1173.36 (Figure 3). Phe3.36 shown that binding of lipid-like ligands to a lipid-specific and Trp6.48 have been described as conformational toggle or GPCR is through a narrow channel between transmembrane trigger switches involved in the initial agonist-induced receptor helices (TMs) 1 and 7 that opens toward the lipid bilayer.26 activation in CB1R34,35 and other GPCRs.36−39 Thus, the We have recently used this channel to design bitopic ligands of pentyl chain of CBD in its allosteric binding mode might CB2R.27 Furthermore, superimposition of the computer model modulate receptor activation. of CB2R in complex with JWH-133 to the crystal structure of Chain Length Determines the NAM or PAM Character CB1R in complex with the antagonist AM6538 shows that of the Allosteric Modulator. Our simulations suggest that AM6538 occupies, in addition to the orthosteric binding site, a the ability of CBD to block the active state of CB2R (NAM “side pocket”28 that is adjacent to JWH-133 near TMs 1 and 7 activity) is due to the insertion of the pentyl chain inside the (Figure S2). Thus, we proposed that CBD, acting as a NAM, hydrophobic pocket between TMs 2, 3, and 7. We have tested binds in an allosteric binding site located at the entrance of the this hypothesis by measuring the activation of CB2R using receptor near TMs 1 and 7. Multiple binding sites have been CBD analogues of different chain lengths. We have synthesized described for CB1R.29 CBD (1a) and CBD analogues 1b−e with decreasing (n = 1, 2, In order to better delineate this allosteric binding site, we and 3) and increasing (n = 5) numbers of methylene units in performed unbiased 1 μs MD simulations of JWH-133 bound the hydrophobic chain with respect to CBD (n = 4) while to the CB2R−Gi complex (Figure S3). Allosteric binding site keeping the rest of the molecule identical (Scheme 1). exploration, conducted on structure snapshots extracted from CBD and analogues have been synthesized by several this simulation, together with molecular docking of CBD into methods, among which the Lewis acid-catalyzed Friedel− these identified cavities (Figure S4) has permitted to propose Crafts reaction of cyclic allylic alcohols with resorcinol the binding mode of CBD into an allosteric binding site close derivatives has given satisfactory results.40 However, the to TMs 1 and 7 (Figure 3). In this computational model, the main drawback of this arylation reaction is the formation of propenyl-methylcyclohexene moiety of CBD points toward the side regioisomers, coming from the attack of the resorcinol at entrance channel between TMs 1 and 7 and the pentyl chain the 4/6 positions that decrease the yield and make the product points toward the intracellular side. In detail, the propane isolation difficult.41 Here, we have followed a practical substituent is located between Val361.35 and Ala2827.36. With approach that avoids the formation of the side regioisomers the aim of experimentally verifying this model, we mutated the by using protected 4,6-dihalo resorcinols in the coupling side chains of Val361.35 (Figure 2C) and Ala2827.36 (Figure reaction.41 The compounds were prepared as depicted in 2D) to the much larger Met side chain. As expected, these Scheme 1. mutations completely impair the NAM effect of CBD on the The functional properties of compounds 1a−1e were JWH-133 agonist (Table 1) by occupying the volume of the evaluated through cAMP assays using HEK293 cells stably proposed allosteric binding site. Moreover, we also mutated expressing CB2R and treating with forskolin to activate adenyl Ser2857.39 to Leu to verify the potential hydrogen bond cyclase (Figure 4 and Table 2). Remarkably, the ethyl or 9356 https://doi.org/10.1021/acs.jmedchem.1c00561 J. Med. Chem. 2021, 64, 9354−9364 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Scheme 1. Synthesis of CBD (1a, n = 4) and CBD Analogues 1b−e with Decreasing (1b−1d, n = 1−3) and Increasing (1e, n = 5) Numbers of Methylene Unitsa a The synthesis began by the Wittig reaction of the ylide of 3a−e with commercially available 3,5-dimethoxybenzaldehyde, 2, to deliver olefins 4a−e as mixtures of Z and E isomers, which were conveniently reduced with hydrogen under pressure to the C-5 alkyl resorcinol derivatives 5a−e.30 Regioselective electrophilic aromatic bromination of 5a−e using 2.3 equiv of N-bromosuccinimide (NBS) in DCM at rt produced exclusively the 4,6-dibrominated products 6a−e in good yields.31 Then, the methyl ether-protecting groups were removed with boron tribromide to generate the key resorcinol intermediates 7a−e which were submitted to the coupling reaction with (1S,4R)-4-isopropenyl-1-methyl-2-cyclohexen-1-ol, 8, under a Lewis acid catalyst.32 For this purpose, different acids were screened (p-TsOH, BF3·OEt2, and AlCl3) and among these, p-TsOH was found to be the best catalyst. Thus, Friedel−Craft alkylation of resorcinols 7a−e with 8 under p-TsOH catalysis in DCM furnished adducts 9a−e as single diastereomers. Finally, reductive didehalogenation using sodium sulfite33 in the presence of Et3N in a mixture of MeOH and H2O at 75 °C delivered the targeted cannabinoids 1a−e. The optical rotation of the prepared CBD was consistent with the literature [[α]D22 = −121.4 (c 1.00, EtOH)32 and [α]D20 = −122.0 (c 1.10, EtOH]. Complete experimental details and analytical data for the synthetized compounds are included in the Experimental Section. Table 2. Modulation of the Agonist Signal of JWH-133 by Designed Allosteric Modulators 1a−1e orthosteric allosteric max. effect site site pEC50a Emaxb (% of JWH-133)c JWH-133 none 7.6 ± 0.1 56.0 ± 1.3 100% JWH-133 1b 7.5 ± 0.2 45.3 ± 1.5 124% PAM (n = 1) JWH-133 1c 7.9 ± 0.2 48.0 ± 1.8 118% PAM (n = 2) Figure 4. Decrease of forskolin-induced cAMP (normalized to 100%), JWH-133 1d 6.7 ± 0.2 64.1 ± 3.2 82% NAM in HEK-293T cells, upon the stimulation of wild-type CB2R with the (n = 3) orthosteric JWH-133 agonist (green line) and in conjunction with JWH-133 1a 6.6 ± 0.2 67.8 ± 3.3 73% NAM CBD analogues with decreasing (1b−1d, n = 1−3) and increasing (n = 4) (1e, n = 5) numbers of methylene units in the hydrophobic chain with JWH-133 1e 6.9 ± 0.2 83.5 ± 2.3 38% NAM respect to CBD (1a, n = 4). Compounds with chains of n = 1−2 are (n = 5) a PAMs (blue lines) and with n = 3−5 are NAMs (red lines). pEC50 (nM). bEmax (%), the maximum inhibition of forskolin- stimulated cAMP levels (normalized to 100%). These values were calculated using nonlinear regression analysis. Data are expressed as propyl chains make compounds 1b and 1c (n = 1 and 2) the mean ± SEM of at least three independent experiments performed PAMs, as they facilitate the decrease of forskolin-induced in triplicates. cThe efficiency (in %) of 1a−1e together with JWH-133 cAMP triggered by JWH-133 (Emax 45.3 or 48.0 vs 56.0). in decreasing cAMP relative to JWH-133 (100%). Compound 1c is more potent than 1b, as the propyl chain of 1c left-shifts the dose−response curve (0.4 log units) relative relative to CBD (Emax 67.8). The influence of ligand chain to the ethyl chain of 1b. The butyl chain makes compound 1d length in the activation of lipid GPCRs has been described.42 (n = 3) a NAM, which is very similar in properties to CBD (n PAM 1c (n = 2) and NAM 1e (n = 5) were modeled in the = 4). In agreement with our hypothesis, the extension of the CB2R−Gi complex and performed unbiased MD simulations to number of methylene units makes the hexyl chain of explore the influence of the chain length in the conformational compound 1e (n = 5) a more efficacious NAM (Emax 83.5), toggle or trigger switches (Figure 5). The shorter chain of 9357 https://doi.org/10.1021/acs.jmedchem.1c00561 J. Med. Chem. 2021, 64, 9354−9364 Journal of Medicinal Chemistry pubs.acs.org/jmc Article reactions were monitored by analytical thin-layer chromatography (TLC) using silica gel 60-precoated aluminum plates (0.20 mm thickness). Flash column chromatography was performed using silica gel Geduran SI 60 (40−63 μm). 