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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Med Chem. Author manuscript; available in PMC 2017 July 28.
Published in final edited form as:
PMCID: PMC5532543
NIHMSID: NIHMS823939

Novel C-Ring-Hydroxy-Substituted Controlled Deactivation Cannabinergic Analogues

Abstract

In pursuit of safer controlled-deactivation cannabinoids with high potency and short duration of action, we report the design, synthesis, and pharmacological evaluation of novel C9- and C11-hydroxy-substituted hexahydrocannabinol (HHC) and tetrahydrocannabinol (THC) analogues in which a seven atom long side chain, with or without 1′-substituents, carries a metabolically labile 2′,3′-ester group. Importantly, in vivo studies validated our controlled deactivation approach in rodents and non-human primates. The lead molecule identified here, namely, butyl-2-[(6aR,9R,10aR)-1-hydroxy-9-(hydroxymethyl)-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl]-2-methylpropanoate (AM7499), was found to exhibit remarkably high in vitro and in vivo potency with shorter duration of action than the currently existing classical cannabinoid agonists.

Graphical Abstract

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INTRODUCTION

Modulation of the two cannabinoid receptors CB11 and CB22 by the plant-derived (−)-Δ9-tetrahydrocannabinol [(−)-Δ9-THC] and its synthetic analogues3 is a promising therapeutic approach to treat an array of indications including pain, inflammation, CNS disorders, and cancer.410 Unfortunately, widespread use of cannabinergic agents in therapy has been hampered due to target related adverse side effects as well as poor pharmacokinetic (PK) properties.11 While seeking to obtain novel THC-based analogues with improved druggability and safety, we reported on a controlled deactivation/detoxification approach. To this effect, we have combined the “soft” analogue/drug concept of enzymatic inactivation with the “depot effect” which is related to the compound’s lipophilicity as well as its tissue distribution and retention.1215 Specifically, we have shown that incorporation of a metabolically labile carboxyester group (soft spot) at strategic positions within the THC structure leads to potent and efficacious CB1 agonists (2, Figure 1) that are bioconverted to inactive metabolites by plasma esterases while the rate of hydrolytic cleavage can be accordingly modulated using stereochemical features adjacent to the ester group (enzymatic effect). Also, it is well established that the pharmacological time of action of a lipophilic drug molecule, such as Δ9- and Δ8-THC, can greatly exceed its biochemical half-life. This is the result of a “depot effect” due to which when the drug molecule is administered, it is sequestered in the fatty tissues before being slowly released into the circulation to start exerting its action. The depot effect is dependent on the compound’s polar characteristics and can be regulated by varying its log P and PSA values. Thus, higher lipophilicity of a molecule is associated with longer residency in fatty tissue, while enhancement of the compound’s polar features is expected to moderate its lipophilic character and reduce the depot effect.1215.

Figure 1
Design of the first and second generation side chain carboxylated cannabinoid analogues, with controllable deactivation and increasing polarity, and structures of the prototype (−)-Δ8-THC-DMH and inactive metabolites.

A major goal of this project is to develop controlled-deactivation cannabinoids with faster onset/offset and shorter duration of action than currently existing THC analogues. Such compounds can be useful as tools to study the pharmacological relevance of the precise, short-term activation of CB receptors and to help develop better designed and more predictable animal models. Toward this goal, we have recently focused on controlled-deactivation cannabinergic templates with enhanced polar characteristics that are associated with lower lipophilicity and, thus, less of the depot effect. In an initial study, we observed that incorporation of polar groups (e.g., cyano and imidazolyl groups) at the carboxyester side chain pharmacophore of THC-like analogues increases its stability toward esterases.13 This suggests that the depot effect and enzymatic actions are not working synergistically for optimal short duration of action when both the soft spot and the polar group are incorporated in the side chain of the molecule. For this reason we sought to design second generation controlled deactivation ligands that retain the metabolically labile ester group at the 2′-position of the side chain while the polar moiety, in the form of a hydroxyl group, is introduced at the C9 or C11 positions of the tricyclic cannabinoid structure (3, Figure 1). The choice of the hydroxyl group and the relative stereochemistry at the C9 is based on our earlier lead optimization and pharmacophore refinement work in the classical cannabinoid prototype.1621 An additional goal of the present work was to explore the validity of our controlled deactivation approach by obtaining information related to the modulation of the depot effect and its role in the in vivo pharmacokinetic profile of the analogues in rodents and monkeys.

All synthesized compounds were characterized biochemically by determining their in vitro CB1 and CB2 receptor binding affinities, functional activities, and assessment of their in vitro metabolic stability toward mouse and rat plasma esterases. The most successful compounds were evaluated for their hypothermic and analgesic effects in rodents, while a key compound was further tested in drug discrimination experiments in squirrel monkeys. Our data show that the C1′-gem-dimethyl analogues exhibit very high affinities for the CB1 and CB2 receptors and that they are susceptible to enzymatic deactivation by plasma esterases while their hydrolytic metabolites showed no or very low cannabinergic activity. In vitro and in vivo characterization suggested that three of the analogues identified in this study are potent CB1/CB2 agonists and exhibit CB-mediated hypothermic effects. The analogue with the most promising activity and short duration of action profile, namely, AM7499 (3b), was further tested in the analgesia assay in mice and drug discrimination experiments in squirrel monkeys. The in vivo data show that our lead compound is a remarkably potent CB receptor agonist with significantly faster onset and shorter duration of action than the less polar and nonhydrolyzable congener 11-OH-Δ8-THC-DMH22 (3g, Table 1). Importantly, drug discrimination experiments in non-human primates validated our controlled deactivation approach in which compound 3b exhibited approximately 3-fold greater potency and approximately 4 times shorter duration of action when compared to non-hydrolyzable congener 11-OH-Δ8-THC-DMH.

Table 1
Affinities (Ki) of C9- and C11-Hydroxy-Substituted Cannabinoid Analogues for CB1 and CB2 Cannabinoid Receptors (±95% Confidence Limits) and Half-Lives (t1/2) of Representative Compounds for Mouse and Rat Plasma Esterases

CHEMISTRY

Synthesis of the 11-OH-(−)-Δ8-THC ester derivative 3a is depicted in Scheme 1. The chiral 4-hydroxymyrtenyl pivalate 6 was prepared in three steps from (1R,5S)-myrtenol (5) using the method by Liddle et al.,23 while carboxylic acid 8 was obtained in two steps from nitrile 7 by following procedures we reported earlier.15 Methyl ester formation produced 9 (72% yield) which was treated with boron tribromide to give the corresponding resorcinol 10 in 71% yield.15,19 Condensation of 10 with the chiral terpenoid alcohol 6 in the presence of boron trifluoride diethyl etherate provided the diester 11 in 40% yield.23,24 Saponification of all ester groups in 11 gave the precursor carboxylic acid 4a (68% yield) which upon treatment with 1-bromobutane and sodium bicarbonate under microwave conditions afforded the respective ester 3a in 51% yield (nonoptimized).14,15,19

Scheme 1a
aReagents and conditions: (a) MeI, K2CO3, acetone, rt, overnight, 72%; (b) BBr3, CH2Cl2, −78 to 0 °C, 2.5 h, 71%; (c) 6, BF3·Et2O, CH2Cl2, −20 °C to rt, 2 h, 40%; (d) NaOH, THF/H2O, reflux, 12 h 68%; (e) Br(CH2 ...

Stereoselective synthesis of the 11-OH-(−)-hexahydrocannabinol ester analogue 3b and the respective carboxylic acid metabolite 4b is shown in Scheme 2. Deprotection of the phenolic hydroxyl groups in 8 led to carboxylic acid 12 (98% yield) which upon microwave assisted esterification gave resorcinol 13 in an excellent yield (92%).13,15 Coupling of 13 with a mixture of chiral terpene diacetates 14 in the presence of p-toluenesulfonic acid (p-TSA) gave norpinanone 15 in moderate yield (27%).25 We had explored a variety of solvents, acids, and conditions in order to improve the yield to no avail. In addition to the desired product 15, this reaction produces a mixture of byproducts where the (+)-apoverbenone (14a) and the resorcinol monoacetate 13a were found to be the major ingredients. We and others have observed that this condensation works well with resorcinols bearing electron donating alkyl side chains with bulky groups at the benzylic position including the 5-(1,1-dimethylheptyl)resorcinol.1820,26 This, coupled with our earlier investigations on the mechanism of this condensation,25 led us to postulate that presence of the carboxyester group in 13 decreases the electron density of the aromatic ring and slows down the rate of the initial Friedel–Crafts allylation reaction. Thus, the acid catalyzed decomposition of diacetates 14 to give 14a and the monoacetylation of 13 to give 13a become important side reactions. Currently studies to circumvent the problem of the moderate yield of this synthetic step are ongoing in our laboratory. Treatment of 15 with catalytic amounts of trimethylsilyl triflate in dichloromethane/nitromethane gave 16 with the required 6aR,10aR stereochemistry in 67% yield.16,19 The free phenolic hydroxyl in 16 was protected as the tert-butyldimethylsilyl (TBS) ether (87% yield). Ketone 17 was exposed to (methoxymethylene)-triphenylphosphorane to give enol ether 18 in 63% yield as a 2:3 mixture of two geometric isomers (18a and 18b; see Experimental Section) based on 1H NMR analysis. Methyl vinyl ether 18 was hydrolyzed with wet trichloroacetic acid (97% yield), and the diastereomeric mixture of C9 aldehydes 19 (5:2 ratio by 1H NMR) was epimerized to produce the β-equatorial isomer 20 in 78% yield.16,18 Reduction with sodium borohydride afforded the intermediate alcohol 21 in 85% yield. Subsequent treatment with tetra-n-butylammonium fluoride gave the final compound 3b in high yield (90%). The metabolite acid 4b was prepared by alkaline hydrolysis of the ester 3b using lithium hydroxide in dioxane/water (85% yield).27

Scheme 2a
aReagents and conditions: (a) BBr3, CH2Cl2, 0 °C to rt, 3.5 h, 98%; (b) NaHCO3, 1-bromobutane, DMF, 165 °C, microwave irradiation, 12 min, 92%; (c) 14, p-TSA, CHCl3, 0 °C to rt, 4 days, 27%; (d) TMSOTf, CH2Cl2/MeNO2, 0 °C ...

The intermediate side chain carboxylated ketone 16 served as the starting point for the synthesis of the C9 alcohols 3c and 3d (Scheme 3). Thus, reduction of the C9 keto group in 16 with sodium borohydride led to the β-equatorial hydroxyl compound 3c along with traces (~4% by 1H NMR) of the respective α-axial isomer (3d).19 This was followed by flash column chromatography purification to give pure 3c in 84% yield. Alkaline hydrolysis of the ester side chain in 16 provided keto acid 22 (63% yield) which upon treatment with sodium borohydride afforded the acid metabolite 4c in 81% yield after purification from traces of the α-axial diastereomer 4d. Reduction of 22 with potassium tri-sec-butyl borohydride (K-selectride) was very selective and gave the α-axial alcohol 4d exclusively (91% yield). Alkylation of the respective carboxylate anion with 1-bromobutane under microwave heating led to the corresponding ester 3d (42% yield). It should be noted that the stereochemistry of the hydroxyl groups of 3c/3d and 4c/4d isomeric pairs was assigned on the basis of 1H NMR (500 MHz) spectral data as in our earlier work with closely related systems.19 Thus, in compounds 3c and 4c, the peak half-width for the C9 protons was found to be 25–26 Hz while in compounds 3d and 4d it was 9.5 Hz (see Experimental Section). This correlates well with an axial C9 proton in the former two compounds and an equatorial C9 proton in the latter two.

Scheme 3a
aReagents and conditions: (a) NaBH4, MeOH, −78 °C, 2 h, 84%; (b) LiOH, dioxane/H2O, rt, 24 h, 63%; (c) NaBH4, MeOH, rt, 1 h, 81%; (d) K-selectride, THF, rt, 2 h, 91%; (e) NaHCO3, 1-bromobutane, DMF, 165 °C, microwave irradiation, ...

Syntheses of the side chain carboxylated analogues lacking the C1′-gem-dimethyl group are summarized in Scheme 4. In a similar fashion, terpenylation of resorcinol 23 (23% yield) was followed by dibenzo[b,d]pyran ring closure to give methyl ester 25 (57% yield). The sequence methyl ester hydrolysis,28 esterification, hydride reduction worked as expected and provided the C9 equatorial alcohol 3e in good overall yield. Sodium borohydride and K-selectride reductions were employed to synthesize the requisite C9 alcohols 3f, 4e, and 4f from the C9 ketones 27 and 26.

Scheme 4a
aReagents and conditions: (a) 14, p-TSA, CHCl3/acetone, 0 °C to rt, 4 days, 23%; (b) TMSOTf, CH2Cl2/MeNO2, 0 °C to rt, 4 h, 57%; (c) NaOH, THF/H2O, rt, 4 h, 63%; (d) NaHCO3, 1-bromobutane, DMF, 165 °C, microwave irradiation, 12 ...

CANNABINOID RECEPTOR AFFINITIES

The abilities of compounds 3af and 4af to displace the radiolabeled CB1/CB2 agonist CP-55,940 from membranes prepared from rat brain (source of CB1) and HEK 293 cells expressing either mouse CB2 or human CB2 were determined as described earlier,13,19 and inhibition constant values (Ki) from the respective competition binding curves are listed in Table 1 in which the nonhydrolyzable prototype 11-OH-Δ8-THC-DMH (3g) and the first generation carboxy-Δ8-THC analogue 2a are included for comparison. The use of two CB2 receptor preparations was aimed at addressing species differences that we observed earlier.29 The compounds included in this study are C9- and C11-hydroxy-substituted hexahydrocannabinol (HHC) and tetrahydrocannabinol (THC) analogues in which a seven atom long side chain, with or without 1′-substituents, carries a 2′,3′-ester group. In agreement with our rational design, the hydrolytic metabolites 4af have no significant affinities for CB1 and CB2 receptors, thus minimizing the possibility of undesirable cannabinoid receptor related side effects.

Examination of the binding data of 11-OH-Δ8-THC-DMH (3g), the first generation carboxyester THC compound 2a, and the side chain carboxylated 11-OH-THC analogue 3a suggests that simultaneous addition of carboxyester functionality at the side chain and hydroxyl group at C11 of the prototype maintains very high binding affinities for both the CB1 and CB2 receptors. This holds true when the tetrahydrocannabinol C-ring is converted to the fully saturated hexahydrocannabinol ring with the hydroxymethyl substituent occupying the equatorial position (analogue 3b). Likewise, the one carbon shorter homologue 3c exhibits somewhat higher binding affinities for both CB receptors. However, conversion of the C9 equatorial hydroxyl group (seen in 3c) to the axial hydroxyl (3d) results in a distinct reduction (approximately 20- to 30-fold) in the binding affinities for CB1 and CB2. This indicates that the relative stereochemistry of the hydroxyl group at C9 can affect the ligand’s affinity within the side chain carboxylated hexahydrocannabinol class of compounds. A comparison of the binding data of the two diastereomers 3e and 3f with their 1′,1′-dimethyl congeners 3c and 3d demonstrates the striking effects of the C1′-gem-dimethyl substitution. Thus, analogues 3c and 3d (0.1 nM < Ki < 7 nM) exhibit 2–3 orders of magnitude higher binding affinities when compared to 3e and 3f that bind weakly at CB receptors (238 nM < Ki < 750 nM).

Overall, this SAR study shows that C9- and C11-hydroxy-substituted hexahydrocannabinol and tetrahydrocannabinol analogues with a 1′,1′-dimethyl-2′-carboxyester group [-C-(CH3)2-C(O)O-] on the side chain pharmacophore exhibit remarkably high affinities for both the CB1 and CB2 receptors. It should be noted that we have observed similar SAR trends with the side chain carboxyester THC analogues.13,15 However, it appears that the HHC scaffold is more sensitive to the presence of the C1′-gem-dimethyl substitution with regard to CB receptor recognition.

