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Recent Patents on CNS Drug Discovery, 2009, 4, 112-136
Naturally Occurring and Related Synthetic Cannabinoids and their
Potential Therapeutic Applications
Ahmed M. Galal1,*, Desmond Slade1, Waseem Gul1,5, Abir T. El-Alfy2, Daneel Ferreira1,3 and
Mahmoud A. Elsohly1,4,5
1
National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS
38677, 2Department of Pharmacology, School of Pharmacy, The University of Mississippi, University, MS 38677,
3
Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, University, MS 38677,
4
Department of Pharmaceutics, School of Pharmacy, The University of Mississippi, University, MS 38677, and 5ElSohly
Laboratories, Inc., 5 Industrial Park Drive, Oxford, MS 38655
Received: October 9, 2008; Accepted: November 3, 2008; Revised: November 17, 2008
Abstract: Naturally occurring cannabinoids (phytocannabinoids) are biosynthetically related terpenophenolic compounds
uniquely produced by the highly variable plant, Cannabis sativa L. Natural and synthetic cannabinoids have been
extensively studied since the discovery that the psychotropic effects of cannabis are mainly due to !9-THC. However,
cannabinoids exert pharmacological actions on other biological systems such as the cardiovascular, immune and
endocrine systems. Most of these effects have been attributed to the ability of these compounds to interact with the
cannabinoid CB1 and CB2 receptors. The FDA approval of Marinol®, a product containing synthetic "9-THC
(dronabinol), in 1985 for the control of nausea and vomiting in cancer patients receiving chemotherapy, and in 1992 as an
appetite stimulant for AIDS patients, has further intensified the research interest in these compounds. This article reviews
patents (2003-2007) that describe methods for isolation of cannabinoids from cannabis, chemical and chromatographic
methods for their purification, synthesis, and potential therapeutic applications of these compounds.
Keywords: Cannabis sativa L, cannabinoids, phytocannabinoids, pharmacological actions, CB1 and CB2 receptors, isolation,
purification, synthesis, therapeutic applications.
INTRODUCTION
Table 1.
Cannabinoids are terpenophenolic secondary plant
metabolites uniquely found in Cannabis sativa L. [1].
Biogenetically they are derived from a mixed origin, with the
3-alkylphenol moiety originating from a polyketide precursor and the tetrahydroisochroman moiety from a monoterpene residue [2]. Cannabinoids found in cannabis are
designated as phytocannabinoids or exogenous cannabinoids
to distinguish them from the eicosanoid endocannabinoids, a
group of arachidonoyl esters and amides that were first
discovered in 1988 in mammalian tissues acting as endogenous cannabinoid receptor ligands with neuromodulatory
action [3].
Cannabis is divided mainly into three phenotypes or
chemotypes: Phenotype I (drug type), with (-)-trans-(6aR,
10aR)-!9-tetrahydrocannabinol (!9-THC) (1) > 0.3% and
cannabidiol (CBD) (2) < 0.5%; an intermediate phenotype II
(intermediate type), with (2) as the major cannabinoid but
with (1) also present at various concentrations; and
phenotype III (fiber type), with especially low (1) content.
The rare phenotypes IV and V have low (1) and (2) content
and high cannabigerol (CBG) (3) content, and undetectable
amounts of any cannabinoid, respectively (Table 1) [4]. All
analyses are based on plant inflorescence dry material.
Although environmental factors play a role in the amount of
cannabinoids present in different parts of the plant at
*
Address correspondence to this author at the National Center for Natural
Products Research, School of Pharmacy, The University of Mississippi,
University, MS 38677, USA; Tel: +1-662-915-5928;
Fax: +1-662-915-5587; E-mail: [email protected]
1574-8898/09 $100.00+.00
Cannabis Phenotypes
Phenotype
(1)
(2)
(2):(1)
(3):(2)
I
Drug
0.5-15%
0.01-0.16%
< 0.02
~ 0.5
II
Intermediate
0.5-5%
0.9-7.3%
0.6-4
~ 0.1
III
Fiber
0.05-0.70%
1.0-13.6%
>5
~ 0.05
IV
CBG
< 0.05%
< 0.5%
-
> 0.5
V
Noncannabinoid
0
0
-
-
different growth stages [5], the tripartite distribution of
(2):(1) ratios in most populations (phenotypes I to III) are
under genetic control [4c].
Not only is the isolation and purification of cannabinoids
challenging owing to the structural, physical and chemical
similarity of these compounds, but synthetic routes are
equally demanding due to low yields and the formation of
by-products while the final products are typically noncrystalline. The critical step in the majority of synthesis
routes yielding (1) [in cannabinoid parlance, synthetic (1) is
called dronabinol] is the condensation of a monoterpene with
a resorcinol derivative such as olivetol (5-pentyl-1,3benzenediol). Presently, condensation of (+)-p-mentha-2,8dien-1-ol with olivetol is used to produce commercial (1). In
addition, the thermodynamic instability of (1) must always
be considered during isolation, synthesis and storage, since
© 2009 Bentham Science Publishers Ltd.
Cannabinoids and Their Therapeutic Applications
this labile compound readily converts to the more stable
regioisomer, !8-THC (4a) [1].
The pharmacological effects of (1) have been unraveled
since the discovery of the endocannabinoid (endogenous
cannabinoid) system, which consists of endogenous ligands
(endocannabinoids), cannabinoid receptors and enzymes
involved in biosynthesis and degradation of the endocannabinoids. The system affects a variety of physiological
processes, e.g. appetite, pain-sensation, mood and memory
[6].
Endocannabinoids are capable of binding to and
functionally activating cannabinoid receptors. At least two
cannabinoid receptors, namely subtype CB1 and CB2, have
been identified, including their primary structure, ligandbinding properties and signal transduction systems. CB1 and
CB2 receptors belong to the large superfamily of G-protein
coupled receptors (GPCR) that couple to guaninenucleotidebinding proteins (heptahelical receptors). Endocannabinoids, unlike classical neurotransmitters, are not
stored in intracellular compartments and act as neuromodulators. They are synthesized as needed on location by
cleavage of their membrane lipid precursors, followed by
release from the cell. Inactivation occurs via intracellular
hydrolyzing enzymes. CB1 receptors, expressed mainly in
the CNS, are responsible for psychoactive effects, while CB2
receptors are expressed in the immune system. CB1
receptors are also expressed by some non-neuronal cells, e.g.
immune cells and peripheral tissues and organs, e.g. heart
and blood vessels. Recent reports indicate that CB2 receptors
are also localized in the CNS, including spinal cord, brain
stem and cortex [7]. There is also mounting evidence for the
existence of additional non-CB1/CB2 cannabinoid receptors
[8], e.g. bioassays conducted with compounds devoid of
noteworthy CB1 or CB2 affinity are sensitive to CB1 or CB2
selective antagonists [6c].
Five different types of endogenous agonists, having
submicromolar affinity for the CB1 and CB2 receptors, have
been identified, namely anandamide (5a), 2-arachidonylglycerol (5b), noladin ether (5c), virodhamine (5d) and
N-arachidonoyldopamine (5e).
Cannabinoid receptor agonists can be divided into four
groups based on their chemical structures. The classical
cannabinoids are ligands with a tetrahydro-6H-benzo[c]
chromene (dibenzopyrane) structure, including phytocannabinoids, e.g. (1), and their synthetic analogs, e.g. HU210 (6).
This group lacks CB1/CB2 selectivity. The non-classical
cannabinoids are bi- and tricyclic analogs of (1) without the
pyran ring, e.g. (-)-CP55940 (7). This group binds to CB1
and CB2 receptors with similar affinity, while displaying
high in vivo activity. The aminoalkylindole cannabimimetic
group of agonists, e.g. (R)-(+)-WIN55212-2 (8) exhibits high
affinity for both receptors, but with CB2 selectivity. The
eicosanoid (arachidonic acid derivative) group includes (5a)(5e).
SR141716A (9), the first specific cannabinoid antagonist,
impedes the action of cannabinoid agonists in vivo at
nanomolar concentrations. It is CB1 selective, but not CB1
specific, blocking both receptors at sufficiently high doses.
SR141716A (9) and (-)-SR144528 (10), CB2 selective
Recent Patents on CNS Drug Discovery, 2009, Vol. 4, No. 2
113
antagonists, may also behave as inverse agonists, i.e., they
block the effects of endocannabinoids and cause an opposite
effect.
In vivo Testing of endocannabinoids produce behavioral
and pharmacological actions associated with other cannabimimetic ligands. Anandamide (5a) produces antinociception, hypothermia, hypomobility, and catalepsy in the mouse
tetrad model, with rapid onset of effects, but with a short
duration of action due to rapid uptake into neurons and
astrocytes and subsequent enzymatic degradation.
Pharmacological investigations of the other major
cannabinoids revealed that (2), a non-psychoactive cannabis
constituent, displayed marked antioxidant and antiinflammatory properties that could be utilized to provide
neuroprotection in acute and chronic neurodegeneration [9].
It also displayed antischizophrenic, antiepileptic [10], anxiolytic, sleep-promoting [9, 11], and potent immunomodulating properties [10]. CBG (3) is a partial agonist at both
CB1 and CB2 receptors [11]. (-)-Trans-!9-tetrahydrocannabivarin (!9-THCV) (11) has been shown to be a strong
antagonist of (5a), a neuromodulator found in animal and
human organs [12]. Some of the aforementioned biological
effects are apparently due to interactions with non-CB1/CB2
receptors [11]. Several 1-O-methyl- and 1-deoxy-"8-THC
analogs have high affinity for the CB2 receptor, but little
affinity for the CB1 receptor, e.g. (-)-trans-(6aR,10aR)-3(1,1-dimethylbutyl)-1-deoxy-"8-THC (JWH133) (12), (-)trans-(6aR,10aR)-3-[(2R)-2-methylbutyl]-1-O-methyl-"8THC (JWH359) (13) and trans-(6aR,10aR)-3-(1,1dimethylhexyl)-1-O-methyl-"8-THC (14). This is in line
with traditional cannabinoid structure-activity relationship
(SAR) requiring a free phenolic hydroxy at C-1 for CB1
receptor interaction [13].
It has been shown that synthetic !8-THCV (4b) and (11)
exhibit in vitro pharmacological properties similar to those
of natural (11), and that they can antagonize (1), the
CB1/CB2 receptor agonist, in vivo. It is, however, important
to realize that (4b) and (11) behave as agonists or antagonists
in a dose dependant manner [14]. As an antiemetic, (4a) has
activity equal to (1) [15]. Numerous synthetic analogs of (1)
have been developed and tested as CB1/CB2 agonists or
antagonists and for potential therapeutic benefits, with 1,1dimethylheptyl and 1,2-dimethylheptyl side-chain homologs
found to be several hundred times more psychoactive than
the natural compound [3].
