Naturally Occurring and Related Synthetic Cannabinoids and Their Potential Therapeutic Applications

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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
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
1
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 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
*
Table 1.
Cannabis Phenotypes
(1) 0.5-15% 0.5-5% 0.05-0.70% < 0.05% 0 (2) 0.01-0.16% 0.9-7.3% 1.0-13.6% < 0.5% 0 (2):(1) < 0.02 0.6-4 >5 (3):(2) ~ 0.5 ~ 0.1 ~ 0.05 > 0.5 -
Phenotype I II III IV V Drug Intermediate Fiber CBG Noncannabinoid
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
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Cannabinoids and Their Therapeutic Applications
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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
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
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11 9 8 7 6a 6 12 13 3 10 10a
Galal et al.
OH
1
R1
1' 2' 3' 5'
OH R1
4'
OH R2 HO (3) CBG: R1 = H, R2 = geranyl (21) CBNRA: R1 = COOH, R2 = neryl (22) CBGA: R1 = COOH, R2 = geranyl R1
O
4 R2
HO (2) CBD: R1 = H (19) CBDA: R1 = COOH
(1) !9-THC: R1 = R2 = H (18a) !9-THCA A: R1 = COOH, R2 = H (18b) !9-THCA B: R1 = H, R2 = COOH
OH OH OH
OH OH
R1 O OH (6) HU210 (7) CP55940
(4a) !8-THC: R1 =
(4b) !8-THCV: R1 =
R1
O OH (5a) anandamide: R1 = N H OH O OH (5b) 2-arachidonylglycerol: R1 = O (5d) virodhamine: R1 =
O O NH2
OH O (5e) N-arachidonoyldopamine: R1 = N H OH
OH OH (5c) noladin ether: R1 = O (27) AM404: R1 =
OH O 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
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()-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]. 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
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 100-110C for 10-20 min. This step removes water and allows for uniform heating of the plant material. The second step involves heating at 115-125C for 4575 min. Care must be taken to avoid thermal degradation of (1) to cannabinol (CBN) (16).
2.
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].
Fig. (1). Supercritical fluid extraction (SFE) diagram.
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 = 31.1C) 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. SFE (CO2) Extraction of Cannabis
Pressure 5800 psi 870 psi
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. 10C 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. 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. Organic modifier concentration: 0-20% (v/w) of total supercritical fluid used. Production of extract: 31-120C, 1015-9862 psi, 0-24 hrs. Decannabinization of marijuana: 25-65C, 5800-7252 psi, 0-24 hrs. Sub- or supercritical fluid flow rate: 20-50 mL/min (80 g marijuana). Plant material with a particle size 1-2 mm results in improved extraction as packaging density is improved. 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.
2.
3. 4. 5. 6. 7. 8.
Purification Purification of cannabis BDS or extract includes techniques such as chromatography, removal of hydrocarbons,
Extraction Supercritical Subcritical
Temperature 60C 10C
Wax Removed 6.8% 3.0%
(1) After Winterization 67.4% 65.0%
Fig. (2). Supercritical fluid chromatography (SFC) diagram.
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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.
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 105C (15 min) and 145C (55 min), followed by SFE (CO2) (10 hrs, 870 psi, 10C, CO2 flow at 1250 kg/hr) [28]. The crude BDS extract (3.5 g) was filtered through a column of activated charcoal, fractionated on

O N N O Cl N N
Galal et al.
Cl
O N N H N N O N H
X (9a) SR141716A: X = Cl (HCl salt) (9b) SR141716A: X = Cl (free base) (29) AM251: X = I (10) SR144528
(8) WIN55212-2
Cl
OH
OMe
O (11) !9-THCV O OMe
O (13) JWH359
(12) JWH133
OH
OH R1
O (14)
(15) nabilone
(16) CBN: R1 = H (23) CBNA: R1 = COOH
OH R1
OH OH
O O (17) CBC: R1 = H (20) CBCA: R1 = COOH (24) abn-!9-THC (25) ajulemic acid OH O
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, -20C, 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
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Table 3.
SFE (CO2) Decannabinization of Marijuana

