1

Cannabis and Bioactive 1

Cannabinoids 2

3

Federica Messina, Ornelio Rosati, Massimo Curini, M. Carla Marcotullio* 4 Department of Pharmaceutical Science, University of Perugia, Via del Liceo, 1 06123, Perugia, 5 Italy 6

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CANNABIS 10

Cannabis is a genus of flowering plants of the Moraceae family [1]. It 11 includes three putative varieties: Cannabis indica, Cannabis sativa, and 12 Cannabis ruderalis, showing some morphological difference from each 13 other, e.g. height of the mature plant, quantity of branches, bushy growth and 14 quantity of bore flowers. Some of these characteristics are shown in Figure 1. 15

16

17 FIGURE 1 Morphologic differences between Cannabis subspecies 18

19 Cannabis is dioecious, flowering herb. Cannabis indica is annual 20

while Cannabis sativa is biannual. The leaf shape is digitate, palmately 21 compound, with serrate leaflets. The first pair of leaves usually have a single 22 leaflet, the number gradually increasing up to usually seven or nine leaflets 23 per leaf, depending on variety and growing conditions. At the top of a 24 flowering plant, this number diminishes to a single leaflet per leaf. The lower 25 leaf pairs usually occur in an opposite leaf arrangement and the upper leaf 26

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pairs in an alternate arrangement on the main stem of a mature plant. The 27 leaves have a peculiar and diagnostic venation pattern [2]. Cannabis 28 normally has imperfect flowers, with staminate "male" and pistillate "female" 29 flowers occurring on separate plants [3]. It is not unusual for individual 30 plants to bear both male and female flowers [4]. Male flowers are usually 31 borne on loose panicles while female flowers are borne on racemes. All 32 known strains of Cannabis are wind-pollinated. 33

The fruit is an achene. Most strains of Cannabis are short day plants, 34 with the possible exception of C. ruderalis which is commonly described as 35 "auto-flowering" and may be day-neutral [5]. Cannabinoids, terpenoids, and 36 other compounds are secreted by glandular trichomes that occur most 37 abundantly on the floral calyxes and bracts of female plants. As a drug it 38 usually comes in the form of dried flower buds (marijuana), resin (hashish), 39 or various extracts collectively known as hashish oil. 40 41

Cannabis in History 42 43 The therapeutic use Cannabis sativa, commonly known as hemp, is known 44 since ancient times. It was certainly cultivated in China in 4000 BC, and is 45 included in the oldest known pharmacopoeia, the Pen Ts'ao Ching, 46 traditionally attributed to the legendary emperor Shen Nung (III millennium 47 BC), where it is recommended against "female’s disorders, gout, rheumatism, 48 malaria, constipation and muscular weakness" [6]. Around 220 AD the great 49 Chinese surgeon Hua T'o describes the use of Cannabis as analgesic and 50 anesthetic. Later, the therapeutic indications were further extended to wounds 51 healing, against emaciation, in the removal of pus, in rheumatism treatment 52 and to reduce fever and anxiety [7, 8, 9] 53

In India, Cannabis is cited in Atharvaveda (II millennium BC) as 54 "plant that releases anxiety", while in the oldest medical textbook of 55 Ayurvedic tradition, based on the doctrine of Sushruta (II millennium BC), it 56 is mentioned as "remedy". Actually, in Indian culture Cannabis plays a 57 peculiar role: as a plant sacred to Shiva, it is used in religious rituals, as 58 intoxicating is widely used in popular culture and finally, as drug is used in 59 various traditional medicines (i.e. Ayurveda, Unani, Tibbi). 60

According to the note written by J. M. Campbell included in Appendix 61 III of the famous Indian Hemp Drugs Commission Report (1893-4), 62 Cannabis is used in the treatment of fever, described as “being active not 63 directly or physically as an ordinary drug, but indirectly or spiritually, 64 calming the angry spirits that the fever is due to", also is described to have 65 many other medicinal virtues [10]. With regard to the Middle East and the 66 Mediterranean area, where the Cannabis has a role as intoxicating and "social 67 drug", there are only few ancient citations of medical interest. Among the 68 others, the Assyrian medical tablets from the library of Ashurbanipal (seventh 69 century BC) report hemp as antidepressant. The most important handbook 70 referring to Cannabis dates back to 70 AD: in "Materia Medica" Dioscorides 71 shows the oldest known depiction of the plant and recommends its use in 72 earache, to reduce edema and against yellow jaundice. A century later 73 according to Galen (II century AD), the most famous physician of the Roma 74

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Empire, hemp preparations are useful against flatulence and a panacea in the 75 treatment of all kinds of pain, admonishing “if you overdo the dose it affects 76 the head, getting into it hot vapors and intoxicants". Throughout the Middle 77 Age and the Renaissance, the most important use of Cannabis is to obtain 78 fibers for rope, textiles and paper [11]. The ropes, rigging and sails of the 79 ships were usually obtained from hemp and this is the main reason why the 80 plant, already widely cultivated in Europe, was imported to America, to the 81 south by Spanish and Portuguese vessels, and to the north by British and 82 French. In this period there are also interesting medical notations from Garcia 83 de Orta, a physician at the service of the Portuguese viceroy in Goa, India. In 84 his "Colloquies on the Simples and Drugs of India" (1563) he mentions the 85 use of Cannabis as an appetite stimulant, as well as a “sleeping adjuvant”, 86 hypnotic, aphrodisiac and euphoric [12]. Unfortunately almost all the copies 87 were burned under the Inquisition. A similar contribution was given shortly 88 after by Cristobal Acosta in the opera " Treatise of the drugs and medicines of 89 the East Indies" (1578) [13]. Later, Englebert Kampfer, medical-botanical-90 historical German diplomat, ambassador of the King of Sweden in Persia and 91 later chief physician of the fleet of the Dutch East India Company (VOC), 92 describes in his "Amenitates exoticarum" (1712) the use of many medicinal 93 plants, including Cannabis [14]. 94

With regards of Europe, Robert Burton in his classic "The anatomy of 95 melancholy" (1621) [15] suggests the possible utility of Cannabis to treat 96 depression. In the famous The Complete Herbal by Nicolas Culpeper (1653) 97 are listed in detail all the at-the-time known medical applications of Cannabis 98 [16]. In 1682, the New London Dispensatory states that cannabis "cure cough 99 and jaundice but fills the head of steam of dizziness". The New English 100 Dispensatory (1764) recommends to boil the hemp’s roots and apply the 101 decoction on the skin to reduce inflammation, as well as to "dry out cancer" 102 and to dissolve the "deposits in the joints" [17]. 103

In 1753 Linnaeus named hemp as Cannabis sativa, considering the 104 existence of a single species [18], while in 1785 Lamarck, on the basis of 105 significant morphological differences, distinguished the genus Cannabis into 106 two distinct species: C. sativa, native to Europe, and C. indica, native to Asia 107 [19]. The Dictionnaire des Sciences Médicales (1812) reports that the part of 108 the plant used for medical purposes in Europe are the seeds [20]. The 109 importance of Cannabis, been always marginal in Western medicine, was 110 definitely increased in result of Napoleon's Egyptian campaign (1798), after 111 which the hashish, essentially an intoxicant and euphoric, became well 112 known in France, especially among intellectual circles such as the famous 113 Club des Hashishins, attended by figures such as the psychiatrist J.J. Moreau 114 de Tours [21] and artists such as Gautier, Dumas, Nerval, Hugo, Baudelaire 115 and Delacroix. The books having the most influence in the West were "On the 116 preparations of the Indian Hemp, or Gunjah" by William B. O'Shaughnessy 117 [22], a British physician serving in India and "De typhus fever ou d'orient a 118 suivi d' essai sur le hachisch" of L. Aubert-Roche [23], in addition to the "Du 119 Hachisch et de l'aliénation mentale " by J. J. Moreau de Tours, published in 120 1845. While Aubert-Roche reports the use of hashish against the plague and 121 Moreau de Tours considers it a useful tool to investigate the mind, it is an 122

