3-(1',1'-Dimethylbutyl)-1-deoxy-Δ8-THC and related compounds: Synthesis of selective ligands for the CB2 receptor

Article abstract of DOI:10.1016/S0968-0896(99)00219-9

The synthesis and pharmacology of 15 1-deoxy-Δ⁸-THC analogues, several of which have high affinity for the CB₂ receptor, are described. The deoxy cannabinoids include 1-deoxy-11-hydroxy-Δ⁸-THC (5), 1-deoxy-Δ⁸-THC (6), 1-deoxy-3-butyl-Δ⁸-THC (7), 1-deoxy-3-hexyl-Δ⁸-THC (8) and a series of 3-(1',1'-dimethylalkyl)-1-deoxy-Δ⁸-THC analogues (2, n=0-4, 6, 7, where n=the number of carbon atoms in the side chain-2). Three derivatives (17-19Scheme 3(a) (C₆H₅)₃PCH₃⁺ Br^-, n-BuLi/THF, 65°C; (b) LiAlH₄/THF, 25°C; (c) KBH(sec-Bu)₃/THF, -78 to 25°C then H₂O₂/NaOH.) of deoxynabilone (16) were also prepared. The affinities of each compound for the CB₁ and CB₂ receptors were determined employing previously described procedures. Five of the 3-(1',1'-dimethylalkyl)-1-deoxy-Δ⁸-THC analogues (2, n=1-5) have high affinity (K(i)=<20nM) for the CB₂ receptor. Four of them (2, n=1-4) also have little affinity for the CB₁ receptor (K(i)=>295nM). 3-(1',1'-Dimethylbutyl)-1-deoxy-Δ⁸-THC (2, n=2) has very high affinity for the CB₂ receptor (K(i)=3.4±1.0nM) and little affinity for the CB₁ receptor (K(i)=677±132nM). Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00219-9

Bioorganic & Medicinal Chemistry 7 (1999) 2905±2914
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3-(1 ,1 -Dimethylbutyl)-1-deoxy-D-THC and Related Compounds:
Synthesis of Selective Ligands for the CB2 Receptor
John W. Hu€man, a,* John Liddle, a Shu Yu, a Mie Mie Aung, b
Mary E. Abood, b Jenny L. Wiley b and Billy R. Martin b
aHoward L. Hunter Laboratory, Clemson University, Clemson, SC 29634-1905, USA
bDepartment of Pharmacology and Toxicology, Medical College of Virginia,
Virginia Commonwealth University, Richmond, VA 23298-0613, USA
Received 18 May 1999; accepted 20 July 1999
AbstractÐThe synthesis and pharmacology of 15 1-deoxy-Á8-THC analogues, several of which have high anity for the CB2
receptor, are described. The deoxy cannabinoids include 1-deoxy-11-hydroxy-Á8-THC (5), 1-deoxy-Á8-THC (6), 1-deoxy-3-butyl-
Á8-THC (7), 1-deoxy-3-hexyl-Á8-THC (8) and a series of 3-(10,10-dimethylalkyl)-1-deoxy-Á8-THC analogues (2, n=0±4, 6, 7, where
n=the number of carbon atoms in the side chain 2). Three derivatives (17±19) of deoxynabilone (16) were also prepared. The
anities of each compound for the CB1 and CB2 receptors were determined employing previously described procedures. Five of the
3-(10,10-dimethylalkyl)-1-deoxy-Á8-THC analogues (2, n=1±5) have high anity (Ki=<20 nM) for the CB2 receptor. Four of them
(2, n=1±4) also have little anity for the CB1 receptor (Ki=>295 nM). 3-(10,10-Dimethylbutyl)-1-deoxy-Á8-THC (2, n=2) has very
high anity for the CB2 receptor (Ki=3.4‹1.0 nM) and little anity for the CB1 receptor (Ki=677‹132 nM). # 1999 Elsevier
Science Ltd. All rights reserved.
Introduction
had an order of magnitude lower anity for each receptor
than we reported.5 This group also reported that two 1-
methoxy cannabinoids, 1-methoxy-Á8-THC-DMH (3)
and 1-methoxy-Á9(11)-THC-DMH (4), had anities for the
CB2 receptor in the 20 nM range, and virtually no anity
for the CB1 receptor.5 This is in contrast to traditional
cannabinoids with a phenolic hydroxyl at C-1 which
have similar anities for both cannabinoid receptors.5,6
Traditional cannabinoid structure±activity relationships
(SAR)1±3 state that a phenolic hydroxyl at C-1 of the
cannabinoid skeleton is necessary for interaction with the
CB1 receptor. However, we reported recently that 3-(10,10-
dimethylheptyl)-1-deoxy-11-hydroxy-Á8-tetrahydrocan-
nabinol (1-deoxy-11-hydroxy-Á8-THC-DMH, deoxy-
HU-210, 1), a traditional cannabinoid lacking the 1-hy-
droxyl, has very high anity for the CB1 receptor
(Ki=1.2‹0.1 nM), and exhibits characteristic cannabi-
noid in vivo pharmacology. Cannabinoid 1 also has
exceptionally high anity for the CB2 receptor
(Ki=0.032‹0.019 nM).4 A second 1-deoxy-cannabinoid,
3-(10,10-dimethylheptyl)-1-deoxy-Á8-THC (1-deoxy-Á8-
THC-DMH, 2, n=5, where n=the number of carbon
atoms in the side chain 2), is also potent in vivo, has sig-
ni®cant anity for the CB1 receptor (Ki=23‹7 nM), and
nearly 10 times higher anity for the CB2 receptor
(Ki=2.9‹1.6 nM). 4 A group at Merck Frosst also descri-
bed 1-deoxy-Á8-THC-DMH; however, they found that it
The unexpected potency of 11-hydroxy-1-deoxy-Á8-
THC-DMH (1) was explained in terms of a possible
interaction of the 11-hydroxyl with Lys 192 of the CB1
receptor.4 It is thought that this amino acid hydrogen
bonds to the phenolic hydroxyl of traditional cannabi-
noids, and in a mutant receptor which lacks Lys 192, the
anities of the potent cannabinoids CP 55,940 and 11-
hydroxy-Á8-THC-DMH were greatly attenuated.7,8 The
potency of 1-deoxy-Á8-THC-DMH (2, n=5) was ratio-
nalized on the basis of molecular modeling studies which
suggested that this compound docked with the receptor
in an orientation such that Lys 192 hydrogen bonds to
the pyran oxygen.4 However, no rationalization was
presented for the very high anities of cannabinoids 1
and 2, n=5, for the CB2 receptor.4
Key words: Cannabinoid; deoxy-cannabinoid; structure±activity rela-
tionships; CB2 receptor.
In view of the selective anity for the CB2 receptor
reported for cannabinoids 1±4, the synthesis of a number
* Corresponding author. Tel.: +1-864-656-3133; fax: +1-864-656-
6613; e-mail: hu€man@clemson.edu
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00219-9

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J. W. Hu€man et al. / Bioorg. Med. Chem. 7 (1999) 2905±2914
of additional 1-deoxy-cannabinoids has been carried out
to obtain selective ligands for the CB2 receptor, and to
explore the SAR of the 1-deoxy-cannabinoids at both
cannabinoid receptors. Three series of compounds were
synthesized which included simple 1-deoxy analogues of
Á8-THC, 1-deoxy-10,10-dimethylalkyl-Á8-THCs, and 1-
deoxynabilone analogues.
dissolving metal reduction of which proceeded stereo-
selectively to provide saturated ketone 11. Ether clea-
vage, followed by treatment with SnCl4, gave cis-fused
ketone 12. Isomerization of ketone 12 with AlCl3 pro-
vided the 1-deoxynabilone analogue (13) in mediocre
yield, plus a mixture of several other products from
which ¯ourene derivative 14 was isolated. Conversion of
ketone 13 to the vinyl tri¯ate, followed by palladium
mediated carbonylation in the presence of methanol,
gave ester 15 which provided alcohol 5 after reduction.
1-Deoxycannabinoids 6±8 were prepared from the cor-
responding Á8-THC analogue by conversion to the
phosphate ester, followed by dissolving metal reduction
as outlined in Scheme 2.4,5
Results
Modeling studies had indicated that in the proposed
orientation of 1-deoxy-Á8-THC-DMH (2, n=5) which
interacts with the CB1 receptor, the dimethylheptyl side
chain has sucient length to reach the hydrophobic
binding pocket of the receptor.4 In designing additional
1-deoxy-cannabinoids it was assumed that decreasing
the length of the alkyl side chain would attenuate the
anity for the CB1 receptor. However, no prediction
could be made concerning the e€ect of this structural
modi®cation on the anity for the CB2 receptor. A ser-
ies of 1-deoxy-10,10-dimethylalkyl-Á8-THCs (2, n=0±7,
where n=the number of carbon atoms in the side
chain 2) was synthesized employing the method used
for the preparation of deoxycannabinoids 2, n=5, and
6±8. The resorcinol precursors were synthesized in four
steps from 2,6-dimethoxyphenol using modi®cations of
the procedure described by Dominianni et al. for the
synthesis of 1,3-dimethoxy-5-(1,1-dimethylheptyl)ben-
zene.10,11 Ether cleavage to the resorcinol employing
boron tribromide, followed by acid catalyzed reaction
with trans-para-menthadienol, provided Á8-THC ana-
logues,12 which were converted to the 1-deoxy-
cannabinoids by reduction of the phosphate ester as
described above for the preparation of 2, n=5, and 6±8
(Scheme 2).
Analogues of Á8-THC included 11-hydroxy-1-deoxy-
Á8-THC (5), 1-deoxy-Á8-THC (6), its 3-butyl (7), and 3-
hexyl analogues (8). As outlined in Scheme 1, alcohol 5
was prepared from 2-bromo-5-pentylmethoxybenzene
(9) and apoverbenone following the protocol employed
in the synthesis of the dimethylheptyl analogue (1).4,9,10
The aryllithium derived from 9 was added to apoverbe-
none to give, after oxidative rearrangement, enone 10,
Scheme 1. (a) BuLi/THF, 0ꢀC then apoverbenone; (b) PDC/CH2Cl2,
25ꢀC; (c) Li/NH3, THF, 78ꢀC; (d) NaSPr/DMF, 120ꢀC; (e) SnCl4/
CH2Cl2, 25ꢀC; (f) AlCl3/CH2Cl2, 25ꢀC; (g) Tf2O, 2,6-di-tert-butyl-4-
methyl-pyridine/CH2Cl2, 40ꢀC; (h) Et3N, Pd(OAc)2, Ph3P, CO,
CH3OH/DMF, 45ꢀC; (i) LiAlH4/Et2O, 0±25ꢀC.