1H NMR and 13C NMR spectra were recorded at 250, 360, and 400 MHz and 90 and 100 MHz, respectively. Proton chemical shifts are reported in parts per million (δ) (CDCl3, δ 7.26 or CD3OD, δ 3.31). Carbon chemical shifts are reported in parts per million (δ) (CDCl3, δ 77.16 or CD3OD, δ 49.00). NMR signals were assigned with the help of heteronuclear single-quantum coherence. Infrared peaks are reported in cm−1. The purity of all final compounds was ≥95% as determined by quantitative one-dimensional (1D) 1H NMR (qHNMR) experiments using dimethylsulfone (DMSO2, 99.8% pure) as the internal calibrant. Melting points were determined on a hot stage and are uncorrected. High-resolution mass spectra were recorded using electrospray ionization (ESI). Optical rotations were measured at 20 ± 3 °C. (Z)- and (E)-1,3-Dimethoxy-5-(pent-1-en-1-yl)benzene (4a). To a suspension of butyltriphenylphosphonium bromide (7.38 g, 18.17 mmol) in anhydrous tetrahydrofuran (THF) (40 mL), n-BuLi (2.5 M in THF, 7.4 mL, 18.50 mmol) was added dropwise at 0 °C. After continuous stirring for 20 min, a solution of 3,5-dimethox- ybenzaldehyde, 2, (2.05 g, 12.32 mmol) in dry THF (60 mL) was slowly added dropwise. Then, the reaction mixture was stirred at room temperature (rt) until the complete consumption of the starting material, TLC (hexanes/EtOAc, 9:1). The reaction was quenched by the slow addition of water (50 mL) and extracted with EtOAc (3 × 40 mL). The combined organic layers were dried over anhydrous Na2SO4, concentrated, and purified by column chromatography (hexanes/EtOAc, 9:1) to give a 1.3:1 mixture of (Z)- and (E)-olefins Figure 5. (A,B) MD simulation snapshots (10 structures collected 4a (1.88 g, 9.11 mmol, 74% yield) as a colorless oil. (E)-4a47 1H every 100 ns) of CB2R−Gi (only the initial structure is shown for NMR (360 MHz, CDCl3): δ 6.54 (d, J4,2 = J6,2 = 2.2 Hz, 2H, H-4, H- clarity) in complex with the JWH-133 agonist (yellow surface) bound 6), 6.40−6.20 (m, 3H, H-2, H-1′, H-2′), 3.82 (s, 6H, 3-OCH3/5- to the orthosteric site and PAM 1c [cyan sticks, (A)] or NAM 1e OCH3), 2.21 (q, J3′,2′/4′ = 7.2 Hz, 2H, H-3′), 1.55−1.45 (m, 2H, H- [salmon sticks, (B)] bound to the allosteric site. Conformational 4′), and 0.99−0.94 (m, 3H, H-5′); 13C NMR (90 MHz, CDCl3): δ toggle/trigger switches are shown in pink spheres (Phe1173.36 and 161.0 (C1, C3), 140.1 (C5), 131.7 (C2′), 129.9 (C1′), 104.1 (C4, C6), W2586.48). Calculated frequency contacts (%) between side-chain 99.2 (C2), 55.4 (2×-OCH3), 35.2 (C3′), 22.6 (C4′), and 13.9 (C5′). residues of CB2R (blue sticks) involved in stable interactions with (Z)-4a: 1H NMR (360 MHz, CDCl3): δ 6.47 (d, J4,2 = J6,2 = 2.2 Hz, PAM 1c or NAM 1e during MD simulations are displayed in squares, 2H, H-4, H-6), 6.40−6.35 (m, 2H, H-2, H-1′), 5.69 (dt, J2′,1′ = 11.7 color-coded according to the shown scale. Contact frequency analysis Hz, J2′,3′ = 7.2 Hz, 1H, H-2′), 2.34 (q, J3′,2′/4′ = 7.3 Hz, 2H, H-3′), was conducted with the GetContacts software package.43 1.50 (q, J4′,2′/3′ = 7.3 Hz, 2H, H-4′), and 0.96 (q, J5′,4′ = 7.3 Hz, 2H, H-5′); 13C NMR (90 MHz, CDCl3): δ 160.6 (C1, C3), 139.8 (C5), 133.7 (C2′), 128.9 (C1′), 107.0 (C4, C6), 98.8 (C2), 55.4 (2×-OCH3), PAM 1c does not interact with Phe1173.36, Cys2887.42, or 30.9 (C3′), 23.3 (C4′), and 14.0 (C5′). Leu2897.43, whereas the longer chain of NAM 1e does. The 1,3-Dimethoxy-5-vinylbenzene (4b). The synthesis of 4b48 was interaction of the long hexyl chain of NAM 1e with Phe1173.36 performed as described for 4a by using methyltriphenylphosphonium favors its inactive trans conformation, in contrast to the active bromide (6.51 g, 18.20 mmol) in dry THF (35 mL), n-BuLi (2.5 M in gauche+ conformation favored by JWH-133 and PAM 1c THF, 7.4 mL, 18.38 mmol), and 2 (2.01 g, 12.08 mmol) in dry THF (Figure S6). (60 mL). Yield 4b: 88% (1.74 g, 10.61 mmol) as a colorless oil: 1H ■ NMR (360 MHz, CHCl3): δ 6.66 (dd, Jtrans = 17.5 Hz, Jcis = 10.9 Hz, CONCLUSIONS 1H, H-1′), 6.57 (d, J4,2 = J6,2 = 2.4 Hz, 2H, H-4, H-6), 6.39 (t, J2,4 = J2,6 = 2.4 Hz, 1H, H-2), 5.73 (dd, Jtrans = 17.5 Hz, Jgem = 0.9 Hz, 1H, It has been suggested that cannabinoid ligands could fill the H-2′a), 5.25 (dd, Jcis = 10.9 Hz, Jgem = 0.9 Hz, 1H, H-2′b), and 3.81 therapeutic gap between opioids and nonsteroidal anti- (s, 6H, 1-OCH3/3-OCH3); 13C NMR (90 MHz, CDCl3): δ 161.0 inflammatories in multiple moderate pain conditions, but (C1, C3), 139.7 (C5), 136.9 (C1′), 114.5 (C2′), 104.4 (C4, C6), 100.1 these compounds can have significant undesirable side effects. (C2), and 55.4 (2×-OCH3). This limitation can be avoided by designing ligands that act on (Z)- and (E)-1,3-Dimethoxy-5-(prop-1-en-1-yl)benzene (4c). CB2R, instead of CB1R, due to their lack of adverse A mixture of olefins (Z)- and (E)-4c was prepared as described for 4a psychotropic effects44 and designing PAMs that increase the by using ethyltriphenylphosphonium bromide (6.70 g, 18.53 mmol) response of the orthosteric endogenous agonist to limit adverse in dry THF (30 mL), n-BuLi (2.5 M in THF, 7.2 mL, 18.05 mmol), and 2 (2.07 g, 12.47 mmol) in dry THF (60 mL). Yield 4c (Z/E, effects.19 Accordingly, we have designed and synthesized in 1:1.2): 99% (2.20 g, 12.34 mmol) as a colorless oil. (E)-4c:48 1H this article PAMs of CB2R using the natural product CBD as a NMR (360 MHz, CD3OD): δ 6.48 (m, 2H, H-4, H-6), 6.37−6.20 (m, scaffold that might be useful for the treatment of pain without 3H, H-2, H-1′, H-2′), 3.76 (s, 6H, 1-OCH3/3-OCH3), and 1.88−1.84 producing tolerance or dependence.45,46 ■ (m, 3H, H-3′); 13C NMR (90 MHz, CD3OD): δ 162.4 (C1, C3), 141.3 (C5), 132.4 (C1′), 126.4 (C2′), 104.9 (C4, C6), 99.9 (C2), 55.6 EXPERIMENTAL SECTION (2×-OCH3), and 18.5 (C3′). (Z)-4c: 1H NMR (360 MHz, CD3OD): Synthetic Procedures and Compound Characterization. δ 6.42 (m, 2H, H-4, H-6), 6.37−6.20 (m, 2H, H-2, H-1′), 5.75 (m, Commercially available reagents were used as received. Solvents 1H, H-2′), 3.75 (s, 6H, 1-OCH3/3-OCH3), and 1.88−1.84 (m, 3H, were dried by distillation over the appropriate drying agents. All H-3′); 13C NMR (90 MHz, CD3OD): δ 162.0 (C1, C3), 140.7 (C5), 9358 https://doi.org/10.1021/acs.jmedchem.1c00561 J. Med. Chem. 2021, 64, 9354−9364 Journal of Medicinal Chemistry pubs.acs.org/jmc Article 131.1 (C1′), 127.7 (C2′), 107.9 (C4, C6), 99.6 (C2), 55.7 (2×-OCH3), J4,2 = J6,2 = 2.2 Hz, 2H, H-4, H-6), 6.31 (m, 1H, H-2), 3.79 (s, 6H, 1- and 14.9 (C3′). OCH3/3-OCH3), 2.54 (t, J1′,2′ = 7.5 Hz, 2H, H-1′), 1.64 (quint, (Z)- and (E)-1-(But-1-en-1-yl)-3,5-dimethoxybenzene (4d). J2′,1′/3′ = 7.5 Hz, 2H, H-2′), and0.95 (t, J3′,2′ = 7.5 Hz, 3H, H-3′); 13C The synthesis of a mixture of olefins (Z)- and (E)-4d was performed NMR (90 MHz, CDCl3): δ 160.7 (C1, C3), 145.3 (C5), 106.9 (C4, as described for 4a by using propyltriphenylphosphonium bromide C6), 97.7 (C2), 55.3 (2×-OCH3), 38.5 (C1′), 24.5 (C2′), and 14.0 (7.10 g, 18.42 mmol) in dry THF (40 mL), n-BuLi (2.5 M in THF, (C3′). 