IN VITRO PLASMA STABILITY STUDIES

Enzymatic labile moieties such as ester, thioester, carbamate, or phosphate groups that are incorporated into the parent drug molecules can be targeted by esterase enzymes expressed through body organs and other tissues as well as blood. Esterases are a heterogeneous group of enzymes that are classified broadly as cholinesterases (including acetylcholinesterases and butyrylcholinesterases), paraoxonases, and carboxyesterases. Human serum albumin also exhibits esterase activity toward phenyl esters such as aspirin (acetylsalicylic acid). Notably, esterase activity is higher in the blood of small rodents than in large animals and humans, while carboxyesterases are found in mice and rat but not in human plasma.13,30 In this study, analogues with the highest binding affinities for CB1 and CB2 were assessed for their in vitro plasma stability toward mouse and rat plasma esterases as detailed in the Experimental Section.13,15 A comparison of the half-lives (t1/2, Table 1) of the C9- and C11-hydroxy-substituted analogues (3a, 3b, 3c, and 3d) with the first generation side chain carboxylated compound 2a shows that incorporation of a polar hydroxyl group at the Cring of the prototype reduces the stabilities of the analogues toward esterases for both the mouse and the rat plasma. In addition, a comparison of the compounds’ half-lives in mouse and rat plasma indicates some species differences with the rat plasma being less active. This observation parallels those we reported earlier for carboxyester THC analogues.15 Interestingly, our stability data show that the isomeric C9 alcohols 3c and 3d have similar half-lives in mouse plasma whereas in rat plasma the equatorial alcohol 3c appears somewhat more stable. In summary, our data validated the soft analogue profile of our more polar, second generation, controlled deactivation cannabinergic ligands that also exhibit shorter metabolic half-lives for esterases when compared to the first generation side chain carboxylated THCs which carry no hydroxy groups at the C9 or C11 positions.

FUNCTIONAL CHARACTERIZATION

We focused on those analogues possessing the highest CB1/CB2 binding affinities (3a, 3b, and 3c), and experiments were carried out by measuring changes in forskolin-stimulated cAMP, as detailed earlier.19,20 Our testing results (Table 2) show that all three compounds potently decreased the levels of cAMP, indicating that within this signaling mechanism these compounds behaved as potent agonists at both the CB1 and CB2 receptors with the 11-OH-HHC ester analogue 3b being the most potent.

Table 2
CB1/CB2 Functional Potencies (EC50) of Selected C9- and C11-Hydroxy-Substituted Cannabinoid Analogues

IN VIVO BEHAVIORAL CHARACTERIZATION

Hypothermia Testing

The hypothermic effects of the side chain carboxyester analogues 3a,31 3b, and 3c with the highest CB1 receptor affinities were compared in rats. Rectal body temperature was measured in isolated rats over a 6 h period following drug injection (detailed procedures are given in Experimental Section). In agreement with our in vitro functional characterization, compounds 3a, 3b, and 3c all decreased core body temperature in a dose-dependent manner, reducing body temperature by 4.7–7.2 °C at the highest doses tested (Figure 2). For comparison, the effects of the nonhydrolyzable congener 11-OH-(−)-Δ8-THC-DMH (3g) are also shown. The ED50 values (in mg/kg, with 95% CI) were 0.04 (0.001, 0.15) for 3a, 0.04 (0.02, 0.1) for 3b, 0.74 (0.05, >10) for 3c, and 0.04 (0.01, 0.12) for 3g. Thus, the 11-OH-analogues 3a and 3b were equipotent with 3g, and 3c was slightly less potent than the other three compounds. All compounds had relatively fast onsets of drug effect. Significant decreases in body temperature typically occurred within 60 min after injection, although peak effects were not obtained until 2–3 h after injection (time course data for all tested doses of each compound are included in Supporting Information, Figure S1). A more detailed examination of the time course of the lowest doses of 3a, 3b, 3c, and 3g that had significant hypothermic effects is summarized in Figure 3. Administration of 0.1 mg/kg of 3a or 3b, carboxyester side chain analogues, resulted in temperature decreases of >4 °C, with significant recovery toward baseline within the 6 h test period. By comparison, doses of 3c and 3g that produced an equivalent reduction in rectal temperature had effects that persisted for more than 6 h.

Figure 2
Effects of 3a, 3b, and 3c, 11-OH-Δ8-THC-DMH (3g), or vehicle (left-most points, above Veh) on rectal body temperature using female Sprague-Dawley rats (n = 6). Abscissa: dose, in mg/kg. Ordinate: change in rectal body temperature from an average ...
Figure 3
Hypothermic effects of select doses of 3a, 3b, 11-OH-Δ8-THC-DMH (3g), and 3c at different times after injection. The doses selected were the lowest doses that decreased temperature by ≥4 °C in female Sprague–Dawley rats ...

Our hypothermia data in rats indicate that compounds 3a and 3b are equipotent and they have similar duration of action profiles. For further studies, the HHC analogue 3b was selected over the THC analogue 3a on the basis of the in vitro cAMP data where it behaves as a slightly more potent agonist for both the CB1 and CB2 receptors.

Analgesia Testing

To confirm the observed pharmacokinetic differences between the side chain carboxyester analogue 3b and the nonhydrolyzable counterpart 3g, we used the CB1 receptor characteristic analgesia assay. Two-way ANOVA suggested a significant interaction between time and drug [F(3, 66) = 28.94; p < 0.001]. The main effect of time was also significant [F(3, 66) = 4.33; p = 0.008]. Post hoc comparisons suggested significant differences between the two drugs (3b and 3g) at 20, 60, and 360 min postinjection (Figure 4). These differences in onset and offset of effect for the two compounds are also reflected by comparisons within each ligand over time. Pairwise comparisons suggested less analgesia at the 360 min time point compared to the three earlier time points as well as a significant difference between the recordings at 60 and 180 min postinjection for compound 3b. Slow onset of effect for compound 3g is suggested by significant differences in analgesia scores at 20 as well as 60 min postinjection and the recordings at the 180 and 360 time points. In keeping with above profile of a fast onset and offset for compound 3b, an additional dose of 1 mg/kg resulted in average MPE scores of 94%, 96%, 97%, and 58% at the four time-points (20, 60, 180, and 360 min postinjection) examined (n = 6; data not shown).

Figure 4
Tail-flick latencies in a hot water-bath (52 °C) after administration of two doses each of compounds 3b and 3g at four time-points (20, 60, 180, and 360 min postadministration) using male CD-1 mice (n = 6). Abscissa: time (min) after injection. Ordinate; ...

Drug Discrimination in Non-Human Primates

In an effort to further explore the validity of our controlled deactivation approach in species beyond rodents, we performed drug discrimination studies in squirrel monkeys (Saimiri sciureus) trained to discriminate 0.01 mg/kg (6aR,9R,10aR)-3-(adamantan-1-yl)-9-(hydr oxymethyl)-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol32 (28, AM4054, see Supporting Information Figure S2) from saline. This compound is a structurally related cannabinoid CB1 full agonist which was synthesized at the Center for Drug Discovery and characterized in our earlier work.33 It should be noted that studies with non-human primates are key to this project because metabolic processes and volumes of drug distribution vary between small rodent and primate species.34

In these experiments, the time courses of the lowest doses of our lead compound 3b (0.001 mg/kg) and the non-hydrolyzable cannabinoid 3g (0.003 mg/kg) that fully substituted for the training dose of the CB1 agonist 28 were compared directly in the same group of subjects (Figure 5; see Experimental Section). Compound 3b produced evidence of CB1-related discriminative-stimulus effects within 15 min (t1/2 = 48 min) and was fully effective in three of four subjects 1 h after injection. Compound 3g was similar in its onset of action (t1/2 = 66 min) but did not achieve a full effect in the group of subjects until 2 h after injection. Notwithstanding the generally comparable onset of behavioral effects, the two compounds varied consid[erably in offset of action. Thus, 3b began losing its CB1 discriminative-stimulus effects 4 h after injection and was completely without effect 16 h following treatment. On the other hand, 3g fully substituted for the training drug stimulus (28) 24 h after injection and was completely without CB1 discriminative-stimulus effects at the 36 h time point. On the basis of calculated values for loss of 50% of full effect, the duration of action for 3b was approximately 4 times shorter than for 3g. Neither compound had appreciable effects on response rates throughout the present time course studies (Figure 5, lower panel).

Figure 5
Time course of CB1 discriminative-stimulus effects of 3b (0.001 mg/kg) and 3g (0.003 mg/kg).

Overall, our in vivo experiments in rats show that compounds 3a, 3b, and 3c have in vivo hypothermia activity and produce similar maximum effects as the structurally related cannabinoid agonist 3g. Among the hydrolyzable compounds tested, analogues 3a and 3b were found to be equipotent and they have similar duration of action profiles. Our antinociception data in mice and drug discrimination experiments in squirrel monkeys confirmed that the more polar 11-OH-carboxyester analogue 3b is equipotent and exhibits significantly shorter duration of action when compared to the nonhydrolyzable standard 3g.

CONCLUSIONS

We recently reported on a controlled deactivation/detoxification approach for obtaining cannabinoids with improved druggability and safety. In these earlier studies we have also provided evidence that the pharmacological half-lives of our selectively detoxified analogues can be controlled by the joint modulation of their relative stabilities for plasma esterases as well as through variation of their polar characteristics and thus to the depot effects. A major goal of this project is to develop controlled deactivation cannabinoid agonists with faster onset/offset and significantly shorter duration of action than the currently existing classical cannabinoids and also to explore the validity of this approach in species beyond rodents including nonhuman primates. To this extent, we report now the design, synthesis, and pharmacological evaluation of second generation, more polar, controlled deactivation cannabinoids. These novel cannabinergic compounds are C9- and C11-hydroxy-substituted hexahydrocannabinol (HHC) and tetrahydrocannabinol (THC) analogues in which the seven atom long side chain, with or without substituents, carries a metabolically labile 2′,3′-ester group. Our in vitro data showed that compounds with a 1′,1′-dimethyl-2′-carboxyester group [-C(CH3)2-C(O)O-] on the side chain pharmacophore exhibit very high affinities for both the CB1 and CB2 receptors and also behave as potent and efficacious CB1/CB2 agonists in the cAMP assay. In agreement with our design principles, the analogues are susceptible to enzymatic hydrolysis by plasma esterases, while their hydrolytic metabolites are inactive. Importantly, for further work toward clinical development, in vivo studies validated our controlled deactivation approach in rodents and non-human primates. The lead molecule identified in this study, namely, butyl-2-[(6aR,9R,10aR)-1-hydroxy-9-(hydroxymethyl)-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl]-2-methylpropanoate (3b), was found to exhibit remarkably high in vitro and in vivo potency with a shorter duration of action than the currently existing classical cannabinoid agonists.

EXPERIMENTAL SECTION

Materials

All reagents and solvents were purchased from Aldrich Chemical Co., unless otherwise specified, and used without further purification. All anhydrous reactions were performed under a static argon atmosphere in flame-dried glassware using scrupulously dry solvents. Flash column chromatography employed silica gel 60 (230–400 mesh). All compounds were demonstrated to be homogeneous by analytical TLC on precoated silica gel TLC plates (Merck, 60 F245 on glass, layer thickness 250 μm), and chromatograms were visualized by phosphomolybdic acid staining. Melting points were determined on a micromelting point apparatus and are uncorrected. IR spectra were recorded on a PerkinElmer Spectrum One FT-IR spectrometer. NMR spectra were recorded in CDCl3, unless otherwise stated, on a Bruker Ultra Shield 400 WB plus (1H at 400 MHz, 13C at 100 MHz) or on a Varian INOVA-500 (1H at 500 MHz, 13C at 125 MHz) spectrometers, and chemical shifts are reported in units of δ relative to internal TMS. Multiplicities are indicated as br (broadened), s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and coupling constants (J) are reported in hertz (Hz). Low- and high-resolution mass spectra were performed in School of Chemical Sciences, University of Illinois at Urbana—Champaign. Mass spectral data are reported in the form of m/z (intensity relative to base = 100). Elemental analyses were obtained in Baron Consulting Co, Milford, CT, and were within ±0.4% of the theoretical values (see Supporting Information). Purities of the tested compounds were determined by elemental analysis or by HPLC (using Waters Alliance HPLC system, 4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water with 8.5% o-phosphoric acid) or by LC/MS analysis using a Waters MicroMass ZQ system [electrospray-ionization (ESI) with Waters-2525 binary gradient module coupled to a photodiode array detector (Waters-2996) and ELS detector (Waters-2424) using a XTerra MS C18, 5 μm, 4.6 mm × 50 mm column and acetonitrile/water] and were >95%.

Butyl 2-[(6aR,10aR)-1-Hydroxy-9-(hydroxymethyl)-6,6-dimethyl-6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-3-yl]-2-methylpropanoate (3a)

A stirred mixture of carboxylic acid 4a (100 mg, 0.29 mmol), bromobutane (119 mg, 0.87 mmol), and sodium bicarbonate (37 mg, 0.43 mmol) in DMF (2 mL) was heated at 165 °C for 12 min using microwave irradiation. The reaction mixture was cooled to room temperature and diluted with water and ethyl acetate. The organic layer was separated, and the aqueous phase was extracted with ethyl acetate. The combined organic layer was washed with brine, dried (MgSO4), and concentrated under reduced pressure. Purification by flash column chromatography on silica gel (5–50% ethyl acetate in hexane) gave 3a (60 mg, 51%) as a light yellow gum. IR (neat) 3292, 2961, 1727, 1704 (s, >C═O), 1619, 1578, 1417, 1328, 1263cm−1; 1H NMR (500 MHz, CDCl3) δ 6.41 (d, J = 2.0 Hz, 1H, 4-H), 6.25 (d, J = 2.0 Hz, 1H, 2-H), 5.73 (m as d, J = 4.5 Hz, 1H, 8-H), 5.42 (s, 1H, ArOH), 4.10–4.02 (d, t and d overlapping, 4H, especially 4.08, d, J = 13.0 Hz,1H, half of an AB system, -CH2-OH, 4.06, t, J = 6.5 Hz, 2H, -C(O)OCH2-, 4.03, d, J = 13.0 Hz, 1H, half of an AB system, -CH2-OH), 3.41 (dd, J = 16.0 Hz, J = 4.5 Hz, 1H, 10α-H), 2.71 (td, J = 11.0 Hz, J = 5.0 Hz, 1H, 10a-H), 2.27–2.20 (m, 1H, 7α-H), 1.93–1.78 (m, 3H, 10β-H, 7β-H, 6a-H), 1.55 (quintet, J = 7.0 Hz, 2H, -CH2- of the side chain), 1.50 (s, 6H, -C(CH3)2-), 1.39 (s, 3H, 6β-Me), 1.26 (quintet, 2H, -CH2- of the side chain), 1.10 (s, 3H, 6α-Me), 0.86 (t, J = 7.5Hz, 3H, 7′-H). 13C NMR (125 MHz CDCl3) δ 177.3 (>C═O), 155.6 (C-1 or C-5), 154.8 (C-5 or C-1), 144.5, 138.3, 121.6, 111.4, 107.3, 105.5, 77.7 (C-6), 67.2 (-O-CH2), 65.0(-OCH2-), 46.2, 45.1, 31.5, 31.4, 30.6, 27.8, 27.7, 26.4, 26.2, 19.1 18.5, 13.8. Mass spectrum (ESI) m/z (relative intensity) 403 (M+ + H, 100). Mass spectrum (EI) m/z (relative intensity) 402 (M+, 100), 301 (53), 217 (38). Exact mass (EI) calculated for C24H34O5 (M+), 402.2406; found 402.2407. HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 11.1min for 3a. Anal. (C24H34O5) C, H.