Scheme 2. (a) HOTs/C6H6, 80ꢀC; (b) NaH/THF, 0ꢀC then
(C2H5O)2P(O)Cl; (c) Li/NH3, THF, 78ꢀC.

J. W. Hu€man et al. / Bioorg. Med. Chem. 7 (1999) 2905±2914
2907
Gareau et al. reported that nabilone methyl ether
showed modest selectivity for the CB2 receptor, and that
the Á9(11)-ole®n (4) derived from it had high anity for
the CB2 receptor (Ki=19.4 nM), but e€ectively no a-
nity for the CB1 receptor (Ki=>20,000).5 Based on the
premise that the lack of the phenolic hydroxyl would
have an e€ect on receptor anity similar to that of
converting the hydroxyl to a methyl ether, 1-deoxy-
nabilone (16) and the derived Á9(11)-ole®n (17) were
prepared (Scheme 3). We had earlier employed deoxy-
nabilone (16) as an intermediate in the synthesis of alcohol
1, and the corresponding Á9(11)-ole®n (17) was prepared
by Wittig reaction of 16 with methylenetriphenyl-
phosphorane. Epimeric alcohols 18 and 19 were obtained
by reduction of 16. Lithium aluminum hydride provided
the equatorial b-isomer (18) as the major product, and
K-Selectride (potassium tri-sec-butyl borohydride) gave
predominantly the axial a-isomer (19).
The anities of deoxycannabinoids 2, 5, 6±8 and 16±19
for the CB1 receptor were determined by measuring
their ability to displace the potent cannabinoid [3H]CP
55,940 from its binding site in a membrane preparation
from rat brain as described by Compton et al.13 A-
nities for the CB2 receptor were determined by measur-
ing the ability of the compounds to displace [3H]CP
55,940 from a cloned human receptor preparation using
the procedure described by Showalter et al.6 The results
of these determinations are summarized in Table 1.
Also included in Table 1 are the receptor anities for
cannabinoids 1 and Á9-THC.
With the exception of 1-deoxy-Á8-THC-DMH (2, n=5)
and its dimethyloctyl homologue (2, n=6), 1-deoxy-
cannabinoids lacking an oxygen function at C-9 or C-11
have little anity for the CB1 receptor. However, 1-
deoxy-10,10-dimethylalkyl-Á8-THCs with side chains of
three to seven carbon atoms (2, n=1±5) have high a-
nity for the CB2 receptor, ranging from Ki=2.9‹
1.6 nM for 2, n=5, to Ki=19‹4 nM for 1-deoxy-10,10-
dimethylhexyl-Á8-THC (2, n=4).
1-Deoxy-10,10-dimethylbutyl-Á8-THC (2, n=2) has only
slight anity for the CB1 receptor (Ki=677‹132 nM),
but has 200-fold selectivity for the CB2 receptor
(Ki=3.4‹1.0 nM). 1-Deoxy-Á8-THC (6) has virtually no
anity for the CB1 receptor (Ki>10,000 nM), but has
signi®cant anity for the CB2 receptor (Ki=32‹9 nM).
Of the 9- and 11-oxygenated cannabinoids, deoxynabil-
one (16) and the derived alcohols (18 and 19) show sig-
ni®cant anity for the CB1 receptor (Table 1).
However, the anity of these compounds for the CB2
receptor are approximately the same as their anities
for the CB1 receptor. In accord with generalizations
Scheme 3. (a) (C6H5)3PCH3+ Br , n-BuLi/THF, 65ꢀC; (b) LiAlH4/
THF, 25ꢀC; (c) KBH(sec-Bu)3/THF, 78 to 25ꢀC then H2O2/NaOH.
Table 1. Receptor anities (mean ‹ SEM) of 1-deoxy-cannabinoids and related compounds
Ki (nM)
Compound
CB1
CB2
Á9-THC
41‹2a
44‹12
36‹10b
44‹17
Á8-THC
1-Deoxy-11-hydroxy-Á8-THC-DMH (1)
3-(10,10-Dimethylethyl)-1-deoxy-Á8-THC (2, n=0)
3-(10,10-Dimethylpropyl)-1-deoxy-Á8-THC (2, n=1)
3-(10,10-Dimethylbutyl)-1-deoxy-Á8-THC (2, n=2)
3-(10,10-Dimethylpentyl)-1-deoxy-Á8-THC (2, n=3)
3-(10,10-Dimethylhexyl)-1-deoxy-Á8-THC (2, n=4)
Deoxy-Á8-THC-DMH (2, n=5)
3-(10,10-Dimethyloctyl)-1-deoxy-Á8-THC (2, n=6)
3-(10,10-Dimethylnonyl)-1-deoxy-Á8-THC (2, n=7)
1-Deoxy-11-hydroxy-Á8-THC (5)
1-Deoxy-Á8-THC (6)
1.2‹0.1c
0.032‹0.019c
58‹13
14‹10
2150‹231
2290‹505
677‹132
399‹76
295‹52
23‹7c
51‹11
178‹2
161‹26
>10,000
2790‹820
1610‹302
44‹2
3.4‹1.0
10‹2
19‹4
2.91‹1.6c
76‹4
449‹98
16‹3
32‹9
3-Butyl-1-deoxy-Á8-THC (7)
54‹8
3-Hexyl-1-deoxy-Á8-THC (8)
Deoxynabilone (16)
273‹63
25‹7
137‹6
5.2‹2.0
23‹7
65‹8, 20‹12d
35‹14, 19‹4d
Deoxy-Á9(11)-THC-DMH (17)
909‹121
7.9‹0.9
28‹3
b-Deoxynabilol (18)
a-Deoxynabilol (19)
Á8-THC-DMH methyl ether (3)
Á9(11)-THC-DMH methyl ether (4)
924‹104, 15,850‹2960d
529‹49, >20,000d
a
ref. 13.
ref. 6.
ref. 4.
ref. 5.
b
c
d

2908
J. W. Hu€man et al. / Bioorg. Med. Chem. 7 (1999) 2905±2914
regarding the steric requirements in the region of C-
9,14,15 1-deoxy-Á9(11)-THC-DMH (17) shows little a-
nity for the CB1 receptor (Ki=909‹121 nM), and only
modest anity for the CB2 receptor (Ki=137‹6 nM).
n=5)4 which have comparable anities for the CB2
receptor. The 10,10-dimethylethyl (2, n=0) and 10,10-
dimethyloctyl (2, n=6) have moderate anity for
the CB2 receptor, while 10,10-dimethylnonyl-1-deoxy-
Á8-THC (2, n=7) has very low anity for the CB2
receptor.
In order to independently evaluate the anities of 1-
methoxy cannabinoids 3 and 4, an e€ort was made to
obtain samples of these ligands. However, samples were
unavailable and an independent synthesis of these com-
pounds was carried out. Methyl ether 3 was prepared by
routine O-methylation of Á8-THC-DMH (K2CO3/
dimethyl sulfate), and ole®n 4 was obtained from nabi-
lone methyl ether by a Wittig reaction with methylen-
triphenylphosphorane. Nabilone was prepared as
described by Tius et al.16 and was converted to the
methyl ether by the procedure employed for the pre-
paration of 3. The anity of each compound was
determined as described above, and the data are inclu-
ded in Table 1. Neither compound had appreciable a-
nity for the CB1 receptor; for 3, Ki=924‹104 nM, and
for 4, Ki=529‹49 nM. These data are considerably
di€erent from those reported by Gareau et al. who
found Ki=>10,000 for both compounds.5 However, it
has been our experience that for compounds with very
low receptor anity, in which Ki approaches or exceeds
the micromolar range, there is considerable variability
in anity data. Also, there are possible di€erences in
methodology; our data were obtained using rat brain
homogenates as described above,13 while the Merck
Frosst group employed a human CB1 preparation of
unspeci®ed origin.5 Both compounds 3 and 4 have
moderate anity (Ki=65‹8 and 35‹14 nM, respec-
tively) for the CB2 receptor. These data are in the same
general range as those reported by Gareau et al., who
found that 3 had Ki=20‹12 nM, and that 4 had
Ki=19‹4 nM. As in the case of the CB1 receptor, these
di€erences may be due to di€erences in methodology in
determining the receptor anities.
For all of the 1-deoxy-cannabinoids listed in Table 1,
with the exception of 1-deoxy-Á8-THC-DMH4 (2, n=5)
and the next higher homologue, 3-(10,10-dimethyloctyl)-
1-deoxy-Á8-THC (2, n=6), only those 1-deoxy-cannabi-
noids with an oxygen functionality at C-9 or C-11, and a
3-(10,10-dimethylheptyl) side chain (1, 16, 18 and 19),
show signi®cant anity for the CB1 receptor. These
data are in accord with the hypothesis reached on the
basis of molecular modeling studies reported previously,
which suggested that the 11-hydroxyl of cannabinoid 1
interacts with Lys 192 of the CB1 receptor in a manner
similar to that of the 1-hydroxyl of traditional cannabi-
noids, and that 1-deoxy-Á8-THC-DMH adopts an
orientation with respect to the receptor in which the
pyran oxygen interacts with Lys 192.4 The modeling
studies indicated that the heptyl side chain of 2, n=5,
has sucient length to reach the lipophilic portion of
the receptor, and presumably the octyl side chain of 2,
n=6, also is of the correct length to interact with the
lipophilic portion of the CB1 receptor. 1-Deoxy-Á8-
THC analogues with a 1,1-dimethylalkyl side chain of
less than seven or more than eight carbon atoms have
relatively little anity for the CB1 receptor; however,
three of the lower homologues (2, n=1±3) which have
little anity for the CB1 receptor (Ki=>650 nM) have
high anity for the CB2 receptor. In particular, 3-(10,10-
dimethylbutyl)-1-deoxy-Á8-THC (2, n=2) has excep-
tionally high anity (Ki=3.4‹1.0 nM) for the CB2
receptor and low anity for the CB1 receptor
(Ki=677‹32 nM).
