Synthesis and pharmacology of 1-deoxy analogs of CP-47,497 and CP-55,940

Article abstract of DOI:10.1016/j.bmc.2007.09.033

A series of 1-deoxy analogs of CP-47,497 (8 and 13, n = 0-7) and 1-deoxy analogs of CP-55,940 (9, n = 0-7) have been synthesized and their affinities for the cannabinoid CB₁ and CB₂ receptors have been determined. Although the majori

Full text of DOI:10.1016/j.bmc.2007.09.033

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 322–335
Synthesis and pharmacology of 1-deoxy analogs
of CP-47,497 and CP-55,940
John W. Huffman,a,* Alicia L. S. Thompson,a Jenny L. Wileyb and Billy R. Martinb
aHoward L. Hunter Laboratory, Clemson University, Clemson, SC 29634-0973, USA
bDepartment of Pharmacology and Toxicology, Medical College of Virginia Campus,
Virginia Commonwealth University, Richmond, VA 23298-0613, USA
Received 12 July 2007; revised 14 September 2007; accepted 19 September 2007
Available online 22 September 2007
Abstract—A series of 1-deoxy analogs of CP-47,497 (8 and 13, n = 0–7) and 1-deoxy analogs of CP-55,940 (9, n = 0–7) have been
synthesized and their affinities for the cannabinoid CB1 and CB2 receptors have been determined. Although the majority of these
compounds exhibit selectivity for the CB2 receptor, none have greater than modest affinity for either receptor. The interactions of
these 1-deoxy nontraditional cannabinoids with the CB2 receptor are discussed.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
laboratory to identify a cannabinoid receptor in rat
brain.9 This G-protein-coupled, transmembrane recep-
tor is now designated as the CB1 receptor and is located
primarily in the central nervous system.10–12
In the years following the elucidation of the structure of
D9-tetrahydrocannabinol (D9-THC, 1) and its recogni-
tion as the principal psychoactive constituent of mari-
juana, a number of analogs were synthesized and
structure–activity relationships (SAR) based upon the
dibenzopyran structure of THC were developed.1–4 In
the early 1980s a group at Pfizer explored the develop-
ment of analgesics using the potent synthetic cannabi-
noid, (ꢀ)-9-nor-9b-hydroxyhexahydrocannabinol (HHC,
2), as a template.5–7 This led to a series of nontraditional
cannabinoids in which the oxygen containing pyran ring
of THC was removed to provide a bicyclic system that
retained the phenolic hydroxyl group of THC and the
9-hydroxyl of HHC. In common with the SAR devel-
oped for traditional cannabinoids, it was found that
potency was maximum with a 1,1-dimethylheptyl substi-
tuent at the 3-position of the aromatic ring and the 9-hy-
droxyl had the b-orientation.8 The least complex
molecule that fulfilled these requirements was CP-
47,497 (3, DMH = 1,1-dimethylheptyl), which was
found to be more potent than THC in vivo. The addi-
tion of a hydroxypropyl group at C-4 of the cyclohexa-
nol ring, as in CP-55,940 (4), led to enhanced potency.
In 1988 [3H]CP-55,940 was used in work from Howlett’s
A second receptor, designated CB2, was originally identi-
fied from macrophages present in the spleen,13 and is ex-
pressed primarily in the immune system.14–18 It has been
suggested that this receptor is responsible for the immu-
nomodulatory effects of cannabinoids,14 a conclusion
that is supported by the fact that these effects are absent
in CB2 receptor knockout mice.19 CB1 and CB2 receptors
are expressed in a variety of cancer cells and both CB1 and
CB2 receptor agonists have been found to inhibit tumor
growth.20,21 CB2 receptors are expressed in C6 glioma
cells22 and both CB1 and CB2 receptors are expressed in
nonmelanoma skin cancer cells.23 There is also evidence
that the CB2 receptor is involved in inflammatory
pain24–31 and it has been implicated in cardioprotection.32
A highly selective CB2 receptor ligand, JWH-133 (10,10-
dimethylbutyl)-D8-tetrahydrocannabinol, 5, causes the
regression of both glioma tumors and nonmelanoma skin
tumors22,23 and very recently the CB2 selective cannabim-
imetic indole AM1241 (6) was found to delay disease pro-
gression in a mouse model of amyotrophic lateral
sclerosis.33 Two recent reviews have pointed out the po-
tential of the endocannabinoid system as a therapeutic
target and indicate that developing new, selective ligands
for the CB2 receptor in particular may lead to the develop-
ment of new useful drugs for the treatment of a number
diseases.34,35
Keywords: CB1 receptor; CB2 receptor; Nontraditional cannabinoids;
1-Deoxy cannabinoids.
*
Corresponding author. Tel.: +1 864 656 3133; fax: +1 864 656
6613; e-mail: huffman@clemson.edu
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.09.033

J. W. Huffman et al. / Bioorg. Med. Chem. 16 (2008) 322–335
323
OH
11
CH3
9
OH
OH
10
8
7
OH
OH
1
2
H3C
H3C
H3C
6
3
O
C5H11
O
C5H11
DMH
H3C
4
5
2
1
3
O
I
CH3
OH
OH
N
H3C
H3C
CH3
NO2
CH3
O
DMH
N
CH3
H3C
HO
4
5
6
OH
OH
OH
CH
CH
H C
CH
CH
CH
CH
3
3
3
3
CH
n
3
O
( )
(CH ) CH
2 n 3
(CH ) CH
2 n
3
H C
3
3
3
3
HO
7
8
9
Most of the CB2 selective agonists currently available are
either 1-methoxy or 1-deoxy analogs of D8-THC or are
indole derivatives.36,37 Among these CB2 selective can-
nabinoids, the 9-hydroxy-1-deoxyhexahydrocannabinols
(7, n = 1–4, both epimers at C-9), which we described re-
cently, are structurally similar to hexahydrocannabinol
(2), which is a very potent cannabinoid.38 Compound 2
was used as a template to develop the potent Pfizer bicy-
clic nontraditional cannabinoids such as CP-47,497 (3)
and CP-55,940 (4) and it appeared promising to employ
hexahydrocannabinols 7 as a template for a series of
1-deoxy analogs of CP-47,497 (8, n = 0–5) and CP-
55,940 (9, n = 0–5). In analogy to the highly CB2 selective
1-deoxy-3-(1,1-dimethylalkyl)-D8-THC series we re-
ported several years ago, of which JWH-133 (5) is an
example, the length of the alkyl side chain in the deoxy
analogs of the Pfizer compounds was to range from
tert-butyl to 1,1-dimethylheptyl.39 During the course of
the present study, the synthetic goals were extended to
include the dimethyloctyl and dimethylnonyl analogs
(8, and 9, n = 6 and 7). The 1-deoxy analog of CP-
55,940 (9, n = 5) was described previously by Melvin
et al. and was reported to have moderate affinity for
the CB1 receptor (Ki = 40.2 13.5 nM), although it
lacked potency in vivo.40 The affinity of this compound
for the CB2 receptor was not reported.
however, the only compound of this type that is com-
mercially available is 1-bromo-4-tert-butylbenzene (10,
n = 0). The synthesis of the other members of this series
from the trifluoromethanesulfonate ether of the corre-
sponding phenol via a borate ester was carried out using
a sequence developed in our laboratory for the conver-
sion of phenols to the corresponding aryl bromide.41
Halogen–metal interconversion was applied to aryl bro-
mides 10, followed by reaction of the organolithium
compound with 3-ethoxy-2-cyclohexen-1-one and mild
acid treatment to provide 3-arylcyclohexeneones 11.
Dissolving metal reduction using Li in liquid NH3 gave
3-arylcyclohexanones 12. Reduction of the carbonyl
group of ketones 12 with NaBH4 gave the racemic cis-
3-arylcyclohexanols (8, n = 0–7) in which the hydroxyl
group has the equatorial conformation. The trans-alco-
hols (13, n = 0–7) with an axial hydroxyl group were
prepared by stereoselective reduction of ketones 12 with
K-Selectrideꢁ (potassium tri-sec-butylborohydride).
The CP-55,940 analogs (9, n = 0–7) were prepared by a
modification of the procedure described by the Pfizer
group for the synthesis of CP-55,940 and its homologs.42
Copper catalyzed conjugate addition of the Grignard re-
agents derived from aryl bromides 10 to 4-(2-propenyl)-
2-cyclohexen-1-one provided racemic ketones 14 as
shown in Scheme 2. 4-(2-Propenyl)-2-cyclohexen-1-one
was prepared from 3-ethoxy-2-cyclohexen-1-one by the
Pfizer procedure and has spectroscopic properties iden-
2. Results
¨
tical to those reported recently by Tanyeli and Ozdemir-
The synthesis of the 1-deoxy CP-47,497 analogs is out-
lined in Scheme 1. This synthesis required a series of
1-bromo-4-(1,1-dimethylalkyl)benzenes (10, n = 0–7);
han.43 Stereoselective NaBH4 reduction of ketones 14
gave alcohols 15, which upon hydroboration–oxidation
provided 1-deoxy CP-55,940 analogs 9.

324
J. W. Huffman et al. / Bioorg. Med. Chem. 16 (2008) 322–335
O
Br
CH3
CH3
a, b, c
d, e
CH3
CH3
(CH2)nCH3
(CH2)nCH3
10
11
f
8
O
OH
g, h
CH3
CH3
(CH2)nCH3
(CH2)nCH3
CH3
CH3
12
13
Scheme 1. Reagents and conditions: (a) n-BuLi, THF ꢀ78 ꢂC; (b) 3-ethoxy-2-cylohexen-1-one, THF, reflux; (c) 10% HCl, 25 ꢂC; (d) Li, NH3, t-BuOH,
Et2O, ꢀ78 ꢂC; (e) NH4Cl, 25 ꢂC; (f) NaBH4, EtOH, 25 ꢂC; (g) K(sec-butyl)3BH, THF, ꢀ78 ꢂC then 25 ꢂC; (h) NaOH, H2O2, EtOH, 25 ꢂC.
O
Br
CH
3
a, b, c
d
CH
CH
3
(CH ) CH
2 n
3
(CH ) CH
CH
2 n
3
3
3
10
14
OH
e, f
CH
CH
3
9
(CH ) CH
2 n
3
3
15
Scheme 2. Reagents and conditions: (a) Mg, THF, 35 ꢂC; (b) CuI, ꢀ20 ꢂC; (c) 4-(2-propenyl)-cyclohexen-1-one, 25 ꢂC; (d) NaBH4, 0–25 ꢂC; (e) BH3–
THF, THF, 25 ꢂC; (f) H2O2, NaOH, H2O.
The affinities of alcohols 8, 9, and 13 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.44 Affinities for
the CB2 receptor were determined by measuring 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.45 The results
of these determinations are summarized in Tables 1
and 2. Also included in Table 1 are the receptor affin-
ities for D8-THC (1) and CP-47,497 (3). The CB1 and
CB2 receptor affinities for CP-55,940 are included in
Table 2.
for either the CB1 or CB2 receptor. The compounds with
a b-hydroxyl group (series 8) have little (8, n = 4–7) or
no affinity (8, n = 0–3) for the CB1 receptor. Further,
none of this series of compounds has high affinity for
the CB2 receptor (maximum affinity: Ki = 231 48 nM
for JWH-324 (8, n = 5)).
The CB1 receptor affinities of most of the 9-a-alcohols
(series 13, Table 1) are similar to those of the 9-b-alco-
hols, having little or no affinity for this receptor. The
one exception is JWH-405 (13, n = 6) with
Ki = 193 3 nM. While CB2 receptor affinities are some-
what better than CB1 affinities for most compounds of
series 13 [exceptions are JWH-232 (13, n = 0) and
JWH-402 (13, n = 7)], they are still only modest at best.
The three 9a-ols (JWH-406, JWH-342, and JWH-405,
13, n = 4, 5, and 6, respectively) have the highest CB2
The data summarized in Table 1 indicate that none of
the CP-47,497 analogs have better than modest affinity