Pressure 1044 psi 1044 psi 1044 psi 2176 psi 5802 psi 5802 psi 5802 psi 5802 psi 6527 psi 6527 psi 6527 psi 6527 psi Temperature 31C 31C 31C 58C 31C 31C 51C 60C 45C 62C 60C 55C Time 0.5 hrs 1.0 hrs 3.5 hrs 4.0 hrs 4.0 hrs 4.0 hrs 5.0 hrs 2.5 hrs 5.0 hrs 6.0 hrs 5.0 hrs 7.0 hrs Marc: (1) 0.24% 0.13% 0.23% 1.88% 0.14% 0.23% 0.22% 0.22% 0.31% 0.12% 0.14% 0.07% Extract: (1) 8.6% 17.4% 41.7% 36.7% 36.5% 40.1% 32.5% 28.6% 28.5% 38.2% 15.9% Extraction of (1) 93.1% 96.2% 93.2% 44.7% 96.0% 93.4% 93.7% 93.5% 90.9% 96.5% 95.9% 97.9%
SFE # SFE 1 SFE 2 SFE 3 SFE 4 SFE 5 SFE 6 SFE 7 SFE 8 SFE 9 SFE 10 SFE 11 SFE 12
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 (4-25C 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, 4C, 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 R1 COOH O R3 R2 R4
R5 R1 COOH HO R3 R2
R4
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
(13) 202 184 16 (1) 1.43 1.14 0.11 (2) + (14) 0.21 0.16 14.86 (11) < 0.00005 < 0.00005 < 0.00005
Cultivar # Cultivar 1 Cultivar 2 Cultivar 3
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
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OR1 O R1O R1O silica gel O n H R1 =
O N
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: 1. 2. Milled cannabis is extracted with a non-polar solvent, e.g. heptane or n-hexane, providing extract rich in (18). 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. The salt is extracted into a third solvent, namely isopropyl ether (IPE). Alternatively, chloroform, diethyl ether or dimethyl ether can also be used. The salt dissolves preferentially in IPE compared to the aqueous solution, while other impurities, such as CBD phenolate, remain in the aqueous phase. The IPE solution is washed with aqueous NaOH/NaCl. The resulting solution is acidified with dilute HCl (pH < 3), followed by florisil treatment to remove any residual solid material. Decarboxylation is achieved by refluxing the IPE solution in aqueous NaOH. The obtained (1) is filtered through charcoal and concentrated to provide crude product, which is stored at -20C. The crude product is purified by reversed-phase chromatography to provide (1) of high purity (99.7%).
Fig. (5). ADMPC coated on macroporous silica gel.
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 5-45C 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.
MeO OMe Si O Si N silica gel
3.
4.
5. 6.
7. 8.
9.
Fig. (6). 2-Ethylpyridine siloxane immobilized on a silica support. Table 5. Purification of Dronabinol (1)
Particle Size 10 !m 20 !m
Stationary Phase 2-Ethylpyridine siloxane ADMPC
Support
CO2/ethanol
(1)
Silica Macroporous silica
92:8 80:20
95% 99.5%
Pressure: 1450 psi. Temperature: 25C. 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,
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seedless, female flowering tops) was decarboxylated (5 min, 93C) 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. 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. 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
of pain and spasticity symptoms associated with MS [43], and the alleviation of neuropathic pain and migraine headaches [44]. 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 patients 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]. 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
OH p-TsCl O (1) (impure) CH2Cl2 / Et3N O
OTs K / t-BuOH N2 / reflux O
OH
!9-THC tosylate
(1) (> 99% purity)
Scheme (1). Purification of (1) via its tosylate.

X R1 OH R1-N=C=X O (1) (impure) CH2Cl2 / Et3N O X = O: !9-THC carbamate X = S: !9-THC thiocarbamate O N H K2CO3 EtOH / H2O O (1) (> 98% purity) OH
Scheme (2). Purification of (1) via its carbamate/thiocarbamate.
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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
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
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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
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
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]. 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). 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], Alzheimers disease [98] and Parkinsons disease [99]. The neuroprotective capacity of cannabinoid-containing plant extracts is described in patent [100]. The data showed
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. 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. 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
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OH OH CBr4 / Ph3P O (32)
Br N OH 1a) 1H-imidazole / xylene 1b) n-BuLi / THF O bromo-intermediate 2) HCl O
N HCl
OH
(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]. 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. 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
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. Appetite Control 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 + OH oxalic acid dihydrate toluene / Et2O OH
p-mentha-2,8-diene-1-ol
olivetol (34)