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effective drug in various mental illnesses (melancholia, including form of 123 obsession and hypomania, and chronic mental illness in general). 124 O'Shaughnessy draws on the vast Indian medical tradition and shows the 125 richest repertoire. After an extensive exploration through the medical 126 literature, O'Shaughnessy reports fully on the use of Cannabis in the 127 following conditions: acute and chronic rheumatism, hydrophobia, cholera, 128 tetanus and infantile convulsions. After a notation on the "delirium" caused 129 by chronic intoxication, he reports the methods used to prepare the extracts 130 and tincture "gunjah", and recommends different dosages in depending on the 131 case. From this period the medical use of Cannabis experienced a certain 132 spread in the West: extracts and tinctures made from Cannabis were 133 displayed on the shelves of pharmacies in Italy and in Europe as in the U.S. 134 until the I World War and beyond [24]. 135

Between 1840 and 1900, were published more than 100 articles on the 136 medical uses of Cannabis as a potent narcotic, analgesic, hypnotic, on its 137 properties in the treatment of convulsions, hysteria, depression and 138 dysmenorrhea”. Cannabis was included on U.S. Dispensatory in 1854 [25] 139 with the following properties: "powerful narcotic”. It is said that also acts as 140 aphrodisiac, stimulates the appetite and occasionally induce a state of trance; 141 produces sleep, relieves spasms, calms nervous restlessness, relieves pain; as 142 an analgesic differs from opium mostly because it not decreases the appetite, 143 does not reduce the secretions and does not cause constipation. Disorders in 144 which it is specially recommended are neuralgia, gout, tetanus, hydrophobia, 145 epidemic cholera, convulsions, chorea, hysteria, mental depression, insanity 146 and uterine bleeding. 147

In 1860, Cannabis is so highly considered as to determine the 148 appointment of a "Committee on Cannabis indica" by the Medical 149 Association of Ohio. In the report published by the Committee (ed. RR 150 M'Meens), is recognized the utility of Cannabis to treat tetanus, neuralgia, 151 postpartum hemorrhage, childbirth pain, dysmenorrhea, convulsions, 152 rheumatism, asthma, psychosis, post-partum depression, chronic cough, 153 gonorrhea, chronic bronchitis, gastric pain, and more. In addition, it is useful 154 as a sleeping pill and as a drug that stimulates the appetite. H.C.J. Wood 155 reports that Cannabis indica is "used primarily for pain relief, to calm states 156 of restlessness and malaise, to alleviate the suffering of incurable diseases, 157 such as tuberculosis at the last stage, and finally as a mild sleeping pill" [26]. 158

The world’s leading medical journal The Lancet in 1887 recommends 159 the use of Cannabis "night and day, and continued for some time" as "the 160 best available remedy in the treatment of persistent headache” [27]. More 161 than twenty years later William Osler, one of the fathers of modern medicine, 162 believed the Cannabis to be "probably the most satisfactory remedy" for 163 migraine [28], while J. Brown writes in the British Medical Journal indicates 164 that Cannabis "should have the first place in the treatment of menorrhagia" 165 [29]. 166

In conformity with the rest of Europe, dates from this period also the 167 first Italian experience and scientific publications on Cannabis, carried out by 168 the most illustrious names of the medical profession of that time, including 169 Giovanni Polli, Carlo Erba, Andrea Verga, Filippo Lussana [24]. In the 170

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Official Italian Pharmacopoeia (FU) were present both the extract and 171 tincture of Cannabis indica. 172 In 1937 in the U.S. was issued the Marijuana Tax Act, signed by President 173 Franklin Delano Roosevelt, which marked the beginning all around the world 174 of what is commonly referred as Prohibition of Cannabis. Starting from the 175 years of American prohibition, scientific studies that take into account the 176 medical use of Cannabis become very rare, and it is only around 1960 that a 177 timid interest awoken and scientific studies on Cannabis and cannabinoids 178 begun to reappear. 179

Up to date hemp is legally grown when used for textile purposes. To 180 satisfy the UN Narcotics Convention, some Cannabis strains have been bred 181 to produce minimal levels of the principal psychoactive constituents. 182 Moreover, a certain therapeutic use of Cannabis and cannabinoids is legal in 183 some parts of the world, but there are still many controversies on the topic 184 and the authorization on the market are spread in a non-homogeneous 185 manner. In the past 30 years the scientific community made extensive studies 186 to understand Cannabis mechanism of action and to develop some 187 “solutions” to take apart the therapeutic effect from the undesired side effects, 188 mainly the psychotropic activity. 189 190 191

THE ENDOCANNABINOID SYSTEM 192 193 The endocannabinoid (EC) system, consists of two G-protein coupled 194 receptors, CB1 and CB2, several arachidonoyl-derived endogenous ligands, 195 named endocannabinoids, the most abundant and well-studied representatives 196 are N-arachidonoylethanolamine (AEA or anandamide) and 2-197 arachidonoylglycerol (2-AG), and different enzymes involved in the 198 biosynthesis and degradation of the endocannabinoids [30]. All components 199 of the latter category belong to the serine hydrolases superfamily and show a 200 very good substrate-selectivity: fatty acid amide hydrolase (FAAH) is 201 responsible for the degradation of anandamide, monoacylglycerol lipase 202 (MAGL) is the key enzyme involved in the hydrolysis of 2-AG and recently 203 α,β-hydrolase-6 and -12 (ABHD-6 and -12), have been identified to 204 participate in the 2-AG hydrolysis in several tissues, especially in the brain 205 [31, 32]. Emerging evidence implicates endocannabinoids in a wide variety 206 of physiological and pathophysiological processes. To date, most drugs used 207 therapeutically that interact with the endocannabinoid system are derived 208 from Cannabis and produce their effects by modulating cannabinoid 209 receptors activity. Regrettably, the psychoactivity of these compounds has 210 prevented their widespread acceptance and application in Western medicine. 211 In the past decade, the components elucidation of the endocannabinoid 212 system and a better understanding of its role have broadened the therapeutic 213 possibilities for its manipulation. For example, cannabinoid receptors can be 214 directly manipulated by ligands that bind or block them or indirectly by 215 molecules that modulate the levels of the endogenous ligands. Studies in 216 these fields have continued apace: during the last two decades two 217 cannabinoid receptors, CB1 and CB2, have been cloned [33], several 218

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endogenous cannabinoids have been identified, and the synthetic and 219 degradative pathways for the endocannabinoids have been partially 220 elucidated [34]. CB1 and CB2 are seven transmembrane G-protein coupled 221 receptors, which signal transduction is prevalently associated to Gi/0 proteins 222 [35]. Cannabinoid receptors are activated by three major groups of ligands, 223 endocannabinoids (produced by the mammalian body), plant cannabinoids 224 (such as THC, produced by the Cannabis plant) and synthetic cannabinoids. 225