J. W. Huffman et al. / Bioorg. Med. Chem. 16 (2008) 322–335
325
Table 1. Receptor affinities (mean SEM) of CP-47,497 analogs (8
and 13), D9-THC (1) and CP-47,497 (3)
JWH-406 has modest affinity for the CB2 receptor, it
is a moderately efficacious partial agonist relative to
CP-55,940 with EC50 = 39.6 17.9 nM and Emax = 66
0.1 % of that produced by CP-55,940.
Compound
Ki (nM)
CB1
CB2
CB1/CB2
1.1
D9-THC (1)
CP-47,497 (3)
41 2a
36 10b
ND
>10,000
The CB1 and CB2 receptor affinities for 1-deoxy-CP-
55940 analogs (series 9, n = 0–7) are summarized in Table
2. Although none of this series of compounds has high
affinity for the CB1 receptor [highest affinity: Ki = 203
12 nM for sample 2 of JWH-337 (9, n = 5)], all of these
compounds have equal or higher CB1 receptor affinities
than the corresponding series of CP-47,497 analogs (8,
n = 0–7). CB2 receptor affinities for the 1-deoxy-CP-
55940 analogs with shorter side chains (series 9, n = 0–
3, Table 2) are poor. In contrast, analogs with longer side
chains (series 9, n = 4–7, Table 2) show modest affinities
for this receptor, ranging from Ki = 118 to 221 nM for
sample 2 of JWH-337 to Ki = 421 36 nM for JWH-385.
2.20 0.47b
>10,000
>10,000
8, n = 0, JWH-231
8, n = 1, JWH-294
8, n = 2, JWH-296
8, n = 3, JWH-323
8, n = 4, JWH-403
8, n = 5, JWH-324
8, n = 6, JWH-404
8, n = 7, JWH-401
13, n = 0, JWH-232
13, n = 1, JWH-295
13, n = 2, JWH-297
13, n = 3, JWH-407
13, n = 4, JWH-406
13, n = 5, JWH-342
13, n = 6, JWH-405
13, n = 7, JWH-402
3972 228
2060 71
639 45
460 28
231 48
672 1.2
1120 40
>10,000
>2.5
>4.9
>16
4.6
>10,000
>10,000
2113 178
2954 191
786 67
1707 82
>10,000
13
1.2
1.5
>10,000
3759 170
1506 191
546 73
>2.7
5.7
3.2
4.8
3.6
1.3
0.7
8626 459
1731 378
1028 81
645 29
215
178 15
154
1077 25
6
The dimethylheptyl homolog, JWH-337 (9, n = 5), had
been reported previously to have moderate affinity for
the CB1 receptor.40 However, in our hands this com-
pound was found to have very little affinity for the
CB1 receptor, with Ki = 547 57 nM. The synthesis
was independently repeated after an interval of several
months and the second sample had somewhat greater
affinity for the CB1 receptor (Ki = 203 12 nM). The
two samples of JWH-337 also had somewhat different
CB2 receptor affinities, with Ki = 238 41 nM for the
first sample and Ki = 118 3 nM for the second. Be-
cause of the considerable difference in receptor affinities
from that reported previously, we were concerned that
our CP-55,940 analogs did not have the correct struc-
ture, although our synthetic protocol followed closely
that described by the Pfizer group.42 The lowest member
of this series, JWH-384 (9, n = 0) is a crystalline solid
and the structure was confirmed by X-ray crystallogra-
phy.47 We have no explanation for the considerable
difference in CB1 receptor affinities obtained in this work
and the data reported in the earlier work.
193
749 56
3
8
a Ref. 44.
b Ref. 45.
Table 2. Receptor affinities (mean SEM) of CP-55,940 analogs (9),
CP-55,940 (4) and 1-deoxy CP-55,940 (9, n = 5) reported by Melvin
et al. (Ref. 40)
Compound
Ki (nM)
CB1
CB2
CB1/CB2
0.8
CP-55,940 (4)
9, n = 5
0.58 0.07a
40.2 13.5b
>10,000
0.69 0.02a
ND
>10,000
9, n = 0, JWH-384
9, n = 1, JWH-343
9, n = 2, JWH-392
9, n = 3, JWH-325
9, n = 4, JWH-344
9, n = 5, JWH-337,
Sample 1
>10,000
1362 112
1782 196
700 15
>7.3
2.1
3795 409
579 79
308 21
0.8
1.4
221 5
238 41
547 57
2.3
9, n = 5, JWH-337,
Sample 2
203 12
118
3
1.7
9, n = 6, JWH-345
9, n = 7, JWH-385
266 18
566 69
173 18
421 36
1.5
1.3
3. Discussion
a Ref. 45.
b Ref. 40.
Several compounds in the 1-deoxy-HHC series, which
served as a template for the CP-47,947 (8 and 13,
n = 0–7) and CP-55,940 (9, n = 0–7) analogs, have the
desirable combination of good affinity for the CB2
receptor and modest to poor affinity for the CB1 recep-
tor.38 However, none of the CP-47,497 and CP-55,940
analogs (8, 13, and 9, respectively) have high affinity
for the CB1 receptor or better than modest affinity for
the CB2 receptor. For example, the greatest selectivity
for the CB2 receptor (13-fold) is found in JWH-324 (8,
n = 5), but this compound still has only modest affinity
for the CB2 receptor with Ki = 231 48 nM.
receptor affinities with Ki = 215 6, 178 8, and
154 8 nM, respectively.
None of these CP-47,497 analogs (Table 1, series 8 and
13) has high affinity for the CB2 receptor; however, the
efficacy of one of them, JWH-406 (13, n = 4), was eval-
uated for [35S]GTPcS binding, a functional assay that
measures G-protein-coupled receptor activation.46 Chi-
nese hamster ovary (CHO) cells stably expressing the
human CB2 receptor were employed in this determina-
tion (see Section 5). The stimulation is normalized to
that produced by a maximally effective concentration
(3 lM) of the standard cannabinoid agonist CP-55,940
(4). CP-55,940 stimulated [35S]GTPcS binding with an
Emax value of 85 6.3% above basal (normalized to
100%) and an EC50 value of 0.69 0.23 nM. Although
In the 1-deoxy-HHC series (7, n = 1–3), the 9b-hydroxy
compounds have higher affinity for both the CB1 and
CB2 receptors than their 9a-epimers.39 Several years
ago, Reggio et al. demonstrated that 9b-substituted
THC analogs were also more potent than their 9a-epi-
mers.48 This conclusion was supported by our observa-

326
J. W. Huffman et al. / Bioorg. Med. Chem. 16 (2008) 322–335
tion that, while 9b-methyl-D7-THC had moderate affin-
ity for the CB1 receptor (Ki = 72 7 nM) and was com-
parable to D9-THC in potency in the mouse tetrad, the
9a-epimer had little affinity for the CB1 receptor with
Ki = 304 131 nM and lacked potency in vivo.49 In con-
trast to these findings, the 9a-isomers have greater affin-
ity than the 9b-hydroxy compounds in the CP-47,497
series presented here. Analysis of the basis for this differ-
ence in optimal orientation of CP-47,497 analogs versus
those of the THC and 1-deoxy-HHC series requires a re-
view of relevant molecular modeling and site-directed
mutagenesis studies carried out on the CB2 receptor.
that these deoxy CP-47,497 and CP-55,940 analogs
interact in a different orientation than CP-55,940 and
traditional cannabinoids in hydrogen bonding to the
CB2 receptor. Based upon docking studies, it was sug-
gested some years ago that the unexpectedly high affinity
(Ki = 23.7 7 nM) of 1-deoxy-3-(10,10-dimethylheptyl)-
D8-THC for the CB1 receptor could be explained if the
orientation of this compound with the receptor was in-
verted relative to that of D9-THC so that lysine
3.28(192) would hydrogen bond to the benzopyran oxy-
gen of 1-deoxy-3-(10,10-dimethylheptyl)-D8-THC.54 In
the case of 1-deoxy-D8-THCs, many of which have very
high affinity for the CB2 receptor,38,39,54–56 it appears
plausible to suggest that they adopt a similar orientation
in binding to the CB2 receptor, which would facilitate
hydrogen bonding with the receptor. For JWH-133 (5)
and other 1-deoxy-3-(1,1-dimethylalkyl)-D8-THCs, CB2
receptor affinity is relatively insensitive to the length of
the alkyl chain. Although the CB1 receptor affinities in
this series decline from 23 nM for the dimethylheptyl
compound to 2290 nM for the dimethylpropyl analog,
the CB2 receptor affinities fall in a very narrow range,
from 2.9 to 19 nM, for the same compounds.39 This
indicates that in their orientation with the CB2 receptor
the alkyl side chain of the 1-deoxy-3-(1,1-dim-
ethylalkyl)-D8-THCs apparently has no significant inter-
actions with the receptor. Although no modeling or
mutagenesis studies have been reported for the
1-deoxy-3-(1,1-dimethylalkyl)-D8-THCs, it is plausible
that their hydrogen bonding interactions with the CB2
receptor involve the benzopyran oxygen serving as a
surrogate for the phenolic hydroxyl of traditional can-
nabinoids and CP-55,940. In the case of the 1-deoxy
CP-47,497 and CP-55,940 analogs, however, there is
no oxygen substituent on the aromatic ring equivalent
to the benzopyran oxygen of the 1-deoxy-D8-THC ana-
logs, leading to diminished hydrogen bonding to the
CB2 receptor.
A number of studies of the interaction of cannabinoid
ligands with the CB2 receptor have been reported utiliz-
ing molecular modeling and/or site-directed mutagenesis
of the CB2 receptor. While many of these studies were
restricted to cannabimimetic indoles or CB2 receptor
antagonists/inverse agonists, several dealt with tradi-
tional cannabinoids and CP-55,940.50–52 Song and Bon-
ner found that binding of CP-55,940 to a mutant CB1
receptor in which a lysine on helix 3 of the receptor
was converted to an alanine, K3.28(192)A, resulted in
loss of affinity of CP-55,940 for the receptor.53 It was
concluded that this loss of affinity was caused by the loss
of a hydrogen bonding interaction between the lysine of
the wild type receptor and the phenolic hydroxyl of CP-
55,940. In a similar mutation of the CB2 receptor in
which the conserved lysine residue, K3.28(109), was con-
verted to alanine, K3.28(109)A, Tao et al. found that
CP-55,940 binding was not attenuated.50 This mutant
CB2 receptor was fully functional as indicated by for-
skolin-stimulated inhibition of cAMP. Molecular mod-
eling suggested that
a hydrogen bonding cluster
consisting of serine 3.31(112) and threonine 3.35(116)
is important for CP-55,940 (4) binding to the CB2 recep-
tor.50 These modeling studies showed that the secondary
hydroxyl group has hydrogen bonding interactions with
serine 3.31(112) and tyrosine 3.35(116). The phenolic hy-
droxyl group interacts with asparagine 7.45(291) and the
primary hydroxyl interacts with lysine 3.28(109). These
modeling results were supported by the observation that
[3H]CP-55,940 showed no appreciable specific binding
to a doubly mutant CB2 receptor, in which the lysine
to alanine (K109A) mutant was further transformed
4. Conclusions
Although the original goal of this work was the synthe-
sis of new selective ligands for the CB2 receptor, none of
the resulting 1-deoxy CP-47,497 and CP-55,940 analogs
have high affinity for the CB2 receptor. The failure of
these compounds to have better than modest affinity
for the CB2 receptor does, however, provide some in-
sight into the manner in which 1-deoxy traditional can-
nabinoids, other traditional cannabinoids, and the Pfizer
nontraditional cannabinoids interact with the CB2
receptor. These results also indicate that the presence
of an oxygen substituent appended to the aromatic ring
of a traditional cannabinoid is probably essential for
CB2 receptor binding.
by substituting serine 3.31(112) with
a
glycine
(K109AS112G), although specific binding of [3H]WIN-
55,212-2 was observed. Similarly, when a tryptophan
residue in the fourth transmembrane domain of the
CB2 receptor was transformed to alanine (W172A) or
leucine (W172L), Rhee et al. found that HU243, a tradi-
tional cannabinoid, did not bind to either mutant recep-
tor.51 Results of a recent modeling study substantially
agreed with the work of Tao and that of Rhee.52
Based upon the site-directed mutagenesis and modeling
studies, it appears probable that CP-55,940 and tradi-
tional cannabinoids interact with the CB2 receptor by
a combination of hydrogen bonding and aromatic stack-
ing.50–52 In contrast to the usual SAR for 9-hydroxy
cannabinoids,38 the 1-deoxy-9a-hydroxy CP-47,497 ana-
logs (13, n = 0–7) have greater affinity for the CB2 recep-
tor than their 9b-epimers (8, n = 0–7, Table 1), implying
5. Experimental
5.1. General
IR spectra were obtained using Nicolet 5DX or Magna
1
spectrometers; H and 13C NMR spectra were recorded

