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(Received for publication, October 16, 1995; and in revised form, February 12, 1996) From the
During a search for novel drugs possessing analgesic properties
but devoid of the psychotropic effects of marijuana, a group of
molecules designated as nonclassical cannabinoids was synthesized by
Pfizer. Of these nonclassical cannabinoids CP-55,940 has received the
most attention principally because it was used as the high affinity
radioligand during the discovery and characterization of the
G-protein-coupled cannabinoid receptor. In an effort to obtain
information on the stereoelectronic requirements at the cannabinoid
receptor active site, we have studied the conformational properties of
CP-55,940 using a combination of solution NMR and computer modeling
methods. Our data show that for the most energetically favored
conformation, (i) the aromatic phenol ring is perpendicular to the
cyclohexane ring, and the phenolic O-H bond is coplanar with the
aromatic ring and points away from the cyclohexyl ring; ii) the
dimethylheptyl chain adopts one of four preferred conformations in all
of which the chain is almost perpendicular to the phenol ring; and iii)
an intramolecular H-bond between the phenolic and hydroxypropyl groups
allows all three hydroxyl groups of CP-55,940 to be oriented toward the
upper face of the molecule. Such an orientation by the OH groups may be
a characteristic requirement for cannabimimetic activity. The psychoactive effects of cannabinoids, particularly
A series of compounds was designed, synthesized, and designated as
nonclassical cannabinoids (NCCs), e.g. CP-55,940 in Fig. 1, which differ from classical cannabinoids by the absence
of a tetrahydropyran ring, e.g.
Figure 1:
Evolution in cannabinoid structures with
progressively enhanced potencies from the naturally occurring
The conformational properties of the NCCs
still remained to be investigated in detail. Of the nonclassical
analogs, one particularly potent and enantioselective derivative,
CP-55,940, has received extensive attention because it was used as a
radiolabel for the identification and characterization of the
cannabinoid receptor(11) . CP-55,940 is structurally similar to
its prototype, CP-47,497, except for having a hydroxypropyl group
(southern aliphatic OH) on the C-ring. This structural modification
incorporated in CP-55,940 enhances CB1 receptor binding potency by
20-fold and enantioselectivity for CB1 receptor binding by
44-fold(12) . As an analgesic, it is 10 times more potent than
CP-47,497 and about 100 times more potent than
In this
report, we examine the conformational properties of CP-55,940 in order
to define within this important ligand those stereoelectronic features
most probably associated with cannabimimetic activity and receptor
binding. To accomplish our goal, we have combined two-dimensional high
resolution NMR and computer modeling techniques to study the
conformational properties of CP-55,940 while paying special attention
to: (i) the relative orientation of the C-ring with respect to the
A-ring; (ii) the conformation of the phenolic Ph-OH group; and (iii)
the conformation of the 1,1-dimethylheptyl side chain.
Molecular mechanics/dynamics
calculations (13, 23) were carried out using a
Biosym-interfaced AMBER force field (24, 34) with the
following three steps: 1) initial structure was minimized to relieve
any overly strained coordinates; 2) molecular dynamics sampling was
performed using the following protocol with time steps of 1 fs: (i)
heat up to 2500 K and equilibrate for 1 ps and (ii) dynamics simulation
at this temperature for 300 ps with atomic coordinate trajectories
recorded every 1 ps; and 3) the 300 frames recorded during the dynamics
run were retrieved and minimized with a two-step minimization, using
the steepest descent method for the first 100 iterations and then the
conjugate gradient method until the maximum derivative was less than
0.001 kcal/mol. The calculations were carried out in a vacuum condition
(default, dielectric constant = 1). A total of 300,000
conformations or frames were sampled during the simulation. In order to
reduce the volume of the output data to a more manageable level,
conformer structures were recorded at 1-ps intervals, thus reducing the
number of structures to be analyzed to 300 frames. For a molecule like
CP-55,940 with several flexible substituents other than the
hydroxypropyl chain, the dynamics calculations would have to sample too
many conformations. Therefore, we imposed restraints on the torsional
angles of certain regions, such as the DMH chain (except for the two
dihedral angles adjacent to the A-ring,
Figure 2:
500
MHz
Figure 3:
Expanded scale of a 500 MHz
two-dimensional COSY-PH-DQF spectrum of CP-55,940 in CDCl
Figure 4:
500 MHz two-dimensional
Figure 5:
500 MHz NOESYPH spectrum in
CDCl
Figure 6:
Molecular graphic representation of six
energetically favored conformations of CP-55,940 on the basis of the
energy minimization of structures occurring along the molecular
dynamics trajectory. The dimethylheptyl side chain is not
displayed.