Diseases of the nervous system are not only diverse, but
also result in approximately 9% of all human deaths [16].
The most widespread immunomediated disease of the central
nervous system (CNS) is multiple sclerosis (MS), with
patients developing inflammation that leads to demyelination
and neuronal dysfunction, resulting in serious clinical
symptoms [17a]. Currently, there is no successful treatment
available for these symptoms, however, clinical studies using
cannabinoids for controlling spastic pain, tremors and
nocturia have yielded promising results [18]. Cannabinoids
have been linked to modulation of neuroinflammation [19]
and are reported to regulate the neuronal and immune
functions [20]. Evidence supporting the role of cannabinoids
in treatment of CNS inflammatory diseases was found in the
regulation of glial cell function, while treatment of
114 Recent Patents on CNS Drug Discovery, 2009, Vol. 4, No. 2
Galal et al.
11
9
10
8
OH
10a
7
1
3
O
12
1'
2'
3'
5'
4'
4
R2
13
R1
R2
R1
6a
6
OH
OH
R1
HO
HO
(1) !9-THC: R1 = R2 = H
(2) CBD: R1 = H
(3) CBG: R1 = H, R2 = geranyl
(18a) !9-THCA A: R1 = COOH, R2 = H
(19) CBDA: R1 = COOH
(21) CBNRA: R1 = COOH, R2 = neryl
(22) CBGA: R1 = COOH, R2 = geranyl
(18b) !9-THCA B: R1 = H, R2 = COOH
OH
OH
OH
OH
O
OH
R1
O
OH
(4a) !8-THC: R1 =
(6) HU210
(7) CP55940
(4b) !8-THCV: R1 =
R1
O
O
NH2
OH
(5a) anandamide: R1 =
N
O
(5d) virodhamine: R1 =
H
OH
OH
O
O
OH
O
(5b) 2-arachidonylglycerol: R1 =
(5e) N-arachidonoyldopamine: R1 =
N
OH
H
OH
OH
O
OH
(5c) noladin ether: R1 =
O
(27) AM404: R1 =
N
H
experimental autoimmune encephalomyelitis (EAE) with
cannabinoids reduced its clinical signs (spasticity, tremors
and paralysis) [19].
Although the link between the nervous and immune
systems has been established in neurological diseases with
an immune element, it is difficult to recognize which system
is controlling the other. This is further complicated by recent
reports that the cannabinoid system is involved in the
regulation of both these systems [20]. There are many factors
that complicate understanding of the cannabinoid system,
e.g. the existence of substantial overlap in the specificity of
CB1 and CB2 ligands [8a] and the fact that many natural and
synthetic cannabinoids bind to both CB1 and CB2 receptors,
making it hard to unambiguously define the role of each
receptor in immune responses. A third factor is that the
pharmacological effects of cannabinoids depend largely on
the density and coupling efficiency of CB1 and CB2 receptors [11, 20]. Although the molecular properties influencing
the psychotropic activity of cannabinoids have been studied
[21], a major obstacle in developing cannabinoid-based
drugs is the difficulty in separating psychotropic from other
medicinally useful effects. Subsequently, the only
cannabinoid-type agent that has been marketed is synthetic
Cannabinoids and Their Therapeutic Applications
(±)-trans-11-nor-9-oxo-3-(1,1-dimethylheptyl)hexahydrocannabinol (nabilone) (15), which is used for the treatment
of chemotherapy-related nausea and vomiting [22].
The tremendous body of work established over the past
three decades on the pharmacology of cannabinoids is
indicative of the potential therapeutic use of these compounds. This has been matched by a steady growth in the
number of cannabinoid-type drugs in development from two
in 1995 to 27 in 2004, with focus on pain, obesity and MS
therapeutic agents [23].
Thus, although a wide diversity of cannabinoid pharmacological effects have been discovered, reviewing the
literature on cannabinoids and the endocannabinoid system
over the past decade leaves no doubt that much is still to be
understood. This review covers the isolation, purification,
synthesis and pharmacology of phytocannabinoids (natural
and related synthetic derivatives), as disclosed in patents
spanning 2003-2007. Patents dealing with cannabis and
cannabinoid formulations were not considered. Also, patents
focusing only on the chemistry of cannabis, including the
total synthesis of cannabinoids and cannabinoid related
compounds, were not considered. Typically, these patents
proposed numerous potential medicinal applications,
however, limited or no pharmacological data was given to
assert these claims.
In this review, the commonly used tetrahydro-6Hbenzo[c]chromene numbering system (sometimes referred to
as a dibenzopyrane system) will be employed in naming the
cannabinoids [see (1)] [24].
ISOLATION AND PURIFICATION
Introduction
Although numerous analytical chromatographic methods
are available that provide qualitative and quantitative
analysis of cannabinoids with baseline separation, preparative separation is much more problematic [25].
Recent Patents on CNS Drug Discovery, 2009, Vol. 4, No. 2
compounds and cause cell disruption, which, in turn, results
in extracts containing finely dispersed solid material.
Extraction of cannabis is typically achieved by organic
solvent (maceration or percolation) or SFE. As a first step,
the plant material, or in some cases the obtained extract, is
decarboxylated to convert all cannabinoid acids into their
neutral form, unless the acid form is the targeted product.
Decarboxylation of the cannabinoid acids in the plant
material, which is a function of time and temperature, is
performed as a multi-step process [28]:
1.
The first step involves exposure of the plant material to
temperatures of 100-110°C for 10-20 min. This step
removes water and allows for uniform heating of the
plant material.
2.
The second step involves heating at 115-125°C for 4575 min. Care must be taken to avoid thermal degradation
of (1) to cannabinol (CBN) (16).
SFE Fig. (1) comprises the use of supercritical fluids to
selectively remove analytes from solid, semisolid and liquid
matrices. A supercritical fluid is a substance at a temperature
and pressure above its thermodynamic critical point, causing
the interface between the liquid and vapor phases to
disappear and improving the solvating power (E°) of the
substance. This critical temperature (Tc) is the highest
temperature at which a gas can be converted into a liquid by
an increase in pressure, and the critical pressure (Pc) is the
highest pressure at which a liquid can be converted into a gas
by increasing the temperature. There is only one phase in the
critical region and it possesses properties of both a gas and a
liquid, e.g., high diffusivity, low viscosity and minimal
surface tension. Varying the extraction temperature and pressure allows for changing the selectivity of the supercritical
fluid [29].
A number of methods are available for obtaining
cannabinoids from plant material [26] or via synthesis [27].
However, these are time- and labor-intensive and generally
not suitable for preparative-scale isolations. This represents a
major obstacle in providing, especially minor, cannabinoids
in sufficient amounts for use as standards or for pharmacological evaluations.
Extraction of cannabinoids includes diverse methods, e.g.
Soxhlet and supercritical fluid extraction (SFE), reflux and
organic solvent extraction, while purification is achieved
through chromatography, activated charcoal treatment, filtration, distillation and chemical derivatization or combinations of these methods.
Extraction
The extraction of biologically active substances from raw
plant material is crucial in the development of natural
medicinal preparations. Improvements of traditional solvent
extraction methods (maceration, percolation, Soxhlet
extraction and steam distillation) include vortex technology,
rotary-pulsation, sonication, pressing, and squeezing. These
methods generally improve the overall extraction, but also
greatly enhance the extraction of high molecular weight
115
Fig. (1). Supercritical fluid extraction (SFE) diagram.
116 Recent Patents on CNS Drug Discovery, 2009, Vol. 4, No. 2
Galal et al.
Advantages of SFE include enhanced extraction efficiency, speed and selectivity due to its gas-like mass transfer
properties and liquid-like solubility properties, environmental benefits, extraction of analytes present in low
concentrations, cleaner extracts and preservation of bioactive
constituents. Disadvantages of SFE include high startup
costs, complicated system optimization, strong dependence
on matrix-analyte interactions and difficult scale-up [30].
Carbon dioxide (CO2) is a popular choice for SFE due to
its low cost, inert nature, low toxicity, non-flammability and
low critical temperature (Tc = 31.1°C) and critical pressure
(Pc = 1070.4 psi). The virtual absence of surface tension
from non-polar supercritical CO2 allows for improved
penetration into plant matrices compared to liquid solvents.
However, polar modifiers, such as ethanol, need to be added
when extracting polar compounds. This method has high
reproducibility and can remove heavy metals and pesticides
from the cannabis matrix. The polarity of subcritical CO2 is
similar to n-hexane, while that of supercritical CO2 is
comparable to toluene or ether.
Extraction of cannabinoids with CO2 is preferably done
under subcritical rather than supercritical conditions by
setting the temperature and pressure below Tc and Pc,
respectively. This provides a botanical drug substance (BDS)
containing active substances selectively from a complex
mixture of compounds as found in a botanical raw material
such as cannabis. Although the density, and therefore E°, of
subcritical CO2 is lower than supercritical CO2, selectivity is
enhanced for cannabinoids since only the most soluble
components are efficiently dissolved in the CO2. This allows
for selective extraction of the lipophilic cannabinoids by the
non-polar CO2, and implies that although supercritical
conditions might improve yields, subcritical conditions
provide much higher sensitivity. The high wax burden under
supercritical conditions indicates loss of selectivity, and
while precipitation at sub-zero temperatures (winterization)
can remove large amounts of wax, this process can be
troublesome as, e.g. the blocking of filters easily occurs.
Table 2.
Subcritical conditions lower the wax burden without
significant loss in cannabinoid yield (Table 2).
SFE conditions are typically as follows: Extraction is
done at ca. 10°C and 870 psi with a CO2 mass flow of 1250
kg/h for 8-10 hrs (60 kg marijuana). A wide variety of
conditions can, however, be employed to obtain the appropriate extract or, in some cases, decannabinized marijuana
for use as a placebo [31]:
1.
Supercritical fluids: CO2, carbon monoxide, ammonia,
nitrous oxide, ethanol, n-pentane, n-hexane, propane,
water, ethane, fluoroform and xenon.
2.
Organic modifiers: Ethanol, methanol, 2-propanol, diethyl ether, ethyl acetate, chloroform, dichloromethane,
carbon tetrachloride, acetonitrile, cyclohexane, acetone,
acetic acid, nitromethane, dioxane, n-hexane, n-pentane
and pyridine.
3.
Organic modifier concentration: 0-20% (v/w) of total
supercritical fluid used.
4.
Production of extract: 31-120°C, 1015-9862 psi, 0-24
hrs.
5.
Decannabinization of marijuana: 25-65°C, 5800-7252
psi, 0-24 hrs.
6.
Sub- or supercritical fluid flow rate: 20-50 mL/min (80
g marijuana).
7.
Plant material with a particle size 1-2 mm results in
improved extraction as packaging density is improved.
8.
