(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
-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) .
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. -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.
Figure 1:
Evolution in cannabinoid structures with
progressively enhanced potencies from the naturally occurring
-THC toward the potent synthetic
analogs.
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
-THC(8, 10) . In an earlier
publication(7, 13) , we reported preliminary results
on the conformational properties of prototype CP-47,497.
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, 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.
Figure 2:
500
MHz H spectrum of CP-55,940 in CDCl
at 298 K in
full and expanded scales.
Figure 3:
Expanded scale of a 500 MHz
two-dimensional COSY-PH-DQF spectrum of CP-55,940 in CDCl solution at 298 K.
Figure 4:
500 MHz two-dimensional 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.
Figure 5:
500 MHz NOESYPH spectrum in
CDCl at 298 K. The NOE interactions for CP-55,940 are
indicated with arrows.
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 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.
Figure 7:
Cross-sections parallel to the F2
dimension sliced through the phenolic hydroxyl resonance in the 200 MHz
two-dimensional 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).
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 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.
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 ( 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.
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 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 2
3 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.
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 -THC. The conformation of
-THC was generated from the x-ray crystallographic
data of
-tetrahydrocannabinolic acid (22) and
energy-minimized using the Biosym program.
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 -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.
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.