J. W. Huffman et al. / Bioorg. Med. Chem. 16 (2008) 322–335
327
on Bruker 300AC and JEOL 500 spectrometers. Mass
spectral analyses were performed on a Hewlett-Packard
5890A capillary gas chromatograph equipped with a
mass sensitive detector. HRMS data were obtained in
the Mass Spectrometry Laboratory, School of Chemical
Sciences, University of Illinois. Ether and THF were dis-
tilled from Na-benzophenone ketyl immediately before
use, and other solvents were purified using standard pro-
cedures. Column chromatography was carried out on
Sorbent Technologies silica gel (32–63 l) using the indi-
cated solvents as eluents. All new compounds were
homogeneous to TLC and 13C NMR. All target com-
pounds were homogeneous to GLC or TLC in two dif-
ferent solvent systems. TLC was carried out using
200 lm silica gel plates with the indicated solvents.
GLC analyses were performed on the Hewlett-Packard
5890A GC/MS using a 60 m carbowax column and
helium gas as a carrier. An initial column temperature
of 60 ꢂC was employed and the temperature was in-
creased at a rate of 1.5 ꢂC/min to a maximum tempera-
ture of 300 ꢂC.
the solvent was removed in vacuo. The orange liquid
was purified by chromatography (petroleum ether/ethyl
acetate, 95:5) to give 0.15 g (99%) of 12 (n = 0): mp
37.5–39.0 ꢂC; IR (neat, m/cmꢀ1) 3090 (w), 3070 (w),
3053 (w), 2957 (s), 2858 (m), 1716 (s), 1520 (m), 1461
(m), 1428 (w), 1360 (m), 1321 (w), 1272 (m), 1230 (w),
1
1122 (m), 1026 (w), 835 (m); H NMR (500 MHz) d
1.32 (s, 9H), 1.72–1.90 (m, 2H), 2.04–2.11 (m, 1H),
2.11–2.19 (m, 1H), 2.32–2.48 (m, 2H), 2.48–2.63 (m,
2H), 2.99 (dddd, J = 4.1, 4.1, 11.9, 11.9 Hz, 1H), 7.16
(d, J = 7.8 Hz, 2H), 7.35 (d, J = 7.4 Hz, 2H); 13C
NMR (75.5 MHz) d 25.5, 31.3, 32.8, 34.4, 41.2, 44.2,
49.0, 125.5, 126.2, 141.3, 149.4, 211.2; MS (EI) m/z (rel
intensity) 230 (27), 216 (18), 215 (100), 187 (2), 173
(4), 145 (7), 129 (6), 117 (7), 91 (6), 69 (8), 57 (11).
5.4. (1R*,3S*)-3-(4-tert-Butylphenyl)cyclohexanol (JWH–
231, 8, n = 0)
To a solution of 0.070 g (0.30 mmol) of ( )-3-(4-tert-
butylphenyl)cyclohexanone in 10 mL of dry ethanol at
0 ꢂC was added 0.091 g (2.4 mmol) of NaBH4. The mix-
ture was warmed to ambient temperature and stirred for
2 h, quenched with 10 mL of 10% HCl, and extracted
with ether. The ethereal extracts were washed with suc-
cessive portions of saturated aqueous NaHCO3, brine
and dried (MgSO4). The solvent was removed in vacuo.
The residue was purified by column chromatography
(petroleum ether/ethyl acetate, 4:1) to give 0.048 g
(68%) of JWH-231 as an off-white solid: mp 95.5–
96.5 ꢂC; 1H NMR (500 MHz) d 1.20–1.35 (m, 11H),
1.37–1.48 (m, 2H), 1.79–1.85 (m, 2H), 1.88 (dp,
J = 3.3, 13.2 Hz, 1H), 2.01–2.07 (m, 1H), 2.13–2.20 (m,
1H), 2.55 (dddd, J = 3.2, 3.2, 12.2, 12.2 Hz, 1H), 3.71
(dddd, J = 4.4, 4.4, 10.8, 10.8 Hz, 1H), 7.14 (d,
J = 8.2 Hz, 2H), 7.32 (d, J = 8.7 Hz, 2H); 13C NMR
(125.8 MHz) d 24.4, 31.4, 33.4, 34.3, 35.3, 42.1, 43.1,
71.0, 125.2, 126.3, 143.1, 148.8; MS (EI) m/z (rel inten-
sity) 232 (17), 218 (20), 217 (100), 215 (11), 199 (14),
171 (2), 157 (4), 145 (7), 131 (13), 117 (9), 105 (4), 91
(12), 57 (16); HRMS: Calcd for C16H24O: 232.1827.
Found: 232.1832.
5.2. 3-(4-tert-Butylphenyl)cyclohex-2-en-1-one (11, n = 0)
To a solution of 0.21 g (0.98 mmol) of 1-bromo-4-tert-
butylbenzene in 5 mL of dry THF at ꢀ78 ꢂC was added
0.75 mL of n-butyllithium (1.6 M solution in cyclohex-
ane, 1.2 mmol) and the mixture was stirred for 30 min.
A solution of 0.14 g (1.0 mmol) of 3-ethoxy-2-cyclohex-
en-1-one in 5 mL of THF was added dropwise and the
reaction mixture was heated at reflux for 2 h. After cool-
ing to ambient temperature, the reaction mixture was
acidified with 10% aqueous HCl and stirred for 30 min
and extracted with three portions of ether. The com-
bined ether layers were washed successively with satu-
rated aqueous NaHCO3, and brine. After drying
(MgSO4) the solvent was removed in vacuo. And the
residue was chromatographed (petroleum ether/ethyl
acetate, 9:1) to give 0.16 g (70%) of 11, n = 0, as an
1
off-white crystalline solid: mp 47.5–49.5 ꢂC; H NMR
(300 MHz) d 1.34 (s, 9H), 2.09–2.20 (m, 2H), 2.44–2.54
(m, 2H), 2.77 (t, J = 6.0 Hz, 2H), 6.44 (s, 1H), 7.44 (d,
J = 8.6 Hz, 2H), 7.50 (d, J = 8.6 Hz, 2H); 13C NMR
(75.5 MHz) d 22.8, 27.9, 31.1, 34.7, 37.2, 124.7, 125.7,
125.8, 135.7, 153.5, 159.5, 199.9; MS (EI) m/z (rel inten-
sity) 228 (44), 213 (100), 185 (21), 171 (35), 157 (6), 144
(27), 128 (8), 115 (12), 92 (5), 78 (10), 67 (11), 57 (16).
5.5. (1R*,3R*)-3-(4-tert-Butylphenyl)cyclohexanol (JWH-
232, 13, n = 0)
To a solution of 0.070 g (0.30 mmol) ( )-3-(4-tert-butyl-
phenyl)cyclohexanone in 10 mL of dry THF at ꢀ78 ꢂC
was added 0.60 mL of K-selectrideꢁ (1.0 M solution in
THF, 0.60 mmol) and the mixture was stirred for 2 h.
The reaction was allowed to warm to ambient tempera-
ture and was stirred for an additional 1 h. To the reac-
tion mixture was added 0.10 mL of water, 0.40 mL of
ethanol, 0.15 mL of 15% NaOH, and 0.15 mL of 30%
hydrogen peroxide and the reaction mixture was stirred
for 5 min. The reaction mixture was extracted with ether
and the ethereal extracts were washed with saturated
brine, dried (MgSO4), and the solvent was removed in
vacuo. The crude product was chromatographed (petro-
leum ether/ethyl acetate, 4:1) to give 0.063 g (89%) of
JWH-232 as an off-white solid: mp 94.0–96.0 ꢂC; 1H
NMR (500 MHz) d 1.31 (s, 9H), 1.36–1.51 (m, 2H),
1.51–1.73 (m, 3H), 1.76–1.86 (m, 2H), 1.86–1.94 (m,
5.3. ( )-3-(4-tert-Butylphenyl)cyclohexanone (12, n = 0)
To 50 mL of liquid NH3 at ꢀ78 ꢂC was added 0.100 g
(14.4 g atom) of lithium shot and the solution was stir-
red for 20 min. A solution of 0.15 g (0.66 mmol) of 3-
(4-tert-butylphenyl)cyclohex-2-en-1-one and 0.049 g
(0.66 mmol) of tert-butanol in 5 mL of dry ether was
added dropwise and the mixture was stirred at ꢀ78 ꢂC
for 10 min. The reaction was quenched with NH4Cl
and the ammonia was evaporated at ambient tempera-
ture. The mixture was diluted with 20 mL of water
and 20 mL of ether. The aqueous layer was diluted with
brine and the product was extracted with ether. The
ethereal solution was washed with successive portions
of 10% HCl and saturated brine, dried (MgSO4), and

