Naturally Occurring and Related Synthetic Cannabinoids and Their Potential Therapeutic Applications

8/3/2019 Naturally Occurring and Related Synthetic Cannabinoids and Their Potential Therapeutic Applications

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

1574-8898/09 $100.00+.00 2009 Bentham Science Publishers Ltd.

Naturally Occurring and Related Synthetic Cannabinoids and theirPotential Therapeutic Applications

Ahmed M. Galal1,*

, Desmond Slade1, Waseem Gul

1,5, Abir T. El-Alfy

2, Daneel Ferreira

1,3and

Mahmoud A. Elsohly1,4,5

1National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS

38677, 2 Department 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 38677,4Department of Pharmaceutics, School of Pharmacy, The University of Mississippi, University, MS 38677, and5ElSohly

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, isolationpurification, synthesis, therapeutic applications.

INTRODUCTION

Cannabinoids are terpenophenolic secondary plantmetabolites uniquely found in Cannabis sativa L. [1].Biogenetically they are derived from a mixed origin, with the3-alkylphenol moiety originating from a polyketide pre-cursor and the tetrahydroisochroman moiety from a mono-

terpene residue [2]. Cannabinoids found in cannabis aredesignated as phytocannabinoids or exogenous cannabinoidsto distinguish them from the eicosanoid endocannabinoids, agroup of arachidonoyl esters and amides that were firstdiscovered in 1988 in mammalian tissues acting as endo-genous cannabinoid receptor ligands with neuromodulatoryaction [3].

Cannabis is divided mainly into three phenotypes orchemotypes: 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 butwith (1) also present at various concentrations; andphenotype III (fiber type), with especially low (1) content.The rare phenotypes IV and V have low (1) and (2) contentand high cannabigerol (CBG) (3) content, and undetectableamounts of any cannabinoid, respectively (Table 1) [4]. Allanalyses are based on plant inflorescence dry material.Although environmental factors play a role in the amount ofcannabinoids 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]

Table 1. 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

VNon-

cannabinoid0 0 - -

different growth stages [5], the tripartite distribution o(2):(1) ratios in most populations (phenotypes I to III) areunder genetic control [4c].

Not only is the isolation and purification of cannabinoidchallenging owing to the structural, physical and chemica

similarity of these compounds, but synthetic routes areequally demanding due to low yields and the formation o by-products while the final products are typically noncrystalline. The critical step in the majority of synthesiroutes yielding (1) [in cannabinoid parlance, synthetic (1) icalled dronabinol] is the condensation of a monoterpene witha resorcinol derivative such as olivetol (5-pentyl-1,3 benzenediol). Presently, condensation of (+)-p-mentha-2,8dien-1-ol with olivetol is used to produce commercial (1). Inaddition, the thermodynamic instability of (1) must alwaybe considered during isolation, synthesis and storage, sinc


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Cannabinoids and Their Therapeutic Applications Recent Patents on CNS Drug Discovery, 2009, Vol. 4, No. 2 113

this labile compound readily converts to the more stableregioisomer, !

8-THC (4a) [1].

The pharmacological effects of (1) have been unraveledsince the discovery of the endocannabinoid (endogenouscannabinoid) system, which consists of endogenous ligands(endocannabinoids), cannabinoid receptors and enzymesinvolved in biosynthesis and degradation of the endocan-nabinoids. The system affects a variety of physiological

processes, e.g. appetite, pain-sensation, mood and memory[6].

Endocannabinoids are capable of binding to andfunctionally activating cannabinoid receptors. At least twocannabinoid receptors, namely subtype CB1 and CB2, have been identified, including their primary structure, ligand-binding properties and signal transduction systems. CB1 andCB2 receptors belong to the large superfamily of G-proteincoupled receptors (GPCR) that couple to guanine-nucleotidebinding proteins (heptahelical receptors). Endo-cannabinoids, unlike classical neurotransmitters, are notstored in intracellular compartments and act as neuro-modulators. They are synthesized as needed on location by

cleavage of their membrane lipid precursors, followed byrelease from the cell. Inactivation occurs via intracellularhydrolyzing enzymes. CB1 receptors, expressed mainly inthe CNS, are responsible for psychoactive effects, while CB2receptors are expressed in the immune system. CB1receptors are also expressed by some non-neuronal cells, e.g.immune cells and peripheral tissues and organs, e.g. heartand blood vessels. Recent reports indicate that CB2 receptorsare also localized in the CNS, including spinal cord, brainstem and cortex [7]. There is also mounting evidence for theexistence of additional non-CB1/CB2 cannabinoid receptors[8], e.g. bioassays conducted with compounds devoid ofnoteworthy CB1 or CB2 affinity are sensitive to CB1 or CB2selective antagonists [6c].

Five different types of endogenous agonists, havingsubmicromolar affinity for the CB1 and CB2 receptors, have been identified, namely anandamide (5a), 2-arachi-donylglycerol (5b), noladin ether (5c), virodhamine (5d) and

N-arachidonoyldopamine (5e).

Cannabinoid receptor agonists can be divided into fourgroups based on their chemical structures. The classicalcannabinoids are ligands with a tetrahydro-6H-benzo[c]chromene (dibenzopyrane) structure, including phytocanna-binoids, e.g. (1), and their synthetic analogs, e.g. HU210 (6).This group lacks CB1/CB2 selectivity. The non-classicalcannabinoids are bi- and tricyclic analogs of (1) without thepyran ring, e.g. (-)-CP55940 (7). This group binds to CB1

and CB2 receptors with similar affinity, while displayinghigh in vivo activity. The aminoalkylindole cannabimimeticgroup of agonists, e.g. (R)-(+)-WIN55212-2 (8) exhibits highaffinity for both receptors, but with CB2 selectivity. Theeicosanoid (arachidonic acid derivative) group includes (5a)-(5e).

SR141716A (9), the first specific cannabinoid antagonist,impedes the action of cannabinoid agonists in vivo atnanomolar concentrations. It is CB1 selective, but not CB1specific, blocking both receptors at sufficiently high doses.SR141716A (9) and (-)-SR144528 (10), CB2 selective

antagonists, may also behave as inverse agonists, i.e., theyblock the effects of endocannabinoids and cause an oppositeeffect.

In vivo Testing of endocannabinoids produce behavioraand pharmacological actions associated with other cannabimimetic ligands. Anandamide (5a) produces antinociception, hypothermia, hypomobility, and catalepsy in the mousetetrad model, with rapid onset of effects, but with a shor

duration of action due to rapid uptake into neurons andastrocytes and subsequent enzymatic degradation.

Pharmacological investigations of the other majorcannabinoids revealed that (2), a non-psychoactive cannabiconstituent, displayed marked antioxidant and antiinflammatory properties that could be utilized to provideneuroprotection 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 bothCB1 and CB2 receptors [11]. (-)-Trans-!

9-tetrahydrocannabivarin (!