In conclusion, 3-(10,10-dimethylbutyl)-1-deoxy-Á8-THC
(2, n=2) and several other 1-deoxy-Á8-THC analogues
have high anity for the CB2 receptor, but little anity
for the CB1 receptor. These compounds are readily
available and should prove to be useful selective ligands
for investigating the role of the CB2 receptor.
From the data summarized in Table 1, it is possible to
develop preliminary SAR for the CB2 receptor based
upon the anities of 1-deoxy-Á8-THC analogues. First,
it is quite apparent that a 10,10-dimethyl group leads to
enhanced anity for the CB2 receptor. A comparison of
the anities of 3-(10,10-dimethylbutyl)-1-deoxy-Á8-THC
(2, n=2), its pentyl (2, n=3) and hexyl (2, n=4) homo-
logues with 1-deoxy-cannabinoids with the same length
side chain but lacking the gem-dimethyl group (6±8)
shows that the 10,10-dimethylbutyl and dimethylhexyl
cannabinoids have approximately 15-fold greater a-
nity for the CB2 receptor than the unsubstituted anal-
ogues. Although 3-butyl-1-deoxy-Á8-THC (7) has
moderate anity for the CB2 receptor (Ki=54‹8 nM),
the 3-hexyl analogue (8) has low anity (Ki=273‹
63 nM). With a pentyl side chain the 10,10-dimethyl-
deoxy-cannabinoid (2, n=3) has threefold greater a-
nity for the CB2 receptor than 1-deoxy-Á8-THC (6).
Experimental
General
IR spectra were obtained using Nicolet 5DX or Magna
1
spectrometers; H and 13C NMR spectra were recorded
on a Bruker 300AC spectrometer. Mass spectral ana-
lyses were performed on a Hewlett±Packard 5890A gas
chromatograph with a mass sensitive detector and
HRMS data were obtained in the Mass Spectrometry
Laboratory, School of Chemical Sciences, University of
Illinois. Ether and THF were distilled from Na±benzo-
phenone ketyl immediately before use, and other sol-
vents were puri®ed using standard procedures. Column
chromatography was carried out on Universal silica gel
(32±63 mm) using the indicated solvents as eluents. All
new compounds were homogeneous to TLC and 13C
In the 10,10-dimethyl-1-deoxy-Á8-THC series, those
compounds with a three to seven carbon side chain (2,
n=1±5) all have high anity for the CB2 receptor
(Ki=<20 nM). The greatest anities are 10,10-dimethyl-
butyl- (2, n=2) and 10,10-dimethylheptyl-Á8-THC (2,

J. W. Hu€man et al. / Bioorg. Med. Chem. 7 (1999) 2905±2914
2909
NMR. All target compounds were homogeneous to
TLC in two di€erent solvent systems.
(2-Methoxy-4-pentylphenyl)-6,6-dimethylbicyclo[3.1.1]-
heptan-2-one (11). To a solution of 0.50 g (71 mg
atoms) of Li in 200 mL of liquid NH3 at 78ꢀC was
added dropwise 2.57 g (8.5 mmol) of enone 10 in 25 mL
of dry ether and 5 mL of dry THF. The reaction mixture
was stirred at 78ꢀC for 1.5 h, quenched by the addition
of solid NH4Cl, and the NH3 evaporated at ambient tem-
perature. The solid residue was taken up in ether, and the
ether solution was washed with successive portions of
10% aqueous HCl, water, saturated aqueous NaHCO3
and water. After drying (MgSO4), the solvent was
removed in vacuo to a€ord an oil which was puri®ed by
¯ash chromatography (petroleum ether:ether 3:1) to give
2.41g (82%) of pure ketone: 1H NMR (300MHz, CDCl3)
d 0.82±0.96 (m, 6H), 1.21±1.41 (m, 4H), 1.46 (s, 3H), 1.51±
1.69 (m, 3H), 2.39±2.65 (m, 4H), 2.70±2.82 (m, 2H), 3.25±
3.41 (m, 1H), 3.60±3.70 (m, 1H), 3.82 (s, 3H), 6.68 (s, 1H),
6.70 (d, J=8.9 Hz, 1H), 6.93 (d, J=7.6 Hz, 1H); 13C
NMR (75.5 MHz, CDCl3) d 14.0, 22.5, 25.3, 26.6, 28.5,
31.1, 31.6, 35.7, 35.8, 39.6, 40.8, 44.1, 55.0, 58.3, 110.3,
119.2, 126.8, 129.6, 142.4, 157.4, 214.2; MS (EI) m/z 314
(31), 243 (34), 204 (100), 147 (29), 115 (21), 83 (70);
HRMS: calcd for C21H30O2 314.2246, found 314.2246.
2-Bromo-5-pentylmethoxybenzene (9). To a solution of
5.30 g (29.7 mmol) of 3-pentylmethoxybenzene17 in
120 mL of freshly distilled cyclohexane at 25ꢀC was
added 23.7 mL (59.3 mmol) of 2.5 M n-butyllithium in
hexanes. The mixture was stirred at re¯ux temperature
for 18 h, cooled and diluted with 60 mL of dry THF.
After cooling to 78ꢀC, 7.10 mL (59.4 mmol) of 1,2-
dibromoethane was added dropwise, and the mixture
was stirred at 78ꢀC for 0.5 h, allowed to warm to
ambient temperature and stirred for an additional 1 h. The
reaction was quenched with water, the phases were sepa-
rated, and the aqueous phase extracted with two portions
of ether. The combined organic layers were washed with
brine, dried (MgSO4) and the solvent removed in vacuo.
Chromatography (petroleum ether:ethyl acetate 50:1)
gave 5.50g (72%) of aryl bromide as a pale yellow oil: bp
160ꢀC/0.5 mm Hg; 1H NMR (300MHz, CDCl3) d 0.89 (t,
J=6.6 Hz, 3H), 1.22±1.41 (m, 4H), 1.52±1.68 (m, 2H),
2.55 (t, J=8.0 Hz, 2H), 3.86 (s, 3H), 6.64 (dd, J=1.6,
7.9 Hz, 1H), 6.71 (d, J=1.7 Hz, 1H), 7.39 (d, J=7.9 Hz,
1H); 13C NMR (75.5 MHz, CDCl3) d 13.9, 22.4, 30.9,
31.3, 35.9, 55.9, 108.3, 112.2, 121.7, 132.7, 143.8, 155.5;
HRMS: calcd for C12H17BrO 256.0463, found 256.0464.
(6aS,10aR)-3-Pentyl-6,6a,7,8,10,10a-hexahydro-6,6-di-
methyl-9H-dibenzo[b,d]pyran-9-one (12). To a stirred
solution of 2.01 g (67.0 mmol) of 80% NaH in 80 mL of
dry DMF was added dropwise at ambient temperature
6.80mL (74.5 mmol) of 1-propanethiol. The mixture was
stirred until it became clear, and a solution of 2.34g
(7.45 mmol) of 4-(2-methoxy-4-pentylphenyl)-6,6-dime-
thylbicyclo[3.1.1]heptan-2-one 11 in 5 mL of DMF was
added dropwise. The mixture was heated with stirring at
120ꢀC for 5 h, cooled, poured into 10% aqueous HCl and
extracted with ether. The extracts were washed with brine,
dried (MgSO4), and concentrated to give an oil which was
chromatographed (petroleum ether:ethyl acetate 5:2) to
give 2.10g (94%) of phenol which was used in the next
4-(2-Methoxy-4-pentylphenyl)-6,6-dimethylbicyclo[3.1.1]-
hept-3-en-2-one (10). To a stirred solution of 7.80 mL
(19.5 mmol) of 2.5 M n-butyllithium in hexanes and
3.20 mL of freshly distilled TMEDA in 10 mL of dry
THF at 78ꢀC was added dropwise a solution of 4.48 g
(17.4 mmol) of 2-bromo-5-pentylmethoxybenzene (9) in
5 mL of dry THF. The reaction mixture was stirred for
0.5 h at 78ꢀC, and 2.18 g (16.0 mmol) of apoverbenone
in 5 mL of dry THF was added dropwise. The mixture
was allowed to warm to room temperature, stirred for
18 h, quenched with saturated aqueous NH4Cl, and
extracted with ether. The ethereal extracts were washed
with brine, dried (MgSO4), and the solvent was removed
in vacuo. The residue was dissolved in 25mL of dry
CH2Cl2 and the solution was added dropwise to a sus-
pension of 3.3 g of PDC in 30mL of dry CH2Cl2. The
reaction mixture was stirred for 2 h at ambient tempera-
ture, diluted with ether, ®ltered, and the dark residue was
washed thoroughly with ether. The combined ether
extracts were washed successively with 10% aqueous
NaOH, water, 10% aqueous HCl, water, saturated aqu-
eous NaHCO3 and water. After drying (MgSO4), the sol-
vent was removed in vacuo to give the crude enone which
was puri®ed by ¯ash chromatography (petroleum ether:
1
step without further puri®cation: H NMR (300MHz,
CDCl3) d 0.78±0.96 (m, 6H), 1.17±1.40 (m, 4H), 1.40±1.72
(m, 6H), 2.46 (t, J=8.1 Hz, 2H), 2.58±2.90 (m, 4H), 3.51±
3.78 (m, 2H), 6.60 (d, J=8.2 Hz, 1H), 6.62 (s, 1H), 6.85 (d,
J=7.6 Hz, 1H); 13C NMR (75.5 MHz, CDCl3) d 13.9,
22.5, 25.4, 26.5, 28.4, 28.8, 31.0, 31.5, 35.3, 35.7, 39.8, 41.0,
43.6, 58.3, 115.2, 118.8, 126.8, 127.6, 142.4, 154.4, 218.0.
To a solution of 1.63 g (8.30 mmol) of phenol in 8 mL of
CHCl3 was added 8.30 mL (8.30 mmol) of 1 M SnCl4 in
CH2Cl2. The reaction was stirred at ambient tempera-
ture for 18 h, poured onto ice and extracted with ether.