7.2 mL, 18.05 mmol), and 2 (2.03 g, 12.19 mmol) in dry THF (60 5-Butyl-1,3-dimethoxybenzene (5d). Compound 5d49 was mL). Yield 4d (Z/E, 1:1.5): 88% (2.05 g, 10.66 mmol) as a colorless prepared as described for 5a by using a solution of 4d (2.05 g, oil: HRMS (ESI+) calcd for [C24H16O2 + H]+ ([M + H]+), 193.1229; 10.66 mmol) and two drops of acetic acid in MeOH (60 mL) and found, 193.1220. IR (ATR): 2960, 1591, 1457, 1204, 1152, 1065, and Pd/C (10% in wt of Pd, 212 mg, 200 μmol). Yield 5d: 98% (2.03 g, 826 cm−1. (E)-4d: 1H NMR (360 MHz, CDCl3): δ 6.52 (d, J2,4 = J6,4 10.44 mmol) as a yellow oil: 1H NMR (360 MHz, CDCl3): δ 6.35 (m, = 2.3 Hz, 2H, H-2, H-6), 6.34 (d, J4,2 = J4,2 = 2.3 Hz, 1H, H-4), 6.37− 2H, H-4, H-6), 6.30 (m, 1H, H-2), 3.78 (s, 6H, 1-OCH3/3-OCH3), 6.22 (m, 2H, H-1′, H-2′), 3.80 (s, 6H, 3-OCH3/5-OCH3), 2.22 (m, 2.56 (t, J1′,2′ = 7.6 Hz, 2H, H-1′), 1.59 (quint, J2′,1′/3′ = 7.6 Hz, 2H, H- 2H, H-3′), and 1.12−1.05 (m, 3H, H-4′); 13C NMR (90 MHz, 2′), 1.36 (sext, J3′,2′/4′ = 7.5 Hz, 2H, H-3′), and 0.93 (t, J4′,3′ = 7.4 Hz, CDCl3): δ 161.0 (C3, C5), 140.1 (C1), 133.3 (C1′/C2′), 128.9 (C1′/ 3H, H-4′); 13C NMR (90 MHz, CDCl3): δ 160.8 (C1, C3), 145.5 C2′), 104.1 (C2, C6), 99.2 (C4), 55.4 (2×-OCH3), 26.1 (C3′), and 13.7 (C5), 106.6 (C4, C6), 97.6 (C2), 55.3 (2×-OCH3), 36.1 (C1′), 33.6 (C4′). (Z)-4d: 1H NMR (360 MHz, CDCl3): δ 6.44 (d, J2,4 = J6,4 = 2.2 (C2′), 22.5 (C3′), and 14.1 (C4′). Hz, 2H, H-2, H-6), 6.37−6.22 (m, 2H, H-4, H-1′), 5.65 (m, 1H, H- 5-Hexyl-1,3-dimethoxybenzene (5e). Compound 5e was 2′), 3.80 (s, 6H, 3-OCH3/5-OCH3), 2.36 (m, 2H, H-3′), and 1.12− prepared as described for 5a by using a solution of 4e (2.00 g, 9.08 1.05 (m, 3H, H-4′); 13C NMR (90 MHz, CDCl3): δ 160.6 (C3, C5), mmol) and two drops of acetic acid in MeOH (70 mL) and Pd/C 139.8 (C1), 135.4 (C2′), 128.3 (C1′), 106.9 (C2, C6), 98.8 (C4), 55.4 (10% in wt of Pd, 200 mg, 187 μmol). Yield 5e: 95% (1.97 g, 9.07 (2×-OCH3), 22.3 (C3′), and 14.6 (C4′). mmol) as a yellow oil: 1H NMR (360 MHz, CDCl3): δ 6.35 (m, 2H, (Z)- and (E)-1-(Hex-1-en-1-yl)-3,5-dimethoxybenzene (4e). A H-4, H-6), 6.30 (s, 1H, H-2), 3.78 (s, 6H, 1-OCH3/3-OCH3), 2.54 (t, mixture of olefins (Z)- and (E)-4e was prepared as described for 4a by J1′,2′ = 7.8 Hz, 2H, H-1′), 1.61 (m, 2H, H-2′), 1.33 (m, 6H, H-3′, H- using pentyltriphenylphosphonium bromide (6.15 g, 14.89 mmol) in 4′, H-5′), and 0.88 (m, 3H, H-6′); 13C NMR (90 MHz, CDCl3): δ dry THF (25 mL), n-BuLi (2.5 M in THF, 6.0 mL, 14.89 mmol), and 160.8 (C1, C3), 145.6 (C5), 106.6 (C4, C6), 97.6 (C5), 55.4 (2×- 2 (1.65 g, 9.93 mmol) in dry THF (85 mL). Yield 4e (Z/E, 1.2:1): OCH3), 36.5 (C1′), 31.9 (C4′), 31.4 (C2′), 29.2 (C3′), 22.8 (C5′), and quant. (2.19 g, 9.93 mmol) as a colorless oil: HRMS (ESI+) calcd for 14.3 (C6′); IR (ATR): 2930, 2856, 1598, 1464, 1210, and 1153 cm−1. [C14H20O2 + H]+ ([M + H]+), 221.1542; found, 221.1535. IR HRMS (ESI+) calcd for [C14H22O2 + H]+ ([M + H]+), 223.1698; (ATR): 2957, 1591, 1458, 1205, 1154, and 1067 cm−1. (E)-4e: 1H found, 223.1693. NMR (360 MHz, CDCl3): δ 6.52 (m, 2H, H-2, H-6), 6.37−6.19 (m, 2,4-Dibromo-1,5-dimethoxy-3-pentylbenzene (6a). To a 3H, H-4, H-1′, H-2′), 3.80 (s, 6H, 3-OCH3/5-OCH3), 2.22 (m, 2H, solution of 5a (515 mg, 2.47 mmol) in CH2Cl2 (10 mL), NBS H-3′), 1.49−1.36 (m, 4H, H-4′, H-5′), and 0.96−0.89 (m, 3H, H-6′); (525 mg, 2.95 mmol) was added, and the reaction mixture was stirred 13 C NMR (90 MHz, CDCl3): δ 160.6 (C3, C5), 139.8 (C1), 131.9 at rt for 1 h. Then, another portion of NBS (499 mg, 2.81 mmol) and (C1′/C2′), 129.7 (C1′/C2′), 104.0 (C2, C6), 99.2 (C4), 55.4 (2×- CH2Cl2 (5 mL) was added and the mixture was stirred for 1 h. After OCH3), 32.8 (C3′), 31.6 (C4′), 22.4 (C5′), and 14.1 (C6′). (Z)-4e: 1H this time, a third portion of NBS (276 mg, 1.55 mmol) and CH2Cl2 NMR (360 MHz, CDCl3): δ 6.46 (m, 2H, H-2, H-6), 6.37−6.19 (m, (3 mL) was added and the mixture was stirred overnight. The 2H, H-2, H-1′), 5.67 (m, 1H, H-2′), 3.80 (s, 6H, 3-OCH3/5-OCH3), reaction was quenched by the slow addition of water (5 mL), and the 2.36 (m, 2H, H-3′), 1.49−1.36 (m, 4H, H-4′, H-5′), and 0.96−0.89 organic layer was washed with water (5 × 15 mL), dried over (m, 3H, H-6′); 13C NMR (90 MHz, CDCl3): δ 160.9 (C3, C5), 140.1 anhydrous Na2SO4, and concentrated under vacuum. The resulting (C1), 133.8 (C1′/C2′), 128.8 (C1′/C2′), 106.9 (C2, C6), 98.7 (C4), solid was purified by flash column chromatography (hexanes/EtOAc, 55.4 (2×-OCH3), 32.3 (C3′), 28.6 (C4′), 22.6 (C5′), and 14.1 (C6′). 9:1) to furnish 6a as a white solid (806 mg, 2.20 mmol, 92% yield): 1,3-Dimethoxy-5-pentylbenzene (5a). A stirred solution of 4a mp 70−71 °C (from CH2Cl2); 1H NMR (360 MHz, CDCl3): δ 6.40 (82 mg, 398 μmol) and two drops of acetic acid in MeOH (3 mL) (s, 1H, H-6), 3.90 (s, 6H, 1-OCH3/5-OCH3), 3.03 (m, 2H, H-1′), were hydrogenated over Pd/C (10% in wt of Pd, 11 mg, 10 μmol) 1.55 (m, 2H, H-2′), 1.39 (m, 4H, H-3′, H-4′), and 0.92 (m, 3H, H- under 1 atm of H2 for 24 h. Then, the catalyst was removed by 5′); 13C NMR (90 MHz, CDCl3): δ 155.9 (C1, C5), 143.5 (C3), 105.4 filtration over Celite and the solvent was evaporated under reduced (C2, C4), 94.9 (C6), 56.7 (2×-OCH3), 37.3 (C1′), 32.0 (C2′), 27.8 pressure to deliver compound 5a49 (82 mg, 389 μmol, 98% yield) as a (C3′), 22.5 (C4′), and 14.2 (C5′); IR (ATR): 2928, 1571, 1449, 1423, yellow oil: 1H NMR (360 MHz, CDCl3): δ 6.37 (m, 2H, H-4, H-6), 1332, 1212, and 1085 cm−1. HRMS (ESI+) calcd for [C13H18Br2O2 + 6.37 (m, 1H, H-2), 3.80 (s, 6H, 1-OCH3/3-OCH3), 2.56 (t, J1′,2′ = 7.6 Na]+ ([M + Na]+), 386.9571; found, 386.9563. Hz, 2H, H-1′), 1.62 (quint, J2′,1′/3′ = 7.6 Hz, 2H, H-2′), 1.38−1.28 2,4-Dibromo-3-ethyl-1,5-dimethoxybenzene (6b). Com- (m, 4H, H-3′, H-4′), and 0.91 (m, 3H, H-5′); 13C NMR (90 MHz, pound 6b was prepared as described for 6a by using a solution of CDCl3): δ 160.7 (C1, C3), 145.5 (C5), 105.4 (C4, C6), 97.6 (C2), 55.3 5b (1.66 g, 7.02 mmol) in CH2Cl2 (20 mL; 10 mL; and 5 mL) and (3-OCH3/5-OCH3), 36.4 (C1′), 31.7 (C3′), 31.1 (C2′), 22.7 (C4′), NBS (1.29 g, 7.22 mmol; 1.50 g, 8.45 mmol; and 0.79 g, 4.43 mmol). and 14.2 (C5′). Yield 6b: 70% (1.60 g, 4.98 mmol) as a pale yellow solid: mp 89−91 1,3-Dimethoxy-5-ethylbenzene (5b). Compound 5b49 was °C (from CH2Cl2); 1H NMR (360 MHz, CDCl3): δ 6.40 (s, 1H, H- prepared as described for 5a by using a solution of 4b (1.67 g, 6), 3.89 (s, 6H, 1-OCH3/5-OCH3), 3.08 (q, J1′,2′ = 7.6 Hz, 2H, H-1′), 10.19 mmol) and two drops of acetic acid in MeOH (60 mL) and and 1.03 (t, J2′,1′ = 7.5 Hz, 3H, H-2′); 13C NMR (90 MHz, CDCl3): δ Pd/C (10% in wt of Pd, 168 mg, 160 μmol). Yield 5b: 70% (1.17 g, 155.9 (C1, C5), 144.3 (C3), 105.1 (C2, C4), 94.9 (C6), 56.6 (2×- 7.01 mmol) as a brown oil: 1H NMR (360 MHz, CDCl3): δ 6.38 (d, OCH3), 30.9 (C1′), and 12.5 (C2′). IR (ATR): 2943, 1422, 1334, J4,2 = J6,2 = 2.1 Hz, 2H, H-4, H-6), 6.31 (t, J2,4 = J2,6 = 2.1 Hz 1H, H- 1092, 1054, and 986 cm−1. 2), 3.79 (s, 6H, 1-OCH3/3-OCH3), 2.60 (q, J1′,2′ = 7.6 Hz, 2H, H-1′), 2,4-Dibromo-1,5-dimethoxy-3-propylbenzene (6c). Com- and 1.23 (t, J2′,1′ = 7.6 Hz, 3H, H-2′); 13C NMR (90 MHz, CDCl3): δ pound 6c was prepared as described for 6a by using a solution of 160.9 (C1, C5), 146.9 (C5), 106.0 (C4, C6), 97.6 (C2), 55.4 (2×- 5c (1.91 g, 10.61 mmol) in CH2Cl2 (40 mL; 20 mL; and 10 mL) and OCH3), 29.3 (C1′), and 15.6 (C2′). NBS (1.91 g, 10.71 mmol; 1.90 g, 10.69 mmol; and 950 mg, 5.34 1,3-Dimethoxy-5-propylbenzene (5c). Compound 5c49 was mmol). Yield 6c: quant. (3.58 g, 10.61 mmol) as a white solid: mp prepared as described for 5a by using a solution of 4c (2.03 g, 11.38 82−84 °C (from CH2Cl2); 1H NMR (360 MHz, CDCl3): δ 6.40 (s, mmol) and two drops of acetic acid in MeOH (70 mL) and Pd/C 1H, H-6), 3.90 (s, 6H, 1-OCH3/5-OCH3), 3.01 (m, 2H, H-1′), 1.58 (10% in wt of Pd, 215 mg, 200 μmol). Yield 5c: 93% (1.91 g, 10.60 (m, 2H, H-2′), and 1.03 (t, J3′,2′ = 7.3 Hz, 3H, H-3′); 13C NMR (90 mmol) as a pale yellow oil: 1H NMR (360 MHz, CDCl3): δ 6.36 (d, MHz, CDCl3): δ 155.9 (C1, C5), 143.2 (C3), 105.4 (C2, C4), 94.9 9359 https://doi.org/10.1021/acs.jmedchem.1c00561 J. Med. Chem. 2021, 64, 9354−9364 Journal of Medicinal Chemistry pubs.acs.org/jmc Article (C6), 56.6 (2×-OCH3), 39.2 (C1′), 21.6 (C2′), and 14.3 (C3′); IR 4,6-Dibromo-5-propylbenzene-1,3-diol (7d). Compound 7d (ATR): 2965, 1570, 1447, 1422, 1329, 1209, and 1081 cm−1. HRMS was prepared as described for 7a by using a solution of 6d (2.00 g, (ESI+) calcd for [C11H14Br2O2 + H]+ ([M + H]+), 336.9439; found, 5.68 mmol) in CH2Cl2 (80 mL) and BBr3 (1 M in CH2Cl2, 22.7 mL, 336.943. 