CB1 is found primarily in the brain, primarily in the basal ganglia and 226 in the limbic system, including the hippocampus. CB1 receptors are absent in 227 the medulla oblongata, the part of the brain stem responsible for respiratory 228 and cardiovascular functions. CB1 are expressed on several types of cell in 229 pituitary gland, thyroid gland, and possibly in the adrenal gland. CB1 are also 230 expressed in several peripheral tissues, such as liver, kidney, skin, lungs, 231 gastrointestinal tract, immune system, adipocytes and bone [36]. They are 232 also found both in male and female reproductive systems. CB1 receptors, 233 besides appearing to be responsible for the euphoric and anticonvulsive 234 effects of Cannabis, are also involved in several other biological activities 235 such as cardiovascular, gastrointestinal, pain, olfaction, anxiety response to 236 novelty and liver de novo lipogenesis [37]. CB2 are mainly found in 237 peripheral tissues and in particular in the immune system. CB2 receptors have 238 been also identified in the CNS, in glial cells and astrocytes, while the 239

expression in neurons is still a debated issue in the scientific community [37]. 240 CB2 receptors are also found throughout the gastrointestinal system, where 241 they modulate intestinal inflammatory response. Thus, CB2 receptor agonists 242 are a potential therapeutic target for inflammatory bowel diseases, such as 243 Crohn’s disease and ulcerative colitis [38]. 244

CB2 receptors are involved in several physio-pathological conditions 245 mainly due to their ability to modulate the immune cells activity. The 246 modulation of CB2 activity is an appealing therapeutic option, because it 247 lacks the typical psychotropic side effects linked to CB1 modulation. The 248 affinity of an individual cannabinoid to each receptor type determines the 249 selectivity of the effect and consequently the risk/benefit ratio of that 250 cannabinoid. Cannabinoids that bind more selectively to CB2 are more 251 desirable for medical purposes. Due to the multiple activity of the CBs 252 receptors, the endocannabinoid system is becoming an interesting target for 253 the treatment of several diseases, among the others neuroinflammation and 254 related pathologies, cancer, endocrine and motor dysfunctions. 255

Endocannabinoids 256

The endogenous ligands known as endocannabinoids are natural agonists of 257 cannabinoid receptors, metabolites of arachidonic acid. The name 258 “Endocannabinoid” was given by a group of Italian researchers in 1995 [39] 259 and identifies a new class of neurotransmitters that share the ability to bind to 260 cannabinoid receptors. 261

The term "endocannabinoid system" identifies the set of CB receptors, 262 their ligands and the all the enzymes and proteins that regulate the 263 concentration of ligands at the receptors. Their discovery comes from the 264

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work of Mechoulam and his colleagues [40]. At the time, they hardly 265 believed in the existence of an endogenous agonist for the CB1 receptor able 266 to promote and activate a biological response of the receptor. The first 267 isolated and identified endocannabinoid was N-arachidonoylethanolamine or 268 anandamide (AEA, 1), followed by 2-arachidonoylglycerol (2-AG, 2), 269 structure are shown in Figure 2. 270 271

NH

OH

O

O

O

OH

OH

1 2

272 FIGURE 2 First isolated endocannabinoids. 273

274 Both are characterized by a portion derived from arachidonic acid 275

condensed with one molecule of ethanolamine or glycerol, respectively. AEA 276 and 2-AG show a different activity at the cannabinoid receptors CB1 and 277 CB2. Anandamide (1) was named after the Sanskrit word "ananda", which 278 means "state of grace” [40]. It preferentially binds the CB1 [41] receptor and 279 exhibits an activity similar to THC’s [42]. It is degraded primarily by the 280 fatty acid amide hydrolase (FAAH) enzyme, which converts AEA into 281 ethanolamine and arachidonic acid. As such, inhibitors of FAAH lead to 282 elevated anandamide levels and are being pursued for therapeutic use [43]. 283 Moreover AEA (1) not only binds CB receptors but, as capsaicin (3), an 284 active ingredient in chili peppers (Figure 3), shows the ability to stimulate the 285 transient receptor potential vanilloid type 1 (TRPV1), which would explain 286 the vasodilator effect of AEA (1). 287 288

NH

O

HO

O

3

289 FIGURE 3 Structure of capsaicin. 290

291 2-AG (2), whose activity in the endocannabinoid system was first 292

described in 1995 [44], is an endogenous agonist of the CB1 receptor. It is an 293 ester formed from the omega-6 fatty acid arachidonic acid and glycerol. It is 294 present at high levels in the central nervous system [45], with cannabinoid 295 neuromodulatory effects. 2-AG is synthesized from arachidonic acid-296 containing diacylglycerol (DAG). Recently in vitro studies suggested that 2-297 AG (2) is able to stimulate higher G-protein activation than anandamide, 298 although the physiological implications of this finding are not yet known 299 [46]. In 2001, a third ether-type endocannabinoid, 2-arachidonyl glyceryl 300 ether (noladin ether, 4), was isolated from porcine brain [47]. It is a stable 301

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analogue of 2-AG (2), and some controversies about its classification among 302 endocannabinoid exists, since other research groups failed to detect it in 303 appreciable amounts from the brain of different mammalian species. 304 305

O

OH

OH

6

4

NH

5

O

NH2

O

O

OH

OH

306 FIGURE 4 Endocannabinoids structures 307

308 Nonetheless, noladin ether (4) actually binds almost selectively to 309

CB1 receptors (Ki = 21.2 nmol/L) causing sedation, hypothermia, intestinal 310 immobility, and mild antinociception in mice. N-Arachidonoyl dopamine 311 [48] (NADA, 5) is an amidic conjugate of arachidonic acid with dopamine 312 and was recently identified as an endogenous capsaicin-like compound that 313 acts as an agonist of the CB1 receptor and TRPV1 ion channels [49]. A fifth 314 endocannabinoid, virodhamine (6) or O-arachidonoyl-ethanolamine (OAE), 315 was discovered in 2002 [50]. It is a non-classic eicosanoid, derived from 316 arachidonic acid and ethanolamine, joined by an ester linkage, the opposite of 317 what found in anandamide (1). Although it is a full agonist at CB2 and a 318 partial agonist at CB1, it behaves as a CB1 antagonist in vivo. Structures are 319 shown in Figure 4.Other ligands are suggested to be endocannabinoids are N-320 dihomo-γ-linolenoyl ethanolamine (7), N-docosatetraenoyl ethanolamine (8), 321 oleamide (9) and N-oleoyl dopamine (OLDA, 10) [51] (Figure 5). 322

323

8

NH

10

NH

O

OH

O

OHNH

OH

O

7

NH2

O

OH

9

324 FIGURE 5 Other molecules that are suggested to be endocannabinoids. 325

326

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CANNABINOIDS 327

328 In general, are called “cannabinoids” several classes of diverse chemical 329 compounds, able to interact with the endocannabinoid system, mainly at 330 receptors level. Cannabinoids can be divided into two main different 331 subclasses: phytocannabinoids, which are natural compounds found in 332 Cannabis and some other plants, and synthetic cannabinoids. 333