328
J. W. Huffman et al. / Bioorg. Med. Chem. 16 (2008) 322–335
1H), 1.94–2.01 (m, 1H), 2.98 (dd, J = 12.4, 12.4 Hz, 1H),
4.19–4.26 (m, 1H), 7.16 (d, J = 8.2 Hz, 2H), 7.32 (d,
J = 8.2 Hz, 2H); 13C NMR (125.8 MHz) d 20.4, 31.4,
32.4, 33.7, 34.3, 36.9, 40.5, 66.9, 125.2, 126.5, 143.9,
148.6; MS (EI) m/z (rel intensity) 232 (13), 217 (28),
214 (28), 199 (100), 171 (5), 157 (21), 145 (8), 131 (15),
117 (12), 105 (5), 91 (15), 77 (5), 57 (18); HRMS: Calcd
for C16H24O: 232.1827. Found: 232.1826.
2.54 (dd, J = 12.0, 12.0 Hz, 1H), 3.71 (dddd, J = 4.2,
4.2, 10.8, 10.8 Hz, 1H), 7.12 (d, J = 8.3 Hz, 2H), 7.25
(d, J = 8.3 Hz, 2H); 13C NMR (75.5 MHz) d 14.7,
17.9, 24.4, 28.9, 33.4, 35.3, 37.3, 42.1, 43.2, 47.1,
71.0, 125.7, 126.2, 142.9, 147.5; MS (EI) m/z (rel inten-
sity) 260 (9), 218 (20), 217 (100), 199 (12), 171 (4), 145
(5), 131 (13), 117 (10), 105 (5), 91 (13), 81 (6), 77 (4);
HRMS: Calcd for C18H28O: 260.2140. Found:
260.2141.
5.6. (1R*,3S*)-3-[4-(1,1-Dimethylpropyl)phenyl]cyclohex-
anol (JWH-294, 8, n = 1)
5.9. (1R*,3R*)-3-[4-(1,1-dimethylbutyl)phenyl]cyclohexa-
nol (JWH-297, 13, n = 2)
This compound was prepared by the procedure used for
the preparation of JWH-231. From 0.060 g (0.24 mmol)
of ( )-3-[4-(1,1-dimethylpropyl)phenyl]cyclohexanone
there was obtained, after chromatography (petroleum
ether/ethyl acetate, 4:1), 0.054 g (89%) of JWH-294 as
This compound was prepared by the procedure used for
the preparation of JWH-232. From 0.060 g (0.23 mmol)
of
( )-3-[4-(1,1-dimethylbutyl)phenyl]cyclohexanone
there was obtained, after chromatography (petroleum
ether/ethyl acetate, 4:1), 0.052 g (86%) of JWH-297 as a
1
an off-white solid: mp 57–58 ꢂC; H NMR (300 MHz)
1
d 0.68 (t, J = 7.4 Hz, 3H), 1.20–1.34 (m, 8H), 1.34–1.52
(m, 2H), 1.62 (q, J = 7.4 Hz, 2H), 1.69–1.96 (m, 3H),
1.96–2.11 (m, 1H), 2.11–2.23 (m, 1H), 2.56 (dd,
J = 12.1, 12.1 Hz, 1H), 3.72 (dddd, J = 4.2, 4.2, 10.8,
10.8 Hz, 1H), 7.13 (d, J = 8.3 Hz, 2H), 7.25 (d,
J = 8.2 Hz, 2H); 13C NMR (75.5 MHz) d 9.1, 24.5,
28.4, 33.4, 35.3, 36.8, 37.5, 42.1, 43.2, 71.0, 125.9,
126.2, 142.9, 147.2; MS (EI) m/z (rel intensity) 246 (6),
218 (17), 217 (100), 199 (4), 171 (4), 157 (1), 145 (5),
131 (12), 117 (8), 105 (4), 91 (11), 81 (6); HRMS: Calcd
for C17H26O: 246.1984. Found: 246.1988.
white solid: mp 63.5–65.0 ꢂC; H NMR (300 MHz) d
0.81 (t, J = 7.2 Hz, 3H), 0.99–1.16 (m, 2H), 1.27 (s,
6H), 1.44 (qd, J = 3.3, 12.1 Hz, 1H), 1.50–1.60 (m, 4H),
1.62–1.73 (m, 2H), 1.73–2.00 (m, 4H), 2.98 (dd,
J = 12.1, 12.1 Hz, 1H), 4.16–4.26 (m, 1H), 7.13 (d,
J = 8.2 Hz, 2H), 7.24 (d, J = 8.3 Hz, 2H); 13C NMR
(75.5 MHz) d 14.7, 17.9, 20.4, 28.9, 32.4, 33.6, 36.8,
37.3, 40.5, 47.1, 66.8, 125.6, 126.3, 143.7, 147.2; MS
(EI) m/z (rel intensity) 260 (8), 242 (3), 218 (17), 217
(100), 199 (57), 185 (2), 171 (6), 145 (5), 131 (14), 117
(11), 105 (6), 91 (14); HRMS: Calcd for C18H28O:
260.2140. Found: 260.2138.
5.7. (1R*,3R*)-3-[4-(1,1-Dimethylpropyl)phenyl]cyclo-
hexanol (JWH-295, 13, n = 1)
5.10. (1R*,3S*)-3-[4-(1,1-Dimethylpentyl)phenyl]cyclo-
hexanol (JWH-323, 8, n = 3)
This compound was prepared by the procedure used for
the preparation of JWH-232. From 0.060 g (0.24 mmol)
of ( )-3-[4-(1,1-dimethylpropyl)phenyl]cyclohexanone
there was obtained after chromatography (petroleum
ether/ethyl acetate, 4:1), 0.055 g (91%) of JWH-295 as
This compound was prepared by the procedure used for
the preparation of JWH-231. From 0.055 g (0.20 mmol)
of ( )-3-[4-(1,1-dimethylpentyl)phenyl]cyclohexanone
there was obtained, after chromatography (petroleum
ether/ethyl acetate, 4:1), 0.043 g (78%) of JWH-323 as
1
an off-white solid: mp 82–83 ꢂC; H NMR (300 MHz)
1
d 0.68 (t, J = 7.4 Hz, 3H), 1.26 (s, 6H), 1.44 (qd,
J = 3.2, 12.0 Hz, 1H), 1.50–1.73 (m, 6H), 1.74–2.00 (m,
4H), 2.98 (dd, J = 12.1, 12.1 Hz, 1H), 4.18–4.28 (m,
1H), 7.14 (d, J = 8.2 Hz, 2H), 7.25 (d, J = 8.2 Hz, 2H);
13C NMR (75.5 MHz) d 9.1, 20.4, 28.4, 32.4, 33.7,
36.8, 37.5, 40.5, 66.8, 125.8, 126.4, 143.8, 146.9; MS
(EI) m/z (rel intensity) 246 (10), 228 (6), 218 (17), 217
(100), 199 (71), 171 (8), 157 (4), 145 (7), 131 (16), 117
(13), 105 (6), 91 (17); HRMS: Calcd for C17H26O:
246.1984. Found: 246.1978.
an off-white solid: mp 46–48 ꢂC; H NMR (300 MHz)
d 0.82 (t, J = 7.2 Hz, 3H), 0.97–1.13 (m, 2H), 1.22 (sex-
tet, J = 7.0 Hz, 2H), 1.23–1.34 (m, 8H), 1.34–1.52 (m,
2H), 1.52–1.63 (m, 2H), 1.74 (br s, 1H), 1.78–1.94 (m,
2H), 1.98–2.11 (m, 1H), 2.11–2.23 (m, 1H), 2.48–2.63
(m, 1H), 3.64–3.79 (m, 1H), 7.12 (d, J = 8.3 Hz, 2H),
7.25 (d, J = 8.3 Hz, 2H); 13C NMR (75.5 MHz) d 14.0,
23.4, 24.5, 26.9, 28.9, 33.4, 35.3, 37.2, 42.1, 43.2, 44.3,
71.0, 125.7, 126.2, 142.9, 147.5; MS (EI) m/z (rel inten-
sity) 274 (4), 218 (16), 217 (100), 199 (4), 171 (3), 145
(6), 131 (13), 117 (11), 105 (6), 91 (14), 81 (6), 77 (4);
HRMS: Calcd for C19H30O: 274.2297. Found: 274.2296.
5.8. (1R*,3S*)-3-[4-(1,1-Dimethylbutyl)phenyl]cyclohexanol
(JWH-296, 8, n = 2)
5.11. (1R*,3R*)-3-[4-(1,1-Dimethylpentyl)phenyl]cyclo-
hexanol (JWH-396, JWH-407, 13, n = 3)
This compound was prepared by the procedure used
for the preparation of JWH-231. From 0.060 g
(0.23 mmol)
of
( )-3-[4-(1,1-dimethylbu-
This compound was prepared by the procedure used for
the preparation of JWH-232. From 0.055 g (0.20 mmol)
tyl)phenyl]cyclohexanone there was obtained, after
chromatography (petroleum ether/ethyl acetate, 4:1),
0.052 g (86%) of JWH-296 as an off-white solid: mp
49–50 ꢂC; 1H NMR (300 MHz) d 0.81 (t, J = 7.2 Hz,
3H), 1.00–1.17 (m, 2H), 1.17–1.34 (m, 8H), 1.34–1.50
(m, 2H), 1.50–1.63 (m, 2H), 1.75–1.93 (m, 2H), 1.94
(br s, 1H), 1.98–2.10 (m, 1H), 2.10–2.22 (m, 1H),
of
( )-3-[4-(1,1-dimethylpentyl)phenyl]cyclohexanone
there was obtained, after chromatography (petroleum
ether/ethyl acetate, 4:1), 0.047 g (85%) of JWH-396 as
a pale yellow oil: 1H NMR (500 MHz) d 0.82 (t,
J = 7.4 Hz, 3H), 1.00–1.08 (m, 2H), 1.21 (sextet,
J = 7.3 Hz, 2H), 1.27 (s, 6H), 1.45 (qd, J = 3.7,