Butyl 2-[(6aR,9R,10aR)-1-Hydroxy-9-(hydroxymethyl)-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl]-2-methylpropanoate (3b)

To a solution of 21 (75 mg, 0.15 mmol) in anhydrous THF (3.6 mL) at −40 °C, under an argon atmosphere, was added tetra-n-butylammonium fluoride (0.3 mL, 0.3 mmol, 1 M solution in anhydrous THF). The reaction mixture was stirred for 30 min at the same temperature and then quenched using a saturated aqueous NH4Cl solution. Extractive isolation with diethyl ether and purification by flash column chromatography on silica gel (20% ethyl acetate in hexane) gave 3b (52 mg, 90% yield) as a white solid. Mp = 59–60 °C. IR (neat) 3380, 2971, 2869, 1727, 1703, 1620, 1577, 1417, 1268, 1141 cm−1; 1H NMR (500 MHz, CDCl3) δ 6.39 (d, J = 2.0 Hz, 1H, Ar-H), 6.22 (d, J = 2.0 Hz, 1H, Ar-H), 5.33 (br s, 1H, ArOH), 4.05 (d, J = 6.0 Hz, 2H, 4′-H), 3.56–3.49 (m, 2H, -CH2OH), 3.23–3.18 (m as br d, J = 13.0 Hz, 1H, C-ring), 2.51–2.44 (m as td, J = 11.0 Hz, J = 2.5 Hz, 1H, C-ring), 2.00–1.94 (m, 1H, C-ring), 1.94–1.88 (m, 1H, C-ring), 1.82–1.72 (m, 1H, C-ring), 1.58–1.44 (quintet, s, and m, overlapping, 9H, -C(CH3)2-, 5′-H, C-ring, especially 1.49, s, 6H, -C(CH3)2-), 1.38 (s, 3H, 6-Me), 1.29–1.22 (m as sextet, J = 7.0 Hz, 2H, 6′-H), 1.18–1.10 (m, 2H, C-ring), 1.07 (s, 3H, 6-Me), 0.86 (t, J = 7.5 Hz, 3H, 7′-H), 0.76 (m as q, J = 11.0 Hz, 1H, C-ring). 13C NMR (100 MHz CDCl3) δ 177.1 (-C(O)O-), 155.2 (ArC-1 or ArC-5), 154.9 (ArC-5 or ArC-1), 144.2 (tertiary aromatic), 111.0 (tertiary aromatic), 107.2 (ArC-2 or ArC-4), 105.3 (ArC-4 or ArC-2), 68.4 (-CH2OH), 64.7 (-OCH2-), 49.3, 46.0, 40.4, 35.0, 33.0, 30.5, 29.7, 27.7, 27.4, 26.2, 26.0, 19.0, 13.6. Mass spectum (ESI) m/z (relative intensity) 405 (M+ + H, 100). Mass spectum (EI) m/z (relative intensity) 404 (M+, 100), 361 (41), 303 (65), 265 (41). Exact mass (ESI) calculated for C24H3705 (M+ + H), 405.2641; found 405.2642. Exact mass (EI) calculated for C24H3605 (M+), 404.2563; found 404.2559. HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 99.5% and retention time of 10.8 min for the title compound.

Butyl 2-[(6aR,9R,10aR)-6a,7,8,9,10,10a-Hexahydro-1,9-dihydroxy-6,6-dimethyl-6H-benzo[c]chromen-3-yl]-2-methylpropanoate (3c)

To a solution of 16 (35 mg, 0.09 mmol) in anhydrous methanol (3 mL) at −78 °C, under an argon atmosphere, was added sodium borohydride (3.4 mg, 0.09 mmol). The reaction mixture was stirred at the same temperature for 2 h and then quenched by the addition of aqueous 2 N HCl solution. The mixture was warmed to room temperature, diluted with water, and extracted with ethyl acetate. The organic layer was washed with brine, dried (MgSO4), and concentrated in vacuo. Purification by flash column chromatography on silica gel (24% acetone in hexane) gave 3c (30 mg, 84% yield) as light yellow gum free from its epimer 3d which is also produced in traces during the reaction (ratio 3c/3d ~ 96:4). IR (neat) 3329, 2971, 2872, 1727, 1704, 1620, 1578, 1418, 1272, 1142 cm−1; 1H NMR (500 MHz, CDCl3) δ 6.38 ( d, J = 2.0 Hz, 1H, ArH), 6.22 (d, J = 2.0 Hz, 1H, ArH), 6.08 (br s, 1H, ArOH), 4.04 (t, J = 6.5 Hz, 2H, 4′-H), 3.86 (dddd, J = 16.0 Hz, J = 15.0 Hz, J = 4.5 Hz, J = 4.0 Hz, 1H, 9ax-H, peak half-width = 26 Hz), 3.51 (dddd, J = 12.0 Hz, J = 4.5 Hz, J = 2.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.47 (ddd, J = 12.0 Hz, J = 11.5 Hz, J = 2.5 Hz, 1H, 10a-H), 2.16 (m as br d, J = 10.0 Hz, 1H, 8eq-H), 1.88 (dddd, J = 13.5 Hz, J = 3.5 Hz, J = 2.5 Hz, J = 2.0 Hz,, 1H, 7eq-H), 1.63 (br s, 1H, OH), 1.57–1.50 (quintet and m, overlapping 3H, 5′-H and 6a-H), 1.49 (s, 6H, -C(CH3)2-), 1.42 (dddd, J = 15.5 Hz, J = 15.0 Hz, J = 13.0 Hz, J = 3.5 Hz, 1H, 8ax-H), 1.38 (s, 3H, 6-Me), 1.25 (sextet, J = 7.0 Hz, 2H, 6′-H), 1.15 (dddd, J = 13.5 Hz, J = 13.0 Hz, J = 12.0 Hz, J = 3.5 Hz, 1H, 7ax-H), 1.12 (ddd, J = 15.5 Hz, J = 12.0 Hz, J =12.0 Hz, 1H, 10ax-H), 1.05 (s, 3H, 6-Me), 0.85 (t, J = 7.5 Hz, 3H, 7′-H). 13C NMR (100 MHz, CDCl3) δ 171.5 (-C(O)O-), 155.0 (ArC), 154.7 (ArC), 144.5 (ArC), 115.4 (ArC), 110.2 (ArC), 107.5 (ArC), 77.2 (C-6), 70.7 (>CH-OH), 64.6 (-OCH2-), 48.3, 46.0, 39.0, 35.6, 33.4, 30.5, 27.8, 26.2, 26.1, 26.0, 19.0, 178, 13.6. Mass spectum (ESI) m/z (relative intensity) 391 (M+ + H, 100). Mass spectum (EI) m/z (relative intensity) 390 (M+, 87), 329 (32), 289 (100, M+ − CH3(CH2)3OC(O)), 265 (37), 177 (61). Exact mass (EI) calculated for C23H3405 (M+), 390.2406; found 390.2408. HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 99.5% and retention time of 9.7 min for the title compound.

Butyl 2-[(6aR,9S,10aR)-6a,7,8,9,10,10a-Hexahydro-1,9-dihydroxy-6,6-dimethyl-6H-benzo[c]chromen-3-yl]-2-methylpropanoate (3d)

The synthesis was carried out as described for 3a using 4d (34 mg, 0.10 mmol), sodium bicarbonate (15 mg, 0.16 mmol), and 1-bromobutane (35 mg, 0.25 mmol) in DMF (1.5 mL) to give 3d (17 mg, 42% yield) as light yellow gum. IR (neat) 3357, 2927, 2873, 1727, 1706, 1620, 1578, 1418, 1262, 1140 cm−1; 1H NMR (500 MHz, CDCl3) δ 6.41 (d, J = 2.0 Hz, 1H, ArH), 6.31 ( d, J = 2.0 Hz,1H, ArH), 4.29 (m as t, J = 3.5 Hz, 1H, 9eq-H, peak half-width = 9.5 Hz), 4.07 (m as t, J = 6.5 Hz, 2H, 4′-H), 3.24 (ddd, J = 14.5 Hz, J = 3.0 Hz, J = 2.5 Hz, 1H, 10eq-H), 2.97 (ddd, J = 12.0 Hz, J = 11.0 Hz, J = 2.5 Hz, 1H, 10a-H), 2.02–1.97 (m as br d, J = 13.5 Hz, 1H, 8eq-H), 1.71–1.47 (m and s overlapping, 12H, 8ax-H, 7ax-H, 7eq-H, 6a-H, 5′-H, -C(CH3)2-, especially 1.51, s, -C(CH3)2-), 1.39 (s, 3H, 6-CH3), 1.38–1.32 (m, 1H, 10ax-H), 1.28 (sextet, J = 7.0 Hz, 2H, 6′-H), 1.07 (s, 3H, 6-CH3), 0.87 (t, J = 7.5 Hz, 3H, 7′-H). Mass spectum (ESI) m/z (relative intensity) 391 (M+ + H, 100), 289 (45, M+ − CH3(CH2)3OC(O)). Mass spectum (EI) m/z (relative intensity) 390 (M+, 57), 329 (55), 289 (32, M+ − CH3(CH2)3OC(O)), 183 (97), 84 (100). Exact mass (EI) calculated for C23H3405 (M+), 390.2406; found 390.2408. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 97.7% and retention time of 5.5 min for the title compound.

Butyl 2-[(6aR, 9R, 10aR)-1,9-Dihydroxy-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl]acetate (3e)

The synthesis was carried out as described for 3c using 27 (58 g, 0.16 mmol) and NaBH4 (6.1 mg, 0.16 mmol) in MeOH (5 mL) and gave 3e (35 mg, 61% yield) as a light yellow gum. Purification by flash column chromatography on silica gel (20–30% acetone in hexane) gave the title compound free from its epimer 3f which is also produced in traces (4–6%) during the reaction. IR (neat) 3428, 2975, 2872, 1729, 1704, 1619, 1578, 1417 cm−1; 1H NMR (500 MHz, CDCl3) δ 6.41 (br s, 1H, ArOH), 6.32 ( d, J = 2.0 Hz, 1H, ArH), 6.22 (d, J = 2.0 Hz, 1H, ArH), 4.08 (t, J = 6.5 Hz, 2H, 4′-H), 3.80 (dddd, J = 16.0 Hz, J = 15.0 Hz, J = 4.5 Hz, J = 4.0 Hz, 1H, 9ax-H, peak half- width = 26 Hz), 3.68 (d, J = 16.0 Hz, 1H, half of an AX system, -CH2-C(O)O-), 3.55 (d, J = 16.0 Hz, 1H, half of an AX system, -CH2-C(O)O-), 3.51 (dddd, J = 12.0 Hz, J = 4.5 Hz, J = 2.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.39 (ddd, J = 12.0 Hz, J = 11.5 Hz, J = 2.5 Hz, 1H, 10a-H), 2.16 (m as br d, J = 10.0 Hz, 1H, 8eq-H), 1.87 (dddd, J = 13.5 Hz, J = 3.5 Hz, J = 2.5 Hz, J = 2.0 Hz, 1H, 7eq-H), 1.59 (quintet, J = 7.5 Hz, 2H, 5′-H), 1.51 (m as td, J = 12.5 Hz, J = 2.5 Hz, 1H, 6a-H), 1.42–1.28 (m, s and sextet overlapping, 6H, 8ax-H, 6′-H, 6-Me), 1.19–1.06 (m, 2H, 7ax-H, 10ax-H), 1.05 (s, 3H, 6-Me), 0.89 (t, J = 7.5 Hz, 3H, 7′-H). 13C NMR (100 MHz CDCl3) δ 171.8 (-C(O)O-), 155.4 (ArC), 154.8 (ArC), 134.2 (ArC), 116.3 (ArC), 110.8 (ArC), 103.6 (ArC), 77.2(C-6), 70.5(>CH-OH), 65.0(-OCH2-), 49.8, 42.0, 39.7, 35.6, 34.7, 30.6, 27.6, 26.21, 19.0, 18.4, 13.6. Mass spectum (ESI) m/z (relative intensity) 363 (M+ + H, 23), 345 (100, M+ − OH), 271 (53). Exact mass (ESI) calculated for C21H31O5 (M+ + H), 363.2171; found 363.2167. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 97.3% and retention time of 7.1 min for the title compound.

Butyl 2-[(6aR,9S,10aR)-1,9-Dihydroxy-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl]acetate (3f)

The synthesis was carried out as described for 4d using 27 (40 mg, 0.11 mmol) and K-selectride (0.44 mL, 1 M solution in THF) in THF (3 mL). Purification by flash column chromatography on silica gel (20–30% acetone in hexane) gave the title compound (21 mg, 52% yield) as light yellow gum free from its epimer 3e which is also produced in traces (3–5%) during the reaction. IR (neat) 3430, 2973, 1729, 1704, 1620, 1578, 1417 cm−1; 1H NMR (500 MHz, CDCl3) δ 6.30 (d, J = 2.0 Hz, 1H, ArH), 6.23 ( d, J = 2.0 Hz,1H, ArH), 5.22 (br s, 1H, ArOH), 4.23 (m as t, J = 2.5 Hz, 1H, 9eq-H, peak half-width = 9.0 Hz), 4.11 (m as td, J = 7.0 Hz, J = 2.5 Hz, 2H, 4′-H), 3.74 (d, J = 16.0 Hz, 1H, half of an AX system, -CH2-C(O)O-), 3.58 (d, J = 16.0 Hz, 1H, half of an AX system, -CH2-C(O)O-), 2.90 (ddd, J = 12.0 Hz, J = 11.0 Hz, J = 2.0 Hz, 1H, 10a-H), 2.51 (ddd, J = 14.5 Hz, J = 3.0 Hz, J = 2.2 Hz, 1H, 10eq-H), 1.98–1.90 (m as br d, J = 13.5 Hz, 1H, 8eq-H), 1.70–1.47 ( m, 6H, 8ax-H, 7ax-H, 7eq-H, 6a-H, 5′-H), 1.42–1.28 (s, m and sextet overlapping, 6H, 10ax-H, 6-CH3, 6′-H, especially 1.36, s, 6-CH3), 1.04 (s, 3H, 6-CH3), 0.89 (t, J = 7.5 Hz, 3H, 7′-H). Mass spectum (ESI) m/z (relative intensity) 363 (M+ + H, 100), 345 (153, M+ − OH). Exact mass (ESI) calculated for C21H3105 (M+ + H), 363.2171; found 363.2173. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 97.8% and retention time of 7.2 min for the title compound.

2-[(6aR,10aR)-1-Hydroxy-9-(hydroxymethyl)-6,6-dimethyl-6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-3-yl]-2-methyl-propanoic Acid (4a)

A stirred mixture of 11 (200 mg, 0.45 mmol) and NaOH (72 mg, 1.8 mmol) in THF/water (1:1 ratio, 8 mL) was refluxed for 16 h under argon. Volatiles were removed under reduced pressure, and the residue was acidified with aqueous 1 N HCl and diluted with ethyl acetate. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate. The combined organic phase was washed with water and brine, dried (MgSO4), and concentrated in vacuo. Purification by flash column chromatography on silica gel (50% acetone in hexane) afforded the title compound in 68% yield (106 mg) as a light yellow gum. IR (neat) 3317, 2976, 2932, 1702, 1620, 1577, 1417, 1265, 1185 cm−1; 1H NMR (500 MHz, CDCl3) δ 7.31–6.40 (br s, 1H, -OH), 6.41 (d, J = 1.5 Hz, 1H, 4-H), 6.30 (d, J = 1.5 Hz, 1H, 2-H), 5.68 (m as d, J = 3.5 Hz, 1H, 8-H), 4.03 (d, J = 12.5 Hz, 1H, half of an AB system, -CH2OH), 3.93 (d, J = 12.5 Hz, 1H, half of an AB system, -CH2OH), 3.47 (br d, J = 14.5Hz, 1H, 10α-H), 2.64 (td, J = 11.5 Hz, J = 4.5 Hz, 1H, 10a-H), 2.16 (m, 1H, 7α-H), 1.83–1.70 (m, 3H, 10β-H, 7β-H, 6a-H), 1.50 (s, 3H, 1′-CH3), 1.47(s, 3H, 1′-CH3) 1.36 (s, 3H, 6β-Me), 1.06 (s, 3H, 6α-Me); 13C NMR (125 MHz CDCl3) δ 180 (>C═O), 155.7 (C-1 or C-5), 154.5 (C-5 or C-1), 144.0, 137.9, 121.4, 111.5, 106.6, 105.4, 76.6 (C-6), 66.7 (-CH2OH), 45.8, 44.9, 31.3, 31.1, 29.6, 27.5, 27.4, 26.1, 18.3. Mass spectrum (ESI) m/z (relative intensity) 347 (M+ + H, 100), 329 (M+ − OH, 33). Mass spectrum (EI) m/z (relative intensity) 346 (M+, 19), 329 (M+ − OH, 8), 285 (10), 247 (15), 84 (100); exact mass (EI) calculated for C20H26O5 (M+), 346.1780; found 346.1780. HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 99.8% and retention time of 6.2 min for 4a.

