Synthesis and testing of novel classical cannabinoids: Exploring the side chain ligand binding pocket of the CB1 and CB2 receptors

Article abstract of DOI:10.1016/S0968-0896(03)00238-4

A series of C3 cyclic side-chain analogues of classical cannabinoids were synthesized to probe the ligand binding pocket of the CB1 and CB2 receptors. The analogues were evaluated for CB1 and CB2 receptor binding affinities relative to Δ⁸-THC. The C3 side-chain geometries of the analogues were studied using high field NMR spectroscopy and quantum mechanical calculations. The results of these studies provide insights into the geometry of the ligand binding pocket of the CB1 and CB2 receptors.

Full text of DOI:10.1016/S0968-0896(03)00238-4

Bioorganic & Medicinal Chemistry 11 (2003) 3121–3132
Synthesis and Testing of Novel Classical Cannabinoids:
Exploring the Side Chain Ligand Binding Pocket of the
CB1 and CB2 Receptors
Asha K. Nadipuram,a Mathangi Krishnamurthy,a Antonio M. Ferreira,b
Wei Lia and Bob M. Moore, IIa,*
aDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee-Memphis,
Memphis, TN 38103, USA
bComputational Research on Materials Institute, Department of Chemistry, University of Memphis,
Memphis, TN 38152, USA
Received 24 January 2003; revised 4 April 2003; accepted 7 April 2003
Abstract—A series of C3 cyclic side-chain analogues of classical cannabinoids were synthesized to probe the ligand binding pocket
of the CB1 and CB2receptors. The analogues were evaluated for CB1 and CB2receptor binding affinities relative to Á8-THC. The
C3 side-chain geometries of the analogues were studied using high field NMR spectroscopy and quantum mechanical calculations.
The results of these studies provide insights into the geometry of the ligand binding pocket of the CB1 and CB2receptors.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
Delta-9-tetrahydrocannabinol (Á9-THC) was isolated
and identified as the major active constituent of mari-
juana in 1964 by Mechoulam and coworkers.1 In the
following decades, the CB1 and CB2receptors were
discovered, characterized and shown to be responsible
for the actions of Á9-THC.2-5 The CB1 and CB2
receptors have since gained attention as potential ther-
apeutic targets for the development of antiobesity,6
anticancer,7 analgesic,8 and antiglaucoma agents.9,10
Efforts to develop therapeutic agents have resulted in
the identification of a number of structurally distinct
classes of compounds that bind to the cannabinoid
receptors, these include the classical cannabinoids
(Á9-THC,
1),
the
non-classical
cannabinoids
(CP55,940, 2),11 the diarylpyrazoles (AM-251, 3),12 and
aminoalkylindoles (WIN-55212, 4).13 By far the most
extensively studied cannabinoid analogues in terms of
the pharmacology and SAR are the classical and non-
classical cannabinoids.
The binding affinity of the classical cannabinoids
(CCBs) and non-classical cannabinoids to the CB1
receptor can generally be defined in terms of a three point
and four point pharmacophore model, respectively.14
*Corresponding author. Tel.: +1-909-448-6085; fax: +1-901-448-
6828; e-mail: bmoore@utmem.edu
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00238-4

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A. K. Nadipuram et al. / Bioorg. Med. Chem. 11 (2003) 3121–3132
The structural elements that comprise the three point
pharmacophore of the CCB analogues are: (1) a phe-
nolic group in the C1 position of the aromatic ring;15,16
(2) an unsaturated Á8 or Á9 C ring with an exocyclic
C11 methyl or hydroxy methyl; alternatively, a satu-
rated C ring containing a 9-b-hydroxyl, 9-b-hydroxy
methyl, or 9-keto functional group;17-20 and (3) a C3
aliphatic side chain ranging from 3 to 7 carbons wherein
heptyl analogues represent the optimum side chain
length. In addition to the basic pharmacophore, sub-
stitution of the C3 side chain with 10,10-dimethyl, 10,20-
dimethyl, and 10,10-ditholane generally enhances the
activity of the CCBs.21ꢀ26
C3-C10 bond. In this study we report the binding affi-
nities of these compounds with the CB1 and CB2
receptors as well as Á8-THC and 10,10-dithiolanepentyl
Á8-THC. Furthermore, the results of 1D and 2D high
field NMR studies and quantum mechanical calcu-
lations are reported as part of our effort to determine
the geometries of the side chains relative to the aromatic
ring and as such the SAR of the ligand binding pocket.
Results
Chemistry
The understanding of the interplay between the
pharmacophoric elements of CCBs and the ligand
binding pocket (LBP) have been significantly refined as
a result of QSAR studies and site directed mutagenesis
of the LBP. Computational studies have identified the
requirement for a hydrogen bond donor/acceptor pair
in the C1 region of CCBs,17,27ꢀ29 a result proposed to
The synthesis of the side chain modified analogues of
Á8-THC was carried out by the acid catalyzed coupling
of cis-Á2-p-menthene-1,8-diol with the appropriately
substituted resorcinol.33 The precursor resorcinols were
synthesized as summarized in Scheme 1, by reacting
3,5-dimethoxybenzonitrile with the Grignard of the
appropriate alkyl or cycloalkyl halide followed by HCl
hydrolysis of the resulting imine to yield ketones 5–8.37
These ketones were then reacted with either ethane-1,2-
correlate with an interaction of the C1 hydroxyl with a
30,31
critical Lys192in the CB1 receptor.
An additional
.
donor/acceptor pair between Tyr275 and the CCBs
containing a hydroxyl in the C9 region may be respon-
sible for the increased CB1 affinity relative to
Á9-THC.32
dithiol in the presence of BF3 Et2O to give the corres-
ponding dithiolanes 9–1233 or reacted with dimethylzinc
and TiCl4 thus yielding the corresponding gem-dimethyl
intermediates 13–15.37 Deprotection of the intermediate
aryl ethers using BBr3 at 0 ꢁC for 12–16 h generated the
5-substituted resorcinols 16–22. Reaction of the resorcinols
with cis-Á2-p-menthene-1,8-diol, prepared from (+)-Á2-
carene according to the method of Prasad and Dev,38 in
the presence of p-toluenesulphonic acid monohydrate gave
the corresponding Á8-THC analogues 23–29.
The intramolecular geometries of the C1 and C9 sub-
stituents are tightly defined by the rigid ring system of
the CCBs, however QSAR studies indicate moderate to
high conformational flexibility in the C3 side
chains.27,33ꢀ35 These studies clearly demonstrate the LBP
of CB1 prefers a hydrophobic substituent at C3 but the
requirement for conformational flexibility remains to be
fully elucidated. Progress to this end has been reported
in studies of a series of conformationally restricted
Á8-THC side chain analogues incorporating methylene
and methyne functionalities35 and 10-cyclopropyl ana-
logues.36 The study suggests that the side chain adopts
an orthogonal geometry relative to the plane of the
aromatic ring with the tail of the side chain folding into
a hydrophobic pocket. Despite the incorporation of
unsaturation into the side chains, considerable flexibility
remains in this set of molecules. The inherent computa-
tional limitations in predicting the conformation of a
flexible side chain, in the absence of X-ray crystallo-
graphic or high resolution NMR data, somewhat limits
the ability to predict the preferred side chain geometry
and LBP steric requirements of the CB receptors.