9-THCV) (11) has been shown to be a strongantagonist of (5a), a neuromodulator found in animal and

human organs [12]. Some of the aforementioned biologicaeffects are apparently due to interactions with non-CB1/CB2receptors [11]. Several 1-O-methyl- and 1-deoxy-"

8-THCanalogs have high affinity for the CB2 receptor, but littleaffinity 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-"

8-

THC (JWH359) (13) and trans-(6aR,10aR)-3-(1,1dimethylhexyl)-1-O-methyl-"

8-THC (14). This is in linewith traditional cannabinoid structure-activity relationship(SAR) requiring a free phenolic hydroxy at C-1 for CB1receptor interaction [13].

It has been shown that synthetic !8-THCV (4b) and (11

exhibit in vitro pharmacological properties similar to thoseof natural (11), and that they can antagonize (1), theCB1/CB2 receptor agonist, in vivo. It is, however, importanto realize that (4b) and (11) behave as agonists or antagonistin a dose dependant manner [14]. As an antiemetic, (4a) haactivity equal to (1) [15]. Numerous synthetic analogs of (1have been developed and tested as CB1/CB2 agonists oantagonists and for potential therapeutic benefits, with 1,1dimethylheptyl and 1,2-dimethylheptyl side-chain homologfound to be several hundred times more psychoactive thanthe natural compound [3].

Diseases of the nervous system are not only diverse, bualso result in approximately 9% of all human deaths [16]The most widespread immunomediated disease of the centra

nervous system (CNS) is multiple sclerosis (MS), withpatients developing inflammation that leads to demyelinationand neuronal dysfunction, resulting in serious clinicasymptoms [17a]. Currently, there is no successful treatmenavailable for these symptoms, however, clinical studies usingcannabinoids for controlling spastic pain, tremors andnocturia have yielded promising results [18]. Cannabinoidhave been linked to modulation of neuroinflammation [19and are reported to regulate the neuronal and immunefunctions [20]. Evidence supporting the role of cannabinoidin treatment of CNS inflammatory diseases was found in theregulation of glial cell function, while treatment o


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()-trans-11-nor-9-oxo-3-(1,1-dimethylheptyl)hexahydro-cannabinol (nabilone) (15), which is used for the treatmentof chemotherapy-related nausea and vomiting [22].

The tremendous body of work established over the pastthree decades on the pharmacology of cannabinoids isindicative of the potential therapeutic use of these compounds. This has been matched by a steady growth in thenumber of cannabinoid-type drugs in development from two

in 1995 to 27 in 2004, with focus on pain, obesity and MStherapeutic agents [23].

Thus, although a wide diversity of cannabinoid phar-macological effects have been discovered, reviewing theliterature on cannabinoids and the endocannabinoid systemover the past decade leaves no doubt that much is still to beunderstood. This review covers the isolation, purification,synthesis and pharmacology of phytocannabinoids (naturaland related synthetic derivatives), as disclosed in patentsspanning 2003-2007. Patents dealing with cannabis andcannabinoid formulations were not considered. Also, patentsfocusing only on the chemistry of cannabis, including thetotal 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 toassert these claims.

In this review, the commonly used tetrahydro-6H-benzo[c]chromene numbering system (sometimes referred toas a dibenzopyrane system) will be employed in naming thecannabinoids [see (1)] [24].

ISOLATION AND PURIFICATION

Introduction

Although numerous analytical chromatographic methodsare available that provide qualitative and quantitative

analysis of cannabinoids with baseline separation, prepa-rative separation is much more problematic [25].

A number of methods are available for obtainingcannabinoids from plant material [26] or via synthesis [27].However, these are time- and labor-intensive and generallynot suitable for preparative-scale isolations. This represents amajor obstacle in providing, especially minor, cannabinoidsin sufficient amounts for use as standards or for phar-macological evaluations.

Extraction of cannabinoids includes diverse methods, e.g.Soxhlet and supercritical fluid extraction (SFE), reflux andorganic solvent extraction, while purification is achievedthrough chromatography, activated charcoal treatment, filt-

ration, distillation and chemical derivatization or combi-nations of these methods.

Extraction

The extraction of biologically active substances from raw plant material is crucial in the development of naturalmedicinal preparations. Improvements of traditional solventextraction methods (maceration, percolation, Soxhletextraction and steam distillation) include vortex technology,rotary-pulsation, sonication, pressing, and squeezing. Thesemethods generally improve the overall extraction, but alsogreatly enhance the extraction of high molecular weight

compounds and cause cell disruption, which, in turn, resultin extracts containing finely dispersed solid material.

Extraction of cannabis is typically achieved by organicsolvent (maceration or percolation) or SFE. As a first stepthe plant material, or in some cases the obtained extract, isdecarboxylated to convert all cannabinoid acids into theineutral form, unless the acid form is the targeted productDecarboxylation of the cannabinoid acids in the plan

material, which is a function of time and temperature, isperformed as a multi-step process [28]:

1. The first step involves exposure of the plant material totemperatures of 100-110C for 10-20 min. This stepremoves water and allows for uniform heating of theplant material.

2. The second step involves heating at 115-125C for 4575 min. Care must be taken to avoid thermal degradationof (1) to cannabinol (CBN) (16).

SFE Fig. (1) comprises the use of supercritical fluids toselectively remove analytes from solid, semisolid and liquidmatrices. A supercritical fluid is a substance at a temperature

and pressure above its thermodynamic critical point, causingthe interface between the liquid and vapor phases todisappear and improving the solvating power (E) of thesubstance. This critical temperature (Tc) is the highestemperature at which a gas can be converted into a liquid byan increase in pressure, and the critical pressure (Pc) is thehighest pressure at which a liquid can be converted into a gaby increasing the temperature. There is only one phase in thecritical region and it possesses properties of both a gas and aliquid, e.g., high diffusivity, low viscosity and minimasurface tension. Varying the extraction temperature and pressure allows for changing the selectivity of the supercriticafluid [29].

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


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116 Recent Patents on CNS Drug Discovery, 2009 , Vol. 4, No. 2 et a

Advantages of SFE include enhanced extraction effi-ciency, speed and selectivity due to its gas-like mass transfer properties and liquid-like solubility properties, environ-mental benefits, extraction of analytes present in lowconcentrations, cleaner extracts and preservation of bioactiveconstituents. Disadvantages of SFE include high startupcosts, complicated system optimization, strong dependenceon matrix-analyte interactions and difficult scale-up [30].

Carbon dioxide (CO2) is a popular choice for SFE due toits low cost, inert nature, low toxicity, non-flammability andlow critical temperature (Tc = 31.1C) and critical pressure(Pc = 1070.4 psi). The virtual absence of surface tensionfrom non-polar supercritical CO2 allows for improvedpenetration into plant matrices compared to liquid solvents.However, polar modifiers, such as ethanol, need to be addedwhen extracting polar compounds. This method has highreproducibility and can remove heavy metals and pesticidesfrom the cannabis matrix. The polarity of subcritical CO2 issimilar to n-hexane, while that of supercritical CO2 iscomparable to toluene or ether.