CO2 flow rate is preferably measured in terms of mass
flow rather than by volume, since the density of the CO2
changes according to the temperature before entering the
pump heads and during compression.
Purification
Purification of cannabis BDS or extract includes techniques such as chromatography, removal of hydrocarbons,
SFE (CO2) Extraction of Cannabis
Extraction
Pressure
Temperature
Wax Removed
(1) After Winterization
Supercritical
5800 psi
60°C
6.8%
67.4%
Subcritical
870 psi
10°C
3.0%
65.0%
Fig. (2). Supercritical fluid chromatography (SFC) diagram.
Cannabinoids and Their Therapeutic Applications
waxes and other non-polar compounds (typically through
winterization), a two-solvent treatment, and filtration
through activated charcoal or florisil, or a combination of
these methods. Chromatographic techniques include
supercritical fluid Fig. (2) [32], reversed-phase (C18) and gel
filtration chromatography (Sephadex LH-20).
Supercritical fluid chromatography (SFC) has several
advantages, e.g., fast, high resolution separations, lower
operating temperatures, gas-like mass transfer and liquid-like
solvating properties.
The BDS or extract usually contains lipid-soluble
material, e.g., hydrocarbons, waxes, glycerides, unsaturated
fatty acids and terpenes, which can be removed through
winterization, followed by filtration or distillation [33].
The two-solvent treatment technique involves sequential
treatment of the extract with two solvents. The polarity of
the first and second solvent should be substantially different,
e.g., methanol and n-pentane. This facilitates the removal of
more and less polar compounds compared to the target
cannabinoid, respectively. The solvents can be used in either
order. This process can be applied to obtain any of the
cannabinoids in free or acid form, while selectivity may be
enhanced by selecting cannabis varieties high in the target
product.
Filtration through activated (porous) charcoal adsorbs
colored impurities in the extract, while filtration through
florisil, which is often used in the purification of pharmaceuticals, removes any residual solid material.
A typical extraction and purification protocol for
cannabinoids comprises the following steps [28, 33-35]:
Step 1: Decarboxylation of the plant material if neutral
cannabinoids are targeted.
Step 2: Solvent or supercritical fluid extraction of the plant
material yielding crude BDS.
Step 3: Winterization of the BDS to remove non-target
compounds.
Step 4: Chromatography of the winterized BDS.
Step 5: Dissolving the purified extract fractions in a first
polar/non-polar solvent, filtering any insoluble material, and
removing the solvent from the filtrate.
Step 6: Dissolving the filtrate in a second non-polar/polar
solvent, filtering any insoluble material and removing the
solvent from the filtrate to obtain a substantially pure
cannabinoid.
Step 7: Optional treatment with activated charcoal or florisil.
Step 8: Optional flash chromatography or recrystallization.
Step 9: Optional chemical derivatization and crystallization
The order of some of these steps is interchangeable,
while some applications do not employ all the steps.
Isolation and Purification Examples
A number of patents [28, 31, 34-35] utilized these steps
(vide supra), or variations thereof, to produce cannabinoid
enriched extracts (preparations) and purified cannabinoids.
Recent Patents on CNS Drug Discovery, 2009, Vol. 4, No. 2
117
Patent [34] describes the use of naturally occurring or
synthetic cannabichromene (CBC)-type compounds and
pharmaceutically acceptable derivatives thereof Fig. (3) in
the treatment of mood disorders such as depression. Extracts
rich in CBC (17) were prepared from cannabis varieties high
in (17) obtained via selective breeding techniques and
incorporated into pharmaceutical dosage forms [28, 33]. The
extract should contain (17) as 5-40% of the total cannabinoid
content. Step 4 in the abovementioned protocol uses
Sephadex LH-20 column chromatography (chloroform/
dichloromethane, 2:1, v/v) for purification, followed by the
two-solvent system (steps 5 and 6) (methanol/n-pentane) to
produce highly enriched (17) (> 98% w/w by TLC, HPLC or
GC).
OH
R1
O
R2
R1: H / COOH
R2: alkyl (C1-C8, branched or linear)
Fig. (3). CBC-type compounds.
Patent [28] describes methods of preparing high purity
cannabinoids (neutral or acids), cannabinoid preparations
and cannabinoid rich extracts from plant material via solvent
extraction, chromatography and recrystallization without the
use of preparative HPLC. A “substantially pure cannabinoid”
and an “enriched extract” are defined as having a chromatographic purity of > 95% and > 80%, respectively, as
determined by HPLC area normalization. Fresh cannabis
plant material contains (1) predominantly as two isomeric
carboxylic acid forms, namely !9-THC acid A (!9-THCA A)
(18a) and !9-THC acid B (!9-THCA B) (18b), with the
latter present in minor quantities. These non-psychoactive
acids are converted into the active (1) during storage and
exposure to heat.
Plant material (100 g) from a phenotype high in (1)
[present as (18) in the plant material] [(18) > 90% of total
cannabinoid content] was extracted with n-hexane/glacial
acetic acid (2 x 1500 mL, 99.9:0.1, v/v), followed by
filtration of the combined extracts and concentration in
vacuo to produce the crude BDS [28]. The crude BDS was
dissolved in the chromatographic eluent (chloroform/dichloromethane, 2:1, ca. 20 mL) and applied to a low pressure
glass column (1560 x 24 mm) packed with Sephadex LH-20
(400 g, stationary phase/sample, 30:1). The collected
fractions (50 mL each) were monitored by TLC. !9-THCA
(18) rich fractions were pooled to give crude (18), which was
sequentially dissolved in methanol and n-pentane, followed
by filtration to remove non-polar and polar compounds,
respectively. Removal of solvent produced (18) as a pale
yellow solid (ca. 5 g, 98% by area normalization).
Plant material (100 g) from a phenotype high in (1) was
decarboxylated at 105°C (15 min) and 145°C (55 min),
followed by SFE (CO2) (10 hrs, 870 psi, 10°C, CO2 flow at
1250 kg/hr) [28]. The crude BDS extract (3.5 g) was filtered
through a column of activated charcoal, fractionated on
118 Recent Patents on CNS Drug Discovery, 2009, Vol. 4, No. 2
O
O
N
Galal et al.
Cl
Cl
O
N
N
N
N
N
N
H
O
O
N
N
H
X
(9a) SR141716A: X = Cl (HCl salt)
(8) WIN55212-2
(10) SR144528
Cl
(9b) SR141716A: X = Cl (free base)
(29) AM251: X = I
OH
OMe
O
O
(11) !9-THCV
O
(13) JWH359
(12) JWH133
O
OMe
OH
OH
R1
O
O
O
(14)
(16) CBN: R1 = H
(15) nabilone
(23) CBNA: R1 = COOH
OH
O
OH
R1
OH
O
O
OH
O
(17) CBC: R1 = H
(20) CBCA: R1 = COOH
(24)
abn-!9-THC
(25) ajulemic acid
Sephadex LH-20 and the fraction rich in (1) sequentially
dissolved in methanol and n-pentane, followed by filtration
to remove non-polar and polar compounds, respectively.
Removal of solvent produced (1) as a semi-solid (1.5 g, 99%
by area normalization).
Patents [28] and [35] describe the isolation and
purification of (2) from plant material. Although (2) was
previously regarded as an inactive constituent of cannabis, it
is now considered an active compound with diverse
pharmacology [9], necessitating a selective process for its
purification. Cannabis varieties with high content of (2) (>
90% of total cannabinoid content) is particularly suitable for
this process. Cannabis plant material, obtained from a
cultivar with high content of (2), was harvested, dried, milled
to a particle size less than 2 mm and decarboxylated. SFE
(CO2) provided a crude BDS extract, which was winterized
(ethanol/BDS, 2:1 v/w, -20°C, 48 hrs), filtered (20 !m filter
followed by activated charcoal column) and the solvent
removed (rotary or thin film evaporation) to produce a (2)
rich extract. Recrystallization from n-pentane provided (2) (>
99% by area normalization) [28].
Patent [31] describes SFE (CO2) of marijuana to provide
compounds with medicinal value or decannabinized
marijuana (cannabinoid-free marijuana) for use as a placebo.
A wide variety of conditions were employed to provide the
decannabinized marijuana Table 3 under relatively mild and
reproducible conditions while maintaining its appea-rance,
color and texture, and lowering the content of (1) to below
Cannabinoids and Their Therapeutic Applications
Table 3.
Recent Patents on CNS Drug Discovery, 2009, Vol. 4, No. 2
119
SFE (CO2) Decannabinization of Marijuana
SFE #
Pressure
Temperature
Time
Marc: (1)
Extract: (1)
Extraction of (1)
SFE 1
1044 psi
31°C
0.5 hrs
0.24%
8.6%
93.1%
SFE 2
1044 psi
31°C
1.0 hrs
0.13%
17.4%
96.2%
SFE 3
1044 psi
31°C
3.5 hrs
0.23%
41.7%
93.2%
SFE 4
2176 psi
58°C
4.0 hrs
1.88%
36.7%
44.7%
SFE 5
5802 psi
31°C
4.0 hrs
0.14%
36.5%
96.0%
SFE 6
5802 psi
31°C
4.0 hrs
0.23%
40.1%
93.4%
SFE 7
5802 psi
51°C
5.0 hrs
0.22%
32.5%
93.7%
SFE 8
5802 psi
60°C
2.5 hrs
0.22%
28.6%
93.5%
SFE 9
6527 psi
45°C
5.0 hrs
0.31%
28.5%
90.9%
SFE 10
6527 psi
62°C
6.0 hrs
0.12%
38.2%
96.5%
SFE 11
6527 psi
60°C
5.0 hrs
0.14%
15.9%
95.9%
SFE 12
6527 psi
55°C
7.0 hrs
0.07%
-
97.9%
Flow: SFE 1-5 = 20 g/min, SFE 6-12 = 30 g/min.
0.5%. The marc obtained from SFE 11 was re-extracted
under similar conditions (SFE 12), but with the addition of
an organic modifier (ethanol). This improved the overall
extraction of (1) to 97.9%, yielding marc with only 0.07%
(1).
A number of patents utilized more specialized techniques, some in combination with the abovementioned
options, to provide cannabis extract or cannabinoids [36].
Patent [36a] describes the medicinal use of acidic
cannabinoids or extracts containing acidic cannabinoids,
including cannabidiolic acid (CBDA) (19), cannabichromenic acid (CBCA) (20), cannabinerolic acid (CBNRA)
(21), cannabigerolic acid (CBGA) (22), cannabinolic acid
(CBNA) (23), and derivatives thereof (Fig. 4). Solvent
extraction represents a suitable method for obtaining the
acidic cannabinoids [26a, 37]. This involves sequential
extraction with a non-polar phase (chloroform or n-hexane)
and a polar phase (methanol or ethanol), while taking
particular care to prevent decarboxylation (4-25°C working
conditions). Lyophilization is therefore the method of choice
for drying the extract. Flower tops obtained from cannabis
varieties were deep-frozen after harvesting, followed by
lyophilization (700 mg) and extraction (chloroform/
methanol, 1:9, 2 x 20 mL). The extraction procedure involves adding methanol (18 mL) to the plant material, sonication (5 min), addition of chloroform (2 mL), sonication (5
min) and extraction with a mechanical stirrer (60 min, 4°C,
250 rpm). The supernatant is removed and the procedure is
repeated. Pooling of supernatants yields the final extract
(Table 4).