To use this
technique, the first step required identification of the two aliphatic
OH resonances that were obscured by other resonances in the
Figure 7:
Cross-sections parallel to the F2
dimension sliced through the phenolic hydroxyl resonance in the 200 MHz
two-dimensional
Several two-dimensional exchange spectra were obtained
using the same parameters while varying the concentration from 0.05 M to 0.0026 M. The one-dimensional cross-section due
to the phenolic OH proton from each of the two-dimensional exchange
spectra are shown in Fig. 7. This allows us to follow the effect
of concentration on the The combined use of two-dimensional NMR and computer
molecular modeling has enabled us to define the conformational
properties of CP-55,940 as discussed below.
Additional information about the relative A/C-ring
orientation was obtained from the NOESYVD spectrum (not shown) using a
similar two-dimensional NOESYPH pulse sequence with four loops of
randomly varied mixing times (800 ± 20 ms). In addition to the
NOE cross-peak between H-5 with H-8a and H-12a, which was also observed
in the previous NOESYPH spectrum, the spectrum had a very weak NOE
cross-peak due to the interaction between the H-5 (
The occurrence of an intramolecular H-bond was also supported by the
result obtained from computer molecular modeling (Fig. 6).
According to the combined experimental and theoretical data, the
intramolecular H-bonding stabilizes the conformation of CP-55,940 by
forming a ten-membered H-bonded ring. Based on these data, the most
preferred conformation having an intramolecular H-bond was conformer IV
in Fig. 8. Such a conformation is also congruent with the
observed nonequivalency of the 3"-CH
Figure 8:
A graphical representation of the
energetically most favored conformation of CP-55,940 stabilized by
intramolecular H-bonding (IV), a low energy H-bonded conformer with all
three OH groups pointing toward the one face of the molecule (II) and
that of
Our data show that the energetically favored conformations have the
following features: (i) the A-ring is approximately perpendicular to
the C-ring; (ii) the proton of Ph-OH points away from the C-ring; and
(iii) the DMH chain randomly adopts one of four possible minimum energy
conformations; however, in all cases, the DMH side chain is almost
perpendicular to the plane of the phenyl ring. It is tempting to
postulate that the dramatic increased potency of cannabinoid analogs
with a 1`,1`-dimethylheptyl side chain when compared with those having
no
Figure 9:
A ligand-membrane-receptor model
representing the trans-membrane diffusion of CP-55,940 en route to
interacting with the cannabinoid receptor. According to our hypothesis,
the ligand preferentially partitions in the membrane bilayer where it
assumes a proper orientation and location allowing for a productive
collision with the active site.
Volume 271,
Number 18,
Issue of May 3, 1996 pp. 10640-10647
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-tetrahydrocannabinol (-THC), (
)are well documented and offer a vexing target for new
therapeutic drug discovery. Potential therapeutic applications include
analgesia, sedation, attenuation of the nausea and vomiting due to
cancer chemotherapy, appetite stimulation, decreasing intraocular
pressure in glaucoma, certain motor or convulsant disorders, and
concentration-time deficits(1, 2) . A most probable
site at which many of the pharmacological effects of cannabimimetics
are induced is now thought to be the cannabinoid receptor (CB). This
receptor type (CB1 and CB2 subtypes are described) is a subgroup of the
G
-protein-coupled seven transmembrane spanning receptor
superfamily(3) . The CB1 receptor subtype is found
predominately in brain(4, 5) , whereas the CB2
receptor subtype is reported only in peripheral tissue(6) . -THC
and(-)9
-OH-hexahydrocannabinol(7, 8) .
