Synthesis and pharmacology of 1-methoxy analogs of CP-47,497

Article abstract of DOI:10.1016/j.bmc.2010.06.054

Three 1-methoxy analogs of CP-47,497 (7, 8, and 19) have been synthesized and their affinities for the cannabinoid CB₁ and CB₂ receptors have been determined. Although these compounds exhibit selectivity for the CB₂ recept

Full text of DOI:10.1016/j.bmc.2010.06.054

Bioorganic & Medicinal Chemistry 18 (2010) 5475–5482
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and pharmacology of 1-methoxy analogs of CP-47,497
a
b
b
c
c
John W. Huffman a, , Seon A. Hepburn , Patricia H. Reggio , Dow P. Hurst , Jenny L. Wiley , Billy R. Martin
*
a Howard L. Hunter Laboratory, Clemson University, Clemson, SC 29634-0973, United States
b Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, NC 27402, United States
c Department of Pharmacology and Toxicology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298-0613, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Three 1-methoxy analogs of CP-47,497 (7, 8, and 19) have been synthesized and their affinities for the
cannabinoid CB1 and CB2 receptors have been determined. Although these compounds exhibit selectivity
for the CB2 receptor none have significant affinity for either receptor. Modeling and receptor docking
studies were carried out, which provide a rationalization for the weak affinities of these compounds
for either receptor.
Received 14 April 2010
Revised 15 June 2010
Accepted 17 June 2010
Available online 22 June 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
CB1 receptor
CB2 receptor
Nontraditional cannabinoids
1-Methoxy cannabinoids
1. Introduction
the CB1 receptor and is expressed primarily in the central nervous
system.10–12
The modern era of the study of cannabinoids began with the elu-
cidation of the structure of the principal psychoactive ingredient in
OH
11
CH3
9
OH
OH
8
7
10
10a
OH
1
OH
marijuana,
D D
9-tetrahydrocannabinol ( 9-THC, 1), by Gaoni and
A
Mechoulam.1 Subsequently, a number of analogs of 1 were synthe-
2
3
1a
H3C
H3C
C
4
H3C
H3C
6
sized and comprehensive structure–activity relationships (SAR)
O
C5H11
O
C5H11
DMH
D
9-THC template.2–4 In the course
5
were developed based upon the
2
of the development of analgesics derived from the potent synthetic
cannabinoid, (À)-9-nor-9b-hydroxyhexahydrocannabinol (HHC, 2),
a series of structurally modified analogs of 2 were prepared.5–7
Although useful analgesics were not developed, a series of non-tra-
ditional cannabinoids was developed in which the oxygen contain-
ing pyran ring of THC was removed to provide a bicyclic system
that retainedthe phenolichydroxyl group of THC and the 9-hydroxyl
of HHC.8 the SAR for traditional cannabinoids specifies maximum
potency with a 1,1-dimethylheptyl substituent at the 3-position of
the aromatic ring and an equatorial b-orientation of the 9-hydroxyl
group.8 These SAR are also valid for these bicyclic non-traditional
cannabinoids and 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. A hydroxypropyl
group at C-4 of the cyclohexanol ring, as in CP-55,940 (4), led to en-
hanced potency and in 1988 [3H]-CP-55,940 was employed by How-
lett’s group to identify a cannabinoid receptor in rat brain.9 This
G-protein-coupled, transmembrane receptor is now designated as
1
3
CH3
OH
OH
OCH3
OH
H3C
H3C
CH3
CH3
CH3
DMH
O
R
HO
4
5
6
In 1993 Munro et al. identified a second receptor in macro-
phages present in the spleen.13 This receptor, designated CB2, is ex-
pressed primarily in the in the immune system and it has been
suggested that it is responsible for the immunomodulatory effects
of cannabinoids,14–19 a conclusion that is supported by the fact that
these effects are absent in CB2 receptor knockout mice.19 There is
increasing evidence that the endocannabinoid system is of very
considerable physiological significance. In particular, CB2 receptors
are expressed in C6 glioma cells20 and both CB1 and CB2 receptors
are expressed in non-melanoma skin cancer cells.21 There is also a
considerable body of evidence that the CB2 receptor is involved in
* Corresponding author. Tel.: +1 864 656 3133; fax: +1 864 656 6613.
E-mail address: huffman@clemson.edu (J.W. Huffman).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.054

5476
J. W. Huffman et al. / Bioorg. Med. Chem. 18 (2010) 5475–5482
inflammatory pain22–29 Two recent reviews have pointed out the
potential of the endocannabinoid system as a therapeutic target
and suggest that developing new, selective ligands for the CB2
receptor may lead to the development of new useful drugs for
the treatment of a number diseases.30,31
As described previously,37 selective demethylation of one of the
methoxy groups of 2-(3,5-dimethoxyphenyl)-2-methylheptane
(11) and 2-(3,5-dimethoxyphenyl)-2-methyloctane (12) using so-
dium thiopropoxide followed by conversion of the phenol to the
corresponding diethyl phosphate and reduction with lithium in
ammonia afforded 2-(3-methoxyphenyl)-2-methylheptane (13)
and 2-(3-methoxyphenyl)-2-methylheptane (14). Reaction of com-
pounds 13 and 14 with bromine in acetic acid provided aryl bro-
mides 9 and 10.37 The corresponding aryllithiums were prepared
via halogen-metal interconversion38 from bromides 9 and 10 using
n-butyllithium, which were reacted with 3-ethoxycyclohex-2-en-
1-one to provide the corresponding tertiary alcohol. Treatment of
the crude reaction products with dilute HCl resulted in hydrolysis
of the enol ether, which eliminated the elements of water to pro-
vide 3-arylcyclohexenones 15 and 16. Lithium ammonia reduction
of these cyclohexenones gave 3-arylcyclohexanones 17 and 18.39
Sodium borohydride reduction40 of 17 and 18 gave, respectively,
(1R*,3S*)3-[2-methoxy-4-(1,1-dimethylhexyl)phenyl]cyclohexanol
(7, JWH-440) and (1R*,3S*)3-[2-methoxy-4-(1,1-dimethylhep-
tyl)phenyl]cyclohexanol (8, JWH 441).41 Both of these compounds
are racemic as indicated by the notation R* and S*, which indicate
relative stereochemistry.41 The equatorial stereochemistry of the
hydroxyl group in alcohols 7 and 8 was assigned based upon the
method of synthesis40 and the 1H NMR spectrum in which the car-
binol proton appears as a multiplet centered about d 3.79 , which is
characteristic of an axial carbinol proton.42
Several years ago we developed a series of 1-deoxy-D
8-THC
analogs, several of which are highly selective for the CB2 recep-
tor.32 One of these compounds, JWH-133 (5) is a full agonist at
the CB2 receptor, has 200-fold selectivity for the receptor and is
inactive in the mouse model of cannabinoid activity.33 This series
of compounds was developed in analogy with 1-deoxy-3-(1,1-dim-
ethylheptyl)-
D
8-THC, which is a moderately selective CB2 receptor
ligand.32,34 Based upon the observation that 1-deoxy-
D
8-THC ana-
logs are highly selective ligands for the CB2 receptor, we hypothe-
sized that 1-deoxy analogs of CP-47,497 (3) and CP-55,940 (4)
would also exhibit useful selectivity for the CB2 receptor and series
of 1-deoxy analogs of both compounds was synthesized and their
pharmacology evaluated.35 In contrast to our hypothesis, none of
these compounds have more than modest affinity for either the
CB1 or CB2 receptor. It appeared possible that the failure of these
compounds to bind to either receptor was due to the lack of the
oxygen atom at C-1 that was present in the Pfizer compounds
and we have now synthesized 1-methoxy analogs of CP-47497
(6) and determined their affinity for the CB1 and CB2 receptors.