J. W. Huffman et al. / Bioorg. Med. Chem. 16 (2008) 322–335
329
12.4 Hz, 1H), 1.54–1.60 (m, 3H), 1.60–1.67 (m, 2H),
1.67–1.72 (m, 1H), 1.77–1.85 (m, 2H), 1.87–1.93 (m,
1H), 1.93–2.00 (m, 1H), 2.98 (dddd, J = 3.4, 3.4, 12.4,
12.4 Hz, 1H), 4.20–4.25 (m, 1H), 7.14 (d, J = 8.2 Hz,
2H), 7.24 (d, J = 8.2 Hz, 2H); 13C NMR (125.8 MHz)
d 14.0, 20.4, 23.4, 26.9, 28.9, 32.4, 33.6, 36.8, 37.2,
40.5, 44.4, 66.9, 125.7, 126.4, 143.7, 147.3; MS (EI) m/z
(rel intensity) 274 (5), 256 (2), 218 (15), 217 (100), 199
(36), 171 (3), 145 (3), 131 (7), 117 (6), 105 (3), 91 (7);
HRMS: Calcd for C19H30O: 274.2297. Found: 274.
2294.
a white solid: mp 59–60 ꢂC; 1H NMR (300 MHz) d
0.84 (t, J = 6.8 Hz, 3H), 0.98–1.13 (m, 2H), 1.13–1.25
(m, 6H), 1.25–1.35 (m, 8H), 1.35–1.52 (m, 2H), 1.52–
1.66 (m, 3H), 1.72–1.96 (m, 2H), 1.98–2.12 (m, 1H),
2.12–2.26 (m, 1H), 2.55 (dd, J = 12.1, 12.1 Hz, 1H),
3.72 (dddd, J = 4.2, 4.2, 10.7, 10.7 Hz, 1H), 7.12 (d,
J = 8.2 Hz, 2H), 7.25 (d, J = 8.2 Hz, 2H); 13C NMR
(75.5 MHz) d 14.0, 22.6, 24.5, 24.6, 28.9, 30.0, 31.7,
33.4, 35.3, 37.3, 42.1, 43.2, 44.6, 71.0, 125.7, 126.2,
142.9, 147.5; MS (EI) m/z (rel intensity) 302 (5), 218
(16), 217 (100), 199 (3), 171 (2), 145 (3), 131 (7), 117
(5), 105 (3), 91 (6), 81 (3); HRMS: Calcd for C21H34O:
302.2610. Found: 302.2617.
5.12. (1R*,3S*)-3-[4-(1,1-Dimethylhexyl)phenyl]cyclo-
hexanol (JWH-403, 8, n = 4)
5.15. (1R*,3R*)-3-[4-(1,1-Dimethylheptyl)phenyl]cyclo-
hexanol (JWH-342, 13, n = 5)
This compound was prepared by the procedure used for
the preparation of JWH-231. From 0.090 g (0.31 mmol)
of
( )-3-[4-(1,1-dimethylhexyl)phenyl]cyclohexanone
This compound was prepared by the procedure used for
the preparation of JWH-232. From 0.070 g (0.23 mmol)
there was obtained, after chromatography (petroleum
ether/ethyl acetate, 4:1), 0.063 g (70%) of JWH-403 as
an off-white solid: mp 50–51 ꢂC; H NMR (500 MHz) d
of
( )-3-[4-(1,1-dimethylheptyl)phenyl]cyclohexanone
1
there was obtained, after chromatography (petroleum
ether/ethyl acetate, 4:1), 0.063 g (89%) of JWH-342 as an
off-white solid: mp 53–54 ꢂC; 1H NMR (500 MHz) d
0.84 (t, J = 6.9 Hz, 3H), 1.01–1.10 (m, 2H), 1.16–1.26 (m,
6H), 1.27 (s, 6H), 1.46 (qd, J = 3.7, 12.4 Hz, 1H), 1.51
(br s, 1H), 1.52–1.60 (m, 3H), 1.60–1.73 (m, 2H), 1.77–
1.85 (m, 2H), 1.87–1.93 (m, 1H), 1.93–2.00 (m, 1H), 2.97
(dddd, J = 3.4, 3.4, 12.4, 12.4 Hz, 1H), 4.19–4.25 (m,
1H), 7.14 (d, J = 8.2 Hz, 2H), 7.24 (d, J = 8.2 Hz, 2H);
13C NMR (125.8 MHz) d 14.1, 20.4, 22.6, 24.6, 28.9,
30.0, 31.8, 32.4, 33.7, 36.9, 37.3, 40.5, 44.6, 66.9, 125.7,
126.4, 143.7, 147.4; MS (EI) m/z (rel intensity) 302 (6),
284 (2), 218 (16), 217 (100), 199 (38), 171 (4), 157 (2), 145
(5), 131 (9), 117 (7), 105 (4), 91 (9), 81 (6); HRMS: Calcd
for C21H34O: 302.2610. Found: 302.2604.
0.81 (t, J = 7.1 Hz, 3H), 1.02–1.11 (m, 2H), 1.13–1.25
(m, 4H), 1.25–1.34 (m, 8H), 1.35–1.47 (m, 2H), 1.53–
1.59 (m, 2H), 1.78–1.84 (m, 1H), 1.89 (dp, J = 3.3,
12.8 Hz, 1H), 1.99–2.06 (m, 1H), 2.12–2.21 (m, 2H),
2.54 (dddd, J = 3.2, 3.2, 12.4, 12.4 Hz, 1H), 3.70 (dddd,
J = 4.3, 4.3, 11.0, 11.0 Hz, 1H), 7.12 (d, J = 8.2 Hz, 2H),
7.24 (d, J = 8.2 Hz, 2H); 13C NMR (125.8 MHz) d 14.0,
22.5, 24.3, 24.4, 28.9, 32.5, 33.4, 35.2, 37.2, 42.1, 43.1,
44.5, 70.9, 125.7, 126.2, 142.9, 147.4; MS (EI) m/z (rel
intensity) 288 (2), 218 (16), 217 (100), 199 (3), 171 (2),
145 (4), 131 (10), 117 (8), 105 (4), 91 (10), 81 (5), 77 (2);
HRMS: Calcd for C20H32O: 288.2453. Found: 288.2455.
5.13. (1R*,3R*)-3-[4-(1,1-Dimethylhexyl)phenyl]cyclo-
hexanol (JWH-406, 13, n = 4)
5.16. (1R*,3S*)-3-[4-(1,1-Dimethyloctyl)phenyl]cyclohex-
anol (JWH-404, 8, n = 6)
This compound was prepared by the procedure used for the
preparation of JWH-232. From 0.090 g (0.31 mmol) of
( )-3-[4-(1,1-dimethylhexyl)phenyl]cyclohexanone there
was obtained, after chromatography (petroleum ether/
ethyl acetate, 4:1), 0.069 g (76%) of JWH-406 as a pale yel-
This compound was prepared by the procedure used for the
preparation of JWH-231. From 0.095 g (0.30 mmol) of
( )-3-[4-(1,1-dimethyloctyl)phenyl]cyclohexanone there
was obtained, after chromatography (petroleum ether/
ethyl acetate, 4:1), 0.076 g (79%) of JWH-404 as a white so-
lid: mp 60–61 ꢂC; 1H NMR (500 MHz) d 0.85 (t,
J = 7.1 Hz, 3H), 1.01–1.10 (m, 2H), 1.14–1.21 (m, 6H),
1.21–1.35 (m, 10H), 1.38–1.49 (m, 2H), 1.53–1.59 (m,
2H), 1.62 (br s, 1H), 1.80–1.86 (m, 1H), 1.89 (dp, J = 3.3,
13.3 Hz, 1H), 2.01–2.09 (m, 1H), 2.14–2.22 (m, 1H), 2.56
(dddd, J = 3.3, 3.3, 12.4, 12.4 Hz, 1H), 3.72 (dddd,
J = 4.3, 4.3, 10.8, 10.8 Hz, 1H), 7.13 (d, J = 8.2 Hz, 2H),
7.25 (d, J = 8.2 Hz, 2H); 13C NMR (125.8 MHz) d 14.1,
22.6, 24.5, 24.7, 28.9, 29.2, 30.3, 31.9, 33.4, 35.3, 37.3,
42.1, 43.2, 44.6, 71.1, 125.7, 126.2, 142.9, 147.6; MS (EI)
m/z (rel intensity) 316 (6), 218 (17), 217 (100), 199 (7), 171
(2), 145 (3), 131 (6), 117 (4), 91 (5), 81 (3), 77 (2); HRMS:
Calcd for C22H36O: 316.2766. Found: 316.2767.
1
low oil: H NMR (500 MHz) d 0.82 (t, J = 7.1 Hz, 3H),
1.02–1.11 (m, 2H), 1.13–1.26 (m, 4H), 1.27 (s, 6H), 1.46
(qd, J = 3.7, 12.4 Hz, 1H), 1.52–1.61 (m, 4H), 1.61–1.73
(m, 2H), 1.77–1.85 (m, 2H), 1.87–1.93 (m, 1H), 1.94–2.00
(m, 1H), 2.97 (dddd, J = 3.4, 3.4, 12.4, 12.4 Hz, 1H),
4.21–4.25 (m, 1H), 7.14 (d, J = 8.2 Hz, 2H), 7.24 (d,
J = 8.2 Hz, 2H); 13C NMR (125.8 MHz) d 14.1, 20.4,
22.5, 24.4, 28.9, 32.4, 32.6, 33.7, 36.9, 37.3, 40.5, 44.6,
66.9, 125.7, 126.4, 143.7, 147.4; MS (EI) m/z (rel intensity)
288 (3), 218 (16), 217 (100), 199 (40), 171 (3), 145 (5), 131 (7),
117 (6), 105 (3), 91 (7); HRMS: Calcd for C20H32O:
288.2453. Found: 288.2451.
5.14. (1R*,3S*)-3-[4-(1,1-Dimethylheptyl)phenyl]cyclo-
hexanol (JWH-324, 8, n = 5)
This compound was prepared by the procedure used for
the preparation of JWH-231. From 0.070 g (0.23 mmol)
of ( )-3-[4-(1,1-dimethylheptyl)phenyl]cyclohexanone
there was obtained, after chromatography (petroleum
ether/ethyl acetate, 4:1), 0.054 g (77%) of JWH-324 as
5.17. (1R*,3R*)-3-[4-(1,1-Dimethyloctyl)phenyl]cyclohex-
anol (13, n = 6)
This compound was prepared by the procedure used
for the preparation of JWH-232. From 0.095 g

