Novel adamantyl cannabinoids as CB1 receptor probes

Article

pubs.acs.org/jmc

Novel Adamantyl Cannabinoids as CB1 Receptor Probes

Ganesh A. Thakur,*^,†,‡Shama Bajaj,^†,§Carol Paronis,^†,‡Yan Peng,^†Anna L. Bowman,^†

Lawrence S. Barak,^∥Marc G. Caron,^∥Demon Parrish,^⊥Jeﬀrey R. Deschamps,^⊥

and Alexandros Makriyannis*^,†,§,‡

^†Center for Drug Discovery, Northeastern University, 116 Mugar Hall, 360 Huntington Avenue, Boston, Massachusetts 02115,

United States

^‡Department of Pharmaceutical Sciences, Northeastern University, 140 The Fenway, 360 Huntington Avenue, Boston, Massachusetts

02115, United States

^§Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115,

United States

^∥Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, United States

^⊥Naval Research Laboratory, Code 6930, 4555 Overlook Avenue, Washington, D.C. 20375, United States

S

* Supporting Information

ABSTRACT: In previous studies, compound 1 (AM411), a 3-

(1-adamantyl) analogue of the phytocannabinoid (−)-Δ⁸-

tetrahydrocannabinol (Δ⁸-THC), was shown to have

improved aﬃnity and selectivity for the CB1 receptor. In

this work, we further explored the role of the 1-adamantyl

group at the C-3 position in a series of tricyclic cannabinoid

analogues modiﬁed at the 9-northern aliphatic hydroxyl

(NAH) position. Of these, 9-hydroxymethyl hexahydrocanna-

binol 11 (AM4054) exhibited high CB1 aﬃnity and full agonist proﬁle. In the cAMP assay, the 9-hydroxymethyl cannabinol

analogue 24 (AM4089) had a partial agonist proﬁle, with high aﬃnity and moderate selectivity for rCB1 over hCB2. In vivo

results in rat models of hypothermia and analgesia were congruent with in vitro data. Our in vivo data indicate that 3-(1-

adamantyl) substitution, within NAH cannabinergics, imparts improved pharmacological proﬁles when compared to the

corresponding, traditionally used 3-dimethylheptyl analogues and identiﬁes 11 and 24 as potentially useful in vivo CB1

cannabinergic probes.

mimetic activity including (a) a phenolic hydroxyl group (PH)

at C-1, (b) a C-3 side chain (SC), (c) 9-OH or 11-OH

northern aliphatic hydroxyl group (NAH) functionalities, and

(d) a southern aliphatic hydroxyl group (SAH) at C-6 (Figure

1).^17,19

INTRODUCTION

■

G-protein-coupled receptors (GPCRs) are the most abundant

class of central nervous system (CNS) receptors in mammals

and are targets of many therapeutic medications.¹(−)-Δ⁹-

Tetrahydrocannabinol (Δ⁹-THC), the main psychoactive

ingredient of cannabis,²produces its biochemical and

pharmacological eﬀects by interacting with two well-charac-

terized GPCRs, CB1 and CB2. CB1 is localized primarily in

brain³and in various tissues in the periphery,⁴⁻⁶whereas CB2

is primarily associated with the immune system^6,7and under

homeostatic conditions is found to a much lesser extent in

CNS.^8,9The subsequent discovery of the endocannabinoids,

represented by arachidonoylethanolamine (anandamide)^10,11

and 2-arachidonoylglycerol (2-AG)^12,13has led to a better

understanding of the physiological and biochemical roles of the

endocannabinoid system.¹⁴During the past decade, numerous

ligands with high aﬃnities and subtype selectivities for both

receptors encompassing several chemotypes were synthesized

and their SAR was explored.¹⁵⁻¹⁸Δ⁹-THC exhibits no receptor

subtype CB1/CB2 selectivity. Also, SAR studies with a number

of synthetic cannabinoids structurally related to Δ⁹-THC have

identiﬁed some key pharmacophores associated with cannabi-

Modifying the phenolic hydroxyl in cannabinoids into a

methoxy group or removing it leads to analogues with severely

reduced CB1 aﬃnities. However, such modiﬁcations produce

only marginal eﬀects on the compounds’ aﬃnities for CB2.²⁰

Additionally, analogues in which the C-1 phenolic OH group is

absent have been shown to exhibit CB2 selectivity, and this

observation has served as the basis for the synthesis of CB2

selective cannabinoids.^21,22The C-3 aliphatic side chain is the

most studied pharmacophore within the tricyclic cannabinoid

template and was shown to have the most drastic eﬀects on

CB1/CB2 aﬃnity and selectivity.^15,17For example, compounds

with a shorter side chain such as those carrying a C-3 butyl

group exhibited enhanced CB2 selectivity,²³whereas, analogues

with longer seven- or eight-carbon side chains were shown to

Received: January 17, 2013

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Figure 1. Structures of representative classical and nonclassical cannabinoids.

a

Scheme 1

a

Reagents and conditions: (a) p-TSA.H₂O, CHCl₃, rt, 3.5 days, 80.5%; (b) TMSOTf, CH₂Cl₂/CH₃NO₂(3:1), 0 °C → 10 °C, 8 h, 61%; (c)

Ph₃PCH₂OMe⁺Cl⁻, nBuLi, THF, −30 °C → 0 °C, 30 min, 96.4%; (d) CCl₃COOH, H₂O, CH₂Cl₂, rt, 45 min, 98%; (e) K₂CO₃, MeOH, rt, 4 h, 78%.

a

Scheme 2

a

Reagents and conditions: (a) NaBH₄, MeOH, 30 min, 92%; (b) K-selectride, THF, −78 °C, 3 h, 64%.

prefer CB1.²⁴⁻²⁶Optimal activity is obtained with the 1′,1′-

dimethylheptyl chain which imparts a 100-fold increase in

potency compared to the n-pentyl side chain of Δ⁹-THC.²⁷

Cannabivarin, a 3-propyl Δ⁹-THC analogue, exhibits poor

binding to both CB receptors but behaves as a functional CB1

antagonist in tissue preparations.²⁸Incorporation of cyclic or

aryl moieties at the 3-position is well tolerated.^26,29−33In

previous work, we have shown that an analogue of (−)-Δ⁸-

THC, an equiactive isomer of the Δ⁹-prototype, carrying a 1-

adamantyl side chain exhibits substantial CB1 aﬃnity,

selectivity, and potency, whereas the 2-adamantyl analogue

shows preference for CB2.^30,34We have also explored other

cyclic side chains such as 3-bornyl and 3-isobornyl,³⁵aromatic

groups,³³and cycloalkyl chain or chains incorporating cyclo-

alkyl groups.^31,32,36The other two NAH and SAH

pharmacophores also appear to play substantial roles in

modulating CB1 and CB2 aﬃnities and selectivities.³⁷⁻⁴¹

We have now explored the SAR of 3-(1-adamantyl)-

substituted cannabinoids with appropriately modiﬁed NAH

substituents and obtained novel analogues with improved

pharmacological proﬁles. This study has identiﬁed some key

cannabinergic probes which can be employed as potent high

eﬃcacy agonists. Additionally, it has led to the identiﬁcation of

two low eﬃcacy cannabinergic compounds which show

promise as CB1 partial agonists.

CHEMISTRY

■

Adamantyl resorcinol 2 was synthesized from 2,6-dimethox-

yphenol in four steps following a previously reported

procedure.^30,42The mixture of chiral terpene diacetates 3,

which was used previously in the stereospeciﬁc synthesis of 9-

oxo-cannabinoids with a 6aR,10aR absolute conﬁguration, was

obtained from commercially available (+)-(1R)-nopinone,

utilizing our earlier reported reaction conditions.^31,38,43,44

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a

Scheme 3

a

Reagents and conditions: (a) NaBH₄, MeOH, rt, 30 min, 93%; (b) I₂, Ph₃P, imidazole, PhH, reﬂux, 1 h, 76%; (c) NaCN, DMSO, rt, 24 h, 72%; (d)

Ph₃PCH₂OMe⁺Cl⁻, nBuLi, THF, −30 °C → 0 °C, 30 min, 86%; (e) CCl₃COOH, H₂O, CH₂Cl₂, rt, 45 min, 96%; (f) NaBH₄, MeOH, rt, 30 min,

98%.

a

Scheme 4

a

Reagents and conditions: (a) NaBH₄, MeOH, rt, 30 min, 95%; (b) Ph₃PCH₂OMe⁺Cl⁻, nBuLi, THF, −30 °C → 0 °C, 30 min; (c) CCl₃COOH,

H₂O, CH₂Cl₂, rt, 45 min, 82% (two steps); (d) NaBH₄, MeOH, rt, 30 min, 92%.

a

Scheme 5

a

Reagents and conditions: (a) BF₃. Et₂O, CH₂Cl₂, −20 °C → rt, 2 h, 32%; (b) LiAlH₄, THF, 0 °C → rt, 2 h, 85%; (c) pyridine, acetic anhydride, rt,

overnight; (d) sulfur, 250 °C, 2 h, 34% (two steps); (e) LiAlH₄, THF, 0 °C → rt, 2 h, 91%.

C

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a

Scheme 6

a

Reagents and conditions: (a) pyridine, acetic anhydride, rt, overnight, 88%; (b) sulfur, 250 °C, 2 h, 37% ; (c) KOH, EtOH, rt, 30 min, 97%.

a

Scheme 7

a

Reagents and conditions: (a) Br₂, 18-crown-6, CH₂Cl₂, 0 °C, 30 min, quantitative; (b) 30, Pd(PPh₃)₄, Ba(OH)₂, DME, H₂O, μW, 25 min, 71.9%;

(c) 9-iodo-9-BBN, CH₂Cl₂, 0 °C, 4 h, then acetic acid, reﬂux, 5 h, 68.8%; (d) CH₃MgI, THF, rt → reﬂux, 2 h; (e) p-TSA·H₂O, CHCl₃, rt, 6 h, 72.5%

for 33 and 56% for 35 (two steps); (f) BBr₃, CH₂Cl₂, reﬂux, 24 h, 85%.

Coupling of resorcinol 2 with 3 in the presence of catalytic p-

toluenesulfonic acid led to norpinanone 4 (Scheme 1) in 81%

yield, which was followed by TMSOTf promoted rearrange-

ment−cyclization to give 5 in 61% yield. Introduction of the C-

9 aldehyde group was accomplished by exposing 5 to

(methoxymethylene)triphenylphosphorane followed by hydrol-

ysis of the resulting enol ether 6 (Scheme 1).³⁹Unlike our

previous report, we found that this reaction did not require the

previous protection of the phenolic OH groups.³⁹The 9-formyl

diastereomeric aldehydes 7 and 8 were obtained in 98% yield as

a 2:1 (β versus α) mixture from 6, the precursor vinyl ether

(1:4 mixture of isomers). Epimerization of the diasteromeric

mixture of aldehydes allowed us to obtain the β-equatorial

isomer 7 in 78% isolated yield.³⁹

analogue 12 in 76% yield by treatment with iodine,

triphenylphosphine, and imidazole.⁴⁵Treatment of iodide 12

with sodium cyanide in DMSO produced the corresponding

cyano analogue 13 in 72% yield. Homologation with

(methoxymethylene)triphenylphosphorane produced almost

exclusively the Z-enol ether 14 (86% yield) as conﬁrmed by

¹H NMR (see Experimental Section). Hydrolysis of 14 with

wet trichloroacetic acid led to aldehyde 15 (96% yield) which

upon reduction with NaBH₄in methanol produced the 9β-

hydroxyethyl analogue 16 (98% yield).

Reduction of 8 with NaBH₄produced the 9α-hydroxymethyl

analogue 17 in 95% yield (Scheme 4). Homologation of 8 with

(methoxymethylene)triphenylphosphorane produced the enol

ether which was hydrolyzed to produce aldehyde 18 in 82%

overall yield. Reduction of 18 with NaBH₄in methanol led to

the 9α-hydroxyethyl analogue 19 in 92% yield.

Analog 22 with a 6aR,10aR absolute stereochemistry was

synthesized from 4-hydroxy myrtenol pivalate (20) (Scheme 5)

which was in turn prepared from (1R,5S)-myrtenol (>98% ee)

by utilizing the modiﬁed procedure reported by Liddle et al.⁴⁶

Condensation of alcohol 20 with resorcinol 2 in anhydrous

dichloromethane at −20 °C in presence of a Lewis acid led to

the desired cannabinoid ester 21 in 32% yield which, upon

reduction with LiAlH₄in THF, aﬀorded tetrahydrocannabinol

22 in 85% isolated yield. To obtain cannabinol analogue 24, the

phenolic pivaloyl intermediate 21 was ﬁrst acetylated by

treatment with pyridine/acetic anhydride and then dehydro-

genated by heating with sulfur at 250 °C to provide the mixed

Aiming for both 9β- and 9α-OH analogues, we used two

diﬀerent routes starting from the 9-keto analogue 5. Reduction

of 5 with sodium borohydride in methanol gave the C-9β

(equatorial; 9) and C-9α (axial; 10) diasteromers as a 96:4

mixture, respectively (Scheme 2). Silica gel ﬂash chromato-

graphic separation provided the pure 9β-isomer 9 in 92% yield.