2. Results
The affinities of alcohols 7 (JWH-440) and 8 (JWH-441) 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 summa-
rized in Table 1. Also included in Table 1 are the receptor affinities for
Typically 1-deoxy and 1-methoxy THC analogs with the highest
affinity for both the CB1 and CB2 receptors are those with a 3-dim-
ethylhexyl or dimethylheptyl substituent32,36 and consequently it
was determined that 1-methoxy-9b-hydroxy dimethylhexyl (7)
and dimethylheptyl (8) CP-47,497 analogs would be the initial syn-
thetic targets (Scheme 1). If either of these compounds exhibited
interesting pharmacology, other compounds in this series were
to be investigated.
D
8-THC (1) and CP-47,497 (3).
The synthesis of the methoxy analogs of CP-47497 as illustrated
in Scheme 1 is a modification of the method employed for the
synthesis of the 1-deoxy analogs, however using 2-(4-bromo-3-
methoxyphenyl)-2-methylheptane (9) and 2-(4-bromo-3-methoxy
phenyl)-2-methyloctane (10) as starting materials rather than the
dimethylalkylbenzenes used for the deoxy analogs.35 We used aryl
bromide 10 a number of years ago in the synthesis of 1-deoxy-11-
As shown in Table 1, both JWH-440 (7) and JWH-441 (8) have
little affinity for either the CB1 or CB2 receptor. Since it was con-
ceivable that the stereochemistry of the secondary hydroxyl group
may have affected the interaction of JWH-440 or JWH-441 with the
receptors, ketone 17 was reduced with L-Selectride, a reagent that
stereoselectively reduces cyclic ketones to the corresponding axial
alcohol,43 to provide racemic (1S*,3S*)3-[2-methoxy-4-(1,1-dim-
ethylhexyl)phenyl]cyclohexanol (19, JWH-442), which has an axial
hydroxyl substituent. The 1H NMR spectrum of this compound is
consistent with the assigned stereochemistry, with the equatorial
carbionol proton as a broadened singlet at d 4.24.42 This methoxy
CP-47,497 analog also has negligible affinity for either the CB1 or
CB2 receptor (Table 1).
hydroxy-3-(1,1-dimethylheptyl)-
cation of that synthesis in the present work.
D
8-THC37 and employed a modifi-
OCH
OCH
OCH
3
3
3
Br
e, f
a, b, c
d
H CO
3
R
R
R
13, 14
11, 12
9, 10
O
OH
O
Table 1
OCH
3
D
9-THC
OCH
3
OCH
g
h
3
Receptor affinities (mean SEM) of CP-47,497 analogs (7, 8, 19, 20, and 21),
(1) and CP-47,497 (3)
R
Compound
Ki (nM)
15, 16
R
R
7, 8
OH
17, 18
CB1
CB2
36 10b
CB1/CB2
1.1
i, j
D
9-THC (1)
41 2a
CP-47,497 (3)
JWH-440 (7)
JWH-441 (8)
JWH-442 (19)
JWH-324 (20)
JWH-342 (21)
9.54 0.35a
4414 693
ND
7, 9, 11, 13, 15, 17, 19 R = 1,1-dimethylhexyl
8, 10, 12, 14, 16, 18 R = 1,1-dimethylheptyl
OCH
3
553 32
808 123
693 80
231 48c
178 15c
8.0
>12
5.9
13
>10,000
19
4123 1319
2954 191c
645 29c
R
Scheme 1. Reagents and conditions: (a) NaSPr, DMF, reflux; (b) (EtO)2PO, CCl4,
Et3N, 25 °C; (c) Li, NH3, À78 °C; (d) Br2, HOAc, 25 °C; (e) n-BuLi, THF À78 °C; (f) 3-
ethoxy-2-cyclohexen-1-one, THF, reflux, then 10% HCl, 25 °C; (g) Li, NH3, THF,
À78 °C; (h) NaBH4, EtOH, 0–25 °C; (i) Li(sec-butyl)3BH, THF, À78 °C then 25 °C; (j)
NaOH, H2O2, EtOH, 25 °C.
3.6
a
b
c
Ref. 44.
Ref. 45.
Ref. 35.

J. W. Huffman et al. / Bioorg. Med. Chem. 18 (2010) 5475–5482
5477
In order to provide a rationale for the failure of not only com-
pounds 7, 8, and 19 but also the deoxy CP-47,497 analogs we re-
ported previously35 to have appreciable affinity for either receptor,
a modeling and receptor docking study was carried out. This study
included JWH-441 (8), JWH-324, (1R*,3S*)3-[4-(1,1-dimethylhep-
tyl)phenyl]cyclohexanol (20) and JWH-342, (1S*,3S*)3-[4-(1,1-dim-
ethylheptyl)phenyl]cyclohexanol (21). The affinities of compounds
20 and 21 for the CB1 and CB2 receptors are included in Table 1.
conformational cost for the ligand to dock at CB2 R* is 6.5 kcal/mol.
In this conformation, JWH-342 (21) can form a hydrogen bond
with S7.39 (O–O distance = 2.62 Å; O–H–O angle = 168°) (see
Fig. 2B). Although JWH-342 (21) incurs a higher energy expense
relative to JWH-324 (20) to dock with CB2 R*, JWH-342 (21) com-
pensates for this higher energy expense with greater Van der
Waals interactions, particularly with S6.58 and A7.36, leading to
an overall interaction energy (À37.3 kcal/mol) that is just slightly
better than that of JWH-324 (20). For JWH-441 (8), it should be
noted that the global minimum energy position of the dimethyl-
heptyl side chain differs from that in JWH-324 (20) and JWH-342
(21). In these ligands one of the methyl groups is in the plane of
the aromatic ring, pointing up (see Fig. 1A and B, green). In JWH-
441, steric repulsion from the methoxy group on Ring C, causes
the methyl to point down, but the methyl does remain in the plane
of the aromatic ring (see Fig. 1C, green). For JWH-441 (8), the C1–
C1a–C10a–C10 torsion angle must shift from its global minimum
position of À117° to 10° and the Ring C methoxy group has to shift
106° out of plane with Ring C (see Fig. 1C, orange) to avoid steric
interference with L6.54, S6.58, S7.39 or the ligand’s A ring. The to-
tal conformational cost for the ligand to dock at CB2 is quite high at
10.4 kcal/mol. In addition to Van der Waals interactions with resi-
dues that line its binding site, JWH-441 (8) can form a hydrogen
bond with S7.39 (O–O distance = 2.67 Å; O–H–O angle = 171°)
(see Fig. 2C). The total interaction energy for JWH-441 (8) at CB2
was found to be À32.6 kcal/mol. Thus, while each of these ligands
can establish some interaction at CB2, the conformational energy
expense incurred when each binds to CB2 will significantly impact
the affinity of each. These results are consistent with the CB2 affin-
ities for these compounds listed in Table 1.