O H2N O (26) oleamide O HO (28) URB597 OH N OH OH OH HO O O (31) DMH-CBD (HU219) (32) dexanabinol (HU211) (33) N HCl (30) CBDV H N OH
Galal et al.
O H2N
OH
OH N OH O (35) O2545 N O
OH
Ph
(34) abn-CBD OH OH OH
(36)
OH
O O (37)
Ph
(38)
(39)
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
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 Alzheimers 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.
127
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]. 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. 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
OMe (4-bromobutoxy)benzene THF / reflux MeO CN MeO O OPh OMe
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]. 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. 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
OH 1) Me2Zn / TiCl4 2) BBr3 HO Br
HO p-TsOH
OH
benzene / 80oC
OH OH TBDMSCl Br Br OTBDMS 1H-imidazole NaH / DMF O N N
(35)

OH OH + OH resorcinol derivative cis-p-mentha-2-ene-1,8-diol OH OH + p-TsOH benzene HO cis-p-mentha-2,8-diene-1-ol CBD-type intermediate X R1 BF3-Et2O O "9-THC analogs X OH p-TsOH benzene / 80oC O "8-THC analogs X R1 OH R1
Galal et al.
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
HO
R1
OH + CO2Et cyclic !-ketoester O POCl3 O O X R1 HO HO MeMgI
OH TFA X R1 O
OH
R1
6-nor-6-oxo-"(6a,10a)-THC intermediate
CBD-type intermediate
"(6a,10a)-THC analogs
Scheme (6). Synthesis of !8-, !9- and !(6a,10a)-THC analogs.

R2 OR3 R3I / K2CO3 O X R1 acetone O 1-alkoxy 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 X R1 R2 OH EtO O P Cl K2CO3 / MeCN MeCN / Li / NH3 (l) / Et2O 1-deoxy derivatives O X OEt R1 R2
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.
129
OH 1) Me2Zn / TiCl4 2) BBr3 HO Ph O
OH
Ph
OMe 1) PhMgBr MeO O H 2) PCC MeO
OMe
(36) p-TsOH benzene / 80oC Ph HO O OH BBr3 HO O Ph O O (37) OH OH
Ph
Scheme (8). Synthesis of (36)-(37). Table 6. CB1 and CB2 Binding Affinities of Natural and Synthetic Cannabinoids, Ki (nM)
Compound (1) (4a) (12) (13) (14) (36) (37) (38) (39) (40) (41b) (42b) (42c) CB1 41 44 677 2918 3134 12.3 297 9 304 160 395 69.2 74.5 CB2 36 44 3.4 13.3 18 0.91 23.6 0.7 0.4 288 11.8 0.7 0.4 CB1/CB2 1.1 1.0 199 219 174 13.5 12.6 12.9 760 0.56 33 98.9 186.3
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-

OMe OMe MeI / THF MeO NH2 MeCOONa MeO 3,5-dimethoxyaniline NHMe 1-bromoheptane NaHCO3 / EtOH MeO N
Galal et al.
OMe 48% HBr AcOH
OH
OH
O HO N CO2Et POCl3 (38) O O N

OH OH Br 1) s-BuLi / TMEDA / -78C N O 2) B(OMe)3 3) H3O+ 4-hydroxy-N,N-diisopropylbenzamide OH OH OH OMe OMe 48% HBr Ac2O N O N (39) BBr3 O N OH N O Pd(PPh3)4 / Ba(OH)2 / DME / H2O B(OH)2 MeO N OMe
O MeO

O O N O OH HCl 9 OMe
O (40)
(41a) (41b)
= =
O 3 4 5 6 HO 2 1 OH
O OH
(42a) [182a] (1R,4R,6R) (unknown double bond geometry) (42b) [182b] cis-(1S,4S,6S) (42c) [182b] cis-(1R,4R,6R) (shown)
(43)

OH OH 2-bromooctane HO phloroglucinol OH KOH / DMF HO O EtO2C COMe
131
EtO2C CO2Et O
OH NaH DMSO
POCl3 / benzene
O OH 1) 4-(2-chloroethyl)morpholine / K2CO3 / DMF O O 2) HCl O
O O N
HCl
O (40)