334

Natural Cannabinoids or Phytocannabinoids 335 336 It is called “phytocannabinoid” any plant-derived natural product capable of 337 either directly interacting with cannabinoid receptors or, in general, with the 338 other constituents of the endocannabinoid system. In the last few years, 339 emerged the evidence that, apart from the terpenophenolic constituents of 340 Cannabis sativa Δ

9-THC and some of its naturally occurring derivatives, 341

several natural cannabinoids from non-Cannabis plant exist. 342

Numbering Systems 343

(−)-trans-Δ9-THC and (−)-trans-Δ

8-THC are the best known natural 344

occurring THCs in the Cannabis hemp, but more isomers of THC are 345 possible by changing the position of the double bond and varying the 346 stereochemistry. There are seven structural isomers of THC, and each has 347 multiple stereoisomeric forms (total 30 stereoisomers). In the number 348 notation of cannabinoids two systems are commonly used: IUPAC 349 dibenzopyran based numbering and monoterpene based numbering (Figure 6) 350 [52]. Both dibenzopyran and monoterpene numerations are accepted and 351 largely used in the literature, albeit in the recent years IUPAC numeration 352 became predominant. 353

For instance, Y. Gaoni and R. Mechoulam in the paper about the 354 isolation and identification of what now is worldwide known as Δ

9-THC (A), 355

refer to it as Δ1-THC (B) [53]. Another example is Δ

6a-THC, in benzopyran 356

numeration, which is commonly known as Δ3-THC, using instead 357

monoterpene numeration. 358 359

OH

CH3

O

H3C

H3C

OH

CH3

O

H3C

H3C

(A) ∆9-THC

Dibenzopyran numbering

(B ) ∆1-THC

Monoterpenoid numbering

11

9

10

10a 1

2

3

45

6

7

8

1a

7

1

2

3 1' 2'

3'

4'

5'6

10

9

8

45

6

360 361

FIGURE 6 THCs Numbering. 362

363

10

Phytocannabinoids from Cannabis sativa 364 365 Cannabis sativa contains hundreds of different secondary metabolites; of 366 these, up to date 85 are considered belonging to the family of cannabinoids, 367 known for their psychotropic effects [54]. 368 369

Classes of Cannabinoids and Biosynthesis 370 371 In 1940, Thomas Wood and colleagues isolated cannabinol CBN (11) from a 372 Cannabis extract; at that time it was thought to be the active psychotropic 373 molecule of the plant. In the meantime Robert Cahn elucidated several 374 fragments of the structure [55]. On the basis of these studies, Alan Todd [56] 375 in England and Roger Adams [57] in the U.S. completed almost 376 simultaneously the synthesis and the chemical characterization of CBN (11) 377 (Figure 7). In 1964 a group of Israeli researchers led by R. Mechoulam 378 isolated and elucidated the structure of the true active ingredient of Cannabis 379 [53], the Δ

9-tetrahydrocannabinol (Δ

9-THC) (12) (Figure 7). CBN (11) is 380

actually the primary product of THC degradation, and there is usually little 381 amount of it in the fresh plant. 382 383

OH

CH3

O

H3C

H3C

12

OH

CH3

O

H3C

H3C

11

384

FIGURE 7 Cannabinol (CBN) and Δ9-tetrahydrocannabinol (Δ9-THC) structures. 385 386

With regards to the biosynthesis [58] shown in Figure 8, all 387 tetrahydrocannabinols have a monoterpenic C10 unit linked to a phenolic ring 388 having a C5 alkyl chain. The phenolic portion is derived from the 389 condensation of hexanoyl-CoA with three molecules of malonyl-CoA, with 390 the formation of a polyketide skeleton that undergoes cyclization, giving 391 origin to pentylresorcylic acid that, after a decarboxylation, leads to olivetol. 392 Olivetol, in turn, undergo to a C-alkylation with geranyl-OPP, giving the not-393 yet-completely cyclized molecule cannabigerol, CBG (13). From the 394 cyclization of the monoterpene unit, via formation of an allylic carbocation 395 and subsequent configuration inversion of the double bond, cannabidiol CBD 396 (14) is formed. At this point the attack of the phenolic group to the double 397 bond leads to the formation of the heterocyclic system of Δ

9-THC (12). 398

399

11

SCoA

O

3 x Malonyl-CoA

Hexanoyl-CoA

OEnzS

O O

O

OH

HO

O

OH

Penthylresorcilic acid

OH

OH

OPP

OH

OH

OH

OH

H

Cannabigerol

OH

OHHO

OH

Cannabidiol∆9−THC

OlivetolGeranyl-OPP

13

1412

400

FIGURE 8 Biosynthesis of Δ9-THC. 401

Depending on the considered breed of Cannabis, the conditions of 402 growth, and the part of the plant harvested, the percentage of 403 phytocannabinoids may vary in a wide range. The phytocannabinoids 404 represented in high percentage in Cannabis are usually tetrahydrocannabinol 405 Δ

9-THC (12), cannabidiol CBD (14), cannabinol CBN (11), cannabigerol 406

CBG (13) and (±) cannabichromene CBC (15) [59] (Figure 9). 407 408

15

O

OH

409 FIGURE 9 Structure of (±)-cannabichromene 410

411 All the currently known phytocannabinoids from Cannabis can be 412

divided into eight subclasses, as shown in Table 1. Among these, Δ9-THC 413

(12) is considered the progenitor of this class of substances and the main 414 responsible for the pharmacological effects of Cannabis, including its 415 psychotropic activity, although other compounds contribute to some of the 416 effects. CBD (14), while failing to elicit the same psychoactive effects of Δ

9-417

THC (12), has antipsychotic, analgesic and anti-inflammatory activity. It is 418

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also able to modulate the action of Δ9-THC in the brain, prolonging the 419

duration of action and limiting side effects [60, 61]. 420 421

Type Skeleton

Cannabigerol-type

CBG ΟΗ

OH

1

2

3

4

5

6

7

8

Cannabichromene-type

CBC

O

OH

1

23

4

5

67

8

Cannabidiol-type

CBD

OH

OH

1

2

34

5

6

1'2'

3'

4'

5'6'

Tetrahydrocannabinol-

and

Cannabinol-type

THC, CBN O

OH

1

2

3

456

6a

10a

10

9

8

7

Cannabielsoin-type

CBE

O

OH

1

23

4

4a

5

5a

67

8

9 9a

9b

iso- THC- type

iso-THC

O

OH

1

2

3

4

5

6

7

8

10

9

Cannabicyclol-type

CBL

OH

O1

1a

2 3

3a

4

5

6

78

8a8b

8c

Cannabicitran-type

CBT

O

O

422 TABLE 1 Main classes of natural cannabinoids from Cannabis. IUPAC numbering system. 423

424

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A recent study carried out at Virginia Commonwealth University 425 shows that CBC (15), whose activity remained unknown for long time, 426 produces the increase of THC levels in the brain and produces some typical 427 effects of cannabinoids when administered at high doses such as decrease of 428 motor activity, antinociception, catalepsy, hypothermia, and anti-edematous 429 effects, but through a mechanism different from the interaction with 430 cannabinoid receptors, as happens with Δ