Although the NCCs possess significant analgesic activity, there was
sufficient data to indicate that these NCC molecules still exhibit the
behavioral effects of the classical analogs(9, 10) .
Key pharmacophores for the NCC analogs include a phenolic hydroxyl
(Ph-OH), an aliphatic side chain attached to the phenyl ring, and a
cyclohexyl ring (C-ring), all of which are also present in the natural
cannabinoid -THC. Two additional pharmacophores, the
northern and southern aliphatic hydroxyl groups, are not found in the
natural cannabinoids but are present in most NCCs. The presence of
these aliphatic hydroxyl groups significantly enhances the analgesic
activity in the NCC series(8) . Consequently, the spatial
arrangement with regard to the molecular pharmacophores should play an
important role in determining CB1 receptor binding affinity and
pharmacological activity.
-THC toward the potent synthetic
analogs.
-THC(8, 10) . In an earlier
publication(7, 13) , we reported preliminary results
on the conformational properties of prototype CP-47,497.
Materials
CP-55,940 was a generous gift from
Pfizer Central Research (Groton, CT). NMR samples were prepared in
CDCl
(99.8 atom %) at concentrations of 0.02 M for H and 0.2 M for 
C, filtered,
degassed, and sealed in 5 mm NMR tubes. Tetramethylsilane was used as
an internal chemical shift reference.NMR Spectra
The high resolution one- and
two-dimensional NMR spectra were obtained on Bruker AMX-500 and
WP-200SY spectrometers. One-dimensional H 500 MHz NMR
spectra were recorded using the acquisition parameters: pulse width,
5.0 µs; spectral width, 4990.0 Hz; data size, 16 k; recycling
delay, 2 s; number of transients, 16; temperature, 298 K.
Two-dimensional phase-sensitive H-H chemical
shift correlation spectra with double quantum filter (DQF-COSY) (14) were obtained at 500 MHz with the acquisition parameters:
90 ° pulse width, 5 µs; spectral width, 4990.0 Hz; recycling
delay, 1.3 s. The data were 512 w in the F1 dimension and 2k in the F2
dimension and were zero-filled in F1 prior to two-dimensional Fourier
transformation to yield a 2k 
2k data matrix. The spectra were
processed using a shift sine-bell window function in both dimensions.
Two-dimensional phase-sensitive H-H nuclear
Overhauser enhancement spectra (NOESYPH) (15) were collected at
500 MHz using the acquisition parameters similar to the DQF-COSY with
the addition of mixing time (800 ms). Two-dimensional chemical exchange
spectra were performed using the same pulse sequence as in NOESYPH (16) to assign the obscured aliphatic OH resonance. In the
spectrum, the positive cross-peaks were observed due to the exchange
among the hydroxyl groups. Two-dimensional H-C
inverse correlated experiments with heteronuclear multiquantum
coherence (HMQC) (17) were performed at 500 MHz (for H) using the inverse detection probe with the acquisition
parameters: 90 ° and 180 ° pulses for H, 8.4 and
16.8 µs, and for C, 12.2 and 24.4 µs; decoupling
high power, 2 dB; decoupling low power, 18 dB; J
value delay time, 3.57 ms; delay for bilinear rotation decoupling
inversion pulse, 0.35 s; increment, 9 µs; number of scans, 8; for H, SI2 = 1024k, TD2 = 512w, SW =
4716.98 Hz; for C, SI1 = 4096k, TD1 = 1024k,
SW = 220.85 ppm. Simulations of one-dimensional H
NMR spectra were performed with Bruker's LAOCOON-based PANIC
software (18) using procedures that we have described
elsewhere(13, 19) .Computer Molecular Modeling
Molecular modeling and
graphic display were performed on an 4D/70GT IRIS Silicon Graphics
workstation using the Biosym InsightII/Discover molecular modeling
package(21) . The structure of CP-55,940 was built from the
molecular model of -THC, which was initially generated
from the x-ray crystallographic data of
-tetrahydrocannabinolic acid (22) and
energy-minimized using the Biosym program. The minimum energy
conformation of -THC was then used as a starting
template on which the structures of CP-55,940 were generated by
deletion and/or addition of atoms at standard bond lengths and bond
angles under the Biosym software.