CH OH
2
OH
OH
H C
CH
CH
3
3
3
NCS
O
DMH
DMH
H C
3
20
21
22
2.1. CB2 docking studies
The minimized structure of each ligand was docked in the re-
fined model of the CB2 receptor in its activated state (R*) in the
same binding site as described for AM841 (22).46 This CB2 receptor
model includes the extracellular and intracellular domains, as well
as the transmembrane helix (TMH) bundle. JWH-324 (20), JWH-
342 (21) and JWH-441 (8) have low CB2 affinities (JWH-324,
Ki = 231 nM; JWH-342, Ki = 178 nM; and JWH-441 Ki = 808 nM). It
was found that a significant conformational energy expense is re-
quired for each of these ligands to dock in an AM841-like confor-
mation at CB2 R*. For all three ligands, this expense includes a
required re-orientation of the dimethylheptyl side chain, as well
as a change in the C1–C1a–C10a–C10 torsion angle, which affects
the relative orientation of the A and C rings. A re-orientation of
the methoxy group is also required for JWH-441 (8) and this addi-
tional expense may be the reason for the very low CB2 affinity of
this compound. Figure 1(A–C) illustrate the conformational differ-
ences between the global minimum energy conformers of JWH-
324, JWH-342 and JWH-441 (shown in green) and the final docked
conformation of each ligand at CB2 (shown in orange). For JWH-
324 (20), the C1–C1a–C10a–C10 torsion angle must shift from its
global minimum position of À117° to 15° (see Fig. 1A, orange).
The total conformational cost for the ligand to dock at CB2 R* is
5.3 kcal/mol. In addition to Van der Waals interactions with resi-
dues that line its CB2 R* binding site, JWH-324 (20) can form a
hydrogen bond with S7.39 (O–O distance = 2.62 Å; O–H–O an-
gle = 170°) (see Fig. 2A). The total interaction energy for JWH-324
(20) at CB2 R* was found to be À36.3 kcal/mol. For JWH-342 (21),
the C1–C1a–C10a–C10 torsion angle must shift from its global
minimum position of À116° to À48° (see Fig. 1B, orange). The total
2.2. CB1 docking studies
The minimized structure of each ligand was docked in the re-
fined model of the CB1 receptor activated (R*) state in the same
binding site as described for HU-210.47 This CB1 receptor model in-
cludes a truncated N-terminus, all extracellular loops and intracel-
lular domains, as well as the transmembrane helix bundle. JWH-
324 (20), JWH-342 (21) and JWH-441 (8) have low/essentially no
CB1 affinity (JWH-324, Ki = 2954 nM; JWH-342, Ki = 645 nM; and
JWH-441 Ki >10,000 nM). Docking studies showed that the A ring
in all three ligands has severe Van der Waals overlaps with S7.39
or K3.28 when docked in the HU-210 binding site at CB1. In addi-
tion, the C ring methoxy group of JWH-441 has severe Van der
Waals overlaps with V3.32, L6.51 and C7.42 or, if the C ring is ro-
tated 180°, the methoxy group has severe Van der Waals overlaps
with F2.57 and L7.43.
Figure 1. A comparison of the global minimum energy conformation (green) versus the docked conformation (orange) of JWH-324 (20), JWH-342 (22) and JWH-441 (8) at
CB2 R* is illustrated here. The conformers have been overlaid on their phenyl rings (Ring C) and are oriented so that the phenyl ring (Ring C) is perpendicular to the plane of
the page, with the dimethylheptyl side chain closest to the viewer and the carbocyclic ring (Ring A) furthest from the viewer (A) For JWH-324 (20), the C1–C1a–C10a–C10
torsion angle must shift from its global minimum position of À117° to 15°. The conformational energy difference between the two conformers 5.3 kcal/mol. (B) For JWH-342
(21), the C1–C1a–C10a–C10 torsion angle must shift from its global minimum position of À116° to À48°. The total conformational cost for the ligand to dock at CB2 is 6.5
kcal/mol. (C) For JWH-441 (8), the C1–C1a–C10a–C10 torsion angle must shift from its global minimum position of À117° to 10° and the Ring C methoxy group has to shift
106° out of plane with Ring C to avoid steric interference with L6.54, S6.58, S7.39 or the ligand’s A ring. The total conformational cost for the ligand to dock at CB2 R* is
therefore quite high at 10.4 kcal/mol.

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J. W. Huffman et al. / Bioorg. Med. Chem. 18 (2010) 5475–5482
Figure 2. JWH-324 (20), JWH-342 (21) and JWH-441(8) are shown here docked at a model of the CB2 activated (R*) state. The view in each case is from the lipid bilayer,
between TMH2 and TMH7 (TMH1 has been omitted for clarity). (A) For JWH-324 (20), in addition to Van der Waals interactions with residues that line its binding site, the
ligand can form a hydrogen bond with S7.39 (O–O distance = 2.62 Å; O–H–O angle = 170°). The total interaction energy for JWH-324 (20) at CB2 R* was found to be À36.3
kcal/mol. (B) For JWH-342 (21), the ligand can form a hydrogen bond with S7.39 (O–O distance = 2.62 Å; O–H–O angle = 168°). Although JWH-342 (21) incurs a higher energy
expense relative to JWH-324 (20) to dock with CB2 R*, JWH-342 (21) compensates for this higher energy expense with greater Van der Waals interactions, particularly with
S6.58 and A7.36, leading to an overall interaction energy (À37.3 kcal/mol) that is just slightly better than that of JWH-324 (20). (C) For JWH-441(8), in addition to Van der
Waals interactions with residues that line its binding site, the ligand can form a hydrogen bond with S7.39 (O–O distance = 2.67 Å; O–H–O angle = 171°). The total interaction
energy for JWH-441(8) at CB2 R* was found to be À32.6 kcal/mol.
sary as well as a re-orientation of the dimethylheptyl side chain.
The conformational energy expense is 5.3 kcal/mol for JWH-324
(20) and 6.5 kcal/mol for JWH-342 (21). In the case of methoxy
analog JWH-441 (8) in addition to re-orientation of the dimethyl-
heptyl side chain and rotation of the C1a–C10a bond, the methoxy
group must rotate out of the plane of the aromatic ring. This results
in a conformational energy expense of 10.4 kcal/mol. The overall
interaction energies of JWH-324 (20), JWH-342 (21) and JWH-
441 (8) at CB2 were found to be À36.3 kcal/mol, À37.3 kcal/mol
and À32.6 kcal/mol, respectively. The trend in interaction energies,
JWH-342 (21) < JWH-324 (20) < JWH-441 (8) is consistent with the
trend in average Ki values for these ligands at CB2. However, the
interaction energy of JWH-342 (21) is only 1.0 kcal/mol better than
that of JWH-324 (20). This small difference is also consistent with
the fact that the Ki values of these two compounds have slightly
overlapping ranges when experimental error is taken into account.
3. Discussion
These methoxy CP-47,497 analogs were designed in analogy to
1-methoxy-3-(dimethylhexyl and dimethylheptyl)-D
8-THC, which
exhibit, respectively, 174- and 12-fold selectivity for the CB2 recep-
tor.36 Both these compounds have from good to high affinity for the
CB2 receptor (Ki = 18 and 57 nM, respectively). Also, both epimers
of the secondary hydroxyl groups of the corresponding 11-nor-9-
hydroxy-hexahydrocannabinols, which have from 4.5 to 33-fold
selectivity for the CB2 receptor, served as templates for the meth-
oxy CP-47,497 analogs.48 These compounds have from 12 nM to
38 nM affinity for the CB2, In earlier work, we synthesized 1-deoxy
analogs of CP-47,497.35 Although all of these compounds exhibit
varying degrees of selectivity for the CB2 receptor, only the dimeth-
ylheptyl and the dimethyloctyl analogs with axial hydroxyl groups
have affinity for the CB2 receptor greater than 200 nM. In that pub-
lication it was suggested that an oxygen substituent on the aro-
matic ring was probably necessary for CB2 receptor binding. This
hypothesis led us to the preparation of the methoxy CP-47,497
analogs described above.