Reduction of 5 with K-selectride at −78 °C led to axial alcohol

10, exclusively in 64% yield. The stereochemistry of the 9-

1

hydroxyl group of 9 and 10 was assigned on the basis of H

NMR spectral data.³⁹

The NAH-functionalized 9β and 9α analogues were

synthesized as shown in Schemes 3 and 4, respectively.

Reduction of 7 with NaBH₄in methanol at room temperature

produced alcohol 11 in 93% yield. The 9β-hydroxylmethyl

group was then converted to the corresponding iodomethyl

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Table 1. Ligand Aﬃnities (K_i) of Adamantyl Cannabinoids for rCB1, mCB2, and hCB2

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Table 1. continued

a

Aﬃnities for rCB1, mCB2, and hCB2 were determined using rat brain (CB1), mouse spleen (CB2) membranes, or HEK293 cell membranes

expressing hCB2 and [³H]CP-55,940 as the radioligand (see Experimental Section). Data were analyzed using nonlinear regression analysis. K_ivalues

b

were obtained from three independent experiments run in duplicate and are expressed as the mean of the three values (SD < 20%). Reported

previously.³⁰

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Figure 2. cAMP data of AM4054 (11, agonist, EC₅₀= 1.29 nM) and AM4089 (24, partial agonist, EC₅₀= 7.97 nM).

ester 23 in a 34% combined yield. Reduction of ester 23 with

LiAlH₄in THF produced alcohol 24 in 91% yield.

properties. However, the 9-keto analogue 5 had substantially

reduced aﬃnities for the rCB1, hCB2, and mCB2 receptors.

Similarly, the hexahydro 9α-OH analogue (10) exhibited a

relatively low aﬃnity proﬁle for all three receptors, while the

9β-OH isomer (9) showed improved CB1 and CB2 aﬃnities

compared to the 9α-isomer but still lower than its Δ⁸-analogue

1. Also, unlike 1, compound 9 exhibited no CB1/CB2

selectivity. Aromatization of the rings C to give the respective

9-methyl (27), 9-OCH₃(33), and 9-OH (35) analogues

resulted in compounds with moderate or low aﬃnities for both

CB receptors.

One-carbon homologation at the 9-position led to

compounds with overall improved CB1/CB2 aﬃnities. The

β-formyl hexahydro analogue 7 had a very similar binding

proﬁle as its α-formyl counterpart 8 both exhibiting moderate

binding proﬁles with modest selectivity for hCB2 (Table 1). Its

cyanomethyl analogue 13 had no observable change in CB1/

CB2 binding proﬁle. Conversely, the corresponding iodomethyl

analogue 12 had severely reduced CB1/CB2 aﬃnities, possibly

reﬂecting unfavorable steroelectronic interactions at CB1 and

CB2 subsites.

The most interesting compounds in the one carbon

homologation series were the hydroxymethyl analogues

(Table 1). The β-hydroxymethyl hexahydro analogue 11 had

the most favorable binding proﬁle while its α-hydroxymethyl

isomer 17 had 6- to 11-fold lower aﬃnities. The corresponding

11-OH tetrahydrocannabinol analogue 22 had a high aﬃnity

binding proﬁle very similar to the hexahydro analogue 11, with

both compounds exhibiting no signiﬁcant CB1/CB2 selectiv-

ities. Finally, 24 the 9-hydroxymethyl analogue with an

aromatized C-ring had the highest CB1 aﬃnity (K_i= 2.1

nM) of the compounds included in this study. It also exhibited

a 22- and 15-fold CB1 selectivity over hCB2 and mCB2,

respectively. Interestingly, compound 21 containing the bulky

pivalate ester at NAH maintains signiﬁcant aﬃnity for CB1

while its CB2 aﬃnity is severely reduced.

Our eﬀort to determine the SAR of 1-adamantyl 9-

substituted cannabinoids was extended to include the 2-carbon

NAH homologues, 9β- (16) and 9α-hydroxyethyl (19), as well

as the 9β- (15) and 9α-formylmethyl (18) and 9β-vinylmethyl

ether (14) analogues. All of these analogues exhibited relatively

unremarkable CB1/CB2 binding proﬁles with intermediate

CB1 and CB2 K_ivalues. Interestingly, only compound 19

exhibited signiﬁcant (17-fold) species selectivity for mCB2 over

hCB2. Among the 2-carbon NAH analogues, the most

interesting was the 9β-hydroxyethyl analogue 16 with a K_i

value of 8.6 nM for CB1 and modest (∼ 4-fold) CB1 over

hCB2 and mCB2 selectivities. Interestingly, all analogues

The C9-methyl cannabinol analogue 27 was prepared

starting from 1, using a previously reported synthetic

approach.⁴⁷The phenolic group of 1 was ﬁrst protected as

the acetate ester 25. Dehydrogenation of 25 by treatment with

sulfur at 250 °C to give 26 (37% yield) was followed by

deprotection to give the phenol 27 in 97% isolated yield

(Scheme 6).

We adopted an alternative approach for the synthesis of the

cannabinol analogues carrying 9-OCH₃(33) or 9-OH groups

(35) (Scheme 7).⁴⁸Dimethoxy resorcinol 28 was monobromi-

nated to provide 2-bromo-5-(1-adamantyl)-1,3-dimethoxyben-

zene 29 in quantitative yields using bromine and 18-crown-6.⁴⁹

Biphenyl 31 was prepared by coupling 2-diisopropyl carbamoyl-

5-methoxyphenyl boronic acid 30 with 29 under our

microwave-accelerated Suzuki reaction conditions.⁴⁸Selective

demethylation of biphenyl 31 with 9-I-9-BBN, followed by

acetic acid-catalyzed intramolecular cyclization gave cannabi-

lactone 32 in a combined 69% isolated yield. Compound 32

was then converted to its 6,6-dimethyl analogue 33 by treating

ﬁrst with methyl magnesium iodide followed by cyclization in

the presence of p-toluenesulfonic acid in a combined 72%

yield.⁴⁸

Our attempt to remove the methyl ether group of 33 under

BBr₃conditions did not proceed smoothly and gave 35 in only

modest yields. As an alternative route, cannabilactone 32 was

demethylated to obtain the bisphenolic lactone 34 (85% yield)

which was then converted to the desired 6,6-dimethyl analogue

35 by treatment with excess methyl magnesium iodide followed

by cyclization in the presence of p-toluenesulfonic acid in a 56%

combined yield.

RESULTS AND DISCUSSION

■

Receptor Binding Studies. The SAR of the novel

adamantyl cannabinoids was examined by measuring their

aﬃnities for the CB1 and CB2 receptors (Table 1). Our

receptor aﬃnity assays included CB2 measurements for both

mouse and human receptors because of species variations

observed in our previous work.^31,48Conversely, CB1 aﬃnities

were measured using only native rCB1 preparations because no

signiﬁcant variations in CB1 between rodent and human

receptors have been observed. The 1-adamantyl cannabinergic

analogues included in this study exhibited binding properties

that were distinct from those of their Δ⁸- and/or C-3 alkyl

counterparts. Modiﬁcation of the Δ⁸-analogues to their 9-keto

and 9-OH counterparts was aimed at obtaining more polar

analogues with improved pharmacokinetic and pharmacological

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Figure 3. Dose−response of compounds on U2OS cell lines permanently expressing β-arrestin2-GFP and CB1 receptors. Eﬃcacy of translocation is

a measure of the ability of compounds to form membrane or cytosolic clusters of cannabinoid receptor complexes in CB1-E /β-arrestin2-GFP upon

exposure to ligand. Note the CB1-E is the CB1 receptor substituted with the human neurokinin-1 receptor tail to enhance arrestin binding.^58,59

Eﬃcacy data were normalized to the β-arrestin2 response of WIN55212-2 (WIN) which is set to 1. Mean eﬃcacy and potency data are presented in

the table. Corresponding 95% conﬁdence intervals for compounds 1, 11, 22, and 24 are respectively (compound, eﬃcacy, potency); (1; 0.43−0.81,

0.51−4.6 μM), (11; 0.65−0.92, 0.037−0.25 μM), (22; 0.54−0.80, 0.083−0.62 μM), and (24; 0.68−0.94, 0.21−0.98 μM). Data were analyzed by

nonlinear regression, N = 3.

containing an aldehyde group at the northern site (7, 8, 15, and

18) exhibited moderate to high binding aﬃnities for hCB2 with

some selectivity over rCB1.

standing of the 3D structures of the novel 9-substituted 3-

adamantyl series. As with our previously reported 1, the

(−)-Δ⁸-THC analogue, the adamantyl moiety occupies a

distinct computational space relative to the tricyclic ring

system.³⁰Rotation around the C3−1-adamantyl bond

encompasses a spherical space that is symmetrically aligned

with the tricyclic cannabinoid component and suggests a

distinctive pharmacophoric subsite within the CB1 receptor. To

further explore 3D diﬀerences between the key compounds

discussed above (5, 9, 11, 22, 24), we compared their

computationally derived structures (Figures 5 and 6). All of

the above four compounds, when properly aligned, exhibit

virtually overlapping phenolic (A ring) and adamantyl rings.

While assuming that the sites of interaction of the above two

moieties are similar for all compounds, we focused our

comparisons on the B and C rings of the tricyclic component.

Functional Characterization Using cAMP and β-

Arrestin Assays. The key high aﬃnity adamantyl ligands

were further tested for their functional potencies which were

obtained by measuring the decrease in forskolin-stimulated

cAMP (compounds 11 and 24) and by their β-arrestin

recruitment assays (compounds 1, 11, 22, and 24) for CB1

receptors. Although all four compounds have similar binding

aﬃnities, their functional proﬁles for CB1 are signiﬁcantly

diﬀerent. In the cAMP assay, 11 was a potent CB1 agonist

(EC₅₀= 1.29 nM), while 24 had good potency (EC₅₀= 7.97

nM) but reduced eﬃcacy, thus qualifying as a CB1 partial

agonist (Figure 2). These results are congruent with their β-

arrestin recruitment data (Figure 3) with compound 1 having

the lowest potency and eﬃcacy of the four and 11 being the

most potent and eﬃcacious. Compounds 22 and 24 exhibited

intermediate potencies. The above functional data are

supported by our in vivo results.

Computational and X-ray Crystallographic Studies.

To establish a structural basis for explaining the aﬃnity and

functional properties of our new analogues while focusing on

CB1 receptor SAR, we sought to obtain the crystal structure of

a representative analogue, as well as computationally derived 3-

D information on our most successful ligands. Of all the novel

cannabinoids studied here, only compound 5, the 9-tetrahydro

analogue, provided crystals suitable for X-rays studies. X-ray

analysis (Figure 4) allowed us to obtain an overall under-

Figure 5. Top view of an overlay of 5 (gray carbons), 9 (orange

carbons), and 11 (green carbons). The overlay was performed with the

ﬂexible ligand alignment tool in Maestro, version 9.3 (Schrodinger,

̈

LLC, New York, NY, 2012).

Our molecular modeling suggests that both the hexahydro-

(11) and tetrahydro- (22) hydroxymethyl analogues have

eﬀectively overlapping B and C rings with their respective

hydroxyl groups capable of engaging in hydrogen-bonding

interactions with a putative corresponding subsite on the

receptor. Conversely, in the 9-keto (5) and 9β-OH (9)

analogues, the respective keto and hydroxyl groups are situated

Figure 4. Thermal ellipsoid plot of compound 5 is shown with the

aromatic A ring perpendicular to the paper.

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Figure 6. Side view of an overlay of 11 (green carbons), 22 (cyan

carbons), and 24 (magenta carbons). The overlay was performed with

the ﬂexible ligand alignment tool in Maestro, version 9.3 (Schrodinger,

̈

LLC, New York, NY, 2012).

in a relative space diﬀerent from that of 11 (Figure 5). It can be

argued that the 11-hydroxymethyl groups in 11 and 22 position

the respective hydroxyl groups in a favorable site within the

receptor, resulting in optimized binding aﬃnities and functional

potencies. This interaction is not available for the 9-keto (5)

and 9β-OH (9) analogues where the position of the keto and β-

OH group does not allow for optimal ligand−receptor (CB1)

interaction, thus explaining their lower CB1 aﬃnities.