and
, in Table 3), the C-ring, and the Ph-OH, which
we found to be similar to that of its earlier studied congener
CP-47,497. To avoid a formation of cis or gauche segments in the DMH side chain and to evade possible chair-boat interconversions in the cyclohexyl ring, a
torsional restraint of 100 kcal/rad
was added to the
corresponding torsion angles in those regions. This effectively
eliminated unnecessary trans-cis or chair-boat conversions. A torsional force was also applied into our dynamics
strategy to restrict the orientation of the phenolic OH on the basis of
the NOE data in which the Ph-OH proton faces the adjacent aromatic H-2
proton. Such an external torque about the specific dihedral angles
tends to force the calculation toward certain restrictions during
dynamics sampling, thus biasing the molecule to the region of interest.
Dihedral drive techniques (13) were performed to calculate
rotational energy barriers with intervals of 5 ° for one-bond
rotation and 10 ° for two-bond rotation. Finally, an additional
torsion force (200 kcal/rad) was applied to restrain the
rotated dihedral angle when applying energy minimization to relax the
whole molecule.
NMR Spectral Assignments
The spectral
assignments of CP-55,940 were initially made by analogy with the
chemical shifts of CP-47,497 reported elsewhere (13) and then
specifically assigned on the basis of the coupling connectivities in
the H-H COSY spectrum and confirmed by the H-C HMQC spectrum. The full and expanded
scales of one-dimensional NMR spectrum are shown in Fig. 2. The
assignments of the cyclohexyl protons were achieved on the basis of
integrated chemical shifts and analysis of the expanded regional
contour plot of the DQF-COSY spectra. A logical starting point is the
resonance of H-9a at 
3.74 ppm, which shows vicinal coupling to
H-10e, H-8e, H-10a, and H-8a. We could assign the proton signals of
H-10e and H-10a at 2.09 ppm and 1.38 ppm with the support
of the cross-peak connectivities of H-9a with H-10a, H-8a, H-10e, and
H-8e. The COSY spectrum (Fig. 3) clearly shows three components
(H-10e, H-8e, and H-11e) under the multiplet at 2.06 ppm, in which
three related strong geminal J couplings, H-10e/a
(F1 = 2.09, F2 = 1.38 ppm), H-8e/a (F1 = 2.05, F2
= 1.53 ppm), and H-11e/a (F1 = 1.98, F2 = 1.13
ppm) can be discerned. In order to confirm the assignments made above,
two-dimensional heteronuclear H-C correlated
experiments were performed to gain information about the H-C
connectivities. Fig. 4shows the two-dimensional H-C HMQC spectra with enhancement of C sensitivity. The cross-peaks serve to identify the H-C
connectivities. The complete assignments of proton and carbon chemical
shifts for CP-55,940 are summarized in Table 1. While comparing
the above assignments with those of CP-47,497(13) , we found
that most of the proton resonances of CP-55,940 are very similar to the
corresponding ones of CP-47,497. Variations, however, could be seen in
the proton chemical shifts of the cyclohexyl ring of CP-55,940 due to
the presence of the hydroxypropyl side chain. Our two-dimensional H-H COSY spectrum (Fig. 3) also showed
that each of the 2"-CH methylene protons had cross-peaks
with the 3"-CH methylene protons. The separation of the two
cross-peaks is about 0.28 ppm, indicating that the two protons of
2"-CH are nonequivalent (see ``Discussion'').