Receptor binding assays
Cell membranes from HEK293 cells transfected with the
human CB1 cannabinoid receptor and membranes from
CHO-K1 cells transfected with the human CB2canna-
binoid receptor were used in the receptor binding
assays. Displacement of [3H]CP 55,940 from the CB1
and CB2receptor preparations by increasing con-
centrations of the Á8-THC analogues 23–29 and
Á8-THC were used to determine the binding affinities of
the conformationally biased probes (Table 1). The Ki
values for Á8-THC at the hCB1 and hCB2receptor
were 28.5 nM and 25.0 nM, respectively (affinity ratio
CB1/CB2=1.14), compared to a reported value of 47.6
nM for the rCB1 and 39.3 for the mCB2(affinity ratio
CB1/CB2=1.21).39 The LBP probes exhibited a 3- to
143-fold enhancement in binding affinity to the receptor
subtypes relative to Á8-THC. The gem-dimethyl ana-
logues 27–29 and the pentyl dithiolane 23 possessed
sub-nanomolar affinities for both the CB1 and CB2
receptors that are comparable to the highly potent
10,10-dimethylheptyl- Á8-THC (DMHT, Ki=0.77
nM).40 Within this group of compounds the 10,10-di-
methylcyclopentyl, 27, had the highest affinity for the
CB1 receptor (Ki=0.34 nM) while the 10,10-dimethyl-
cycloheptyl, 29, exhibited greater affinity for the CB2
receptor (Ki=0.22 nM). Despite the relatively high affi-
nity, none of the compounds showed significant selec-
tivity between receptor subtypes.
To better understand the conformational and steric
requirements of the hydrophobic pocket of the LBP
interacting with the C3 side chain of CCBs, we designed
and synthesized a series of Á8-THC analogues restricted
in both the orientation and flexibility of the side chain.
In order to probe the size and depth of the pocket,
cycloalkyl groups of 5, 6 and 7 carbons, that is, cyclic
equivalents of highly active CCBs, were appended to the
C10 position to restrict the degrees of freedom within the
side chain. The C10 position was further substituted
with either a 10,10-dimethyl or a 10,10-dithiolane func-
tionality in an effort to bias the rotation about the

A. K. Nadipuram et al. / Bioorg. Med. Chem. 11 (2003) 3121–3132
3123
Scheme 1. Reagents and conditions: (a) RMgBr, THF, reflux, 3 h; (b) 6N HCl, reflux; (c)BF3–Et2O, ethanedithiol, CH2Cl2, rt, overnight; (d)
Me2Zn, TiCl4; (e) BBr3, 0 ꢁC; (f) cis-Menth-2-ene-1,8,diol, p-TsOH, benzene, 80 ꢁC.
The substitution of the 10,10-dimethyl for the
10,10-dithiolane group unexpectedly resulted in
decreased affinity for both the CB1 and CB2receptors.
This series did not parallel the affinities observed within
the synthesized 10,1,-dimethyl analogues and had dis-
receptor. The overall results of the receptor binding
studies on both series suggest that we have developed
Á8-THC analogues that will aid in the development of
the SAR of the LBP with respect to the CCBs.
tinctly higher Ki0s than 10,10-dithiolaneheptyl- Á8-THC
NMRand molecular modeling studies
33
(DTHT, Ki=0.32nM).
Within this series of com-
pounds the carbon equivalent of the later, 25, had a Ki
of 1.86 nM suggesting the structural requirements of the
receptors are related to steric constraints. The
10,10-dithiolanepentyl analogue 23 had a 4.7-fold and 11
fold increased affinity for the CB1 and CB2receptors,
respectively, relative to the carbon equivalent 24. These
data combined with the 2- to 28-fold decrease in affinity
of 24–26, relative to 27–29, for the CB1 suggest that this
series may help define the steric limitations of the LBP.
Furthermore, based on the steric requirements of the
dithiolane series, modest receptor subtype selectivity is
observed when considering the 1.76 nM Ki of 26 for the
CB1 receptor and the 2.74 nM Ki of 24 for the CB2
The binding affinities of the dimethyl-cycloalkyl ana-
logues for both the CB1 and CB2receptors are com-
parable to that of the highly potent 10,10-dimethylheptyl
THC (DMHT, Ki=0.77nM) analogue. Molecular
modeling analysis of these compounds shows that the
linear dimension of DMHT is 7.72A compared to 4.55
A for 28, which is the cyclic carbon equivalent of
DMHT. The difference of 3.17 A in the linear length of
the side chain suggested that there is a region in the LBP
that accommodates the C3 substituent which can be
characterized as a hydrophobic ellipsoid. It remains
unclear if this is the same pocket that accommodates the
side chain of linear analogues; however, the potential
existence of an ellipsoid pocket made it important to
characterize the relative geometry of the cyclic side
chain with respect to the tricyclic ring system. One facet
of this effort utilized 1D and 2D high field NMR
spectroscopy to assess the relative side chain geometries;
furthermore, extensive NOESY studies on 28 were con-
ducted to obtain distance constraints for use in mole-
cular modeling.
Table 1. Binding affinities of Á8-THC and analogues 23–29 for the
CB1 and CB2receptors
Compd
CB1 Ki (nM)a
CB2 Ki (nM)a
Ratio CB1/CB2
Á8-THC
28.5 (ꢂ3.3)
0.85 (ꢂ0.02)
9.49 (ꢂ2.42)
1.86 (ꢂ0.71)
1.76 (ꢂ0.56)
0.34 (ꢂ0.04)
0.57 (ꢂ0.05)
0.94 (ꢂ0.05)
25.0 (ꢂ4.8)
0.58 (ꢂ0.03)
2.74 (ꢂ1.10)
1.05 (ꢂ0.41)
6.62( ꢂ0.92)
0.39 (ꢂ0.06)
0.65 (ꢂ0.04)
0.22 (ꢂ0.01)
1.14
1.47
3.46
1.77
0.27
0.84
0.87
4.65
23
24
25
26
27
28
29
The chemical shifts of the constituent protons in ana-
logues 23–29 were first assigned utilizing 1D, gHSQC,
gHMBC, and gCOSY experiments. Based on these
assignments the relative spatial orientations of the pro-
tons were examined via 2D NOESY experiments. In all
the derivatives a network of NOEs interconnecting the
aThe Ki values for Á8-THC and the analogues were obtained from
nꢃ2independent experiments run in triplicate showing the standard
error of the mean in parentheses.

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A. K. Nadipuram et al. / Bioorg. Med. Chem. 11 (2003) 3121–3132
tricyclic ring system protons were observed. The
NOESY experiments on 27–29 show strong NOEs
between the H2and H4 protons of the aromatic ring
and the dimethyl and cycloalkyl methyne protons (Fig. 1);
furthermore, the methylene units alpha to the methyne
of the cycloalkyls show medium NOEs to the aromatic
protons. A similar pattern is observed with respect to
the dithiolane probes 24–26 with the notable addition of
two weak NOEs to one each of the dithiolane methyl-
enes. The presence of these NOEs to the dithiolane
methylene and the multiplet between 3.07–3.33 ppm,
that is, diastereotopic protons (Fig. 2), observed for these
protons suggests that the ring flexibility is restrained
and as such the protons reside in unique regions of the
aromatic shielding/desheilding cone. The NOE patterns
and intensities observed for the LBP probes suggest a
conformational bias of the C3 side chains.
NMR data. The resulting conformations show a single
constraint violation between the cyclohexane methyne
and one of the aromatic protons, most likely due to inter-
conversion between the 41 and 319 degree torsional angles
for the C3–C10–C20–C30 (t2) bond, representing the two
torsional populations observed in the simulated anneal-
ing studies (Fig. 4). Multiple torsional driving utilizing
grid searches failed to predict these geometries suggesting
that electronic effects were responsible for the con-
formations observed in the NMR studies. Therefore,
quantum mechanical calculations were employed to
examine the electronic effects driving the structural bias.