22.7 mmol). Yield 7d: 73% (1.34 g, 4.13 mmol) as a gray solid: mp 2,4-Dibromo-3-butyl-1,5-dimethoxybenzene (6d). Com- 96−97 °C (from CH2Cl2); 1H NMR (360 MHz, CDCl3): δ 6.65 (s, pound 6d was prepared as described for 6a by using a solution of 1H, H-2), 5.65 (s, 2H, OH-1/OH-3), 2.94 (m, 2H, H-1′), 1.49 (m, 5d (2.03 g, 10.44 mmol) in CH2Cl2 (45 mL; 20 mL; and 10 mL) and 4H, H-2′, H-3′), and 0.98 (t, J4′,3′ = 7.1 Hz, 3H, H-4′); 13C NMR (90 NBS (1.94 g, 10.90 mmol; 1.98 g, 11.12 mmol; and 963 mg, 5.41 MHz, CDCl3): δ 152.7 (C1, C3), 141.5 (C5), 104.2 (C4, C6), 100.8 mmol). Yield 6d: 92% (3.39 g, 9.64 mmol) as a pale brown solid: mp (C2), 37.5 (C1′), 30.3 (C2′), 22.9 (C3′), and 14.0 (C4′); IR (ATR): 64−66 °C (from CH2Cl2); 1H NMR (360 MHz, CDCl3): δ 6.40 (s, 3432, 3224, 2962, 1582, 1424, 1260, and 1145 cm−1. HRMS (ESI−) 1H, H-6), 3.90 (s, 6H, 1-OCH3/5-OCH3), 3.03 (m, 2H, H-1′), 1.50 calcd for [C10H12Br2O2 − H]− ([M − H]−), 320.9126; found, (m, 2H, H-2′, H-3′), and 0.97 (t, J4′,3′ = 7.0 Hz, 3H, H-4′); 13C NMR 320.9138. (90 MHz, CDCl3): δ 155.9 (C1, C5), 143.4 (C3), 105.4 (C2, C4), 94.8 4,6-Dibromo-5-hexylbenzene-1,3-diol (7e). Compound 7e (C6), 55.6 (2×-OCH3), 37.1 (C1′), 30.3 (C2′), 23.0 (C3′), and 14.0 was prepared as described for 7a by using a solution of 6e (1.00 g, (C4′); IR (ATR): 2960, 1571, 1449, 1423, 1335, 1211, 1082, and 2.63 mmol) in CH2Cl2 (50 mL) and BBr3 (1 M in CH2Cl2, 10.5 mL, 1052 cm−1. 10.50 mmol).Yield 7e: 91% (846 mg 2.40 mmol) as a gray solid: mp 2,4-Dibromo-3-hexyl-1,5-dimethoxybenzene (6e). Com- 55−53 °C (from CH2Cl2); 1H NMR (360 MHz, CDCl3): δ 6.65 (s, pound 6e was prepared as described for 6a by using a solution of 1H, H-2), 5.67 (s, 2H, OH-1/OH-3), 2.92 (m, 2H, H-1′), 1.52 (m, 5e (1.94 g, 8.72 mmol) in CH2Cl2 (40 mL; 20 mL; and 10 mL) and 2H, H-2′), 1.43 (m, 2H, H-3′), 1.33 (m, 4H, H-4′, H-5′), and 0.90 NBS (1.86 g, 10.47 mmol; 1.86 g, 10.47 mmol; and 1.0 g, 5.62 mmol). (m, 3H, H-6′); 13C NMR (90 MHz, CDCl3): δ 152.6 (C1, C3), 141.6 Yield 6e: 91% (3.01 g, 7.91 mmol) as a pale orange solid: mp 63−65 (C5), 104.2 (C4, C6), 100.8 (C2), 37.8 (C1′), 31.6 (C4′), 29.4 (C3′), °C (from CH2Cl2); 1H NMR (360 MHz, CDCl3): δ 6.41 (s, 1H, H- 28.1 (C2′), 22.9 (C5′), and 14.2 (C6′); IR (ATR): 3427, 3173, 2926, 6), 3.91 (s, 6H, 1-OCH3/5-OCH3), 3.04 (m, 2H, H-1′), 1.55 (m, 2H, 1578, 1421, 1341, and 1231 cm−1. HRMS (ESI−) calcd for H-2′), 1.45 (m, 2H, H-3′), 1.36 (m, 4H, H-4′, H-5′), and 0.91 (m, [C12H16Br2O2 − H]− ([M − H]−), 348.9439; found, 348.9449. 3H, H-6′); 13C NMR (90 MHz, CDCl3): δ 155.9 (C1, C5), 143.4 4,6-Dibromo-2-[(1R,6R)-6-isopropenyl-3-methylcyclohex-2- (C3), 105.4 (C2, C4), 94.8 (C6), 55.6 (2×-OCH3), 37.4 (C1′), 31.6 en-1-yl]-5-pentylbenzene-1,3-diol (9a). A mixture of 7a (152 mg, (C4′), 29.5 (C3′), 28.1 (C2′), 22.8 (C5′), and 14.3 (C6′); IR (ATR): 450 μmol), (1S,4R)-1-methyl-4-(prop-1-en-2-yl)cyclohex-2-en-1-ol, 8 2928, 2857, 1569, 1450, 1422, 1343, 1209, and 1088 cm−1. HRMS (82 mg, 540 μmol), and magnesium sulfate (136 mg, 1.16 mmol) in (ESI+) calcd for [C14H20Br2O2 + H]+ ([M + H]+), 378.9908; found, dry CH2Cl2 (5 mL) was cooled to −35 °C in a N2/Ar atmosphere. 378.9905. Then, p-toluenesulfonic acid monohydrate (43 mg, 225 μmol) was 4,6-Dibromo-5-pentylbenzene-1,3-diol (7a). A solution of added in one portion and the resulting mixture was stirred for 5 h at BBr3 (1 M in CH2Cl2, 2.5 mL, 2.5 mmol) was slowly added to a −35 °C. After this time, the reaction was stirred at rt overnight. The solution of compound 6a (230 mg, 628 μmol) in CH2Cl2 (12 mL) at reaction was quenched with a solution of tribasic potassium −10 °C. The reaction mixture was stirred at rt overnight. The reaction phosphate (401 mg, 1.89 mmol) in water (7 mL), and the layers was quenched by the slow addition of water (16 mL), and the organic were separated. The aqueous layer was extracted with CH2Cl2 (5 layer was separated. The aqueous layer was extracted with CH2Cl2 (3 mL), and the combining layers were separated. The volatiles were × 8 mL), and the combined organic layer was dried over Na2SO4, removed under pressure, and the resulting oil was purified by flash filtered, and concentrated under vacuum. The resulting crude solid column chromatography (hexanes/EtOAc, 100:0 → 100:1) to afford product was purified by flash column chromatography (hexanes/ 9a10 (137 mg, 0.29 mmol, 64% yield) as a yellow oil: [α]D20 −100.1 EtOAc, 2:1) to furnish 7a (193 mg, 570 μmol, 91% yield) as a gray (c 6.5, EtOH); 1H NMR (360 MHz, CDCl3): δ 6.55 (br s, 1H, OH- solid: mp 65−66 °C (from CH2Cl2); 1H NMR (360 MHz, CDCl3): δ 1′/OH-3′), 5.66 (br s, 2H, OH-1′/OH-3′), 5.46 (s, 1H, H-2), 4.53 (s, 6.65 (s, 1H, H-2), 5.72 (s, 2H, OH-1/OH-3), 2.92 (m, 2H, H-1′), 1H, H-10trans), 4.41 (m, 1H, H-10cis), 4.07 (dm, 1H, H-1), 2.92 (m, 1.55 (m, 2H, H-2′), 1.40 (m, 4H, H-3′, H-4′), and 0.93 (m, 3H, H- 2H, H-1″), 2.55 (m, 1H, H-6), 2.22 (m, 1H, H-4), 2.07 (m, 1H, H-4), 5′); 13C NMR (90 MHz, CDCl3): δ 152.6 (C1, C3), 141.5 (C5), 104.2 1.81−1.74 (m, 2H, H-5), 1.77 (s, 3H, H-7), 1.68 (s, 3H, H-9), 1.58− (C4, C6), 100.9 (C2), 37.2 (C1′), 31.9 (C2′), 27.9 (C3′), 22.9 (C4′), 1.49 (m, 2H, H-2″), 1.43−1.38 (m, 4H, H-3″, H-4″), and 0.92 (m, and 14.2 (C5′); IR (ATR): 3427, 3217, 2927, 1577, 1426, 1230, and 3H, H-5″); 13C NMR (90 MHz, CDCl3): δ 151.6 (C3′), 150.5 (C1′), 1164 cm−1. HRMS (ESI−) calcd for [C11H14Br2O2 − H]− ([M − 147.2 (C8), 139.9 (C5′), 139.5 (C3), 123.3 (C2), 115.7 (C2′), 111.6 H]−), 334.9282; found, 334.9291. (C10), 104.4 (C4′, C6′), 45.9 (C6), 38.1 (C1), 37.6 (C1″), 31.9 (C4″), 4,6-Dibromo-5-ethylbenzene-1,3-diol (7b). Compound 7b 30.4 (C4), 28.2 (C5), 27.9 (C3″), 23.9 (C7), 22.5 (C4″), 19.0 (C9), and was prepared as described for 7a by using a solution of 6b (1.40 g, 14.2 (C5″); IR (ATR): 3497, 3395, 2924, 1599, 1429, 1356, and 1248 4.32 mmol) in CH2Cl2 (70 mL) and BBr3 (1 M in CH2Cl2, 17.2 mL, cm−1. 24.69 mmol). Yield 7b: 82% (1.05 g, 3.53 mmol) as a gray solid: mp 4,6-Dibromo-5-ethyl-2-[(1R,6R)-6-isopropenyl-3-methylcy- 105−107 °C (from CH2Cl2); 1H NMR (360 MHz, CDCl3): δ 6.66 clohex-2-en-1-yl]benzene-1,3-diol (9b). Compound 9b was (s, 1H, H-2), 5.68 (s, 2H, OH-1/OH-3), 2.99 (q, J1′,2′ = 7.4 Hz, 2H, prepared as described for 9a by using a mixture of 7b (804 mg, H-1′), and 1.16 (t, J2′,1′ = 7.4 Hz, 3H, H-2′); 13C NMR (90 MHz, 2.72 mmol), 8 (476 mg, 2.99 mmol), and MgSO4 (818 mg, 6.79 CDCl3): δ 152.7 (C1, C3), 142.5 (C5), 103.9 (C4, C6), 100.9 (C2), mmol) in dry CH2Cl2 (70 mL) and p-TsOH (258 mg, 1.35 mmol). 31.3 (C1′), and 12.5 (C2′); IR (ATR): 3476, 3240, 1583, 1429, 1336, Yield 9b: 71% (829 mg, 1.83 mmol) as a yellow oil: [α]D20 −47.3 (c 1231, and 1165 cm−1. HRMS (ESI−) calcd for [C8H8Br2O2 − H]− 1.1, CHCl3); 1H NMR (360 MHz, CDCl3): δ 6.57 (br s, 1H, OH-1′/ ([M − H]−), 292.8813; found, 292.8821. OH-3′), 5.56 (br s, 2H, OH-1′/OH-3′), 5.46 (s, 1H, H-2), 4.53 (s, 4,6-Dibromo-5-propylbenzene-1,3-diol (7c). Compound 7c41 1H, H-10trans), 4.41 (m, 1H, H-10cis), 4.07 (dm, J1,6 = 10.2 Hz, 1H, H- was prepared as described for 7a by using a solution of 6c (2.04 g, 1), 2.98 (m, J1″,2″ = 7.5 Hz, 2H, H-1″), 2.55 (m, 1H, H-6), 2.22 (m, 6.89 mmol) in CH2Cl2 (80 mL) and BBr3 (1 M in CH2Cl2, 24.0 mL, 1H, H-4), 2.08 (m, 1H, H-4), 1.81−1.74 (m, 2H, H-5), 1.77 (s, 3H, 24.00 mmol). Yield 7c: 85% (1.82 g, 5.87 mmol) as a gray solid: mp H-7), 1.68 (s, 3H, H-9), and 1.15 (t, J2″,1″ = 7.5 Hz, 3H, H-2″); 13C 89−91 °C (from CH2Cl2); 1H NMR (360 MHz, CDCl3): δ 6.66 (s, NMR (90 MHz, CDCl3): δ 151.7 (C3′), 150.2 (C1′), 147.3 (C8), 1H, H-2), 5.65 (s, 2H, OH-1/OH-3), 2.91 (m, 2H, H-1′), 1.59 (t, 140.5 (C5′), 140.1 (C3), 123.2 (C2), 115.7 (C2′), 111.6 (C10), 104.3 J2′,1′/3′ = 7.5 Hz, 2H, H-2′), and 1.04 (t, J3′,2′ = 7.4 Hz, 3H, H-3′); 13C (C4′, C6′), 45.9 (C6), 38.0 (C1), 31.2 (C5), 30.4 (C4), 28.2 (C1″), 23.9 NMR (90 MHz, CDCl3): δ 152.7 (C1, C3), 141.3 (C5), 104.3 (C4, (C7), 19.0 (C9), and 12.6 (C2″); IR (ATR): 3491, 2925, 1737, 1596, C6), 100.9 (C2), 39.6 (C1′), 21.7 (C2′), and 14.2 (C3′); IR (ATR): 1410, 1244, 1204, and 890 cm−1. HRMS (ESI−) calcd for 3422, 3155, 2968, 1581, 1423, 1339, and 1238 cm−1. [C18H22Br2O2 − H]− ([M − H]−), 426.9908; found, 426.9918. 