2-[(6aR,9R,10aR)-1-Hydroxy-9-(hydroxymethyl)-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl]-2-methylpropanoic Acid (4b)

The synthesis was carried out as described for 22 using 3b (20 mg, 0.05 mmol) and lithium hydroxide (12 mg, 0.5 mmol) in dioxane/water (2.8 mL, 1:1 ratio). Workup and purification by flash column chromatography on silica gel (60% ethyl acetate in hexane) gave 4b (14.5 mg, 85% yield) as a yellow gum. IR (neat) 3321, 2975, 2932, 1702, 1621, 1577, 1417, 1264, 1185 cm−1; 1H NMR (500 MHz, CDCl3) δ 6.42 (d, J = 2.0 Hz, 1H, Ar-H), 6.28 (d, J = 2.0 Hz, 1H, Ar-H), 3.53 (dd, J = 10.5 Hz, J = 5.5 Hz, half of an AB system, 1H, -CH2OH), 3.45 (dd, J = 10.5 Hz, J = 8.0 Hz, half of an AB system, 1H, -CH2OH), 3.29–3.22 (m as br d, J = 13.0 Hz, 1H, Cring), 2.44–2.37 (m as td, J = 11.0 Hz, J = 2.5 Hz, 1H, C-ring), 1.90–1.82 (m as br d, J = 9.5 Hz, 2H, C-ring), 1.78–1.68 (m, 1H, C-ring), 1.49–1.40 (m and s, overlapping, 7H, -C(CH3)2-, C-ring, especially 1.48, s, 6H, -C(CH3)2-), 1.35 (s, 3H, 6-Me), 1.12–1.10 (m, 2H, C-ring), 0.98 (s, 3H, 6-Me), 0.74 (m as q, J = 11.0 Hz, 1H, C-ring). 13C NMR (100 MHz CDCl3) δ 175.6 (-C(O)O-), 154.9 (ArC-1 or ArC-5), 155.0 (ArC-1 and ArC-5), 143.7 (tertiary aromatic), 111.4 (tertiary aromatic), 107.4 (ArC-2 or ArC-4), 105.3 (ArC-4 or ArC-2), 68.4 (-CH2OH), 49.2, 45.9, 40.4, 34.8, 33.0, 29.5, 27.6, 27.4, 26.1, 19.0. Mass spectrum (ESI) m/z (relative intensity) 349 (M+ + H, 100). LC/MS analysis (Waters MicroMass ZQ system) showed purity of 97.5% and retention time of 4.2 min for the title compound.

2-[(6aR,9R,10aR)-6a,7,8,9,10,10a-Hexahydro-1,9-dihydroxy-6,6-dimethyl-6H-benzo[c]chromen-3-yl]-2-methylpropanoic Acid (4c)

The synthesis was carried out as described for 3c using 22 (55 mg, 0.16 mmol) and sodium borohydride (25 mg, 0.66 mmol) in methanol (6 mL) at room temperature and gave the title compound as a light yellow gum in 81% yield (43 mg). IR (neat) 3338, 2975, 1695, 1622, 1418 cm−1; 1H NMR (500 MHz, CD3OD) δ 6.35 ( d, J = 2.0 Hz, 1H, ArH), 6.27 (d, J = 2.0 Hz, 1H, ArH), 3.75 (dddd, J = 15.5 Hz, J = 15.0 Hz, J = 4.5 Hz, J = 4.0 Hz, 1H, 9ax-H, peak half-width = 25 Hz), 3.53 (dddd, J = 12.0 Hz, J = 4.5 Hz, J = 2.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.45 (ddd, J = 12.0 Hz, J = 11.5 Hz, J = 2.5 Hz, 1H, 10a-H), 2.12 (m as br d, J = 11.0 Hz, 1H, 8eq-H), 1.90 (dddd, J = 13.5 Hz, J = 3.5 Hz, J = 2.5 Hz, J = 2.0 Hz,, 1H, 7eq-H), 1.50–1.34 (s, s and m, overlapping, 11H, -C(CH3)2-, 6a-H, 10ax-H, 6-Me, especially 1.47, s, -C(CH3)2- and 1.34, s, 6-Me), 1.21 (dddd, J = 15.5 Hz, J = 15.0 Hz, J = 13.0 Hz, J = 3.5 Hz, 1H, 8ax-H), 1.05 (s, 3H, 6-Me), 0.97 (dddd, J = 13.5 Hz, J = 13.0 Hz, J = 12.0 Hz, J = 3.5 Hz, 1H, 7ax-H). Mass spectum (ESI) m/z (relative intensity) 335 (M+ + H, 100), 317 (54, M+ − OH), 289 (84, M+ − COOH). Mass spectum (EI) m/z (relative intensity) 334 (M+, 97), 316 (79), 273 (97), 233 (100), 149 (87). Exact mass (EI) calculated for C19H2605 (M+), 334.1780; found 334.1777. HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.3% and retention time of 5.4 min for the title compound. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 98.4% and retention time of 4.2 min for the title compound.

2-[(6aR,9S,10aR)-6a,7,8,9,10,10a-Hexahydro-1,9-dihydroxy-6,6-dimethyl-6H-benzo[c]chromen-3-yl]-2-methylpropanoic Acid (4d)

K-selectride (0.96 mL, 1 M solution in THF) was added under an argon atmosphere to a solution of 22 (80 mg, 0.24 mmol) in dry THF (12 mL) at room temperature. The reaction mixture was stirred at the same temperature for 2 h, and then it was quenched with aqueous 1 N HCl solution and diluted with ethyl acetate. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate. The combined organic phase was washed with water and brine, dried (MgSO4), and concentrated under reduced pressure. Purification by flash column chromatography on silica gel (38% acetone in hexane) gave compound 4d (73 mg, 91% yield) as light yellow gum. IR (neat) 3327, 2978, 1697, 1622, 1420 cm−1; 1H NMR (500 MHz, CD3OD) δ 6.34 (d, J = 2.0 Hz, 1H, ArH), 6.27 ( d, J = 2.0 Hz,1H, ArH), 4.13 (m as t, J = 3.5 Hz, 1H, 9eq-H, peak half-width = 9.5 Hz), 3.40 (ddd, J = 14.5 Hz, J = 3.0 Hz, J = 2.5 Hz, 1H, 10eq-H), 2.94 (ddd, J = 12.0 Hz, J = 11.0 Hz, J = 2.5 Hz, 1H, 10a-H), 1.96–1.91 (m, 1H, 8eq-H), 1.71–1.46 ( m and s overlapping, 10H, 8ax-H, 7ax-H, 7eq-H, 6a-H, -C(CH3)2-, especially 1.47, s, -C(CH3)2-), 1.36 (s, 3H, 6-CH3), 1.24–1.16 (m, 1H, 10ax-H), 1.09 (s, 3H, 6-CH3). Mass spectum (ESI) m/z (relative intensity) 335 (M+ + H, 100), 317 (27, M+ − OH), 289 (29, M+ − COOH). Mass spectum (EI) m/z (relative intensity) 334 (M+, 37), 316 (29), 273 (42), 233 (19), 149 (100). Exact mass (EI) calculated for C19H2605 (M+), 334.1780; found 334.1784. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 96.7% and retention time of 4.3 min for the title compound.

2-[(6aR,9R,10aR)-1,9-Dihydroxy-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl]acetic Acid (4e)

The synthesis was carried out as described for 3c using 26 (25 mg, 0.082 mmol) and NaBH4 (12.4 mg, 0.32 mmol) in MeOH (5 mL) at room temperature and gave 4e (17 mg, 65% yield) as light yellow gum. Purification by flash column chromatography on silica gel (40% acetone in hexane) gave the title compound free from its epimer 4f which is also produced in traces (4–6%) during the reaction. IR (neat) 3350, 2973, 1694, 1622, 1419 cm−1; 1H NMR (500 MHz, CDCl3) δ 6.32 ( d, J = 2.5 Hz, 1H, ArH), 6.21 (d, J = 2.5 Hz, 1H, ArH), 3.78 (dddd, J = 16.0 Hz, J = 15.0 Hz, J = 4.5 Hz, J = 4.0 Hz, 1H, 9ax-H, peak half-width = 26 Hz), 3.62 (d, J = 16.0 Hz, 1H, half of an AB system, -CH2-C(O)O-), 3.54 (d, J = 16.0 Hz, 1H, half of an AB system, -CH2-C(O)O-), 3.49 (dddd, J = 12.0 Hz, J = 4.5 Hz, J = 2.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.39 (ddd, J = 12.0 Hz, J = 11.5 Hz, J = 2.5 Hz, 1H, 10a-H), 2.14 (m as br d, J = 10.0 Hz, 1H, 8eq-H), 1.87 (dddd, J = 13.5 Hz, J = 3.5 Hz, J = 2.5 Hz, J = 2.0 Hz,, 1H, 7eq-H), 1.51 (m as td, J = 12.5 Hz, J = 2.5 Hz, 1H, 6a-H), 1.39–1.28 (m and s overlapping, 4H, 8ax-H, 6-Me), 1.18–1.06 (m, 2H, 7ax-H, 10ax-H), 1.00 (s, 3H, 6-Me). Mass spectum (ESI) m/z (relative intensity) 307 (M+ + H, 98), 289 (100, M+ − OH). Exact mass (ESI) calculated for C17H2305 (M+ + H), 307.1545; found 307.1549. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 96.7% and retention time of 3.6 min for the title compound.

2-[(6aR,9S,10aR)-1,9-Dihydroxy-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl]acetic Acid (4f)

The synthesis was carried out as described for 4d using 26 (20 mg, 0.065 mmol) and K-selectride (0.65 mL, 1 M solution in THF) in THF (5 mL). Purification by flash column chromatography on silica gel (40% acetone in hexane) gave the title compound (18 mg, 90% yield) as light yellow gum free from its epimer 4e which is also produced in traces (3–5%) during the reaction. IR (neat) 3337, 2972, 1695, 1622, 1419 cm−1; 1H NMR (500 MHz, CDCl3 + CD3OD) δ 6.31 (d, J = 2.0 Hz, 1H, ArH), 6.22 ( d, J = 2.0 Hz,1H, ArH), 4.21 (m as br S, 1H, 9eq-H, peak half-width = 9.0 Hz), 3.70 (d, J = 16.0 Hz, 1H, half of an AB system, -CH2-C(O)O-), 3.57 (d, J = 16.0 Hz, 1H, half of an AB system, -CH2-C(O)O-), 2.90 (ddd as td, J = 12.0 Hz, J = 2.0 Hz, 1H, 10a-H), 2.49 (br d, J = 14.0 Hz, 1H, 10eq-H), 1.98–1.90 (m as br d, J = 13.5 Hz, 1H, 8eq-H), 1.70–1.23 (m and s overlapping, 8H, 8ax-H, 7ax-H, 7eq-H, 6a-H, 10ax-H and 6-CH3, especially 1.36, s, 6-CH3), 1.04 (s, 3H, 6-CH3). Mass spectum (ESI) m/z (relative intensity) 307 (M+ + H, 65), 289 (100, M+ − OH). Exact mass (ESI) calculated for C17H2305 (M+ + H), 307.1545; found 307.1540. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 96.5% and retention time of 3.7 min for the title compound.

Methyl 2-(3,5-Dimethoxyphenyl)-2-methylpropanoate (9)

A suspension of carboxylic acid 8 (11.4 g, 50.9 mmol), MeI (36.2 g, 225 mmol), and K2CO3 (10.6 g, 76 mmol) in anhydrous acetone (200 mL) was stirred overnight at room temperature under argon. Insoluble materials were filtered off, and the filtrate was evaporated under reduced pressure. The residue was diluted with diethyl ether and water, and the ethereal layer was separated. The aqueous layer was extracted with diethyl ether, and the combined organic phase was washed with water and brine, dried (MgSO4), and concentrated in vacuo. Purification by flash column chromatography on silica gel (5–20% AcOEt in hexane) gave the title compound as colorless oil in 72% yield (8.75 g). IR (neat) 3412, 2962, 2932, 1728 (s, >C═O), 1702 cm−1; 1H NMR (500MHz, CDCl3) δ 6.47 (d, J = 2.0 Hz, 2H, ArH), 6.33 (t, J = 2.0 Hz, 1H, ArH), 3.79 (s, 6H, OMe), 3.66 (s, 3H, COOMe), 1.33 (s, 6H, -C(CH3)2-). 13C NMR (100 MHz CDCl3) δ 181.8 (>C═O), 160.7, 146.2, 104.5, 98.4, 55.3 (-OMe), 46.3, 26.2. Mass spectrum (ESI) m/z (relative intensity) 239 (M+ + H, 100).

Methyl 2-(3,5-Dihydroxyphenyl)-2-methylpropanoate (10)

To a stirred solution of methyl ester 9 (5.0 g, 21.0 mmol) in dry CH2Cl2 (210 mL) at −78 °C, under an argon atmosphere was added boron tribromide (65.1 mL of 1 M solution in CH2Cl2). Following the addition, the reaction temperature was gradually raised over a period of 1 h to 0 °C, and the stirring was continued at that temperature until the reaction was completed (2.5 h). Unreacted boron tribromide was destroyed by the addition of MeOH at −78 °C. The resulting mixture was warmed to room temperature, and volatiles were removed in vacuo. The residue was dissolved in diethyl ether and washed with water and brine and dried (MgSO4). Solvent evaporation and purification by flash column chromatography on silica gel (10–40% AcOEt in hexane) gave 10 as a pale yellow semisolid material in 71% yield (3.1 g). IR (neat) 3180, 1725 (s, >C═O), 1701cm−1; 1H NMR (500MHz, CDCl3) δ 6.41 (d, J = 2.0 Hz, 2H, ArH), 6.24 (t, J = 2.0 Hz, 1H, ArH), 5.67 (br s, 2H, OH), 3.66 (s, 3H, COOMe), 1.52 (s, 6H, -C(CH3)2-). 13C NMR (100 MHz CDCl3) δ 177.6 (>C═O), 156.9, 147.5, 105.5, 101.4, 52.5 (-OMe), 46.4, 26.2. Mass spectrum (ESI) m/z (relative intensity) 211 (M+ + H, 100).