9-THC [62]. 431

432

Phytocannabinoids Beyond the Cannabis 433 434 Recently, phytocannabinoids are found to occur in several plant species 435 different from Cannabis, including Echinacea purpurea, Echinacea 436 angustifolia, Echinacea pallida, Acmella oleracea, Helichrysum 437 umbraculigerum, and Radula marginata. According to the definition, these 438 molecules are phytocannabinoids as showing binding affinity at cannabinoid 439 receptors, but they show chemical structures far different from THCs (Figure 440 10). 441

The best known cannabinoids that are not derived from Cannabis are 442 the lipophilic alkylamides (16, 17) from Echinacea species [63]. They 443 exhibited a selective binding CB2 affinity (Ki values CB2 = 60 nM and 57 444 nM, CB1 > 2000 nM and > 6000 nM, respectively) [64]. In the recent years 445 at least 25 different alkylamides (dodeca-2E,4E,8Z,10E/Z-tetraenoic-acid-446 isobutylamides) have been identified [64, 65]. In Echinacea species, 447 cannabinoids are found throughout the plant structure, but are most 448 concentrated in the roots and flowers [66, 67]. Certain Echinacea N-449 alkylamides also inhibit anandamide reuptake in vitro [68]. Tea (Camellia 450 sinensis) catechins have a certain affinity for both human cannabinoid 451

receptors, CB1 (Ki ≈ 40 nM) and CB2 (Ki = 100 to 300 nM) [69]. 452

Epigallocathechin-O-gallate (18) showed a moderate CB1 affinity (Ki = 33.6 453

µM), while binding to CB2 was weaker with inhibition constants exceeding 454

50 µM. β-caryophyllene (19) is a component from the essential oil of 455

Cannabis and other medicinal plants and has also been identified as a 456 selective agonist of peripheral CB2-receptors, in vivo (Ki CB2 = 155.0 nM, 457 CB1 > 1000 nM). It has beed described as a “widespread dietary 458 cannabinoid” [70]. Falcarinol (20), a fatty alcohol found in carrots and 459 relatively widespread in Apiacee, showed a non-selective CB1 affinity (Ki 460 values CB1 = 0.59 µM, CB2 = 2.1 µM) [71]. 461

Rutamarin (21), from Ruta graveolens [72] exhibited a selective CB2 462 affinity (Ki values CB2 < 10 µM, CB1 > 100 µM). Yangonin (22), a 463 kavalactone from Kava plant (Piper methysticum) has been shown to possess 464 significant CB1 binding affinity (Ki CB1 = 0.72 μM, CB2 > 10 µM) [73]. 465 Diindoylmethane (DIM, 23) (3,3-diindolylmethane metabolite from indole-3-466 carbinol), relatively widespread in Brassica genus, was found to be a CBs 467

partial agonist with a certain CB2 selectivity (Ki values CB2 ≈ 1 µM, CB1 = 468

4.3 µM) [74]. As THCs, most of the non-Cannabis-phytocannabinoids are 469 nearly insoluble in water but are soluble in lipids, alcohols, and other non-470 polar organic solvents. 471

14

NH

O

16

H H

19

HO

20

OO

O

O

O

21

O O

O

OH

OH

OH

OH

HO

OH

OH

OH

18

O

O

O

O

22

NH

NH

23

NH

O

17

472 FIGURE 10 Phytocannabinoids beside Cannabis. Molecules interacting with CB receptors 473

474 Beside molecules that bind to CB receptors, there are some other molecules 475

that are suggested to exert certain cannabimimetic effects not by interacting directly 476 with cannabinoid receptors, but with the enzymes within the EC system (structures 477 in Figure 11). Pristimerin (24) is a naturally occurring terpenoid that potently 478 inhibits MAGL (IC50 = 93.0 nM) through a rapid, reversible, and noncompetitive 479 action. Pristimerin (conc. 1 µM) significantly increases 2-AG levels in isolated rat 480 neurons, indicating that it inhibits endogenous MAGL in cultured cells. Moreover, it 481 does not increase levels of palmitoyl ethanolamide, suggesting that pristimerin does 482 not affect the activity of fatty acid amide hydrolase (FAAH) [75]. Euphol (25), a 483 tetracyclic triterpene alcohol, commonly found in Euphorbia spp, inhibits MAGL 484

activity with high potency (IC50 = 315.0 nM) [75]. Kaempferol (26) is a natural 485 flavonol almost widespread in nature. It has been found to concentration-486 dependently inhibit FAAH [76], thus anandamide hydrolysis, in rat brain 487 homogenates (IC50value < 1 µM). β-amyrin (27) is a pentacyclic triterpene that 488

15

exerts its analgesic and anti-inflammatory pharmacological effects via indirect 489 cannabimimetic mechanisms by inhibiting the degradation of the endocannabinoid 490 2-AG without interacting directly with CB receptors. It mainly inhibits MAGL and 491 inhibits ABHD-6 and -12, without affecting FAAH [77]. N-acylethanolamines (28) 492 (NAEs) from plants are reported to not interact with CB receptors, but they have 493 been shown to inhibit FAAH, thus leading to an increase in endocannabinoid tone. 494 N-linoleoylethanolamide and N-oleoylethanolamide, are found in chocolate 495 (Theobroma cacao) and also other plants [78]. 496 497

CO2CH3

O

HO24

OHO

OH O

OH

OH

25

HO

27

R NH

OH

O

R= linoleoyl, oleoyl, palmitoyl

28

H

HO

26

498

FIGURE 11 Phytocannabinoids beside Cannabis. Molecules not directly interacting with CB receptors. 499 500

There are several other natural products that have been suggested to exert 501 some cannabimimetic effects, but the data supporting this hypothesis are not 502 exhaustive or accurate [79]. 503 504 505

Synthetic Cannabinoids 506 507

Since the discovery of Δ9-THC (12), the pharmaceutical industry undertook several 508

studies for the development of synthetic analogues, in an attempt to create 509 compounds that retained the biological activity of natural cannabinoids but devoid 510 of psychoactive side effects. These new molecules included not only compounds 511 structurally similar to the already known phytocannabinoids, but also compounds 512 with different chemical scaffolds. Synthetic cannabinoids include a variety of 513

16

distinct chemical structures and can be divided into two main classes: the classical 514 cannabinoids, structurally related to THCs, and the nonclassical cannabinoids 515 (cannabimimetics) structurally unrelated to THCs. 516

Examples of classical cannabinoids synthetic analogues are HU-210 (29), 517 JWH-133 (30), Nabilone (31) and Dronabinol (32). Cannabinoids defined as "non-518 classical" include bicyclic and tricyclic structures, among these CP 47,497 (33) and 519 its analogues [80]. The group of aminoalkylindoles comprises compounds that do 520

not share any structural similarity with ∆9-THC; among these WIN 55,212-2 (34), 521

AM-1241 (35), JWH-015 (36) are examples. Structures are shown in Figure 12. 522 JWH aminoalkylindoles series is a group of more of 450 molecules, 523

including naphtoylindoles, naphtylmethylindoles, naphtylmethylhindanes and 524 phenylindoles [81], designed and synthesized by John W. Huffman [82] and his 525 research group at Clemson University and up to date is the most numerous class of 526 synthetic cannabinoids. Other famous series of non-classical cannabinoids are AMs, 527 synthesized by Alexandros Makriyannis and coll., CPs, a group cannabinoid 528 receptor agonist drug, developed by Pfizer in the 1980s [83] and HUs prepared by a 529 group led by Professor Raphael Mechoulam at the Hebrew University. 530 531 532