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J. W. Huffman et al. / Bioorg. Med. Chem. 16 (2008) 322–335
(0.30 mmol) of ( )-3-[4-(1,1-dimethyloctyl)phenyl]cyclo-
hexanone there was obtained, after chromatography
(petroleum ether/ethyl acetate, 4:1), 0.079 g (83%) of
JWH-405 as a white solid: mp 58–59 ꢂC; 1H NMR
(500 MHz) d 0.85 (t, J = 7.1 Hz, 3H), 1.02–1.10 (m,
2H), 1.13–1.21 (m, 6H), 1.21–1.26 (m, 2H), 1.27 (s,
6H), 1.45 (qd, J = 3.2, 12.4 Hz, 1H), 1.52–1.60 (m,
4H), 1.60–1.72 (m, 2H), 1.77–1.85 (m, 2H), 1.87–
1.93 (m, 1H), 1.93–2.00 (m, 1H), 2.97 (dddd,
J = 3.4, 3.4, 12.4, 12.4 Hz, 1H), 4.20–4.25 (m, 1H),
7.14 (d, J = 8.2 Hz, 2H), 7.24 (d, J = 8.2 Hz, 2H);
13C NMR (125.8 MHz) d 14.1, 20.4, 22.6, 24.7,
28.9, 29.2, 30.3, 31.9, 32.4, 33.7, 36.8, 37.3, 40.5,
44.6, 66.9, 125.7, 126.4, 143.7, 147.4; MS (EI) m/z
(rel intensity) 316 (2), 218 (16), 217 (100), 199 (32),
171 (3), 145 (2), 131 (6), 117 (4), 105 (2), 91 (4),
81 (3); HRMS: Calcd for C22H36O: 316.2766. Found:
316.2773.
5.20. ( )-3-Ethoxy-6-(2-propenyl)-2-cyclohexen-1-one
To a solution of 50 mmol of LDA, prepared from
20.0 mL of n-butyllithium (2.5 M in hexanes) and
7.4 mL (53 mmol) of diisopropylamine in 5 mL of dry
THF at ꢀ78 ꢂC under nitrogen, was added dropwise
6.72 g of 3-ethoxy-2-cyclohexen-1-one (47.9 mmol) in
5 mL of THF. The reaction was stirred for 30 min and
17.5 mL (100 mmol) of HMPA and 8.9 mL (100 mmol)
of 3-bromo-1-propene were added successively. The
reaction was warmed to ambient temperature and stir-
red for 1.5 h, quenched with water, and most of the sol-
vent was removed in vacuo. The remaining mixture was
diluted with 300 mL of ice water and extracted with
ether. The ether extracts were washed with water, dried
(MgSO4), and the solvent was removed in vacuo. The
bright yellow liquid was distilled in vacuo to give
6.08 g (70%) of ( )-3-ethoxy-6-(2-propenyl)-2-cyclohex-
en-1-one as a yellow liquid: 1H NMR (300 MHz) d
1.36 (t, J = 7.0 Hz, 3H), 1.63–1.78 (m, 1H), 2.00–2.32
(m, 3H), 2.38–2.47 (m, 2H), 2.57–2.70 (m, 1H), 3.90
(qd, J = 0.8, 6.8 Hz, 2H), 4.99–5.12 (m, 2H), 5.32 (s,
1H), 5.69–5.87 (m, 1H); 13C NMR (75.5 MHz) d 13.8,
25.5, 27.9, 33.7, 44.4, 63.9, 101.9, 116.2, 136.1, 176.6,
200.0; MS (EI) m/z (rel intensity) 180 (52), 165 (5), 151
(9), 139 (6), 123 (7), 112 (58), 97 (6), 84 (100), 69 (79),
55 (22). These data agree in all respects with those
reported previously.42
5.18. (1R*,3S*)-3-[4-(1,1-Dimethylnonyl)phenyl]cyclo-
hexanol (JWH-401, 8, n = 7)
This compound was prepared by the procedure used for
the preparation of JWH-231. From 0.075 g (0.23 mmol)
of
( )-3-[4-(1,1-dimethylnonyl)phenyl]cyclohexanone
there was obtained, after chromatography (petroleum
ether/ethyl acetate, 4:1), 0.036 g (48%) of JWH-401 as
a white solid: mp 52–53 ꢂC; 1H NMR (500 MHz) d
0.86 (t, J = 7.1 Hz, 3H), 1.01–1.10 (m, 2H), 1.16–1.23
(m, 8H), 1.23–1.35 (m, 10H), 1.38–1.49 (m, 2H), 1.53–
1.59 (m, 2H), 1.66 (br s, 1H), 1.80–1.86 (m, 1H), 1.89
(dp, J = 3.3, 13.3 Hz, 1H), 2.02–2.08 (m, 1H), 2.14–
2.21 (m, 1H), 2.56 (dddd, J = 3.3, 3.3, 12.4, 12.4 Hz,
1H), 3.73 (dddd, J = 4.3, 4.3, 11.0, 11.0 Hz, 1H), 7.13
(d, J = 8.2 Hz, 2H), 7.25 (d, J = 8.2 Hz, 2H); 13C
NMR (125.8 MHz) d 14.1, 22.6, 24.5, 24.7, 28.9, 29.3,
29.5, 30.3, 31.8, 33.4, 35.3, 37.3, 42.1, 43.2, 44.6, 71.1,
125.8, 126.2, 142.9, 147.6; MS (EI) m/z (rel intensity)
330 (2), 218 (15), 217 (100), 199 (2), 171 (2), 145 (3),
131 (7), 117 (5), 91 (6), 81 (4), 57 (6); HRMS: Calcd
for C23H38O: 330.2923. Found: 330.2921.
5.21. ( )-4-(2-Propenyl)-2-cyclohexen-1-one
A solution of 6.00 g (33.3 mmol) of ( )-3-ethoxy-6-(2-
propenyl)-2-cyclohexen-1-one in 15 mL of dry ether
was added to 0.65 g (17 mmol) of LiAlH4 in 50 mL of
ether at 0 ꢂC, and the mixture was stirred for 1 h. The
reaction was quenched with 70 mL of 2 M aqueous
HCl, stirred for 30 min, and extracted with ether. The
ethereal extracts were washed with saturated NaHCO3,
dried (MgSO4), and the solvent was removed in vacuo.
The yellow liquid was distilled to give 3.55 g (78%) of
( )-4-(2-propenyl)-2-cyclohexen-1-one as a pale yellow
liquid: 1H NMR (300 MHz) d 1.62–1.80 (m, 1H),
2.03–2.18 (m, 1H), 2.24 (t, J = 7.1 Hz, 2H), 2.39 (dd,
J = 4.8, 12.4 Hz, 1H), 2.47 (t, J = 4.7 Hz, 1H), 2.49–
2.58 (m, 1H), 5.12 (d, J = 12.5 Hz, 2H), 5.71–5.89 (m,
1H), 5.99 (dd, J = 2.4, 10.2 Hz, 1H), 6.88 (d,
J = 10.2 Hz, 1H); 13C NMR (75.5 MHz) d 28.3, 35.6,
36.7, 38.7, 117.3, 129.0, 135.1, 153.9, 199.4; GC/MS
(EI) m/z (rel intensity) 136 (10), 118 (13), 108 (7), 95
(24), 79 (100), 67 (96), 53 (21). These data agree in all re-
spects with those reported previously.43
5.19. (1R*,3R*)-3-[4-(1,1-Dimethylnonyl)phenyl]cyclo-
hexanol (JWH-402, 13, n = 7)
This compound was prepared by the procedure used for
the preparation of JWH-232. From 0.075 g (0.23 mmol)
of
( )-3-[4-(1,1-dimethylnonyl)phenyl]cyclohexanone
there was obtained, after chromatography (petroleum
ether/ethyl acetate, 4:1), 0.050 g (66%) of JWH-402 as
a pale yellow oil: 1H NMR (500 MHz) d 0.86 (t,
J = 6.8 Hz, 3H), 1.02–1.10 (m, 2H), 1.13–1.23 (m, 8H),
1.23–1.30 (m, 8H), 1.45 (qd, J = 3.6, 12.4 Hz, 1H),
1.51–1.60 (m, 4H), 1.61–1.72 (m, 2H), 1.77–1.87 (m,
2H), 1.87–1.93 (m, 1H), 1.93–2.00 (m, 1H), 2.97 (dddd,
J = 3.4, 3.4, 12.2, 12.2 Hz, 1H), 4.20–4.25 (m, 1H), 7.14
(d, J = 8.2 Hz, 2H), 7.24 (d, J = 8.2 Hz, 2H); 13C NMR
(125.8 MHz) d 14.1, 20.4, 22.6, 24.7, 28.9, 29.3, 29.5,
30.3, 31.8, 32.4, 33.7, 36.8, 37.3, 40.5, 44.6, 66.9, 125.7,
126.4, 143.7, 147.4; MS (EI) m/z (rel intensity) 330 (4),
312 (1), 218 (15), 217 (100), 199 (34), 171 (3), 145 (4),
131 (9), 117 (7), 105 (4), 91 (8); HRMS: Calcd for
C23H38O: 330.2923. Found: 330.2930.
5.22. (3R*,4S*)-3-(4-tert-Butylphenyl)-4-(2-propenyl)-
cyclohexanone 3 (14, n = 0)
A solution of 0.43 g (2.0 mmol) of 1-bromo-4-tert-butyl-
benzene in 10 mL of THF and a crystal of iodine were
added under N2 to 0.10 g (4.1 mmol) of Mg ribbon.
The mixture was warmed slightly and stirred for 1.5 h,
cooled to ꢀ20 ꢂC and 0.060 g (0.32 mmol) of CuI was
added followed by 0.28 g (2.0 mmol) of ( )-4-(2-prope-
nyl)-2-cyclohexen-1-one in 2 mL of THF. The reaction
was stirred for 2 h at ambient temperature, quenched