9360 https://doi.org/10.1021/acs.jmedchem.1c00561 J. Med. Chem. 2021, 64, 9354−9364 Journal of Medicinal Chemistry pubs.acs.org/jmc Article 4,6-Dibromo-2-[(1R,6R)-6-isopropenyl-3-methylcyclohex-2- 100:1 → 10:1) to deliver 1a (38 mg, 12 μmol, 43% yield) as a light- en-1-yl]-5-propylbenzene-1,3-diol (9c). Compound 9c50 was yellow oil: [α]D20 −122.0 (c 1.1, EtOH), −51.3 (c 1.7, CHCl3); 1H prepared as described for 9a by using a mixture of 7c (383 g, 1.23 NMR (400 MHz, CD3OD): δ 6.07 (s, 2H, H-4′, H-6′), 5.28 (s, 1H, mmol), 8 (242 mg, 1.59 mmol), and MgSO4 (372 mg, 3.09 mmol) in H-2), 4.46 (s, 1H, H-10trans), 4.42 (m, 1H, H-10cis), 3.93 (dm, J1,6 = dry CH2Cl2 (6 mL) and p-TsOH (118 mg, 620 μmol). Yield 9c: 70% 8.7 Hz, 1H, H-1), 2.89 (m, 1H, H-6), 2.37 (t, J1″,2″ = 7.5 Hz, 2H, H- (380 mg, 860 μmol) as a yellow oil: [α]D20 −61.8 (c 0.5, CHCl3); 1H 1″), 2.18 (m, 1H, H-4), 1.99 (m, 1H, H-4), 1.74 (m, 2H, H-5), 1.67 NMR (360 MHz, CDCl3): δ 6.56 (br s, 1H, OH-1′/OH-3′), 5.56 (br (s, 3H, H-7), 1.63 (s, 3H, H-9), 1.53 (quint, J2″,1″/3″ = 7.5 Hz, 2H, H- s, 2H, OH-1′/OH-3′), 5.46 (s, 1H, H-2), 4.53 (s, 1H, H-10trans), 4.40 2″), 1.29 (m, 4H, H-3″,H-4″), and 0.89 (t, J5″,4″ = 6.9 Hz, 3H, H-5″); (m, 1H, H-10cis), 4.07 (dm, 1H, H-1), 2.90 (m, 2H, H-1″), 2.55 (m, 13 C NMR (100 MHz, CD3OD): δ 157.5 (C3′), 150.3 (C8, C1′), 142.7 1H, H-6), 2.20 (m, 1H, H-4), 2.07 (m, 1H, H-4), 1.81−1.74 (m, 2H, (C5′), 134.1 (C3), 127.3 (C2), 115.9 (C2′), 110.5 (C10), 108.3 (C4′, H-5), 1.77 (s, 3H, H-7), 1.68 (s, 3H, H-9), 1.58 (sext, J2″,1″/3″ = 7.3 C6′), 46.3 (C6), 37.5 (C1), 36.6 (C1″), 32.7 (C3″), 32.0 (C2″), 31.7 Hz, 2H, H-2″), and 1.02 (t, J3″,2″ = 7.3 Hz, 3H, H-3″); 13C NMR (90 (C5), 30.7 (C4), 23.7 (C7), 23.6 (C4″), 19.5 (C9), and 14.4 (C5″); IR MHz, CDCl3): δ 151.6 (C3′), 149.9 (C1′), 147.2 (C8), 139.9 (C5′), (ATR): 3424, 2926, 1632, 1586, 1447, 1218, and 891 cm−1. All 139.3 (C3), 123.2 (C2), 115.7 (C2′), 111.6 (C10), 104.5 (C4′, C6′), spectral data are in agreement with the literature.12 45.9 (C6), 39.5 (C1″), 38.0 (C1), 30.4 (C4), 28.2 (C5), 23.9 (C7), 21.7 5-Ethyl-2-[(1R,6R)-6-isopropenyl-3-methylcyclohex-2-en-1- (C2″), 18.9 (C9), and 14.2 (C3″); IR (ATR): 3493, 3387, 2925, 1598, yl]benzene-1,3-diol (1b). Compound 1b was prepared as described 1428, 1325, 1245, 1099, and 889 cm−1. for 1a by using a solution of 9b (709 mg, 1.65 mmol) in MeOH (7 4,6-Dibromo-5-butyl-2-[(1R,6R)-6-isopropenyl-3-methylcy- mL), a solution of Na2SO3 (551 g, 4.37 mmol) and L-ascorbic acid clohex-2-en-1-yl]benzene-1,3-diol (9d). Compound 9d was (43.6 mg, 250 μmol) in H2O (7 mL), and Et3N (830 μL, 5.94 mmol). prepared as described for 9a by using a mixture of 7d (663 mg, Yield 1b: 63% (282 mg, 1.04 mmol) as a light-brown oil: [α]D20 2.05 mmol), 8 (365 mg, 2.39 mmol), and MgSO4 (615 mg, 5.11 −86.9 (c 1.9, CHCl3); 1H NMR (400 MHz, CD3OD): δ 6.10 (s, 2H, mmol) in dry CH2Cl2 (11 mL) and p-TsOH (199 mg, 1.05 mmol). H-4′, H-6′), 5.28 (s, 1H, H-2), 4.48 (s, 1H, H-10trans), 4.44 (m, 1H, Yield 9d: 56% (522 mg, 1.14 mmol) as a yellow oil: [α]D20 −63.1 (c H-10cis), 3.93 (dm, J1,6 = 8.7 Hz, 1H, H-1), 2.92 (m, 1H, H-6), 2.42 1.9, CHCl3); 1H NMR (360 MHz, CDCl3): δ 6.57 (br s, 1H, OH-1′/ (q, J1″,2″ = 7.6 Hz, 2H, H-1″), 2.18 (m, 1H, H-4), 1.99 (m, 1H, H-4), OH-3′), 5.56 (br s, 2H, OH-1′/OH-3′), 5.46 (s, 1H, H-2), 4.53 (s, 1.74 (m, 2H, H-5), 1.68 (s, 3H, H-7), 1.64 (s, 3H, H-9), and 1.15 (t, 1H, H-10trans), 4.40 (m, 1H, H-10cis), 4.06 (dm, 1H, H-1), 2.92 (m, J2″,1″ = 7.6 Hz, 3H, H-2″); 13C NMR (100 MHz, CD3OD): δ 157.5 2H, H-1″), 2.54 (m, 1H, H-6), 2.20 (m, 1H, H-4), 2.09 (m, 1H, H-4), (C3′), 150.3 (C8, C1′), 144.1 (C5′), 134.1 (C3), 127.3 (C2), 115.9 1.81−1.74 (m, 2H, H-5), 1.77 (s, 3H, H-7), 1.68 (s, 3H, H-9), 1.57− (C2′), 110.5 (C10), 107.7 (C4′, C6′), 46.3 (C6), 37.4 (C1), 31.7 (C5), 1.42 (m, 4H, H-2″, H-3″), and 0.97 (t, J4″,3″ = 7.0 Hz, 3H, H-4″); 13C 30.8 (C4), 29.5 (C1″), 23.7 (C7), 19.5 (C9), and 15.8 (C2″); IR NMR (90 MHz, CDCl3): δ 151.8 (C3′), 150.0 (C1′), 147.3 (C8), (ATR): 3405, 2925, 1628, 1582, 1439, 1215, and 888 cm−1. HRMS 140.1 (C5′), 139.5 (C3), 123.2 (C2), 115.7 (C2′), 111.6 (C10), 104.4 (ESI+) calcd for [C18H24O2 + H]+ ([M + H]+), 273.1855; found, (C4′, C6′), 45.9 (C6), 38.1 (C1), 37.4 (C1″), 30.4 (C4), 30.4 (C2″), 273.1849. 28.2 (C5), 23.9 (C7), 22.9 (C3″), 19.0 (C9), and 14.0 (C4″); IR 2-[(1R,6R)-6-Isopropenyl-3-methylcyclohex-2-en-1-yl]-5- (ATR): 3494, 2924, 1600, 1429, 1328, 1247, and 892 cm−1. HRMS propylbenzene-1,3-diol (1c). Compound 1c13 was prepared as (ESI−) calcd for [C20H26Br2O2 − H]− ([M − H]−), 455.0221; found, described for 1a by using a solution of 9c (321 mg, 723 μmol) in 455.0228. MeOH (4 mL), a solution of Na2SO3 (262 g, 2.26 mmol) and L- 4,6-Dibromo-5-hexyl-2-[(1R,6R)-6-isopropenyl-3-methylcy- ascorbic acid (37 mg, 210 μmol) in H2O (4 mL), and Et3N (410 μL, clohex-2-en-1-yl]benzene-1,3-diol (9e). Compound 9e was 2.94 mmol). Yield 1c: 50% (103 mg, 360 μmol) as a brown oil: prepared as described for 9a by using a mixture of 7e (629 mg, [α]D20 −138.3 (c 2.3, EtOH), −72.23. (c 0.5, CHCl3); 1H NMR 1.79 mmol), 8 (299 mg, 1.97 mmol), and MgSO4 (537 mg, 4.47 (400 MHz, CD3OD): δ 6.09 (s, 2H, H-4′, H-6′), 5.30 (s, 1H, H-2), mmol) in dry CH2Cl2 (7 mL) and p-TsOH (170 mg, 893 μmol). 4.47 (s, 1H, H-10trans), 4.44 (m, 1H, H-10cis), 3.94 (dm, J1,6 = 8.6 Hz, Yield 9e: 64% (555 mg, 1.14 mmol) as a yellow oil: [α]D20: −56.5 (c 1H, H-1), 2.90 (m, 1H, H-6), 2.36 (t, J1″,2″ = 7.5 Hz, 2H, H-1″), 2.18 1.6, CHCl3); 1H NMR (360 MHz, CDCl3): δ 6.57 (br s, 1H, OH-1′/ (m, 1H, H-4), 1.99 (m, 1H, H-4), 1.74 (m, 2H, H-5), 1.68 (s, 3H, H- OH-3′), 5.67 (br s, 2H, OH-1′/OH-3′), 5.47 (s, 1H, H-2), 4.53 (s, 7), 1.64 (s, 3H, H-9), 1.57 (sext, J2″,1″/3″ = 7.5 Hz, 2H, H-2″), and 1H, H-10trans), 4.40 (m, 1H, H-10cis), 4.06 (dm, 1H, H-1), 2.91 (m, 0.90 (t, J3″,2″ = 7.5 Hz, 3H, H-3″); 13C NMR (100 MHz, CD3OD): δ 2H, H-1″), 2.56 (m, 1H, H-6), 2.23 (m, 1H, H-4), 2.08 (m, 1H, H-4), 157.4 (C3’), 150.2 (C8, C1′), 142.4 (C5′), 134.3 (C3), 127.2 (C2), 1.81−1.74 (m, 2H, H-5), 1.77 (s, 3H, H-7), 1.68 (s, 3H, H-9), 1.57− 115.9 (C2′), 110.5 (C10), 108.3 (C4′, C6′), 46.3 (C6), 38.8 (C1″), 37.4 1.49 (m, 2H, H-2″), 1.43−1.34 (m, 6H, H-3″, H-4″, H-5″), and 0.91 (C1), 31.6 (C5), 30.7 (C4), 25.4 (C2″), 23.7 (C7), 19.5 (C9), and 14.2 (m, 3H, H-6″); 13C NMR (90 MHz, CDCl3): δ 151.7 (C3′), 150.0 (C3″); IR (ATR): 3412, 2924, 1629, 1534, 1443, 1217, and 889 cm−1. (C1′), 147.3 (C8), 140.1 (C5′), 139.6 (C3), 123.2 (C2), 115.7 (C2′), HRMS (ESI+) calcd for [C19H26O2 + H]+ ([M + H]+), 287.2011; 111.6 (C10), 104.6 (C4′, C6′), 46.0 (C6), 38.1 (C1), 37.7 (C1″), 31.7 found, 287.2009. (C4″), 30.4 (C4), 29.4 (C4″), 28.2 (C5), 28.2 (C2″), 23.9 (C7), 22.8 5-Butyl-2-[(1R,6R)-6-isopropenyl-3-methylcyclohex-2-en-1- (C5″), 19.0 (C9), and 14.2 (C6″); IR (ATR): 3499, 3394, 2924, 1593, yl]benzene-1,3-diol (1d). Compound 1d14 was prepared as 1428, 1246, and 890 cm−1. HRMS (ESI−) calcd for [C22H30Br2O2 − described for 1a by using a solution of 9d (292 mg, 637 μmol) in H]− ([M − H]−), 483.0534; found, 483.0537. MeOH (4 mL), a solution of Na2SO3 (229 mg, 1.97 mmol) and L- 2-[(1R,6R)-6-Isopropenyl-3-methylcyclohex-2-en-1-yl]-5- ascorbic acid (31 mg, 176 μmol) in H2O (4 mL), and Et3N (350 μL, pentylbenzene-1,3-diol (1a). To a solution of 9a (130 mg, 280 2.51 mmol). Yield 1d: 56% (108 mg, 360 μmol) as a brown oil: μmol) in methanol (1.5 mL) was added a solution of sodium sulfite [α]D20 −56.1 (c 1.0, CHCl3); 1H NMR (400 MHz, CD3OD): δ 6.08 (92 mg, 730 μmol) and L-ascorbic acid (7 mg, 40 μmol) in water (1.5 (s, 2H, H-4′, H-6′), 5.28 (s, 1H, H-2), 4.47 (s, 1H, H-10trans), 4.43 mL). To the pink suspension formed, triethylamine (140 μL, 1.2 (m, 1H, H-10cis), 3.93 (dm, J1,6 = 8.6 Hz, 1H, H-1), 2.91 (m, 1H, H- mmol) was added in one portion. The resulting mixture was heated to 6), 2.39 (t, J1″,2″ = 7.6 Hz, 2H, H-1″), 2.18 (m, 1H, H-4), 1.99 (m, 75 °C for 24 h. After cooling to rt, the reaction mixture was partially 1H, H-4), 1.74 (m, 2H, H-5), 1.68 (s, 3H, H-7), 1.64 (s, 3H, H-9), concentrated under reduced pressure to remove most of the methanol 1.53 (quint, J2″,1″/3″ = 7.6 Hz, 2H, H-2″), 1.34 (sext, J3″,2″/4″ = 7.4 Hz, and volatiles. The pH of the remaining aqueous phase was adjusted to 2H, H-2″), and 0.92 (t, J4″,3″ = 7.4 Hz, 3H, H-4″); 13C NMR (100 2 with hydrochloric acid 5% w/w. Hexane (10 mL) was added, and MHz, CD3OD): δ 157.5 (C3′), 150.3 (C8, C1′), 142.6 (C5’), 134.0 the mixture was stirred for 15 min. The layers were separated, and the (C3), 127.2 (C2), 115.9 (C2′), 110.5 (C10), 108.3 (C4′, C6′), 46.3 aqueous phase was extracted with hexane (2 × 10 mL). The (C6), 37.4 (C1), 36.3 (C1″), 34.6 (C2″), 31.7 (C5), 30.8 (C4), 23.7 combined organic layers were washed with brine (25 mL), dried over (C7), 23.4 (C3″), 19.5 (C9), and 14.3 (C4″); IR (ATR): 3429, 2925, Na2SO4, and then evaporated under reduced pressure. The obtained 1629, 1583, 1441, 1213, and 1025 cm−1. HRMS (ESI+) calcd for oil was purified by flash column chromatography (hexanes/EtOAc, [C20H28O2 + H]+ ([M + H]+), 301.2168; found, 301.2163. 