Extraction of cannabinoids with CO2 is preferably done

under subcritical rather than supercritical conditions bysetting the temperature and pressure below Tc and Pc,respectively. This provides a botanical drug substance (BDS)containing active substances selectively from a complexmixture of compounds as found in a botanical raw materialsuch as cannabis. Although the density, and therefore E, ofsubcritical CO2 is lower than supercritical CO2, selectivity isenhanced for cannabinoids since only the most solublecomponents are efficiently dissolved in the CO2. This allowsfor selective extraction of the lipophilic cannabinoids by thenon-polar CO2, and implies that although supercriticalconditions might improve yields, subcritical conditionsprovide much higher sensitivity. The high wax burden undersupercritical conditions indicates loss of selectivity, and

while precipitation at sub-zero temperatures (winterization)can remove large amounts of wax, this process can betroublesome as, e.g. the blocking of filters easily occurs.

Subcritical conditions lower the wax burden withousignificant loss in cannabinoid yield (Table 2).

SFE conditions are typically as follows: Extraction idone at ca. 10C and 870 psi with a CO2 mass flow of 1250kg/h for 8-10 hrs (60 kg marijuana). A wide variety oconditions can, however, be employed to obtain the appro priate extract or, in some cases, decannabinized marijuanfor use as a placebo [31]:

1. Supercritical fluids: CO2, carbon monoxide, ammonianitrous oxide, ethanol, n-pentane, n-hexane, propanewater, ethane, fluoroform and xenon.

2. Organic modifiers: Ethanol, methanol, 2-propanol, diethyl ether, ethyl acetate, chloroform, dichloromethanecarbon tetrachloride, acetonitrile, cyclohexane, acetoneacetic acid, nitromethane, dioxane, n-hexane, n-pentaneand pyridine.

3. Organic modifier concentration: 0-20% (v/w) of totasupercritical fluid used.

4. Production of extract: 31-120C, 1015-9862 psi, 0-24hrs.

5. Decannabinization of marijuana: 25-65C, 5800-7252psi, 0-24 hrs.

6. Sub- or supercritical fluid flow rate: 20-50 mL/min (80g marijuana).

7. Plant material with a particle size 1-2 mm results inimproved extraction as packaging density is improved.

8. CO2 flow rate is preferably measured in terms of massflow rather than by volume, since the density of the COchanges according to the temperature before entering thepump heads and during compression.

Purification

Purification of cannabis BDS or extract includes techniques such as chromatography, removal of hydrocarbons

Table 2. SFE (CO2) Extraction of Cannabis

Extraction Pressure Temperature Wax Removed (1) After Winterization

Supercritical 5800 psi 60C 6.8% 67.4%

Subcritical 870 psi 10C 3.0% 65.0%

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


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waxes and other non-polar compounds (typically throughwinterization), a two-solvent treatment, and filtrationthrough activated charcoal or florisil, or a combination ofthese methods. Chromatographic techniques includesupercritical fluid Fig. (2) [32], reversed-phase (C18) and gelfiltration chromatography (Sephadex LH-20).

Supercritical fluid chromatography (SFC) has severaladvantages, e.g., fast, high resolution separations, lower

operating temperatures, gas-like mass transfer and liquid-likesolvating properties.

The BDS or extract usually contains lipid-solublematerial, e.g., hydrocarbons, waxes, glycerides, unsaturatedfatty acids and terpenes, which can be removed throughwinterization, followed by filtration or distillation [33].

The two-solvent treatment technique involves sequentialtreatment of the extract with two solvents. The polarity ofthe first and second solvent should be substantially different,e.g., methanol and n-pentane. This facilitates the removal ofmore and less polar compounds compared to the targetcannabinoid, respectively. The solvents can be used in eitherorder. This process can be applied to obtain any of the

cannabinoids in free or acid form, while selectivity may beenhanced by selecting cannabis varieties high in the targetproduct.

Filtration through activated (porous) charcoal adsorbscolored impurities in the extract, while filtration throughflorisil, which is often used in the purification of pharma-ceuticals, removes any residual solid material.

A typical extraction and purification protocol forcannabinoids comprises the following steps [28, 33-35]:

Step 1: Decarboxylation of the plant material if neutralcannabinoids 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-targetcompounds.

Step 4: Chromatography of the winterized BDS.

Step 5: Dissolving the purified extract fractions in a firstpolar/non-polar solvent, filtering any insoluble material, andremoving the solvent from the filtrate.

Step 6: Dissolving the filtrate in a second non-polar/polarsolvent, filtering any insoluble material and removing thesolvent from the filtrate to obtain a substantially purecannabinoid.

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 cannabinoidenriched extracts (preparations) and purified cannabinoids.

Patent [34] describes the use of naturally occurring orsynthetic cannabichromene (CBC)-type compounds and pharmaceutically acceptable derivatives thereof Fig. (3) inthe treatment of mood disorders such as depression. Extractrich in CBC (17) were prepared from cannabis varieties highin (17) obtained via selective breeding techniques andincorporated into pharmaceutical dosage forms [28, 33]. Theextract should contain (17) as 5-40% of the total cannabinoid

content. Step 4 in the abovementioned protocol useSephadex LH-20 column chromatography (chloroformdichloromethane, 2:1, v/v) for purification, followed by thetwo-solvent system (steps 5 and 6) (methanol/n-pentane) toproduce highly enriched (17) (> 98% w/w by TLC, HPLC orGC).

R1: H / COOH

R2: alkyl (C1-C8, branched or linear)Fig. (3). CBC-type compounds.

Patent [28] describes methods of preparing high puritycannabinoids (neutral or acids), cannabinoid preparationand cannabinoid rich extracts from plant material via solvenextraction, chromatography and recrystallization without theuse of preparative HPLC. A substantially pure cannabinoidand an enriched extract are defined as having a chromatographic purity of > 95% and > 80%, respectively, adetermined by HPLC area normalization. Fresh cannabi plant material contains (1) predominantly as two isomericcarboxylic 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-psychoactiveacids are converted into the active (1) during storage andexposure to heat.

Plant material (100 g) from a phenotype high in (1[present as (18) in the plant material] [(18) > 90% of totacannabinoid content] was extracted with n-hexane/glaciaacetic acid (2 x 1500 mL, 99.9:0.1, v/v), followed byfiltration of the combined extracts and concentration invacuo to produce the crude BDS [28]. The crude BDS wadissolved in the chromatographic eluent (chloroform/dichloromethane, 2:1, ca. 20 mL) and applied to a low pressureglass 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 wasequentially dissolved in methanol and n-pentane, followed by filtration to remove non-polar and polar compoundsrespectively. Removal of solvent produced (18) as a paleyellow solid (ca. 5 g, 98% by area normalization).

Plant material (100 g) from a phenotype high in (1) wadecarboxylated at 105C (15 min) and 145C (55 min)followed by SFE (CO2) (10 hrs, 870 psi, 10C, CO2 flow a1250 kg/hr) [28]. The crude BDS extract (3.5 g) was filteredthrough a column of activated charcoal, fractionated on

O

OH

R2

R1


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Sephadex LH-20 and the fraction rich in (1) sequentiallydissolved 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) waspreviously regarded as an inactive constituent of cannabis, itis now considered an active compound with diverse pharmacology [9], necessitating a selective process for itspurification. Cannabis varieties with high content of (2) (>90% of total cannabinoid content) is particularly suitable forthis process. Cannabis plant material, obtained from acultivar 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 filtefollowed by activated charcoal column) and the solvenremoved (rotary or thin film evaporation) to produce a (2rich extract. Recrystallization from n-pentane provided (2) (>99% by area normalization) [28].