Quantum mechanical calculations
It seemed reasonable to assume that the gem-dimethyl
would adopt a conformer that would maximize the
interaction between the conformationally biased side
chain and the aromatic ring. However, the conforma-
tion of the cyclohexyl analogue 28 predicted by NMR
and molecular dynamics could not be explained based
on molecular mechanics calculations, that is, electro-
static and steric analysis. To address this issue, semi-
empirical and density functional theory (DFT) calcula-
tions have been employed to critically evaluate the
potential conformers and determine the importance of
electronic contributions in the experimental results.
A qualitative analysis of the NOE intensities combined
with model building suggest that cycloalkyl functional-
ities extend away from the aromatic ring and do not
fold over the ring in solution, that is, the distinct
absence of NOEs between the H40-H60and H2/H4 pro-
tons (Fig. 1). To more accurately examine the relative
ring orientation, NOESY experiments using mixing
times from 300–700 ms over 100 ms intervals were car-
ried out on 28. This analogue was selected for investi-
gation because it is the carbon equivalent of DMHT.
The spectra exhibited 41 clearly resolved NOEs with a
subpopulation of 8 signals arising from the cyclohexyl
to the H2and H4 protons. Integration of those non-
overlapping unambiguously assigned peaks utilizing
TRIPOS TRIAD software followed by constraint gen-
eration using MARDIGRAS resulted in a web of 26
distance constraints (Fig. 3).41 Utilizing these con-
straints, the model was subjected to 1 ns of constrained
molecular dynamics to determine the populations of
torsional conformers associated with the experimental
Figure 2. Partial 1D NMR spectra of 25 showing the diastereotopic
methylene protons of the dithiolane group.
Figure 3. Stereoscopic view of 28 showing the NOE constraints
(dashed yellow lines) derived from the 2D NOESY experiments. Car-
bon atoms are shown in white, hydrogen atoms in cyan and oxygen
atoms are red.
Figure 1. Partial 2D NOESY spectra (300 ms mixing time) of 28
showing the NOEs between the aromatic protons H2/H4 and the
methylene, H30/H70, and methyne proton, H20, on the cyclohexyl ring.

A. K. Nadipuram et al. / Bioorg. Med. Chem. 11 (2003) 3121–3132
3125
Figure 5. Calculated PM3 potential energy surface of 28 as a function
of torsional parameters t1and t2. Energies are the calculated heat of
formation in Hartrees. The AM1 potential energy surface (omitted)
has identical qualitative features.
an 180ꢁ rotation about the t1 bond was identified in the
same manner (designated A0, B0, etc.). The range of
values for the t2 dihedral is 56 to 305 degrees for the
quantum mechanical calculations whereas the dynamics
study predicted a range of 41 to 319. The relative ener-
gies for the six lowest energy conformations at the
B3LYP/6-31G(p,d) level43,44 are given in Table 2. Note
that the largest energy difference between these struc-
tures is only 0.6 kcal/mol (roughly 8.8 milliHartrees)
suggesting that all conformers are thermally accessible.
Observed deviations between the molecular modeling
results and the quantum mechanical results are small
enough, that is, within 10%, to justify agreement
between the two methods.
Figure 4. Stereoscopic views of 28 showing the two cyclohexyl side-
chain conformations identified in the molecular dynamics studies.
Panel A shows the side chain geometry looking down the plane of the
tricycle ring system and panel B is a view looking into the plane of the
ring sysem. Carbon atoms are shown in white, hydrogen atoms in cyan
and oxygen atoms are red.
Geometry optimizations were performed using both the
AM1 and PM3 semi-empirical parameterizations with
the GAMESS computational chemistry package.42 The
stationary points were confirmed by calculating the
Hessian at the optimized geometries. Using these opti-
mized structures, the potential energy surface (PES) was
calculated using both AM1 and PM3 as a function of
the rotations around the C2–C3–C10–C20 (t1) and
C3–C10–C20–C30 (t2) bonds with all other geometric
parameters held constant. Both semi-empirical potential
energy surfaces show that there is relatively free rotation
about the t1 and t2 bonds (Fig. 5). More pronounced
‘ridges’ on the PM3 surface result from more diffuse
electron densities produced with the PM3 para-
meterization relative to AM1. The four large peaks in
the PES are the result of near collisions between a
methylene hydrogen on the cyclohexyl group and the C2
hydrogen of the THC moiety. The most severe of these
collisions arise from a 1.03 A separation between two
hydrogen atoms.
However, the NMR data suggests that there is a pre-
ferred conformation in the solvent phase, suggesting
that the electronic contributions may be important.
Figure 6, panel B shows the HOMO orbital diagram
(from the B3LYP/6-31G(p,d) calculations) for the gem-
dimethyl analogue 28. In this conformation there is an
orbital lobe associated with the hydrogen atom bonded
to the C30 carbon atom. The shape of this lobe suggests
an anti-bonding sigma orbital and indicates a repulsive
interaction between the hydrogen atom and the electron
Table 2. Relative energy differences (kcal/mol) between the six lowest
energies on the potential energy surface for the gem-dimethyl analogue
(26)
There are six regions on the PES that represent local
minima for analogue 28 which can be designated by
ordered pairs of angles according to (t1, t2) as A(141,56),
B(259,301), C(270,180), D(323,56), E(40,305), and
F(108,190). Geometry optimizations at the PM3 level
were performed to identify the local minima nearest to
each of these points followed by a DFT energy calcula-
tion using the B3LYP functional and a 6-31G(p,d) basis
set. In addition, the associated conformer obtained by
Conformationa
ÁE (kcal/mol)
F0 (101,60)
B (259,301)
A (141,56)
A0 (124,193)
F (108,190)
D0 (302,193)
0.55
0.52
0.38
0.09
0.05
0.00
aNumbers in parenthesis are the ordered pairs of angles (t1,t2).

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A. K. Nadipuram et al. / Bioorg. Med. Chem. 11 (2003) 3121–3132
Discussion
The objective of this research was to design and syn-
thesize novel Á8-THC analogues as probes of the side
chain LBP pocket of the CB1 and CB2receptors. It was
hypothesized that cycloalkyl side chains that contain
identical carbon numbers as known high affinity ligands
but that have reduced linear dimensions may provide
insight into the side chain pocket geometry. To this end
seven novel Á8-THC analogues were synthesized and
tested for binding affinity to the human CB1 and CB2
receptors. The dimethylcycloalkyl analogues 27–29 had
Ki values (0.34 to 0.94 nM) comparable to those of
highly potent CB1 analogues such as DHMT (0.77 nM)
and 10,20-dimethylheptyls (0.46–0.84 nM). In contrast,
the introduction of the 10,10-dithiolane ring to the
cycloalkyl analogues decreased receptor binding as com-
pared to the dimethyl series. The notable exception is 23
wherein a 33- and 47-fold increase in binding to the CB1
and CB2receptors, respectively, is observed relative to
Á8-THC. Caution must also be exercised when comparing
receptor binding data from different species and that
obtained from different receptor preparations; however,
the affinity ratio for the hCB1 to rCB1 found in our
assays was 1.67 while the ratio for the hCB2to mCB2
was 1.57. Factoring in this ratio our compounds remain
comparable to other known high affinity CB ligands.