Patent [36b] describes a preparative SFC process for
purifying natural or synthetic (1) utilizing a derivatized
polysaccharide solid chiral stationary phase immobilized on
substrates such as silica gel, alumina or ceramics. Com-
R5
R5
R1
R1
COOH
R4
O
COOH
R2
R4
HO
R3
R2
R3
R1, R2, R3: OH / H / alkyl (C1-C10, branched or linear)
R4: OH / alkyl (C1-C3, branched or linear)
R5: H / CnH2nOH / CnH2nCOOH (n = 0, 1 or 2) / alkyl (C1-C3,
branched or linear)
Fig. (4). Acidic cannabinoids.
Table 4.
Extraction of Various Cannabis Cultivars
Cultivar #
(13)
(1)
(2) + (14)
(11)
Cultivar 1
202
1.43
0.21
< 0.00005
Cultivar 2
184
1.14
0.16
< 0.00005
Cultivar 3
16
0.11
14.86
< 0.00005
Concentration of cannabinoids determined by LC/MS/MS (mg/g dry weight).
mercially available examples include Chiralpak AD, containing
amylose
tris(3,5-dimethylphenylcarbamate)
(ADMPC) coated on macroporous silica gel (10 !m), and
Chiralpak IA, containing ADMPC immobilized on macroporous silica gel (5 !m) (Diacel Chemical Co.) Fig. (5) [38].
Encapsulation of the derivatized polysaccharide, which is not
120 Recent Patents on CNS Drug Discovery, 2009, Vol. 4, No. 2
OR1
Galal et al.
since cannabinoids other than (18) are also extracted.
Production of (1) is achieved by converting the acid to a
carboxylate salt under basic conditions and extracting the
salt using a solvent that preferentially dissolves the salt
compared to the free cannabinoids, e.g., a basic aqueous
solution. This prevents the simultaneous extraction of
contaminants such as (2) and (16). The process consists of
the following steps:
O
R1 =
O
N
O
R1O
R1O
H
n
silica gel
Fig. (5). ADMPC coated on macroporous silica gel.
1.
Milled cannabis is extracted with a non-polar solvent,
e.g. heptane or n-hexane, providing extract rich in (18).
bonded to the substrate, may prevent conversion of (1) to
(4a). The mobile phase is a mixture of CO2 and one or more
modifiers such as ethanol, acetonitrile or dichloromethane
(CO2/modifier, 85:15 to 75:25, w/v). Optionally, a second
chromatographic step can also be employed wherein an
achiral stationary phase, e.g., 2-ethylpyridine siloxane
immobilized on a silica support Fig. (6), is used in
conjunction with a CO2/ethanol mobile phase (CO2/modifier,
95:5 to 90:10, w/v). This step is especially useful for the
removal of abnormal !9-THC (abn-!9-THC) (24) produced
during the synthesis of dronabinol (1) [27a, 36b]. The order
of the two chromatographic steps is not critical, however, it
is recommended to perform the achiral stationary phase step
first in order to prevent degradation of the ADMPC chiral
stationary phase. The SFC column dimensions vary from
0.5-50 cm diameter and 5-50 cm length, with particle size
between 5-50 !m. Separations are performed at 5-45°C at
elevated pressures (1160 to 4350 psi) and with 10-4000
g/min flow rates, depending on column size. UV detection is
especially suitable for cannabis constituents. In an example
of a scaled-up two-step purification of a crude synthetic
mixture, (1) was isolated with a purity of > 99% Table 5.
2.
The acid is converted to a sodium carboxylate under pH
control (pH 12.7-13.2) by extraction into an aqueous
dilute NaOH solution. A pH > 13.2 results in a three
layer system, with the carboxylate salt forming an oily
layer between the bottom aqueous and top organic
layers. A pH < 12.7 results in incomplete extraction of
the carboxylate salt and high levels of CBD phenolate in
the aqueous phase. Emulsion formation can be reduced
by adding NaCl (1%) to the extraction solvent.
3.
The salt is extracted into a third solvent, namely
isopropyl ether (IPE). Alternatively, chloroform, diethyl
ether or dimethyl ether can also be used.
4.
The salt dissolves preferentially in IPE compared to the
aqueous solution, while other impurities, such as CBD
phenolate, remain in the aqueous phase.
5.
The IPE solution is washed with aqueous NaOH/NaCl.
6.
The resulting solution is acidified with dilute HCl (pH <
3), followed by florisil treatment to remove any residual
solid material.
7.
Decarboxylation is achieved by refluxing the IPE
solution in aqueous NaOH.
8.
The obtained (1) is filtered through charcoal and concentrated to provide crude product, which is stored at
-20°C.
9.
The crude product is purified by reversed-phase
chromatography to provide (1) of high purity (99.7%).
MeO OMe
Si
O
Si
N
silica gel
Fig. (6). 2-Ethylpyridine siloxane immobilized on a silica support.
Table 5.
Purification of Dronabinol (1)
Stationary
Phase
Particle
Size
Support
CO2/ethanol
(1)
2-Ethylpyridine
siloxane
10 !m
Silica
92:8
95%
ADMPC
20 !m
Macroporous
silica
80:20
99.5%
Pressure: 1450 psi. Temperature: 25°C. Flow: 40 g/mL. Column: 25 x 2.1 cm.
Patent [36c] describes a method for producing (1) by
converting (18) found in cannabis extract to a sodium salt by
pH manipulation, followed by extraction into a polar solvent.
The purified (18) is subsequently converted to (1) and
optionally purified via esterification or chromatography.
Although extraction of cannabinoids under pH control has
been described [39], solvent selection can be problematic
Patent [36d] describes the preparation of cannabis
extracts utilizing time-sensitive selective partial extraction
by keeping the solvent in contact with the cannabis for less
time than is needed to reach an equilibrium of dissolved
cannabinoids in solvent. Shortening the solvent-cannabis
contact time during extraction allows for the preparation of
extracts low in non-therapeutic compounds and enriched in
target therapeutic compounds such as (1), found in the
glandular trichomes. The obtained extract contains less high
molecular weight tars and oils. The composition of the
extract may be varied by choice of solvent and extraction
time. The spectrum of solvents that are applicable include
non-polar solvents, such as heptane and n-hexane through
polar solvents such as ethanol and propanol. Extraction time
should be between one and 180 s. A 30 s extraction with
absolute ethanol provides 55-60% of the total soluble
material in the first pass. Mechanical disruption of the plant
material and agitation during extraction is not recommended
since these increase solubilization of non-therapeutic compounds. The extract can be further purified by chromatography, distillation and filtration. Cannabis (2.27 kg, dried,
Cannabinoids and Their Therapeutic Applications
Recent Patents on CNS Drug Discovery, 2009, Vol. 4, No. 2
of pain and spasticity symptoms associated with MS [43],
and the alleviation of neuropathic pain and migraine
headaches [44].
seedless, female flowering tops) was decarboxylated (5 min,
93°C) and ethanol (7.6 L, 200 proof) was added to the
material contained in a muslin cloth basket. Extraction time
was approximately 30 s with 75% solvent recovery, yielding
extract (225 g) after solvent evaporation. No data on the
potency of (1) thus recovered was given.
MS is among the most common neurological disorders in
young adults. The symptoms of the disease are primarily
caused by impairment in neuron impulse conduction due to
loss of myelin most commonly initiated by an autoimmune
response. The clinical symptoms of MS include muscle
spasms, pain, ataxia, tremors, weakness, paralysis, constipation, loss of bladder control and speech impediments.
These symptoms typically progress by age and have a high
impact on the patient’s daily life [43]. The use of cannabis in
the management of MS symptoms is documented in ancient
traditional medicine [45]. Recently, MS patients who have
been self medicating with cannabis reported improvement of
symptoms, e.g. pain, spasticity, tremors and depression [46],
triggering several clinical trials to evaluate the effectiveness
of cannabis and cannabinoids in treating MS symptoms [43].
In the majority of cases, administration of cannabinoids
(natural or synthetic analogs) resulted in improvement of
several symptoms of MS, particularly spasticity, muscle
pain, ataxia, tremors and bladder control [47]. The
improvements were principally documented by subjective
patient data. In some cases, objective test results supported
the improvement reported by patients [48]. However,
differences were observed in the actions of orally administered versus inhaled cannabinoids, which might be
explained by the variable absorption of orally administered
cannabinoids [47a, 49].
Chemical Derivatization
Patent [40] describes methods to produce stable
crystalline cannabinoid derivatives, e.g. tosylates. Crystallization is employed to produce derivatives with targeted
purity, while facile hydrolysis yields high purity cannabinoids (Scheme 1).
Patent [41] describes a method for separating tetrahydrocannabinol isomers (regio- and stereoisomers) via
crystallization of their carbamate or thiocarbamate derivatives (Scheme 2).
PHARMACOLOGY
Cannabinoid-based patent applications during 2003-2007
have, amongst others, addressed three aspects: 1) synthesis
of non-psychoactive compounds, with primary emphasis on
CB2 selective analogs, 2) synthesis of water soluble
derivatives of (1) for therapeutic applications and to enhance
bioavailability, and 3) pharmacological evaluation of
cannabinoids (natural and synthetic) for various therapeutic
applications. The pharmacological actions most commonly
explored included analgesic (pain), antidepressant, antiemetic, neuroprotection, glaucoma treatment and appetite
control.
Patent [50] describes the potential use of cannabis extract
or dronabinol (1) in MS patients. The 15 week study was
divided into four phases. The first phase (weeks 1-5)
constituted the dose-titration phase whereby patients
increased their daily intake of the study medication by one
capsule twice daily at weekly intervals. During the second,
or plateau, phase (weeks 6-13), the patients were maintained
on a stable dose, while in the third phase (week 14), the
Analgesic (Pain)
Despite the historical use of cannabis in relieving pain,
its therapeutic application as an analgesic was constrained by
reports of adverse effects [42]. The past decade has,
however, witnessed resurgence in the use of cannabis for
pain relief. The key applications considered are management
OTs
OH
OH
p-TsCl
CH2Cl2 / Et3N
O
(1) (impure)
K / t-BuOH
N2 / reflux
O
O
(1) (> 99% purity)
!9-THC tosylate
Scheme (1). Purification of (1) via its tosylate.