H spectrum of CP-55,940 in CDCl at 298 K in
full and expanded scales.
solution at 298 K.
H-C inverse correlation spectrum (HMQC) of
CP-55,940 in CDCl at 300 K (C one-dimensional
external spectrum is not displayed at F1 dimension). A, the
downfield region showing the aromatic resonances. B, the
expanded scale of the upfield region showing H-C coupling for the aliphatic
resonances.
Coupling Constant Measurements
Coupling constants
were measured and refined using a method described
elsewhere(13) . J and J values were first approximated from the two-dimensional DQF-COSY
spectra, which offered a better resolution of cross-peaks(25) .
The chemical shift values (Table 1) and approximated J values were then used as the starting point for an iterative
simulation of the subspectra thus allowing us to refine J and J values (Table 2). J values so obtained were then
incorporated into the Karplus equation (18) as we have
described elsewhere(13, 19) to calculate the
corresponding dihedral angles (Table 2), which indicated a
typical chair conformation of the C-ring.
NOE Interactions
NOE is one of the most important
NMR parameters used in conformational analysis because the magnitude of
the NOE is inversely proportional to the sixth power of the interproton
distance in space (I
r
). Typically, an observed NOE cross-peak
indicates that the two protons are near in space within a distance of
3.0 Å and exhibit through space coupling with each other. Beyond
the 3.0 Å range, the NOE is very weak, and the effect becomes
barely detectable at 4.5 Å(26) . To obtain the NOE values
for the protons of CP-55,940, the two-dimensional H-H phase-sensitive NOESY spectrum was obtained (Fig. 5). Our data show an NOE cross-peak between the phenol
hydroxyl proton ( 5.28 ppm) and the adjacent aromatic H-2 proton
( 6.68 ppm), indicating that these two protons are spatially near
and coupled through a dipole-dipole interaction. As will be discussed
later, this result is used to assign the orientation of the phenolic OH
proton. Another two NOE cross-peaks were identified as being due to
spatial coupling between H-5 with H-8a and H-12a, indicating that H-5
is near H-8a and H-12a. Such cross-peaks provide important information
regarding the preferred orientation of the A-ring with respect to the
C-ring as shown in Fig. 5. The NOE cross-peak pattern that was
observed between the DMH side chain and the aromatic ring protons (Fig. 5) indicates that the conformation of the DMH chain of
CP-55,940 is very similar to one observed for CP-47,497, in which the
DMH side chain is almost perpendicular to the plane of phenolic
ring(13) .
at 298 K. The NOE interactions for CP-55,940 are
indicated with arrows.
Computational Results
Molecular dynamics/mechanics
was used to search for the preferred conformations of CP-55,940 and to
examine the possibility of intramolecular H-bonding between hydroxyl
groups. The main advantage of using molecular dynamics is that it
simulates molecular motion at high temperature, thus increasing the
probability of inducing conformational transitions past any possible
high energy barriers. This would ensure sampling of all possible minima
while avoiding the risk of a situation in which the molecular mechanics
calculation might lead to a local rather than the global energy
minimum. Dynamics simulations were performed with time steps of 1 fs
for 300 ps. The data were recorded at 1-ps intervals, and a total of
300 frames of conformers were sampled. Molecular mechanics energy
minimizations were carried out for each of the 300 conformers while
releasing all torsional angle constraints. This operation resulted in a
convergence of these conformers into several families. Of these
families, the six energetically lowest are shown in Fig. 6.
Conformers II, III, IV, V, and VI allow for intramolecular H-bonding
between the hydroxypropyl and the phenolic hydroxyl groups if we assume
that hydroxyl groups separated by approximately 2 Å will form a
H-bond. Such H-bonding imparts greater stability to each of the five
conformers. The most stable of these is conformer IV, which is also the
global energy minimum. Conformer II, with the Ph-OH/3"-OH hydrogen bond
on the opposite face of the molecule, has only 0.28 kcal/mol higher
energy than that of conformer IV. The summary of the calculated
structural features of CP-55,940 is given in Table 3.