[(6aR, 10aR)-1-Hydroxy-3-(1-methoxy-2-methyl-1-oxopropan-2-yl)-6,6-dimethyl-6a,7,10,10a-tetrahydro-6H-benzo[c]-chromen-9-yl]methyl Pivalate (11)

To a stirred solution of resorcinol 10 (1.0 g, 4.76 mmol) and pivalate ester 6 (1.32 g, 5.24 mmol) in anhydrous CH2Cl2 (24 mL) at −20 °C, under an argon atmosphere, was added boron trifluoride diethyl etherate (3.53 mL, 28.6 mmol) dropwise. Stirring was continued at −20 °C for 1 h and then at room temperature for 1 h. The reaction mixture was quenched with saturated aqueous NaHCO3 solution and diluted with CH2Cl2. The organic layer was separated, and the aqueous layer was extracted with CH2Cl2. The combined organic phase was washed with water and brine, dried (MgSO4), and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (15% ethyl acetate in hexane) to give 11 (846 mg, 40% yield) as a light yellow gum. Rf = 0.3 (20% acetone in hexane). IR (neat) 3418, 2954, 1725 (s, >C═O), 1705cm−1; 1H NMR (500MHz, CDCl3) δ 6.43 (d, J = 2.0 Hz, 1H, 4-H), 6.31 (d, J = 2.0 Hz, 1H, 2-H), 5.77 (m as d, J = 4.0 Hz, 1H, 8-H), 4.53 (d, J = 13.0 Hz, 1H, half of an AB system, -CH2O-), 4.50 (d, J = 13.0 Hz, 1H, half of an AB system, -CH2O-), 3.67 (s, 3H, -OMe), 3.42 (dd, J = 16.0 Hz, J = 4.0 Hz, 1H, 10α-H), 2.73 (td, J = 11.0 Hz, J = 5.0 Hz, 1H, 10a-H), 2.26 (m as d, J = 16.5 Hz, 1H, 7α-H), 1.96–1.82 (m, 3H, 10β-H, 7β-H, 6a-H), 1.52 (brs, 6H, -C(CH3)2-), 1.41 (s, 3H, 6β-Me), 1.23 (brs, 9H, -C(CH3)3) 1.13 (s, 3H, 6α-Me). Mass spectrum (ESI) m/z (relative intensity) 445 (M+ + H, 90), 385 (M+ − COOMe, 45), 342 (M+ − (CH3)3COOH, 100), 283 (M + − (CH3)3COOH, –COOCH3, 51).

2-(3,5-Dihydroxyphenyl)-2-methylpropanoic Acid (12)

To a stirred solution of 8 (2.54 g, 11.33 mmol) in dry CH2Cl2 (75 mL) at −78 °C, under an argon atmosphere, was added boron tribromide (39.6 mL, 39.6 mmol, 1 M solution in CH2Cl2). Following the addition, the reaction mixture was gradually warmed to room temperature and stirred for 3 h. Unreacted boron tribromide was quenched by the addition of methanol and ice at 0 °C. The resulting mixture was warmed to room temperature, and volatiles were removed in vacuo. The residue was diluted with diethyl ether and washed with saturated aqueous NaHCO3 solution, water, and brine. The organic layer was dried (MgSO4), filtered, and concentrated under reduced pressure. Purification by flash column chromatography on silica gel (40% diethyl ether in hexane) gave 12 (2.17 g, 98% yield) as white solid with physical, spectral, and analytical data identical to those reported earlier.15

Butyl 2-(3,5-Dihydroxyphenyl)-2-methylpropanoate (13)

A stirred mixture of 12 (2.0 g, 10.20 mmol), bromobutane (2.1 g, 15.30 mmol), and sodium bicarbonate (1.05 g, 12.24 mmol) in anhydrous DMF (3 mL) was heated at 165 °C for 12 min using microwave irradiation. The reaction mixture was cooled to room temperature and diluted with water and ethyl acetate. The organic layer was washed with brine, dried (MgSO4), and concentrated in vacuo. The residue was chromatographed on silica gel (50% ethyl acetate in hexane) to give 13 (2.5 g, 92% yield) as a light brown viscous oil. IR (neat) 3370, 2963, 1698, 1600, 1465, 1271, 1142 cm−1; 1H NMR (500 MHz, CDCl3) δ 6.40 (d, J = 2.0 Hz, 2H, ArH), 6.23 (t, J = 2.0 Hz, 1H, ArH), 5.10 (s, 2H, OH), 4.06 (t, J = 7.0 Hz, 2H, 4-H), 1.55 (quintet, J = 7.5 Hz, 2H, 5′-H), 1.51 (s, 6 H, -C(CH3)2-), 1.28 (sextet, J = 7.0 Hz, 2H, 6′-H), 0.87 (t, J = 7.5 Hz, 3H, 7′-H). 13C NMR (100 MHz CDCl3) δ 177.7 (-C(O)O-), 156.9 (ArC-3 and ArC-5), 147.4 (ArC-1), 105.5 (ArC-4 and ArC-6), 101.5 (ArC-2), 65.3 (-OCH2-), 46.5, 30.4, 26.1, 18.9, 13.5.

Butyl 2-(4-((1R,2R,5R)-6,6-Dimethyl-4-oxobicyclo[3.1.1]-heptan-2-yl)-3,5-dihydroxyphenyl)-2-methylpropanoate (15)

To a degassed solution of 13 (2.2 g, 8.73 mmol) and diacetates 1419,25 (8.55 g, ~85% pure by 1H NMR, 30.55 mmol) in CHCl3 (88 mL) at 0 °C, under an argon atmosphere, was added p-toluenesulfonic acid monohydrate (2.32 g, 12.22 mmol). The mixture was warmed to room temperature and stirred for 4 days to ensure complete formation of the product. The reaction mixture was diluted with diethyl ether and washed sequentially with water, saturated aqueous NaHCO3, and brine. The organic phase was dried over MgSO4, and the solvent was removed under reduced pressure. The residue was chromatographed on silica gel (43% diethyl ether in hexane) to give 15 as a white crystalline solid (914 mg, 27% yield); Rf = 0.4 (40% diethyl ether in hexane); mp 70–71 °C. IR (neat) 3362, 2959, 2873, 1727, 1683, 1619, 1589, 1421, 1265, 1143 cm−1; 1H NMR (500 MHz, CDCl3) δ 6.31 (s, 2H, ArH), 5.50 (s, 2H, OH), 4.06 (t, J = 7.0 Hz, 2H, 4′-H), 3.95 (t, J = 7.5 Hz, 1H, 4-H), 3.50 (dd, J = 19.0 Hz, J = 8.0 Hz, 1H, 3α-H), 2.59 (dd, J = 19.0 Hz, J = 8.5 Hz, 1H, 3β-H), 2.58 (t, J = 5.0 Hz, 1H, 1-H), 2.50 (m, 1H, 7α-H), 2.46 (d, J = 10.5 Hz, 1H, 7β-H), 2.27 (t, J = 5.5 Hz, 1H, 5-H), 1.56 (quintet, J = 7.5 Hz, 2H, 5′-H), 1.50 (s, 6H, -C(CH3)2-), 1.36 (s, 3H, 6-Me), 1.28 (sextet, J = 7.0 Hz, 2H, 6′-H), 0.99 (s, 3H, 6-Me), 0.87 (t, J = 7.5 Hz, 3H, 7′H). 13C NMR (100 MHz CDCl3) δ 213.3 (>C═O), 177.1 (-C(O)O-), 155.4 (ArC-3 and ArC-5), 143.9 (tertiary aromatic), 115.0 (tertiary aromatic), 106.1 (ArC-2 and ArC-6), 65.1 (-OCH2-), 58.0, 46.8, 46.0, 42.1, 37.6, 30.4, 29.4, 26.1, 26.0, 24.4, 22.1, 19.0, 13.6. Mass spectrum (ESI) m/z (relative intensity) 389 (M+ + H, 100). Mass spectrum (EI) m/z (relative intensity) 388 (M+, 25), 305 (32), 287 (20), 243 (19), 177 (21), 84 (100). Exact mass (EI) calculated for C23H32O5 (M+), 388.2250; found 388.2252. HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 99.7% and retention time of 8.9 min for 15.

Butyl 2-(3-Acetoxy-5-hydroxyphenyl)-2-methylpropanoate (13a)

The title compound was also isolated during the chromatographic purification of 15 as a colorless oil in 25% yield (641 mg). Rf = 0.5 (40% diethyl ether in hexane); IR (neat) 3417, 2962, 1770, 1728, 1702, 1609, 1592, 1261, 1142 cm−1; 1H NMR (500 MHz, CDCl3) δ 6.69 (t, J = 2.0 Hz, 1H, Ar-H), 6.62 (t, J = 2.0 Hz, 1H, Ar-H), 6.51 (t, J = 2.0 Hz, 1H, Ar-H), 6.78 (br s, 1H, OH), 4.06 (t, J = 7.0 Hz, 2H, 4′-H), 2.29 (s, 3H, OC(O)CH3), 1.55 (quintet, J = 7.5 Hz, 2H, 5′-H), 1.53 (s, 6H, -C(CH3)2-), 1.26 (sextet, J = 7.0 Hz, 2H, 6′-H), 0.87 (t, J = 7.5 Hz, 3H, 7′-H). 13C NMR (125 MHz CDCl3) δ 176.5 (-C(O)O-), 169.4 (-C(O)O-), 156.4, 151.4, 147.4, 111.2, 110.8, 107.6, 65.0 (-OCH2-), 46.5, 30.5, 26.2, 21.2, 19.0, 13.6. Mass spectrum (ESI) m/z (relative intensity) 317 (M++Na, 20), 295 (M+ + H, 55), 193 (100). LC/MS analysis (Waters MicroMass ZQ system) showed purity of 97.8% and retention time of 4.6 min for the title compound.

(+)-Apoverbenone (14a)

The title compound was also isolated during the chromatographic purification of 15 as a colorless oil (1.7 g). Spectroscopic data were reported earlier.27

Butyl 2-[(6aR,10aS)-1-Hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl]-2-methylpropanoate (16)

To a stirred solution of 15 (850 mg, 2.20 mmol) in anhydrous CH2Cl2/CH3NO2 (3:1 mixture, 44 mL) at 0 °C, under an argon atmosphere, was added trimethylsilyl trifluoromethanesulfonate (2.3 mL, 0.3 M solution in CH3NO2, 0.66 mmol). Stirring was continued for 3 h while the temperature was allowed to rise to 25 °C. The reaction mixture was quenched using 1:1 solution of saturated NaHCO3 and brine and extracted with diethyl ether. The organic layer was washed with brine, dried (MgSO4), and concentrated under reduced pressure. Purification by flash column chromatography on silica gel (30% acetone in hexane) gave 16 as a white foam (570 mg, 67% yield). Mp 35–36 °C. IR (neat) 3356, 2958, 1727, 1697, 1620, 1579, 1419, 1259, 1142 cm−1; 1H NMR (500 MHz, CDCl3) δ 6.41 (d, J = 2.0 Hz, 1H, Ar-H), 6.30 (br d, J = 2.0 Hz, 1H, Ar-H), 6.23 (br s, 1H, OH), 4.06 (m as td, J = 7.0 Hz, J = 2.0 Hz, 2H, 4′-H), 3.96 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.87 (m as td, J = 12.5 Hz, J = 4.0 Hz, 1H, 10a-H), 2.64–2.57 (m, 1H, 8eq-H), 2.50–2.40 (m, 1H, 8ax-H), 2.20–2.08 (m, 2H, 10ax-H, 7eq-H), 1.95 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.59–1.51 (m, 3H, 7ax-H, 5′-H), 1.50 (s, 6H, -C(CH3)2-), 1.47 (s, 3H, 6-Me), 1.26 (sextet, J = 7.5 Hz, 2H, 6′-H), 1.11 (s, 3H, 6-Me), 0.86 (t, J = 7.5 Hz, 3H, 7′-H). 13C NMR (125 MHz CDCl3) δ 213.3 (>C═O), 177.1 (-C(O)O-), 155.4 (ArC-1 or ArC-5), 154.8 (ArC-5 or ArC-1), 145.4 (tertiary aromatic), 109.4 (tertiary aromatic), 107.2 (ArC-2 or ArC-4), 105.6 (ArC-4 or ArC-2), 77.1 (C-6), 64.9 (-OCH2-), 47.6, 46.4, 45.2, 41.0, 34.9, 30.7, 28.1, 27.1, 26.5, 26.3, 19.2, 19.1, 13.9. Mass spectrum (EI) m/z (relative intensity) 388 (M+, 79), 305 (43), 287 (100), 245 (98), 177 (94). Exact mass (EI) calculated for C23H32O5 (M+), 388.2250; found 388.2246. HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.3% and retention time of 10.8 min for 16. Anal. (C23H32O5) C, H.

Butyl 2-{(6aR,10aR)-1-[(tert-Butyldimethylsilyl)oxy]-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]-chromen-3-yl}-2-methylpropanoate (17)

To a solution of 16 (550 mg, 1.41 mmol) in anhydrous DMF (9.5 mL) under an argon atmosphere were added sequentially imidazole (675 mg, 9.92 mmol), DMAP (172 mg, 1.41 mmol), and TBDMSCl (1.46 g, 9.72 mmol). The reaction mixture was stirred at room temperature for 12 h and then quenched by the addition of brine and extracted with diethyl ether. The organic phase was dried (MgSO4) and evaporated under reduced pressure. Purification by flash column chromatography on silica gel (20% ethyl acetate in hexane) afforded 620 mg (87% yield) of 17 as a colorless oil. IR (neat) 2959, 1715, 1612, 1567, 1415, 1253, 1096 cm−1; 1H NMR (500 MHz, CDCl3) δ 6.46 (d, J = 2.0 Hz, 1H, Ar-H), 6.35 (br d, J = 2.0 Hz, 1H, Ar-H), 6.24 (br s, 1H, OH), 4.04 (m as td, J = 7.0 Hz, J = 2.0 Hz, 2H, 4′-H), 3.76 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.71 (m as td, J = 12.5 Hz, J = 4.0 Hz, 1H, 10a-H), 2.59–2.40 (m, 1H, 8eq-H), 2.45–2.36 (m, 1H, 8ax-H), 2.17–2.11 (m, 2H, 10ax-H, 7eq-H), 1.93 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.56–1.51 (m, 3H, 7ax-H, 5′-H), 1.49 (s, 6H, -C(CH3)2-), 1.46 (s, 3H, 6-Me), 1.25 (sextet, J = 7.5 Hz, 2H, 6′-H), 1.09 (s, 3H, 6-Me), 0.99 (s, 9H, Si(Me)2CMe3), 0.85 (t, J = 7.5 Hz, 3H, 7′-H), 0.23 (s, 3H, Si(Me)2CMe3), 0.15 (s, 3H, Si(Me)2CMe3). 13C NMR (100 MHz CDCl3) δ 210.1 (>C═O), 176.4 (-C(O)O-), 154.5 (ArC-1 or ArC-5), 154.4 (ArC-5 or ArC-1), 144.8 (tertiary aromatic), 113.2 (tertiary aromatic), 109.6 (ArC-2 or ArC-4), 108.1 (ArC-4 or ArC-2), 64.6 (-OCH2-), 47.7, 46.1, 45.4, 40.7, 35.0, 30.5, 27.7, 26.8, 26.2, 26.1, 25.9, 25.8, 19.0, 18.6, 18.3, 13.6, −3.7, −4.1. Mass spectrum (ESI) m/z (relative intensity) 503 (M+ + H, 100). Exact mass (ESI) calculated for C29H47O5Si (M+ + H), 503.3193; found 503.3195. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 99.2% and retention time of 6.2 min for the title compound.