In the partial agonist 24 the C-ring is aromatic and has the

hydroxymethyl group oriented in a distinct pharmacophoric

space which allows it to interact favorably with the same CB1

subsite. However, this interaction involves a slightly diﬀerent

but distinct orientation compared to its other two high eﬃcacy

agonist hydroxymethyl congeners, 11 and 22 (Figure 6). To

explain the observed functional diﬀerences, we propose that the

hydrogen bonding interaction of high eﬃcacy agonists 11 and

22 with the activated form of CB1 is signiﬁcantly more

favorable than that of the partial agonist 24.

Earlier work from our laboratory based on mutational data

with an 9-hydroxymethyl analogue, (−)-9β-hydroxymethyl-3-

(1′,1′-dimethylheptyl)-hexahydrocannabinol (AM4056) had

identiﬁed Ser7.39 (383) in helix-7 as the site of interaction of

this very potent high eﬃcacy agonist.⁵⁰On the basis of the

work presented here, we can postulate that this Ser7.39 (383) is

the putative site for interaction with the hydroxyl groups in 11

and 22. Conversely, compounds 5 and 9 are unable to access

this favorable interaction while 24 can do this, however, with a

reduced H-bonding strength.

In Vivo Evaluation. In vivo evaluation aimed at comparing

the CB1 potencies and eﬃcacies of the novel CB1 high eﬃcacy

and potent agonist 11 with its partial agonist counterpart 24

and the previously reported parent 1-adamantyl cannabinoid 1.

The work included measurement of ligand-induced hypo-

thermia and analgesia in rats. Body temperature was measured

in isolated rats over a 6 h period following drug injection.

Compound 11 decreased core body temperature in a dose-

dependent manner, with a dose of 1.0 mg/kg reducing body

temperature up to 6.4 0.4 °C from an average baseline of

38.02 0.18 °C (Figure 7). At this dose, the onset of drug

eﬀects occurred within 60−90 min after injection, although

peak eﬀects were obtained at 240−360 min after injection.

Compound 1 did not have hypothermic eﬀects up to a dose of

10.0 mg/kg, and a 10.0 mg/kg dose of compound 24 decreased

■

●

▲

Figure 7. Eﬀects of 1 ( ), 11 ( ), and 24 ( ) on antinociception

(top graph; A) and body temperature (bottom graph; B). Symbols

represent the group mean SEM (n = 5−7 rats), and the open circles

to the left indicate the eﬀects of vehicle pretreatment. Abscissa: dose,

in mg/kg; ordinate: (A) Percentage of the maximum possible

antinociceptive eﬀect, (B) change in body temperature. Asterisks

indicate eﬀects that are signiﬁcantly diﬀerent from vehicle, *p < 0.05,

*** p < 0.001.

following drug injection. Prior to drug administration, the

average baseline tail-ﬂick latency for individual groups was 2.3 s;

with a range from 1.8 to 2.6 s. Compound 11 increased tail-ﬂick

latency over the same dose range as that for hypothermic eﬀects

and with a similar time course. Doses of 0.1−1.0 mg/kg

compound 1 had signiﬁcant antinociceptive eﬀects, with a mean

( 95% cx CL) ED₅₀value of 0.05 mg/kg (0.01, 0.1). The onset

of the antinociceptive eﬀects occurred between 60 and 120 min

after injection, and these eﬀects generally increased over the 6 h

test period. In comparison, although both 1 and 24 tended to

increase tail-ﬂick latency, neither had signiﬁcant antinociceptive

eﬀects up to doses of 10.0 mg/kg. The results obtained with 1

seemingly contradict previous reports of the eﬀects of this drug

in rats; however, this likely reﬂects slight diﬀerences in

procedures, as the absolute magnitude of hypothermic eﬀects

reported here are similar to those reported earlier.^51,52

CONCLUSION

■

We have sought to further probe the SAR of 3-(1-adamantyl)

cannabinoids and supplement previous work involving the 3-(1-

adamantyl) Δ⁸-THC analogue 1 to develop novel ligands as

useful CB1 pharmacological tools with improved pharmaco-

logical proﬁles. Of the new analogues synthesized, the most

interesting were those carrying a hydroxymethyl group at the 9-

position. In vitro functional characterization assays found both

compound 11 and 22 to be high eﬃcacy CB1 agonists while 24

exhibited the proﬁle of a partial CB1 agonist in cAMP assay.

Initial in vivo characterization showed that compound 11 dose-

dependently produced hypothermia and antinociception, two

eﬀects often associated with cannabinoid agonist activity

(Figure 8). In comparison, compound 1 had reduced behavioral

colonic temperature by 4.1

measured using a tail-ﬂick procedure over a 6 h period

0.1 °C. Antinociception was

I

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Figure 8. Eﬀects of compounds 1, 11, and 24 at diﬀerent times after injection on body temperature (left panels, A, C, and E) and antinociception

(right panels, B, D, and F). Abscissae: time (in minutes) after injection; left ordinates: change in body temperature; right ordinates: percentage of the

maximum possible antinociceptive eﬀect. Asterisks indicate eﬀects that are signiﬁcantly diﬀerent from vehicle, *p < 0.05.

visualized under ultraviolet (UV) light by phosphomolybdic acid

staining or by anisaldehyde reagent staining. Flash column

chromatography employed silica gel 60 (230−400 mesh). Melting

points were determined on a micromelting point apparatus and are

uncorrected. ¹H NMR spectra were recorded in CDCl₃, unless

otherwise stated, on a Varian 500 MHz. Chemical shifts are recorded

in parts per million (δ) relative to internal TMS. Multiplicities are

indicated as br (broadened), s (singlet), d (doublet), t (triplet), q

(quartet), quint (quintet), sept (septet), or m (multiplet). 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-Chamapaign. Mass spectral data are reported in the

form of m/z (intensity relative to base = 100). Purity of all material

was determined to be at least 95% from HPLC.

eﬀects, and 24 produced only hypothermia up to a dose of 10

mg/kg (Figure 7). Due to its improved pharmacological

proﬁles when compared to the highly long acting side chain

counterparts, 11 should prove to be a very useful CB1 in vivo

probe. Additionally, in view of the paucity of available CB1

partial agonists, 24 will be of value as such a ligand in

cannabinoid research.

EXPERIMENTAL SECTION

■

Chemistry. All commercial chemicals and solvents were purchased

from Aldrich Chemicals and Co., unless otherwise speciﬁed, and were

used without further puriﬁcation. All moisture-sensitive reactions were

performed under a static atmosphere of nitrogen or argon in oven-

dried or ﬂame-dried glasswares. The progress of the reaction was

monitored by thin layer chromatography (TLC) using commercially

prepared silica gel 60 F₂₅₄glass-backed plates. All compounds were

(4R)-4-[4-(1-Adamantyl)-2,6-dihydroxyphenyl]-6,6-dimeth-

yl-2-norpinanone (4). To a degassed solution of 2 (6.50 g, 26.60

mmol) and diacetates 3 (8.86 g, 37.20 mmol; 10.19 g, 87% pure

diacetates 3 were used) in CHCl₃(267 mL) at 0 °C under an argon

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atmosphere was added p-toluenesulfonic acid monohydrate (7.085 g,

37.25 mmol). The reaction mixture was warmed to room temperature

and stirred for 3.5 days. The reaction mixture was diluted with ether

and washed sequentially with water, saturated aqueous NaHCO₃, and

brine. The organic phase was dried (MgSO₄), and the solvent was

removed under reduced pressure to give crude product as a brown oil.

Recrystallization from CH₂Cl₂and hexane (2:3) gave 4 as a white

crystalline solid (7.95 g, 80.5% yield): mp = 284−286 °C. R_f= 0.4

(EtOAc/hexane = 30/70) ¹H NMR (500 MHz, CDCl₃+ 1 drop

DMSO) δ: 6.50 (br s, 2H, ArOH), 6.38 (s, 2H, 3-H and 5-H, ArH),

4.00 (t, J = 8.0 Hz, 1H, 4-H), 3.63 (dd, J = 19.0 Hz, J = 8.0 Hz, 1H, 3α-

H), 2.60−2.52 (m, 3H, especially 2.56, dd, J = 17.0 Hz, J = 8.5 Hz, 1H,

3β-H), 2.50−2.44 (m, 1H), 2.28 (t, J = 5.0 Hz, 1H, 5-H), 2.06 (br s,

3H, Ad-H), 1.82 (br s, 6H, Ad-H), 1.77 (br d, J = 12.5 Hz, 3H, Ad-H),

1.70 (br d, J = 12.5 Hz, 3H, Ad-H), 1.35 (s, 3H, 6-CH₃), 0.99 (s, 3H,

6-CH₃). HRMS (ESI) calculated for C₂₅H₃₃O₃: calculated 381.2430;

found 381.2433.

70 mL of CH₂Cl₂at room temperature was added wet trichloroacetic

acid (4.84 g, 29.6 mmol in 5 mL of water). The resulting solution was

stirred at room temperature for 45 min, quenched with saturated

NaHCO₃, and diluted with water. The organic layer separated, and the

aqueous phase was extracted with CH₂Cl₂(2×). The combined

organic layer was washed with water, brine (1×), dried (MgSO₄), and

concentrated to give crude aldehyde. Puriﬁcation by ﬂash chromatog-

raphy on silica gel (EtOAc/hexane = 7/93 → 15/85) gave a aldehyde

mixture as a white foam (1.15 g, 98% yield; ratio of α:β epimers = 1:2).

1

β-isomer (7): R_f= 0.49 (EtOAc/hexane = 30/70). H NMR (500

MHz, CDCl₃) δ: 9.65 (s, 1H, CHO), 6.40 (d, J = 1.5 Hz, 1H, ArH),

6.26 (d, J = 1.5 Hz, 1H, ArH), 5.55 (br s, 1H, ArOH), 3.52 (d, J = 13.0

Hz, 1H), 2.56−2.45 (m, 2H), 2.15−2.08 (m, 1H), 2.06−1.96 (m, 4H,

especially 2.03, br s, 3H), 1.81 (br s, 6H), 1.74 (d, J = 12.5 Hz, 3H), 1.

68 (d, J = 11.5 Hz, 3H), 1.52−1.35 (m, 5H, especially 1.39, s, 3H, 6β-

CH₃), 1.16 (qd, J = 13.0 Hz, J = 4.0 Hz, 1H), 1.09 (s, 3H, 6α-CH₃),

1.06 (q, J = 12.0 Hz, 1H); HRMS (ESI) calculated for C₂₆H₃₅O₃:

calculated 395.2586; found 395.2582. α-isomer (8): R_f= 0.35

(6aR,10aR)-3-(1-Adamantyl)-6,6a,7,8,10,10a-hexahydro-1-

hydroxy-6,6-dimethyl-9H-dibenzo[b,d]pyran-9-one (5). To a

solution of 4 (3.95 g, 10.38 mmol) in anhydrous CH₂Cl₂/CH₃NO₂

(3:1, 260 mL) at 0 °C under an argon atmosphere was added

trimethylsilyl triﬂuoromethanesulfonate (13.84 mL, 0.3 M solution in

CH₃NO₂, 4.152 mmol), and the resulted mixture was stirred at 10 °C

for 8 h. The reaction was quenched with saturated aqueous NaHCO₃/

brine (1:1), and diethyl ether was added. The organic phase was

separated, the aqueous phase was extracted with diethyl ether, and the

combined organic phase was washed with brine and dried over

MgSO₄. Solvent evaporation and puriﬁcation by ﬂash column

chromatography on silica gel (acetone/hexane = 20/80) aﬀorded 5

as white crystalline solid (2.41 g, 61% yield): mp = 263−264 °C. R_f=

1

(EtOAc/hexane = 20/80) H NMR (500 MHz, CDCl₃) δ: 9.86 (s,

1H, CHO), 6.40 (d, J = 2.0 Hz, 1H, ArH), 6.31 (d, J = 2.0 Hz, 1H,

ArH), 4.95 (s, 1H, ArOH), 3.56 (m as dd, J = 14.0 Hz, J = 2.5 Hz,

1H), 2.64 (m as br s, 1H), 2.41 (m as dd, J = 14.0 Hz, J = 2.0 Hz, 1H),

2.31 (td, J = 11.5 Hz, J = 3.0 Hz, 1H), 2.05 (br s, 3H, Ad-H), 1.84 (br

s, 6H, Ad-H), 1.82−1.64 (m, 8H), 1.52 (td, J = 11.5 Hz, J = 2.5 Hz,

1H),, 1.41 (ddd, J = 13.5 Hz, J = 11.5 Hz, J = 5.0 Hz, 1H), 1.36 (s, 3H,

6β-CH₃), 1.08 (qd, J = 13.0 Hz, J = 4.0 Hz, 1H), 0.99 (s, 3H, 6α-

CH₃). HRMS (ESI) calculated for C₂₆H₃₅O₃: calculated 395.2586;

found 395.2585.