The most significant difference in CB1 affinity occurs
within the cyclopentyl and pentyl compounds 23, 24,
and 27. Interestingly, 23 and 27 have high affinities for
the CB1 receptor in contrast to 24 that has an 11- to 28-
fold decrease in CB1 affinity. The physical basis for the
increased Ki for 24 may reflect the limited rotation of t1
due the dithiolane ring that thus prevents the cyclo-
pentyl group from optimizing interactions with the
receptor. This limitation to rotation about t1 and t2 is
not predicted based on the NMR and quantum
mechanical calculations on 28 thus suggesting that 27
can adopt geometries that maximize receptor inter-
actions. In particular, despite the conformational flexi-
bility about t2, the steric bulk of the cyclohexyl group
prevents free rotation about t1. Analogue 23 is not
restricted by the steric constraints associated with the
cyclic analogues and as such can adopt receptor favor-
able conformations. In contrast to the affinities of 24–26
for the CB1 receptor the dithiolane ring does not appear
to be as well tolerated by the CB2receptor, that is, a 7-
and 33-fold decrease in affinity for 24 and 26. This
combined with the data for CB1 affinity suggests that
the LBP side chain requirements between the receptor
subtypes are sensitive to steric bulk.
Figure 6. Orbital diagrams of the calculated HOMO at the B3LYP/
6-31G(p,d) level of theory. The shape of the orbital lobes centered on
the cyclohexyl hydrogen atoms is suggestive of antibonding ꢀ orbitals
associated with hydrogen 1s atomic orbitals. Part A demonstrates a
repulsive interaction between two of the cyclohexyl hydrogen atoms
and the aromatic orbital lobes on ring A whereas Part B shows only
one such interaction. There is noticeable distortion of the orbital lobes
associated with ring A, which supports a significant repulsion between
the cyclohexyl hydrogen atoms and the electron density associated
with the aromatic ring.
density of the aromatic ring. An examination of the
shape of the orbital lobes associated with the aromatic
ring shows a deformation associated with this hydrogen
interaction. Figure 6, panel A shows the HOMO orbital
diagram for the same compound after a rotation of 180
degrees about the t2 angle and a subsequent reoptimi-
zation as described previously. In this case we notice
two such repulsions between the aromatic ring and the
nearest hydrogen atom causing an increase in energy of
0.28 kcal/mol. Although small, this energy could be
sufficient to cause conformational preference in the sol-
vent phase. This feature can be observed because of the
quality of the B3LYP functional and the presence of
diffuse functions in the 6-31G(p,d) basis set. By com-
parison with the experimental results we can surmise
that the bias in the NMR spectrum is the result of
electronic interactions between the aromatic ring and
the terminal hydrogens of the cyclohexyl group, favor-
ing conformations that minimize the number of such
repulsions.
By far the most important finding of this study is that
cycloalkyl side chains bind to both the CB1 and CB2
receptors with affinities comparable to the linear chain-
Á8-THC analogues, for example, DMHT. An analysis
of the maximum linear dimensions for the cyclic side
chain analogues range from 3.90 A for 27 to 4.99 A for
29 compared to 7.72A for DMHT. These distances
combined with the receptor binding data suggest the
existence of an ellipsoidal hydrophobic pocket that may
or may not represent the pocket occupied by the linear

A. K. Nadipuram et al. / Bioorg. Med. Chem. 11 (2003) 3121–3132
3127
side chain analogues. The orientation of the C3 side
chain in solution with respect to the tricyclic ring system
projects 124 degrees from the plane of the aromatic ring
base on NMR and quantum mechanical calculations.
The results of our studies provide insights into the
solution conformations of the Á8-THC analogues,
which may reflect the LBP spatial constraints of these
CB ligands. The orientation of the side C3 side chain in
the LBP cannot be conclusively defined since ligands do
bind receptors in conformations different from solution
or crystal structures. Furthermore, the analogue affi-
nities cannot be correlated with ligand efficacy since a
number of high affinity ligands exhibit poor efficacy in
functional assays.45 Functional assays on this series of
compounds will ultimately permit us to refine the struc-
tural requirements of the LBD of the CB receptors and
properties of these analogues.
1-(3,5-Dimethoxy-phenyl)-pentan-1-one (5). To Mg
turnings (1.12g, 46.7 mmol), dried in an oven for 1 h,
and dry THF (32mL) was added 1-butylbromide (4.46
g, 32.5 mmol) and allowed to react at reflux for half an
hour. After formation of the Grignard, 3,5-dimethoxy-
benzonitrile (4 g, 24.5 mmol) was added and the mixture
was refluxed for 4 h. The reaction was cooled with ice
for 15 min followed by the slow addition of 40 mL of
6N HCl and then the mixture was refluxed for 16 h. The
THF was removed and the residue dissolved in EtOAc
(60 mL) and 6N HCl (15 mL). The layers were sepa-
rated, the aqueous layer extracted with EtOAc (4ꢄ20
mL). The combined EtOAc extracts were extracted and
washed with saturated NaHCO3, water and brine. After
drying the organic phase was concentrated and the
residue resolved on silica gel eluting with EtOAc/hex-
anes (5:25), to yield 3.52 g (64.7%) of compound 5 as a
white solid. Rf=0.43 (Hexane/ethyl acetate 9:1); IR
(KBr pellet) 2956, 1601, 1206, 1067, 755 cmꢀ1; 1H NMR
d 7.09 (d, J=2.31 Hz, 2H), 6.64 (t, J=2.31 Hz, 1H),
3.84 (s, 6H), 2.92 (t, J=7.4 Hz, 2H), 1.71 (q, J=7.71
Hz, 2H), 1.40 (s, J=7.71 Hz, 2H), 0.95 (t, J=7.32Hz,
3H); 13C NMR d 200.51, 161.10, 139.33, 106.17, 105.29,
55.83, 38.70, 26.84, 22.71, 14.17; MS: (ESI, Pos.) m/z
245 ([M+23]+).
Conclusion
We have synthesized restricted side chain analogues of
Á8-THC as probes of the LBP of the CB1 and CB2
receptors. Analogues 27–29 have affinities for the CB1
and CB2receptors comparable to known high affinity
CCB ligands while the dithiolane derivatives 24 and 26
have reduced affinity. The receptor binding data suggest
that the side chain binding pocket for this series of
compounds can be characterized as a hydrophobic
ellipsoid. Though the functional potency need not cor-
relate with binding affinities, the Ki values give an indi-
cation of part of the receptor structure and provide for
improvement on the CCB structure to produce higher
affinity analogues. The determination of the functional
activity of the reported compounds combined with
SAR studies on the CCBs should provide invaluable
insights into the development of this class of CB
receptor analogues. Furthermore, the aforementioned
studies may help clarify if the ellipsoid pocket is the
same pocket occupied by the linear side chain CB
analogues.
Utilizing the appropriate alkylbromides the following
compounds were similarly prepared from 3,5-di-
methoxybenzonitrile.
Cyclopentyl-(3,5-dimethoxy-phenyl)-methanone (6). Yield
2.90 g (50.2%) as a clear oil. Rf=0.58 (hexane/ethyl
acetate 9:1); IR (KBr neat) 2956, 1604, 1204, 1067, 755
1
cmꢀ1; H NMR d 7.11 (d, J=2.31 Hz, 2H), 6.63 (t,
J=2.29 Hz, 1H), 3.83 (s, 6H), 3.70–3.60 (m, 1H), 1.95–
1.87 (m, 4H), 1.75–1.59 (m, 4H); 13C NMR d 202.71,
161.06, 139.23, 106.57, 105.11, 55.80, 46.72, 30.35,
26.54; MS: (ESI, Pos.) m/z 257 ([M+23]+).
Cyclohexyl-(3,5-dimethoxy-phenyl)-methanone (7). Yield
19.3 g (63.3%) as a clear oil. Rf=0.43 (hexane/ethyl
acetate 9:1); IR (KBr neat) 2936, 1594, 1200, 1059, 734
;
cmꢀ1 1H NMR d 7.08 (d, J=2.4 Hz, 2H), 6.63 (t,
J=2.25 Hz, 1H), 3.83 (s, 6H), 3.24–3.14 (m, 1H), 2.05–
1.71 (m, 5H), 1.54–1.19 (m, 5H); 13C NMR d 203.73,
161.12, 138.61, 106.35, 104.92, 55.77, 45.99, 30.39,
29.73, 27.12, 26.17, 26.05; MS: (ESI, Pos.) m/z 271
([M+23]+).