Butyl 2-{(6aR,10aR)-1-[(tert-Butyldimethylsilyl)oxy]-9-(methoxymethylene)-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo [c] chromen-3-yl}-2-methylpropanoate (18)

(Methoxymethyltriphenylphosphonium chloride (2.18 g, 6.36 mmol) was suspended in 32 mL of dry benzene. Sodium tert-amylate (688 mg, 6.25 mmol) was added, and the reaction mixture was stirred for 5 min at 0 °C. Intermediate 17 (532 mg, 1.06 mmol) was dissolved in minimum amount of dry benzene and transfered to the solution of the orange ylide. The reaction mixture was stirred at room temperature for 3 h and then quenched with saturated aqueous NH4Cl solution and diluted using ethyl acetate. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate. The combined organic layer was washed with brine, dried (MgSO4), and concentrated in vacuo. The residue was chromatographed on silica gel (20% acetone in hexane) to give two geometrical isomers 18a and 18b (353 mg, 63% yield) as a colorless oil in the ratio of 2:3, respectively, as determined by 1H NMR analysis. IR (neat) 2957, 1728 (>C═O), 1611, 1565, 1414, 1251, 1138, 1097 cm−1; 1H NMR (500 MHz, CDCl3) δ 6.43 (d, J = 2.0 Hz, 1H, Ar-H, 18b), 6.41 (d, J = 2.0 Hz, 1H, Ar-H, 18a), 6.34 (d, J = 2.0 Hz, 1H, Ar-H, 18a), 6.32 (d, J = 2.0 Hz, 1H, Ar-H, 18b), 5.85 (s, 1H, ═CHOMe, 18b), 5.82 (s, 1H, ═CHOMe, 18a), 4.19–4.14 (m as dd, J = 13.5 Hz, J = 3.5 Hz, 1H, C-ring, 18a), 4.10–3.96 (m, 2H, 4′-H, 18a and 2H, 4′-H, 18b), 3.56 (s, 3H, OMe, 18b), 3.53 (s, 3H, OMe, 18a), 3.45–3.39 (m as dd, J = 13.5 Hz, J = 3.5 Hz, 1H, C-ring, 18b), 2.96–2.88 (m as br d, J = 14.0 Hz, 1H, C-ring, 18b), 2.34–2.27 (m, 1H, C-ring of 18a and 1H, C-ring of 18b), 2.22–2.16 (m, 1H, C-ring, 18a), 2.09–2.00 (m, 1H, C-ring, 18a), 1.91–1.84 (m, 1H, C-ring of 18a and 1H, C-ring of 18b), 1.82–1.74 (m, 1H, C-ring, 18b), 1.67–1.51 (m, 2H, C-ring of 18a, 2H, C-ring of 18b, 2H, 5′-H of 18a and 2H, 5′-H of 18b), 1.49 (s, 6H, -C(CH3)2-, 18a), 1.48 (s, 6H, -C(CH3)2-, 18b), 1.38 (s, 3H, 6-Me, 18b), 1.37 (s, 3H, 6-Me, 18a), 1.30–1.22 (sextet and sextet overlapping, J = 7.5 Hz, 2H, 6′-H, 18a and 2H, 6′-H, 18b), 1.12–0.96 (m, s, s, s and s, overlapping, 1H, C-ring of 18a, 1H, C-ring of 18b, 3H, 6-Me of 18a, 3H, 6-Me of 18b, 9H, -Si(Me)2CMe3, of 18a, 9H, -Si(Me)2CMe3, of 18b especially 1.02, s, -Si(Me)2CMe3, of 18b and 0.99, s, -Si(Me)2CMe3, of 18a), 0.85 (t, J = 7.5 Hz, 3H, 7′-H of 18a and 3H, 7′-H of 18b), 0.24 (s, 3H, -Si(Me)2CMe3, 18b), 0.23 (s, 3H, -Si(Me)2CMe3, 18a), 0.18 (s, 3H, -Si(Me)2CMe3, 18a), 0.15 (s, 3H, -Si(Me)2CMe3, 18b). 13C NMR (100 MHz CDCl3) δ 176.7 (-C(O)O-), 176.6 (-C(O)O-), 155.2 (ArC), 154.8 (ArC), 154.7 (ArC), 154.6 (ArC), 144.0 (ArC), 143.9 (ArC), 140.7 (ArC), 140.2 (ArC), 116.5 (ArC), 115.7 (ArC), 114.9 (ArC), 114.7 (ArC), 109.7 (ArC), 105.5 (ArC), 108.0 (ArC), 107.8 (ArC), 64.5 (-OCH2-), 59.3 (-OMe), 59.0 (-OMe), 49.6, 49.5, 46.0, 37.1, 35.9, 34.1, 30.5, 30.3, 29.0, 27.8, 27.7, 27.6, 26.2, 26.1, 26.0, 25.0, 19.0, 18.8, 18.5, 18.3, 13.6, −3.6, −3.8, −3.9, −4.0. Mass spectrum (ESI) m/z (relative intensity) 531 (M+ + H, 100). Exact mass (ESI) calculated for C31H51O5Si (M+ + H), 531.3506; found 531.3499. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 98.9% and retention time of 8.4 min for the title compound.

Butyl 2-{(6aR,10aR)-1-[(tert-Butyldimethylsilyl)oxy]-9-formyl-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]-chromen-3-yl}-2-methylpropanoate (19)

To the stirred solution of 18 (255 mg, 0.48 mmol) in CH2Cl2 (16 mL) under an argon atmosphere was added wet trichloroacetic acid (391 mg, 2.40 mmol). The reaction mixture was stirred at room temperature for 2.5 h and then quenched with saturated aqueous NaHCO3 solution and diluted with diethyl ether. The organic layer was separated, and the aqueous layer was extracted with diethyl ether. The combined organic phase was washed with water and brine, dried (MgSO4), and evaporated. The residue consisted of a mixture of epimeric aldehydes 19a and 19b (241 mg, 97% yield) in the ratio of 5:2, respectively, as determined by 1H NMR analysis, and it was used into the next step as such. IR (neat) 2932, 2711 (CHO), 1726 (>C═O), 1611, 1566, 1414, 1253, 1140, 1064 cm−1; 1H NMR (500 MHz, CDCl3) δ 9.89 (s, 1H, 9α-CHO, 19b), 9.62 (s, 1H, 9β-CHO, 19a), 6.44 (d, J = 2.0 Hz, 1H, Ar-H, 19a), 6.41 (d, J = 2.0 Hz, 1H, Ar-H, 19b), 6.34 (d, J = 2.0 Hz, 1H, Ar-H, 19b), 6.33 (d, J = 2.0 Hz, 1H, Ar-H, 19a), 4.10–3.97 (m, 2H, 4′-H, 19a and 2H, 4′-H, 19b), 3.68–3.62 (m as br d, J = 14.0 Hz, 1H, Cring, 19b), 3.50–3.44 (m as br d, J = 13.5 Hz, 1H, C-ring, 19a), 2.66–2.61 (m, 1H, C-ring, 19b), 2.46–2.33 (m, 2H, C-ring, 19a and 2H, Cring, 19b), 2.31–2.24 (m, 1H, C-ring, 19b), 2.14–2.08 (m, 1H, Cring, 19a), 2.02–1.96 (m, 1H, C-ring, 19a), 1.78–1.71 (m, 1H, C-ring, 19b), 1.58–1.41 (m, s, and s, overlapping, 2H, 5′-H of 19a, 2H, 5′-H of 19b, 3H, C-ring of 19a, 2H, C-ring of 19b, 6H, -C(CH3)2- of 19a, 6H, -C(CH3)2- of 19b), 1.38 (s, 3H, 6-Me, 19a), 1.35 (s, 3H, 6-Me, 19b), 1.29–1.21 (sextet and sextet overlapping, J = 7.5 Hz, 2H, 6′-H of 19a and 2H, 6′-H of 19b), 1.20–1.11 (m, 1H, C-ring of 19a, 1H, Cring of 19b), 1.06 (s, 3H, 6-Me, 19a), 1.00 (s and s overlapping, 3H, 6-Me of 19b and 9H, -Si(Me)2CMe3 of 19a), 0.97 (s, 9H, -Si(Me)2CMe3, 19b), 0.86 (t, J = 7.5 Hz, 3H, 7′-H, of 19a and 3H, 7′-H, of 19b), 0.27 (s, 3H, -Si(Me)2CMe3, 19b), 0.25 (s, 3H, Si(Me)2CMe3, 19a), 0.24 (s, 3H, Si(Me)2CMe3, 19b), 0.14 (s, 3H, Si(Me)2CMe3, 19a). Mass spectrum (ESI) m/z (relative intensity) 517 (M+ + H, 100) for both peaks at 6.2 and 6.5 min. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 97.8% and retention time of 6.2 and 6.5 min for the two epimeric aldehydes in the ratio of 2:5, respectively.

Butyl 2-{(6aR,9R,10aR)-1-[(tert-Butyldimethylsilyl)oxy]-9-formyl-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]-chromen-3-yl}-2-methylpropanoate (20)

To a solution of 19 (200 mg, 0.39 mmol) in ethanol (9.5 mL) under an argon atmosphere was added potassium carbonate powder (269 mg, 1.95 mmol), and the mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with diethyl ether, and solid materials were filtered off. The filtrate was washed with brine, dried (MgSO4), and concentrated under reduced presure. Purification by flash column chromatography on silica gel (15% diethyl ether in hexane) gave 20 (156 mg, 78% yield) as a colorless oil. IR (neat) 2932, 2711 (CHO), 1726 (>C═O), 1611, 1566, 1414, 1253, 1140, 1064 cm−1; 1H NMR (500 MHz, CDCl3) δ 9.62 (s, 1H, 9β-CHO), 6.44 (d, J = 2.0 Hz, 1H, Ar-H), 6.33 (d, J = 2.0 Hz, 1H, Ar-H), 4.10–3.98 (m, 2H, 4′-H), 3.50–3.44 (m as br d, J = 13.5 Hz, 1H, C-ring), 2.46–2.36 (m, 2H, C-ring), 2.14–2.08 (m, 1H, C-ring), 2.02–1.96 (m, 1H, C-ring), 1.58–1.42 (m, s, and s, overlapping, 2H, 5′-H, 3H, C-ring, 6H, -C(CH3)2-, especially 1.50, s, -C(CH3)2- and 1.49, s, -C(CH3)2-), 1.38 (s, 3H, 6-Me), 1.26 (sextet, J = 7.5 Hz, 2H, 6′-H), 1.20–1.12 (m, 1H, C-ring), 1.06 (s, 3H, 6-Me), 1.00 (s, 9H, -Si(Me)2CMe3), 0.86 (t, J = 7.5 Hz, 3H, 7′-H), 0.25 (s, 3H, Si(Me)2CMe3), 0.14 (s, 3H, Si(Me)2CMe3). 13C NMR (100 MHz CDCl3) δ 203.4 (>C═O), 176.5 (-C(O)O-), 154.7 (ArC-1 or ArC-5), 154.6 (ArC-5 or ArC-1), 144.5 (tertiary aromatic), 113.8 (tertiary aromatic), 109.4 (ArC-2 or ArC-4), 108.1 (ArC-4 or ArC-2), 64.5 (-OCH2-), 50.5, 49.0, 46.0, 35.4, 30.5, 30.1, 27.5, 26.9, 26.2, 26.1, 25.9, 25.8, 19.0, 18.8, 18.1, 13.6, −3.6, −4.2. Mass spectrum (ESI) m/z (relative intensity) 517 (M+ + H, 100). Exact mass (ESI) calculated for C30H49O5Si (M+ + H), 517.3349; found 517.3351. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 96.7% and retention time of 6.5 min for the title compound.

Butyl 2-{(6aR,9R,10aR)-1-[(tert-Butyldimethylsilyl)oxy)-9-(hydroxymethyl)-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl}-2-methylpropanoate (21)

Sodium borohydride (57 mg, 1.55 mmol) was added to a stirred solution of aldehyde 20 (100 mg, 0.19 mmol) in ethanol (4.8 mL) at 0 °C under argon. After 30 min, the reaction was quenched with saturated ammonium chloride solution and volatiles were removed in vacuo. The residue was dissolved in ethyl acetate, and water was added. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate. The combined organic phase was washed with water and brine, dried (MgSO4), and evaporated. Purification by flash column chromatography on silica gel (30% diethyl ether in hexane) gave 21 (84 mg, 85% yield) as a colorless viscous oil. IR (neat) 3430, 2932, 1729 (>C═O), 1611, 1566, 1414, 1253, 1140, 1064 cm−1; 1H NMR (500 MHz, CDCl3) δ 6.43 (d, J = 2.0 Hz, 1H, Ar-H), 6.31 (d, J = 2.0 Hz, 1H, Ar-H), 4.09–3.97 (m, 2H, 4′-H), 3.54 (dd, J = 10.0 Hz, J = 5.5 Hz, half of an AB system, 1H, -CH2OH), 3.45 (dd, J = 10.0 Hz, J = 7.0 Hz, half of an AB system, 1H, -CH2OH), 3.18–3.12 (m as br d, J = 13.0 Hz, 1H, C-ring), 2.40–2.32 (m as td, J = 11.0 Hz, J = 2.5 Hz, 1H, C-ring), 2.04–1.98 (m, 1H, C-ring), 2.94–1.88 (m, 1H, C-ring), 1.76–1.68 (m, 1H, C-ring), 1.58–1.43 (m, s, and s, overlapping, 9H, -C(CH3)2-, 5′-H, C-ring, especially 1.49, s, 3H, -C(CH3)2-, and 1.48, s, 3H, -C(CH3)2-), 1.37 (s, 3H, 6-Me), 1.29–1.22 (m as sextet, J = 7.0 Hz, 2H, 6′-H), 1.18–1.10 (m, 2H, C-ring), 1.05 (s, 3H, 6-Me), 0.99 (s, 9H, -Si(Me)2CMe3), 0.86 (t, J = 7.5 Hz, 3H, 7′-H), 0.76 (m as q, J = 11.0 Hz, 1H, C-ring), 0.23 (s, 3H, Si(Me)2CMe3), 0.12 (s, 3H, Si(Me)2CMe3). 13C NMR (100 MHz CDCl3) δ 176.6 (-C(O)O-), 154.7 (ArC-1 or ArC-5), 154.6 (ArC-5 or ArC-1), 144.0 (tertiary aromatic), 114.8 (tertiary aromatic), 109.5 (ArC-2 or ArC-4), 108.0 (ArC-4 or ArC-2), 68.4 (-CH2OH), 64.5 (-OCH2-), 49.6, 46.0, 40.5, 35.5, 33.1, 30.5, 29.7, 27.6, 27.5, 26.1, 25.9, 19.0, 18.8, 18.2, 13.6, −3.6, −4.3. Mass spectrum (ESI) m/z (relative intensity) 519 (M+ + H, 100). Exact mass (ESI) calculated for C30H51O5Si (M+ + H), 519.3506; found 519.3507. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 98.3% and retention time of 6.2 min for the title compound.

2-[(6aR,10aR)-6a,7,8,9,10,10a-Hexahydro-1-hydroxy-6,6-dimethyl-9-oxo-6H-benzo[c]chromen-3-yl]-2-methylpropanoic Acid (22)

To a stirred solution of 16 (430 mg, 1.1 mmol) in dioxane/H2O (1:1 ratio, 20 mL) at room temperature, under an argon atmosphere, was added lithium hydroxide (80 mg, 3.3 mmol). Stirring was continued for 8 h, and then the reaction mixture was quenched with 1 N HCl and diluted with ethyl acetate. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate. The combined organic phase was washed with water and brine, dried (MgSO4), and concentrated under vacuo. The crude product was chromatographed on silica gel (48% acetone in hexane) to give 22 (232 mg, 63% yield) as a white solid. Mp 153–154 °C; IR (neat) 3340, 2978, 1695, 1620, 1417 cm−1; 1H NMR (500 MHz, CDCl3 + CD3OD) δ 6.39 (d, J = 1.5 Hz, 1H, ArH), 6.34 (d, J = 1.5, 1H, ArH), 4.35 (br s, 1H, OH), 3.94 (ddd, J = 15.0, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.86 (m as td, J = 12.5, J = 3.5 Hz, 1H, 10a-H), 2.62–2.58 (m, 1H, 8eq-H), 2.48 - 2.41 (m, 1H, 8ax-H), 2.19 - 2.04 (m, 2H, 10ax-H, 7eq-H), 1.94 (m as td, J = 12.2, J = 3.0 Hz, 1H, 6a-H), 1.57–1.48 (m, s and s, overlapping, 7H, 7ax-H, -C(CH3)2-, especially 1.50, s, and 1.49, s, -C(CH3)2-), 1.47 (s, 3H, 6-CH3), 1.11 (s, 3H, 6-CH3). 13C NMR (125 MHz CDCl3) δ 213.2 (>C═O), 180.1 (-C(O)O-), 155.1 (ArC-1 or ArC-5), 154.7 (ArC-5 or ArC-1), 144.3 (tertiary aromatic), 109.7 (tertiary aromatic), 107.1 (ArC-2 or ArC-4), 105.6 (ArC-4 or ArC-2), 65.9, 47.2, 45.7, 44.8, 40.7, 34.6, 30.3, 27.8, 26.8, 25.7, 18.9. Mass spectum (ESI) m/z (relative intensity) 333 (M+ + H, 100). Exact mass (ESI) calculated for C19H2505 (M+ + H), 333.1702; found 333.1700. HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 97.5% and retention time of 6.5 min for the title compound.