(6aR,9R,10aR)-3-(Adamantan-1-yl)-1-hydroxy-6,6-dimethyl-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromene-9-carbalde-

hyde (7). The aldehyde mixture (1.0 g, 2.53 mmol) was dissolved in

70 mL of methanol and added via cannula to powdered K₂CO₃(1.75

g, 12.67 mmol). After the reaction mixture was stirred for 4 h at room

temperature, methanol was removed under reduced pressure, diluted

with water, and extracted with ethyl acetate (3×). Combined organic

extracts washed with brine and dried (MgSO₄). Evaporation of

volatiles under reduced pressure gave a crude product that was puriﬁed

by column chromatography (EtOAc/hexane = 7/93 → 15/85) to give

pure β-aldehyde 7 (0.78 g, 78% yield) as a white foam.

1

0.55 (ethylacetate/hexane = 40/60). H NMR (500 MHz, CDCl₃) δ:

6.74 (br s, 1H, ArOH), 6.41 (d, J = 2.0 Hz, 1H, ArH), 6.37 (d, J = 2.0

Hz, 1H, ArH), 4.07 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.5 Hz, 1H,

10eq-H), 2.90 (ddd, J = 13.5 Hz, J = 12.5 Hz, J = 4.0 Hz, 1H, 10a-H),

2.66−2.59 (m, 1H, 8eq-H), 2.47 (ddd, J = 15.0 Hz, J = 13.0 Hz, J = 7.0

Hz, 1H, 8ax-H), 2.20−2.12 (m, 2H, 10ax-H, 7eq-H), 2.05 (br s, 3H,

Ad-H), 1.97 (td, J = 12.0 Hz, J = 2.5 Hz, 1H, 6a-H), 1.85 (br s, 6H,

Ad-H), 1.76 (d, J = 12.5 Hz, 3H, Ad-H), 1.71 (d, J = 12.5 Hz, 3H, Ad-

H), 1.53 (dddd, J = 15.0 Hz, J = 13.0 Hz, J = 12.5 Hz, J = 5.0 Hz, 1H,

7ax-H), 1.47 (s, 3H, 6-CH₃), 1.13 (s, 3H, 6-CH₃); HRMS (ESI)

calculated for C₂₅H₃₃O₃: calculated 381.2430; found 381.2433.

Tricyclic Methyl Enol Ether (6). To a suspension of

(methoxymethyl)triphenylphosphonium chloride (8.12 g, 23.7

mmol) in 90 mL of anhydrous THF at −30 °C was added a solution

of n-BuLi in THF (9.21 mL, 23.02 mmol, 2.5 M in hexane). The

resulting blood red colored solution was warmed to 0 °C over a period

of 15 min. A solution of ketone 5 (1.288 g, 3.38 mmol) in anhydrous

THF (60 mL) was then added through cannula, keeping the reaction

temperature at 0 °C. The resulting solution was stirred for 15 min and

then quenched by addition of water and stirred for 30 min till the

solution turns colorless. The reaction mixture was diluted with ether,

organic phase separated, and the aqueous phase extracted with ether

(2×). Combined organic extracts was washed with brine and dried

(MgSO₄). Puriﬁcation by ﬂash chromatography on silica gel (EtOAc/

hexane = 2/98 → 15/85) gave 6 as a white foam (1.33 g, 96.4%, 1:4

mixture of geometric isomers). R_f= 0.50 (EtOAc/hexane = 20/80).

¹H NMR (500 MHz, CDCl₃) δ: 6.41 (d, J = 2.0 Hz, 1H, ArH), 6.40

(d, J = 2.0 Hz, 1H, ArH), 6.26 (d, J = 2.0 Hz, 1H, ArH), 6.24 (d, J =

2.0 Hz, 1H, ArH), 5.93 (t as br s, 1H),5.85 (t as br s, 1H), 4.79 (s, 1H

ArOH), 4.65 (s, 1H, ArOH), 4.18−4.12 (m, 1H), 3.59 (s, 3H, OCH₃),

3.58 (s, 3H, OCH₃), 3.47−3.40 (m), 2.92 (br d, J = 11.5 Hz, 1H), 2.41

(td, J = 12.0 Hz, J = 3.5 Hz, 2H),, 2.08−2.02 (m, 6H, Ad-H, both

isomers), 1.93−1.86 (m, 2H), 1.85 (br s, 12H, Ad-H, both isomers),

1.76 (d, J = 12.5 Hz, 6H, Ad-H, both isomers) 1.70 (d, J = 12.0 Hz,

6H, Ad-H, both isomers) 1.65−1.58 (m, 2H), 1.39 (s, 3H, 6β-CH₃),

1.38 (s, 3H, 6β-CH₃), 1.06 (s, 6H, 6α-CH₃of both isomers). HRMS

(ESI) calculated for C₂₇H₃₇O₃: calculated 409.2743; found 409.2735.

(6aR,10aR)-3-(Adamantan-1-yl)-1-hydroxy-6,6-dimethyl-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromene-9-carbalde-

hyde (7 and 8). To a solution of enol ether 6 (1.21 g, 2.96 mmol) in

(6aR,9R,10aR)-3-(Adamantan-1-yl)-6,6-dimethyl-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromene-1,9-diol (9).

To a solution of ketone 5 (500 mg, 1.31 mmol) in 15 mL of methanol

at room temperature was added NaBH₄(198 mg, 5.25 mmol)

portionwise. The reaction mixture was stirred for 30 min and

quenched with 10% acetic acid, and the mixture diluted with ethyl

acetate. The aqueous phase was extracted twice with ethyl acetate, and

the combined organic extract was washed with brine and dried

(MgSO₄). The solvent was evaporated, and the crude product was

chromatographed (EtOAc/hexane = 40/60 → 50/50) to produce pure

β-alcohol 9 as a white solid (463 mg, 92%). R_f= 0.30 (EtOAc/hexane

1

= 70/30). H NMR (500 MHz, CDCl₃) δ: 6.39 (d, J = 2.0 Hz, 1H),

6.23 (d, J = 2.0 Hz, 1H), 5.40 (s, 1H, OH), 3.90−3.82 (m, 1H, 9ax-H,

peak half-width =21 Hz), 3.49 (m as br d, J = 14.0 Hz, 1H, 10eq-H),

2.48 (ddd, J = 12.0 Hz, J = 11.5 Hz, J = 3.0 Hz, 1H, 10a-H), 2.17 (m as

br d, J = 11.0 Hz, 1H, 8eq-H), 2.05 (br s, 3H, Ad-H), 1.88 (dq, J =

13.5 Hz, J = 3.5 Hz, 1H, 7eq-H), 1.83 (br s, 6H, Ad-H), 1.75 (d, J =

12.5 Hz, 3H, Ad-H), 1.70 (d, J = 12.5 Hz, 3H, Ad-H), 1.64 (br s, 1H,

OH), 1.49 (ddd, J = 12.0 Hz, J = 11.5 Hz, J = 2.5 Hz, 1H, 6a-H),

1.45−1.34 (m and s overlapping, 4H, 8ax-H, 6β-CH₃especially 1.38, s,

3H, 6β-CH₃), 1.20−1.08 (m, 2H, 7ax-H and 10ax-H), 1.07 (s, 3H, 6α-

CH₃). HRMS (ESI) calculated for C₂₅H₃₅O₃: calculated 383.2586;

found 383.2590.

(6aR,9S,10aR)-3-(Adamantan-1-yl)-6,6-dimethyl-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromene-1,9-diol

(10). To a solution of ketone 5 (400 mg, 1.05 mmol) in anhydrous

THF (21 mL) at −78 °C under argon was added K-selectride (7.35

mL, 1.0 M solution in THF) over a period of 10 min, and the resulting

solution was stirred at the same temperature for 3 h. The reaction was

quenched by addition of water, warmed to room temperature, and

diluted with ether. The organic layer separated, washed with 1 M HCl,

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water, and brine, and dried (MgSO₄). Evaporation of volatiles gave

crude product that was puriﬁed by column chromatography (ethyl

acetate/hexane = 40/60 → 50/50) to give 10 as a white foam (257

ArH), 4.81 (s, 1H, ArOH), 3.31 (m as dd, J = 12.5 Hz, J = 2.0 Hz, 1H,

10eq-H), 2.50 (ddd as dt, J = 11.5 Hz, J = 2.5 Hz, 1H, 10a-H), 2.35

(dd, J = 17.0 Hz, J = 6.0 Hz, 1H, −CH₂CN), 2.29 (dd, J = 17.0 Hz, J =

7.0 Hz, 1H, −CH₂CN), 2.09−2.02 (m, 4H, especially 2.05, br s, 3H,

Ad-H), 2.00−1.90 (m, 2H), 1.83 (br s, 6H, Ad-H), 1.75 (d, J = 12.5

Hz, 3H, Ad-H), 1.70 (d, J = 11.5 Hz, 3H, Ad-H), 1.49 (td, J = 12.0 Hz,

J = 2.5 Hz, 1H, 6a-H), 1.38 (s, 3H, 6β-CH₃), 1.32−1.11 (m, 2H), 1.08

(s, 3H, 6α-CH₃), 0.95 (q, J = 12.5 Hz, 1H, 9ax-H). HRMS (ESI)

calculated for C₂₇H₃₆NO₂: calculated 406.2746; found 406.2747.

(6aR,9R,10aR)-3-(Adamantan-1-yl)-9-((Z)-2-methoxyvinyl)-

6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]-

chromen-1-ol (14). To a suspension of (methoxymethyl)-

triphenylphosphonium chloride (5.21 g, 15.21 mmol) in 90 mL of

anhydrous THF at −30 °C was added a solution of n-BuLi in THF

(5.88 mL, 14.70 mmol, 2.5 M in hexane). The resulting blood red

colored solution was warmed to 0 °C over a period of 15 min. A

solution of aldehyde 7 (1.0 g, 2.53 mmol) in anhydrous THF (30 mL)

was then added through cannula, keeping the reaction temperature at

0 °C. The resulting solution was stirred for 15 min and then quenched

by addition of water and stirred for 30 min till the solution turned

colorless. The reaction mixture was diluted with diethyl ether, the

organic phase separated, and the aqueous phase extracted with ether

(2×). Combined organic extracts was washed with brine and then

dried (MgSO₄). Puriﬁcation by ﬂash chromatography on silica gel

(EtOAc/hexane = 5/95 → 20/80) gave 14 as a white foam (0.92 g,

86% yield). R_f= 0.47 (EtOAc/hexane = 20/80). ¹H NMR (500 MHz,

CDCl₃) δ: 6.39 (d, J = 2.0 Hz, 1H, ArH), 6.24 (d, J = 2.0 Hz, 1H,

ArH), 5.82 (d, J = 6.5 Hz, 1H, CHCH-OMe), 4.78 (s, 1H, ArOH),

4.22 (dd, J = 9.0 Hz, J = 6.5 Hz, 1H, CHCH−OMe), 3.60 (s, 3H,

OCH₃), 3.05 (m as br d, J = 12.5 Hz, 1H, 10eq-H), 2.72−2.62 (m,

1H), 2.51 (td, J = 12.0 Hz, J = 2.5 Hz, 1H), 2.04 (br s, 3H, Ad-H),

1.92−1.80 (m, 8H, especially 1.84, s, 6H, Ad-H), 1.78−1.66 (m, 7H,

especially 1.75, d, J = 12.5 Hz, 3H, Ad-H and 1.69, d, J = 11.5 Hz, 3H,

Ad-H), 1.48 (t, J = 11.0 H, 1H, 6aH), 1.37 (s, 3H, 6β-CH₃), 1.22−1.11

(m, 1H), 1.07 (s, 3H, 6α-CH₃), 0.88−0.83 (m, 1H). HRMS (ESI)

calculated for C₂₈H₃₉O₃: calculated 423.2899 found 423.2896.