Experimental
All chemicals and reagents were purchased from Sigma-
Aldrich or Fisher Scientific Inc. Anhydrous solvents
were prepared by distillation over sodium metal or cal-
cium hydride just prior to use. All reactions were carried
out under dry conditions and under an argon atmo-
sphere. Silica Gel 60, 200–425 mesh was used for flash
Cycloheptyl-(3,5-dimethoxy-phenyl)-methanone (8). Yield
11.0 g (45.6%) as a clear oil. Rf=0.58 (hexane/ethyl
acetate 9:1); IR (KBr neat), 2941, 1592, 1201, 1063, 757
;
cmꢀ1 1H NMR d 7.07 (d, J=2.4 Hz, 2H), 6.64 (t,
chromatography. H NMRs, 13C NMRs and 2D spec-
1
tra were obtained on a Varian 500 Inova MHz NMR
and were consistent with the assigned structures. All
NMR were recorded in CDCl3 unless otherwise speci-
fied. Routine mass spectra were determined on a Bruker
ESQUIRE Ion Trap LC/MS(n) system while HRMS
were measured at the Mass Spectrometry Center, Uni-
versity of Tennessee, Knoxville. IR spectra were mea-
sured on a Perkin–Elmer Model 1605 FT infrared
spectrophotometer. Thin layer chromatography was
performed on silica gel plates (Merck TLC plates, silica
J=2.4 Hz, 1H), 3.84 (s, 6H), 3.40–3.32 (m, 1H), 1.96–
1.52(m, 1H2 ).
13C NMR d 204.16, 161.12, 138.72,
106.40, 104.96, 55.80, 46.99, 46.66, 31.59, 31.13, 28.55,
28.36, 28.08, 27.04; MS: (ESI, Pos.) m/z 285 ([M+23]+).
2-Butyl-2-(3,5-dimethoxy-phenyl)-[1,3]dithiolane (9). To
a stirred solution of 5 (3.52g, 15.9 mmol) in anhydrous
CH2Cl2 (58 mL); was added BF3–Et2O (0.58 mL, 4.8
mmol) and ethane-1,2-dithiol (2.71 g, 28.8 mmol) and
stirred at room temperature for 16 h. The organic phase
gel 60, F254
)

3128
A. K. Nadipuram et al. / Bioorg. Med. Chem. 11 (2003) 3121–3132
was then extracted with 10% NaOH (20 mL) followed
by water and brine. The organic phase was dried, con-
centrated and the residue resolved over silica gel eluting
with EtOAc/hexanes (5:10) to yield 4.50 g (95.2%) of 9
as a colorless oil. Rf=0.51 (hexane/ethyl acetate 9:1); IR
The combined organic extracts were washed with satu-
rated NaHCO3, water and brine, dried and con-
centrated. The residue was resolved over silica gel
EtOAc/hexanes (1:9) to yield 2.75 g (65.0%) of 13 as a
colorless oil. Rf=0.50 (hexane/ethyl acetate 95:5); IR
(neat) 2955, 1457, 1422, 1205, 1067, 831 cmꢀ1; 1H NMR
d 6.53 (d, J=2.4 Hz, 2H), 6.30 (t, J=2.25, 1H), 3.79 (s,
6H), 1.55–1.38 ( m, 7H), 1.25 (s, 6H), 1.21–1.15 (m, 2H);
13C NMR d 160.50, 153.36, 105.30, 96.76, 55.45, 51.80,
39.90, 27.94, 25.97, 25.82; MS: (ESI, Pos.) m/z 249
([M+H]+).
1
(neat) 2955, 1205, 1067 694 cmꢀ1; H NMR d 6.89 (d,
J=2.22 Hz, 2H), 6.34 (t, J=2.21 Hz, 1H), 3.81 (s, 6H).
3.40–3.20 (m, 4H), 2.33 (t, J=7.53 2H), 1.31–1.20 (m,
4H), 0.85 (t, J=6.78 Hz, 3H). 13C NMR d 160.56,
148.04, 106.01, 98.74, 74.58, 55.60, 45.92, 39.33, 30.23,
23.03, 14.13; MS: (ESI, Pos.) m/z 299 (M+).
Utilizing the appropriate arylketones the following
dithiolanes were similarly prepared.
Utilizing the appropriate arylketones the following
10,10-gem-dimethyl compounds were similarly prepared.
2-Cyclopentyl-2-(3,5-dimethoxy-phenyl)-[1,3]dithiolane
(10). Yield 2.92 g (87%) as an oil. Rf=0.52(hexane/
ethyl acetate 92:8); IR (neat) 2955, 1206, 1066, 694
1-(1-Cyclohexyl-1-methyl-ethyl)-3,5-dimethoxy-benzene
(14). Yield 3.69 g (70.3%) as an oil. Rf=0.55 (hexane/
ethyl acetate 95:5); IR (neat) 2932, 1457, 1422, 1208,
1
1
cmꢀ1; H NMR d 6.98 (d, J=2.19 Hz, 2H), 6.34 (t,
1066, 702cm ꢀ1; H NMR d 6.48 (d, J=2.4 Hz, 2H),
J=2.25 Hz, 1H), 3.80 (s, 6H), 3.35–3.09 (m, 4H),
2.82–2.71 (m, 1H), 1.80–1.43 (m, 8H). 13C NMR d
160.03, 149.37, 106.37, 98.35, 79.474, 55.35, 52.14,
38.79, 31.17, 25.73; MS: (ESI, Pos.) m/z 333 ([M+23]+).
6.35 (t, J=2.1 Hz, 1H), 3.80 (s, 6H), 1.72–1.55 (m, 6H),
1.45–1.39 (m, 1H), 1.21 (s, 6H), 1.18–1.05 (m, 2H),
0.97–0.85 (m, 2H); 13C NMR d 160.52, 153.48, 105.35,
96.64, 55.44, 49.28, 41.06, 28.18, 27.44, 26.96, 25.50;
MS: (ESI, Pos.) m/z 263 ([M+H]+).
2-Cyclohexyl-2-(3,5-dimethoxy-phenyl)-[1,3]dithiolane (11).
Yield 16.8 g (89.6%) as an oil. Rf=0.43 (hexane/ethyl
1-(1-Cycloheptane-1-methyl-ethyl)-3,5-dimethoxy-benzene
(15). Yield 3.61 g (72.3%) as an oil. Rf=0.50 (hexane/
ethyl acetate 95:5) IR (neat) 2934, 1455, 1422, 1206,
1
acetate 92:8); IR (neat) 2930, 1198, 1062, 698 cmꢀ1; H
NMR d 6.94 (d, J=2.4 Hz, 2H), 6.33 (t, J=2.25 Hz,
1H), 3.80 (s, 6H), 3.33–3.09 (m, 4H), 2.17–2.09 (m, 1H),
1.92–1.88 (m, 2H), 1.73–1.58 (m, 3H), 1.27–0.97 (m,
5H). 13C NMR d 160.20, 148.17, 107.00, 98.59, 80.97,
55.60, 50.71, 39.07, 31.18, 26.91, 26.35; MS: (ESI, Pos.)
m/z 347 ([M+23]+).