The ether extracts were washed with successive portions
of 10% aqueous HCl, water, saturated aqueous
NaHCO3, brine and dried (MgSO4). The solvent was
removed in vacuo to a€ord, after chromatography (pet-
roleum ether:ethyl acetate 2:1), 1.55 g (81%) of ketone as
an oil: 1H NMR (300 MHz, CDCl3) d 0.88 (t, J=6.9 Hz,
3H), 1.18±1.40 (m, 5H), 1.34 (s, 3H), 1.43 (s, 3H), 1.50±
1.73 (m, 3H), 1.96±2.43 (m, 4H), 2.49 (t, J=8.0 Hz, 2H),
2.64±2.84 (m, 1H), 3.58±3.77 (m, 1H), 6.60 (d, J=1.2 Hz,
1H), 6.70 (dd, J=8.0, 1.4 Hz, 1H), 7.13 (d, J=7.9 Hz,
1H); 13C NMR (75.5 MHz, CDCl3) d 13.8, 22.4, 23.0,
26.3, 26.6, 30.6, 31.4, 34.3, 35.3, 39.6, 40.1, 43.9, 75.6,
116.6, 117.2, 120.7, 126.9, 143.0, 152.6, 210.1; MS (EI)
1
ether 3:1) to give 2.57g (47%) of pure enone: H NMR
(300MHz, CDCl3) d 0.90 (t, J=6.7 Hz, 3H), 1.11 (s, 3H),
1.21±1.43 (m, 4H), 1.54 (s, 3H), 1.57±1.68 (m, 2H), 2.26 (d,
J=9.2 Hz, 1H), 2.61 (t, J=7.9 Hz, 2H), 2.68±2.77 (m,
1H), 2.83±2.95 (m, 1H), 3.00±3.07 (m, 1H), 3.81 (s, 3H),
6.06 (s, 1H), 6.73 (s, 1H), 6.79 (d, J=7.8 Hz, 1H), 7.11 (d,
J=7.7 Hz, 1H); 13C NMR (75.5 MHz, CDCl3) d 14.0,
22.4, 26.8, 31.0, 31.5, 36.1, 41.2, 49.2, 54.0, 55.2, 57.8,
111.3, 120.7, 121.9, 125.7, 128.3, 146.4, 156.8, 168.2, 204.4;
MS (EI) m/z 312 (78), 269 (44), 242 (100), 213 (25), 185
(36), 115 (29); HRMS: calcd for C21H28O2 312.2089,
found 312.2090.

2910
J. W. Hu€man et al. / Bioorg. Med. Chem. 7 (1999) 2905±2914
m/z 300 (100), 285 (51), 257 (39), 217 (85), 134 (51);
HRMS: calcd for C20H28O2 300.2089, found 300.2090.
6.72 (dd, J=8.0, 1.5 Hz, 1H), 7.03 (d, J=7.8 Hz, 1H);
13C NMR (75.5 MHz, CDCl3) d 14.0, 18.9, 22.5, 25.8,
27.6, 30.9, 31.5, 32.5, 33.7, 35.5, 41.9, 76.2, 117.2, 117.4,
120.4, 120.7, 126.2, 143.3, 148.0, 152.6.
(6aR,10aR)-3-Pentyl-6,6a,7,8,10,10a-hexahydro-6,6-di-
methyl-9H-dibenzo[b,d] pyran-9-one (13). To a solution
of 1.47 g (4.89 mmol) of cis-ketone 12 in 10 mL of dry
CH2Cl2 at ambient temperature was added 1.95 g
(14.7 mmol) of AlCl3, and the reaction mixture was
stirred for 2 h. The mixture was poured onto ice,
extracted with three portions of ether, and the combined
extracts were washed with 10% aqueous HCl, brine,
saturated aqueous NaHCO3, brine and dried (MgSO4).
The solvent was removed in vacuo to give an oil which
was chromatographed (petroleum ether:ethyl acetate 4:1)
to give 0.066 g of recovered cis-ketone and 0.590 g (42%
based on recovered starting material) of trans-ketone as a
To a solution of 0.490 g (1.13 mmol) of tri¯ate in 4 mL of
dry DMF were added 0.320 mL (2.26 mmol) of Et3N,
0.0255 g (0.11 mmol) of Pd(OAc)2, 0.0595 g (0.226 mmol)
of triphenylphosphine and 1.90 mL of methanol. The
reaction ¯ask was purged for 10 min with CO, then
stirred under an atmosphere of CO at 45ꢀC for 18 h.
The reaction mixture was poured into water, and
extracted with ether. After drying (MgSO4), the solvent
was removed in vacuo and the residue was puri®ed by
¯ash chromatography (petroleum ether:ether 10:1) to give
0.219 g (57%) of methyl ester: 1H NMR (300MHz,
CDCl3) d 0.88 (t, J=6.7 Hz, 3H), 1.16 (s, 3H), 1.40 (s, 3H),
1.20±1.82 (m, 7H), 1.90±2.16 (m, 2H), 2.32±2.47 (m, 1H),
2.52 (t, J=7.9 Hz, 2H), 2.58±2.73 (m, 1H), 3.10±3.28 (m,
1H), 3.76 (s, 3H), 6.64 (s, 1H), 6.72 (d, J=7.7 Hz, 1H),
7.04 (m, 1H), 7.18 (d, J=7.9 Hz, 1H); 13C NMR
(75.5 MHz, CDCl3) d 14.0, 18.9, 22.5, 27.6, 28.0, 30.6,
30.8, 31.5, 31.7, 35.5, 42.2, 51.6, 76.2, 116.8, 120.4, 12.7,
126.6, 129.8, 138.2, 142.6, 152.6, 167.4; MS (EI) m/z 342
(95), 230 (27), 215 (100), 177 (63), 159 (27), 119 (32);
HRMS: calcd for C22H30O3 342.2195, found 342.2196.
1
pale yellow oil: H NMR (300MHz, CDCl3) d 0.88 (t,
J=6.9 Hz, 3H), 1.16 (s, 3H), 1.49 (s, 3H), 1.23±1.43 (m,
4H), 1.43±1.67 (m, 3H), 1.82±1.96 (m, 1H), 2.09±2.63 (m,
6H), 2.78±2.92 (m, 1H), 3.03±3.17 (m, 1H), 6.66 (s, 1H),
6.70 (d, J=8.0 Hz, 1H), 6.98 (d, J=7.8 Hz, 1H); 13C
NMR (75.5 MHz, CDCl3) d 14.0, 19.8, 22.5, 27.2, 28.1,
30.8, 31.5, 35.4, 35.8, 40.8, 45.7, 45.9, 76.9, 116.9, 120.4,
120.6, 125.4, 143.2, 152.7, 210.0; MS (EI) m/z 300 (100),
217 (96), 190 (63), 177 (52), 161 (66); HRMS: calcd for
C20H28O2 300.2089, found 300.2090.
Further elution with the same solvent pair a€orded 0.65g
of a mixture of several compounds with very close pola-
rities. Repeated chromatography provided 0.042 g of the
major component of the mixture, ¯ourene derivative 14:
1H NMR (300MHz, CDCl3) d 0.90 (t, J=6.8 Hz, 3H),
0.96 (s, 3H), 1.36 (s, 3H), 1.22±1.41 (m, 4H), 1.48±1.66 (m,
2H), 1.66±1.84 (m, 1H), 1.88±2.11 (m, 2H), 2.31±2.57 (m,
4H), 2.57±2.71 (m, 1H), 2.98±3.16 (m, 1H), 3.50±3.65 (m,
1H), 6.11 (s, 1H, disappears when shaken with D2O), 6.44
(s, 1H), 6.58 (s, 1H); 13C NMR (75.5 MHz, CDCl3) d
32;14.0, 22.3, 22.5, 23.2, 26.0, 31.3, 31.7, 35.9, 41.4, 43.5,
44.8, 46.3, 57.4, 114.0, 114.5, 124.9, 144.0, 152.3, 155.6,
213.6; MS (EI) m/z: 300 (60), 285 (48), 244 (37), 243 (42),
229 (100), 173 (22), 145 (15), 55 (62).
(6aR,10aR)-9-Hydroxymethyl-3-pentyl-6a,7,10,10a-tet-
rahydro - 6,6 - dimethyl - 6H - dibenzo[b,d]pyran (5). To a
stirred suspension of 0.090 g (2.37 mmol) of LiAlH4 in
10mL of dry ether was added dropwise at 0ꢀC a solution
of 0.201 g (0.587 mmol) of ester 15 in 7 mL of dry ether.
The reaction mixture was stirred at ambient temperature
for 18 h, quenched with water, and acidi®ed to pH 3. The
phases were separated, and the aqueous phase was extrac-
ted with ether. The combined organic extracts were washed
with brine, dried (MgSO4), and the solvent was removed in
vacuo. The crude product was puri®ed by ¯ash chromato-
graphy (petroleum ether:ether 1:1) to give 0.172g (92%) of
alcohol as a colorless oil: Rf 0.32 (petroleum ether:ether
1
1:1), 0.61 (petroleum ether:ethyl acetate 1:1); H NMR
(300 MHz, CDCl3) d 0.88 (t, J=6.9 Hz, 3H), 1.15 (s, 3H),
1.39 (s, 3H), 1.23±1.45 (m, 4H), 1.50±1.67 (m, 2H), 1.67±
2.31 (m, 5H), 2.51 (t, J=7.6 Hz, 2H), 2.68±2.86 (m, 2H),
4.05 (m, 2H), 5.69±5.78 (m, 1H), 6.63 (d, J=1.4 Hz, 1H),
6.69 (dd, J=7.9, 1.5 Hz, 1H), 7.11 (d, J=7.9 Hz, 1H); 13C
NMR (75.5 MHz, CDCl3) d 13.9, 19.0, 22.5, 27.1, 27.6,
30.8, 31.5, 31.9, 32.1, 35.4, 43.0, 66.8, 76.6, 116.8, 120.3,
121.5, 122.4, 126.4, 136.9, 142.4, 152.7; MS (EI) m/z 314
(100), 281 (28), 253 (35), 215 (95), 177 (63); HRMS: calcd
for C21H30O2 314.2246, found 314.2246.