2-((6aR,9R,10aR)-3-(Adamantan-1-yl)-1-hydroxy-6,6-dimeth-

yl-6a,7,8, 9,10,10a-hexahydro-6H-benzo[c]chromen-9-yl)-

acetaldehyde (15). To a solution of enol ether 14 (800 mg, 1.893

mmol) in 40 mL of CH₂Cl₂at room temperature was added wet

trichloroacetic acid (1.546 g, 9.47 mmol in 5 mL of water). The

resulting solution was stirred at room temperature for 45 min,

quenched with saturated NaHCO₃, and diluted with water. The

aqueous phase was extracted with CH₂Cl₂(2×), and the combined

organic extracts were washed with water and brine (1×), dried

(MgSO₄), and concentrated to give crude aldehyde. Puriﬁcation by

ﬂash chromatography on silica gel (EtOAc/hexane = 15/85 → 40/60)

gave 15 as a white foam (743 mg, 96% yield). R_f= 0.24 (EtOAc/

hexane = 20/80). ¹H NMR (500 MHz, CDCl₃) δ: 9.79 (s, 1H, CHO),

6.40 (d, J = 1.5 Hz, 1H, ArH), 6.24 (d, J = 1.5 Hz, 1H, ArH), 4.64 (br

s, 1H, ArOH), 3.18 (m as br d, J = 12.5 Hz, 1H, 10eq-H), 2.52 (ddd as

td, J = 11.5 Hz, J = 3.0 Hz, 1H, 10a-H), 2.44−2.32 (m, 2H), 2.22−1.92

(m, 1H), 2.04 (br s, 3H, Ad-H), 1.97−1.86 (m, 2H), 1.82 (br s, J = 2.5

Hz, 6H, Ad-H), 1.78−1.64 (m, 7H, especially 1.75, d, J = 12.0 Hz, 3H,

Ad-H and 1.69, d, J = 12.0 Hz, 3H, Ad-H), 1.48 (td, J = 11.5 Hz, J =

2.5 Hz, 1H, 6a-H), 1.37 (s, 3H, 6β-CH₃), 1.29−124 (m, 1H), 1.17 (br

t, J = 10.0 Hz, 1H), 1.08 (s, 3H, 6α-CH₃). HRMS (ESI) calculated for

C₂₇H₃₇O₃: calculated 409.2743; found 409.2740

1

mg, 64% yield). R_f= 0.35 (diethyl ether/hexane = 70/30). H NMR

(500 MHz, CDCl₃) δ: 6.67 (br s, 1H, OH), 6.39 (d, J = 2.0 Hz, 1H,

ArH), 6.36 (d, J = 2.0 Hz, 1H, ArH), 4.29 (s, 1H, 9eq-H; peak half-

width =10 Hz), 3.25 (m as dd, J = 14.5 Hz, J = 2.5 Hz, 1H, 10eq-H),

2.95 (m as t, J = 10.5 Hz, 1H), 2.71 (br s, 1H, OH), 2.06−1.94 (m and

br s overlapping, 4H, especially 2.01, br s, 3H, Ad-H), 1.81 (br s, 6H),

1.78−1.60 (m and doublets overlapping, 8H, especially 1.73, d, J =

12.0 Hz, 3H, Ad-H and 1.67, d, J = 11.5 Hz, 3H, Ad-H), 1.55−1.47

(m, 2H), 1.39−1.32 (m, 4H especially 1.37, s, 3H, 6β-CH₃), 1.03 (s,

3H, 6α-CH₃). HRMS (ESI) calculated for C₂₅H₃₅O₃: calculated

383.2586; found 383.2580.

(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 (11). To a solution of aldehyde 7 (1.50 g, 3.80 mmol) in 45 mL of

methanol at room temperature was added NaBH₄(0.575 g, 15.21

mmol) portionwise. The reaction mixture was stirred for 30 min and

quenched with 10% acetic acid, and the mixture was diluted with ether.

The aqueous phase was extracted twice with ethyl acetate, and the

combined organic extract was washed with brine and dried (MgSO₄).

The solvent was evaporated, and the crude was chromatographed

(EtOAc/hexane = 40/60 → 50/50) to produce pure β-alcohol 11 as a

white solid (1.41 g, 93% yield). R_f= 0.42 (EtOAc/hexane = 50/50).

¹H NMR (500 MHz, CDCl₃) δ: 6.40 (d, J = 2.0 Hz, 1H, ArH), 6.24

(d, J = 2.0 Hz, 1H, ArH), 4.78 (s, 1H, ArOH), 3.57−3.46 (m, 2H),

3.24−3.16 (m as br d, J = 12.5 Hz, 1H), 2.48 (ddd as dt, J = 11.0 Hz, J

= 2.5 Hz, 1H, 10a-H), 2.05 (br s, 3H, Ad-H), 2.00−1.89 (m, 2H), 1.84

(br s, 6H, Ad-H), 1.80−1.67 (m, 7H, especially 1.75, d, J = 12.5 Hz,

3H, Ad-H and 1.70, d, J = 12.5 Hz, 3H, Ad-H), 1.48 (td, J = 11.0 Hz, J

= 2.5 Hz, 1H, 6a-H), 1.38 (s, 3H, 6β-CH₃), 1.36 (br s, 1H, OH),

1.18−1.10 (m, 2H), 1.08 (s, 3H, 6α-CH₃), 0.82 (q, J = 12.0 Hz, 1H,

9ax-H). HRMS (ESI) calculated for C₂₆H₃₇O₃: calculated 397.2743;

found 397.2736.

(6aR,9R,10aR)-3-(Adamantan-1-yl)-9-(iodomethyl)-6, 6-di-

methyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol

(12). To a solution of 11 (250 mg, 0.63 mmol) in 10 mL anhydrous

benzene under argon was added triphenylphosphine (331 mg, 1.26

mmol), imidazole (258 mg, 3.78 mmol) and iodine (320 mg, 1.26

mmol), and the resulting solution was reﬂuxed for 1 h. The mixture

was cooled to room temperature, diluted with ether, washed with

water, aqueous sodium thiosulfate, and brine, dried (MgSO₄), and

evaporated under reduced pressure. Puriﬁcation by ﬂash chromatog-

raphy (EtOAc/hexane = 2/98 → 10/90) gave 12 as white solid (243

1

mg, 76%). R_f= 0.41 (EtOAc/hexane = 5/95). H NMR (500 MHz,

CDCl₃) δ: 6.40 (d, J = 2.0 Hz, 1H, ArH), 6.23 (d, J = 2.0 Hz, 1H,

ArH), 4.71 (s, 1H, ArOH), 3.28 (m as dd, J = 13.0 Hz, J = 2.0 Hz, 1H,

10eq-H), 3.22 (dd, J = 9.0 Hz, J = 6.0 Hz, 1H, −CH₂I), 3.14 (dd, J =

9.0 Hz, J = 7.0 Hz, 1H, −CH₂I), 2.49 (ddd as dt, J = 11.0 Hz, J = 2.5

Hz, 1H, 10a-H), 2.10−2.02 (m and br s overlapping, 4H, especially

2.05, br s, 3H, Ad-H), 1.94−1.88 (m, 1H), 1.83 (br s, 6H, Ad-H),

1.79−1.64 (m, 7H, especially 1.75, d, J = 12.5 Hz, 3H, Ad-H and 1.70,

d, J = 11.5 Hz, 3H, Ad-H), 1.46 (td, J = 11.0 Hz, J = 2.5 Hz, 1H, 6a-

H), 1.38 (s, 3H, 6β-CH₃), 1.19−1.10 (m, 2H), 1.07 (s, 3H, 6α-CH₃),

0.88 (q, J = 12.0 Hz, 1H, 9ax-H). HRMS (ESI) calculated for

C₂₆H₃₆O₂I: calculated 507.1760; found 507.1761.

2-((6aR,9R,10aR)-3-(Adamantan-1-yl)-1-hydroxy-6,6-dimeth-

yl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-9-yl)-

acetonitrile (13). To a solution of 12 (70 mg, 0.138 mmol) in

DMSO (4 mL) at room temperature under an argon atmosphere was

added NaCN (34 mg, 0.691 mmol). After being stirred at the same

temperature for 24 h, the reaction mixture was cooled to 0 °C and

diluted with water and ethyl acetate. The organic layer separated and

the aqueous layer extracted with EtOAc (3×). Combined organic layer

was washed with water and brine, dried (MgSO₄), and evaporated

under reduced pressure. Puriﬁcation by ﬂash chromatography on silica

gel (EtOAc/hexane = 20/80 → 50/50) gave 13 as a white foam (40.5

mg, 72%)). R_f= 0.49 (EtOAc/hexane = 30/70). ¹H NMR (500 MHz,

CDCl₃) δ: 6.40 (d, J = 2.0 Hz, 1H, ArH), 6.23 (d, J = 2.0 Hz, 1H,

(6aR,9R,10aR)-3-(Adamantan-1-yl)-9-(2-hydroxyethyl)-6,6-

dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-

ol (16). To a solution of aldehyde 15 (100 mg, 0.245 mmol) in 10 mL

of methanol at room temperature was added NaBH₄(46.3 mg, 1.224

mmol) portionwise. The reaction mixture was stirred for 30 min and

quenched with 10% acetic acid, and the mixture was diluted with ether.

The aqueous phase was extracted twice with ethyl acetate, and the

combined organic extract was washed with brine and dried (MgSO₄).

The solvent was evaporated, and the crude was chromatographed

(EtOAc/hexane = 30/70 → 70/30) to produce pure β-alcohol 16 as a

1

white solid (98 mg, 98%). R_f= 0.41 (EtOAc/hexane = 50/50). H

NMR (500 MHz, CDCl₃) δ: 6.43 (d, J = 2.0 Hz, 1H, ArH), 6.27 (d, J

L

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= 2.0 Hz, 1H, ArH), 4.92 (s, 1H, ArOH), 3.82−3.70 (m, 2H), 3.17 (m

as br d, J = 13.5 Hz, 1H, 10eq-H), 2.46 (ddd as td, J = 11.5 Hz, J = 2.5

Hz, 1H, 10a-H), 2.05 (br s, 3H, Ad-H), 1.96−1.82 (m, 8H especially

1.83, br s, 6H, Ad-H), 1.79−1.66 (m, 7H, especially 1.75, d, J = 12.5

Hz, 3H, Ad-H and 1.70, d, J = 11.5 Hz, 3H, Ad-H), 1.58−1.51 (m,

2H), 1.48 (td, J = 11.5 Hz, J = 2.5 Hz, 1H, 6a-H), 1.37 (s, 3H, 6β-

CH₃)1.25 (br s, 1H, OH), 1.16−1.05 (m and s overlapping, 5H,

especially 1.07, s, 3H, 6α-CH₃), 0.82 (q, J = 12.5 Hz, 1H). HRMS

(ESI) calculated for C₂₇H₃₉O₃: calculated 411.2899; found 411.2904.

(6aR,9S,10aR)-3-(Adamantan-1-yl)-9-(hydroxymethyl)-6,6-

dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-

ol (17). To a solution of aldehyde 8 (200 mg, 0.507 mmol) in 15 mL

of methanol at room temperature was added NaBH₄(96 mg, 2.53

mmol) portionwise. The reaction mixture was stirred for 30 min and

quenched with 10% acetic acid, and the mixture was diluted with ethyl

acetate. The aqueous phase was extracted twice with ethyl acetate, and

the combined organic extract was washed with brine and dried

(MgSO₄). The solvent was evaporated and the crude was chromato-

graphed (EtOAc/hexane = 30/70 → 60/40) to produce pure alcohol

17 (191 mg, 95% yield). R_f= 0.41 (EtOAc/hexane = 50/50). ¹H NMR

(500 MHz, CDCl₃) δ: 6.38 (d, J = 2.0 Hz, 1H, ArH), 6.31 (d, J = 2.0

Hz, 1H, ArH), 6.09 (s, 1H, ArOH), 3.90 (dd as br t, J = 10.5 Hz, 1H,

CH₂OH), 3.74 (dd, J = 10.5 Hz, J = 7.0 Hz, 1H, CH₂OH), 3.17 (m as

dd, J = 14.0 Hz, J = 1.5 Hz, 1H, 10eq-H), 2.51−2.45 (m, 2H), 2.11 (br

s, 1H), 2.05 (br s, 3H, Ad-H), 1.83 (br s, 6H, Ad-H), 1.78−1.60 (m,

9H, especially 1.75, d, J = 12.0 Hz, 3H, Ad-H and 1.70, d, J = 12.0 Hz,

3H, Ad-H), 1.51 (td, J = 11.5 Hz, J = 2.5 Hz, 1H, 6a-H), 1.33 (s, 3H,

6β-CH₃), 1.29−1.08 (m, 2H), 0.93 (s, 3H, 6α-CH₃); HRMS (ESI)

calculated for C₂₆H₃₇O₃: calculated 397.2743; found 397.2751.