It is apparent from the experimentally determined receptor
binding data that, although the hypothesis that an oxygen substi-
tuent on the aromatic ring was necessary for CB2 binding appeared
to be eminently reasonable, the lack of affinity of methoxy CP-
47,497 analogs JWH-440 (7), JWH-441 (8) and JWH-442 (19) for
the CB2 receptor indicates that this hypothesis is not correct. In or-
der to provide a rationale for the lack of affinity of not only the
methoxy CP-47,497 analogs as well as the deoxy CP-47,497 ana-
logs reported previously,35 molecular modeling and docking stud-
ies were carried out for deoxy analogs JWH-324 (20), JWH-342
(21) and methoxy analog JWH-441 (8). These three compounds
have negligible affinity for the CB1 receptor and the docking stud-
ies indicate that the saturated carbocyclic ring of these compounds
has severe steric clashes with serine 7.39 and lysine 3.28 of the CB1
receptor. In addition the methoxy group of JWH-441 (8) has severe
clashes with valine 3.32, leucine 6.51 and cysteine 7.42. Rotation of
the aromatic ring leads to alternative steric clashes with phenylal-
anine 2.57 and leucine 7.43. These severe steric clashes of these
compounds with the amino acids of the CB1 receptor are consistent
with the very weak CB1 receptor affinities of JWH-441 (8), JWH-
324 (20), and JWH-342 (21).
4. Conclusions
A priori it appeared that 1-deoxy and 1-methoxy analogs of the
very potent bicyclic Pfizer non-traditional cannabinoids would
provide compounds that were selective ligands for the CB2 recep-
tor, just as various similar analogs of
D
8-THC (1) and 11-nor-9-
hydroxyhexahydrocannabinol (2) have good CB2 receptor affinity
with little affinity for the CB1 receptor. However, the lack of signif-
icant affinity for these analogs of CP-47,497 indicates that this
hypothesis is not correct. CP-47,497 and related compounds have
a phenolic hydroxyl group, which permits energetically significant
hydrogen bonding interactions with the receptors, which are lack-
ing in the deoxy and methoxy analogs. It appears that the rigid
structure of the dibenzopyran system of the classical cannabinoids
that provided the nucleus of the CB2 selective ligands based upon
that template provides a superior platform for the synthesis of
structurally modified ligands for the CB2 receptor.
5. Experimental
5.1. General
1H and 13C NMR spectra were recorded on Bruker 300AC and
JEOL 500 spectrometers. Mass spectral analyses were performed
on a Shimadzu QP2010 capillary gas chromatograph/mass spec-
trometer equipped with a mass sensitive detector at 1.01 kV.
HRMS data were obtained in the Mass Spectrometry Laboratory,
School of Chemical Sciences, University of Illinois. Ether and THF
were distilled from Na-benzophenone ketyl immediately before
The two 1-deoxy CP-47,497 analogs, JWH-324 (20) and JWH-
342 (21) both have modest affinities for the CB2 receptor
(231 48 and 178 15 nM, respectively). For docking of these
two compounds with the CB2 receptor model, rotation about the
C1a–C10a bond between the aromatic and saturated rings is neces-

J. W. Huffman et al. / Bioorg. Med. Chem. 18 (2010) 5475–5482
5479
use, and other solvents were purified using standard procedures.
Column chromatography was carried out on Sorbent Technologies
13C NMR (125.77 MHz, CDCl3) d 14.1, 22.7, 24.6, 28.9, 32.6, 37.5,
44.7, 55.0, 96.6, 104.8, 118.1, 128.6, 151.7, 159.2; MS (EI) m/z (rel
intensity) 57 (40), 205 (100), 220(50).
silica gel (32–63 l) using the indicated solvents as eluents. All new
compounds were homogeneous to TLC and 13C NMR. All target
compounds were homogeneous to GLC or TLC in two different sol-
5.4. 2-(4-Bromo-3-methoxyphenyl)-2-methylheptane (9)
vent systems and GLC. TLC was carried out using 200
plates with the indicated solvents.
lm silica gel
To a solution of 0.281 g (1.27 mmol) of 13 in 0.5 mL of glacial
acetic acid was slowly added 0.065 mL (1.27 mmol) of bromine
in 0.5 mL of acetic acid at ambient temperature. The solution was
stirred for 2 h at ambient temperature, diluted with 5 mL of water
and 3 mL of aqueous NaHCO3. The reaction mixture was extracted
with two portions of ether and the ethereal extracts were washed
with brine, dried (MgSO4), and concentrated in vacuo. The resul-
tant orange oil was purified by flash chromatography (petroleum
ether/ether, 9:1) to give 0.201 g (79%) of 2-(4-bromo-3-methoxy-
5.2. 2-(3-methoxy-5-hydroxyphenyl)-2-methylheptane
To a suspension of 1.4 g (34 mmol, 60% dispersion in oil) of NaH
in 14 mL of dry DMF under argon, was added dropwise 4.2 mL
(46 mmol) of 1-propanethiol, and the reaction was stirred for
30 min at ambient temperature. To this mixture was added
1.32 g (5.28 mmol) of 2-(3,5-dimethoxyphenyl)-2-methylhep-
tane32 (11) in 7 mL of dry DMF, and the resultant solution was
heated at reflux for 5 h, cooled to ambient temperature and poured
into 40 mL of 1 M HCl. The solution was extracted with three por-
tions of ether and the ethereal extracts were washed with succes-
sive portions of aqueous NaHCO3, brine, dried (MgSO4) and
concentrated in vacuo. The crude product was purified by flash
chromatography (petroleum ether/ether, 8:2) to afford 0.96 g
(77%) of 2-(3-methoxy-5-hydroxyphenyl)-2-methylheptane as a
colorless oil; 1H NMR (500 MHz, CDCl3) d 0.81 (t, J = 7.0 Hz, 3H),
0.97–1.15 (m, 2H), 1.16–1.24 (m, 4H), 1.26 (s, 6H), 1.56 (t,
J = 6.0 Hz, 2H), 3.79 (s, 3H), 6.28 (s, 1H), 6.36 (s, 1H), 6.45 (s, 1H);
13C NMR (125.77 MHz, CDCl3) d 14.1, 22.7, 24.5, 28.7, 32.6, 37.6,
44.5, 55.6, 96.6, 105.2, 105.9, 152.9, 156.1, 160.2; MS (EI) m/z (rel
intensity) 121 (37), 149 (100), 236 (60).
phenyl)-2-methylheptane (9) as
a
colorless oil; 1H NMR
(500 MHz, CDCl3) d 0.87 (t, J = 7.0 Hz, 3H), 0.96–1.11 (m, 2H),
1.12–1.24 (m, 4H), 1.25 (s, 6H), 1.60 (t, J = 6, 2H), 3.90 (s, 3H),
6.81 (dd, J = 2.2, 2 Hz, 1H), 6.84 (d, J = 2.2 Hz, 1H) ; 7.43 (d,
J = 8 Hz, 1H); 13C NMR (125.77 MHz, CDCl3) d 14.1, 22.5, 24.3,
28.9, 29.7, 32.5, 37.9, 44.5, 56.1, 110.1, 119.7, 132.5, 151.1, 155.4;
MS (EI) m/z (rel intensity) 91 (80), 148 (90), 199 (70), 227 (94),
298 (60).