1
1067, 701 cmꢀ1; H NMR d 6.50 (d, J=2.5 Hz, 2H),
6.29 (t, J=2.25 Hz, 1H), 3.80 (s, 6H), 1.73–1.69 (m,
1H), 1.65–1.50 (m, H), 1.48–1.38 (m, H), 1.37–1.28 (m,
H), 1.18 (s, 6H); 13C NMR d 160.33, 153.83, 104.94,
96.41, 55.21, 49.21, 42.03, 29.52, 28.06, 27.95, 25.19;
MS: (ESI, Pos.) m/z 277 ([M+H]+).
2-Cycloheptyl-2-(3,5-dimethoxy-phenyl)-[1,3]dithiolane
(12). Yield 3.60 g (79.2%) as an oil. Rf=0.56 (hexane/
ethyl acetate 92:8); IR (neat) 2925, 1206, 1067, 832, 693
5-(2-Butyl-[1,3]dithiolan-2-yl)-benzene-1,3-diol (16). Boron
tribromide (32.9 mL of 1M soln, 32.9 mmol) was added
to a solution of 9 (4.51 g, 3.23 mmol) in CH2Cl2 (546
mL) under argon at ꢀ78 ꢁC. The reaction temperature
was then raised slowly to 0 ꢁC over a period of 3 h.
Stirring was continued at 0 ꢁC for 12–14 h or until
completion of reaction. Unreacted boron tribromide
was destroyed by adding methanol, solvent removed
and the residual oil diluted with diethyl ether. The
organic phase was washed with saturated NaHCO3,
water and brine, dried and concentrated. The residue
was resolved over silica gel eluting with diethylether/
hexanes (4:6) to yield 0.675 g (74.1%) of 16 as a waxy
cmꢀ1 1H NMR d 6.94 (d, J=2.4 Hz, 2H), 6.32 (t,
;
J=2.25 Hz, 1H), 3.80 (s, 6H), 3.31–3.10 (m, 4H), 2.40–
2.32 (m, 1H), 1.96–1.30 (m, 12H). 13C NMR d 160.38,
148.94, 106.52, 98.48, 81.99, 70.21, 55.60, 51.27, 45.22,
39.34, 32.82, 29.80, 28.04, 27.77; MS: (ESI, Pos.) m/z
361 ([M+23]+).
61-(1-Cyclopentyl-1-methyl-ethyl)-3,5-dimethoxy-benzene
(13). In a dry three-necked flask equipped with an
addition funnel was added anhydrous CH2Cl2 (80 mL)
and cooled to ꢀ40 ꢁC. A 1 M solution of TiCl4 in
CH2Cl2 (102mL, 102mmol) was transferred to the
addition funnel and added slowly to the cold CH2Cl2
solution maintaining a temperature of ꢀ40 ꢁC. The
solution was cooled to ꢀ50 ꢁC and via the addition
funnel a 2M solution of dimethylzinc in toluene (51 mL,
102mmol) was added as rapidly as possible, maintain-
ing the temperature between ꢀ40 and ꢀ50 ꢁC. Upon
completion of the addition, the viscous red solution was
stirred vigorously for 10 min, after which a solution of 6
(4.01 g, 17.1 mmol) in dry CH2Cl2 (20 mL) was added
rapidly maintaining the temperature between ꢀ45 and
ꢀ35 ꢁC. The temperature was then allowed to rise
slowly to ꢀ10 ꢁC over 2h with constant stirring. The
mixture was poured into ice/water (200 mL) and the
aqueous layer was extracted with CH2Cl2 (4ꢄ50 mL).
1
solid. Rf=0.28 (hexane/ethyl acetate 8:2) H NMR d
6.79 (d, J=2.5 Hz, 2H), 6.23 (t, J=2H, 1H), 5.30 (br s,
2H), 3.38–3.19 (m, 4H), 2.25 (t, J=9.75 Hz, 2H), 1.29–
1.18 (m, 4H), 0.83 (t, J=7 Hz, 3H); 13C NMR d 156.24,
148.45, 107.23, 101.50, 73.90, 45.50, 39.01, 29.96, 22.74,
13.86; MS: (ESI, Neg.) m/z 269 ([MꢀH]ꢀ).
Utilizing the appropriate dimethoxy-aryl-30-substituted
precursors the following resorcinol compounds were
similarly prepared.
5-(2-Cyclopentyl-[1,3]dithiolan-2-yl)-benzene-1,3-diol (17).
Yield 0.67 g (74.1%) as a waxy solid. Rf=0.28 (hexane/
1
ethyl acetate 8:2); H NMR d 6.89 (d, J=2.4 Hz, 2H),
6.23 (t, J=2.1 Hz, 1H), 5.68 (br s, 2H), 3.34–3.07 (m,

A. K. Nadipuram et al. / Bioorg. Med. Chem. 11 (2003) 3121–3132
3129
4H), 2.78–2.67 (m, 1H), 1.71–1.38 (m, 8H); 13C NMR d
156.26, 150.31, 108.13, 101.59, 79.28, 52.27, 39.02,
31.39, 25.94; MS: (ESI, Pos.) m/z 305 ([M+23]+).
4H), 3.22–3.18 (m, 1H), 2.73–2.68 (m, 1H), 2.31–2.27
(m, 2H), 2.17–2.18 (m, 1H), 1.86–1.79 (m, 3H), 1.71 (s,
3H), 1.40 (s, 3H), 1.29–1.23 (m, 6H), 1.12 (s, 3H), 0.86–
0.84 (m, 3H); 13C NMR d 154.81, 154.65, 144.23,
134.95, 119.53, 112.11, 109.55, 106.69, 77.17, 73.98,
45.87, 44.97, 39.40, 37.11, 36.03, 31.83, 30.18, 28.09,
27.81, 23.73, 23.04, 18.80, 14.17; HRMS (FAB),
m/z, calcd for C23H32O2S2, 404.1844, experimental
404.1844.
5-(2-Cyclohexyl-[1,3]dithiolan-2-yl)-benzene-1,3-diol (18).
Yield 0.853 g (62.2%) as a waxy solid. Rf=0.27 (hexane/
1
ethyl acetate 8:2); H NMR d 6.78 (d, J=0.9 Hz, 2H),
6.19 (t, J=1.95 Hz, 1H), 4.14 (br s, 2H), 3.32–3.07 (m,
4H), 2.15–2.06 (m, 1H), 1.96–1.58 (m, 4H), 1.19–1.00
(m, 4H); 13C NMR d 156.95, 147.94, 107.39, 101.12,
80.58, 60.83, 50.56, 38.82, 30.92, 26.77, 26.24, 21.08,
14.17; MS: (ESI, Neg.) m/z 295 ([MꢀH]ꢀ).
The synthesis of the 30-substituted Á-THC analogues
8
was carried out using the appropriately substituted
rescorcinol analogues.
5-(2-Cycloheptyl-[1,3]dithiolan-2-yl)-benzene-1,3-diol (19).
Yield 0.582g (41.5%) as a waxy solid. Rf=0.28 (hexane/
ethyl acetate 8:2); H NMR d 6.85 (d, J=2.4 Hz, 2H),
6.22 (t, J=2.25 Hz, 1H), 5.16 (br s, 2H), 3.31–3.07 (m,
4H), 2.35–2.28 (m, 1H), 1.97–1.29 (m, 12H); 13C NMR
d 156.07, 149.33, 107.65, 101.25, 50.92, 39.00, 32.55,
27.70, 27.43; MS: (ESI, Pos.) m/z 333 ([M+23]+).
3- (2 - Cyclopentyl - [1,3]dithiolan - 2-yl)- 6,6,9- trimethyl-
6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-1-ol (24).
Yield 55 mg (14.6%) as a light yellow, waxy solid.