The solvent was evaporated, and the crude was chromatographed

(EtOAc/hexane = 30/70 → 70/30) to produce pure β-alcohol 19 (92

1

mg, 92% yield). R_f= 0.40 (EtOAc/hexane = 50/50). H NMR (500

MHz, CDCl₃) δ: 6.88 (br s, 1H, ArOH), 6.38 (d, J = 1.5 Hz, 1H,

ArH), 6.34 (d, J = 1.5 Hz, 1H, ArH), 4.02−3.92 (m, 1H, CH₂OH),

3.90−3.80 (m, 1H, CH₂OH), 2.96 (br d, J = 13.5 Hz, 1H, 10eq-H),

2.71 (br t, J = 10.5 Hz, 1H, 10a-H), 2.45 (br s, 1H, OH), 2.10−1.96

(m, 4H, especially 2.04, br s, 3H, Ad-H), 1.85 (br s, 6H, Ad-H), 1.78−

1.52 (m, 11H, especially 1.74, d, J = 12.0 Hz, 3H, Ad-H and 1.69, d, J =

13.0 Hz, 3H, Ad-H), 1.42−1.20 (m and s overlapping 5H, especially

1.36, s, 3H, 6β-CH₃), 1.08 (s, 3H, 6α-CH₃), 0.92−0.82 (m, 1H).

HRMS (ESI) calculated for C₂₇H₃₉O₃: calculated 411.2899; found

411.2903.

((6aR,10aR)-3-(Adamantan-1-yl)-1-hydroxy-6,6-dimethyl-

6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-9-yl)methyl Piv-

alate (21). To a solution of resorcinol 2 and pivalate ester 20

(0.413 g, 1.64 mmol) in anhydrous dichloromethane (40 mL) at −20

°C under argon atmosphere was added boron triﬂuoride etherate (1.03

mL, 8.18 mmol). The mixture was allowed to warm up to room

temperature and then stirred for further 2 h. The mixture was washed

with brine and dried (MgSO₄), and the solvent was evaporated under

reduced pressure. The crude product was then puriﬁed by ﬂash

chromatography on silica gel (EtOAc/hexane = 5/95 → 15/85) to

give the cannabinoid ester 21 as a white foam (0.25 g, 32% yield); R_f=

0.38 (EtOAc/hexane = 15/85). ¹H NMR (500 MHz, CDCl₃): δ: 6.43

(d, J = 2.0 Hz, 1H, ArH), 6.27 (d, J = 2.0 Hz, 1H, ArH), 5.75 (d, J =

4.5 Hz, 1H, −CHC<), 4.75 (s, 1H, ArOH), 4.50 (s, 2H), 3.35 (dd, J

= 16.0 Hz, J = 4.5 Hz, 1H), 2.71 (td, J = 11.0 Hz, J = 4.5 Hz, 1H),

2.28−2.20 (m, 1H), 2.05 (br s, 3H, Ad-H), 1.92−1.80 (m, 8H,

especially 1.84, br s, 6H, Ad-H), 1.79−1.68 (m, 7H, especially 1.75, d, J

= 12.0 Hz, 3H, Ad-H and 1.70, d, J = 11.5 Hz, 3H, Ad-H), 1.39 (s, 3H,

6β-CH₃), 1.21 (s, 9H), 1.12 (s, 3H, 6α-CH₃). HRMS (ESI) calculated

for C₃₁H₄₃O₄: calculated 479.3161; found 479.3156.

2-((6aR,9S,10aR)-3-(Adamantan-1-yl)-1-hydroxy-6,6-dimeth-

yl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-9-yl)-

acetaldehyde (18). To a suspension of (methoxymethyl)-

triphenylphosphonium chloride (4.17 g, 12.17 mmol) in 80 mL of

anhydrous THF at −30 °C was added a solution of n-BuLi in THF

(4.70 mL, 11.76 mmol, 2.5 M in hexane). The resulting blood red

colored solution was warmed to 0 °C over a period of 15 min. A

solution of aldehyde 8 (0.8 g, 2.03 mmol) in anhydrous THF (20 mL)

was then added through a cannula, keeping the reaction temperature at

0 °C. The resulting solution was stirred for 30 min and then quenched

by addition of water and stirred for 30 min till the solution turned

colorless. The reaction mixture was diluted with diethyl ether, the

organic phase separated, and the aqueous phase extracted with ether

(2×). Combined organic extracts was washed with brine and then

dried (MgSO₄). The residue was dissolved in 20 mL of CH₂Cl₂, wet

trichloroacetic acid (1.66 g, 10.15 mmol in 7 mL of water) was added,

and the resulting solution was stirred at room temperature for 45 min.

The reaction was quenched with saturated NaHCO₃. The organic

layer separated and aqueous phase was extracted with CH₂Cl₂(2×),

and the combined organic extracts were washed with water and brine

(1×), dried (MgSO₄), and concentrated to give crude aldehyde.

Puriﬁcation by ﬂash chromatography on silica gel (EtOAc/hexane =

20/80 → 40/60) gave aldehyde 18 as a white foam (657 mg, 82%

(6aR,10aR)-3-(Adamantan-1-yl)-9-(hydroxymethyl)-6, 6-di-

methyl-6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-1-ol (22).

To a solution of pivalate ester 21 (125 mg, 0.261 mmol) in anhydrous

THF (20 mL) at 0 °C under argon was added a solution of LiAlH₄in

THF (1.05 mL, 1.045 mmol, 1.0 M in THF), and the resulting mixture

was stirred at same temperature for 2 h. The reaction was quenched by

addition of water and extracted with ethyl acetate (3×). Combined

organic layer was washed with brine, dried (MgSO₄), and concentrated

under reduced pressure. Puriﬁcation of the crude by ﬂash

chromatography on silica gel (EtOAc/hexane = 30/70 → 70/30)

gave allylic alcohol 22 as a white solid (88 mg, 85% yield). R_f= 0.50

1

(EtOAc/hexane = 50/50). H NMR (500 MHz, CDCl₃) δ: 6.40 (d, J

= 1.5 Hz, 1H, ArH), 6.28 (d, J = 1.5 Hz, 1H, ArH), 6.12 (s, 1H,

ArOH), 5.73 (d, J = 4.5 Hz, 1H, −CHC<), 3.53−3.42 (m, 2H),

2.68 (td, J = 11.0 Hz, J = 4.5 Hz, 1H), 2.29 (br s, 1H), 2.25−2.15 (m,

1H), 2.03 (br s, 3H, Ad-H), 1.90−1.76 (m, 10H, especially 1.81, br s,

6H, Ad-H), 1.75 (d, J = 12.5 Hz, 3H, Ad-H), 1.69 (d, J = 11.5 Hz, 3H,

Ad-H), 1.37 (s, 3H, 6β-CH₃), 1.05 (s, 3H, 6α-CH₃). HRMS (ESI)

calculated for C₂₆H₃₅O₃: calculated 395.2586; found 395.2583.

(1-Acetoxy-3-(adamantan-1-yl)-6,6-dimethyl-6H-benzo[c]-

chromen-9-yl)methyl Pivalate (23). To a solution of 21 (1.0 g,

2.089 mmol) in dry pyridine (5 mL) at 0 °C was added acetic

anhydride (0.97 mL, 10.45 mmol), the resulting solution was warmed

to room temperature and stirred overnight. The reaction was cooled to

0 °C, quenched by addition of water, and diluted with ether. The

organic layer was separated and the aqueous layer extracted with ether

(3×). Combined organic layer was washed with water (2×), aqueous

NaHCO₃, and brine, dried (MgSO₄), and evaporated in vacuo. The

crude was mixed with sulfur (0.67 g, 20.89 mmol), and the resulting

solid mixture was heated to 250 °C for 2 h. The reaction mixture was

cooled to room temperature, dissolved in ethyl acetate, ﬁltered, and

washed with water and brine, dried (MgSO₄), and concentrated.

Puriﬁcation of crude by ﬂash chromatography on silica gel (EtOAc/

hexane = 5/95 → 20/80) yielded 23 as a white solid (0.37 g, 34%

1

overall yield). R_f= 0.24 (EtOAc/hexane = 20/80). H NMR (500

MHz, CDCl₃) δ: 9.84 (d, J = 2 Hz, 1H, CHO), 6.39 (d, J = 2.0 Hz,

1H, ArH), 6.24 (d, J = 2.0 Hz, 1H, ArH), 4.97 (br s, 1H, ArOH), 3.00

(m as br dd, J = 14.0 Hz, J = 1.5 Hz 1H, 10eq-H), 2.76 −2.66 (m, 1H),

2.64−2.52 (m, 3H), 2.04 (br s, 3H, Ad-H), 1.83 (br s, 6H, Ad-H),

1.80−1.64 (m, 8H, especially 1.75, d, J = 13.0 Hz, 3H, Ad-H and 1. 69,

d, J = 10.5 Hz, 3H, Ad-H), 1.52 (td, J = 12.0 Hz, J = 2.5 Hz, 1H, 6a-

H), 1.44−1.32 (m and s overlapping, 4H, especially 1.37, s, 3H, 6β-

CH₃), 1.30−1.18 (m, 2H), 1.08 (s, 3H, 6α-CH₃); HRMS (ESI)

calculated for C₂₇H₃₇O₃: calculated 409.2743; found 409.2742.

(6aR,9S,10aR)-3-(Adamantan-1-yl)-9-(2-hydroxyethyl)-6,6-

dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-

ol (19). To a solution of aldehyde 18 (100 mg, 0.245 mmol) in 10 mL

of methanol at room temperature was added NaBH₄(46.3 mg, 1.224

mmol) portionwise. The reaction mixture was stirred for 30 min and

quenched with 10% acetic acid, and the mixture diluted with ether.

The aqueous phase was extracted twice with ethyl acetate, and the

combined organic extract was washed with brine and dried (MgSO₄).

1

overall yield). R_f= 0.50 (EtOAc/hexane = 15/85). H NMR (500

MHz, CDCl₃) δ: 7.96 (s, 1H, ArH), 7.25 (br s, 2H, ArH), 6.89 (d, J =

2.0 Hz, 1H, ArH), 6.73 (d, J = 2.0 Hz, 1H, ArH), 5.10 (s, 2H, CH₂O-

M

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Piv), 2.35 (s, 3H, CH₃COO), 2.09 (s, 3H, Ad-H), 1.90 (br s, 6H, Ad-

H), 1.78 (d, J = 12.0 Hz, 3H, Ad-H), 1.73 (d, J = 11.5 Hz, 3H, Ad-H),

1.62 (s, 6H), 1.23 (s, 9H). HRMS (ESI) calculated for C₃₃H₄₁O₅:

calculated 517.2954; found 517.2958.

= 12.0 Hz, 3H, Ad-H), 1.73 (d, J = 11.5 Hz, 3H, Ad-H), 1.60 (s, 6H, 2

× CH₃). HRMS (ESI) calculated for C₂₆H₃₁O₂: calculated 375.2324;

found 375.2318.

5-(1-Adamantyl)-2-bromo-1,3-dimethoxybenzene (29). Bro-

mine (0.38 mL, 7.34 mmol) was added dropwise to a stirred solution

of 28 (5-(1-adamantyl)-1,3-dimethoxybenzene)³⁰(2.0 g, 7.34 mmol)

and 18-crown-6 (0.194 g, 0.734 mmol) in 74 mL of anhydrous CH₂Cl₂

at 0 °C. The reaction mixture was stirred at 0 °C for 30 min and

quenched by addition of saturated aqueous sodium bisulﬁte solution.

The organic layer separated, washed with water and brine, and then

dried (MgSO₄). Evaporation of volatiles under reduced pressure gave

29 as white solid (2.58 g, quantitative) which was >98% pure by NMR

3-(Adamantan-1-yl)-9-(hydroxymethyl)-6,6-dimethyl-6H-

benzo[c]chromen-1-yl Acetate (24). To a solution of ester 23 (150

mg, 0.29 mmol) in anhydrous THF (20 mL) at 0 °C under argon was

added a solution of LiAlH₄in THF (0.32 mL, 0. 32 mmol, 1.0 M in

THF), and the resulting mixture was stirred at same temperature for 2

h. The reaction was quenched by addition of water and extracted with

ethyl acetate (3×). Combined organic layer was washed with brine,

dried (MgSO₄), and concentrated under reduced pressure. Puriﬁcation

of the crude by ﬂash chromatography on silica gel (EtOAc/hexane =

5/95 → 15/85) gave alcohol 24 as a white solid (103 mg, 91% yield).