Methyl 2-{4-[(1R,2R,5R)-6,6-Dimethyl-4-oxobicyclo[3.1.1]-heptan-2-yl]-3,5-dihydroxyphenyl}acetate (24)

The synthesis was carried out as described for 15, using 23 (4.9 g, 27.0 mmol), diacetates 14 (11.4 g, 47.8 mmol), and p-TSA (5.7 g, 30.0 mmol) in 74 mL of CHCl3/acetone (7:3 ratio) and gave 24 as a white gum in 23% yield (2.0 g). IR (neat) 3344, 2940, 2873, 1719, 1682, 1611, 1460, 1261, 1140 cm−1; 1H NMR (500MHz, CDCl3) δ 6.95 (brs, 2H, OH), 6.26 (d, J = 2.0 Hz, 1H, ArH), 6.19 (d, J = 2.0 Hz, 1H, ArH), 3.67 (s, 3H, OMe), 3.63 (dd, J = 18.5 Hz, J = 8.0 Hz,1H, 3α-H), 3.58 (d, J = 15.2 Hz,1H, half of an AB system, -CH2C(O)O-), 3.53 (d, J = 15.2 Hz, 1H, half of an AB system, -CH2(O)O-), 3.45, (t, J = 8.5 Hz, 1H, 4-H), 2.63 (d, J = 10.5 Hz, 1H, 7β-H), 2.56 (t, J = 5.5 Hz, 1H, 1-H), 2.48–2.34 (m, 2H, 7α-H, 3β-H), 2.06 (t, J = 5.5 Hz, 1H, 5-H), 1.31 (s, 3H, 6-Me), 0.93 (s, 3H, 6-Me). 13C NMR (100 MHz CDCl3) δ 218.7 (>C═O), 173.0 (-C(O)O-), 156.5 (ArC), 154.7 (ArC), 135.3, 119.9, 111.1, 103.9, 57.9 (-OCH3), 52.4, 47.0, 42.5, 40.0, 37.7, 33.0, 26.1, 24.1, 21.9. Mass spectum (ESI) m/z (relative intensity) 319 (M+ + H, 100), 287 (M+ − MeOH, 35). LC/MS analysis (Waters MicroMass ZQ system) showed purity of 98.7% and retention time of 3.9 min for the title compound.

Methyl 2-[(6aR,10aR)-1-Hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl]acetate (25)

The synthesis was carried out as described for 16, using 24 (950 mg, 2.98 mmol) and TMSOTf (3.5 mL, 0.89 mmol of a 0.3 M solution in MeNO2) in 30 mL of anhydrous CH2Cl2/MeNO2 (3:1 ratio) and gave 25 (540 mg, 57% yield) as a white foam. IR (neat) 3332, 2960, 1727, 1697, 1620, 1420, 1257, 1140 cm−1; 1H NMR (500 MHz, CDCl3) δ 6.33 (d, J = 2.5 Hz, 1H, ArH), 6.24 (d, J = 2.5 Hz, 1H, ArH), 5.53 (br s, 1H, OH), 3.71 (s, 3H, OMe), 3.63 (d, J = 16.5 Hz, 1H, half of an AB system, -CH2C(O)O-), 3.56 (d, J = 16.5 Hz,1H, half of an AB system, -CH2C(O)O-), 2.99 (ddd, J = 15.5 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.85, (m as td, J = 12.0 Hz, J = 3.5 Hz, 1H, 10a-H), 2.64–2.56 (m, 1H, 8eq-H), 2.48–2.38 (m, 1H, 8ax-H), 2.24 (m as t, J = 13.5 Hz 1H, 7eq-H), 2.18–2.11 (m, 1H, 10ax-H), 2.01 (m as d, J = 12.5 Hz, J = 3.0 Hz, 1H, 6a-H), 1.55–1.44 (m and s, overlapping, 4H, 7ax-H, 6-Me), 1.08 (s, 3H, 6-Me). Mass spectum (ESI) m/z (relative intensity) 319 (M+ + H, 100). Exact mass (ESI) calculated for C18H2305 (M+ + H), 319.1545; found 319.1547.

2-[(6aR,10aR)-1-Hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl]acetic Acid (26)

The synthesis was carried out as described for 4a using 25 (450 mg, 1.41 mmol), sodium hydroxide (226 mg, 5.7 mmol) in 32 mL of THF/H2O (1:1 ratio) and gave 26 (270 mg, 63% yield) as a white foam. IR (neat) 3345, 2980, 1725, 1694, 1620, 1417 cm−1; 1H NMR (500MHz, CDCl3) δ 6.20 (d, J = 2.5 Hz, 1H, ArH), 6.05 (d, J = 2.5 Hz, 1H, ArH), 3.47 (d, J = 16.0 Hz, 1H, half of an AB system, -CH2C(O)O-), 3.43 (d, J = 16.0 Hz, 1H, half of an AB system, -CH2C(O)O-), 2.90 (ddd, J = 15.5 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.82, (m as td, J = 12.0 Hz, J = 3.5 Hz, 1H, 10a-H), 2.46–2.34 (m, 2H, 8eq-H, 8ax-H), 2.16–2.02 (m, 2H, 7eq-H, 10ax-H), 1.91 (m as td, J = 12.5 Hz, J = 3.0 Hz, 1H, 6a-H), 1.48–1.37 (m, 1H, 7ax-H), 1.33 (s, 3H, 6-Me), 0.97 (s, 3H, 6-Me). Mass spectum (ESI) m/z (relative intensity) 305 (M+ + H, 46), 259 (55, M+ − COOH), 149 (100).

Butyl 2-[(6aR,10aR)-1-Hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl]acetate (27)

The synthesis was carried out as described for 3a using 26 (170 mg, 0.56 mmol), sodium bicarbonate (70 mg, 0.82 mmol), and 1-bromobutane (185 mg, 1.34 mmol) in DMF (4 mL) and gave 27 (109 mg, 54% yield) as a white foam. IR (neat) 3345, 2960, 1726, 1697, 1620, 1420, 1257 cm−1; 1H NMR (500MHz, CDCl3) δ 6.34 (d, J = 2.5 Hz, 1H, ArH), 6.23 (d, J = 2.5 Hz, 1H, ArH), 5.59 (s, 1H, OH), 4.11 (m as octet, J = 4.0 Hz, 2H, -C(O)OCH2-), 3.62 (d, J = 16.5 Hz, 1H, half of an AB system, -CH2C(O)O-), 3.54 (d, J = 16.5 Hz,1H, half of an AB system, -CH2C(O)O-), 2.99 (br d, J = 15.0 Hz, 1H, 10eq-H), 2.86, (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 10a-H), 2.64–2.56 (m, 1H, 8eq-H), 2.48–2.38 (m, 1H, 8ax-H), 2.24 (m as t, J = 13.0 Hz, 1H, 7eq-H), 2.18–2.10 (m, 1H, 10ax-H), 2.00 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.61 (quintet, J = 6.5 Hz, 2H, 5′-H), 1.55–1.44 (m and s overlapping, 4H, 7ax-H, 6-Me), 1.35 (sextet, J = 7.5 Hz, 2H, 6′-H), 1.08 (s, 3H, 6-Me), 0.91 (t, J = 7.5 Hz, 3H, 7′-H). 13C NMR (100 MHz, CDCl3) δ 209.6 (>C═O) 171.5 (-C(O)O-), 155.2 (ArC), 155.1 (ArC), 134.2 (ArC), 115.4 (ArC), 111.1 (ArC), 103.9 (ArC), 77.2 (C-6), 65.1 (-OCH2-), 48.9, 48.2, 40.4, 39.3, 35.6, 30.5, 27.6, 26.5, 19.0, 19.3, 13.6. Mass spectrum (ESI) m/z (relative intensity) 361 (M+ + H, 100). Exact mass (ESI) calculated for C21H29O5 (M+ + H), 361.2015; found 361.2012. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 98.3% and retention time of 4.5 min for the title compound.

Radioligand Binding Assays

The affinities (Ki) of the new compounds for rat CB1 receptor as well as for mouse and human CB2 receptors were obtained by using membrane preparations from rat brain or HEK293 cells expressing either mCB2 or hCB2 receptors, respectively, and [3H]CP-55,940 as the radioligand, as previously described.13,19 Results from the competition assays were analyzed using nonlinear regression to determine the IC5035 values for the ligand; Ki values were calculated from the IC50 (Prism by GraphPad Software, Inc.). Each experiment was performed in triplicate, and Ki values determined from three independent experiments and are expressed as the mean of the three values.

cAMP Assay.19,20

HEK293 cells stably expressing rCB1 or hCB2 receptors were used for the studies. The cAMP assay was carried out using PerkinElmer’s Lance ultra cAMP kit following the protocol of the manufacturer. Briefly, the assays were carried out in 384-well plates using 1000–1500 cells/well. The cells were harvested with non-enzymatic cell dissociation reagent Versene, washed once with HBSS, and resuspended in the stimulation buffer. The various concentrations of the test compound (5 μL) in forskolin (2 μM final concentration) containing stimulation buffer were added to the plate followed by the cell suspension (5 μL). Cells were stimulated for 30 min at room temperature. Eu-cAMP tracer working solution (5 μL) and Ulight-anti-cAMP working solution (5 μL) were then added to the plate and incubated at room temperature for 60 min. The data were collected on a PerkinElmer Envision instrument. The EC50 values were determined by nonlinear regression analysis using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA).

Plasma Stability.13

Compounds or their proposed metabolites were diluted (200 μM) in mouse or rat plasma and incubated at 37 °C, 100 rpm. At various time points, samples were taken, diluted 1:4 in acetonitrile, and centrifuged to precipitate the proteins. The resulting supernatant was analyzed by HPLC. 4-Nitrophenyl butyrate was used as a control in each experiment. In vitro plasma half-lives were determined using exponential decay calculations in Prism (GraphPad).

HPLC Analysis

Chromatographic separation was achieved using a Supelco Discovery C18 (4.6 mm × 250 mm) column on a Waters Alliance HPLC system. Mobile phase consisted of acetonitrile (A) and a mixture of 60% water (acidified with 8.5% o-phosphoric acid) and 40% acetonitrile (B). Gradient elution started with 5% A, transitioning to 95% A over 10 min, and holding for 5 min before returning to starting conditions; run time was 15 min, the flow rate was 1 mL/min, and UV detection was used at each compound’s maximal absorbance (204 and 230 nM).

Methods for Characterization of in Vivo Effects

A. Rodents.13,36

Subjects

For hypothermia testing, female Sprague-Dawley rats (n = 6/group) weighing between 250 and 350 g (Charles River, Wilmington MA) were used. Rats were tested repeatedly with at least 5 days intervening between drug sessions. Experiments occurred at approximately the same time (10:00 am to 5:00 pm) during the light portion of the daily light/dark cycle. Outside of experimental sessions, rats were pair housed (2/cage) in a climate controlled vivarium with unrestricted access to food and water. For tail-flick withdrawal (analgesia) testing, male CD-1 mice (n = 6/group), weighing between 30 and 35 g (Charles River, Wilmington MA), were used. Mice were housed 4/cage in a climate controlled vivarium with unrestricted access to food and water and acclimated to these conditions for at least a week before any experimental manipulations occurred. Analgesia testing took place between 11:00 am and 7:00 pm. Mice were used once.

Procedures

Temperature was recorded using a thermistor probe (model 401, Measurement Specialties, Inc., Dayton, OH) inserted into the rectum at a depth of 6 cm and secured to the tail with micropore tape. Rats were minimally restrained and isolated in 38 cm × 50 cm × 10 cm plastic stalls. Temperature was read to the nearest 0.01 °C using a Thermometer (model 4000A, Measurement Specialties, Inc.).

Two baseline temperature measures were recorded at 15 min intervals, and drugs were injected immediately after the second baseline was recorded. After injection, temperature was recorded every 30 min for 3 h and every hour thereafter for a total of 6 h. The change in temperature was determined for each rat by subtracting temperature readings from the average of the two baseline measures. Analgesia testing utilized a thermostatically controlled 2 L water bath commercially available from VWR International where the water temperature was set at 52 °C (±0.5 °C). The tail was immersed into the water at a depth of 2 cm and the withdrawal latency recorded by a commercially available stopwatch (Fisher Scientific), allowing measurements in seconds and 1/100 s. Cut-off was set at 10 s to minimize the risk of tissue damage. A test session consisted of 5 recordings, the first of which constituted the baseline recording. Injections occurred immediately after the baseline recording, and the remaining recordings took place 20, 60, 180, and 360 min postadministration. Prior to this testing, the animals had been accustomed to the procedure for three consecutive “mock” sessions where the water was held at 38 °C, i.e., average body temperature of mice; no tail-flicks are elicited by this water temperature. The third “mock” session also included an ip injection of vehicle (10 mL/kg). The tail-flick withdrawal latencies are expressed as a percentage of maximum possible effect (% MPE), according to the formula % MPE = [(test latency minus baseline latency) divided by (10 minus baseline latency)] times 100.

Drugs

For hypothermia testing, 11-OH-(−)-Δ8-THC-DMH (3g) and compounds 3a, 3b, and 3c were initially dissolved in a solution of 20% ethanol, 20% alkamuls, and 60% saline and were further diluted with saline. Injections were administered sc in a volume of 1.0 mL/kg. For tail-flick withdrawal (analgesia) testing, 3g and 3b were initially dissolved in 2% dimethyl sulfoxide, 4% Tween-80, and 4% propylene glycol before saline was slowly added just prior to the 10 mL/kg ip administration. All suspensions were freshly prepared for analgesia testing.

Data Analysis

Dose- and time-effect functions for hypothermia testing were analyzed using, respectively, one-way or two-way repeated measures ANOVA procedures followed by Bonferroni’s post hoc test; p was set at ≤0.05. ED50 values were calculated using linear regression of the hypothermia dose-effect functions for compounds 11-OH(−)-Δ8-THC-DMH (3g) and compounds 3a, 3b, and 3c. Two-way repeated measures ANOVA was applied to the tail-flick latency data depicted in Figure 4. Statistical analyses were performed using the software package GraphPad Prism 5.03 (GraphPad Software, San Diego, CA).

B. Non-Human Primates

Subjects

Four adult male squirrel monkeys (Saimiri sciureus) that were trained in the behavioral and pharmacological procedures used here served as subjects (see below). Experimental sessions were conducted 5 days a week (Monday–Friday). Each subject had previously received acute intramuscular (im) injections of 0.01 mg/kg of the cannabinoid 28 in training sessions at least twice weekly and other cannabinergic compounds in test sessions conducted no more than once weekly; no test compounds were administered for at least 7 days prior to the present studies. The experimental protocol for the present studies was approved by the Institutional Animal Care and Use Committee at McLean Hospital. Subjects were maintained in a facility licensed by the U.S. Department of Agriculture and in accordance with the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (National Research Council, 2003).