X
R1
O
OH
N
CH2Cl2 / Et3N
(1) (impure)
OH
H
R1-N=C=X
O
K2CO3
O
X = O: !9-THC carbamate
X = S: !9-THC thiocarbamate
Scheme (2). Purification of (1) via its carbamate/thiocarbamate.
121
EtOH / H2O
O
(1) (> 98% purity)
122 Recent Patents on CNS Drug Discovery, 2009, Vol. 4, No. 2
participants reduced their intake by one capsule twice daily
until they were completely off of the medication. At the end
of the last phase (week 15), the patients were subjected to
final assessment of the effectiveness of medication by
measuring the change in spasticity using the Ashworth score
[51]. While no significant improvement was observed in
spasticity, cannabis treatment caused significant improvement in mobility, and the patients reported an overall
improvement in symptoms of pain, sleep quality and muscle
spasms. A decrease in the incidences of MS relapses was
also observed in patients treated with either the cannabis
extract or dronabinol (1).
In addition to cannabinoid monotherapy, combined
treatment with (1) and (2) for five weeks was effective in
alleviating the central neuropathic pain associated with MS
[52]. A two-year follow-up study conducted as an extension
of the five week randomized trial was aimed at evaluating
the long term efficacy and tolerability of this combined
formulation [53], commercially available in the UK as
Sativex®. Patients titrated their dosage while maintaining
their existing level of analgesia and reported any adverse
effects they experienced. The study showed that combined
treatment with (1) and (2) was effective in pain relief up to
two years, with 92% of the patients reporting at least one
adverse effect, most commonly nausea and dizziness. Since
MS is regarded as a relapsing chronic inflammatory disease
of the CNS [54], the beneficial antiinflammatory effects of
cannabinoids, especially (2), could provide much needed MS
symptom relief.
Patent [55] describes the possible use of ajulemic acid
(25), a synthetic derivative of trans-(6aR,10aR)-11-nor-9carboxy-!8-THC (!8-THC-11-oic acid), for the management
of pain and inflammation in MS patients, as supported by a
lengthy review of experimental and clinical data.
The clinical benefits reported for the use of cannabinoids
in MS patients are supported by experimental studies in
animal models of MS [56]. Administration of (1) or (4a) in
rat or pig delayed the onset and reduced the severity of
clinical and histological signs of experimentally induced
autoimmune encephalomylitis (EAE). The involvement of
the CB1 and CB2 receptors in improving the symptoms of
MS exerted by cannabinoid agonists was studied by using a
mouse autoimmune model of MS [57]. The studies revealed
that the cannabinoid agonists (1) and (8) suppressed the
tremor and spasticity exhibited by mice. The effect was blocked by CB1 and CB2 selective antagonists, suggesting the
contribution of both receptor types in mediating these
actions.
Cannabinoids are also employed for alleviating severe
and chronic pain that is either centrally or peripherally
mediated. Patent [58] describes the potential use of a
“cannabis-based medicine extract” for the treatment of
peripheral neuropathic pain. The study showed that acute
administration of a cannabinoid-containing plant extract with
a (2)/(1) ratio of 24:1 was effective in relieving neuropathic
pain induced in animal models. Chronic constriction injury
(CCI) to the sciatic nerve was surgically induced in the
animals one week prior to cannabis extract administration.
Animals administered the cannabis extract showed a marked
decreased pain response in both thermal hyperalgesia and
Galal et al.
mechanical allodynia pain models. Similarly, repeated daily
administration of the cannabis extract resulted in effective
relief of the neuropathic pain in a CCI animal model. In both
cases, the plant extract was more effective than the
administration of either (1) or (2) alone.
Moreover, a follow-up patent [59] describes the results of
a six week, double blind, randomized, parallel group placebo-controlled study with the patients receiving cannabisbased medicinal extracts containing (1)/(2) (1:1) combined
with the regular analgesic drug prescribed to the patients.
The data revealed that patients administered the cannabis
extract along with their analgesic drug(s) showed a statistically significant improvement in their symptoms compared
to the patients treated with the analgesic drug(s) alone, and
proved to be a well tolerated and effective adjunct therapy,
particularly in patients unresponsive to existing analgesic
medications. An added benefit of cannabis-based therapy
revealed by the study was a significant improvement in the
patients’ quality of life as evidenced by an improved pain
disability index (PDI) and relief from sleep disturbances.
Further employment of the analgesic effect of cannabinoids has been described for the treatment of pain and
inflammation associated with arthritis. In a seven week,
multi-center, double blind, randomized clinical study, the
efficacy of cannabis-based medicine in relieving rheumatoid
arthritis associated pain was evaluated [60]. Using equal
amounts of (1) and (2), patients used an oromucosal spray to
deliver the medication and titrated the dose until the
optimum efficacy of pain relief was achieved. Data collected
from the study supported a therapeutic value of cannabinoids
in arthritis. Cannabis-based medicine caused significant
reductions in morning pain and disease activity score, and a
significant improvement in sleep quality, supporting the
potential use of cannabinoids for the management and relief
of arthritis symptoms.
Among the emerging therapeutic applications of cannabis
is the management of migraine headaches [61]. Migraine is
considered a serious public health issue that affects an
estimated 23 million Americans [62]. Despite the development of the serotonin 1D agonist, sumatriptan, in the
early 1990s, several problems are associated with its use,
e.g., poor oral availability, ineffectiveness during the “aura”
phase, cardiovascular side effects and frequent recurrence of
attacks. In addition, approximately 30% of patients taking it
discontinued its use due to lack of efficacy, headache recurrence, cost, and/or side effects [63]. A need for alternative
migraine treatment medications is therefore apparent.
Anecdotal reports have suggested the potential use of
marijuana for migraine headaches and despite the lack of
conclusive clinical data, experimental research studies have
shed some light regarding the potential role of cannabinoids
in migraine treatment. The cannabinoid agonists (5a), (7)
and (8) inhibit the 5-HT3 receptor-mediated current in rat
nodose ganglion neurons [64]. The role of this receptor in
emetic and pain responses has been well documented [65].
Additional evidence was provided by the finding that the
posterior ventrolateral periaqueductal gray (PAG) is an
important brain area for the antinociceptive action of
cannabinoids [66]. The PAG is the brain anatomic region
commonly thought to be involved in migraine generation
[67]. Patent [68] proposed to conduct a clinical study in
Cannabinoids and Their Therapeutic Applications
order to assess the use of dronabinol (1) for treatment of
moderate to severe migraine attacks. The proposed doses
were 1.2, 2.4 and 3.6 mg/kg to be delivered using a pressurized metered dose inhaler (MDI). However, no clinical
data were provided in the patent application.
Antidepressant
Mood disorders are among the most debilitating disease
groups, affecting approximately 9-20% of the population.
Currently, the principal medical treatment for depression
focuses on drugs that enhance the levels of brain
monoamines in accordance with the established monoamine
hypothesis of depression [69]. However, these drugs suffer
from major drawbacks, e.g. efficacy, onset of action and side
effects, necessitating the quest for new and improved
antidepressants [70]. It is well recognized that one of the
components of the complex experience elicited by cannabis
in humans is mood elevation [71]. The notion that these
mood-elevating properties of cannabis could be utilized to
treat depression was introduced in the mid nineteenth
century. Since then, evidence began to accumulate outlining
the role of the endocannabinoid system in the etiology and
treatment of depression, supporting the fact that many
patients report benefits from using cannabis to alleviate
depression [72]. However, the exact actions exhibited by
manipulation of the endocannabinoid system are still unclear
and confounded by findings that both the activation of
endocannabinoid transmission [73] and blockade of CB1
receptors exert antidepressant-like actions in established
animal models of depression, e.g. the forced swim and tail
suspension tests [74].
Direct enhancement of CB1 receptor activity by
administration of the CB1 agonists (6) or oleamide (cis-9octadecenamide) (26) resulted in antidepressant-like effects
in animal models comparable to the tricyclic antidepressant,
desipramine [73b]. Indirect stimulation of the CB1 receptors
by administration of the uptake inhibitor AM404 (27) also
caused potent antidepressant effects. Inhibition of fatty acid
amide hydrolase enzyme (FAAH) by administration of
URB597 (28) leads to potent antidepressant-like action in the
rat forced swim test and the mouse tail suspension test [74a],
emphasizing the role of the endocannabinoid system as a
potential target for the management of depression. This
hypothesis is, however, in conflict with the findings that
blockage of the CB1 receptors leads to antidepressant-like
actions in animal models, since administration of the CB1
receptor antagonists AM251 (29) and SR141716A
(rimonabant hydrochloride) (9a) elicited antidepressant
effects in mice [74b, 75]. In accordance with these findings,
several studies reported neurochemical changes induced by a
CB1 receptor antagonist that correspond to antidepressant
action. These changes include enhanced efflux of noradrenaline, 5-hydroxytryptamine and dopamine in various brain
regions [76].
In support of the role of cannabinoids for the treatment of
depression, two patents [34, 77] described the potential
antidepressant-like actions of (3) and (17) in the rodent tail
suspension test. Data provided showed significant dose
dependent enhancement of mice activity in the test as well as
an increase in the force of struggling behavior as compared
to the established antidepressant drug, imipramine (30
Recent Patents on CNS Drug Discovery, 2009, Vol. 4, No. 2
123
mg/kg). Both parameters confirm potential antidepressant
action for (3) and (17) when administered acutely at doses
equal to or greater than 40 mg/kg, i.p.
Although preclinical and some clinical data suggest the
involvement of the endocannabinoid system in depression,
and hence the possible application of cannabinoids in
treatment of this disorder, it is evident that further studies are
needed to better elucidate the role of endocannabinoids in the
neurobiology of depression as well as the therapeutic benefit
of cannabinoids.
Antiemetic
Nausea and vomiting are among the most distressing side
effects of cancer chemotherapy and may interfere with the
successful completion of cancer treatment. The medicinal
use of marijuana for the treatment of nausea and emesis has
been evaluated in several clinical trials [78a]. In a double
blind randomized trial, the effectiveness of (1) in the
management of nausea in 55 cancer patients suffering from a
variety of neoplasms was reported [78b]. The patients were
selected based on reporting severe chemotherapy-induced
nausea and vomiting. The trial showed that (1) was effective
as an antiemetic against several chemotherapeutic drugs
including cyclophosphamide, fluorouracil and doxorubicin
hydrochloride. A survey of more than 1000 cancer
specialists revealed that 44% recommend (1) or cannabis to
at least one of their patients [79]. The primary driving force
behind the use of cannabis in antiemetic therapy for cancer
patients is the unresponsiveness of many patients to the
widely used 5-HT3 receptor antagonist, ondansetron.