5.5. 3-[2-Methoxy-4-(1,1-dimethylhexyl)phenyl]cyclohex-2-en-
1-one (15)
To
a solution of 0.100 g (0.336 mmol) of 2-(4-bromo-3-
methoxyphenyl)-2-methylheptane (9) in 3 mL of dry THF at
À78 °C under argon was added 0.16 mL (0.403 mmol, 2.5 M solu-
tion in cyclohexane) of n-butyllithium and the mixture was stirred
for 30 min. A solution of 0.048 g (0.336 mmol) of 3-ethoxycyclo-
hex-en-1-one in 2 mL of dry THF was added dropwise and the solu-
tion was heated for 4 h at reflux. After cooling to ambient
temperature the reaction was diluted with 15 mL of 10% aqueous
HCl, stirred for 30 min and extracted with two portions of ether.
The combined ethereal layers were washed with saturated aque-
ous NaHCO3, brine, dried (MgSO4) and concentrated in vacuo.
The residue was chromatographed (petroleum ether/ether, 3:2)
to give 0.075 g (71%) of 15 as a yellow oil: 1H NMR (500 MHz,
CDCl3) d 0.82 (t, J = 7.0 Hz, 3H), 0.98–1.26 (m, 6H), 1.27 (s, 6H),
1.59 (t, J = 6.0, 2H), 2.05–2.13 (m, 2H), 2.21–2.29 (m, 2H), 2.74 (t,
J = 5.8 Hz, 2H), 3.86 (s, 3H), 6.22 (s, 1H), 6.85 (s, 1H), 6.91 (d,
J = 8.0 Hz, 1H), 7.13 (d, J = 8 Hz, 1H); 13C NMR (125.77 MHz, CDCl3)
d 14.1, 22.6, 24.4, 24.6, 28.9, 29.7, 32.4, 34.8, 37.2, 38.6, 44.7, 55.4,
112.9, 118.2, 120.4, 126.2, 133.2.2, 153.7, 155.2, 156.1, 199.6 : MS
(EI). m/z (rel intensity) 243 (100), 314 (16).
5.3. 2-(3-methoxyphenyl)-2-methylheptane (13)
To a solution of 1.56 g (6.61 mmol) of 2-(3-hydroxyphenyl-5-
methoxyphenyl)-2-methylheptane and 1.08 mL (8.42 mmol) of
diethyl phosphite in 4 mL of CCl4 at 0 °C was added dropwise 1 mL
(7.17 mmol) of triethylamine. The solution was stirred at 0 °C for
1 h, allowed to warm to ambient temperature and stirred for 7 h at
ambient temperature. The mixture was diluted with CH2Cl2 and
washed with successive solutions of H2O, 1 M aqueous NaOH, H2O,
1 M HCl, and H2O. The organic layer was dried (MgSO4), concen-
trated in vacuo and purified by flash chromatography (petroleum
ether/ether, 7:3) to give 2.01 g (93%) of 2-(3-hydroxyphenyl-5-
methoxyphenyl)-2-methylheptane diethyl phosphate as a red oil,
which was used in the next step without further purification: 1H
NMR (500 MHz, CDCl3) d 0.82 (t, J = 7.0 Hz, 3H), 0.98–1.14 (m, 2H),
1.15–1.25 (m, 4H), 1.27 (s, 6H), 1.39 (t, J = 7.1 Hz, 6H), 1.56 (t,
J = 6.0 Hz, 2H), 3.79 (s, 3H), 4.21–4.26 (m, 4H), 6.64 (s, 1H), 6.71 (s,
1H), 6.78 (s, 1H); 13C NMR (125.77 MHz, CDCl3) d 13.9, 15.9, 16.8,
22.6, 24.6, 28.6, 31.7, 37.6, 44.3, 55.5, 102.2, 102., 109.2,
109.9,151.3,152.7, 160.1; MS (EI) m/z (rel intensity) 245 (60), 273
(30), 302 (100), 372 (50).
5.6. 3-[2-Methoxy-4-(1,1-dimethylhexyl)phenyl]cyclohexanone
(17)
To 49 mL of liquid NH3 at À78 °C was added 0.052 g (7.49 g
atom) of lithium shot and the solution was stirred for 15 min. A
solution of 0.83 g (2.23 mmol) 2-(3-hydroxyphenyl-5-methoxy-
phenyl)-2-methylheptane diethyl phosphate in 10 mL of dry THF
was added dropwise, and the reaction was stirred at À78 °C for
3 h. The reaction was quenched by the addition of solid NH4Cl
and the NH3 was evaporated overnight at ambient temperature.
The solid residue was taken up in 15 mL of H2O and extracted with
two portions of ether. The ethereal extracts were washed with 10 %
aqueous HCl and brine, dried (MgSO4), and the solvent removed in
vacuo. The yellow oil was purified by flash chromatography (petro-
leum ether/ether, 9:1) to give 0.35 g (78%) of 13 as a colorless oil:
1H NMR (500 MHz, CDCl3) d 0.88 (t, J = 7.0 Hz, 3H), 0.97–1.11 (m,
2H), 1.12–1.25 (m, 4H), 1.26 (s, 6H), 1.57 (t, J = 6.0 Hz, 2H), 3.81
(s, 3H), 6.69–6.72 (m, 1H), 6.88–6.91 (m, 2H), 7.18–7.23 (m, 1H);
To 20 ml of liquid ammonia at À78 °C was added 0.004 g
(0.579 g atom) of lithium shot and the solution was stirred for
10 min. A solution of 0.072 g (0.23 mmol) of 3-[2-methoxy-4-
(1,1-dimethylhexyl)phenyl]cyclohex-2-en-1-one (15) in 25 mL of
dry THF was slowly added and the mixture was stirred at À78 °C
for 30 min. The reaction was quenched by the addition of NH4Cl
and the ammonia was evaporated at ambient temperature. The
mixture was diluted with 10 mL of H2O and extracted with two
portions of ether. The ethereal extracts were washed with brine,
dried (MgSO4) and concentrated in vacuo. The yellow oil was puri-
fied by flash chromatography (petroleum ether/ether, 3:2) to give
0.056 g (78%) of 17 as a colorless oil: 1H NMR (500 MHz, CDCl3) d
0.82 (t, J = 7.1 Hz, 3H), 1.01–1.27 (m, 6H), 1.28 (s, 6H), 1.53–1.60
(m, 2H), 1.71–1.81 (m, 1H), 1.81–1.89 (m, 1H), 2.05–2.14 (m,
1H), 2.12–2.18 (m, 1H), 2.33–2.42(m, 1H), 2.42–2.48 (m, 1H),

5480
J. W. Huffman et al. / Bioorg. Med. Chem. 18 (2010) 5475–5482
2.48–2.56 (m, 1H), 2.57–2.63 (m, 1H), 2.99 (dddd, J = 3.8, 3.9, 11.9,
11.9 Hz, 1H), 3.84 (s, 3H) 7.14 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 8.2 Hz,
2H); 13C NMR (125.77 MHz, CDCl3) d 14.1, 22.5, 24.3, 25.6, 28.9,
32.5, 32.8, 37.4, 41.2, 44.2, 44.6, 49.0, 55.4, 113.3, 119.1, 126.2,
126.6, 148.2, 159.6, 211.4 : MS (EI). m/z (rel intensity) 60 (25),
245 (100), 316 (17).