9361 https://doi.org/10.1021/acs.jmedchem.1c00561 J. Med. Chem. 2021, 64, 9354−9364 Journal of Medicinal Chemistry pubs.acs.org/jmc Article 5-Hexyl-2-[(1R,6R)-6-isopropenyl-3-methylcyclohex-2-en-1- with an assay buffer containing 0.1% DMSO (24 °C, 30 μL/well). yl]benzene-1,3-diol (1e). Compound 1e was prepared as described Hereafter, the sensor plate was scanned, and a baseline optical for 1a by using a solution of 9e (26 mg, 53 μmol) in MeOH (1 mL), a signature was recorded for 10 min before adding 10 μL of the solution of Na2SO3 (19 mg, 167 μmol) and L-ascorbic acid (4 mg, 14 selective antagonists for 30 min, followed by the addition of 10 μL of μmol) in H2O (1 mL), and Et3N (30 μL, 214 μmol). Yield 1e: 63% the selective agonists; all test compounds were diluted in the assay (11 mg, 33 μmol) as a brown oil: [α]D20: −50.2 (c 0.5, CHCl3); 1H buffer. Then, DMR responses were monitored for at least 5000 s in an NMR (400 MHz, CD3OD): δ 6.09 (s, 2H, H-4′, H-6′), 5.30 (s, 1H, EnSpire Multimode Plate Reader (PerkinElmer, Waltham, MA, USA) H-2), 4.47 (s, 1H, H-10trans), 4.43 (m, 1H, H-10cis), 3.94 (dm, J1,6 = by a label-free technology. Results were analyzed using EnSpire 8.7 Hz, 1H, H-1), 2.89 (m, 1H, H-6), 2.38 (t, J1″,2″ = 7.6 Hz, 2H, H- Workstation Software v 4.10. 1″), 2.18 (m, 1H, H-4), 1.99 (m, 1H, H-4), 1.74 (m, 2H, H-5), 1.68 Molecular Docking and MD Simulations. The CB2R- (s, 3H, H-7), 1.64 (s, 3H, H-9), 1.54 (m, 2H, H-2″), 1.30 (m, 6H, H- AM12033-Gi cryo-EM structure (PDB id 6KPF)18 (missing residues 3″, H-4″, H-5″), and 0.89 (m, 3H, H-6″); 13C NMR (100 MHz, 55−181 and 233−239 of αi- were built from the CB2R-WIN55, 212- CD3OD): δ 157.4 (C3′), 150.2 (C8, C1′), 142.6 (C5′), 134.3 (C3), 2-Gi structure, 6PT0,17 using MODELLER v9.25) was used in 127.3 (C2), 115.8 (C2′), 110.6 (C10), 108.3 (C4′, C6′), 46.3 (C6), 37.4 docking studies and MD simulations. JWH-133 and CBD were (C1), 36.6 (C1″), 32.9 (C4″), 32.3 (C3″), 31.7 (C5), 30.6 (C4), 30.0 docked into the orthosteric binding cavity (Figure S1), and CBD, (C2″), 23.8 (C5″), 23.7 (C7), 19.5 (C9), and 14.5 (C6″); IR (ATR): PAM 1c, and NAM 1e were docked into the allosteric binding cavity 3438, 2923, 1629, 1583, 1444, 1217, 1027, and 888 cm−1. HRMS (Figure S4) using the Molecular Operating Environment software (ESI+) calcd for [C22H32O2 + H]+ ([M + H]+), 329.2481; found, (Chemical Computing Group Inc., Montreal, Quebec, Canada). 329.2477. These structures were embedded in a lipid bilayer box, constructed cAMP Determination Assays. Determination of cAMP levels in using PACKMOL-memgen,51 containing 1-palmitoyl-2-oleoyl-sn- HEK-293T cells transiently expressing CB2R (1 μg of cDNA) was glycero-3-phosphocholine, cholesterol , water molecules (TIP3P), performed using the Lance-Ultra cAMP kit (PerkinElmer). 2 h before and monoatomic Na+ and Cl− ions (see Figure S3 for details). MD initiating the experiment, the medium was substituted by a serum-free simulation of these systems was performed with GROMACS 201952 medium. Then, transfected cells were dispensed in white 384-well (Figures S3 and S5). microplates at a density of 3000 cells per well and incubated for 15 min at rt with compounds, followed by 15 min incubation with forskolin, and 1 h more with homogeneous time-resolved fluorescence (HTRF) assay reagents. Fluorescence at 665 nm was analyzed on a ■ ASSOCIATED CONTENT * Supporting Information sı PHERAstar Flagship microplate reader equipped with an HTRF The Supporting Information is available free of charge at optical module (BMG Labtech). Data analysis was made based on the https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00561. fluorescence ratio emitted by the labeled cAMP probe (665 nm) over Computer models of JWH-133 and CBD bound to the the light emitted by the europium cryptate-labeled anti-cAMP orthosteric site of CB2R; superimposition of the antibody (620 nm). A standard curve was used to calculate cAMP computer model of CB2R in complex with JWH-133 concentration. Forskolin-stimulated cAMP levels were normalized to to the crystal structure of CB1R in complex with 100% ERK1/2 Phosphorylation Assays. HEK-293T cells were grown AM6538; MD simulations of JWH-133, JWH-133 + on transparent Biocat poly-D-lysine 96-well plates (Deltalab) and kept CBD, JWH-133 + 1c, and JWH-133 + 1e bound to the at an incubator for 24 h. Then, cells were transiently transfected with CB2R-Gi complex; docking of CBD to the CB2R-Gi 1 μg of cDNA coding for CB2R or mutant receptors and incubated for complex; 1H and 13C NMR spectra of the new 48 h at 37 °C in a 5% CO2 humid atmosphere. 2 h before initiating compounds; and 1D 1H NMR (qHNMR) experiments the experiment, the medium was substituted by a serum-free medium. of compounds 1a−e (PDF) Cells were stimulated at 25 °C for 7 min with vehicles or agonists in SMILES of JWH-133 and compounds 1a−e (CSV) the serum-free Dulbecco’s modified Eagle’s medium. After that, cells Molecular complex data of CB2R + JWH-133 + CBD were washed twice with cold phosphate-buffered saline before the (PDB) addition of lysis buffer (30 μL/well) and incubated for 15 min at 25 °C on a Heidolph Titramax 100 shaker. 10 μl of each cell lysate was transferred to white ProxiPlate 384-well microplates (PerkinElmer; Waltham, MA, USA). ERK1/2 phosphorylation was determined using ■ AUTHOR INFORMATION Corresponding Authors an AlphaScreenSureFire kit (PerkinElmer, Waltham, MA, US): 5 μL/ Ramon Alibés − Departament de Química, Universitat well of acceptor beads was added. Plates, protected from light, were Autònoma de Barcelona, Bellaterra 08193, Barcelona, incubated for 2 h at 25 °C. Finally, 5 μL/well of donor beads was added and plates, protected from light, were incubated for 2 h before Spain; orcid.org/0000-0002-7997-2691; analysis. Fluorescence was determined using an EnSpire Multimode Email: Ramon.Alibes@uab.cat Plate Reader (PerkinElmer, Waltham, MA, USA). The value of Leonardo Pardo − Laboratory of Computational Medicine, reference (100%) was that achieved in the absence of any treatment Biostatistics Unit, Faculty of Medicine, Universitat Autònoma (basal). The effect of ligands was given in percentage with respect to Barcelona, Bellaterra 08193, Barcelona, Spain; orcid.org/ the basal value. 0000-0003-1778-7420; Email: Leonardo.Pardo@uab.es Dynamic Mass Redistribution Assays. Cell mass redistribution Rafael Franco − Centro de Investigación en Red, Enfermedades induced upon receptor activation was detected by illuminating with Neurodegenerativas (CIBERNED), Instituto de Salud Carlos polychromatic light the underside of a biosensor and measuring the III, 28031 Madrid, Spain; Department of Biochemistry and changes in the wavelength of the reflected monochromatic light that is a sensitive function of the index of refraction. The magnitude of the Molecular Biomedicine, Faculty of Biology, Universitat de wavelength shift (in picometers) is directly proportional to the Barcelona, 08028 Barcelona, Spain; orcid.org/0000- amount of mass redistribution. 