Patent [80] describes a clinical trial that examined the
antiemetic efficacy of orally administered dronabinol (1),
either alone or in combination with ondansetron, when
administered prior to chemotherapy. Although the data
showed a significant antiemetic effect for dronabinol (1),
comparable to that of ondansetron, the combination therapy
showed less efficacy than either drug alone. Clinical use of
(1) or cannabis in the management of emesis in cancer
patients was supported by animal studies, confirming the
antiemetic action of (1) and providing ample evidence that
this action is mediated via the CB1 receptor. !9-THC (1)
dose dependently reduced vomiting induced by cisplatin in
the least shrew animal model [81], while the antiemetic
effect was completely reversed by the CB1 antagonist (9a)
but not the CB2 antagonist (10). Similarly, potent antiemetic
action of (1) against emesis induced by 5-hydroxytryptophan
[82] and dopamine D2/D3 agonists [83] has been shown.
The potential therapeutic value of (1) is, however, highly
restricted by its psychoactive effects. The search for related
compounds that lack psychoactivity while retaining the
medicinal antiemetic effect is ongoing.
!8-THC (4a) demonstrates enhanced antiemesis effect
against radiation-induced vomiting in the least shrew when
compared to (1) [84], supporting a clinical study showing
that (4a) unequivocally inhibits chemotherapy-induced
emesis in children, an effect not observed for (1) [85]. The
antiemetic effect of non-psychoactive (2) has also been
investigated [9b]. CBD (2) suppressed lithium-induced
vomiting in the house musk shrew through a biphasic effect,
with doses of 5 and 10 mg/kg suppressing vomiting and
124 Recent Patents on CNS Drug Discovery, 2009, Vol. 4, No. 2
Galal et al.
higher doses potentiating the lithium-induced effect. The
lack of psychoactive properties of (2) together with its
antiemetic effect makes this compound highly appealing for
the management of nausea and emesis [86].
that the extracts significantly reduced the influx of calcium
ions induced by N-methyl-D-aspartic acid (NMDA) in rat
hippocampal cultured cells, suggesting neuroprotection
against acute as well as long term treatment with NMDA.
Several patents have been filed in the last five years
regarding the potential use of cannabinoids as antiemetic
and/or antinausea drugs, ranging from those describing
methods to synthesize or extract promising non-psychoactive
cannabinoids to those investigating the antiemetic efficacy of
these compounds in animal models. Patent [87] focuses on
the cultivation of specific phenotypes of cannabis with the
intention of obtaining cannabis extract enriched in (2), (19)
and cannabidivarin (CBDV) (30) content. The efficacy of
extracts high in (1) and (2), respectively, was compared in a
motion sickness-induced emesis animal model. In
accordance with other findings, the data provided showed a
U-shaped dose response for the antiemetic action of the
extract high in (2).
An expansion on the neuroprotective capability of
cannabinoids was described in patent [101] where the
potential use of the non-psychotropic synthetic cannabinoid
dexanabinol (HU211) (32) [enantiomer of (6)] for the
prevention and management of mild cognitive impairment
was examined. The neuroprotective capability of (32) was
demonstrated by its ability to cause a dramatic decrease in
the number of necrotic foci induced by a high dose of
Cremophor EL®/ethanol in a rat brain (50 mg/kg, i.v.). The
patent claims that pretreatment with the invented pharmaceutical composition of non-psychotropic cannabinoids
notably prevented cognitive impairment associated with
secondary brain injury induced in two animal models
(transient occlusion of the vertebral and carotid arteries
resulting in global ischemia and microemboli injection in
rats). The compounds tested had previously established
neuroprotective and antiinflammatory properties, prompting
the authors to describe the potential use of these compounds
in the prevention of “post-operative, disease/drug-induced,
virally-induced, as well as neonatal cognitive impairment”.
Furthermore, the usefulness of these compounds in
mitigating or delaying the progression of mild cognitive
impairment to chronic neurodegeneration, is discussed.
Patent [88] addresses the use of (2) and its synthetic
homolog dimethylheptyl-CBD (DMH-CBD) (HU219) (31)
for the treatment of nausea. Using the conditioned rejection
reaction measure of nausea in rats, a non-vomiting species, it
was demonstrated that (2) (5 mg/kg, i.p.) and (31)
suppressed the establishment and expression of lithium
chloride induced conditioned rejection reactions. Previous
data have established the role of CB1 receptors in the
antinausea effect elicited by (1) [89]. Since (2) and (31) have
weak binding affinities to the CB1 receptors, it is postulated
that the antinausea effect might occur by enhancing the
levels of (5a). However, further studies are needed to
delineate such mechanism.
Patent [90] proposed the use of a combination
formulation comprised of (2) and (4a) in a ratio of 1:2-10 as
an antiemetic pharmaceutical preparation. The combined use
is suggested to provide a potent antiemetic effect with
diminished psychoactive properties. The potential antiemetic
value of non-psychoactive !8-THC-11-oic acid and several
of its synthetic analogs, such as (25), was described in
patents [91, 92]. The combined use of these compounds with
other
antiemetics,
including
antihistamines
and
anticholinergic drugs, was proposed in these patents.
Neuroprotection
The neuroprotective value of cannabinoids has recently
been the subject of intensive research based on their
antiexcitotoxic, antiinflammatory and antioxidant properties
under various experimental conditions [93]. Several studies
have demonstrated that cannabinoid receptor activation
protects cerebellar and hippocampal neurons against damage
induced by excitotoxic insults [94], hypoxia or glucose
deprivation [95]. This was supported by in vivo animal
studies that revealed that these compounds possess superb
protection against neuronal loss following cerebral ischemia
[95], excitotoxicity [96] or acute brain trauma [97].
Additionally, these protective effects have been demonstrated in models of acute and chronic neurodegenerative
conditions including MS [17b], Alzheimer’s disease [98] and
Parkinson’s disease [99].
The neuroprotective capacity of cannabinoid-containing
plant extracts is described in patent [100]. The data showed
Several analogs of (32) were synthesized and evaluated
for their neuroprotective and antiinflammatory actions [102],
demonstrating an array of pharmacological actions that
support their neuroprotective capacity. They all act as
NMDA non-competitive antagonists (IC50 values of 0.35-100
!M), suggesting their potential protective value against
glutamate excitotoxicity. Further in vitro and in vivo
evaluation of these compounds revealed that the C-11
derivatives of trans-(6aR,10aR)-3-(1,1-dimethylheptyl)-!8 THC (PRS-211 series) possessed marked neuroprotective
action attributed to their antiinflammatory and antioxidant
effects. The PRS-211 derivatives significantly inhibited the
production of PGE2, TNF-" and NO in LPS-induced macrophage cell cultures, with the antiinflammatory action confirmed by using the mouse ear edema model. Furthermore,
the cerebroprotective effects of the C-11 1H-imidazole
derivative (33) (Scheme 3) were assessed in rats following
head trauma, indicating a significant decrease in edema and
neurological deficits as well as significant reduction of brain
lesion and neurological deficits in middle cerebral artery
occlusion studies.
Although clinical neuroprotection seems to be an exciting
prospect for cannabinoids, clinical data are definitely lacking
and its potential will probably require a substantial time
frame for assessment.
Glaucoma Treatment
Glaucoma is an eye disease primarily caused by a rise in
intraocular eye pressure (IOP) and is usually classified into
primary and secondary glaucoma. In both cases an
accumulation of the aqueous humor in the anterior chamber
increases intraocular pressure which, if untreated, may lead
to damage of the optic nerve head and loss of eyesight. With
Cannabinoids and Their Therapeutic Applications
Recent Patents on CNS Drug Discovery, 2009, Vol. 4, No. 2
Br
OH
N
OH
OH
1a) 1H-imidazole / xylene
CBr4 / Ph3P
(32)
N HCl
OH
1b) n-BuLi / THF
2) HCl
O
O
125
O
bromo-intermediate
(33)
Scheme (3). Synthesis of (33).
the recent emergence of the neuroprotective merit of
cannabinoids, it was presumed that the protective value may
extend to the eye and could retard the progressive damage to
the optic nerve [103].
capacity of these compounds to the retina, particularly the
retinal ganglionic cells. Accordingly, these compounds may
be useful in the treatment of glaucoma or for the prevention
of retinal ganglion cell loss.
Patent [104] claims that (2) offers potent neuroprotective
action to the mammalian eye. Abnormal CBD (abn-CBD)
(34) (Scheme 4) demonstrates protective action against
excitatory amino acid toxicity in cultured rat hippocampal
neuronal cells, extending to retinal or optic nerve cells
injured by a deleterious stressor, principally associated with
glaucoma or diabetes. The patent also describes a potent
ocular hypotensive effect of (34) and its homologs/
derivatives. The CBD derivatives tested were administered
topically to the eyes of normotensive and laser induced
unilaterally ocular hypertensive monkeys. The data show
that the abnormal CBD derivatives lower intraocular pressure. However, these compounds fail to increase the
uveoscleral outflow. Hence, the patent focuses on combining
these abnormal CBD derivatives with an agent that enhances
the aqueous outflow from the eye to increase the effectiveness in the treatment of glaucoma and ocular hypertension.
Appetite Control
Patent [105] describes several water and lipid soluble
analogs of (1) and (5a). in vitro Binding to CB1 and CB2
receptors resulted in six cannabinoid analogs with CB1 and
CB2 binding affinities in the 3-300 nM range. These
compounds exhibited typical cannabimimetic activity in the
mouse tetrad assay, except that they lacked hypothermic
action. Topical application of the compounds caused
significant reduction of IOP when tested in the rat glaucoma
model. Combination therapy using (8) and trans-(6aR,10aR)3-[5-(1H-imidazol-1-yl)-1,1-dimethylpentyl]-!8-THC
(O2545) (35) with timolol revealed a significant synergistic
effect in reducing IOP as well as prolonging the duration of
action. Furthermore, results support a high neuroprotective
OH
The stimulatory effect of cannabis on feeding has been
primarily attributed to the psychoactive constituent (1). In
1992 the FDA approved the use of dronabinol (1) to stimulate appetite in AIDS patients suffering from wasting
syndrome. This triggered further research interest in the
effects exerted by other cannabinoid constituents on food
intake and energy expenditure [106]. Experimental data have
proven that the enhanced feeding behavior elicited by (1) is
mediated via the CB1 receptors located both centrally and
peripherally [107]. A recent interest has developed in
cannabinoid receptor antagonists since several studies have
reported that they might be useful in reducing appetite.
Rimonabant (9a or 9b), the CB1 receptor inverse agonist,
attenuates the hyperphagic effects of cannabinoid agonists,
induces hypophagia when administered alone and suppresses
appetite [108]. These findings were surprising since they
expanded the scope of therapeutic application of cannabinoid
antagonists to include obesity and related disorders.