(d, J = 8.0 Hz, 1H), 7.11 (d, J = 8.0 Hz, 1H), 13C NMR (125.77 MHz,
CDCl3) d 14.1, 22.7, 24.6, 24.7, 28.9, 29.7, 30.0, 31.9, 34.9, 35.6,
37.7, 42.1, 44.7, 55.4, 71.3, 108.4, 118.0, 125.8, 131.6, 148.9,
156.8; MS (EI). m/z (rel intensity) 247 (18), 315 (43), 333 (99);
HRMS calcd for C22H37O2 333.2794, found 333.2687.
5.10. Receptor binding experiments
5.7. (1R*,3S*)3-[2-Methoxy-4-(1,1-dimethylhexyl)phenyl]
cyclohexanol (JWH 440, 7)
5.10.1. Materials
Frozen whole brains of male Sprague–Dawley rats were ob-
tained 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 was 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 (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).
To a solution of 0.054 g (0.17 mmol) of 3-[2-methoxy-4-(1,1-
dimethylhexyl)phenyl]cyclohexanone (17) in 6 mL of dry ethanol
at 0 °C, was slowly added 0.056 g (0.177 mmol) of NaBH4. The mix-
ture was warmed to ambient temperature, stirred for 2 h and
quenched by the addition of 10 mL of 10% HCl. The solution was
extracted with two portions of ether and the ethereal extracts were
washed with saturated NaHCO3, brine, dried (MgSO4) and concen-
trated in vacuo. The yellow oil was chromatographed (petroleum
ether/ether, 1:1) to give 0.038 g (68%) of JWH 440 (7) as a colorless
oil; 1H NMR (500 MHz, CDCl3) d 0.88 (t, J = 7.0 Hz, 3H), 1.01–1.28
(m, 6H), 1.29 (s, 6H), 1.45–1.52 (m, 2H), 1.57–1.63 (m, 4H), 1.78–
1.92 (m, 2H), 2.04–2.19 (m, 2H), 2.99 (t, J = 12.0 Hz, 1H), 3.75–
3.83 (m, 1H), 3.81 (s, 3H), 7.84 (s, 1H), 6.87 (d, J = 8.0 Hz, 1H),
7.11 (d, J = 8 Hz, 1H); 13C NMR (125.77 MHz, CDCl3) d 14.1, 22.6,
24.4, 24.6, 28.9, 29.7, 31.9, 32.6, 34.9, 35.6, 37.7, 42.1, 44.7, 55.4,
71.3, 108.4, 117.9, 125.9, 131.2, 148.9, 156.4 ; MS (EI). m/z (rel
intensity) 233 (33), 301 (65), 319 (99); HRMS calcd for C21H35O2
319.2637, found 319.2629.
5.10.2. Development of hCB2–CHO cell line
Chinese hamster ovary cells were maintained in Dulbecco’s
modified Eagle’s medium (DMEM) with 10% fetal clone II and 5%
CO2 at 37 °C in a Forma incubator. Cell lines were created by trans-
fection of CB2pcDNA3 into CHO cells by the Lipofectamine reagent.
Stable transformants 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 anal-
ysis. 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 °C in a Forma incubator.
5.8. (1S*,3S*)3-[2-Methoxy-4-(1,1-dimethylhexyl)phenyl]
cyclohexanol (JWH 442, 19)
To a solution of 0.020 g (0.063 mmol) of 3-[2-methoxy-4-(1,1-
dimethylhexyl)phenyl]cyclohexanone (17) in 1 mL of dry THF at
À78 °C was added 0.27 mL (0.266 mmol, 1.0 M solution in THF)
of L-Selectride and the mixture was stirred for 3 h, warmed to
ambient temperature and stirred for 4 h. To the reaction mixture
was added 4 mL of H2O, 3 mL of ethanol, 1 ml of 1 M aqueous
NaOH and 1 mL of 30% aqueous H2O2. The reaction mixture was
stirred for 30 min, extracted with two portions of ether and the
ethereal layers were washed with brine, dried (MgSO4), and con-
centrated in vacuo. The yellow oil was purified by flash chromatog-
raphy (petroleum ether/ether, 1:1) to give 0.011 g (55%) of JWH
442 (19); 1H NMR (500 MHz) d 0.88 (t, J = 7.0 Hz, 3H), 1.01–1.28
(m, 6H), 1.29 (s, 6H), 1.45–1.52 (m, 2H), 1.57–1.63 (m, 4H), 1.78–
1.92 (m, 2H), 2.04–2.19 (m, 2H), 3.37 (t, J = 12.0 Hz, 1H), 4.24 (bs,
1H), 3.81 (s, 3H), 6.83 (s, 1H), 6.87 (d, J = 8.0 Hz, 1H), 7.11 (d,
J = 8.0 Hz, 1H); 13C NMR (125.77 MHz, CDCl3) d 14.1, 22.6, 24.4,
24.6, 28.9, 29.7, 31.9, 32.4, 34.9, 35.6, 37.7, 42.1, 44.7, 55.4, 67.2,
108.4, 117.9, 125.8, 131.9, 148.7, 156.4; MS (EI) m/z (rel intensity)
233 (33), 301 (65), 319 (99); HRMS calcd for C21H35O2 319.2637,
found 319.2630.
5.10.3. 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 sucrose, 2 mM EDTA, 5 mM
MgCl2, pH 7.4). The homogenate was centrifuged at 1600Âg
(10 min), the supernatant saved, and the pellet washed three times
in solution A with subsequent centrifugation. The combined super-
natants were centrifuged at 100,000Âg (60 min). The (P2 mem-
brane) 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 concentra-
tion of approximately 1 mg/mL. The tissue preparation was divided
into equal aliquots, frozen on dry ice, and stored at À70 °C.
5.11. Competition binding assays
5.9. (1R*,3S*)3-[2-Methoxy-4-(1,1-dimethylheptyl)phenyl]
cyclohexanol (JWH-441, 8)
5.11.1. CB1 assay
[3H]CP-55,940 binding to P2 membranes was conducted as
described elsewhere,49 except whole brain (rather than cortex
only) was used. CP-55,940 and all cannabinoid analogs were pre-
pared 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%). Displacement curves were generated by incu-
bating drugs with 1 nM of [3H]CP-55,940. [3H]CP-55,940 bound to
rat brain membranes 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 tripli-
cate, and the results represent the combined data from three
individual experiments.
2-(3,5-dihydroxyphenyl)-2-methyl octane, provided by Norac
Pharma, was converted to 1R*,3S*)3-[2-methoxy-4-(1,1-dimethyl-
heptyl)phenyl]cyclohexanol (8) by the procedure described for
the synthesis of JWH-440. From 2.2 g (9.31 mmol) of 2-(3,5-dihy-
droxyphenyl)-2-methyl octane there was obtained 0.126 g (5.7%
for eight steps) of pure JWH-441 after flash chromatography
(petroleum ether/ether, 1:1); 1H NMR (500 MHz)
J = 7.0 Hz, 3H), 1.01–1.28 (m, 8H), 1.29 (s, 6H), 1.45–1.52 (m, 4H),
1.57–1.63 (m, 2H), 1.79–1.94 (m, 2H), 2.03–2.17 (m, 2H), 2.99 (t,
J = 12.0 Hz, 1H), 3.74–3.83 (m, 1H), 3.82 (s, 3H), 6.83 (s, 1H), 6.87
d 0.89 (t,

J. W. Huffman et al. / Bioorg. Med. Chem. 18 (2010) 5475–5482
5481
5.11.2. CB2 assay
and omega dihedrals of each CB2 TMH. To preserve the loop confor-
mations, non-moving fixed atom restraints as implemented in
Macromodel were applied to the loop backbone atoms. The full
receptor/ligand complex was energy minimized with the Polak-
Ribier conjugate gradient method until an energy gradient of
0.1 kcal/mol Å2 was reached. Approximately 500 steps were re-
quired to reach the specified gradient since the receptor/ligand
complex was already very near a minimum in each case.