48 h before the assay, HEK-293T cells 0003-2549-4919; Email: rfranco123@gmail.com, were transiently transfected with 1 μg of cDNA coding for CB2R or rfranco@ub.edu mutant receptors. HEK-293T cells were seeded in 384-well sensor microplates to obtain 70−80% confluent monolayers constituted by Authors approximately 10,000 cells/well. Prior to the assay, cells were washed Gemma Navarro − Department of Biochemistry and twice with an assay buffer (HBSS with 20 mM HEPES, pH 7.15, and Physiology, Faculty of Pharmacy and Food Sciences, 1% BSA) (SigmaAldrich, St. Louis, MO, US) and incubated for 2 h Universitat de Barcelona, 08028 Barcelona, Spain; Centro de 9362 https://doi.org/10.1021/acs.jmedchem.1c00561 J. Med. Chem. 2021, 64, 9354−9364 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Investigación en Red, Enfermedades Neurodegenerativas (4) Devinsky, O.; Patel, A. D.; Cross, J. H.; Villanueva, V.; Wirrell, E. (CIBERNED), Instituto de Salud Carlos III, 28031 Madrid, C.; Privitera, M.; Greenwood, S. M.; Roberts, C.; Checketts, D.; Spain VanLandingham, K. E.; Zuberi, S. M.; Group, G. S. Effect of Angel Gonzalez − Laboratory of Computational Medicine, Cannabidiol on Drop Seizures in the Lennox-Gastaut Syndrome. N. Engl. J. Med. 2018, 378, 1888−1897. Biostatistics Unit, Faculty of Medicine, Universitat Autònoma (5) Billakota, S.; Devinsky, O.; Marsh, E. Cannabinoid Therapy in Barcelona, Bellaterra 08193, Barcelona, Spain Epilepsy. Curr. Opin. Neurol. 2019, 32, 220−226. Adrià Sánchez-Morales − Departament de Química, (6) Fasinu, P. S.; Phillips, S.; ElSohly, M. A.; Walker, L. A. Current Universitat Autònoma de Barcelona, Bellaterra 08193, Status and Prospects for Cannabidiol Preparations as New Barcelona, Spain Therapeutic Agents. Pharmacotherapy 2016, 36, 781−796. Nil Casajuana-Martin − Laboratory of Computational (7) Morales, P.; Reggio, P. H. CBD: A New Hope? ACS Med. Chem. Medicine, Biostatistics Unit, Faculty of Medicine, Universitat Lett. 2019, 10, 694−695. Autònoma Barcelona, Bellaterra 08193, Barcelona, Spain (8) Ibeas Bih, C.; Chen, T.; Nunn, A. V. W.; Bazelot, M.; Dallas, M.; Marc Gómez-Ventura − Departament de Química, Whalley, B. J. Molecular Targets of Cannabidiol in Neurological Universitat Autònoma de Barcelona, Bellaterra 08193, Disorders. Neurotherapeutics 2015, 12, 699−730. Barcelona, Spain (9) Morales, P.; Reggio, P. H.; Jagerovic, N. An Overview on Medicinal Chemistry of Synthetic and Natural Derivatives of Arnau Cordomí − Laboratory of Computational Medicine, Cannabidiol. Front. Pharmacol. 2017, 8, 422. Biostatistics Unit, Faculty of Medicine, Universitat Autònoma (10) Thomas, A.; Baillie, G. L.; Phillips, A. M.; Razdan, R. K.; Ross, Barcelona, Bellaterra 08193, Barcelona, Spain R. A.; Pertwee, R. G. Cannabidiol Displays Unexpectedly high Félix Busqué − Departament de Química, Universitat Potency as an Antagonist of CB1 and CB2 Receptor Agonists in vitro. Autònoma de Barcelona, Bellaterra 08193, Barcelona, Spain Br. J. Pharmacol. 2007, 150, 613−623. Complete contact information is available at: (11) McPartland, J. M.; Duncan, M.; Di Marzo, V.; Pertwee, R. G. https://pubs.acs.org/10.1021/acs.jmedchem.1c00561 Are Cannabidiol and Delta(9)-tetrahydrocannabivarin Negative Modulators of the Endocannabinoid System? A Systematic Review. Br. J. Pharmacol. 2015, 172, 737−753. Author Contributions (12) Laprairie, R. B.; Bagher, A. M.; Kelly, M. E. M.; Denovan- G.N., A.G., and A.S.M. authors contributed equally. G.N., A.C., Wright, E. M. Cannabidiol is a Negative Allosteric Modulator of the L.P., and R.F. devised the project concept and designed Cannabinoid CB1 Receptor. Br. J. Pharmacol. 2015, 172, 4790−4805. experiments. A.G. and N.C.M. contributed with computational (13) Martínez-Pinilla, E.; Varani, K.; Reyes-Resina, I.; Angelats, E.; simulations. A.S.M. and M.G.V. synthesized compounds, Vincenzi, F.; Ferreiro-Vera, C.; Oyarzabal, J.; Canela, E. I.; Lanciego, supervised by F.B. and R.A. G.N. performed the biochemical J. L.; Nadal, X.; Navarro, G.; Borea, P. A.; Franco, R. Binding and and molecular assays. R.A., L.P., and R.F. wrote the paper with Signaling Studies Disclose a Potential Allosteric Site for Cannabidiol contributions from all other authors. All authors contributed to in Cannabinoid CB2 Receptors. Front. Pharmacol. 2017, 8, 744. the data analysis and have given approval to the final version of (14) McPartland, J. M.; Glass, M.; Pertwee, R. G. Meta-analysis of the manuscript. Cannabinoid Ligand Binding Affinity and Receptor Distribution: Interspecies Differences. Br. J. Pharmacol. 2007, 152, 583−593. Funding (15) Navarro, G.; Reyes-Resina, I.; Rivas-Santisteban, R.; Sánchez de We acknowledge the financial support from the Spanish Medina, V.; Morales, P.; Casano, S.; Ferreiro-Vera, C.; Lillo, A.; Ministry of Economy and Innovation with FEDER funds Aguinaga, D.; Jagerovic, N.; Nadal, X.; Franco, R. Cannabidiol Skews (projects SAF2017-84117-R, RTI2018-098830-B-I00, Biased Agonism at Cannabinoid CB1 and CB2 Receptors with smaller PID2019-106403RB-I00, and PID2019-109240RB-I00). Effect in CB1-CB2 Heteroreceptor Complexes. Biochem. Pharmacol. Notes 2018, 157, 148−158. The authors declare no competing financial interest. (16) Li, X.; Hua, T.; Vemuri, K.; Ho, J.-H.; Wu, Y.; Wu, L.; Popov, ■ P.; Benchama, O.; Zvonok, N.; Locke, K. a.; Qu, L.; Han, G. W.; Iyer, M. R.; Cinar, R.; Coffey, N. J.; Wang, J.; Wu, M.; Katritch, V.; Zhao, ACKNOWLEDGMENTS S.; Kunos, G.; Bohn, L. M.; Makriyannis, A.; Stevens, R. C.; Liu, Z.-J. A.S.M. acknowledges the Universitat Autònoma de Barcelona Crystal Structure of the Human Cannabinoid Receptor CB2. Cell for its predoctoral grant. ■ 2019, 176, 459−467. (17) Xing, C.; Zhuang, Y.; Xu, T.-H.; Feng, Z.; Zhou, X. E.; Chen, ABBREVIATIONS M.; Wang, L.; Meng, X.; Xue, Y.; Wang, J.; Liu, H.; McGuire, T. F.; GPCR, G-protein-coupled receptor; CB2R, cannabinoid CB2 Zhao, G.; Melcher, K.; Zhang, C.; Xu, H. E.; Xie, X.-Q. Cryo-EM Structure of the Human Cannabinoid Receptor CB2-Gi Signaling receptor; CB1R, cannabinoid CB1 receptor; CBD, cannabidiol; Complex. Cell 2020, 180, 645−654. THC, Δ9-tetrahydrocannabinol; NAM, negative allosteric (18) Hua, T.; Li, X.; Wu, L.; Iliopoulos-Tsoutsouvas, C.; Wang, Y.; modulator; PAM, positive allosteric modulator; MD, molecular Wu, M.; Shen, L.; Brust, C. A.; Nikas, S. P.; Song, F.; Song, X.; Yuan, dynamic; TM, transmembrane helix ■ S.; Sun, Q.; Wu, Y.; Jiang, S.; Grim, T. W.; Benchama, O.; Stahl, E. L.; Zvonok, N.; Zhao, S.; Bohn, L. M.; Makriyannis, A.; Liu, Z.-J. REFERENCES Activation and Signaling Mechanism Revealed by Cannabinoid (1) Iffland, K.; Grotenhermen, F. An Update on Safety and Side Receptor-Gi Complex Structures. Cell 2020, 180, 655−665. Effects of Cannabidiol: A Review of Clinical Data and Relevant (19) Polini, B.; Cervetto, C.; Carpi, S.; Pelassa, S.; Gado, F.; Ferrisi, Animal Studies. Cannabis Cannabinoid Res. 2017, 2, 139−154. R.; Bertini, S.; Nieri, P.; Marcoli, M.; Manera, C. Positive Allosteric (2) White, C. M. A Review of Human Studies Assessing Modulation of CB1 and CB2 Cannabinoid Receptors Enhances the Cannabidiol’s (CBD) Therapeutic Actions and Potential. J. Clin. Neuroprotective Activity of a Dual CB1R/CB2R Orthosteric Agonist. Pharmacol. 2019, 59, 923−934. Life 2020, 10, 333. (3) Giacoppo, S.; Bramanti, P.; Mazzon, E. Sativex in the (20) Petrucci, V.; Chicca, A.; Glasmacher, S.; Paloczi, J.; Cao, Z.; Management of Multiple Sclerosis-Related Spasticity: An Overview Pacher, P.; Gertsch, J. Pepcan-12 (RVD-hemopressin) is a CB2 of the last Decade of Clinical Evaluation. Mult. Scler. Relat. Disord. Receptor Positive Allosteric Modulator Constitutively Secreted by 2017, 17, 22−31. Adrenals and in Liver upon Tissue Damage. Sci. Rep. 2017, 7, 9560. 