Patent [31] describes SFE (CO2) of marijuana to providecompounds with medicinal value or decannabinizedmarijuana (cannabinoid-free marijuana) for use as a placeboA wide variety of conditions were employed to provide thedecannabinized marijuana Table 3 under relatively mild andreproducible conditions while maintaining its appea-rancecolor and texture, and lowering the content of (1) to below

O

OH

(11) !9-THCV

O

(12) JWH133

OMe

O

(13) JWH359

OH

O

R1

(16) CBN: R1 = H

(23) CBNA: R1 = COOH

O

(14)

OMeOH

O

O

(15) nabilone

OH

O

(25) ajulemic acid

OHO

O OH

(24) abn-!9-THC

O

OH

R1

(17) CBC: R1 = H

(20) CBCA: R1 = COOH

N

O

N

O

O

(8) WIN55212-2

N

NO

N

H

ClCl

X

N

(9a) SR141716A: X = Cl (HCl salt)

(9b) SR141716A: X = Cl (free base)

(29) AM251: X = I

N

NO

N

H

Cl (10) SR144528


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0.5%. The marc obtained from SFE 11 was re-extractedunder similar conditions (SFE 12), but with the addition ofan organic modifier (ethanol). This improved the overallextraction of (1) to 97.9%, yielding marc with only 0.07%(1).

A number of patents utilized more specialized tech-niques, some in combination with the abovementionedoptions, to provide cannabis extract or cannabinoids [36].

Patent [36a] describes the medicinal use of acidiccannabinoids or extracts containing acidic cannabinoids,

including cannabidiolic acid (CBDA) (19), cannabi-chromenic acid (CBCA) (20), cannabinerolic acid (CBNRA)(21), cannabigerolic acid (CBGA) (22), cannabinolic acid(CBNA) (23), and derivatives thereof (Fig. 4). Solventextraction represents a suitable method for obtaining theacidic cannabinoids [26a, 37]. This involves sequentialextraction with a non-polar phase (chloroform orn-hexane)and a polar phase (methanol or ethanol), while takingparticular care to prevent decarboxylation (4-25C workingconditions). Lyophilization is therefore the method of choicefor drying the extract. Flower tops obtained from cannabisvarieties were deep-frozen after harvesting, followed bylyophilization (700 mg) and extraction (chloroform/methanol, 1:9, 2 x 20 mL). The extraction procedure invol-

ves adding methanol (18 mL) to the plant material, soni-cation (5 min), addition of chloroform (2 mL), sonication (5min) and extraction with a mechanical stirrer (60 min, 4C,250 rpm). The supernatant is removed and the procedure isrepeated. Pooling of supernatants yields the final extract(Table 4).

Patent [36b] describes a preparative SFC process for purifying natural or synthetic (1) utilizing a derivatizedpolysaccharide solid chiral stationary phase immobilized onsubstrates such as silica gel, alumina or ceramics. Com-

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), andChiralpak IA, containing ADMPC immobilized on macroporous silica gel (5 !m) (Diacel Chemical Co.) Fig. (5) [38]Encapsulation of the derivatized polysaccharide, which is no

Table 3. SFE (CO2) Decannabinization of Marijuana

SFE # Pressure Temperature Time Marc: (1) Extract: (1) Extraction of (1)

SFE 1 1044 psi 31C 0.5 hrs 0.24% 8.6% 93.1%

SFE 2 1044 psi 31C 1.0 hrs 0.13% 17.4% 96.2%

SFE 3 1044 psi 31C 3.5 hrs 0.23% 41.7% 93.2%

SFE 4 2176 psi 58C 4.0 hrs 1.88% 36.7% 44.7%

SFE 5 5802 psi 31C 4.0 hrs 0.14% 36.5% 96.0%

SFE 6 5802 psi 31C 4.0 hrs 0.23% 40.1% 93.4%

SFE 7 5802 psi 51C 5.0 hrs 0.22% 32.5% 93.7%

SFE 8 5802 psi 60C 2.5 hrs 0.22% 28.6% 93.5%

SFE 9 6527 psi 45C 5.0 hrs 0.31% 28.5% 90.9%

SFE 10 6527 psi 62C 6.0 hrs 0.12% 38.2% 96.5%

SFE 11 6527 psi 60C 5.0 hrs 0.14% 15.9% 95.9%

SFE 12 6527 psi 55C 7.0 hrs 0.07% - 97.9%

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

O

R5

R4

R1

R2

COOH

R3

HO

R5

R1

R2

COOH

R3

R4


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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 moremodifiers such as ethanol, acetonitrile or dichloromethane(CO2/modifier, 85:15 to 75:25, w/v). Optionally, a secondchromatographic step can also be employed wherein anachiral stationary phase, e.g., 2-ethylpyridine siloxaneimmobilized on a silica support Fig. (6), is used inconjunction with a CO2/ethanol mobile phase (CO2/modifier,95:5 to 90:10, w/v). This step is especially useful for theremoval of abnormal !

9-THC (abn-!9-THC) (24) produced

during the synthesis of dronabinol (1) [27a, 36b]. The orderof the two chromatographic steps is not critical, however, itis recommended to perform the achiral stationary phase stepfirst in order to prevent degradation of the ADMPC chiralstationary phase. The SFC column dimensions vary from0.5-50 cm diameter and 5-50 cm length, with particle size between 5-50 !m. Separations are performed at 5-45C atelevated pressures (1160 to 4350 psi) and with 10-4000g/min flow rates, depending on column size. UV detection isespecially suitable for cannabis constituents. In an exampleof a scaled-up two-step purification of a crude syntheticmixture, (1) was isolated with a purity of > 99% Table 5.

Fig. (6). 2-Ethylpyridine siloxane immobilized on a silica support.

Table 5. Purification of Dronabinol (1)

Stationary

Phase

Particle

SizeSupport CO2/ethanol (1)

2-Ethylpyridine

siloxane10 !m Silica 92:8 95%

ADMPC 20 !mMacroporous

silica80:20 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) byconverting (18) found in cannabis extract to a sodium salt bypH manipulation, followed by extraction into a polar solvent.The purified (18) is subsequently converted to (1) andoptionally purified via esterification or chromatography.Although extraction of cannabinoids under pH control has been described [39], solvent selection can be problematic

since cannabinoids other than (18) are also extractedProduction of (1) is achieved by converting the acid to acarboxylate salt under basic conditions and extracting thesalt using a solvent that preferentially dissolves the salcompared to the free cannabinoids, e.g., a basic aqueousolution. This prevents the simultaneous extraction ocontaminants such as (2) and (16). The process consists othe following steps:

1. Milled cannabis is extracted with a non-polar solvente.g. heptane orn-hexane, providing extract rich in (18).

2. The acid is converted to a sodium carboxylate under pHcontrol (pH 12.7-13.2) by extraction into an aqueousdilute NaOH solution. A pH > 13.2 results in a threelayer system, with the carboxylate salt forming an oilylayer between the bottom aqueous and top organiclayers. A pH < 12.7 results in incomplete extraction othe carboxylate salt and high levels of CBD phenolate inthe aqueous phase. Emulsion formation can be reducedby adding NaCl (1%) to the extraction solvent.