Binding was assayed by a modification of Compton et al.44
CP-55,940 and all cannabinoid analogs were prepared by suspen-
sion in assay buffer from a 1 mg/mL ethanolic stock without evap-
oration of the ethanol (final concentration of no more than 0.4%).
The incubation was initiated by the addition of 40–50 lg mem-
brane protein to silanized tubes containing [3H]CP-55,940
(102.9 Ci/mmol) and a sufficient volume of buffer C (50 mM Tris–
HCl, 1 mM EDTA, 3 mM MgCl2, and 5 mg/mL fatty acid free BSA,
5.12.4. Energy expense assessments for docked ligands
To calculate the energy difference between the global minimum
energy conformation of each compound and its final docked con-
formation after energy minimization of the ligand/receptor com-
plex, single point energies of the final docked conformation and
the global minimum structure were calculated using the OPLS2005
forcefield. A distance dependent dielectric, 8.0 Å extended non-
bonded cutoff, 20.0 Å electrostatic cutoff, and 4.0 Å H-bond cutoff
were used in the calculation.
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. Fol-
lowing incubation (30 °C 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 buffer D, which was also
filtered, and the filters subsequently rinsed twice with 4 mL of
ice cold buffer D. Before radioactivity was quantitated by liquid
scintillation spectrometry, filters were shaken for 1 h in 5 mL of
scintillation fluid. [3H]CP-55,940 bound to hCB2–CHO cells mem-
branes with a KD value of 0.45 0.07 nM and a Bmax value of
2.93 0.06 pmol/mg.
5.12.5. Assessment of pairwise interaction energies
After defining the atoms of a ligand in the final energy mini-
mized CB2 R* complex as one group (group 1) and the atoms corre-
sponding to a residue that lines the binding site in the final ligand/
CB2 R* energy minimized complex as another group (group 2),
Macromodel (version 9.6) was used to output the pair-wise inter-
action energy (coulombic and van der Waals) for a given pair of
atoms. The pairs corresponding to group 1 (ligand) and group 2
(residue of interest) were then summed to yield the interaction en-
ergy between the ligand and that residue. A total interaction en-
ergy for each ligand with CB2 R* was calculated by summing the
pairwise interaction energies for all residues in the binding site
of that ligand and adding to this sum, the conformational energy
expense for the ligand (see Section 5.12.4 above).
5.11.3. Data analysis
Competition assays were conducted with 1 nM [3H]CP-55,940
and 6 concentrations (0.1 nM to 10 lM displacing ligands). Dis-
placement IC50 values were originally determined by unweighted
least-squares linear regression of log concentration-percent dis-
placement data and then converted to Ki values using the method
of Cheng and Prusoff.50 All experiments were performed in tripli-
cate and repeated 3–6 times. All data are reported as mean
values SEM.
5.12. Computational methods
5.12.6. CB1 receptor docking studies
The C ring atoms of each ligand were superimposed on the
HU-210 C ring in our hCB1 receptor model.47 Modifications to ligand
torsions were performed using interactive computer graphics to at-
tempt fitting each ligand into the HU-210 binding site. It was not
possible to eliminate steric overlaps for any of these compounds.
Therefore, no subsequent energy minimization was performed.
5.12.1. Conformational analysis
A conformational search for each ligand using the MCMM/LCMS
method as implemented in Macromodel 9.6 (Schrödinger LLC, NY,
USA) was conducted. The OPLS2005 forcefield was used with a dis-
tance dependent dielectric and nonbonded cutoffs of 20 Å, 8.0 Å,
and 4.0 Å, respectively, for electrostatic, VdW, and H-bond interac-
tions. 50,000 structures were evaluated within a 10 kcal/mol win-
dow above the lowest energy structure found. All rotatable bonds
were varied during the conformational search. Elimination of
duplicate structures was performed by considering structures to
be different if the maximum atom deviation for any pair of corre-
sponding atoms exceeded 0.5 Å.
Acknowledgments
The work at Clemson was supported by grant DA003590 to
J.W.H., that at Virginia Commonwealth University by grant
DA003672 to J.L.W. and that at the University of North Carolina at
Greensboro by grants DA003934 and DA021358 all from the
National Institute on Drug Abuse.
5.12.2. CB2 receptor docking studies
The C ring atoms of each ligand were superimposed on the
AM841 C ring in our hCB2 receptor model.44 Modifications to li-
gand torsions were performed using interactive computer graphics
in order to fit the compound into the AM841 binding site. Steric
overlaps were eliminated using interactive computer graphics.
AM841 was then removed from the complex in preparation for
subsequent minimization.
References and notes
1. Gaoni, Y.; Mechoulam, R. J. Am. Chem. Soc. 1964, 86, 1648.
2. Razdan, R. K. Pharmacol. Rev. 1986, 38, 75.
3. Rapaka, R. S.; Makriyannis, A. Structure–Activity Relationships of the
Cannabinoids, NIDA Research Monograph 79; National Institute on Drug
Abuse: Rockville, MD, 1987.
4. Mechoulam, R.; Devane, W. A.; Glaser, R. In Marijuana/Cannabinoids:
Neurobiology and Neurophysiology; Murphy, L., Bartke, A., Eds.; CRC Press:
Boca Raton, 1992; pp 1–33.
5.12.3. Ligand/CB2 receptor minimization
Minimization of the hCB2-receptor bundle-JWH compound
complex was performed in Macromodel 9.6 (Schrödinger LLC, NY,
USA) using the OPLS2005 forcefield with a distance dependent
dielectric and nonbonded cutoffs of 20 Å, 8.0 Å, and 4.0 Å, respec-
tively, for electrostatic, VdW, and H-bond interactions. All residues
except D2.50(80), K3.28(109), and D(275) were neutralized during
the minimization. To preserve the TMH backbone conformation,
torsional restraints of 100 kcal/mol were applied to the phi, psi,
5. Johnson, M. R.; Melvin, L. S.; Milne, G. M. Life Sci. 1982, 31, 1703.
6. Weissman, A.; Milne, G. M.; Melvin, L. S., Jr. J. Pharmacol. Exp. Ther. 1982, 223,
516.
7. Melvin, L. S.; Johnson, M. R.; Herbert, C. A.; Milne, G. M.; Weissman, A. A. J. Med.
Chem. 1984, 27, 67.
8. Johnson, M. R.; Melvin, L. S. In Cannabinoids as Therapeutic Agents; Mechoulam,
R., Ed.; CRC Press: Boca Raton, FL, 1986; pp 121–145.
9. Devane, W. A.; Dysarz, F. A.; Johnson, M. R.; Melvin, L. S.; Howlett, A. C. Mol.
Pharmacol. 1988, 34, 605.