(6aR,10aR)-9-Carbomethoxy-3-pentyl-6a,7,10,10a-tetra-
hydro-6,6-dimethyl-6H-dibenzo[b,d]pyran (15). To a stir-
red solution of 0.602 g (2.93 mmol) of 2,6-di-tert-butyl-
4-methylpyridine in 3 mL of dry CH2Cl2 was quickly
added 0.370 g (2.20 mmol) of tri¯uoromethanesulfonic
anhydride. After stirring for 5 min, a solution of 0.440 g
(1.46 mmol) of ketone 13 in 2.5 mL of dry CH2Cl2 was
added dropwise, and the reaction was stirred at re¯ux for
18 h. The solvent was removed in vacuo, and the residue
was taken up in petroleum ether. The precipitated solid
was ®ltered o€, and the ®ltrate was washed with cold
10% aqueous HCl, and brine. After drying (MgSO4) the
solvent was removed in vacuo to give an oil which was
puri®ed by ¯ash chromatography (petroleum ether:ether
10:1) to a€ord 0.514 g (81%) of tri¯ate as an oil which
was used in the subsequent step without further pur-
i®cation: 1H NMR (300 MHz, CDCl3) d 0.88 (t,
J=6.0 Hz, 3H), 1.16 (s, 3H), 1.42 (s, 3H), 1.12±1.38 (m,
3H), 1.48±1.68 (m, 3H), 1.74±1.88 (m, 1H), 1.90±2.19 (m,
1H), 2.28±2.46 (m, 2H), 2.52 (t, J=7.6 Hz, 2H), 2.80±
3.05 (m, 2H), 5.80±5.90 (m, 1H), 6.65 (d, J=1.3 Hz, 1H),
8
1-Deoxy-D-tetrahydrocannabinol (6). To a stirred solu-
tion of 0.495 g (1.58 mmol) of Á8-THC in 2 mL of dry
THF at 0ꢀC was added, in portions, 0.071 g (2.4 mmol)
of 80% NaH (suspension in mineral oil). The resulting
mixture was stirred for 10 min at 0ꢀC and 0.460 mL
(3.18 mmol) of diethyl chlorophosphate was added
dropwise. The mixture was allowed to warm to room
temperature and stirred for 1 h. The reaction mixture was
diluted with ether and the ethereal solution was washed
with 10% NaOH, brine, and dried (MgSO4). Concentra-
tion a€orded an oil which was chromatographed using

J. W. Hu€man et al. / Bioorg. Med. Chem. 7 (1999) 2905±2914
2911
petroleum ether:ethyl acetate (3:1) to give 0.558 g (79%) of
pure (TLC) phosphate ester which was used in the sub-
sequent step without further puri®cation. The phosphate
ester was reduced as described above for the preparation
of ketone 11. From 0.301 g (0.669mmol) of ester there was
obtained 0.173 g (88%) of pure deoxy-Á8-THC (6) after
puri®cation by ¯ash chromatography (petroleum ether:
ethyl acetate 20:1). Rf 0.50 (hexanes:ethyl acetate 50:1),
acid was stirred at 50ꢀC for 3 h and at ambient tempera-
ture for an additional 14h. The reaction mixture was
poured onto ice, extracted with CH2Cl2, and the extracts
were washed with water, saturated aqueous NaHCO3 and
dried (MgSO4). The solvent was removed in vacuo to give
4.2 g (88%) of a brown oil which was used in the sub-
sequent step without further puri®cation.
1
0.61 (hexanes:ethyl acetate 20:1); H NMR (300MHz,
The phenol was converted to 2-methyl-2-(3,5-dime-
thoxyphenyl)propane via the phosphate ester using the
procedure described above for the preparation of deoxy-
cannabinoid 6. From 4.40g (21.0 mmol) of phenol there
was obtained 3.5 g (86%) of 2-methyl-2-(3,5-dimethoxy-
phenyl)propane as a pale yellow oil: 1H NMR (300MHz,
CDCl3) d 1.31 (s, 9H), 3.80 (s, 6H), 6.32 (t, J=2.2 Hz, 1H),
6.56 (d, J=2.2 Hz, 2H); 13C NMR (75.5 MHz, CDCl3) d
31.2, 34.9, 55.1, 96.7, 104.0, 153.8, 160.5.
CDCl3) d 0.88 (t, J=6.4 Hz, 3H), 1.12 (s, 3H), 1.36 (s, 3H),
1.19±1.47. (m, 4H), 1.51±2.02 (m, 5H), 1.71 (s, 3H), 2.06±
2.19 (m, 1H), 2.50 (t, J=7.9 Hz, 2H), 2.44±2.76 (m, 2H),
5.42 (br s, 1H), 6.63 (s, 1H), 6.67 (d, J=7.7 Hz, 1H), 7.07
(d, J=7.8 Hz, 1H); 13C NMR (75.5 MHz, CDCl3) d 14.0,
19.1, 22.5, 23.4, 27.5, 27.6, 30.9, 31.6, 32.1, 35.5, 36.6, 42.8,
76.5, 116.8, 119.9, 120.1, 122.7, 126.3, 133.3, 142.1, 152.8;
MS (EI) m/z 298 (33), 255 (21), 243 (11), 230 (21), 215
(100), 185 (25), 177 (26), 173 (11), 159 (17); HRMS: calcd
for C21H30O 298.2297, found 298.2295.
0
0
8
(1 ,1 -Dimethylethyl)-D-tetrahydrocannabinol. To 1.75g
(9.0 mmol) of 2-methyl-2-(3,5-dimethoxyphenyl)propane
at 0ꢀC was added 22.0 mL (22.0 mmol) of 1.0 M BBr3 in
CH2Cl2. The reaction mixture was warmed to ambient
temperature, stirred for 18h and carefully poured into ice
water. After extraction with CH2Cl2, the organic extracts
were washed with water, and dried (MgSO4). The sol-
vent was removed to give 1.50 g (100%) of crude sub-
stituted resorcinol as a brown oil which was used in the
next step without further puri®cation: A mixture of
1.50 g (9.0 mmol) of crude resorcinol, 1.40 g (9.2 mmol)
of trans-p-menthadienol and 0.20 g of toluenesulfonic
acid monohydrate in 100 mL of benzene was heated at
re¯ux for 3 h. After cooling, the mixture was diluted with
ether, washed with brine and dried (MgSO4). The solvent
was removed in vacuo to give a brown gum which was
chromatographed using petroleum ether:ethyl acetate
(19:1) to a€ord 2.1 g (77%) of cannabinoid as a viscous
pale yellow oil: Rf 0.38 (petroleum ether:ethyl acetate 9:1),
0.58 (petroleum ether:ethyl acetate 4:1); 1H NMR
(300MHz, CDCl3) d 1.12 (s, 3H), 1.23 (s, 9H), 1.40 (s,
3H), 1.70 (s, 3H), 1.75±1.94 (m, 3H), 2.14 (m, 1H), 2.72
(dt, J=4.6, 10.8 Hz, 1H), 3.22 (dd, J=4.6, 16.3 Hz, 1H),
5.04 (s, 1H), 5.43 (d, J=4.0 Hz, 1H), 6.30 (d, J=1.6 Hz,
1H), 6.46 (d, J=1.6 Hz, 1H); 13C NMR (75.5 MHz,
CDCl3) d 18.5, 23.5, 27.6, 27.8, 31.1, 31.4, 34.3, 35.9, 44.8,
76.7, 104.9, 107.3, 110.3, 119.3, 134.7, 151.1, 154.4, 154.5.
MS (EI) m/z (rel intensity) 301 (30), 300 (90), 217 (100).
8
1-Deoxy-3-butyl-D-tetrahydrocannabinol (7). 3-Butyl-Á8-
tetrahydrocannabinol18 was converted to 1-deoxy-3-
butyl-Á8-tetrahydrocannabinol by the procedure used
in the preparation of deoxy-Á8-THC. From 0.42g
(1.40 mmol) of 3-butyl-Á8-THC there was obtained
0.47g (77%) of pure phosphate ester. From 0.301 g
(0.669mmol) of phosphate ester there was obtained 0.22g
(84%) of deoxy-cannabinoid 7 as a colorless oil after
chromatography (petroleum ether:ethyl acetate 200:1). Rf
0.37 (petroleum ether:ethyl acetate 100:1), 0.77 (petroleum
1
ether:ethyl acetate 25:1); H NMR (300MHz, CDCl3) d
0.95 (t, J=7.3 Hz, 3H), 1.18 (s, 3H), 1.28±1.46 (m, 2H),
1.42 (s, 3H), 1.56±1.98 (m, 5H), 1.76 (s, 3H), 2.16±2.22 (m,
1H), 2.56 (t, J=7.8 Hz, 2H), 2.58±2.76 (m, 2H), 5.48 (br s,
1H), 6.66 (d, J=1.2 Hz, 1H), 6.73 (dd, J=1.2, 7.8 Hz,
1H), 7.14 (d, J=7.8 Hz, 1H); 13C NMR (75.5 MHz,
CDCl3) d 13.9, 19.1, 22.4, 23.4, 27.5, 27.7, 32.1, 33.3, 35.2,
36.6, 42.8, 76.7, 116.9, 119.9, 120.2, 122.8, 126.4, 133.4,
142.2, 152.8; MS (EI) m/z 285 (25), 284 (90), 201 (100);
[a]2d0 122ꢀ (c 0.88, CHCl3); HRMS: calcd for C20H28O
284.2140, found 284.2135.
8
1-Deoxy-3-hexyl-D-tetrahydrocannabinol (8). 3-Hexyl-
Á8-tetrahydrocannabinol19 was converted to the 1-
deoxy analogue by the procedure described above.
From 0.56 g (1.70 mmol) of 3-hexyl-Á8-THC there was
obtained 0.285 g (54% for two steps) of pure deoxy-
cannabinoid 8 after ¯ash chromatography (petroleum
ether:ethyl acetate 20:1): Rf 0.25 (petroleum ether:ether
50:1), 0.69 (petroleum ether:ether 20:1): 1H NMR
(300 MHz, CDCl3) d 0.88 (t, J=6.4 Hz, 3H), 1.24 (s,
3H), 1.36 (s, 3H), 1.71 (s, 3H), 1.19±1.47 (m, 6H), 1.51±
2.02 (m, 5H), 2.06±2.19 (m, 1H), 2.50 (t, J=7.9 Hz, 2H),
2.44±2.76 (m, 2H), 5.38±5.46 (m, 1H), 6.63 (s, 1H), 6.67
(d, J=7.7 Hz, 1H), 7.07 (d, J=7.8 Hz, 1H); 13C NMR
(75.5 MHz, CDCl3) d 14.0, 19.1, 22.5, 23.4, 27.4, 27.6,
29.1, 30.9, 31.6, 32.1, 35.5, 36.6, 42.8, 76.5, 116.8, 119.9,
120.1, 122.7, 126.3, 133.3, 142.1, 152.8; [a]2d0 160ꢀ;
HRMS: calcd for C22H32O 312.2453, found 312.2452.