3. The salt is extracted into a third solvent, namelyisopropyl ether (IPE). Alternatively, chloroform, diethy

ether or dimethyl ether can also be used.

4. The salt dissolves preferentially in IPE compared to theaqueous solution, while other impurities, such as CBDphenolate, 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 99% purity)

OH

O

O

O

(1) (impure) X = O: !9-THC carbamateX = S: !9-THC thiocarbamate

OH

O

(1) (> 98% purity)

R1-N=C=X

CH2Cl2 / Et3N

N

X

R1

H

K2CO3

EtOH / H2O


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participants reduced their intake by one capsule twice dailyuntil they were completely off of the medication. At the endof the last phase (week 15), the patients were subjected tofinal assessment of the effectiveness of medication bymeasuring the change in spasticity using the Ashworth score[51]. While no significant improvement was observed inspasticity, cannabis treatment caused significant impro-vement in mobility, and the patients reported an overall

improvement in symptoms of pain, sleep quality and musclespasms. A decrease in the incidences of MS relapses wasalso observed in patients treated with either the cannabisextract or dronabinol (1).

In addition to cannabinoid monotherapy, combinedtreatment with (1) and (2) for five weeks was effective inalleviating the central neuropathic pain associated with MS[52]. A two-year follow-up study conducted as an extensionof the five week randomized trial was aimed at evaluatingthe long term efficacy and tolerability of this combinedformulation [53], commercially available in the UK asSativex. Patients titrated their dosage while maintainingtheir existing level of analgesia and reported any adverseeffects they experienced. The study showed that combinedtreatment with (1) and (2) was effective in pain relief up totwo years, with 92% of the patients reporting at least oneadverse effect, most commonly nausea and dizziness. SinceMS is regarded as a relapsing chronic inflammatory diseaseof the CNS [54], the beneficial antiinflammatory effects ofcannabinoids, especially (2), could provide much needed MSsymptom relief.

Patent [55] describes the possible use of ajulemic acid(25), a synthetic derivative of trans-(6aR,10aR)-11-nor-9-carboxy-!

8-THC (!8-THC-11-oic acid), for the management

of pain and inflammation in MS patients, as supported by alengthy review of experimental and clinical data.

The clinical benefits reported for the use of cannabinoids

in MS patients are supported by experimental studies inanimal models of MS [56]. Administration of (1) or (4a) inrat or pig delayed the onset and reduced the severity ofclinical and histological signs of experimentally inducedautoimmune encephalomylitis (EAE). The involvement ofthe CB1 and CB2 receptors in improving the symptoms ofMS exerted by cannabinoid agonists was studied by using amouse autoimmune model of MS [57]. The studies revealedthat the cannabinoid agonists (1) and (8) suppressed thetremor and spasticity exhibited by mice. The effect was bloc-ked by CB1 and CB2 selective antagonists, suggesting thecontribution of both receptor types in mediating theseactions.

Cannabinoids are also employed for alleviating severeand chronic pain that is either centrally or peripherallymediated. Patent [58] describes the potential use of acannabis-based medicine extract for the treatment of peripheral neuropathic pain. The study showed that acuteadministration of a cannabinoid-containing plant extract witha (2)/(1) ratio of 24:1 was effective in relieving neuropathicpain induced in animal models. Chronic constriction injury(CCI) to the sciatic nerve was surgically induced in theanimals one week prior to cannabis extract administration.Animals administered the cannabis extract showed a markeddecreased pain response in both thermal hyperalgesia and

mechanical allodynia pain models. Similarly, repeated dailyadministration of the cannabis extract resulted in effectiverelief of the neuropathic pain in a CCI animal model. In bothcases, the plant extract was more effective than theadministration of either (1) or (2) alone.

Moreover, a follow-up patent [59] describes the results oa six week, double blind, randomized, parallel group placebo-controlled study with the patients receiving cannabis

based medicinal extracts containing (1)/(2) (1:1) combinedwith the regular analgesic drug prescribed to the patientsThe data revealed that patients administered the cannabisextract along with their analgesic drug(s) showed a statistically significant improvement in their symptoms comparedto the patients treated with the analgesic drug(s) alone, andproved to be a well tolerated and effective adjunct therapy particularly in patients unresponsive to existing analgesimedications. An added benefit of cannabis-based therapyrevealed by the study was a significant improvement in the patients quality of life as evidenced by an improved paindisability index (PDI) and relief from sleep disturbances.

Further employment of the analgesic effect of canna

binoids has been described for the treatment of pain aninflammation associated with arthritis. In a seven weekmulti-center, double blind, randomized clinical study, theefficacy of cannabis-based medicine in relieving rheumatoidarthritis associated pain was evaluated [60]. Using equaamounts of (1) and (2), patients used an oromucosal spray todeliver the medication and titrated the dose until theoptimum efficacy of pain relief was achieved. Data collectedfrom the study supported a therapeutic value of cannabinoidin arthritis. Cannabis-based medicine caused significanreductions in morning pain and disease activity score, and asignificant improvement in sleep quality, supporting thepotential use of cannabinoids for the management and relieof arthritis symptoms.

Among the emerging therapeutic applications of cannabiis the management of migraine headaches [61]. Migraine iconsidered a serious public health issue that affects anestimated 23 million Americans [62]. Despite the development of the serotonin 1D agonist, sumatriptan, in theearly 1990s, several problems are associated with its usee.g., poor oral availability, ineffectiveness during the auraphase, cardiovascular side effects and frequent recurrence oattacks. In addition, approximately 30% of patients taking idiscontinued its use due to lack of efficacy, headache recurrence, cost, and/or side effects [63]. A need for alternativemigraine treatment medications is therefore apparentAnecdotal reports have suggested the potential use omarijuana for migraine headaches and despite the lack o

conclusive clinical data, experimental research studies haveshed some light regarding the potential role of cannabinoidin migraine treatment. The cannabinoid agonists (5a), (7and (8) inhibit the 5-HT3 receptor-mediated current in ranodose ganglion neurons [64]. The role of this receptor inemetic and pain responses has been well documented [65]Additional evidence was provided by the finding that the posterior ventrolateral periaqueductal gray (PAG) is aimportant brain area for the antinociceptive action ocannabinoids [66]. The PAG is the brain anatomic regioncommonly 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 ofmoderate to severe migraine attacks. The proposed doseswere 1.2, 2.4 and 3.6 mg/kg to be delivered using a pres-surized metered dose inhaler (MDI). However, no clinicaldata 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 depressionfocuses on drugs that enhance the levels of brainmonoamines in accordance with the established monoaminehypothesis of depression [69]. However, these drugs sufferfrom major drawbacks, e.g. efficacy, onset of action and sideeffects, necessitating the quest for new and improvedantidepressants [70]. It is well recognized that one of thecomponents of the complex experience elicited by cannabisin humans is mood elevation [71]. The notion that thesemood-elevating properties of cannabis could be utilized totreat depression was introduced in the mid nineteenthcentury. Since then, evidence began to accumulate outliningthe role of the endocannabinoid system in the etiology and

treatment of depression, supporting the fact that many patients report benefits from using cannabis to alleviatedepression [72]. However, the exact actions exhibited bymanipulation of the endocannabinoid system are still unclearand confounded by findings that both the activation ofendocannabinoid transmission [73] and blockade of CB1receptors exert antidepressant-like actions in establishedanimal models of depression, e.g. the forced swim and tailsuspension tests [74].