Further Investigation of Intramolecular
H-Bonding
Intramolecular H-bonding is among the most important
interactions in biological molecules and is an internal cohesive force
that can play an important role in determining the geometry and mode of
recognition and association of biological molecules(27) . In
many instances, the presence of one or more H-bonds can be critical in
determining the more stable conformer. Experimentally, the presence of
H-bonding can be determined using solution NMR
spectroscopy(28) . NMR experiments designed to study
intramolecular H-bonding generally rely on the premise that
intramolecular H-bonding is less concentration-dependent than
intermolecular H-bonding. In an intramolecular H-bond system, the
proton participating in the hydrogen bond becomes more positive in its
electronic character. As a consequence, this proton is deshielded
compared with a nonhydrogen bonded proton, causing its signal to shift
to a downfield in the NMR spectrum(28) . This is usually the
basis for measuring H-bonding using H NMR.H spectrum of CP-55,940 in CDCl (Fig. 2), thus preventing a concentration dependence
study. We approached this problem by obtaining the spectra in different
solvents and with the use of two-dimensional exchange experiments. The
two-dimensional exchange spectrum had two strong positive cross-peaks
generated through chemical exchange among the three OH groups and
appearing at F1 = 1.55, F2 = 5.08 ppm and F1 =
1.24, F2 = 5.08 ppm, respectively. Differentiation of these two
sets of positive cross-peaks could be accomplished by slicing out the
cross-section plot of the two-dimensional exchange spectrum along the
F2 dimension to obtain three positive peaks (Fig. 7). Of these,
the most downfield positive peak at 5.08 ppm is due to the
phenolic OH proton, whereas the most upfield situated peak centered at
1.24 ppm is due to the 3"-hydroxypropyl OH proton appearing as a
triplet due to scalar coupling with the adjacent methylene protons (Fig. 7A). The peak at 1.55 ppm was readily
assigned to the cyclohexyl 9-OH, whereas the peak at 1.64 ppm was
shown to be due to an impurity in the commercial CDCl solvent. The use of the two-dimensional H-H exchange experiment thus allowed us to
overcome the difficulty in identifying the two aliphatic OH peaks that
overlapped with the methylene peaks in the one-dimensional
spectrum.
H chemical exchange spectrum of CP-55,940
in CDCl at 298 K with the varied concentrations of 0.0026 (A), 0.026 (B), and 0.05 M (C). The
negative peak at 6.67 ppm is the NOE peak attributed to the
dipolar coupling of H-2 with Ph-OH. The asterisk indicates the
peak at 1.64 ppm that is due to an impurity from the CDCl solvent).
H chemical shifts of all three OH
resonances. The results showed that the broad Ph-OH singlet at
5.08 ppm was the most concentration-dependent with a concentration
coefficient of 10.8 ppm/mol; the 9-OH proton had a coefficient of 5.01
ppm/mol, whereas the 3"-OH triplet showed only a modest shift with a
concentration coefficient of 2.3 ppm/mol. These results may be
interpreted to mean that the 3"-OH proton is possibly engaged in
intramolecular H-bonding with the phenolic OH and is thus less prone to
chemical exchange or intermolecular H-bonding, whereas the phenolic OH
hydrogen is more available for such interactions.
Orientation of the Phenolic OH
The observed NOE
cross-peak between the phenolic hydroxyl proton and the adjacent
aromatic H-2 indicates that these two protons are spacially near each
other and thus coupled through a dipole-dipole interaction (Fig. 5). Such a result implies that in its preferred
conformation, the Ph-OH proton points away from the cyclohexyl ring and
toward the H-2 proton. This result was also supported by our
computational data showing that the phenolic O-H-bond lies in the plane
of the aromatic ring and is oriented toward H-2. These findings are
consistent with earlier results with the classical cannabinoid
-THC (19, 29, 35) and the
nonclassical cannabinoid prototype CP-47,497(13) .Orientation of the DMH Chain
The similar NOE
cross-peak pattern observed as in the case of CP-47,497 (13) implies the possibility of several interconverting DMH
side chain conformers that are time averaged on the NMR time scale.