1-Deoxy-3-(10,10 -dimethylethyl)-D-tetrahydrocannabinol
8
(2, n=0). Dimethyl-ethyl-Á8-THC was converted to the
corresponding deoxy-cannabinoid by the procedure used
for the preparati0on of deoxy-Á8-THC. From 1.0 g
(3.3 mmol) of 3-(1 ,10-dimethylethyl)-Á8-THC there was
obtained 1.0 g (69%) of phosphate ester. Reduction of
0.70g (1.61 mmol) of this phosphate ester gave 0.35g
(77%) of deoxy-cannabinoid as a yellow solid, mp 101±
103ꢀC, after chromatography (petroleum ether:ethyl ace-
tate 100:1): Rf 0.39 (petroleum ether:ethyl acetate 100:1),
0.71 (petroleum ether:ethyl acetate 25:1); 1H NMR
(300MHz, CDCl3) d 1.19 (s, 3H), 1.32 (s, 9H), 1.42 (s,
3H), 1.69±2.03 (m, 3H) 1.72 (s, 3H), 2.15 (m, 1H), 2.61±
2.76 (m, 2H), 5.48 (br s, 1H), 6.86 (d, J=1.9 Hz, 1H), 6.94
(dd, J=1.9, 8.1 Hz, 1H), 7.17 (d, J=8.1 Hz, 1H); 13C
NMR (75.5 MHz, CDCl3) d 19.2, 23.5, 27.5, 27.7, 31.3,
2 Methyl-2-(3,5-dimethoxyphenyl)propane. A solution of
1.7 g (23.0 mmol) of tert-butyl alcohol, 3.5 g (22.7 mmol)
of 2,6-dimethoxyphenol and 4.0 mL of methanesulfonic

2912
J. W. Hu€man et al. / Bioorg. Med. Chem. 7 (1999) 2905±2914
32.0, 34.4, 36.4, 42.8, 76.8, 114.1, 117.1, 119.9, 122.6, 126.2,
133.4, 150.6, 152.6; MS (EI) m/z (rel intensity) 284 (30),
201 (30), 57 (100); [a]d20 168ꢀ (c 1.30, CHCl3); anal. calcd
for C20H28O: C, 84.45; H, 9.92; found: C, 84.39; H, 10.00.
(75.5 MHz, CDCl3) d 14.1, 19.2, 22.5, 23.5, 24.3, 27.5,
27.7, 28.8, 28.9, 32.0, 32.6, 36.5, 37.3, 42.9, 44.5, 76.7,
114.6, 117.6, 119.9, 122.4, 126.0, 133.4, 149.4, 152.5; MS
(EI) m/z (rel intensity) 340 (43), 270 (75), 269 (100), 257
(49); [a]2d0 134ꢀ (c 1.33, CHCl3); HRMS: calcd for
C24H36O 340.2766, found 340.2765.
0
0
8
1-Deoxy-3-(1 ,1 -dimethylpropyl)-D-tetrahydrocannabinol
(2, n=1). Deoxy-cannabinoid 2, n=1, was obtained as
a colorless oil after chromatography (petroleum ether:-
ethyl acetate 100:1): Rf 0.36 (petroleum ether:ethyl ace-
1-Deoxy-3-(10,10 -dimethyloctyl)-D-tetrahydrocannabinol
8
(2, n=6). Pure deoxy-cannabinoid 2, n=6, was
obtained as an oil after ¯ash chromatography (petroleum
ether:ethyl acetate 20:1): Rf 0.40 (hexanes:ethyl acetate
100:1), 0.60 (hexanes:ethyl acetate 25:1); 1H NMR
(300MHz, CDCl3) d 0.85 (t, J=7.1 Hz, 3H), 1.15 (s, 3H),
1.25 (s, 6H), 1.39 (s, 3H), 1.72 (s, 3H), 0.97±2.06 (m, 15H),
2.06±2.24 (m, 1H), 2.51±2.74 (m, 2H), 5.44 (s, 1H), 6.76 (d,
J=1.7 Hz, 1H), 6.83 (dd, J=8.0, 1.7 Hz, 1H), 7.11 (d,
J=8.1 Hz, 1H); 13C NMR (75.5 MHz, CDCl3) d 14.1,
19.2, 22.6, 23.5, 24.7, 27.5, 27.7, 28.8, 29.2, 30.3, 31.9, 32.0,
36.5, 37.3, 42.8, 44.5, 76.7, 114.6, 117.6, 119.9, 122.4,
126.0, 133.4, 149.4, 152.5; MS (EI) m/z (relative intensity)
369 (26), 285 (14), 269 (100), 185 (13); [a]2d0 142ꢀ (c 1.33,
CHCl3); HRMS: calcd for C26H40O 368.3079, found
368.3080.
1
tate 100:1), 0.72 (petroleum ether:ethyl acetate 25:1); H
NMR (300MHz, CDCl3) d 0.69 (t, J=7.3, 3H), 1.16 (s,
3H), 1.25 (s, 6H), 1.40 (s, 3H), 1.60 (q, J=7.3 Hz, 2H),
1.67±1.97 (m, 6H), 2.13 (m, 1H), 2.58±2.73 (m, 2H), 5.45
(br s, 1H), 6.77 (d, J=2.0 Hz, 1H), 6.85 (dd, J=2.0,
8.1 Hz, 1H), 7.14 (d, J=8.1 Hz, 1H); 13C NMR
(75.5 MHz, CDCl3) d 9.2, 19.2, 23.5, 27.5, 27.7, 28.3, 32.0,
36.5, 36.8, 37.6, 42.8, 76.7, 114.8, 117.8, 119.9, 122.5,
126.0, 133.4, 149.1, 152.5; MS (EI) m/z (relative intensity)
298 (10), 167 (100); [a]2d0 143ꢀ (c 1.25, CHCl3); HRMS:
calcd for C21H30O 298.2297, found 298.2297.
1-Deoxy-3-(10,10-dimethylbutyl)-D-tetrahydrocannabinol
8
(2, n=2). Deoxy-cannabinoid 2, n=2, was obtained as a
colorless oil after chromatography (petroleum ether:ethyl
acetate 100:1): Rf 0.42 (petroleum ether:ethyl acetate
100:1), 0.81 (petroleum ether:ethyl acetate 25:1); 1H NMR
(300MHz, CDCl3) d 0.84 (t, J=7.3, 3H), 1.08±1.20 (m,
2H), 1.23 (s, 3H), 1.28 (s, 6H), 1.42 (s, 3H), 1.53±1.59 (m,
2H), 1.66±2.21 (m, 3H) 1.75 (s, 3H), 2.15 (m, 1H), 2.60±
2.75 (m, 2H), 5.48 (br s, 1H), 6.78 (d, J=1.8 Hz, 1H), 6.78
(dd, J=1.8, 8.0 Hz, 1H), 7.15 (d, J=8.0 Hz, 1H); 13C
NMR (75.5 MHz, CDCl3) d 14.8, 17.9, 19.2, 23.4, 27.5,
27.7, 28.8, 28.9, 32.0, 36.5, 37.4, 42.8, 47.0, 76.7, 114.6,
117.6, 119.9, 122.4, 126.0, 133.3, 149.3, 152.5; MS (EI) m/z
(rel intensity) 312 (30), 269 (45), 57 (100); [a]2d0 185ꢀ
(c 1.65, CHCl3); HRMS: calcd for C22H32O 312.2453,
found 312.2451.
1-Deoxy-3-(10,10-dimethylnonyl)-D-tetrahydrocannabinol
8
(2, n=7). Pure deoxy-cannabinoid 2, n=7, was obtained
as an oil after ¯ash chromatography (petroleum ether:
ethyl acetate 20:1): Rf 0.62 (hexanes:ethyl acetate 25:1),
1
0.43 (hexanes:ethyl acetate 100:1); H NMR (300MHz,
CDCl3) d 0.86 (t, J=6.9 Hz, 3H), 1.16 (s, 3H), 1.25 (s, 6H),
0.98±1.35 (m, 15H), 1.39 (s, 3H), 1.48±1.61 (m, 2H), 1.61±
2.06 (m, 3H), 2.06±2.24 (m, 1H), 2.52±2.76 (m, 2H), 5.43
(s, 1H), 6.76 (d, J=1.8 Hz, 1H), 6.83 (dd, J=8.1, 2.1 Hz,
1H), 7.11 (d, J=8.1 Hz, 1H); 13C NMR (75.5 MHz,
CDCl3) d 4.1, 19.2, 22.7, 23.5, 24.7, 27.5, 27.7, 28.8, 29.3,
30.4, 31.9, 32.0, 36.5, 37.3, 42.8, 44.5, 76.7, 114.6, 117.6,
119.9, 122.4, 126.0, 133.4, 149.5, 152.5; MS (EI) m/z (rel
intensity) 383 (33), 299 (11), 269 (100), 201 (20); [a]d20
150ꢀ (c 0.48, CHCl3); HRMS: calcd for C27H42O
382.3236, found 382.3237.
0
0
8
1-Deoxy-3-(1 ,1 -dimethylpentyl)-D-tetrahydrocannabinol
(2, n=3). Deoxy-cannabinoid 2, n=3, was obtained
from 3-(10,10-dimethylpentyl-Á8-tetrahydrocannabinol20
as a colorless oil after ¯ash chromatography (petroleum
ether:ethyl acetate 20:1): Rf 0.50 (hexanes:ethyl acetate
50:1), 0.61 (hexanes:ethyl acetate 20:1); 1H NMR
(300MHz, CDCl3) d 0.84 (t, J=7.1 Hz, 3H), 1.15 (s, 3H),
1.25 (s, 6H), 1.38 (s, 3H), 1.71 (s, 3H), 1.00±2.05 (m, 9H),
2.07±2.22 (m, 1H), 2.52±2.75 (m, 2H), 5.43 (s, 1H), 6.76 (d,
J=1.7 Hz, 1H), 6.83 (dd, J=8.1, 1.7 Hz, 1H), 7.10 (d,
J=8.1 Hz, 1H); 13C NMR (75.5 MHz, CDCl3) d 14.0,
19.1, 23.4, 23.5, 26.9, 27.5, 27.7, 28.8, 32.0, 36.5, 37.2, 42.8,
44.3, 76.6, 114.6, 117.6, 119.9, 122.3, 126.0, 133.3, 149.3,
152.5; MS (EI) m/z (rel intensity) 326 (23), 269 (100), 243
(18); [a]2d0 152ꢀ (c 1.24, CHCl3); HRMS: calcd for
C23H34O 326.2610, found 326.2609.