J. W. Huffman et al. / Bioorg. Med. Chem. 16 (2008) 322–335
331
with 15 mL of saturated NH4Cl, and the product was
extracted with three portions of ether. The combined
ether layers were washed with saturated NH4Cl, brine
and dried (MgSO4). The solvent was removed in vacuo
and the crude product was chromatographed (petroleum
ether/ethyl acetate, 95:5) to give 0.39 g (71%) of
(3R*,4S*)-3-(4-tert-butylphenyl)-4-(2-propenyl)cyclo-
hexanone as a clear pale yellow liquid: 1H NMR
(500 MHz) d 1.31 (s, 9H), 1.41–1.52 (m, 1H), 1.69 (dt,
J = 8.6, 13.8 Hz, 1H), 1.98–2.10 (m, 2H), 2.22 (dq,
J = 4.3, 13.8 Hz, 1H), 2.44–2.49 (m, 2H), 2.49–2.60 (m,
2H), 2.68 (td, J = 5.0, 11.2 Hz, 1H), 4.92 (d, J = 17.4
Hz, 1H), 4.97 (d, J = 10.1 Hz, 1H), 5.67 (dddd,
J = 5.8, 8.4, 10.1, 17.1 Hz, 1H), 7.10 (d, J = 8.2 Hz,
2H), 7.33 (d, J = 8.2 Hz, 2H); 13C NMR (125.8 MHz)
d 30.8, 31.3, 34.4, 37.3, 41.0, 41.1, 49.3, 49.5, 116.6,
125.6, 126.8, 136.1, 140.0, 149.6, 210.9; MS (EI) m/z
(rel intensity) 270 (7), 255 (44), 228 (70), 214 (23), 213
(100), 185 (10), 145 (64), 131 (20), 117 (28), 105 (12),
91 (29), 79 (18).
acetate, 1:1) to give 0.18 g (84%) of JWH-384 as a white
crystalline solid: mp 134.8–135.8 ꢂC; 1H NMR
(500 MHz) d 0.84–0.93 (m, 1H), 1.08–1.16 (m, 1H), 1.23–
1.28 (m, 2H), 1.28–1.34 (m, 10H), 1.34–1.61 (m, 5H), 2.00
(dq, J = 3.5, 13.5 Hz, 1H), 2.03–2.12 (m, 2H), 2.22–2.30
(m, 1H), 3.39–3.51 (m, 2H), 3.70 (dddd, J = 4.4, 4.4, 10.8,
10.8 Hz, 1H), 7.06 (d, J = 8.2 Hz, 2H), 7.29 (d,
J = 8.2 Hz, 2H); 13C NMR (125.8 MHz) d 29.3, 29.8,
29.9, 31.4, 34.3, 35.4, 41.2, 44.6, 48.3, 63.1, 70.8, 125.3,
127.0, 141.7, 148.9; MS (EI) m/z (rel intensity) 290 (12),
272 (46), 257 (20), 145 (85), 131 (74), 117 (54), 57 (100);
HRMS: Calcd for C19H30O2: 290.2246. Found: 290.2253.
5.25. (1R*,3R*,4R*)-3-[4-(1,1-Dimethylpropyl)phenyl]-4-
(3-hydroxypropyl)cyclohexanol (JWH-343, n = 1)
The title compound was prepared using the procedure
employed for the preparation of JWH-384. From
0.16 g (0.56 mmol) of (1R*,3R*,4S*)-3-[4-(1,1-dimethyl-
propyl)phenyl]-4-(2-propenyl)cyclohexanol there was
obtained, after chromatography (petroleum ether/ethyl
acetate, 1:1), 0.15 g (88%) of JWH-343 as a pale yellow
5.23. (1R*,3R*,4S*)-3-(4-tert-Butylphenyl)-4-(2-prope-
nyl)cyclohexanol (15, n = 0)
1
liquid: H NMR (500 MHz) d 0.66 (t, J = 7.3 Hz, 3H),
0.80–0.95 (m, 1H), 1.06–1.15 (m, 1H), 1.16–1.30 (m,
8H), 1.30–1.58 (m, 5H), 1.61 (q, J = 7.5 Hz, 2H), 1.99
(dq, J = 3.5, 13.5 Hz, 1H), 2.04–2.12 (m, 2H), 2.22–
2.29 (m, 1H), 3.38–3.49 (m, 2H), 3.71 (dddd, J = 4.1,
4.1, 11.0, 11.0 Hz, 1H), 7.06 (d, J = 8.2 Hz, 2H), 7.23
(d, J = 8.2 Hz, 2H); 13C NMR (125.8 MHz) d 9.1,
28.4, 29.3, 29.8, 30.0, 35.5, 36.9, 37.5, 41.3, 44.6, 48.3,
63.1, 70.8, 126.0, 127.0, 141.6, 147.3; MS (EI) m/z (rel
intensity) 314 (11), 276 (20), 275 (100), 257(24), 239
(11), 207 (16), 197 (14), 171 (14), 157 (11), 145 (27),
131 (34), 121 (15), 91 (14), 71 (9), 55 (10); HRMS: Calcd
for C20H32O2: 304.2402. Found: 304.2411.
To a solution of 0.38 g (1.4 mmol) of (3R*,4S*)-3-(4-tert-
butylphenyl)-4-(2-propenyl)cyclohexanone in 5 mL of
methanol at 0 ꢂC was added 0.053 g (1.4 mmol) of
NaBH4. The reaction was stirred at ambient tempera-
ture for 30 min, diluted with 15 mL of saturated brine,
and extracted with three portions of ether. The com-
bined ether layers were washed with brine, dried
(MgSO4), and the solvent was removed in vacuo. The
crude product was chromatographed (petroleum
ether/ethyl acetate, 9:1) to give 0.21 g (55%) of
(1R*,3R*,4S*)-3-(4-tert-Butylphenyl)-4-(2-propenyl)cyclo-
1
hexanol as a pale yellow liquid: H NMR (500 MHz) d
1.04–1.14 (m, 1H), 1.31 (s, 9H), 1.33–1.41 (m, 1H),
1.47–1.62 (m, 3H), 1.89 (br s, 1H), 1.91–1.99 (m, 2H),
2.03–2.10 (m, 2H), 2.23–2.30 (m, 1H), 3.68 (dddd,
J = 4.3, 4.3, 11.0, 11.0 Hz, 1H), 4.86 (d, J = 17.0 Hz,
1H), 4.91 (d, J = 10.1 Hz, 1H), 5.63 (dddd, J = 6.0,
8.2, 10.4, 16.8 Hz, 1H), 7.07 (d, J = 8.2 Hz, 2H), 7.30
(d, J = 8.2 Hz, 2H); 13C NMR (125.8 MHz) d 29.7,
31.4, 34.3, 35.4, 37.9, 41.4, 44.5, 47.8, 70.8, 115.8,
125.2, 127.1, 137.0, 141.6, 148.9; MS (EI) m/z (rel inten-
sity) 272 (16), 257 (36), 230 (41), 212 (79), 197 (58), 145
(38), 131 (28), 117 (41), 91 (38), 79 (17), 67 (17), 57 (100).
5.26. (1R*,3R*,4R*)-3-[4-(1,1-Dimethylbutyl)phenyl]-4-
(3-hydroxypropyl)cyclohexanol (JWH-392, 9, n = 2)
The title compound was prepared using the procedure em-
ployed for the preparation of JWH-384. From 0.22 g
(0.73 mmol)
of
(1R*,3R*,4S*)-3-[4-(1,1-dimethylbu-
tyl)phenyl]-4-(2-propenyl)cyclohexanol there was ob-
tained, after chromatography (petroleum ether/ethyl
acetate, 1:1), 0.20 g (86%) of JWH-392 as a pale yellow li-
1
quid: H NMR (500 MHz) d 0.81 (t, J = 7.3 Hz, 3H),
0.84–0.91 (m, 1H), 1.01–1.16 (m, 3H), 1.24–1.30 (m, 8H),
1.30–1.52 (m, 5H), 1.52–1.57 (m, 3H), 1.99 (dq, J = 3.4,
13.5 Hz, 1H), 2.04–2.12 (m, 2H), 2.22–2.29 (m, 1H), 3.38–
3.49 (m, 2H), 3.71 (dddd, J = 4.4, 4.4, 10.8, 10.8 Hz, 1H),
7.05 (d, J = 8.2 Hz, 2H), 7.23 (d, J = 8.7 Hz, 2H); 13C
NMR (125.8 MHz) d 14.8, 18.0, 28.9, 29.4, 29.9, 30.0,
35.5, 37.4, 41.3, 44.6, 47.2, 48.4, 63.1, 70.8, 125.8, 127.0,
141.6, 147.7; MS (EI) m/z (rel intensity) 318 (12), 276
(24), 275 (100), 257 (31), 239 (10), 207 (21), 171 (15), 157
(18), 145 (31), 131 (48), 121 (26), 91 (15); HRMS: Calcd
for C21H34O2: 318.2559. Found: 318.2557.
5.24. (1R*,3R*,4R*)-3-(4-tert-Butylphenyl)-4-(3-hydroxy-
propyl)cyclohexanol (JWH-384, 9, n = 0)
To a solution of 0.20 g (0.73 mmol) of (1R*,3R*,4S*)-3-(4-
tert-butylphenyl)-4-(2-propenyl)cyclohexanol in 5 mL of
dry THF at 0 ꢂC under N2 was added 1.5 mL (1.5 mmol)
of BH3-THF (1.0 M in THF). The reaction was stirred at
ambient temperature for 45 min, cooled to 0 ꢂC, and
0.20 mL of water, 0.45 mL of 2 M aqueous NaOH, and
0.25 mL (2.0 mmol) of 30% H2O2 were added. The reaction
mixture was stirred at ambient temperature for 45 min,
quenched with 50 mL of brine, and extracted with ether.
The ethereal solution was washed with brine, dried
(MgSO4), and the solvent was removed in vacuo. The crude
product was chromatographed (petroleum ether/ethyl
5.27. (1R*,3R*,4R*)-3-[4-(1,1-Dimethylpentyl)phenyl]-4-
(3-hydroxypropyl)cyclohexanol (JWH-325, 9, n = 3)
The title compound was prepared using the procedure
employed for the preparation of JWH-384. From

332
J. W. Huffman et al. / Bioorg. Med. Chem. 16 (2008) 322–335
0.19 g (0.60 mmol) of (1R*,3R*,4S*)-3-[4-(1,1-dimethyl-
pentyl)phenyl]-4-(2-propenyl)cyclohexanol there was
obtained, after chromatography (petroleum ether/ethyl
acetate, 1:1), 0.15 g (75%) of JWH-325 as a pale yellow
5.30. (1R*,3R*,4R*)-3-[4-(1,1-Dimethyloctyl)phenyl]-4-
(3-hydroxypropyl)cyclohexanol (JWH-345, 9, n = 6)
The title compound was prepared using the procedure
employed for the preparation of JWH-384. From
0.13 g (0.36 mmol) of (1R*,3R*,4S*)-3-[4-(1,1-dimethy-
loctyl)phenyl]-4-(2-propenyl)cyclohexanol there was ob-
tained, after chromatography (petroleum ether/ethyl
acetate, 1:1), 0.12 g (88%) of JWH-345 as a pale yellow
1
liquid: H NMR (500 MHz) d 0.81 (t, J = 7.3 Hz, 3H),
0.85–0.91 (m, 1H), 0.97–1.05 (m, 2H), 1.05–1.16 (m,
1H), 1.20 (sextet, J = 7.3 Hz, 2H), 1.23–1.30 (m, 8H),
1.30–1.53 (m, 5H), 1.53–1.60 (m, 3H), 1.98 (dq,
J = 3.4, 13.5 Hz, 1H), 2.03–2.11 (m, 2H), 2.20–2.28 (m,
1H), 3.35–3.48 (m, 2H), 3.69 (dddd, J = 4.4, 4.4, 11.0,
11.0 Hz, 1H), 7.05 (d, J = 8.2 Hz, 2H), 7.22 (d,
J = 8.2 Hz, 2H); 13C NMR (125.8 MHz) d 14.0, 23.3,
26.9, 28.8, 29.3, 29.8, 29.9, 35.4, 37.2, 41.3, 44.4, 44.5,
48.3, 63.0, 70.7, 125.8, 126.9, 141.6, 147.6; MS (EI) m/
z (rel intensity) 332 (7), 276 (21), 275 (100), 257 (24),
239 (10), 207 (21), 171 (18), 157(19), 145 (36), 131 (65),
121 (19), 117 (22), 91 (23), 57 (20); HRMS: Calcd for
C22H36O2: 332.2715. Found: 332.2707.
1
liquid: H NMR (500 MHz) d 0.85 (t, J = 7.1 Hz, 3H),
0.87–0.91 (m, 1H), 0.98–1.06 (m, 2H), 1.06–1.14 (m,
1H), 1.14–1.22 (m, 8H), 1.22–1.32 (m, 8H), 1.32–1.51
(m, 3H), 1.51–1.58 (m, 5H), 1.98 (dq, J = 3.2, 13.8 Hz,
1H), 2.02–2.13 (m, 2H), 2.19–2.29 (m, 1H), 3.35–3.48
(m, 2H), 3.69 (dddd, J = 4.4, 4.4, 10.8, 10.8 Hz, 1H),
7.05 (d, J = 8.2 Hz, 2H), 7.22 (d, J = 8.2 Hz, 2H); 13C
NMR (125.8 MHz) d 14.1, 22.6, 24.7, 28.8, 29.2, 29.3,
29.8, 29.9, 30.2, 31.8, 35.4, 37.3, 41.3, 44.5, 44.6, 48.3,
63.1, 70.8, 125.8, 126.9, 141.6, 147.6; MS (EI) m/z (rel
intensity) 374 (3), 276 (20), 275 (100), 257 (22), 239
(9), 207 (16), 197 (10), 171 (13), 157 (12), 145 (27), 131
(44), 121 (12), 117 (15), 91 (14), 55 (12); HRMS: Calcd
for C25H42O2: 374.3185. Found: 374.3182.
5.28. (1R*,3R*,4R*)-3-[4-(1,1-Dimethylhexyl)phenyl]-4-
(3-hydroxypropyl)cyclohexanol (JWH-344, 9, n = 4)
The title compoundwas prepared using the procedure em-
ployed for the preparation of JWH-384. From 0.18 g
(0.55 mmol) of (1R*,3R*,4S*)-3-[4-(1,1-dimethylhexyl)-
phenyl]-4-(2-propenyl)cyclohexanol there was obtained
after, chromatography (petroleum ether/ethyl acetate,
5.31. (1R*,3R*,4R*)-3-[4-(1,1-Dimethylnonyl)phenyl]-4-
(3-hydroxypropyl)cyclohexanol (JWH-385, 9, n = 7)
1
1:1), 0.17 g (90%) of JWH-344 a pale yellow liquid: H
The title compound was prepared using the procedure
employed for the preparation of JWH-384. From
0.12 g (0.32 mmol) of (1R*,3R*,4S*)-3-[4-(1,1-dim-
NMR (300 MHz) d 0.83 (t, J = 6.9 Hz, 3H), 0.87–0.99
(m, 1H), 0.99–1.12 (m, 2H), 1.12–1.26 (m, 5H), 1.26–
1.34 (m, 8H), 1.34–1.51 (m, 4H), 1.51–1.64 (m, 4H),
1.96–2.16 (m, 3H), 2.19–2.35 (m, 1H), 3.36–3.53 (m,
2H), 3.71 (dddd, J = 4.2, 4.2, 10.8, 10.8 Hz, 1H), 7.07 (d,
J = 8.4 Hz, 2H), 7.24 (d, J = 8.1 Hz, 2H); 13C NMR
(75.5 MHz) d 14.0, 22.5, 24.3, 28.9, 29.4, 29.9, 30.0,
32.5, 35.5, 37.3, 41.4, 44.6 · 2, 48.4, 63.1, 70.8, 125.8,
127.0, 141.6, 147.7; MS (EI) m/z (rel intensity) 346 (12),
328 (4), 276 (21), 275 (100), 257 (25), 239 (8), 197 (10),
171 (14), 145 (26), 131 (41), 117 (17), 91 (15); HRMS:
Calcd for C23H38O2: 346.2871. Found: 346.2868.
ethylnonyl)phenyl]-4-(2-propenyl)cyclohexanol
there
was obtained, after chromatography (petroleum ether/
ethyl acetate, 1:1), 0.12 g (95%) of JWH-385 as a pale
yellow liquid: 1H NMR (500 MHz)
d 0.86 (t,
J = 6.9 Hz, 3H), 0.87–0.91 (m, 1H), 0.99–1.06 (m, 2H),
1.06–1.14 (m, 1H), 1.14–1.23 (m, 10H), 1.23–1.29 (m,
8H), 1.29–1.52 (m, 5H), 1.52–1.60 (m, 3H), 1.99 (dq,
J = 3.2, 13.8 Hz, 1H), 2.04–2.11 (m, 2H), 2.21–2.29 (m,
1H), 3.37–3.49 (m, 2H), 3.70 (dddd, J = 4.1, 4.1, 11.0,
11.0 Hz, 1H), 7.05 (d, J = 8.2 Hz, 2H), 7.22 (d,
J = 8.2 Hz, 2H); 13C NMR (125.8 MHz) d 14.1, 22.6,
24.7, 28.9, 29.3, 29.4, 29.5, 29.8, 30.0, 30.3, 31.8, 35.5,
37.3, 41.3, 44.6, 44.7, 48.3, 63.1, 70.8, 125.8, 127.0,
141.6, 147.7; MS (EI) m/z (rel intensity) 388 (3), 276
(18), 275 (100), 257 (20), 239 (8), 197 (8), 171 (11), 157
(10), 145 (21), 131 (33), 121 (11), 117 (11), 91 (10), 55
(10); HRMS: Calcd for C26H44O2: 388.3341. Found:
388.3350.
5.29. (1R*,3R*,4R*)-3-[4-(1,1-Dimethylheptyl)phenyl]-4-
(3-hydroxypropyl)cyclohexanol (JWH-337, 9, n = 5)
The title compound was prepared using the procedure em-
ployed for the preparation of JWH-384. From 0.18 g
(0.52 mmol) of (1R*,3R*,4S*)-3-[4-(1,1-dimethylhep-
tyl)phenyl]-4-(2-propenyl)cyclohexanol there was ob-
tained, after chromatography (petroleum ether/ethyl
acetate, 1:1), 0.16 g (84%) of JWH-337 as a pale yellow li-
5.32. Receptor binding experiments
1
quid: H NMR (500 MHz) d 0.83 (t, J = 6.9 Hz, 3H),
0.86–0.91 (m, 1H), 0.99–1.06 (m, 2H), 1.06–1.14 (m,
1H), 1.14–1.26 (m, 6H), 1.26–1.33 (m, 8H), 1.33–1.52
(m, 4H), 1.52–1.58 (m, 4H), 1.99 (dq, J = 3.4, 13.3 Hz,
1H), 2.04–2.12 (m, 2H), 2.21–2.29 (m, 1H), 3.37–3.49
(m, 2H), 3.70 (dddd, J = 4.2, 4.2, 11.0, 11.0 Hz, 1H),
7.05 (d, J = 8.2 Hz, 2H), 7.22 (d, J = 8.2 Hz, 2H); 13C
NMR (125.8 MHz) d 14.1, 22.6, 24.6, 28.9, 29.3, 29.7,
29.8, 30.0, 31.7, 35.5, 37.3, 41.3, 44.6, 44.7, 48.3, 63.1,
70.8, 125.8, 127.0, 141.6, 147.7; MS(EI) m/z (rel intensity)
360 (9), 342 (3), 276 (20), 275 (100), 257 (26), 239 (8), 197
(12), 171 (18), 157 (16), 145 (34), 131 (53), 117 (24);
HRMS: Calcd for C24H40O2: 360.3028. Found: 360.3028.
5.32.1. Materials. Frozen whole brains of male Sprague–
Dawley rats were obtained from Harlan (Dublin, VA).
CP-55,940 was provided by Pfizer (Groton, CT).
[3H]CP-55,940 was purchased from NEN Life Science
Products, Inc. (Boston, MA). Lipofectamine reagent
was purchased from Life Technologies (Gaithersburg,
MD). Human CB2 cDNA was provided by Dr. Sean
Munro (MRC Lab, Cambridge, UK). DMEM and
geneticin were purchased from Gibco BRL (Grand
Island, NY). Fetal clone II was purchased from Hyclone
Laboratories, Inc. (Logan, UT). Aquasil was purchased
from Pierce (Rockford, IL). GF/C glass-fiber filters