This conclusion was confirmed by our molecular modeling studies, which
showed that the DMH side chain of CP-55,940 has an equal probability of
existing in one of four minimum energy conformations, having a
dihedral angle of 60 °, -60 °, 120
°, and -120 °, respectively, and equal
to 60 ° and -60 °, respectively (Table 3). In all
the above conformations, the DMH side chain is almost perpendicular to
the plane of the phenyl ring. In these conformations, the 3`-CH protons of the DMH side chain are located either above or below
the plane of the aromatic ring. This conformational feature would
account for the observed upfield shift (0.16 ppm versus 4`,
5`) of the 3` protons, which can thus be attributed to the shielding
effect of the phenyl ring.Relative Orientation of the A- and C-rings
Two NOE
cross-peaks were assigned to the spatial coupling of H-5 with H-8a and
H-12a. Such cross-peaks are congruent with a calculated preferred
conformation in which the planes of the two rings are almost
perpendicular to each other and the Ph OH bond points down in the same
plane as the axial H-7 and toward the 
-face of the cyclohexyl ring (Fig. 5). 7.02 ppm) and
H-7a ( 2.72 ppm) protons. The presence of such an additional weak
peak suggests the existence of two rotamers, a major one in which the
phenolic OH is positioned toward the -phase of the C-ring
( 
60 °) and a minor one in which the OH
group faces up toward the -face of the C-ring ( 120°). The calculated relative energy difference between
the above two conformations was found to be only 0.34 kcal/mol. The two
rotamer populations interpreted on the basis of the extra NOE
cross-peak is also congruent with the calculated high rotational energy
barrier of CP-55,940 (20.5 kcal/mol), whereas the value for CP-47,497
is 9.7 kcal/mol, and no minor NOE between H-5 and H-7a was observed.
Rotation about the C7-C6 bond on the NMR time scale was also indicated
by a broad peak for H-7a with hardly discernible splitting in the
one-dimensional H NMR spectrum (Fig. 2) at room
temperature. When the spectrum of CP-55,940 was obtained at a higher
temperature (345 K), the peak for the H-7a resonance became a sharper
and narrower triplet of triplets as in the case of CP-47,497, which
indicates a faster rotational motion about the C7-C6 bond of CP-55,940
at a high temperature. Such an interpretation may also explain the
apparent disappearance of the aromatic carbon C5 signal in the
one-dimensional C proton-decoupled broad band spectrum
even when a delay as long as 120 s was used. Furthermore, the C5 carbon
did not appear in the distortionless enhancement polarization transfer
spectra and was not represented by a cross-peak in the hetero-COSY
spectra. It was only with the help of a H-C
inverse detection HMQC spectrum, which is normally 10-100 times
more sensitive than a conventional C-detected hetero-COSY
spectrum(17) , that a cross-peak due to 5-CH could be observed.