Direct enhancement of CB1 receptor activity byadministration of the CB1 agonists (6) or oleamide (cis-9-octadecenamide) (26) resulted in antidepressant-like effectsin animal models comparable to the tricyclic antidepressant,

desipramine [73b]. Indirect stimulation of the CB1 receptors by administration of the uptake inhibitor AM404 (27) alsocaused potent antidepressant effects. Inhibition of fatty acidamide hydrolase enzyme (FAAH) by administration ofURB597 (28) leads to potent antidepressant-like action in therat 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. Thishypothesis is, however, in conflict with the findings that blockage of the CB1 receptors leads to antidepressant-likeactions in animal models, since administration of the CB1receptor antagonists AM251 (29) and SR141716A(rimonabant hydrochloride) (9a) elicited antidepressanteffects in mice [74b, 75]. In accordance with these findings,

several studies reported neurochemical changes induced by aCB1 receptor antagonist that correspond to antidepressantaction. These changes include enhanced efflux of noradre-naline, 5-hydroxytryptamine and dopamine in various brainregions [76].

In support of the role of cannabinoids for the treatment ofdepression, two patents [34, 77] described the potentialantidepressant-like actions of (3) and (17) in the rodent tailsuspension test. Data provided showed significant dosedependent enhancement of mice activity in the test as well asan increase in the force of struggling behavior as comparedto the established antidepressant drug, imipramine (30

mg/kg). Both parameters confirm potential antidepressanaction for (3) and (17) when administered acutely at doseequal to or greater than 40 mg/kg, i.p.

Although preclinical and some clinical data suggest theinvolvement of the endocannabinoid system in depressionand hence the possible application of cannabinoids intreatment of this disorder, it is evident that further studies arneeded to better elucidate the role of endocannabinoids in the

neurobiology of depression as well as the therapeutic benefiof cannabinoids.

Antiemetic

Nausea and vomiting are among the most distressing sideeffects of cancer chemotherapy and may interfere with thesuccessful completion of cancer treatment. The medicinause of marijuana for the treatment of nausea and emesis ha been evaluated in several clinical trials [78a]. In a doubl blind randomized trial, the effectiveness of (1) in themanagement of nausea in 55 cancer patients suffering from avariety of neoplasms was reported [78b]. The patients wereselected based on reporting severe chemotherapy-inducednausea and vomiting. The trial showed that (1) was effectiveas an antiemetic against several chemotherapeutic drugincluding cyclophosphamide, fluorouracil and doxorubicinhydrochloride. A survey of more than 1000 cancespecialists revealed that 44% recommend (1) or cannabis toat least one of their patients [79]. The primary driving forcebehind the use of cannabis in antiemetic therapy for cance patients is the unresponsiveness of many patients to thwidely used 5-HT3 receptor antagonist, ondansetron.

Patent [80] describes a clinical trial that examined theantiemetic efficacy of orally administered dronabinol (1)either alone or in combination with ondansetron, whenadministered prior to chemotherapy. Although the datashowed a significant antiemetic effect for dronabinol (1)

comparable to that of ondansetron, the combination therapyshowed less efficacy than either drug alone. Clinical use o(1) or cannabis in the management of emesis in cance patients was supported by animal studies, confirming thantiemetic action of (1) and providing ample evidence thathis action is mediated via the CB1 receptor. !

9-THC (1

dose dependently reduced vomiting induced by cisplatin inthe least shrew animal model [81], while the antiemeticeffect was completely reversed by the CB1 antagonist (9abut not the CB2 antagonist (10). Similarly, potent antiemeticaction of (1) against emesis induced by 5-hydroxytryptophan[82] and dopamine D2/D3 agonists [83] has been shownThe potential therapeutic value of (1) is, however, highlyrestricted by its psychoactive effects. The search for related

compounds that lack psychoactivity while retaining themedicinal antiemetic effect is ongoing.

!8-THC (4a) demonstrates enhanced antiemesis effec

against radiation-induced vomiting in the least shrew whencompared to (1) [84], supporting a clinical study showingthat (4a) unequivocally inhibits chemotherapy-inducedemesis in children, an effect not observed for (1) [85]. Theantiemetic effect of non-psychoactive (2) has also beeninvestigated [9b]. CBD (2) suppressed lithium-inducedvomiting in the house musk shrew through a biphasic effectwith doses of 5 and 10 mg/kg suppressing vomiting and


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higher doses potentiating the lithium-induced effect. Thelack of psychoactive properties of (2) together with itsantiemetic effect makes this compound highly appealing forthe management of nausea and emesis [86].

Several patents have been filed in the last five yearsregarding the potential use of cannabinoids as antiemeticand/or antinausea drugs, ranging from those describingmethods to synthesize or extract promising non-psychoactive

cannabinoids to those investigating the antiemetic efficacy ofthese compounds in animal models. Patent [87] focuses onthe cultivation of specific phenotypes of cannabis with theintention of obtaining cannabis extract enriched in (2), (19)and cannabidivarin (CBDV) (30) content. The efficacy ofextracts high in (1) and (2), respectively, was compared in amotion sickness-induced emesis animal model. Inaccordance with other findings, the data provided showed aU-shaped dose response for the antiemetic action of theextract high in (2).

Patent [88] addresses the use of (2) and its synthetichomolog 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, itwas demonstrated that (2) (5 mg/kg, i.p.) and (31)suppressed the establishment and expression of lithiumchloride induced conditioned rejection reactions. Previousdata have established the role of CB1 receptors in theantinausea effect elicited by (1) [89]. Since (2) and (31) haveweak binding affinities to the CB1 receptors, it is postulatedthat the antinausea effect might occur by enhancing thelevels of (5a). However, further studies are needed todelineate such mechanism.

Patent [90] proposed the use of a combinationformulation comprised of (2) and (4a) in a ratio of 1:2-10 asan antiemetic pharmaceutical preparation. The combined useis suggested to provide a potent antiemetic effect withdiminished psychoactive properties. The potential antiemeticvalue of non-psychoactive !

8-THC-11-oic acid and severalof its synthetic analogs, such as (25), was described inpatents [91, 92]. The combined use of these compounds withother antiemetics, including antihistamines andanticholinergic drugs, was proposed in these patents.

Neuroprotection

The neuroprotective value of cannabinoids has recently been the subject of intensive research based on theirantiexcitotoxic, antiinflammatory and antioxidant propertiesunder various experimental conditions [93]. Several studieshave demonstrated that cannabinoid receptor activation

protects cerebellar and hippocampal neurons against damageinduced by excitotoxic insults [94], hypoxia or glucosedeprivation [95]. This was supported by in vivo animalstudies that revealed that these compounds possess superbprotection against neuronal loss following cerebral ischemia[95], excitotoxicity [96] or acute brain trauma [97].Additionally, these protective effects have been demons-trated in models of acute and chronic neurodegenerativeconditions including MS [17b], Alzheimers disease [98] andParkinsons disease [99].