Apparatus

During experimental sessions, subjects were seated in a Plexiglas chair within a ventilated sound- and light-attenuating chamber.33 The front panel of the chair was outfitted with two response levers that were positioned 6 cm left and right of center. Each lever-press with a force of at least 0.25 N closed a microswitch, produced an audible click, and was recorded as a response. Red stimulus lights were mounted behind the transparent front panel of the chair, approximately 10 cm above each response lever. Before each session, a shaved portion of each subject’s tail was coated with electrode paste and placed under brass electrodes for the delivery of brief, low-intensity current (see below). Experimental events and data collection were controlled by Med Associates (St. Albans, VT) interfacing equipment and operating software.

Behavioral Procedure

The subjects were previously trained to discriminate the presession administration of 0.01 mg/kg of the cannabinoid agonist 28 or its vehicle (20% ethanol/20% emulphor/60% saline) by responding on one of two levers. Briefly, subjects initially were trained to terminate visual stimuli associated with the delivery of brief, low-intensity current (200 ms; 3 mA) across the electrodes by depressing one of the two response levers; the inactive lever was removed to facilitate early training. The active lever varied until subjects reliably terminated visual stimuli by responding on either lever. Subsequently, both levers were present in all sessions and the active lever was signaled only by a presession injection: one lever was active only following the im injection of the cannabinoid CB1 agonist 28 (0.01 mg/kg, im), whereas the other lever was active only following im injection of vehicle; right and left lever assignments were counterbalanced among subjects. Under terminal conditions, each training session began with a 10 min timeout period during which all lights were extinguished and responding had no programmed consequences. After the timeout period, two red stimulus lights above each lever were illuminated and completion of 10 consecutive responses [fixed ratio (FR) 10] on the active lever extinguished all stimulus lights and initiated a 50 s timeout. Responses on the inactive lever reset the FR requirement. Current delivery was scheduled for delivery every 10 s until either the FR 10 was completed on the correct lever or 30 s elapsed, whichever came first.

Drug Testing

Testing was conducted to determine the extent to which different doses of drugs substituted for the training compound, i.e., produced responding on the training drug-associated response lever. Tests for the time course of substitution of compounds 3b and 3g to the training stimulus were conducted when a subject’s discrimination performance was at least 90% accurate for four of the last five training sessions and on the immediately preceding session. Procedurally, test sessions differed from training sessions in two ways. First, 10 consecutive responses on either lever extinguished the stimulus lights and associated program of current delivery and initiated the 50 s timeout. Second, no current deliveries were scheduled during test sessions so as to preclude possible stimulus-induced enhancement of responding. Other schedule contingencies were unchanged. In initial experiments, dose-ranging procedures were employed to quickly establish the lowest dose that fully substituted for (≥90% responding on the CB1 lever) the 0.01 mg/kg of the CB1 agonist 28. In these procedures, each subject received one of several doses of each drug (0.0001–0.003 mg/kg) 60 min prior to the test session; doses were chosen based on data from receptor binding studies and previous work in rodents (3b; see above) or monkeys (3g). Next, the lowest effective dose of each drug was studied in all subjects. Results indicated that 0.003 mg/kg 3g was not fully effective in any subject at 60 min whereas 0.001 mg/kg 3b was fully effective in three of four subjects at this time point. Both drugs fully substituted for 28 at 120 min after treatment. In a follow-up experiment, 0.0003 mg/kg 3b and 0.001 mg/kg 3g failed to fully substitute for 28 in all subjects; consequently, subsequent studies were conducted with 0.001 mg/kg 3b and 0.003 mg/kg 3g. To assess behavioral onset and time course of action of these doses of the two drugs, experiments with each drug were arranged to include multiple sequential test sessions that began 15, 30, 60, 120, 240, 480, and 960 min after injection. The effects of 3g were additionally determined 36 and 48 h after administration.

Data Analysis

The two primary dependent measures at the time points examined in the present experiments were response distribution across the two levers and overall response rate. Response distribution (percent 28 lever) was calculated by dividing the number of responses on the lever associated with the injection of 28 by the total number of responses (excluding any responses during timeout periods). Response rate was calculated by dividing the total number of responses on both levers by the total session time (excluding all timeout periods). Doses of drugs were considered to substitute fully when response distribution was 90% 28 lever responding and response rates were >0.2 responses/s. Regression analysis was used to compare the onset and offset of action for 3b and 3g; for this measure, group data were used to calculate the times when 50% of responding occurred on the 28 lever, i.e., before and following full substitution for the training drug stimulus.

Supplementary Material

Elemental analysis results for compounds 3a and 16, chemical structure of (6aR,9R,10aR)-3-(adamantan-1-yl)-9-(hydroxymethyl)-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (28), and time course of hypothermic effects for all tested doses

Molecular formula strings and some data

Acknowledgments

This work was supported by grants from the National Institute on Drug Abuse to A.M. (Grants DA009158, DA007215), J.B. (Grant DA026795), and T.U.C.J. (Grant DA09064).

ABBREVIATIONS USED

CB1
cannabinoid receptor 1
CB2
cannabinoid receptor 2
(−)-Δ9-THC
(−)-Δ9-tetrahydrocannabinol
HHC
hexahydrocannabinol
THC
tetrahydrocannabinol
CNS
central nervous system
PK/PD
pharmacokinetic/pharmacodynamic
SAR
structure–activity relationship
HEK293
human embryonic kidney cell line
log P
octanol–water partition coefficient
PSA
polar surface area
NMR
nuclear magnetic resonance
HPLC
high-performance liquid chromatography
MPE
maximum posible effect

Footnotes

Notes

The authors declare no competing financial interest.

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmed-chem.6b00717.

Elemental analysis results for compounds 3a and 16, chemical structure of (6aR,9R,10aR)-3-(adamantan-1-yl)-9-(hydroxymethyl)-6,6-dimethyl-6a,7,8,9,10,10a-hexahy-dro-6H-benzo[c]chromen-1-ol (28), and time course of hypothermic effects for all tested doses of 3a, 3b, 3c, and 11-OH-Δ8-THC-DMH (3g) (PDF)

Molecular formula strings and some data (CSV)

References

1. Devane WA, Dysarz FA, 3rd, Johnson MR, Melvin LS, Howlett AC. Determination and characterization of a cannabinoid receptor in rat brain. Mol Pharmacol. 1988;34:605–613. [PubMed]
2. Munro S, Thomas KL, Abu-Shaar M. Molecular characterization of a peripheral receptor for cannabinoids. Nature. 1993;365:61–65. [PubMed]
3. Makriyannis A, Nikas SP, Thakur GA, Pavlopoulos S. Cannabinoid receptors as therapeutic targets. Curr Pharm Des. 2006;12:1751–1769. [PubMed]
4. Han S, Thatte J, Buzard DJ, Jones RM. Therapeutic utility of cannabinoid receptor type 2 (CB2) selective agonists. J Med Chem. 2013;56:8224–8256. [PubMed]
5. Hwang J, Adamson C, Butler D, Janero DR, Makriyannis A, Bahr BA. Enhancement of endocannabinoid signaling by fatty acid amide hydrolase inhibition: a neuroprotective therapeutic modality. Life Sci. 2010;86:615–623. [PMC free article] [PubMed]
6. Karst M, Wippermann S, Ahrens J. Role of cannabinoids in the treatment of pain and (painful) spasticity. Drugs. 2010;70:2409–2438. [PubMed]
7. Lu D, Vemuri VK, Duclos RI, Jr, Makriyannis A. The cannabinergic system as a target for anti-inflammatory therapies. Curr Top Med Chem. 2006;6:1401–1426. [PubMed]
8. Pacher P, Batkai S, Kunos G. The endocannabinoid system as an emerging target of pharmacotherapy. Pharmacol Rev. 2006;58:389–462. [PMC free article] [PubMed]
9. Pertwee RG. The diverse CB1 and CB2 receptor pharmacology of three plant cannabinoids: delta9-tetrahydrocannabinol, cannabidiol and delta9-tetrahydrocannabivarin. Br J Pharmacol. 2008;153:199–215. [PMC free article] [PubMed]
10. Vemuri VK, Makriyannis A. Medicinal chemistry of cannabinoids. Clin Pharmacol Ther. 2015;97:553–558. [PMC free article] [PubMed]
11. Grotenhermen F. Pharmacokinetics and pharmacodynamics of cannabinoids. Clin Pharmacokinet. 2003;42:327–360. [PubMed]
12. Makriyannis A. 2012 Division of Medicinal Chemistry Award Address. Trekking the cannabinoid road: a personal perspective. J Med Chem. 2014;57:3891–3911. [PMC free article] [PubMed]
13. Nikas SP, Sharma R, Paronis CA, Kulkarni S, Thakur GA, Hurst D, Wood JT, Gifford RS, Rajarshi G, Liu Y, Raghav JG, Guo JJ, Jarbe TU, Reggio PH, Bergman J, Makriyannis A. Probing the carboxyester side chain in controlled deactivation (−)-delta(8)-tetrahydrocannabinols. J Med Chem. 2015;58:665–681. [PMC free article] [PubMed]
14. Sharma R, Nikas SP, Guo JJ, Mallipeddi S, Wood JT, Makriyannis A. C-ring cannabinoid lactones: a novel cannabinergic chemotype. ACS Med Chem Lett. 2014;5:400–404. [PMC free article] [PubMed]
15. Sharma R, Nikas SP, Paronis CA, Wood JT, Halikhedkar A, Guo JJ, Thakur GA, Kulkarni S, Benchama O, Raghav JG, Gifford RS, Jarbe TU, Bergman J, Makriyannis A. Controlled-deactivation cannabinergic ligands. J Med Chem. 2013;56:10142–10157. [PMC free article] [PubMed]
16. Busch-Petersen J, Hill WA, Fan P, Khanolkar A, Xie XQ, Tius MA, Makriyannis A. Unsaturated side chain beta-11-hydroxyhexahydrocannabinol analogs. J Med Chem. 1996;39:3790–3796. [PubMed]
17. Dixon DD, Sethumadhavan D, Benneche T, Banaag AR, Tius MA, Thakur GA, Bowman A, Wood JT, Makriyannis A. Heteroadamantyl cannabinoids. J Med Chem. 2010;53:5656–5666. [PMC free article] [PubMed]
18. Drake DJ, Jensen RS, Busch-Petersen J, Kawakami JK, Concepcion Fernandez-Garcia M, Fan P, Makriyannis A, Tius MA. Classical/nonclassical hybrid cannabinoids: southern aliphatic chain-functionalized C-6beta methyl, ethyl, and propyl analogues. J Med Chem. 1998;41:3596–3608. [PubMed]
19. Nikas SP, Alapafuja SO, Papanastasiou I, Paronis CA, Shukla VG, Papahatjis DP, Bowman AL, Halikhedkar A, Han X, Makriyannis A. Novel 1′,1′-chain substituted hexahydrocannabinols: 9beta-hydroxy-3-(1-hexyl-cyclobut-1-yl)-hexahydrocannabinol (AM2389) a highly potent cannabinoid receptor 1 (CB1) agonist. J Med Chem. 2010;53:6996–7010. [PMC free article] [PubMed]
20. Ogawa G, Tius MA, Zhou H, Nikas SP, Halikhedkar A, Mallipeddi S, Makriyannis A. 3′-Functionalized adamantyl cannabinoid receptor probes. J Med Chem. 2015;58:3104–3116. [PMC free article] [PubMed]
21. Harrington PE, Stergiades IA, Erickson J, Makriyannis A, Tius MA. Synthesis of functionalized cannabinoids. J Org Chem. 2000;65:6576–6582. [PubMed]
22. Devane WA, Breuer A, Sheskin T, Jarbe TU, Eisen MS, Mechoulam R. A novel probe for the cannabinoid receptor. J Med Chem. 1992;35:2065–2069. [PubMed]
23. Liddle J, Huffman JW. Enantioselective synthesis of 11-hydroxy-(1 ′S,2′ R)-dimethylheptyl-Delta(8)-THC, a very potent CB1 agonist. Tetrahedron. 2001;57:7607–7612.
24. Guo Y, Abadji V, Morse KL, Fournier DJ, Li X, Makriyannis A. (−)-11-Hydroxy-7′-isothiocyanato-1′,1′-dimethylheptyl-delta 8-THC: a novel, high-affinity irreversible probe for the cannabinoid receptor in the brain. J Med Chem. 1994;37:3867–3870. [PubMed]
25. Nikas SP, Thakur GA, Parrish D, Alapafuja SO, Huestis MA, Makriyannis A. A concise methodology for the synthesis of (−)-Delta(9)-tetrahydrocannabinol and (−)-Delta(9)-tetrahydrocannabivarin metabolites and their regiospecifically deuterated analogs. Tetrahedron. 2007;63:8112–8123.
26. Archer RA, Blanchard WB, Day WA, Johnson DW, Lavagnino ER, Ryan CW, Baldwin JE. Cannabinoids 0.3. Synthetic approaches to 9-ketocannabinoids - total synthesis of nabilone. J Org Chem. 1977;42:2277–2284. [PubMed]
27. Nikas SP, D’Souza M, Makriyannis A. Enantioselective synthesis of (10S)- and (10R)-methylanandamides. Tetrahedron. 2012;68:6329–6337. [PMC free article] [PubMed]
28. Shelnut EL, Nikas SP, Finnegan DF, Chiang N, Serhan CN, Makriyannis A. Design and synthesis of novel prostaglandin E ethanolamide and glycerol ester probes for the putative prostamide receptor(s) Tetrahedron Lett. 2015;56:1411–1415. [PMC free article] [PubMed]
29. Khanolkar AD, Lu D, Ibrahim M, Duclos RI, Jr, Thakur GA, Malan TP, Jr, Porreca F, Veerappan V, Tian X, George C, Parrish DA, Papahatjis DP, Makriyannis A. Cannabilactones: a novel class of CB2 selective agonists with peripheral analgesic activity. J Med Chem. 2007;50:6493–6500. [PubMed]
30. Fukami T, Yokoi T. The emerging role of human esterases. Drug Metab Pharmacokinet. 2012;27:466–477. [PubMed]
31. Chopda GR, Vemuri VK, Sharma R, Thakur GA, Makriyannis A, Paronis CA. Diuretic effects of cannabinoid agonists in mice. Eur J Pharmacol. 2013;721:64–69. [PMC free article] [PubMed]
32. Thakur GA, Bajaj S, Paronis C, Peng Y, Bowman AL, Barak LS, Caron MG, Parrish D, Deschamps JR, Makriyannis A. Novel adamantyl cannabinoids as CB1 receptor probes. J Med Chem. 2013;56:3904–3921. [PMC free article] [PubMed]
33. Kangas BD, Delatte MS, Vemuri VK, Thakur GA, Nikas SP, Subramanian KV, Shukla VG, Makriyannis A, Bergman J. Cannabinoid discrimination and antagonism by CB(1) neutral and inverse agonist antagonists. J Pharmacol Exp Ther. 2013;344:561–567. [PubMed]
34. Berry LM, Li C, Zhao Z. Species differences in distribution and prediction of human V(ss) from preclinical data. Drug Metab Dispos. 2011;39:2103–2116. [PubMed]
35. Cheng Y, Prusoff WH. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50% inhibition (I50) of an enzymatic reaction. Biochem Pharmacol. 1973;22:3099–3108. [PubMed]
36. Paronis CA, Nikas SP, Shukla VG, Makriyannis A. Delta(9)-Tetrahydrocannabinol acts as a partial agonist/antagonist in mice. Behav Pharmacol. 2012;23:802–805. [PMC free article] [PubMed]
37. Thakur GA, Nikas SP, Li C, Makriyannis A. Structural requirements for cannabinoid receptor probes. Handb Exp Pharmacol. 2005;168:209–246. [PubMed]
38. Felder CC, Joyce KE, Briley EM, Mansouri J, Mackie K, Blond O, Lai Y, Ma AL, Mitchell RL. Comparison of the pharmacology and signal transduction of the human cannabinoid CB1 and CB2 receptors. Mol Pharmacol. 1995;48:443–450. [PubMed]