1
1
Rf=0.22 (petroleum ether/diethyl ether 9:1) H NMR d
6.86 (d, J=1.5 Hz, 1H), 6.70 (d, J=2Hz, 1H), 5.43 (d,
J=4 Hz, 1H), 4.76 (br s, 1H), 3.33–3.12(m, 5H), 2.77–
2.66 (m, 1H), 2.15–2.12 (m, 1H), 1.88–1.78 (m, 3H), 1.70
(s, 3H), 1.68–1.67 (m, 2H), 1.60–1.59 (m, 3H), 1.53–1.42
(m, 5H), 1.38 (s, 3H), 1.10 (s, 3H); 13C NMR d 154.55,
154.33, 146.82, 134.95, 119.53, 111.90, 110.12, 107.31,
79.00, 52.40, 44.98, 39.12, 36.06, 31.83, 31.41, 31.34,
28.08, 27.80, 25.91, 23.71, 18.77; HRMS (FAB),
m/z, calcd for C24H32O2S2, 416.1844, experimental
416.1841.
5-(1-Cyclopentyl-1-methyl-ethyl)-benzene-1,3-diol (20).
Yield 1.29 g (72.9%) as a viscous oil. Rf=0.28 (hex-
1
ane:diethyl ether 6:4); H NMR d 6.44 (d, J=2.1 Hz,
2H), 6.20 (t, J=2.1 Hz, 1H), 5.70 (br s, 2H), 2.02–1.98
(m, 1H), 1.57–1.35 (m, 6H), 1.18 (s, 6H); 13C NMR d
156.22, 154.66, 105.10, 99.83, 53.24, 50.33, 36.21, 24.99,
24.75; MS: (ESI, Neg.) m/z 219 ([MꢀH]ꢀ).
5-(1-Cyclohexyl-1-methyl-ethyl)-benzene-1,3-diol
(21).
3 - (2 - Cyclohexyl - [1,3]dithiolan - 2 - yl) - 6,6,9 - trimethyl-
6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-1-ol (25).
Yield 193 mg (22.2%) as a light yellow solid. Rf=0.22
Yield 1.11 g (61.9%) as a viscous oil. Rf=0.28 (hex-
ane:diethyl ether 6:4); 1H NMR d 6.38 (d, J=2Hz, 2H),
6.17 (t, J=2.25 Hz, 1H), 4.82 (br s, 2H), 1.71–1.68 (m,
2H), 1.63–1.60 (m, 1H), 1.53–1.51 (m, 2H), 1.42–1.36
(m, 1H), 1.17 (s, 6H), 1.16–1.03(m, 3H), 0.94–0.86 (m,
2H); 13C NMR d 156.32, 154.45, 106.56, 100.02, 49.21,
40.85, 28.12, 27.39, 26.91, 25.39, 14.40; MS: (ESI, Neg.)
m/z 233 ([MꢀH]ꢀ).
1
(petroleum ether/diethyl ether 9:1) H NMR d 6.81 (d,
J=2Hz, 1H), 6.65 (d, J=2Hz, 1H), 5.44 (d, J=5 Hz,
1H), 4.79 (br s, 1H), 3.28–3.10 (m, 5H), 2.72–2.66 (m,
1H), 2.16–2.06 (m, 2H), 1.93–1.79 (m, 5H), 1.72–1.67
(m, 1H), 1.70 (s, 3H), 1.61–1.58 (m, 1H), 1.29–1.02 (m,
6H), 1.38 (s, 3H), 1.11 (s, 3H); 13C NMR d 154.48,
154.25, 145.36, 134.97, 119.54, 111.94, 110.44, 107.62,
80.35, 77.11, 66.14, 50.61, 44.99, 39.14, 36.07, 31.85,
31.07, 31.00, 28.10, 27.83, 26.88, 26.36, 23.74, 18.81,
15.50, 11.94; HRMS (FAB), m/z, calcd for C25H34O2S2,
430.2000, experimental 430.2000.
5-(1-Cycloheptyl-1-methyl-ethyl)-benzene-1,3-diol (22).
Yield 0.442g (24.7%) as a viscous oil. Rf=0.26 (hex-
ane:diethyl ether 6:4); 1H NMR d 6.40 (d, J=2Hz, 2H),
6.17 (t, J=2Hz, 1H), 4.74 (br s, 2H), 1.68–1.50 (m, 7H),
1.48–1.39 (m, 2H), 1.35–1.25 (m, 2H), 1.15 (s, 6H),
1.14–1.08 (m, 2H); 13C NMR d 156.15, 154.78, 106.13,
99.74, 49.22, 41.79, 29.46, 28.01, 27.88, 25.06; MS: (ESI,
Neg.) m/z 247 ([MꢀH]ꢀ).
3- (2 - Cycloheptyl - [1,3]dithiolan - 2-yl)- 6,6,9- trimethyl-
6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-1-ol (26).
Yield 61 mg (14.2%) as a light yellow solid. Rf=0.22
1
(petroleum ether/diethyl ether 9:1) H NMR d 6.82(d,
3-(2-Butyl-[1,3]dithiolan-2-yl)-6,6,9-trimethyl-6a,7,10,10a-
tetrahydro-6H-benzo[c]chromen-1-ol (23). To a solution
of resorcinol 16 (650 mg, 2.4 mmol) in dry benzene (20
mL) was added cis-Menth-2-ene-1,8-diol (408 mg, 2.4
mmol) followed by the addition of p-toluenesulfonic
acid monohydrate (19 mg, 0.099 mmol). The reaction
mixture was stirred at 80 ꢁC for 4 h. The reaction mix-
ture was cooled and diluted with ether and washed well
with saturated NaHCO3, water and brine. After drying
it was concentrated and the residue resolved on silica gel
(1.9ꢄ25 cm), eluting with 3% diethylether–petroleum
ether to yield 254 mg (26.3%) of 23 as a light yellow,
waxy solid. Rf=0.22 (petroleum ether/diethyl ether 9:1)
1H NMR d 6.77 (d, J=2Hz, 1H), 6.63 (d, J=1.5 Hz,
1H), 5.44 (d, J=4 Hz, 1H), 4.82(s, 1H), 3.37–3.24 (m,
J=1.5 Hz, 1H), 6.67 (d, J=1.5 Hz, 1H), 5.426 (d,
J=4.5 Hz, 1H), 4.88 (br s, 1H), 3.28–3.11 (m, 5H),
2.72–2.66 (m, 1H), 2.33–2.28 (m, 1H), 2.16–2.13 (m,
1H), 1.93–1.79 (m, 7H), 1.70 (s, 3H), 1.39 (s, 3H), 1.11
(s, 3H), 1.93–1.28 (m, 8H); 13C NMR d 154.32, 154.18,
145.80, 134.69, 119.25, 111.59, 109.66, 106.87, 81.16,
76.85, 50.93, 44.67, 39.06, 35.78, 32.54, 32.47, 31.57,
27.78, 27.71, 27.54, 27.48, 23.46, 18.52; HRMS (FAB),
m/z, calcd for C26H36O2S2, 444.2157, experimental
444.2170.
3 - (1 - Cyclopentyl - 1 - methyl - ethyl) - 6,6,9 - trimethyl-
6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-1-ol
(27).