The neuroprotective capacity of cannabinoid-containingplant extracts is described in patent [100]. The data showed

that the extracts significantly reduced the influx of calciumions induced by N-methyl-D-aspartic acid (NMDA) in rahippocampal cultured cells, suggesting neuroprotectionagainst acute as well as long term treatment with NMDA.

An expansion on the neuroprotective capability ocannabinoids was described in patent [101] where thepotential use of the non-psychotropic synthetic cannabinoiddexanabinol (HU211) (32) [enantiomer of (6)] for the

prevention and management of mild cognitive impairmenwas examined. The neuroprotective capability of (32) wademonstrated by its ability to cause a dramatic decrease inthe number of necrotic foci induced by a high dose oCremophor EL/ethanol in a rat brain (50 mg/kg, i.v.). The patent claims that pretreatment with the invented phamaceutical composition of non-psychotropic cannabinoidnotably prevented cognitive impairment associated withsecondary brain injury induced in two animal model(transient occlusion of the vertebral and carotid arterieresulting in global ischemia and microemboli injection inrats). The compounds tested had previously establishedneuroprotective and antiinflammatory properties, promptingthe authors to describe the potential use of these compoundin the prevention of post-operative, disease/drug-inducedvirally-induced, as well as neonatal cognitive impairmentFurthermore, the usefulness of these compounds inmitigating or delaying the progression of mild cognitiveimpairment to chronic neurodegeneration, is discussed.

Several analogs of (32) were synthesized and evaluatedfor their neuroprotective and antiinflammatory actions [102]demonstrating an array of pharmacological actions thasupport their neuroprotective capacity. They all act aNMDA non-competitive antagonists (IC50 values of 0.35-100!M), suggesting their potential protective value againsglutamate excitotoxicity. Further in vitro and in vivoevaluation of these compounds revealed that the C-1

derivatives of trans-(6aR,10aR)-3-(1,1-dimethylheptyl)-!8

THC (PRS-211 series) possessed marked neuroprotectiveaction attributed to their antiinflammatory and antioxidaneffects. The PRS-211 derivatives significantly inhibited theproduction of PGE2, TNF-" and NO in LPS-induced macro phage cell cultures, with the antiinflammatory action confirmed by using the mouse ear edema model. Furthermorethe cerebroprotective effects of the C-11 1H-imidazolederivative (33) (Scheme 3) were assessed in rats followinghead trauma, indicating a significant decrease in edema andneurological deficits as well as significant reduction of brainlesion and neurological deficits in middle cerebral arteryocclusion studies.

Although clinical neuroprotection seems to be an excitingprospect for cannabinoids, clinical data are definitely lackingand its potential will probably require a substantial timeframe for assessment.

Glaucoma Treatment

Glaucoma is an eye disease primarily caused by a rise inintraocular eye pressure (IOP) and is usually classified into primary and secondary glaucoma. In both cases accumulation of the aqueous humor in the anterior chambeincreases intraocular pressure which, if untreated, may leadto damage of the optic nerve head and loss of eyesight. With


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the recent emergence of the neuroprotective merit ofcannabinoids, it was presumed that the protective value mayextend to the eye and could retard the progressive damage tothe optic nerve [103].

Patent [104] claims that (2) offers potent neuroprotectiveaction to the mammalian eye. Abnormal CBD (abn-CBD)(34) (Scheme 4) demonstrates protective action againstexcitatory amino acid toxicity in cultured rat hippocampalneuronal cells, extending to retinal or optic nerve cellsinjured by a deleterious stressor, principally associated withglaucoma or diabetes. The patent also describes a potentocular hypotensive effect of (34) and its homologs/derivatives. The CBD derivatives tested were administeredtopically to the eyes of normotensive and laser inducedunilaterally ocular hypertensive monkeys. The data showthat the abnormal CBD derivatives lower intraocular pres-sure. However, these compounds fail to increase theuveoscleral outflow. Hence, the patent focuses on combiningthese abnormal CBD derivatives with an agent that enhancesthe aqueous outflow from the eye to increase the effec-tiveness in the treatment of glaucoma and ocular hyper-tension.

Patent [105] describes several water and lipid soluble

analogs of (1) and (5a). in vitro Binding to CB1 and CB2receptors resulted in six cannabinoid analogs with CB1 andCB2 binding affinities in the 3-300 nM range. Thesecompounds exhibited typical cannabimimetic activity in themouse tetrad assay, except that they lacked hypothermicaction. Topical application of the compounds causedsignificant reduction of IOP when tested in the rat glaucomamodel. 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 synergisticeffect in reducing IOP as well as prolonging the duration ofaction. Furthermore, results support a high neuroprotective

capacity of these compounds to the retina, particularly theretinal ganglionic cells. Accordingly, these compounds maybe useful in the treatment of glaucoma or for the preventionof retinal ganglion cell loss.

Appetite Control

The stimulatory effect of cannabis on feeding has been primarily attributed to the psychoactive constituent (1). In1992 the FDA approved the use of dronabinol (1) to stimulate appetite in AIDS patients suffering from wasting

syndrome. This triggered further research interest in theeffects exerted by other cannabinoid constituents on foodintake and energy expenditure [106]. Experimental data haveproven that the enhanced feeding behavior elicited by (1) imediated via the CB1 receptors located both centrally and peripherally [107]. A recent interest has developed cannabinoid receptor antagonists since several studies havereported that they might be useful in reducing appetiteRimonabant (9a or 9b), the CB1 receptor inverse agonistattenuates the hyperphagic effects of cannabinoid agonistsinduces hypophagia when administered alone and suppresseappetite [108]. These findings were surprising since theyexpanded the scope of therapeutic application of cannabinoidantagonists to include obesity and related disorders

Consequently, a two year randomized, double blindplacebo-controlled clinical trial was conducted to assess thefficacy and safety of (9b) in reducing body weight andimproving the cardiometabolic risk factors in overweight oobese patients, showing that it caused a modest but sustainedreduction in body weight and favorable changes incardiometabolic risk factors [109].

Patent [110] proposed the combined use of a CB1receptor antagonist and a peroxisome proliferator activatedreceptor alpha (PPAR") agonist to reduce body weight. Therationale behind using a PPAR" agonist stems from theabundant literature advocating its role in all aspects of lipid

Scheme (3). Synthesis of (33).


OH

O

(32)

Br

CBr4 / Ph3P

1a) 1H-imidazole / xylene

1b) n-BuLi / THF

2) HCl

(33)bromo-intermediate

OH

O

OH

OH

O

NN HCl

OH

OH

OH

(34)

OH

OHoxalic acid dihydrate

toluene / Et2O

+

olivetolp-mentha-2,8-diene-1-ol


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Several research efforts have focused on the preparation ofwater soluble derivatives of cannabinoids with subsequent pharmacological evaluation of their binding affinities toreceptors [114].