1-Deoxy-3-(1,1-dimethylheptyl)-D9(11)-THC (17). To a
stirred solution of 1.84 g (5.15 mmol) of methyl-
triphenylphosphonium bromide in 20 mL of dry THF,
at ambient temperature, was added dropwise 1.90 mL
(4.75 mmol) of 2.5 M n-butyllithium in hexanes. After
stirring for 5 min, a solution of 0.166 g (0.466 mmol) of
deoxynabilone (16) in 3 mL of dry THF was added
dropwise. The reaction mixture was stirred at ambient
temperature for 2 h, and heated at re¯ux with stirring
for 18 h. After cooling, the reaction was quenched with
water, and extracted with three portions of ether. The
combined extracts were washed with brine and dried
(MgSO4). The solvent was removed in vacuo to give an oil
which was chromatographed (petroleum ether:ether 30:1)
to provide 0.137 g (82%) of 17: Rf 0.25 (hexanes), 0.55
(hexanes:ethyl acetate 40:1); 1H NMR (300MHz, CDCl3)
d 0.80 (t, J=6.9 Hz, 3H), 1.09 (s, 3H), 1.21 (s, 6H), 0.92±
1.29 (m, 9H), 1.38 (s, 3H), 1.45±1.62 (m, 3H), 1.81±2.16
(m, 3H), 2.32±2.49 (m, 2H), 2.89±3.06 (m, 1H), 4.72 (s,
1H), 4.77 (s, 1H), 6.71 (d, J=1.8 Hz, 1H), 6.79 (dd,
J=1.8, 8.0 Hz, 1H), 7.09 (d, J=8.1 Hz, 1H); 13C NMR
(75.5 MHz, CDCl3) d 14.1, 20.1, 22.6, 24.6, 28.1, 28.6,
1-Deoxy-3-(10,10-dimethylhexyl)-D-tetrahydrocannabinol
8
(2, n=4). Pure deoxy-cannabinoid 2, n=4, was obtained
as a yellow oil: Rf 0.39 (hexanes:ethyl acetate 100:1), 0.58
(hexanes:ethyl acetate 25:1); 1H NMR (300MHz, CDCl3)
d 0.82 (t, J=7.1 Hz, 3H), 1.18 (s, 6H), 1.01±2.04 (m, 14H),
1.39 (s, 3H), 1.68 (s, 3H), 2.07±2.23 (m, 1H), 2.51±2.77 (m,
2H), 5.44 (s, 1H), 6.76 (d, J=2.0 Hz, 1H), 6.83 (dd, J=
8.1, 2.0 Hz, 1H), 7.11 (d, J=8.0 Hz, 1H); 13C NMR

J. W. Hu€man et al. / Bioorg. Med. Chem. 7 (1999) 2905±2914
2913
28.8, 30.0, 31.7, 34.4, 36.7, 37.3, 39.3, 44.5, 46.7, 77.0,
108.7, 114.5, 117.4, 121.5, 125.2, 147.5, 149.6, 152.6; MS
(EI) m/z (relative intensity) 354 (26), 335 (29), 269 (100),
185 (9); [a]2d5 14.0ꢀ (c 3.0, CHCl3); HRMS: calcd for
C25H38O 354.2923, found 354.2921.
distilled acetone was added 1.3 mL (13.8 mmol) of
dimethyl sulfate. The resulting mixture was re¯uxed
overnight, cooled to ambient temperature and diluted
with distilled water. The acetone was removed under
reduced pressure and the aqueous residue extracted
three times with ether. The combined organic extracts
were washed with water, brine and dried (MgSO4).
Concentration and chromatography (petroleum ether:
ethyl acetate 100:1) gave the methyl ether as a colorless
oil: Rf 0.34 (petroleum ether), 0.74 (petroleum ether:
ethyl acetate 9:1); 1H NMR (300 MHz, CDCl3) d 0.85 (t,
J=6.9 Hz, 3H), 1.10 (s, 3H), 1.05±1.35 (m, 8H), 1.25 (s,
6H), 1.39 (s, 3H), 1.62 (m, 2H) 1.74 (s, 3H), 1.75±1.90
(m, 3H), 2.14 (m, 1H), 2.67 (dt, J=4.8, 10.9 Hz, 1H),
3.17 (dd, J=4.0, 17.6 Hz, 1H), 3.81 (s, 3H), 5.42 (d,
J=4.2 Hz, 1H), 6.38 (d, J=1.6 Hz, 1H), 6.43 (d,
J=1.6 Hz, 1H); 13C NMR (75.5 MHz, CDCl3) d 14.1,
18.4, 22.7, 23.5, 24.6, 27.6, 28.0, 28.8, 30.0, 31.8, 36.2,
37.7, 44.5, 45.1, 55.0, 76.7, 100.6, 108.3, 111.6, 119.2,
135.0, 149.6, 154.0, 158.7; MS (EI) m/z (relative inten-
sity) 384 (40), 300 (100).
(6aR,10aR)-3-(1,1-Dimethylheptyl)-6,6a,7,8,10,10a-hexa-
hydro-6,6-dimethyl-9H-dibenzo[b,d]pyran-9-ꢀ-ol (18). To
a stirred solution of 0.238 g (0.668 mmol) of deoxy-
nabilone (16) in 2 mL of dry THF at 78ꢀC was added
0.051 g (1.3 mmol) of LiAlH4. The reaction mixture was
stirred at 78ꢀC for 2 h, warmed to ambient tempera-
ture and stirred for 18 h. After quenching with water,
and acidi®cation with 10% HCl, the reaction mixture
was extracted with ether. The ether extracts were
washed with brine, dried (MgSO4), and the solvent
removed in vacuo to give an o€ white gum. Chromato-
graphy (petroleum ether:ether 1:2) gave 0.177 g (72%)
of 18 as an o€ white solid: mp 110±111ꢀC; Rf 0.13
(hexanes:ethyl acetate 5:1), 0.49 (hexanes:ethyl acetate
1
1:2); H NMR (300 MHz, CDCl3) d 0.84 (t, J=6.9 Hz,
3H), 0.97±1.48 (m, 12H), 1.14 (s, 3H), 1.24 (s, 6H), 1.41
(s, 3H), 1.48±1.59 (m, 2H), 1.64 (br s, 1H), 1.84±1.96 (m,
1H), 2.07±2.22 (m, 1H), 2.39±2.55 (m, 1H), 2.65±2.80
(m, 1H), 3.72±3.89 (m, 1H), 6.74 (d, J=1.8 Hz, 1H),
6.83 (dd, J=1.8, 8.1 Hz, 1H), 7.10 (d, J=8.1 Hz, 1H);
13C NMR (75.5 MHz, CDCl3) d 14.1, 20.2, 22.7, 24.6,
25.8, 28.2, 30.0, 31.8, 33.7, 35.4, 37.3, 39.8, 44.5, 46.2,
70.6, 77.1, 114.6, 117.5, 121.0, 125.1, 149.8, 152.7; MS
(EI) m/z (relative intensity) 340 (21), 321 (48), 255 (100),
235 (12), 213 (11); [a]2d5 29.9ꢀ (c 1.10, CHCl3); HRMS:
calcd for C24H38O2 358.2872, found 358.2871.
Nabilone methyl ether. Nabilone16 was converted to the
corresponding methyl ether by the procedure described
above for the preparation of 3-(10,10-dimethylheptyl)-
Á8-tetrahydrocannabinol methyl ether. From 0.18 g
(0.48 mmol) of nabilone there was obtained 0.14 g
(75%) of methyl ether after chromatography (petroleum
1
ether:ethyl acetate 9:1). The H and 13C NMR spectra
were identical to those of a sample prepared by an
alternative route.10
3-(10,10-Dimethylheptyl)-D9(11)-tetrahydrocannabinol meth-
yl ether (4). Reaction of 0.10 g (0.26 mmol) of nabilone
methyl ether with the ylide derived from 0.925 g
(2.59 mmol) methyl triphenylphosphonium bromide was
carried out as described above for the preparation of 17,
to give 0.40 g (40%) of methyl ether 4 as a colorless oil
after chromatography (petroleum ether:ethyl acetate
19:1): Rf 0.38 (hexane), 0.64 (petroleum ether:ethyl ace-
tate 9:1); 1H NMR (300 MHz, CDCl3) d 0.83 (t,
J=6.9 Hz, 3H), 1.04 (s, 3H), 1.08±1.19 (m, 9H), 1.23 (s,
6H), 1.39 (s, 3H), 1.53 (m, 2H), 1.60±1.80 (m, 2H), 1.92
(m, 1H), 2.20 (m, 1H), 2.43 (dt, J=3.1, 11.5 Hz, 2H), 3.63
(d, J=11.5 Hz, 1H), 3.81 (s, 3H), 4.72 (s, 1H), 4.77 (s,
1H), 6.36 (d, J=1.6 Hz, 1H), 6.40 (d, J=1.6 Hz, 1H); 13C
NMR (75.5 MHz, CDCl3) d 14.1, 19.0, 22.7, 24.6, 27.8,
28.8, 28.9, 30.0, 31.8, 35.0, 36.7, 37.7, 39.1, 44.5, 48.9,
55.1, 76.7, 100.7, 108.2, 108.6, 110.8, 148.8, 149.8, 154.1,
158.6; MS (EI) m/z (relative intensity) 384 (40), 300 (100).