Yield 334 mg (41.6%) as a light yellow solid. Rf=0.32
(petroleum ether/diethyl ether 95:5); 1H NMR d 6.42(d,

3130
A. K. Nadipuram et al. / Bioorg. Med. Chem. 11 (2003) 3121–3132
J=1 Hz, 1H), 6.26 (d, J=2Hz, 1H), 5.43 (d, J=5 Hz,
1H), 4.66 (br s, 1H), 3.21–3.17 (m, 1H), 2.72–2.67 (m,
1H), 2.16–2.13 (m, 1H), 2.06–1.99 (m, 1H), 1.94–1.76
(m, 6H), 1.70 (s, 3H), 1.39 (s, 3H), 1.22–1.13 (m, 2H),
1.11 (s, 3H), 1.18 (s, 6H); 13C NMR d 154.8, 154.45,
150.68, 135.00, 119.58, 110.38, 108.60, 106.03, 76.90,
51.77, 45.11, 39.22, 36.25, 31.75, 28.13, 27.90, 27.86,
25.85, 25.76, 23.74, 18.76; HRMS (FAB), m/z, calcd for
C24H34O2, 354.2558, experimental 354.2566.
for NOE constraints wherein; x<2.5 A was +/ꢀ 0.1 A;
xꢅ3.0 A was +/ꢀ 0.2A ; xꢅ3.5 A was +/ꢀ 0.3 A; and
xꢃ3.5 was +/ꢀ 0.4 A.
A four-step simulated annealing using 1 fs time steps
and the constraints generated by MARDIGRAS was
performed on 28 as follows: (1) 1 ps dynamics at 300 K;
(2) 1 ps heating to 500 K; (3) another 1 ps heating phase
to 700 K; (4) a 1 ns equilibration to 500 K. Additional
parameters included the Tripos force field with
Gasteiger-Huckel charges, an 8 A nonbonding cutoff,
and distance dependent dielectric constant function.
The experimentally obtained NOE distance constraints
were applied during all steps of the dynamics runs, and
the aromatic carbons were defined as aggregates to
maintain the ring geometry. The molecular geometry
was sampled at 1000 fs intervals during phase (1) of the
dynamics runs and once during the heating and cooling
periods. A total of 1007 conformations were collected
for further analysis and these were subjected to twenty
dynamics simulations each to obtain average con-
formations. These average conformations were then
3 - (1 - Cyclohexyl - 1 - methyl - ethyl) - 6,6,9 - trimethyl-
6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-1-ol (28).
Yield 298 mg (37.9%) as a light yellow solid. Rf=0.33
1
(petroleum ether/diethyl ether 95:5) H NMR d 6.37 (d,
J=2Hz 1H), 6.22(d, J=1.5 Hz, 1H), 5.43 (d, J=5 Hz,
1H), 4.65(br s, 1H), 3.21–3.17 (m, 1H), 2.72–2.67 (m,
1H), 2.18–2.13 (m, 1H), 1.91–1.77 (m, 3H), 1.71(s, 3H),
1.67–1.67 (m, 2H), 1.61–1.50 (m, 4H), 1.39 (s, 3H),
1.41–1.36 (m, 1H), 1.16 (s, 3H), 1.15 (s, 3H), 1.10–1.03
(m, 3H), 0.92–0.75 (m, 2H); 13C NMR d 154.32, 154.23,
150.54, 134.76, 119.34, 110.08, 108.44, 105.80, 76.66,
48.84, 44.87, 40.155, 36.03, 31.51, 27.89, 27.63,
27.18, 26.72, 25.26, 24.93, 23.50, 18.53; HRMS (FAB),
m/z, calcd for C25H36O2, 368.2715, experimental
368.2715.
minimized with
a
gradient tolerance of 0.005
Aꢀ1 without defined aggregates or experi-
.
ꢀ1
.
kcal mol
mental NOE distance constraints to obtain the final
average conformations.
3 - (1 - Cycloheptyl - 1 - methyl - ethyl) - 6,6,9 - trimethyl-
6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-1-ol (29).
Yield 341 mg (42.9%) as a light yellow waxy solid.
Quantum mechanical calculations
1
Rf=0.33 (petroleum ether/diethyl ether 95:5) H NMR
Quantum mechanical calculations were performed using
the GAMESS computational chemistry package on a
SGI Origin 2000 with 8 processors and 4GB of memory.
Molecular orbital surfaces were calculated from the
B3LYP/6-31G(p,d) results using a version of MOL-
DEN46 modified at The University of Memphis. Poten-
tial energy surfaces were calculated using the AM1 and
PM3 semi-empirical wavefunctions, as were the final
geometries optimizations.
d 6.39 (d, J=1.5 Hz, 1H), 6.2(d, J=1.5 Hz, 1H), 5.43
(d, J=4.5 Hz, 1H), 4.67 (br s, 1H), 3.21–3.17 (m, 1H),
2.72–2.67 (m, 1H), 2.16–2.10 (m, 1H), 1.91–1.79 (m,
3H), 1.70 (s, 3H), 1.67–1.51 (m, 8H), 1.47–1.41 (m, 2H),
1.38–1.26 (m, 3H), 1.14 (s, 6H), 1.11 (s, 3H), 1.12–1.07
(m, 1H); 13C NMR d 154.40, 154.29, 151.11, 134.77,
119.35, 110.05, 109.76, 108.31, 105.67, 76.65, 49.13,
44.89, 41.36, 36.05, 31.54, 29.48, 29.45, 28.16, 28.05,
27.94, 27.90, 27.87, 27.63, 25.15, 24.93, 23.50, 22.95,
18.52; HRMS (FAB), m/z, calcd for C26H38O2,
382.2872, experimental 382.2878.
Receptor binding assays
Cell membranes from HEK293 cells transfected with the
human CB1 receptor (Lot #1929, Bmax: 1.7 pmol/mg
protein, Kd for [3H]CP 55,940 binding: 186 pM) and
membranes from CHO-K1 cells transfected with the
human CB2receptor (Lot #1930, Bmax: 3.3 pmol/mg
protein, Kd for [3H]CP 55,940 binding: 0.12nM) were
purchased from Perkin–Elmer Life Sciences, Inc.
[3H]CP 55,940 having a specific activity of 120 Ci/mmol
was obtained from Perkin–Elmer Life Sciences, Inc. All
other chemicals and reagents were obtained from
Sigma-Aldrich. The assays were carried out in 96-well
plates obtained from Millipore, Inc. fitted with glass
fiber filters (hydrophilic, GFC filters) having a pore size
of 1.2 m. The filters were soaked with 0.05% poly-
ethyleneimine solution and washed 5ꢄ with deionized
water prior to carrying out the assays. The filtrations
were carried out on a 96-well vacuum manifold (Milli-
pore Inc.), the filters punched out with a pipette tip
directly into scintillation vials at the end of the experi-
ment and vials filled with 5 mL scintillation cocktail
Ecolite (+) (Fisher Scientific). Counting was carried out
on a Beckmann Scintillation Counter model LS6500.
NMRstudies and molecular modeling
All spectra were acquired at 23 ꢁC and 500 MHz on a
Varian Inova-500 spectrometer using a 5 mm HCN tri-
ple resonance probe. Both proton and carbon chemical
shifts were referenced to the residual solvent peak of
DMSO (2.49 ppm for proton and 40 ppm for carbon).
For two-dimensional NOESY measurements, a total of
512fids were recorded for the indirect dimension, with a
2second recycle delay. The TRIAD NMR package
within the Sybyl software was used for data processing
and analysis. Peaks in the NOESY spectra were
assigned and integrated using TRIAD standard func-
tions. MARDIGRAS was then used to generate dis-
tance constraints for 28 using these peak integrals.
Results from each of the five mixing times gave very
similar distance constraints, hence each distance con-
straint was averaged over the five mixing times to get
the final set of distance constraints for the molecule. The
resulting constraints were then examined to ensure that
the error in distances conformed to established errors

A. K. Nadipuram et al. / Bioorg. Med. Chem. 11 (2003) 3121–3132
3131
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mM EDTA, 0.5 mg/mL fatty acid free bovine serum
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Acknowledgements
This research was supported by the College of Phar-
macy, University of Tennessee Health Sciences Center.
We would like to thank Dr. Henry Kurtz for his valu-
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