10. Huffman, J. W.; Lainton, J. A. H. Curr. Med. Chem. 1996, 3, 101.

5482
J. W. Huffman et al. / Bioorg. Med. Chem. 18 (2010) 5475–5482
11. Herkenham, M.; Lynn, A. B.; Little, M. D.; Johnson, M. R.; Melvin, L. S.; De Costa,
D. R.; Rice, K. C. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 1932.
12. Pertwee, R. G. Curr. Med. Chem. 1999, 6, 635.
31. Pacher, P.; Batkai, S.; Kunos, G. Pharmacol. Rev. 2006, 58, 389.
32. Huffman, J. W.; Liddle, J.; Yu, S.; Aung, M. M.; Abood, M. E.; Wiley, J. L.; Martin,
B. R. Bioorg. Med. Chem. 1999, 7, 2905.
13. Munro, S.; Thomas, K. L.; Abu-Shar, M. Nature (London) 1993, 365, 61.
14. Klein, T. W.; Friedman, H.; Specter, S. J. Neuroimmunol. 1998, 83, 102.
15. Galiègue, S.; Mary, S.; Marchand, J.; Dussossoy, D.; Carrière, D.; Carayon, P.;
Bouaboula, M.; Shire, D.; Le Fur, G.; Casellas, P. Eur. J. Biochem. 1995, 232, 54.
16. Condie, R.; Herring, A.; Koh, W. S.; Lee, M.; Kaminski, N. E. J. Biol. Chem. 1996,
271, 13175.
17. Pettit, D. A.; Anders, D. L.; Harrison, M. P.; Cabral, G. A. Adv. Exp. Med. Biol. 1996,
402, 119–129.
18. Schatz, A. R.; Lee, M.; Condie, R. B.; Pulaski, J. T.; Kaminski, N. E. Toxicol. Appl.
Pharmacol. 1997, 142, 278.
19. Buckley, N. E.; McCoy, K. L.; Mezey, E.; Bonner, T.; Zimmer, A.; Felder, C. C.;
Glass, M.; Zimmer, A. Eur. J. Pharmacol. 2000, 396, 141.
20. Sanchez, C.; de Ceballos, M. L.; Gómez del Pulgar, T.; Rueda, D.; Corbacho, C.;
Velasco, G.; Galve-Roperh, I.; Huffman, J. W.; Ramón y Cajal, S.; Guzmán, M.
Cancer Res. 2001, 61, 578.
33. WIley, J. L.; Jefferson, R. G.; Griffin, G.; Liddle, J.; Yu, S.; Huffman, J. W.; Martin,
B. R. Pharmacology 2002, 66, 89.
34. Gallant, M.; Dufresne, C.; Gareau, Y.; Guay, D.; Leblanc, Y.; Prasit, P.; Rochette,
C.; Sawyer, N.; Slipetz, D. M.; Tremblay, N.; Metters, K. M.; Labelle, M. Bioorg.
Med. Chem. Lett. 1996, 6, 2263.
35. Huffman, J. W.; Thompson, A. L. S.; Wiley, J. L.; Martin, B. R. Bioorg. Med. Chem.
2008, 16, 322.
36. Huffman, J. W.; Bushell, S. M.; Miller, J. R. A.; Wiley, J. L.; Martin, B. R. Bioorg.
Med. Chem. 2002, 10, 4119.
37. (a) Huffman, 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; (B)
Yu, S. Ph.D. dissertation, Clemson University, 1997.; (c) Itagaki, N.; Sugahara, T.;
Iwabuchi, Y. Org. Lett. 2005, 7, 4181.
38. Jones, R. G.; Gilman, H. Org. React. 1951, 6, 339.
39. Caine, D. Org. React. 1976, 23, 1.
21. Casanova, M. L.; Blazquez, C.; Martinez-Palacio, J.; Villanueva, C.; Fernandez-
Acenero, M. J.; Huffman, J. W.; Jorcano, J. L.; Guzman, M. J. Clin. Invest. 2003,
111, 43.
22. Hanus, L.; Breuer, A.; Tchilibon, S.; Shiloah, S.; Goldenberg, D.; Horowitz, M.;
Pertwee, R. G.; Ross, R. A.; Mechoulam, R.; Fride, E. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 1422.
40. House, H. O. Modern Synthetic Reactions; W. A. Benjamin: Menlo Park, CA, 1972.
pp 62–65.
41. The notation R* or S* indicates relative configuration in racemic
compounds. See: Eliel, E. L.; Wilen, S. H.; Mander, L. N. Stereochemistry
of Organic Compounds; John Wiley and Sons: New York, 1994. pp 1205–
1206.
23. Conti, S.; Costa, B.; Colleoni, M.; Parolaro, D.; Giagnoni, G. Br. J. Pharmacol. 2002,
135, 181.
42. Bhacca, N. S.; Williams, D. H. Applications of NMR Spectroscopy in Organic
Chemistry; Holden-Day: San Francisco, 1966. pp 77–83.
24. (a) Malan, T. P.; Ibrahim, M. M.; Vanderah, T. W.; Makriyannis, A.; Porreca, F.
Chem. Phys. Lipids 2002, 121, 191; (b) Malan, T. P.; Ibrahim, M. M.; Deng, H.; Liu,
Q.; Main, H. P.; Vanderah, T.; Porreca, F.; Makriyannis, A. Pain 2001, 93, 239.
25. Ibrahim, M. M.; Deng, H.; Zvonok, A.; Cockayne, D. A.; Kwan, J.; Mata, H. P.;
Vanderah, T. W.; Lal, J.; Porreca, F.; Makriyannis, A. M.; Malan, T. P. Proc. Natl.
Acad. Sci. U.S.A. 2003, 100, 10529.
26. Zhang, J.; Hoffert, C.; Vu, H. K.; Groblewski, T.; Ahmad, S.; O’Donnell, D. Eur. J.
Neurosci. 2003, 2750.
27. Kehl, L. J.; Hamamoto, D. T.; Wacnik, P. W.; Croft, D. L.; Norsted, B. D.; Wilcox,
G. L.; Simone, D. A. Pain 2003, 103, 175.
28. Clayton, N.; Marshall, F. H.; Bountra, C.; O’Shaughnessy, C. T. Pain 2002, 96, 253.
29. Giblin, G. M. P.; O’Shaughnessy, C. T.; Naylor, A.; Mitchell, W. L.; Eatherton, A. J.;
Slingsby, B. P.; Rawlings, D. A.; Goldsmith, P.; Brown, A. J.; Haslam, C. P.;
Clayton, N. M.; Wilson, A. W.; Chessel, I. P.; Wittington, A. R.; Green, R. J. Med.
Chem. 2007, 50, 2597.
43. Brown, H. C.; Krisnamurthy, S. J. Am. Chem. Soc. 1972, 94, 7159.
44. 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.
45. Showalter, V. M.; Compton, D. R.; Martin, B. R.; Abood, M. E. J. Pharmacol. Exp.
Ther. 1996, 278, 989.
46. Pei, Y.; Mercier, R. W.; Anday, J. K.; Thakur, G. A.; Zvonok, A. M.; Hurst, D.;
Reggio, P. H.; Janero, D. R.; Makriyannis, A. Chem. Biol. 2008, 15, 1207.
47. Kapur, A.; Hurst, D. P.; Fleischer, D.; Whitnell, R.; Thakur, G. A.; Makriyannis, A.;
Reggio, P. H.; Abood, M. E. Mol. Pharmacol. 2007, 71, 1512.
48. Marriott, K. C.; Huffman, J. W.; Wiley, J. L.; Martin, B. R. Bioorg. Med. Chem.
2006, 14, 2386.
49. 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.
50. Cheng, Y. C.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.
30. Mackie, K. Annu. Rev. Pharmacol. Toxicol. 2006, 46, 101.