(6aR,10aR)-3-(1,1-Dimethylheptyl)-6,6a,7,8,10,10a-hexa-
hydro-6,6-dimethyl-9H-dibenzo[b,d]pyran-9-ꢁ-ol (19). To
a solution of 0.252 g (0.707 mmol) of deoxynabilone
in 2 mL of dry THF at 78ꢀC was added 1.50 mL
(1.50 mmol) of 1.0 M K-selectride in THF. The reaction
mixture was stirred at 78ꢀC for 2 h and at ambient
temperature for 1 h. A solution of 1 mL of water in 5 mL
of EtOH was added, followed by 2 mL of 15% aqueous
NaOH and 2 mL of 30% H2O2. The mixture was extrac-
ted with ether, the ether extracts were washed with brine,
dried (MgSO4) and the solvent removed at reduced pres-
sure to a€ord an oil. Chromatography (petroleum ether:
ether 1:1) gave 0.199 g (79%) of 19 as an oil: Rf 0.26
(hexanes:ethyl acetate 5:1), 0.52 (hexanes:ethyl acetate
1
1:2); H NMR (300MHz, CDCl3) d 0.84 (t, J=7.0 Hz,
3H), 0.99±1.31 (m, 8H), 1.17 (s, 3H), 1.24 (s, 6H), 1.40 (s,
3H), 1.35±1.68 (m, 7H), 1.84±2.00 (m, 1H), 2.05 (s, 1H),
2.45±2.60 (m, 1H), 2.81±2.96 (m, 1H), 4.26 (s, 1H), 6.74 (d,
J=1.7 Hz, 1H), 6.81 (dd, J=1.9, 8.1 Hz, 1H), 7.07 (d,
J=8.0 Hz, 1H); 13C NMR (75.5 MHz, CDCl3) d 14.0,
20.2, 21.5, 22.6, 24.6, 27.8, 28.8, 29.0, 30.0, 31.7, 32.6, 37.2,
37.6, 44.5, 47.0, 66.0, 77.0, 114.5, 117.3, 121.8, 125.1,
149.4, 152.9; MS (EI) m/z (relative intensity) 340 (32), 321
(76), 255 (100), 251 (19); [a]2d5 19.4ꢀ (c 4.90, CHCl3);
HRMS: calcd for C24H38O2 358.2872, found 358.2871.
Receptor binding assays
CB1 assay. [3H]CP-55,940 (KD=690 nM) binding to P2
membranes was conducted as described elsewhere,21
except whole brain (rather than cortex only) was used.
Displacement curves were generated by incubating
drugs with 1 nM of [3H]CP-55,940. The assays were
performed in triplicate, and the results represent the
combined data from three individual experiments.
3-(10,10-Dimethylheptyl)-D-tetrahydrocannabinol methyl
8
ether (3). To a suspension of 2.0 g (14.5 mmol) of anhy-
drous potassium carbonate and 0.25 g (0.68 mmol) of
3-(10,10-dimethylheptyl)-Á8-THC in 10 mL of freshly
CB2 assay. Human embryonic kidney 293 cells were
maintained in Dulbecco0s modi®ed Eagle0s medium

2914
J. W. Hu€man et al. / Bioorg. Med. Chem. 7 (1999) 2905±2914
(DMEM) with 10% fetal clone II (HyClone, Logan,
UT) and 5% CO2 at 37ꢀC in a Forma incubator. Cell
lines were created by transfection of CB2pcDNA3 into
293 cells by the Lipofectamine reagent (Life Technolo-
gies, Gaithersburg, MD). The human CB2 cDNA was
provided by Dr. Sean Munro (MRC, Cambridge, Eng-
land). Stable transformants were selected in growth
medium containing geneticin (1 mg/mL reagent, Life
Technologies, Gaithersburg, MD). Colonies of about
500 cells were picked (about 2 weeks post transfection)
and allowed to expand, then tested for expression of
receptor mRNA by Northern blot analysis. Cell lines
containing moderate to high levels of receptor mRNA
were tested for receptor binding properties. Transfected
cell lines were maintained in DMEM with 10% fetal
clone II plus 0.3±0.5 mg/mL geneticin and 5% CO2 at
37ꢀC in a Forma incubator.
Displacement IC50 values were originally determined by
unweighted least-squares linear regression of log con-
centration±percent displacement data and then con-
verted to Ki values using the method of Cheng and
Pruso€.22
Acknowledgements
The work at Clemson was supported by grant
DA03590, and that at Virginia Commonwealth Uni-
versity by grants DA03672 (B.R.M.) and DA05274
(M.E.A.), all from the National Institute on Drug
Abuse.
References
1. Razdan, R. K. Pharmacol. Rev. 1986, 38, 75.
2. Rapaka, R. S.; Makriyannis, A. Structure±Activity Rela-
tionships of the Cannabinoids. NIDA Research Monograph 79,
National Institute on Drug Abuse, Rockville, MD, 1987.
3. Melvin, L. S.; Milne, G. M.; Johnson, M. R.; Subramian,
B.; Wilken, G. H.; Howlett, A. C. Mol. Pharmacol. 1993, 44,
1008.
4. Hu€man, J. W.; Yu, S.; Showalter, V.; Abood, M. E.;
Wiley, J. L.; Compton, D. R.; Martin, B. R.; Bramblett, R.
D.; Reggio, P. H. J. Med. Chem. 1996, 39, 3875.
5. Gareau, Y.; Dufresne, C.; Gallant, M.; Rochette, C.; Saw-
yer, N.; Slipetz, D. M.; Tremblay, N.; Weech, P. K.; Metters,
K. M.; Labelle, M. Bioorg. Med. Chem. Lett. 1996, 6, 189.
6. Showalter, V. M.; Compton, D. R.; Martin, B. R.; Abood,
M. E. J. Pharmacol. Exp. Ther. 1996, 278, 989.
7. Song, Z. H.; Bonner, T. I. Mol. Pharmacol. 1996, 49, 891.
8. Chin, C.; Lucas-Lenard, J.; Abadji, V.; Kendall, D. A. J.
Neurochem. 1998, 70, 366.
9. Hu€man, J. W.; Zhang, X.; Wu, M.-J.; Joyner, H. H.;
Pennington, W. T. J. Org. Chem. 1991, 56, 1481.
10. Hu€man, J. W.; Joyner, H. H.; Lee, M. D.; Jordan, R. D.;
Pennington, W. T. J. Org. Chem. 1991, 56, 2081.
11. Dominianni, S. J.; Ryan, C. W.; De Armitt, C. W. J. Org.
Chem. 1977, 42, 344.
12. Petrzilka, T.; Sikemeier, C. Helv. Chim. Acta 1967, 50,
1416.
13. Compton, D. R.; Rice, K. C.; De Costa, B. R.; Razdan, R.
K.; Melvin, L. S.; Johnson, M. R.; Martin, B. R. J. Pharmacol.
Exp. Ther. 1993, 265, 218.
The current assay is a modi®cation of Compton et al.13
Cells were harvested in phosphate-bu€ered saline con-
taining 1 mM EDTA and centrifuged at 500 g. The cell
pellet was homogenized in 10 mL of solution A (50 mM
Tris±HCl, 320 mM sucrose, 2 mM EDTA, 5 mM
MgCl2, pH 7.4). The homogenate was centrifuged at
1600 g (10 min), the supernatant saved, and the pellet
washed three times in solution A with subsequent cen-
trifugation. The combined supernatants were cen-
trifuged at 100,000 g (60 min). The (P2 membrane) pellet
was resuspended in 3 mL of bu€er B (50 mM Tris±HCl,
1 mM EDTA, 3 mM MgCl2, pH 7.4) to yield a protein
concentration of approximately 1 mg/mL. The tissue
preparation was divided into equal aliquots, frozen on
dry ice, and stored at 70ꢀC. Binding was initiated by
the addition of 40±50 mg membrane protein to silanized
tubes containing [3H]CP-55,940 (102.9 Ci/mmol) and a
sucient volume of bu€er C (50 mM Tris±HCl, 1 mM
EDTA, 3 mM MgCl2, and 5 mg/mL fatty acid free BSA,
pH 7.4) to bring the total volume to 0.5 mL. The addi-
tion of 1 mM unlabeled CP-55,940 was used to assess
nonspeci®c binding. Following incubation (30ꢀC for
1 h), binding was terminated by the addition of 2 mL of
ice cold bu€er D (50 mM Tris±HCl, pH 7.4, plus 1 mg/
mL BSA) and rapid vacuum ®ltration through What-
man GF/C ®lters (pretreated with polyethyleneimine
(0.1%) for at least 2 h). Tubes were rinsed with 2 mL of
ice cold bu€er D, which was also ®ltered, and the ®lters
subsequently rinsed twice with 4 mL of ice cold bu€er
D. Before radioactivity was quantitated by liquid scin-
tillation spectrometry, ®lters were shaken for 1 h in
5 mL of scintillation ¯uid.
14. Reggio, P. H.; Panu, A. M.; Miles, S. J. Med. Chem. 1993,
36, 1761.
15. Reggio, P. H.; Greer, K. V.; Cox, S. M. J. Med. Chem.
1989, 32, 1630.
16. Tius, M. A.; Kawakami, J. K.; Hill, W. G. A.; Makriyan-
nis, A. Chem. Commun. 1996, 2085.
17. Alles, G. A.; Icke, R. N.; Feigen, G. A. J. Am. Chem. Soc.
1942, 64, 2031.
18. Smith, R. M. J. Forensic Sci. 1997, 42, 610.
19. Edery, H.; Grunfeld, Y.; Porath, G.; Ben-Zvi, Z.; Shani,
A.; Mechoulam, R. Arzneim.-Forsch. 1972, 22, 1995.
20. Petrzilka, T.; Hae¯iger, W.; Sikemeier, C. Helv. Chim.
Acta 1969, 52, 1102.
21. Martin, B. R.; Compton, D. R.; Thomas, B. F.; Prescott,
W. R.; Little, P. J.; Razdan, R. K.; Johnson, M. R.; Melvin, L.
S.; Mechoulam, R.; Ward, S. J. Pharmacol. Biochem. Behav.
1991, 40, 471.
CP-55,940 and all cannabinoid analogues were prepared
by suspension in assay bu€er from a 1 mg/mL ethanolic
stock without evaporation of the ethanol (®nal con-
centration of no more than 0.4%). When anandamide
was used as a displacing ligand, experiments were per-
formed in the presence of phenylmethylsulfonyl ¯uor-
ide (50 mM). Competition assays were conducted with
1 nM [3H]CP-55,940 or 1 nM [3H]SR141716A and 6
concentrations (0.1 nM to 10 mM displacing ligands).
22. Cheng, Y. C.; Pruso€, W. H. Biochem. Pharmacol. 1973,
22, 3099.