Consequently, a two year randomized, double blind,
placebo-controlled clinical trial was conducted to assess the
efficacy and safety of (9b) in reducing body weight and
improving the cardiometabolic risk factors in overweight or
obese patients, showing that it caused a modest but sustained
reduction in body weight and favorable changes in
cardiometabolic risk factors [109].
Patent [110] proposed the combined use of a CB1
receptor antagonist and a peroxisome proliferator activated
receptor alpha (PPAR") agonist to reduce body weight. The
rationale behind using a PPAR" agonist stems from the
abundant literature advocating its role in all aspects of lipid
OH
OH
+
OH
oxalic acid dihydrate
toluene / Et2O
p-mentha-2,8-diene-1-ol
OH
olivetol
(34)
Scheme (4). Synthesis of (34).
126 Recent Patents on CNS Drug Discovery, 2009, Vol. 4, No. 2
Galal et al.
O
O
H2N
H2N
H
O
(26) oleamide
OH
N
O
HO
(30) CBDV
(28) URB597
OH
N
OH
N HCl
OH
OH
HO
O
O
(32) dexanabinol (HU211)
(31) DMH-CBD (HU219)
(33)
OH
OH
OH
N
N
O
OH
Ph
O
(35) O2545
(36)
(34) abn-CBD
OH
OH
OH
Ph
O
O
O
OH
N
O
O
N
O
(37)
(38)
metabolism [111]. The proposed PPAR! agonists were
oleoylethanolamide and its homologs, or clofibrate and its
derivatives. Similarly, the combined use of a CB1 antagonist
and an FAAH inhibitor was proposed.
The potential use of (2) as a CB1/CB2 inverse agonist to
reduce weight is elucidated in patent [112]. Binding of (2) to
CB1 and CB2 receptors was characterized utilizing the
[35S]GTP"S {[35S]guanosine 5'-("-thiotriphosphate)} binding
assay, indicating that it antagonizes the activation of both
CB1 and CB2 receptors by (7). However, by itself, (2)
behaves as an inverse agonist at the CB1 receptors in mouse
brain membranes. In vivo studies have demonstrated that the
administration of plant extracts high in (2) causes a dose
dependent reduction in body weight gain at 15 and 50
mg/kg/day dose rates from 1 to 104 weeks of administration.
Furthermore, the same doses significantly reduced the
amount of food consumed by both male and female animals
over the course of the experiment. The patent thus claims
that (2), acting as an inverse agonist, is highly suitable for
use in the prevention and treatment of various disease
(39)
conditions that require a cannabinoid receptor inverse
agonist, including obesity, epilepsy and schizophrenia.
Patent [113] describes the use of one or more cannabinoids in the treatment of diseases and conditions, e.g.
obesity, schizophrenia, epilepsy and Alzheimer’s disease,
benefiting from neutral antagonism of the CB1 receptors.
The majority (ca. 85%) of all known G-protein coupled
receptor (GPCR) antagonists are inverse agonists. The
relatively rare neutral antagonists affect only ligand-dependent receptor activation and have no effect on constitutive
receptors. The possible advantage of a neutral antagonist
versus an inverse antagonist is that fewer side effects should
occur since it would not supplement the consequences of
CB1 receptor constitutive activity. The patent claims that
(11) is a neutral competitive antagonist of the CB1 and CB2
receptors.
Generation of Water Soluble Cannabinoids
The high lipophilicity of cannabinoids has always been a
major hindrance to their full pharmacological evaluation.
Cannabinoids and Their Therapeutic Applications
Recent Patents on CNS Drug Discovery, 2009, Vol. 4, No. 2
Several research efforts have focused on the preparation of
water soluble derivatives of cannabinoids with subsequent
pharmacological evaluation of their binding affinities to
receptors [114].
cannabinoid receptor, is also critical [118]. The SAR for the
CB2 receptor has not been elucidated as extensively as for
the CB1 receptor. It is, however, clear that beneficial CB2
selectivity requires not only moderate to high affinity at the
CB2 receptor, but also low affinity and efficacy at the CB1
receptor [117].
Patent [115] describes the synthesis of a series of analogs
of (1) with higher water solubility and bioavailability. The
compounds were evaluated for binding to CB1 and CB2
receptors using Chinese Hamster Ovary (CHO) and Human
Embryonic Kidney 293 (HEK 293) cells, respectively. Most
of the compounds tested had affinities to both the CB1 and
CB2 receptors. The 1H-imidazol-1-yl analog (35) (Scheme
5) [116] possessed high CB1 receptor agonist affinity and
similar efficacy to the synthetic cannabinoid (7) in the
functional GTP!S [guanosine 5'-(!-thiotriphosphate)] assay.
In addition, the high pharmacological potency of this
compound in the mouse behavioral tetrad assay emphasized
its action as a cannabinoid agonist. The patent claims that
such compounds could have potential therapeutic applications in the treatment of disorders involving the CB1 and
CB2 receptors, e.g. appetite loss, pain, MS, nausea, vomiting
and epilepsy.
The objectives of patent [119] were to develop "8-, "9and "(6a,10a)-THC analogs as CB1/CB2 receptors agonists or
antagonists for potential treatment of illnesses mediated by
these receptors. The fact that the binding properties of (4a)
are similar to those of (1), in addition to the enhanced
stability and less expensive total synthesis of the former
compound, makes (4a) an attractive alternative when
designing derivatives [120]. The "8- and "9-THC analogs
were prepared by reacting resorcinol derivatives with cis-pmentha-2-ene-1,8-diol and cis-p-mentha-2,8-diene-1-ol,
respectively. The "(6a,10a)-THC analogs were prepared by
reacting resorcinol derivatives with a cyclic #-ketoester via
6-nor-6-oxo-"(6a,10a)-THC and CBD-type intermediates
(Scheme 6). The synthesis of 1-deoxy and 1-alkoxy
derivatives are also described (Scheme 7). A number of "8 THC analogs with phenyl side-chains were synthesized,
including (36)-(37) (Scheme 8), and their CB1/CB2 binding
affinities assessed using membrane preparations of the
human receptors transfected into HEK 293 Epstein-Barr
nuclear antigen (EBNA) cells. Receptor binding assays were
carried out using (7) and (8) as the competing radioactive
ligand and for determining non-specific binding, respectively
[121]. The CB1 (12-297 nM) and CB2 (0.9-86 nM) binding
affinities of the analogs were comparable to those of (4a)
(Table 6), with (36) exhibiting good binding affinities for
both the CB1 and the CB2 receptors and (37) exhibiting
decreased binding affinity for the CB1 receptor.
Enhancement of Cannabinoid Receptor Selectivity
Over the past few years, a growing body of evidence has
accumulated supporting the role of the CB2 receptors in the
immunomodulatory, anticancer and antiinflammatory effects
of cannabinoids. These findings, in addition to the fact that
the CB1 receptors mediate the psychotropic activities of
cannabinoids, have lead to extensive interest in the
development of highly selective CB2 ligands [117].
The basic structural parameters required for cannabinoid
binding affinity to the CB1 receptor are the following: 1) a
free hydroxy at C-1, 2) a "(8,9) or "(9,10) double bond, and an
exocyclic C-11 methyl or C-11 hydroxymethyl, or a
hexahydrocannabinol skeleton with a 9#-hydroxy, 9#hydroxymethyl or 9-keto functionality, and 3) a C3-C7
aliphatic side-chain at C-3. Substitution of the C-3 side-chain
with 1,1-dimethyl, 1,2-dimethyl or 1,1-dithiolane moieties
generally enhances the cannabinoid activity. Several studies
indicated that the ligand binding pocket of CB1 prefers a
hydrophobic substituent at C-3, however, the requirements
for conformational flexibility are still unresolved. Hydrogen
bonding between the C-1 hydroxy and the side-chain
nitrogen of Lys192 in transmembrane helix 3 of the
Patent [122] describes the synthesis of fluorescent
derivatives of cannabinoids for use as biosensors, molecular
probes and imaging agents, and to provide temporal, spatial
and dynamic data on receptor-ligand interactions. Radiochemical methods for investigating the cannabinoid system
and cannabimimetic molecules have several disadvantages,
e.g., high cost, handling and disposal difficulties, and
potential health hazards. Fluorescence methods circumvent
some of these shortcomings in addition to being more
accurate, sensitive, efficient, safe and generally less costly.
This alternative methodology provides an additional tool to
OMe
OMe
OH
THF / reflux
CN
HO
1) Me2Zn / TiCl4
(4-bromobutoxy)benzene
MeO
OPh
MeO
2) BBr3
Br
HO
OH
p-TsOH
benzene / 80oC
O
OH
OH
OTBDMS
O
N
1H-imidazole
TBDMSCl
Br
O
Br
NaH / DMF
N
O
(35)
Scheme (5). Synthesis of (35).
127
128 Recent Patents on CNS Drug Discovery, 2009, Vol. 4, No. 2
Galal et al.
OH
OH
HO
OH
X
+
R1
benzene / 80oC
O
OH
resorcinol derivative
X = C(CH3)2, C[-Y(CH2)nY-), CH2, C(O), Y = S or O
p-TsOH
"8-THC
cis-p-mentha-2-ene-1,8-diol
X
R1 = C3 to C8 cycloalkyl, thiophenyl, furanyl, pyrrolyl, pyridinyl, pyrimidinyl,
R1
pyrrolidinyl, biphenyl, 2-napthyl or thiazolyl
analogs
OH
OH
OH
p-TsOH
+
BF3-Et2O
benzene
HO
cis-p-mentha-2,8-diene-1-ol
R1
X
O
CO2Et
MeMgI
O
cyclic !-ketoester
OH
OH
POCl3
O
O
X
R1
"9-THC analogs
CBD-type intermediate
OH
+
X
TFA
R1
HO
6-nor-6-oxo-"(6a,10a)-THC intermediate
HO
X
R1
CBD-type intermediate
O
X
R1
"(6a,10a)-THC analogs
Scheme (6). Synthesis of !8-, !9- and !(6a,10a)-THC analogs.
R2
R2
R2
OR3
O
O
X
R1
EtO
OH
R3I / K2CO3
P
OEt
Cl
acetone
O
X
R1
K2CO3 / MeCN
O
X
R1
MeCN / Li / NH3 (l) / Et2O
1-alkoxy derivatives
1-deoxy derivatives
X = C(CH3)2, C[-Y(CH2)nY-), CH2, C(O), Y = S or O
R1 = C3 to C8 cycloalkyl, thiophenyl, furanyl, pyrrolyl, pyridinyl, pyrimidinyl, pyrrolidinyl,
biphenyl, 2-napthyl or thiazolyl
R2 = Me, OMe, (CH2)mCOOH, (CH2)mCOH
R3 = alkyl
Scheme (7). Synthesis of 1-deoxy and 1-alkoxy derivatives.
study the interactions between macromolecules (such as an
enzyme or a receptor) and their ligands. Fluorescent ligands
are prepared by covalently linking the parent ligand to the
fluorescent moiety to make the new ligands detectable and
measurable by fluorescence detectors. A major drawback to
this method is the possible reduced potency of the new
fluorescent ligand compared to the parent ligand. The patent
describes the synthesis of cannabinoid analogs containing
lactone moieties, rendering the compounds endogenously
fluorescent (390-502 nm), e.g. (38)-(40). The synthesis of
(38)-(40) utilized 3,5-dimethoxy-aniline (Scheme 9), 4hydroxy-N,N-diisopropylbenzamide (Scheme 10) and
phloroglucinol (Scheme 11), respectively as starting materials. The crucial structural feature for the key pharmacophore of these 1-oxo-3-substituted-benzo[c]chromen-6-ones
is the presence of a carbonyl moiety replacing the 6,6dimethyl residue found in the phytocannabinoids [123].