OH

CH3

O

H3C

H3C

12 H3C CH3

OH

O

H3C

H3C

HO

29

N

O

H3C CH3

O

H3C

H3C

30

H3C CH3

OH

O

H3C

H3C

O

OH

CH3

OH3C

31

H3C

32

OH

OH

CH3H3C

33

N

O

N

O

O

34

N

O

N

I

O2N

35 36

533 FIGURE 12 Examples of classical cannabinoids, non-classical cannabinoids and aminoalkylindoles 534

535 536 537 538

17

First Generation of Synthetic Cannabinoids: Non-Selective CB1/CB2 Agonists 539 540 The first analogues of THC, including HU-210 (29) and CP 47,497 (33), were 541 synthesized in the 80s. Their introduction allowed the location and identification of 542 the responses evoked by THC analogues and, subsequently, during the 90s, the 543 discovery of CB1 and CB2 receptors. These first generation of cannabinoids are 544 basically designed with the specific purpose to act as agonists, increasing the 545 binding affinity directly to the receptors. They have a high affinity for the receptors, 546 10-100 folds higher than natural cannabinoids, but are totally non selective [84]. 547

The cannabinoids isolated from Cannabis show usually tricyclic structures 548 characterized by a phenol bearing, in meta to the hydroxyl group, an alkyl chain of 549 five carbon atoms (n-pentylic), a central pyran ring and a monounsaturated 550 cyclohexyl. The addition of another hydroxyl group on the alkyl side chain or on the 551 cyclohexyl moiety lead to poorly active or inactive compounds [85]. However, the 552 hydroxylation of the 11-methyl group linked to the cyclohexyl leads to a metabolite 553 having a CB receptor affinity higher than Δ

9-THC. The replacement of the 11-554

methyl group with a hydroxyl gives a similar product characterized by an enhanced 555 antinociceptive activity [86]. A series of bicyclic and tricyclic analogues of Δ

9-THC 556

have been developed by Pfizer in an effort to prepare non-opioid analgesics, derived 557 from the potent synthetic cannabinoid (-)-9-nor-9β-hydroxyhexahydrocannabinol 558 (HHC, 37, Figure 13). HHC (37) is a synthetic cannabinoid derivative which 559 resulted from early modifications to the structure of THC [87]. HHC is active in its 560 own right with similar potency to THC, but further simplification and variation of 561 this parent structure lead to more potent, yet structurally simpler derivatives [88] 562 such as CP 47,497 (33) [89] and CP 55,940 (38, Figure 13) [90], which after several 563 steps of modification have become quite structurally distinct from THCs, while 564 HHC (37) on the other hand is still substantially similar in structure to THCs. 565 566

OH

OH

HO38

OH

O

OH

CH3

CH3

37

N

N

O

O

O

39

567

FIGURE 13 Non selective CB1/CB2 agonists 568

569

18

SAR for classical cannabinoids remains valid for these non-traditional 570 bicyclic cannabinoid and indicates a maximum activity when bearing a 1,1-571 dimethylheptyl substituent in C-3 position of the aromatic ring and a β orientated 9-572 hydroxhyl group [91]. The simplest non-classical cannabinoid that meets these 573 requirements is CP 47,497 (33), which has been shown to be more potent in vivo 574 than Δ

9-THC. CP 55,940 (38) shows a higher activity, basically due to the presence 575

of a hydroxypropyl group linked to C-4 of cyclohexanol. This molecule was used in 576 1988 by Howlett’s group to identify the first cannabinoid receptor in the rat brain, 577 then called CB1 receptor. An independent research for new antinociceptive 578 compounds carried out by Sterling Winthrop, starting from Pravadoline (39) also 579 known as WIN 48,098, an anti-inflammatory and analgesic drug, led to the 580 optimization of aminoalkylindoles structurally distinct from THC. 581

The structure of Pravadoline (39, Figure 13) is related to the best-known 582 NSAID indomethacin. It was developed in 1980 as a non-ulcerogenic anti-583 inflammatory drug and non-steroidal inhibitor of prostaglandin synthesis, but it was 584 never marketed as a result of its toxicity. Pravadoline (39) showed unexpectedly 585 strong analgesic effects at doses ten times lower than the expected, which therefore 586 could not be explained by its action as an inhibitor of COX. Moreover, the absence 587 of blockade of this activity by opioid antagonists such as naloxone led to the 588 conclusion that Pravadoline (39) was a cannabinoid agonist, the first compound of a 589 new class: the aminoalkylindoles [92]. The most studied compound belonging to the 590 aminoalkylindoles and the prototype of this class is WIN 55,212-2 (34) which is a 591 full agonist at the CB receptors (Ki CB1 = 1.9 nM and CB2 = 0.28 nM) showing 592 much higher affinity at these receptors than Δ

9-THC (12) (Ki CB1 = 41.0 CB2 = 593

36.0 nM) [93]. Notably, the first generation of synthetic cannabinoids show a 594 limited or no CB1/CB2 selectivity. 595 596

Second Generation of Synthetic Cannabinoids: Selective CB1/CB2 597

Agonists/Antagonists/Inverse Agonists 598

599

Selective CB2 Agonist 600

601 Starting from the revolutionary molecule WIN 55,212-2 (34) Huffman, concluded 602 that "a simple alkyl chain could replace the aminoalkyl group" and, with some 603 modifications, gave birth to hundreds of compounds, named JWH after him, mainly 604 characterized by their high binding affinity to the CB receptors. JWH-018 (40) is the 605 prototype of the series [94]. JWH-018 (40) is a full agonist of both the CB1 and CB2 606 cannabinoid receptors (Ki CB1 = 9.00 nM, CB2 = 2.94 nM) and shows the typical in 607 vivo pharmacological activity of cannabinoids. Recently it has been shown that also 608 the metabolites of synthetic full agonists JWH-018 (40) and JWH-073 (41), 609 structures in Figure 14, are potent agonists of CB2 receptor, and bind to it with high 610 affinity [95]. 611

JWH-015 (36), is a naphtoylindole showing certain CB2 selectivity [96]. It 612 binds to CB2 receptors with a Ki = 13.8 nM while binds at CB1 with a Ki= 383 nM, 613 meaning that it binds almost 28x more strongly to CB2 than CB1 and was therefore 614 one of the first compounds to show a certain CB2 selectivity. JWH-015 (36) still 615 displays some CB1 activity at reasonable concentrations [97]. 616 617

19

N

O

40

N

O

41

618 FIGURE 14 Examples of JWHs structures 619

620 A series of analogues of 1-deoxy-Δ

8-THC were later developed by the same 621

research group, many of them resulting to be highly selective for the CB2 receptor. 622 The prototype of the series is JWH-133 (30), a potent CB2 agonist that displays 623 approximately 200-fold selectivity over CB1 (Ki = 3.4 nM and 677 nM respectively) 624 [98]. In vivo, JWH-133 (30) reduces spasticity in a murine autoimmune model of 625 multiple sclerosis [99]. The superior selectivity of this CB2 agonist makes it an 626 important tool in the study of the physiological function of CB2 receptors. 627