1

and used for the next reaction without further puriﬁcation. H NMR

(500 MHz, CDCl₃) δ: 6.59 (s, 2H, ArH), 3.91 (s, 6H, 2 x OCH₃), 2.11

(br s, 3H, Ad-H), 1.91 (br s, 6H, Ad-H), 1.81 (d, J = 12.5 Hz, 3H, Ad-

H), 1.76 (d, J = 12.0 Hz, 3H, Ad-H). HRMS (ESI) for C₁₈H₂₄BrO₂:

calculated 351.0960; found, 351.0959.

1

M. P. = 134−135 °C. R_f= 0.48 (EtOAc/hexane = 50/50). H NMR

(500 MHz, CDCl₃) δ: 8.42 (s, 1H, ArH), 7.32−7.20 (m, 2H, ArH),

6.60 (d, J = 1.5 Hz, 1H, ArH), 6.45 (d, J = 1.5 Hz, 1H, Ar−H), 5.53 (s,

1H, ArOH), 4.73 (br s, 2H, CH₂OH), 2.08 (br s, 3H, Ad-H), 1.88 (br

s, 6H, Ad-H), 1.78 (d, J = 12.5 Hz, 3H, Ad-H), 1.73 (d, J = 11.5 Hz,

3H, Ad-H), 1.61 (s, 6H, 2xCH₃), 1.26 (br s, 1H, OH). HRMS (ESI)

calculated for C₂₆H₃₁O₃: calculated 391.2273; found 391.2268.

(6aR,10aR)-3-(Adamantan-1-yl)-6,6,9-trimethyl-6a,7,10,10a-

tetrahydro-6H-benzo [c]chromen-1-yl Acetate (25). To a

solution of 1 (800 mg, 2.113 mmol) in dry pyridine (5 mL) at 0

°C was added acetic anhydride (1.0 mL, 10.57 mmol), and the

resulting solution was warmed to room temperature and stirred

overnight. The reaction was cooled to 0 °C, quenched by addition of

water, and diluted with ether. The organic layer was separated and the

aqueous layer extracted with ether (3×). Combined organic layer was

washed with water (2×), aqueous NaHCO₃, and brine, dried,

(MgSO₄) and evaporated in vacuo. Puriﬁcation of the crude by ﬂash

chromatography on silica gel (EtOAc/hexane = 5/95 → 15/85) gave

acetoxy 25 as a white foam (782 mg, 88% yield). R_f= 0.47 (EtOAc/

hexane = 10/90). ¹H NMR (500 MHz, CDCl₃) δ: 6.71 (d, J = 2.0 Hz,

1H, ArH), 6.56 (d, J = 2.0 Hz, 1H, ArH), 5.43 (br d, J = 4.5 Hz, 1H,

CHC<), 2.72 (dd, J = 17.0 Hz, J = 5.0 Hz, 1H), 2.61 (td, J = 11.0

Hz, J = 4.5 Hz, 1H), 2.29 (s, 3H, CH₃CO), 2.17−2.09 (m, 1H), 2.06

(br s, 3H, Ad-H), 1.96−1.84 (m, 7H, especially 1.86, br s, 6H, Ad-H),

1.83−1.64 (m, 11H), 1.38 (s, 3H, 6β-CH₃), 1.11 (s, 3H, 6α-CH₃).

HRMS (ESI) calculated for C₂₈H₃₇O₃: calculated 421.2743; found

421.2739.

4′-(Adamantan-1-yl)-N,N-diisopropyl-2′,5,6′-trimethoxy-

[1,1′-biphenyl]-2-carboxamide (31). Argon was bubbled through a

mixture of boronic acid 30 (0.715 g, 2.562 mmol),⁴⁸29 (0.75 g, 2.135

mmol), Ba(OH)₂.8H₂O (1.01 g, 3.203 mmol), 2.5 mL of water, and

16 mL of dimethoxyethane for 10 min. The Pd(PPh₃)₄(0.247 g, 0.213

mmol) catalyst was added to the mixture while argon bubbling was

maintained through the mixture, and degassing was continued for an

additional 5 min. The reaction mixture was microwaved for 25 min at

160 °C in a CEM Discover apparatus. Then the mixture was cooled to

room temperature and ﬁltered through a short Celite pad. The ﬁltrate

was concentrated, and Et₂O was added. The ether layer was washed

with water and brine and dried (MgSO₄). Evaporation of solvent

under reduced pressure gave a crude product which was puriﬁed by

ﬂash chromatography (EtOAc/hexane: 30/70 → 40/60) on silica gel

to aﬀord biphenyl 31 as a white solid (0.776 g, 71.9% yield). R_f= 0.50

1

(EtOAc/hexane = 20/80). H NMR (500 MHz, CDCl₃) δ: 7.25 (d, J

= 9.0 Hz, 1H, ArH), 6.86 (dd, J = 9.0 Hz, J = 3.0 Hz, 1H, ArH), 6.80

(d, J = 3.0 Hz, 1H, ArH), 6.58 (s, 1H, ArH), 6.56 (s, 1H, ArH), 3.80

(s, 3H, OMe), 3.73 (s, 3H, OMe), 3.72 (s, 3H, OMe), 3.68 (sept, J =

6.5 Hz, 1H, (CH₃)₂CH), 3.17 (sept, J = 6.5 Hz, 1H, (CH₃)₂CH), 2.12

(br s, 3H, Ad-H), 1.94 (br s, 6H, Ad-H), 1.84−1.74 (m, 6H, Ad-H),

1.45 (d, J = 6.5 Hz, 3H, (CH₃)₂CH), 1.07 (d, J = 6.5 Hz, 3H,

(CH₃)₂CH), 0.91 (d, J = 6.5 Hz, 3H, (CH₃)₂CH), 0.55 (d, J = 6.5 Hz,

3H, (CH₃)₂CH), HRMS (ESI) for C₃₂H₄₄NO₄: calculated 506.3270;

found 506.3268.

3-(Adamantan-1-yl)-6,6,9-trimethyl-6H-benzo[c]chromen-1-

yl Acetate (26). Compound 25 (600 mg, 1.427 mmol) was mixed

with sulfur (457 mg, 14.27 mmol), and the resulting solid mixture was

heated at 250 °C for 2 h. The reaction mixture was cooled to room

temperature, dissolved in ethyl acetate, ﬁltered, and washed with water

and brine, dried (MgSO₄), and concentrated. Puriﬁcation of the crude

by ﬂash chromatography on silica gel (EtOAc/hexane = 5/95 → 15/

85) gave 26 as a white solid (220 mg, 37% yield). R_f= 0.40 (EtOAc/

hexane = 10/90). ¹H NMR (500 MHz, CDCl₃) δ: 7.80 (s, 1H, ArH),

7.13 (d, J = 8.0 Hz, 1H, ArH), 7.08 (dd, J = 8.0 Hz, J = 1.0 Hz, 1H,

ArH), 6.88 (d, J = 2.0 Hz, 1H, ArH), 6.71 (d, J = 2.0 Hz, 1H, ArH),

2.36 (s, 3H), 2.32 (s, 3H), 2.09 (br s, 3H, Ad-H), 1.90 (br s, 6H, Ad-

H), 1.78 (d, J = 12.0 Hz, 3H, Ad-H), 1.73 (d, J = 11.5 Hz, 3H, Ad-H),

1.60 (s, 6H, 2xCH₃). HRMS (ESI) calculated for C₂₈H₃₃O₃: calculated

417.2430; found 417.2424.

3-(-Adamantan-1-yl)-6,6,9-trimethyl-6H-benzo[c]chromen-

1-ol (27). To a stirred solution of 26 (110 mg, 0.264 mmol) in 10 mL

of EtOH at room temperature was added an aqueous solution of KOH

(59.3 mg, 1.06 mmol, in 3 mL of water), and the resulting solution was

stirred at room temperature for 30 min. The reaction mixture was

neutralized by addition of 1 N HCl solution and extracted with ethyl

acetate (3×). The combined organic layer was washed with water and

brine, dried (MgSO₄), and concentrated. Puriﬁcation by ﬂash

chromatography on silica gel (EtOAc/hexane = 5/95 → 20/80)

gave 27 as a white solid (96 mg, 97% yield). R_f= 0.50 (EtOAc/hexane

= 20/80). ¹H NMR (500 MHz, CDCl₃) δ: 8.15 (s, 1H, ArH), 7.14 (d,

J = 8.0 Hz, 1H, ArH), 7.07 (dd, J = 8.0 Hz, J = 1.0 Hz, 1H, ArH), 6.60

(d, J = 2.0 Hz, 1H, ArH), 6.44 (d, J = 2.0 Hz, 1H, ArH), 5.11 (s, 1H,

ArOH), 2.38 (s, 3H), 2.08 (br s, 3H), 1.89 (br s, 6H, Ad-H), 1.78 (d, J

3-(1-Adamantyl)-1-hydroxy-9-methoxy-6H-benzo[c]-

chromen-6-one (32). A solution of 31 (1.20 g, 2.37 mmol) in 25 mL

of anhydrous CH₂Cl₂was cooled to 0 °C, and 9-iodo-9-BBN (9.50

mL, 1.0 M solution in hexane) was added dropwise. The reaction

mixture was stirred at 0 °C for 4 h. It was then warmed to rt and

concentrated under reduced pressure, and the residue was dissolved in

anhydrous diethyl ether (50 mL). To this mixture was added 10 mL of

ethanolamine solution (1.0 M in ether). The reaction mixture was

stirred for 40 min and then ﬁltered through a short Celite column. The

ﬁltrate was concentrated and dissolved in 10 mL of glacial acetic acid.

The reaction mixture was reﬂuxed for 5 h and then cooled to room

temperature, and water was added cautiously to the mixture at 0 °C

followed by addition of ether (50 mL). The organic layer separated,s

washed with water, 15% aqueous NaHCO₃, water, and brine, and then

dried over MgSO₄. Evaporation of volatiles under reduced pressure

gave crude product that was chromatographed (EtOAc/hexane, 10/90

→ 40/60) on silica gel to give 32 as a white solid (0.615 g, 68.8%

yield). R_f= 0.42 (EtOAc/hexane = 30/70); ¹H NMR (CDCl₃) δ: 8.57

(d, J = 2.5 Hz, 1H, ArH), 8.34 (d, J = 8.5 Hz, 1H, ArH), 7.55 (s, 1H,

ArOH), 7.05 (dd, J = 9.0 Hz, J = 2.5 Hz, 1H, ArH), 6.94 (d, J = 2.0 Hz,

1H, ArH), 6.80 (d, J = 2.0 Hz, 1H, ArH), 3.96 (s, 3H, OCH₃), 2.12 (br

s, 3H, Ad-H), 1.90 (br s, 6H, Ad-H), 1.81 (d, J = 12.5 Hz, 3H, Ad-H),

1.75 (d, J = 12.5 Hz, 3H, Ad-H). HRMS (ESI) for C₂₄H₂₅O₄:

calculated 377.1753; found 377.1751.

3-(1-Adamantyl)-9-methoxy-6,6-dimethyl-6H-benzo[c]-

chromen-1-ol (33). To a solution of 32 (0.4 g, 1.06 mmol) in

anhydrous THF (22 mL) was added methylmagnesium iodide (1.77

mL, 3.0 M solution in ether, 5.30 mmol) at room temperature under

N

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Journal of Medicinal Chemistry

Article

an argon atmosphere. The reaction mixture was stirred at room

temperature for 30 min and then reﬂuxed for 2 h. The reaction was

cooled to room temperature and quenched by addition by saturated

aqueous NH₄Cl (30 mL). THF was removed, and the residue was

dissolved in diethyl ether (50 mL). The organic phase separated,

washed with water and brine, and dried (MgSO₄). Evaporation of

volatiles under reduced pressure gave the crude intermediate that was

used without further puriﬁcation in the subsequent cyclization

reaction. The crude was dissolved in CHCl₃(15 mL), and p-

toluenesulfonic acid monohydrate (50 mg; 0.262 mmol) was added

under argon atmosphere. The reaction was stirred at room

temperature for 6 h and then treated with 10 mL of water. The

organic phase was separated, washed with saturated aqueous NaHCO₃,

water, and brine, and then dried (MgSO₄). Solvent removal under

reduced pressure gave the crude product that was chromatographed

(EtOAc/hexane = 10/90 → 20/80) to give 33 as a white crystalline

solid (0.3g mg, 72.5% overall yield). R_f= 0.39 (EtOAc/hexane = 20/

80).¹H NMR (500 MHz, CDCl₃) δ: 7.99 (d, J = 3.0 Hz, 1H), 7.16 (d,

J = 9.0 Hz, 1H), 6.79 (dd, J = 9.0 Hz, J = 3.0 Hz, 1H), 6.60 (d, J = 2.0

Hz, 1H), 6.43 (d, J = 2.0 Hz, 1H), 5.18 (br s, 1H, ArOH), 3.84 (s, 3H,

OCH₃), 2.08 (br s, 3H, Ad-H), 1.88 (br d, J = 2.5 Hz, 6H, Ad-H), 1.78

(d, J = 12.0 Hz, 3H, Ad-H), 1.73 (d, J = 12.0 Hz, 3H, Ad-H), 1.60 (s,

6H, 2 × CH₃). HRMS (ESI) for C₂₆H₃₁O₃: calculated 391.2273;

found 391.2279.