The compounds showed strong fluorescence and high
cannabinoid receptor affinity as tested in rat forebrain (CB1)
and mouse spleen (CB2) membrane preparations. Binding
affinity was represented by the inhibition constant, Ki (nM),
while binding selectivity was calculated as the ratio of
Ki(CB1)/Ki(CB2) Table 6. The lower the Ki value, the higher
the binding affinity, while high (>>> 1) and low (<<< 1)
CB1/CB2 ratios indicates CB2 and CB1 selectivity,
respectively. The disclosed compounds showed high CB1
(38) and CB2 (39) affinities, with some of the compounds
displaying high CB2 (39) and CB1 (40) selectivity.
Cannabinoids and Their Therapeutic Applications
Recent Patents on CNS Drug Discovery, 2009, Vol. 4, No. 2
129
OH
OH
1) Me2Zn / TiCl4
2) BBr3
OMe
Ph
HO
(36)
OMe
p-TsOH
benzene / 80oC
1) PhMgBr
H
MeO
2) PCC
Ph
O
Ph
MeO
O
HO
O
OH
OH
OH
BBr3
Ph
HO
Ph
O
O
O
(37)
Scheme (8). Synthesis of (36)-(37).
Table 6. CB1 and CB2 Binding Affinities of Natural and Synthetic Cannabinoids, Ki (nM)
Compound
CB1
CB2
CB1/CB2
(1)
41
36
1.1
(4a)
44
44
1.0
(12)
677
3.4
199
(13)
2918
13.3
219
(14)
3134
18
174
(36)
12.3
0.91
13.5
(37)
297
23.6
12.6
(38)
9
0.7
12.9
(39)
304
0.4
760
(40)
160
288
0.56
(41b)
395
11.8
33
(42b)
69.2
0.7
98.9
(42c)
74.5
0.4
186.3
Patent [124] describes the synthesis of a series of tetraand hexahydrocannabinol analogs that exhibit preferential
CB2 binding (Scheme 12) [125]. The compounds displayed
high CB2 and low CB1 affinity, with CB2 selectivity (13,
41b) Table 6. The selective CB2 agonist (41b) was tested in
the formalin model of inflammatory pain in mice, indicating
significant antinociceptive activity, emphasizing the
potential therapeutic role of CB2 agonists in the treatment of
pain and inflammation.
Patent [126] describes the synthesis of 1-O-methyl-, 1deoxy-11-hydroxy- and 11-hydroxy-1-O-methyl-!8-THC
derivatives with CB2 receptor selectivity (12, 14) (Scheme
13) Table 6 [127]. Compound (12) displayed significantly
enhanced CB2 activity and selectivity ascribed through SAR
to the 1,1-dimethylbutyl side-chain at C-4. The 1-O-methyl
series displayed low CB1 affinity, while the 11-hydroxy-1O-methyl series displayed intermediate affinity for both
receptors, indicating the SAR importance of the 11-hydroxy
moiety. The length of the C-3 alkyl side-chain is also critical
in determining receptor affinity, with five carbons a
minimum requirement for significant CB1 affinity. However,
for the 1-deoxy-!8-THC derivatives, a reduction in sidechain length did not significantly reduce CB2 receptor
affinity. Affinity to the receptors was evaluated in rat whole
brain (CB1) and HEK 293 cell (CB2) membrane
preparations [125a].
In patent [128], several novel bicyclic and tricyclic
(hexahydrocannabinol) cannabinoid analogs were synthesized, e.g. (42)-(43) (Scheme 14). A linear C-3 alkyl sidechain is an essential pharmacophore in classical canna-
130 Recent Patents on CNS Drug Discovery, 2009, Vol. 4, No. 2
Galal et al.
OMe
OMe
OMe
1-bromoheptane
MeI / THF
MeO
NH2
48% HBr
NaHCO3 / EtOH
MeCOONa
MeO
MeO
NHMe
AcOH
N
3,5-dimethoxyaniline
OH
OH
O
HO
N
O
O
CO2Et
N
POCl3
(38)
Scheme (9). Synthesis of (38).
OH
OH
OMe
Br
1) s-BuLi / TMEDA / -78°C
N
B(OH)2
2) B(OMe)3
O
3) H3O+
N
MeO
N
O
Pd(PPh3)4 / Ba(OH)2 / DME / H2O
4-hydroxy-N,N-diisopropylbenzamide
OH
OH
OH
N
OH
OMe
OMe
48% HBr
BBr3
Ac2O
O
MeO
O
O
N
N
N
(39)
Scheme (10). Synthesis of (39).
O
O
N
O
O
O
OH
HCl
9
O
O
(40)
O
(41a)
=
(41b)
=
O
3
4
5
OMe
2
OH
OH
1
6
HO
(42a) [182a] (1R,4R,6R) (unknown double bond geometry)
(42b) [182b] cis-(1S,4S,6S)
(42c) [182b] cis-(1R,4R,6R) (shown)
O
(43)
Cannabinoids and Their Therapeutic Applications
Recent Patents on CNS Drug Discovery, 2009, Vol. 4, No. 2
131
OH
2-bromooctane
HO
OH
EtO2C
COMe
OH
EtO2C
OH
NaH
CO2Et
DMSO
KOH / DMF
HO
O
POCl3 / benzene
O
O
phloroglucinol
O
O
O
OH
N
O
HCl
1) 4-(2-chloroethyl)morpholine /
K2CO3 / DMF
O
O
O
2) HCl
O
O
(40)
Scheme (11). Synthesis of (40).
OMe
OMe
HO
OMe
HO
MeSO3H
HO
diethyl phosphite
CCl4 / Et3N
MeO
MeO
H2O3PO
NH3 (l) / Li
THF
MeO
MeMgBr
O
O
O
OMe
OH
/ Pb(OAc)4
SnCl4 / CHCl3
MeO
p-TsOH
HO
OH
OMe
K-Selectride / THF
O
O
O
OH
(41a)
OMe
MeI / K2CO3
acetone
O
OH
O
OMe
LiAlH4 / THF
O
Scheme (12). Synthesis of (41).
binoids (i.e., phytocannabinoids and their synthetic derivatives) and is considered crucial for cannabimimetic
activity. A C-9 carbonyl is also known to enhance cannabinoid potency. The analogs were tested for CB1 (rat
forebrain membranes) and CB2 (mouse spleen) receptor
binding affinity [129]. The bicyclic analogs had affinity
values ranging from 31-224 nM for the CB1 and 0.2-77 nM
for the CB2 receptors, while the hexahydrocannabinol analogs showed CB1 and CB2 binding affinity ranging between
0.1-12 and 0.2-14 nM, respectively. Compounds (42b) and
(42c) displayed enhanced CB2 selectivity (Table 6).
(41b)
Adverse Effects
The adverse effects associated with the use of medical
marijuana or cannabinoids should also be considered [42]. A
number of review studies have concluded that, although,
short-term use has a number of modest adverse effects, the
effects of long-term use has not been fully investigated. In
addition, statistical evidence points to the possible
occurrence of dependence in regular heavy users of cannabis.
This is connected to a withdrawal syndrome impairing the
ability to stop use in a significant number of cases. However,
132 Recent Patents on CNS Drug Discovery, 2009, Vol. 4, No. 2
Galal et al.
OMe
MeI / KOH / DMF
R1
O
(14) R1 =
OH
OH
O
R1
1) SeO2 / EtOH
1) NaH / THF
2) (EtO)2P(O)Cl
O
R1
2) LiAlH4 / THF
O
3) NH3 (l) / Li / THF
R1
1-deoxy-11-hydroxy-!8-THC analogs
(12) R1 =
OH
OMe
OPiv
OPiv
OH
HO
OH
BF3-OEt2 / CH2Cl2
+
1) MeI / KOH / DMF
O
R1
2) LiAlH4 / THF
R1
OH
O
R1
R1 =
n (n = 0-5)
11-hydroxy-1-O-methyl-!8-THC analogs
Scheme (13). Synthesis of (12), (14), 1-deoxy-11-hydroxy- and 11-hydroxy-1-O-methyl-!8-THC analogs.
OMe
OMe
Br
CN
MeO
OMe
O
Br
K[N(SiMe3)2] / THF
MeO
Ph3P=CH(CH2)3CH3
THF
H
!-I-9-BBN
O
O
OH
HO
hexanes
MeO
OH
OH
nopinone diacetates
TfOTMS
p-TsOH / CHCl3
MeNO2 / CH2Cl2
HO
(42)
O
(43)
Scheme (14). Synthesis of (42)-(43).
nothing is known regarding the risk of cannabis dependence
in the context of long-term supervised medical use.
CURRENT & FUTURE DEVELOPMENTS
Over the past three decades, the field of cannabinoid
research, including chemistry, pharmacology and therapeutic
applications, has witnessed unprecedented progress due to
extensive studies that have been conducted on phytocannabinoids and their synthetic derivatives. This was fueled
by the discovery of the CB1 and CB2 cannabinoid receptors,
which mediate a plethora of biological effects in the human
body. A major obstacle, however, remains an understanding
of the structural parameters responsible for separating the
unwanted psychotropic activity from other useful pharmacological effects, which could lead to the design of nonpsychoactive therapeutic agents. The existence of novel
cannabinoid receptors mediating non-CB1/CB2 effects may
explain observed pharmacological properties not attributable
to these known GPCRs. This could also play a valuable role
in future cannabinoid-based drug design.
Cannabinoids and Their Therapeutic Applications
ACKNOWLEDGEMENTS
This project was supported in part by Contract Number
N01DA-5-7746 from the National Institute on Drug Abuse,
and by Grant Number 5P20RR021929 from the National
Center for Research Resources. The content is solely the
responsibility of the authors and does not necessarily
represent the official views of the National Center for
Research Resources or the National Institutes of Health.
CONFLICT OF INTEREST
Recent Patents on CNS Drug Discovery, 2009, Vol. 4, No. 2
[10]
[11]
[12]
[13]
[14]
The authors have no conflicts of interest to declare.
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