OMe HO HO MeO MeSO3H HO MeO OMe diethyl phosphite CCl4 / Et3N H2O3PO MeO OMe NH3 (l) / Li THF
MeMgBr O
O OMe O / Pb(OAc)4 MeO p-TsOH HO OH SnCl4 / CHCl3
OH OMe K-Selectride / THF O O OH O OMe (41a)
MeI / K2CO3 acetone
OH O OMe LiAlH4 / THF O
(41b)
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).
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,
Galal et al.
OMe
MeI / KOH / DMF
R1
(14) R1 = OH
OH O R1 1) NaH / THF 2) (EtO)2P(O)Cl 3) NH3 (l) / Li / THF (12) R1 = O R1 1) SeO2 / EtOH 2) LiAlH4 / THF O R1
1-deoxy-11-hydroxy-!8-THC analogs
OH OMe
OPiv OH + HO R1 OH O R1 R1 = OPiv BF3-OEt2 / CH2Cl2 OH 1) MeI / KOH / DMF 2) LiAlH4 / THF O
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 Br MeO O H Ph3P=CH(CH2)3CH3 THF MeO OMe !-I-9-BBN hexanes
MeO
CN
K[N(SiMe3)2] / THF
O OH nopinone diacetates HO p-TsOH / CHCl3 HO (42) OH TfOTMS MeNO2 / CH2Cl2
O OH
(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.
Recent Patents on CNS Drug Discovery, 2009, Vol. 4, No. 2 [10] [11] [12] [13]
133
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 The authors have no conflicts of interest to declare. REFERENCES
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Naturally Occurring and Related Synthetic Cannabinoids and Their Potential Therapeutic Applications

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Recent Patents on CNS Drug Discovery, 2009, 4, 112-136

Phenotype I II III IV V Drug Intermediate Fiber CBG Noncannabinoid

Cannabinoids and Their Therapeutic Applications

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HO (2) CBD: R1 = H (19) CBDA: R1 = COOH

(1) !9-THC: R1 = R2 = H (18a) !9-THCA A: R1 = COOH, R2 = H (18b) !9-THCA B: R1 = H, R2 = COOH

R1 O OH (6) HU210 (7) CP55940

O OH (5a) anandamide: R1 = N H OH O OH (5b) 2-arachidonylglycerol: R1 = O (5d) virodhamine: R1 =

OH OH (5c) noladin ether: R1 = O (27) AM404: R1 =

Cannabinoids and Their Therapeutic Applications

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Fig. (1). Supercritical fluid extraction (SFE) diagram.

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Extraction Supercritical Subcritical

Temperature 60C 10C

Wax Removed 6.8% 3.0%

(1) After Winterization 67.4% 65.0%

Fig. (2). Supercritical fluid chromatography (SFC) diagram.

Cannabinoids and Their Therapeutic Applications

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O (11) !9-THCV O OMe

(16) CBN: R1 = H (23) CBNA: R1 = COOH

Cannabinoids and Their Therapeutic Applications

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SFE (CO2) Decannabinization of Marijuana

Flow: SFE 1-5 = 20 g/min, SFE 6-12 = 30 g/min.

Cultivar # Cultivar 1 Cultivar 2 Cultivar 3

Concentration of cannabinoids determined by LC/MS/MS (mg/g dry weight).

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OR1 O R1O R1O silica gel O n H R1 =

Fig. (5). ADMPC coated on macroporous silica gel.

Stationary Phase 2-Ethylpyridine siloxane ADMPC

Silica Macroporous silica

Cannabinoids and Their Therapeutic Applications

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OH p-TsCl O (1) (impure) CH2Cl2 / Et3N O

OTs K / t-BuOH N2 / reflux O

(1) (> 99% purity)

Scheme (1). Purification of (1) via its tosylate.

Scheme (2). Purification of (1) via its carbamate/thiocarbamate.

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Cannabinoids and Their Therapeutic Applications

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Cannabinoids and Their Therapeutic Applications

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OH OH CBr4 / Ph3P O (32)

Br N OH 1a) 1H-imidazole / xylene 1b) n-BuLi / THF O bromo-intermediate 2) HCl O

Scheme (3). Synthesis of (33).

OH OH + OH oxalic acid dihydrate toluene / Et2O OH

Scheme (4). Synthesis of (34).

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Cannabinoids and Their Therapeutic Applications

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OH OH TBDMSCl Br Br OTBDMS 1H-imidazole NaH / DMF O N N

Scheme (5). Synthesis of (35).

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OH + CO2Et cyclic !-ketoester O POCl3 O O X R1 HO HO MeMgI

Scheme (6). Synthesis of !8-, !9- and !(6a,10a)-THC analogs.

Scheme (7). Synthesis of 1-deoxy and 1-alkoxy derivatives.