J. W. Huffman et al. / Bioorg. Med. Chem. 16 (2008) 322–335
333
(2.4 cm) were purchased from Baxter (McGaw Park,
IL). Polyethylenimine and bovine serum albumin were
purchased from Sigma Chemical Co. (St. Louis, MO).
Scintillation vials and Budget Solve scintillation fluid
were purchased from RPI Corp. (Mount Prospect, IL).
a sufficient volume of buffer 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 addition of 1 lM unlabeled CP-55,940
was used to assess nonspecific binding. Following
incubation (30 ꢂC for 1 h), binding was terminated by
the addition of 2 mL of ice cold buffer D (50 mM
Tris–HCl, pH 7.4, plus 1 mg/mL BSA) and rapid
vacuum filtration through Whatman GF/C filters
(pretreated with polyethyleneimine (0.1%) for at
least 2 h). Tubes were rinsed with 2 mL of ice cold buf-
fer D, which was also filtered, and the filters subse-
quently rinsed twice with 4 mL of ice cold buffer D.
Before radioactivity was quantitated by liquid scintilla-
tion spectrometry, filters were shaken for 1 h in 5 mL
of scintillation fluid. [3H]CP-55,940 bound to hCB2-
CHO cells membranes with a KD value of 0.45
0.07 nM and a Bmax value of 2.93 0.06 pmol/mg.
5.32.2. Development of hCB2-CHO cell line. Chinese
hamster ovary cells were maintained in Dulbecco’s mod-
ified Eagle’s medium (DMEM) with 10% fetal clone II
and 5% CO2 at 37 ꢂC in a Forma incubator. Cell lines
were created by transfection of CB2pcDNA3 into
CHO cells by the Lipofectamine reagent. Stable trans-
formants were selected in growth medium containing
geneticin (1 mg/mL, reagent). 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 37 ꢂC in a For-
ma incubator.
5.33.3. Data analysis. Competition assays were con-
ducted with 1 nM [3H]CP-55,940 and 6 concentrations
(0.1 nM to 10 lM displacing ligands). Displacement
IC50 values were originally determined by unweighted
least-squares linear regression of log concentration-per-
cent displacement data and then converted to Ki values
using the method of Cheng and Prusoff.58 All experi-
ments were performed in triplicate and repeated 3–6
times. All data are reported as mean values SEM.
5.32.3. Membrane preparation. hCB2-CHO cells were
harvested in phosphate-buffered saline containing
1 mM EDTA and centrifuged at 500g. Cell pellets (for
CB2) or whole rat brains (for CB1) were homogenized
in 10 mL of solution A (50 mM Tris–HCl, 320 mM su-
crose, 2 mM EDTA, 5 mM MgCl2, pH 7.4). The
homogenate was centrifuged at 1600g (10 min), the
supernatant saved, and the pellet washed three times
in solution A with subsequent centrifugation. The com-
bined supernatants were centrifuged at100,000g (60 min).
The (P2 membrane) pellet was resuspended in 3 mL of
buffer 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 ꢀ70 ꢂC.
5.34. [35S]GTPcS binding experiments
5.34.1. Materials. All chemicals were from Sigma (St.
Louis, MO) except the following: [35S]GTPcS (1250 Ci/
mmol) was purchased from New England Nuclear
Group (Boston, MA), GTPcS from Boehringer Mann-
heim (New York, NY), and DMEM/F-12 from Fischer
Scientific (Pittsburgh, PA). Whatman GF/B glass fiber
filters were purchased from Fisher Scientific (Pittsburgh,
PA).
5.33. Competition binding assays
5.33.1. CB1 assay. [H3]CP-55,940 binding to P2 mem-
branes was conducted as described elsewhere,57 except
whole brain (rather than cortex only) was used. CP-
55,940 and all cannabinoid analogs were prepared by
suspension in assay buffer from a 1 mg/mL ethanolic
stock without evaporation of the ethanol (final concen-
tration of no more than 0.4%). Displacement curves
were generated by incubating drugs with 1 nM of
[3H]CP-55,940. [3H]CP-55,940 bound to rat brain mem-
branes with a KD value of 0.68 0.07 nM and a Bmax
value of 1.7 0.11 pmol/mg. The assays were performed
in triplicate, and the results represent the combined data
from three individual experiments.
5.34.2. Membrane preparations. Chinese hamster ovary
(CHO) cells stably expressing the human CB2 receptor
(CB2-CHO) were cultured in a 50:50 mixture of DMEM
and Ham F-12 supplemented with 100 U/mL penicillin,
100 Bg/mL streptomycin, 0.25 mg/mL G418, and 5%
fetal calf serum. Cells were harvested by replacement
of the media with cold phosphate-buffered saline con-
taining 0.4% EDTA followed by agitation. Membranes
were prepared by homogenization of cells in 50 mM
Tris–HCl, 3 mM MgCl2, 1 mM EGTA, pH 7.4, centrifu-
gation at 50,000g for 10 min at 4 ꢂC, and resuspension in
the same buffer at 1.5 mg/mL. Membranes were stored
at ꢀ80 ꢂC until use.
5.34.3. [35S]GTPcS binding assay. Prior to assays, sam-
ples were thawed on ice, centrifuged at 50,000g for
10 min at 4 ꢂC, and resuspended in assay buffer
(50 mM Tris–HCl (pH 7.4), 3 mM MgCl2, 0.2 mM
EGTA, and 100 mM NaCl). Reactions containing 10g
of membrane protein were incubated for 1.5 h at 30 ꢂC
in assay buffer containing 10 lM GDP, 0.1 nM
[35S]GTPcS, 0.1% bovine serum albumin, and various
5.33.2. CB2 assay. Binding was assayed by a modifica-
tion of Compton et al.44 CP-55,940 and all cannabi-
noid analogs were prepared by suspension in assay
buffer from
a 1 mg/mL ethanolic stock without
evaporation of the ethanol (final concentration of no
more than 0.4%). The incubation was initiated by the
addition of 40–50 lg membrane protein to silanized
tubes containing [3H]CP-55,940 (102.9 Ci/mmol) and

334
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The work at Clemson was supported by Grant DA03590
to J.W.H., that at Virginia Commonwealth University
by grant DA03672 to B.R.M. both from the National
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