3-(1-Adamantyl)-1,9-dihydroxy-6H-benzo[c]chromen-6-one

(34). To a suspension of 32 (105 mg, 0.279 mmol) in anhydrous

CH₂Cl₂(15 mL) was added a solution of boron tribromide (0.56 mL,

1.0 M in CH₂Cl₂) at room temperature under an argon atmosphere.

The reaction mixture was stirred at the same temperature for 30 min

and then reﬂuxed for 24 h. The reaction was then cooled and

quenched by addition of ice−water and diluted with ethyl acetate. The

aqueous layer was extracted with ethyl acetate (2×), and the combined

organic layer was washed with 15% aq NaHCO₃, water, and brine and

then dried (MgSO₄). Evaporation of volatiles under reduced pressure

gave a crude product that was chromatographed (EtOAc/hexane =

30/70 → 80/20) to give 34 as a white solid (86 mg, 85% yield). R_f=

0.45 (EtOAc/hexane = 50/50). ¹H NMR (500 MHz, CDCl₃) δ: 10.76

(br s, 2H, ArOH), 8.49 (d, J = 2.0 Hz), 8.10 (d, J = 9.0 Hz, 1H), 6.97

(dd, J = 9.0 Hz, J = 2.5 Hz, 1H), 6.88 (d, J = 2.0 Hz, 1H), 6.80 (d, J =

2.0 Hz, 1H), 2.07 (br s, 3H), 1.86 (br s, 6H), 1.80−1.70 (m, 6H).

HRMS (ESI) for C₂₃H₂₃O₄: calculated 363.1596; found 363.1598.

3-(1-Adamantyl)-6,6-dimethyl-6H-benzo[c]chromene-1,9-

diol (35). This compound was prepared analogously to 33, starting

from 34 (0.30 g, 0.828 mmol) in THF (16 mL), methylmagnesium

iodide (1.38 mL, 3.0 M solution in ether, 4.14 mmol), and cyclization

using p-TSA·H₂O (50 mg, 0.262 mmol). Puriﬁcation of crude by ﬂash

chromatography on silica gel (EtOAc/hexane = 10/90 → 20/80) gave

35 as a white crystalline solid (180 mg, 56% overall yield). R_f= 0.42

(EtOAc/hexane = 30/70).¹H NMR (500 MHz, CDCl₃) δ: 7.92 (d, J =

2.5 Hz, 1H), 7.09 (d, J = 9.0 Hz, 1H), 6.73 (dd, J = 9.0 Hz, J = 2.5 Hz,

1H), 6.58 (d, J = 1.5 Hz, 1H), 6.40 (d, J = 1.5 Hz, 1H), 5.58 (br s, 1H,

ArOH), 5.30 (br s, 1H, ArOH), 2.05 (br s, 3H, Ad-H), 1.84 (br d, J =

2.0 Hz, 6H, Ad-H), 1.76 (d, J = 12.5 Hz, 3H, Ad-H), 1.70 (d, J = 12.5

Hz, 3H, Ad-H), 1.58 (s, 6H, 2 × CH₃). HRMS (ESI) for C₂₅H₂₉O₃:

calculated 377.2117; found 377.2114.

tracer and Ulight-anti-cAMP were added to the plate and incubated at

room temperature for 1 h prior to detection via PerkinElmer Envision;

data were analyzed using GraphPad Prism 5.0 software.³¹

β-Arrestin2 Translocation Assay. U2OS cells stably expressing

the CB1-E cannabinoid receptors and β-arrestin2-GFP were split into

glass-bottom 384 well plates (MGB101-1-2-LG, MatriCal, Spokane,

WA) at a density of 8000 cells/30 μL media/well using a Multidrop

384 dispenser (Thermo Fisher Scientiﬁc, Waltham, MA). The plates

were incubated overnight at 37 °C in 5% CO₂. The following day,

culture medium was changed to 30 μL/well of clear minimum Eagle’s

medium (MEM) with 10 mM HEPES, and then the cells were treated

with a serial concentration of test compounds with WIN55212-2 as a

positive control. A set of serial diluted 4× concentration of each

compound (10 mM in DMSO) was prepared in the same medium and

applied to cells at a volume of 10 μL (ﬁnal DMSO concentration

<1%). The cells were incubated with compound for 40 min at 37 °C

prior to ﬁxation with an equal volume of PBS containing 2%

paraformaldehyde (Sigma, St. Louis, MO). Plates were stored at 4 °C

until analysis. β-arrestin2-GFP aggregates were identiﬁed as

described.⁵⁷Dose response curves were analyzed by nonlinear

regression techniques using GraphPad Prism version 5.0 (GraphPad

Software, La Jolla, CA), and data were ﬁtted to sigmoidal dose−

response curves to obtain EC₅₀and eﬃcacy values.

Single-Crystal X-ray Diﬀraction Analysis of 5. A clear rod of

dimensions 0.33 × 0.07 × 0.06 mm²was mounted on a MiteGen

MicroMesh using a small amount of Cargille Immersion Oil. Data

were collected on a Bruker three-circle platform diﬀractometer

equipped with a SMART APEX II/Platinum 135 CCD detector.

The crystals were irradiated using graphite monochromated Mo K_α

radiation (λ = 0.71073). An Oxford Cobra low temperature device was

used to keep the crystals at a constant 113(2) K during data collection.

Data collection was performed, and the unit cell was initially reﬁned

using APEX2 [v2010.3-0].⁶⁰Data reduction was performed using

SAINT [v7.68A]⁶¹and XPREP [v2008/2].⁶²Corrections were applied

for Lorentz, polarization, and absorption eﬀects using SADABS

[v2008/1].⁶³The structure was solved and reﬁned with the aid of

the programs in the SHELXTL-plus [v2008/4] system of programs.⁶⁴

The full-matrix least-squares reﬁnement on F²included atomic

coordinates and anisotropic thermal parameters for all non-H atoms.

The H atoms were included using a riding model.

C₂₈H₃₈O₄, FW = 438.58, hexagonal, P3₁, a = 12.9968(13) Å, b=

12.9968(13) Å, c = 49.145(10) Å, α = 90°, β = 90°, γ = 120°, V =

7189.2(18) Å³, Z = 12, ρ_calc= 1.216 mg/m³, μ = 0.079 mm⁻¹, F(000)

= 2472, R₁= 0.0644 for 12705 observed (I > 2σI) reﬂections and

0.1018 for all 18166 reﬂections, goodness-of-ﬁt = 1.054, 1165

parameters.

In Vivo Studies. Subjects. Female Sprague−Dawley rats (n = 5−

7/group), weighing between 235 and 350 g (Charles River,

Wilmington, MA). Rats were tested repeatedly with at least seven

days intervening between drug sessions. Outside of experimental

sessions, rats were group housed (2/cage) in a climate-controlled

vivarium with unrestricted access to food and water.

Procedure. The temperature was recorded using a thermistor probe

(Model 401, Measurement Specialties, Inc., Dayton, OH) inserted to a

depth of 7 cm and secured to the tail with micropore tape. Rats were

minimally restrained and isolated in 38 × 50 × 10 cm plastic stalls.

The temperature was read to the nearest 0.01 °C using a thermometer

(Model 4000A, Measurement Specialties, Inc.). Two baseline

temperature measurements 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 1 h 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.

Radioligand Binding Assays: rCB1, hCB2, and mCB2. All

compounds synthesized were tested for their ability to bind to CB1

and CB2 receptors using rat brain or HEK293 cell membranes

expressing hCB2 membrane preparations, respectively, as previously

described via competition-equilibrium binding using [³H]CP-

55,940.⁵³⁻⁵⁵The results were analyzed using nonlinear regression to

determine the actual IC₅₀of the ligand by GraphPad Prism 5.0

Software (GraphPad, San Diego, CA), and the K_ivalues were

56

calculated from the IC_50.

cAMP Assay. HEK-293 cells transfected with rCB1, mCB2, or

hCB2 receptor were used with the PerkinElmer Lance ultra cAMP kit

following the protocol of the manufacturer. Brieﬂy, the assays were

carried out in 384-well format using 1000 cells/well. Test compounds

were added to wells containing stimulation buﬀer and 2 μM forskolin

followed by cell suspension. After 30 min stimulation, the Eu-cAMP

Antinociception was measured using a modiﬁed version of the tail-

ﬂick procedure of D’Amour and Smith.⁶⁵Radiant heat from a halogen

lamp was focused on the tail using a commercial tail-ﬂick apparatus

(model no. LE7106, Harvard Apparatus, Holliston, MA); movement

of the tail activated a photocell, tuning oﬀ the lamp and a reaction

timer. The lamp intensity was adjusted to yield baseline values of 2−3

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Journal of Medicinal Chemistry

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s, and a maximum latency of 6.0 s was imposed to avoid damage to the

tail. Two baseline tail-ﬂick latencies were obtained in each rat at 10

min intervals, and drugs were injected immediately after the second

baseline was recorded. Tail-ﬂick responses were recorded at 30, 60,

120, 180, and 360 min after injection.

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Drugs. All compounds were initially dissolved in a solution of 20%

ethanol, 20% emulphor, and 60% saline and were further diluted with

saline. Injections were administered sc in a volume of 1.0 mL/kg.

Data Analysis. For each rat, the two baseline values recorded prior

to drug injection were averaged to obtain a single baseline value.

Temperatures recorded after drug injection are expressed as a change

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followed by Dunnett’s multiple comparison t test; p was set at

<0.05, and statistical analyses were performed using GraphPad Prism

5.03 (GraphPad Software, San Diego, CA).

ASSOCIATED CONTENT

■

S

* Supporting Information

Crystal data and structure reﬁnement, atomic coordinates and

equivalent isotropic displacement parameters, bond lengths and

angles, anisotropic displacement parameters, hydrogen coor-

dinates and isotropic displacement parameters, and torsion

angles for compound 5. This material is available free of charge

via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

■

Corresponding Author

*(G.A.T.) Phone: +1-617-373-8163; fax: +1-617-373-8886; e-

mail: g.thakur@neu.edu. (A.M.) Phone: +1-617-373-4200; fax:

+1-617-373-7493; e-mail: a.makriyannis@neu.edu.

Notes

The authors declare no competing ﬁnancial interest.

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ACKNOWLEDGMENTS

■

This work was supported by grants from the National Institute

on Drug Abuse (DA07215 (A.M.), DA3801 (A.M.), PO1

DA09158 (A.M.), DA027113 (G.A.T.), and P30-DA029925

(L.S.B.; M.G.C.). The authors also thank the Oﬃce of Naval

Research (Award No. N00014-11-AF-0-0002). We are thankful

to Jodi Wood for her help with radioligand binding and cAMP

assays.

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ABBREVIATIONS USED

■

CB1, cannabinoid receptor 1; CB2, cannabinoid receptor 2;

hCB1 human cannabinoid 1; Δ⁸-THC, (−)-Δ⁸-tetrahydrocan-

nabinol; GPCRs, G-protein-coupled receptors; 2-AG, 2-

arachidonoylglycerol; SAR, structure−activity relationship;

NAH, northern aliphatic hydroxyl; SAH, southern aliphatic

hydroxyl; TMSOTf, trimethylsilyl triﬂuoromethanesulfonate;

cAMP, cyclic adenosine monophosphate; HEPES, (4-(2-

hydroxyethyl)-1-piperazineethanesulfonic acid; MPE, maximum

possible eﬀect

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cannabinoid activity. Life Sci. 1990, 47, 2173−2184.

(20) Gareau, Y.; Dufresne, C.; Gallant, M.; Rochette, C.; Sawyer, N.;

Slipetz, D. M.; Tremblay, N.; Weech, P. K.; Metters, k. M.; Labelle, M.

Structure activity relationship of tetrahydrocannabinol analogues on

P

dx.doi.org/10.1021/jm4000775 | J. Med. Chem. XXXX, XXX, XXX−XXX

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human cannabinoid receptors. Bioorg. Med. Chem. Lett. 1996, 6, 189−

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receptor. Curr. Pharm. Des. 2000, 6, 1323−1337.

(22) Huffman, J. W. CB2 receptor ligands. Mini Rev. Med. Chem.

2005, 5, 641−649.

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