From the observation that the analogues of 1-deoxy-Δ8-THC are highly 628

selective ligands for the CB2 receptor, it has been suggested by Huffman and 629 colleagues that the 1-deoxy analogues of CP 47,497 (33) and CP 55,940 (38) could 630 show the same important selectivity for the CB2 receptor. On these premises, series 631 of 1-deoxy analogues of both compounds have been synthesized and 632 pharmacologically evaluated. JWH-324 (42), shown in Figure 15, is a representative 633 example of the series. In contrast to what hypothesized, none of these compounds 634 showed a significant affinity at both CB receptors. It was concluded that the CB 635 binding inability of 1-deoxy analogues is due to the lack of oxygen at C-1 on the 636 aromatic ring. Also CP 47,497 (33) methoxy derivatives, JWH-440 (43), JWH-441 637 (44) e JWH-442 (45) (Figure 15), albeit showing a certain CB2 selectivity, resulted 638 to be scarcely binding to both the CB subtype receptors [100]. 639

20

OH

43

OCH3

H3C CH3

OH

44

OCH3

H3C CH3

OH

45

OCH3

H3C CH3

OH

42

640 641

FIGURE 15 JWH-324 and methoxy derivatives. 642 643

This synthetic effort resulted to be significant because led to the conclusion 644 that the phenolic hydroxyl group bore by CP 47,497 (33) and related compounds, 645 lead to more significant interactions with the receptor binding site, possibly through 646 hydrogen bonds. This may not occur for deoxy and methoxy analogues. It seems, 647 moreover, that the rigid structure of the classic cannabinoid benzopyran system that 648 provided the nucleus for the design the CB2-selective ligands, provide also a more 649 suitable model for the further design and synthesis of structurally modified ligands, 650 selective for the CB2 receptor. 651

Recently, starting from the results of a study reporting that THC and other 652 cannabinoids can increase the activity of native and recombinant glycine receptors 653 (GlyRs) through a CB1- and CB2-independent mechanism [101], new deoxy-Δ

9-654

THC analogues, have been investigated (Figure 16). Deoxy-THC (46) and deoxy-655 CBD (47) bind strongly to subunits α1 and α3 of GlyRs, enhancing the capability of 656 these receptors to attenuate nociceptive signals directed to the brain [102]. In 657 addition, the deoxy analogues (46) and (47) show a significant reduction in binding 658 affinity to the CB1 receptor, while keeping constant high CB2 affinity (Figure 16). 659 660

46

C5H11

CH3

O

H3C

H3CC5H11

OH

CH3

C5H11

CH3

47 48 661

FIGURE 16 THC-deoxy analogs 662

663

21

When all the oxygen atoms are removed from Δ9-THC, the resulting dideoxy 664

CBD (48) does not show any affinity for both CB subtype receptors. The reduced 665 affinity of deoxy analogues (46) and (47) at CB1 receptor seems to suggest that even 666 the psychoactive effects may be mitigated, showing the prospect of new cannabinoid 667 analgesics without the CNS side effects, typical of cannabinoid drugs [103]. 668

The research team led by David Burdick examined the C-1 position of Δ9-669

THC as the primary modification site, in order to obtain selective CB2 agonists, 670 useful in the treatment of neuropathic pain. In particular, they focused in reducing 671 the lipophilicity by synthesizing analogues bearing polar groups or heterocycles in 672 order to increase the hydrophilicity of these compounds and at the same time to 673 reduce the CNS penetration. Compounds CB2 selective and characterized by a 674 reduced brain penetration would make possible to minimize potential side effects 675 [104]. A series of amine, amide, thiol and aminomethyl Δ

9-THC analogues were 676

synthesized and their affinity at both cannabinoid receptors tested in vitro. This 677 study, albeit led to important consideration about synthetic cannabinoid SAR, did 678 not give any significant molecule in terms of CB2 selectivity. 679

The search for selective agonists for the CB2 receptor was carried out in the 680 same years in the Abott laboratories by Jennifer M. Frost, starting from indole 681 cannabinoid ligands introduced by Sterling-Winthrop and developed by Huffman. 682 This project led to the discovery of a new class of aminoalkylindole analgesics. The 683 Frost group later developed a series of 3-acylindoles with a high affinity for CB2 684 receptors and a good selectivity over CB1. A variety of 3-acyl substituents were 685 investigated and the tetramethylcyclopropyl group was found to lead to high affinity 686 CB2 agonists, as (49) and (50) (Figure 17) displaying Ki values of 4.4 nM and 0.21 687 nM with an affinity over CB1 of 205 and 57 fold, respectively [105]. Among the 688 most relevant contributions after Huffman’s pioneering work in the 689 aminoalkylindoles field, Makriyannis and colleagues have designed and synthesized 690 a series of compounds, including the well-known ligand AM-1241 (35) [106]. This 691 molecule shows a peculiar affinity to opioid receptors, not observed in the other 692 CB2 selective ligands [107]. Interestingly, AM-1241 (35) may be a “protean 693 agonist” as it has been reported to behave as an agonist in tissues in which CB2 694 receptors are naturally expressed but not in tissues in which CB2 receptors have 695 been artificially inserted and overexpressed genetically [108]. 696 697

49

N

50

N

O

O

N

O

O

698 699

FIGURE 17 3-Acylindoles CB2 selective 700 701

Other notable selective CB2 agonists are the GlaxoSmithKline compound 702 GW-405,833 (51), which behaves as a potent partial agonist at the CB2 receptor 703 [109], HU-308 (52) developed at Hebrew University of Jerusalem by Mechoulam 704

22

group, showing a selectivity of 5000 fold for CB2 over CB1 [110], and the Merck-705 Frost compounds L-759,633 (53) and L-759,656 (54) with selectivity of 163 and 414 706 fold for CB2 over CB1, respectively [111]. Structures are shown in Figure 18. 707 708

51

O

53

52

HO

O

O

N

O

N

O

O

Cl

Cl

O

O

O

54

709 FIGURE 18 Selective CB2 agonists 710

711

712

Selective CB1 Agonist 713

714

Given the crucial activity of endocannabinoids AEA (1) and 2-AG (2) in the 715 modulation of the endocannabinoid system response and the interest in finding 716 molecules able to interact selectively with different targets within the 717 endocannabinoid system, it was decided to take as lead compounds endogenous 718 molecules as model for drug design, with the aim of synthesizing new selective 719 ligands for cannabinoid receptors. In the development of the first selective CB1 720 agonists, the starting point was the endocannabinoid AEA (1), whose marginal 721 selectivity for the CB1 receptor can be significantly enhanced by inserting a fluorine 722 or chlorine atom on the C2’ terminal, resulting in arachidonoyl-2´-chloroethylamide 723 (ACEA, 55) [112], replacing the terminal hydroxyl group, or by inserting a methyl 724 group in C1’ or C2’ position to form (R)-(+)-methananandamide (56) [113]. The 725 obtained alkyl chain can be used for cyclization, as in case of N-726 arachidonoylcyclopropylamine (ACPA, 57) [114]. Structures are displayed in Figure 727 19. 728 729