We attribute the low intensity of the C5 resonance in the C spectrum to a broadening of this peak to the high
rotational barrier around the C
-C
bond. The
detailed investigation will be published elsewhere.Conformation of the Southern Hydroxypropyl
Chain
According to the one-dimensional and two-dimensional COSY H spectra of CP-55,940, the 3"-CH protons are
nonequivalent. This is demonstrated by the features of the 3"-CH multiples at 3.47 ppm in the one-dimensional spectrum (Fig. 2) and the presence of two cross-peaks due to coupling of
each of the 3"-CH protons with the vicinal
2"-CH. The above two spectra clearly show that the two
3"-CH protons have different chemical shifts that differ by
= 0.05 ppm. Theoretically, the two 3"-CH protons should be equivalent because the low rotational energy
barrier would allow the C2"-C3" bond to rotate freely and average out
between the three rotamers. Therefore, the above observations imply
some rotional restriction around the C2"-C3" bond, possibly
because of hydrogen bond formation between the 3"-OH and Ph-OH. Such a
postulation is congruent with the earlier data regarding the
spectroscopic properties of the three hydroxyl groups in CP-55,940. protons because the
ten-membered H-bonded ring would prevent free rotation in the
hydroxypropyl chain. Our computational studies also revealed other
conformers, especially conformer II in Fig. 8. Conformer II,
differing from the IV by 0.28 kcal/mol, has all three hydroxyl groups
pointing toward the same side of the molecule. Based on earlier (30, 31) studies from our laboratory, we postulate
that a conformation such as II would be favored when these compounds
partition into the cellular membrane because it would allow all three
hydroxyls to interact with the polar side of the bilayer at the
interface, whereas the other nonpolar parts of the molecule are
associated with the hydrophobic bilayer chains. Such a conformer shares
several stereochemical features of -THC, whose
preferred conformation is depicted in Fig. 8(19, 22, 29, 35) . As
can be seen, the A- and C-rings of CP-55,940 can be superimposed on
those of -THC if the plane of its C-ring is rotated by
75 °, a process that requires an expenditure of 23 kcal/mol in
energy as described elsewhere(13) . The approximately 2 orders
of magnitude higher potency of CP-55,940 when compared with
-THC can then be attributed to the additional
structural features present in this molecule. They include a longer
chain length, two ,-dimethyl substituents in the side chain,
the presence of a 9-hydroxy, and a southern aliphatic
hydroxypropyl group. All of these additional pharmacophores are
expected to enhance the affinity of CP-55,940 for the cannabinoid
receptor.
-THC. The conformation of
-THC was generated from the x-ray crystallographic
data of -tetrahydrocannabinolic acid (22) and
energy-minimized using the Biosym program.
Conclusions
In this study, the high resolution NMR
experiments provided detailed information on the conformation of
CP-55,940 in solution and allowed us to identify a major preferred
conformer as well as the presence of a minor rotamer. Molecular
modeling studies confirmed that the preferred conformation in solution
was indeed the low energy conformer but also suggested at least one
other low energy conformer that may be relevant to biological activity. -methyl substitution may be at least in part associated with
the respective enforced conformational properties of the DMH side
chain. Our results also show that the most energetically favored
conformation for CP-55,940 is the one with Ph-OH pointing down toward
the -face of the C-ring. This conformation is stabilized through
the formation of an intramolecular H-bond between the southern
hydroxypropyl group and the Ph-OH as shown in Fig. 8(IV).
However, this energetically most favored conformer does not necessarily
represent the preferred conformation at the active site. In this
regard, our studies showed that another almost equienergetic conformer
(II in Fig. 8), differing from IV by only 0.28 kcal/mol, may
represent the pharmacophoric conformation. We might postulate that
because of its amphipathic properties, CP-55,940 incorporates into
biological membranes in its pharmacophoric conformation and assumes an
orientation that allows all three hydroxyl groups to face the polar
side of the bilayer, whereas in the bilayer, the cannabinoid ligand
undergoes lateral diffusion and approaches the cannabinoid receptor in
an orientation highly favorable for a productive collision with its
binding site. This general hypothesis for the ligand-membrane-receptor
systems has been discussed elsewhere (31, 32, 33) and is diagrammatically
represented for CP-55,940 in Fig. 9. Currently, we are carrying
out the further investigation of molecular dynamic and conformational
properties for this ligand in a model membrane system.
)-THC,(-)--tetrahydrocannabinol;
NCC, nonclassical cannabinoids; Ph-OH, phenolic hydroxyl; C-ring,
cyclohexyl ring; A-ring, phenol ring; COSY, correlation spectroscopy;
DQF, double quantum filter; NOESYPH, phase-sensitive H-H nuclear Overhauser enhancement
spectroscopy; HMQC, heteronuclear multiquantum coherence; DMH,
1,1-dimethylheptyl; NOE, nuclear Overhauser effect; H-bond, hydrogen
bond.
We thank Dr. Susan S. Pochapsky for professional
support.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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