Patent [115] describes the synthesis of a series of analogsof (1) with higher water solubility and bioavailability. Thecompounds were evaluated for binding to CB1 and CB2receptors using Chinese Hamster Ovary (CHO) and Human

Embryonic Kidney 293 (HEK 293) cells, respectively. Mostof the compounds tested had affinities to both the CB1 andCB2 receptors. The 1H-imidazol-1-yl analog (35) (Scheme5) [116] possessed high CB1 receptor agonist affinity andsimilar efficacy to the synthetic cannabinoid (7) in thefunctional GTP!S [guanosine 5'-(!-thiotriphosphate)] assay.In addition, the high pharmacological potency of thiscompound in the mouse behavioral tetrad assay emphasizedits action as a cannabinoid agonist. The patent claims thatsuch compounds could have potential therapeutic appli-cations in the treatment of disorders involving the CB1 andCB2 receptors, e.g. appetite loss, pain, MS, nausea, vomitingand epilepsy.

Enhancement of Cannabinoid Receptor Selectivity

Over the past few years, a growing body of evidence hasaccumulated supporting the role of the CB2 receptors in theimmunomodulatory, anticancer and antiinflammatory effectsof cannabinoids. These findings, in addition to the fact thatthe CB1 receptors mediate the psychotropic activities ofcannabinoids, have lead to extensive interest in thedevelopment of highly selective CB2 ligands [117].

The basic structural parameters required for cannabinoidbinding affinity to the CB1 receptor are the following: 1) afree hydroxy at C-1, 2) a "(8,9) or"(9,10) double bond, and anexocyclic C-11 methyl or C-11 hydroxymethyl, or ahexahydrocannabinol skeleton with a 9#-hydroxy, 9#-

hydroxymethyl or 9-keto functionality, and 3) a C3-C7aliphatic side-chain at C-3. Substitution of the C-3 side-chainwith 1,1-dimethyl, 1,2-dimethyl or 1,1-dithiolane moietiesgenerally enhances the cannabinoid activity. Several studiesindicated that the ligand binding pocket of CB1 prefers ahydrophobic substituent at C-3, however, the requirementsfor conformational flexibility are still unresolved. Hydrogen bonding between the C-1 hydroxy and the side-chainnitrogen of Lys192 in transmembrane helix 3 of the

cannabinoid receptor, is also critical [118]. The SAR for theCB2 receptor has not been elucidated as extensively as fothe CB1 receptor. It is, however, clear that beneficial CB2selectivity requires not only moderate to high affinity at theCB2 receptor, but also low affinity and efficacy at the CB1receptor [117].

The objectives of patent [119] were to develop "8-, "9

and "(6a,10a)-THC analogs as CB1/CB2 receptors agonists o

antagonists for potential treatment of illnesses mediated bythese receptors. The fact that the binding properties of (4aare similar to those of (1), in addition to the enhancedstability and less expensive total synthesis of the formercompound, makes (4a) an attractive alternative whendesigning derivatives [120]. The "

8- and "

9-THC analog

were prepared by reacting resorcinol derivatives with cis-pmentha-2-ene-1,8-diol and cis-p-mentha-2,8-diene-1-olrespectively. The "(6a,10a)-THC analogs were prepared byreacting resorcinol derivatives with a cyclic #-ketoester via6-nor-6-oxo-"

(6a,10a)-THC and CBD-type intermediate

(Scheme 6). The synthesis of 1-deoxy and 1-alkoxyderivatives are also described (Scheme 7). A number of"

8

THC analogs with phenyl side-chains were synthesizedincluding (36)-(37) (Scheme 8), and their CB1/CB2 bindingaffinities assessed using membrane preparations of thehuman receptors transfected into HEK 293 Epstein-Barnuclear antigen (EBNA) cells. Receptor binding assays werecarried out using (7) and (8) as the competing radioactiveligand and for determining non-specific binding, respectively[121]. The CB1 (12-297 nM) and CB2 (0.9-86 nM) bindingaffinities of the analogs were comparable to those of (4a(Table 6), with (36) exhibiting good binding affinities fo both the CB1 and the CB2 receptors and (37) exhibitingdecreased binding affinity for the CB1 receptor.

Patent [122] describes the synthesis of fluorescenderivatives of cannabinoids for use as biosensors, molecular

probes and imaging agents, and to provide temporal, spatiaand dynamic data on receptor-ligand interactions. Radiochemical methods for investigating the cannabinoid systemand cannabimimetic molecules have several disadvantagese.g., high cost, handling and disposal difficulties, and potential health hazards. Fluorescence methods circumvensome of these shortcomings in addition to being moreaccurate, sensitive, efficient, safe and generally less costlyThis alternative methodology provides an additional tool to


OHOMe

CN

(4-bromobutoxy)benzene

THF / reflux

MeO

OMe

MeOOPh

O

1) Me2Zn / TiCl4

2) BBr3

OH

HO

Br

HO

p-TsOH

benzene / 80o

C

OH

OBr

OTBDMS

OBr

TBDMSCl 1H-imidazole

NaH / DMF

(35)

OH

ON

N


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Patent [124] describes the synthesis of a series of tetra-and hexahydrocannabinol analogs that exhibit preferentialCB2 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 inthe formalin model of inflammatory pain in mice, indicatingsignificant antinociceptive activity, emphasizing thepotential therapeutic role of CB2 agonists in the treatment ofpain and inflammation.

Patent [126] describes the synthesis of 1-O-methyl-, 1-deoxy-11-hydroxy- and 11-hydroxy-1-O-methyl-!

8-THCderivatives with CB2 receptor selectivity (12, 14) (Scheme13) Table 6 [127]. Compound (12) displayed significantlyenhanced CB2 activity and selectivity ascribed through SARto the 1,1-dimethylbutyl side-chain at C-4. The 1-O-methyl

series displayed low CB1 affinity, while the 11-hydroxy-1

O-methyl series displayed intermediate affinity for bothreceptors, indicating the SAR importance of the 11-hydroxy

moiety. The length of the C-3 alkyl side-chain is also criticain determining receptor affinity, with five carbons aminimum requirement for significant CB1 affinity. Howeverfor the 1-deoxy-!

8-THC derivatives, a reduction in side

chain length did not significantly reduce CB2 receptoaffinity. Affinity to the receptors was evaluated in rat whole brain (CB1) and HEK 293 cell (CB2) membrapreparations [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

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

OHHO

OH

O

OMe

MeOH

O

OMe

MeOPh

O

1) PhMgBr

2) PCC

1) Me2Zn / TiCl4

2) BBr3

OH

HOPh

OH

HOPh

O

BBr3

OH

O

Ph

Ph

O

(36)

(37)

p-TsOH

benzene / 80oC


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ACKNOWLEDGEMENTS

This project was supported in part by Contract NumberN01DA-5-7746 from the National Institute on Drug Abuse,and by Grant Number 5P20RR021929 from the NationalCenter for Research Resources. The content is solely theresponsibility of the authors and does not necessarilyrepresent the official views of the National Center forResearch Resources or the National Institutes of Health.

CONFLICT OF INTEREST

The authors have no conflicts of interest to declare.

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Naturally Occurring and Related Synthetic Cannabinoids and Their Potential Therapeutic Applications