9363 https://doi.org/10.1021/acs.jmedchem.1c00561 J. Med. Chem. 2021, 64, 9354−9364 Journal of Medicinal Chemistry pubs.acs.org/jmc Article (21) Gado, F.; Di Cesare Mannelli, L.; Lucarini, E.; Bertini, S.; (36) Pellissier, L. P.; Sallander, J.; Campillo, M.; Gaven, F.; Cappelli, E.; Digiacomo, M.; Stevenson, L. A.; Macchia, M.; Queffeulou, E.; Pillot, M.; Dumuis, A.; Claeysen, S.; Bockaert, J.; Tuccinardi, T.; Ghelardini, C.; Pertwee, R. G.; Manera, C. Pardo, L. Conformational Toggle Switches implicated in Basal Identification of the First Synthetic Allosteric Modulator of the Constitutive and Agonist-Induced Activated States of 5-hydroxytrypt- CB2 Receptors and Evidence of Its Efficacy for Neuropathic Pain amine-4 Receptors. Mol. Pharmacol. 2009, 75, 982−990. Relief. J. Med. Chem. 2019, 62, 276−287. (37) Ersoy, B. A.; Pardo, L.; Zhang, S.; Thompson, D. A.; (22) Schröder, R.; Schmidt, J.; Blättermann, S.; Peters, L.; Janssen, Millhauser, G.; Govaerts, C.; Vaisse, C. Mechanism of N-terminal N.; Grundmann, M.; Seemann, W.; Kaufel, D.; Merten, N.; Drewke, Modulation of Activity at the Melanocortin-4 Receptor GPCR. Nat. C.; Gomeza, J.; Milligan, G.; Mohr, K.; Kostenis, E. Applying Label- Chem. Biol. 2012, 8, 725−730. Free Dynamic Mass Redistribution Technology to Frame Signaling of (38) Pérez-Benito, L.; Doornbos, M. L. J.; Cordomí, A.; Peeters, L.; G protein-Coupled Receptors Noninvasively in Living Cells. Nat. Lavreysen, H.; Pardo, L.; Tresadern, G. Molecular Switches of Protoc. 2011, 6, 1748−1760. Allosteric Modulation of the Metabotropic Glutamate 2 Receptor. (23) González, A.; Perez-Acle, T.; Pardo, L.; Deupi, X. Molecular Structure 2017, 25, 1153−1162. Basis of Ligand Dissociation in beta-Adrenergic Receptors. PLoS One (39) Navarro, G.; Gonzalez, A.; Campanacci, S.; Rivas-Santisteban, 2011, 6, No. e23815. R.; Reyes-Resina, I.; Casajuana-Martin, N.; Cordomí, A.; Pardo, L.; (24) Kruse, A. C.; Ring, A. M.; Manglik, A.; Hu, J.; Hu, K.; Eitel, K.; Franco, R. Experimental and Computational Analysis of Biased Hübner, H.; Pardon, E.; Valant, C.; Sexton, P. M.; Christopoulos, A.; Agonism on full-length and a C-terminally Truncated Adenosine A2A Felder, C. C.; Gmeiner, P.; Steyaert, J.; Weis, W. I.; Garcia, K. C.; Receptor. Comput. Struct. Biotechnol. J. 2020, 18, 2723−2732. Wess, J.; Kobilka, B. K. Activation and Allosteric Modulation of a (40) Dethe, D. H.; Erande, R. D.; Mahapatra, S.; Das, S.; Kumar, B. Muscarinic Acetylcholine Receptor. Nature 2013, 504, 101−106. V. Protecting Group free Enantiospecific Total Syntheses of (25) Audet, M.; Stevens, R. C. Emerging Structural Biology of Lipid Structurally Diverse Natural Products of the Tetrahydrocannabinoid G protein-Coupled Receptors. Protein Sci. 2019, 28, 292−304. family. Chem. Commun. 2015, 51, 2871−2873. (26) Stanley, N.; Pardo, L.; Fabritiis, G. D. The pathway of Ligand (41) Dialer, L.; Petrovic, L.; Weigl, U. Process for the Production of entry from the Membrane Bilayer to a Lipid G protein-Coupled Cannabidiol and delta-9-tetrahydrocannabinol. WO 2017011210 A1, Receptor. Sci. Rep. 2016, 6, 22639. 2017. (27) Morales, P.; Navarro, G.; Gómez-Autet, M.; Redondo, L.; (42) Troupiotis-Tsaïlaki, A.; Zachmann, J.; González-Gil, I.; Fernández-Ruiz, J.; Pérez-Benito, L.; Cordomí, A.; Pardo, L.; Franco, Gonzalez, A.; Ortega-Gutiérrez, S.; López-Rodríguez, M. L.; Pardo, R.; Jagerovic, N. Discovery of Homobivalent Bitopic Ligands of the L.; Govaerts, C. Ligand Chain length Drives Activation of Lipid G Cannabinoid CB2 Receptor. Chemistry 2020, 26, 15839−15842. protein-Coupled Receptors. Sci. Rep. 2017, 7, 2020. (28) Hua, T.; Vemuri, K.; Pu, M.; Qu, L.; Han, G. W.; Wu, Y.; Zhao, (43) https://getcontacts.github.io/ (accessed Jan 2021). S.; Shui, W.; Li, S.; Korde, A.; Laprairie, R. B.; Stahl, E. L.; Ho, J.-H.; (44) Dhopeshwarkar, A.; Mackie, K. CB2 Cannabinoid Receptors as Zvonok, N.; Zhou, H.; Kufareva, I.; Wu, B.; Zhao, Q.; Hanson, M. A.; a Therapeutic Target-what does the Future hold? Mol. Pharmacol. Bohn, L. M.; Makriyannis, A.; Stevens, R. C.; Liu, Z.-J. Crystal 2014, 86, 430−437. Structure of the Human Cannabinoid Receptor CB1. Cell 2016, 167, (45) Slivicki, R. A.; Xu, Z.; Kulkarni, P. M.; Pertwee, R. G.; Mackie, 750−762. K.; Thakur, G. A.; Hohmann, A. G. Positive Allosteric Modulation of (29) Saleh, N.; Hucke, O.; Kramer, G.; Schmidt, E.; Montel, F.; Cannabinoid Receptor Type 1 Suppresses Pathological Pain Without Lipinski, R.; Ferger, B.; Clark, T.; Hildebrand, P. W.; Tautermann, C. Producing Tolerance or Dependence. Biol. Psychiatry 2018, 84, 722− S. Multiple Binding Sites Contribute to the Mechanism of Mixed 733. Agonistic and Positive Allosteric Modulators of the Cannabinoid CB1 (46) Cabañero, D.; Ramírez-López, A.; Drews, E.; Schmöle, A.; Otte, Receptor. Angew. Chem., Int. Ed. Engl. 2018, 57, 2580−2585. D. M.; Wawrzczak-Bargiela, A.; Huerga Encabo, H.; Kummer, S.; (30) Solórzano, R.; Tort, O.; García-Pardo, J.; Escribà, T.; Lorenzo, Ferrer-Montiel, A.; Przewlocki, R.; Zimmer, A.; Maldonado, R. J.; Arnedo, M.; Ruiz-Molina, D.; Alibés, R.; Busqué, F.; Novio, F. Protective Role of Neuronal and Lymphoid Cannabinoid CB2 Versatile iron−catechol-based Nanoscale Coordination Polymers with Receptors in Neuropathic Pain. Elife 2020, 9, No. e55582. Antiretroviral Ligand Functionalization and their use as Efficient (47) Lesch, B.; Toräng, J.; Nieger, M.; Bräse, S. The Diels-Alder Carriers in HIV/AIDS Therapy. Biomater. Sci. 2019, 7, 178−186. Approach towards Cannabinoids. Synthesis 2005, 1888−1900. (31) Zysman-Colman, E.; Arias, K.; Siegel, J. S. Synthesis of (48) Roberts, J. C.; Pincock, J. A. Methoxy-substituted Stilbenes, Arylbromides from Arenes and N-bromosuccinimide (NBS) in Styrenes, and 1-arylpropenes: Photophysical Properties and Photo- Acetonitrile-A convenient Method for Aromatic Bromination. Can. additions of Alcohols. J. Org. Chem. 2006, 71, 1480−1492. J. Chem. 2009, 87, 440−447. (49) Poldy, J.; Peakall, R.; Barrow, R. A. Synthesis of Chiloglottones- (32) Kinney, W. A.; McDonnell, M. E.; Zhong, H. M.; Liu, C.; Yang, semiochemicals from Sexually Deceptive Orchids and their Ollinators. L.; Ling, W.; Qian, T.; Chen, Y.; Cai, Z.; Petkanas, D.; Brenneman, D. Org. Biomol. Chem. 2009, 7, 4296−4300. E. Discovery of KLS-13019, a Cannabidiol-Derived Neuroprotective (50) Usami, N.; Okuda, T.; Yoshida, H.; Kimura, T.; Watanabe, K.; Yoshimura, H.; Yamamoto, I. Synthesis and Pharmacological Agent, with Improved Potency, Safety, and Permeability. ACS Med. Evaluation in Mice of Halogenated Cannabidiol Derivatives. Chem. Chem. Lett. 2016, 7, 424−428. Pharm. Bull. 1991, 47, 1641−1645. (33) Tomanová, M.; Jedinák, L.; Cankař, P. Reductive Dehaloge- (51) Schott-Verdugo, S.; Gohlke, H. PACKMOL-Memgen: A nation and Dehalogenative Sulfonation of Phenols and Hetero- Simple-To-Use, Generalized Workflow for Membrane-Protein-Lipid- aromatics with Sodium Sulfite in an Aqueous Medium. Green Chem. Bilayer System Building. J. Chem. Inf. Model. 2019, 59, 2522−2528. 2019, 21, 2621−2628. (52) Abraham, M. J.; Murtola, T.; Schulz, R.; Páll, S.; Smith, J. C.; (34) McAllister, S. D.; Hurst, D. P.; Barnett-Norris, J.; Lynch, D.; Hess, B.; Lindahl, E. GROMACS: High Performance Molecular Reggio, P. H.; Abood, M. E. Structural Mimicry in class A G protein- Simulations throughMmulti-level Parallelism from Laptops to Super- Coupled Receptor Rotamer Toggle Switches: the importance of the computers. SoftwareX 2015, 1−2, 19−25. F3.36(201)/W6.48(357) Interaction in Cannabinoid CB1 Receptor Activation. J. Biol. Chem. 2004, 279, 48024−48037. (35) Krishna Kumar, K.; Shalev-Benami, M.; Robertson, M. J.; Hu, H.; Banister, S. D.; Hollingsworth, S. A.; Latorraca, N. R.; Kato, H. E.; Hilger, D.; Maeda, S.; Weis, W. I.; Farrens, D. L.; Dror, R. O.; Malhotra, S. V.; Kobilka, B. K.; Skiniotis, G. Structure of a Signaling Cannabinoid Receptor 1-G Protein Complex. Cell 2019, 176, 448− 458. 9364 https://doi.org/10.1021/acs.jmedchem.1c00561 J. Med. Chem. 2021, 64, 9354−9364