23

NH

O

Cl

NH

O

55 56

NH

O

OH

57

NH

O

58

NH

OH

O

59

CN

O

H

OH

OH

H

60

730 FIGURE 19 CB1 selective agonists 731

732 Notably, the most important result of the insertion of a methyl in C1 or C2 is 733

an increasing in the resistance of these compounds to the action of hydrolytic 734 enzyme FAAH. (R)-(+)-methananandamide (56) was synthesized by Alexandros 735 Makriyannis group with the specific purpose to have metabolically more stable 736 analogues. The endogenous eicosanoid cannabinoid AEA (1) does not contain any 737 chiral centres, but chirality seems to play an important role at the receptor affinity. 738 For example with regards to methanandamide, the (R)-(+)-isomer (56) displays nine 739 times greater affinity for CB1 receptors than the (S)-(–)-isomer [115]. The 740 elimination of hydroxyl group (58) does not improve the affinity at the receptor 741 (CB1 Ki = 73 nM), either when compared to the parent oxygenated molecule nor to 742 AEA (1). Together with O-1812 (59) [116] the most potent CB1-selective agonists 743 so far developed are ACEA (55) and ACPA (57), both of which exhibit reasonably 744 high CB1 efficacy. 745

In conclusion, anandamide derivatives with modifications at the amide 746 moiety, in particular those that increase lipophilicity, bind to the CB1 receptor with 747 a higher affinity than the parent molecule. In the meantime, this change reduces the 748 affinity at the CB2 receptor. However, unlike methananandamide (56), neither 749 ACEA (55) nor ACPA (57) show any sign of resistance to enzymatic degradation, 750 since they remain susceptible to FAAH mediated hydrolysis. 751

Recently, a novel putative CB1 full agonist named AM-4054 (60) [117] has 752 been developed by same Makriyannis group. AM-4054 (60), structurally an 753 analogue of classical cannabinoids (Figure 19), produced effects consistent with 754 CB1 agonism in the cannabinoid tetrad of tasks in rats, including induction of 755 analgesia, catalepsy, hypothermia, and locomotor suppression, at a dose range (0.15-756 1.25 mg/kg); this effect was reversed by a selective CB1 inverse agonist. 757

Albeit there would be many potential therapeutic uses of CB1 agonist-based 758 protocols, in general, CB1 full agonists are unlikely to be developed as drugs unless 759 they are peripherally selective, due to the strong psychotropic effect mediated by 760

24

CB1 receptor itself, but they are indeed potent pharmacological tools for assessment 761 of cannabinoid receptor function. 762

Focusing on this issue and starting from what reported in 1985 by Press and 763 Birnberg, stating that benzopyran [4,3-c] pyrazoles are devoid of neuroleptic 764 activity, a series of chromenopyrazolic derivatives have been designed in an attempt 765 to develop new non-psychoactive cannabinoids [118]. 766

The chromenopyrazoles were designed in analogy to CBN (11), with the 767 scope to develop new non-psychoactive cannabinoids that do not cross the blood–768 brain barrier, but act on peripherally located cannabinoid receptors. The ligand 769 binding studies on the generated molecules resulted in Ki values of 4.5–28.5 nM for 770 the 1,1-dimethylheptyl analogues (61-64) at CB1 receptors (Figure 20). The same 771 molecules showed no affinity for CB2 receptors (Ki > 40000 nM). 772

O

OHNHN

61

O

OHNN

62

O

OHNN

63

O

OHNN

64

773 FIGURE 20 CB1 selective 1,1-dimethylheptyl-chromenpyrazole derivatives 774

775

Selective CB2 Receptor Antagonists/Inverse Agonists 776

777 The most notable CB2-selective antagonists/inverse agonist is SR144,528 (65), 778 developed by Sanofi-Aventis [119]. SR144,528 (65) is potent and highly selective 779 (Ki of 0.6 nM at CB2 and 400 nM at CB1) and behaves as inverse agonist [119] that 780 can itself produce inverse cannabimimetic effects at CB2 receptors. It has also been 781 found to be an inhibitor of acyl-coenzymeA-cholesterol acyltransferase, an effect 782 that appears to be independent from its action on CB2 receptors [120]. Another 783 example of CB2 antagonist/inverse agonist is 6-Iodopravadoline (66, Figure 21), 784 also known as AM-630. This molecule has also been reported to reverse CP 55,940 785 (38) induced inhibition of forskolin-stimulated cyclic AMP production by human 786 CB2-transfected CHO cell preparations at concentrations in the nanomolar range 787 (EC50 = 129 nM) and to enhance forskolin-stimulated cyclic AMP production by the 788 same cell line when administered by itself (EC50 = 230 nM) [111]. AM-630 (66) is 789 also notable as one of the first indole derived cannabinoid ligands substituted at the 790 6-position of the indole ring.

This finding resulted subsequently to be important in 791

determining affinity and efficacy at both the CB1 and CB2 receptors [121] and has 792 led to the development of a large number of related derivatives [122, 123]. 793

25

67

N

N

NH

Cl

O

65 66

N

69

I

N

O

O

O

NBr

N

O

O

O

NH

O

O

O

O NHO

O

68

S

OH

O

O

N

OH

794 FIGURE 21 CB2 antagonist/inverse agonists. 795

796 Other molecules that are worth to be mentioned are WIN 54,461 (67), and 797

JTE-907 (68). WIN 54,461(67, 6-Bromopravadoline) basically is an AM-630 (66) 798 analogue, bearing a bromine atom instead of a iodine at the 6 position of the indole 799 ring [124], JTE-907 (68) is a highly selective CB2 receptor inverse agonist [125] 800 that binds CB2 human receptors with high affinity (Ki = 35.9 nM) and produces anti-801 inflammatory effects in vivo [126]. Recently, during a systematic screening of Food 802 and Drug Administration (FDA)-approved drugs libraries in search of potential 803 ligands for the CB2 receptor, Raloxifene (69) has been identified as a CB2 inverse 804 agonist [127]. This also provides important novel mechanisms of actions to explain 805 the known therapeutic effects of Raloxifene (69). Structures are shown in Figure 21. 806 807

Select ive CB1 Receptor Antagonists/Inverse Agonists 808

809 The first CB1 receptor antagonist/inverse agonist to be developed was the 810 diarylpyrazole, SR141716A (70) (Ki = 1.98 nM) [128], which showed a 100 fold 811

26

selectivity for CB1 over CB2 [129]. Also known as Rimonabant or under the 812 commercial name Acomplia, it was the first selective CB1 receptor blocker to be 813 approved for use anywhere in the world. It was approved for use in 38 countries 814 including E.U. as an anorectic anti-obesity drug, but it was rejected for approval for 815 use in the United States. Recently it has been withdrawn from the market [130] due 816 to potentially serious side effects. Other notable CB1-selective antagonists are AM-817 251 (71), AM-281 (72), and LY-320135 (73) (Figure 22), the latter having slightly 818 less affinity for CB1 receptors than the aforementioned three. 819

N

N

NH

N

O

Cl

Cl

Cl

N

N

NH

N

O

Cl

Cl

I

N

N

NH

N

O

O

Cl

Cl

I

OO

OCN

O

7071

72

73

820

FIGURE 22 CB1 antagonist/inverse agonists. 821 822

AM-251 (71) (Ki = 7.49 nM for CB1) is a diarylpyrazole structurally related 823 to SR141716A (70). It is 306 fold selective at CB1 over CB2 receptors and 824 suppresses food intake and food-reinforced behaviour in rats. AM-281 (72) is also a 825 Rimonabant analogue, and it is a potent antagonist/inverse agonist for CB1 receptor, 826 with a Ki =12 nM. AM-281 displays 350 fold selectivity over CB2 [131]. It has no 827 effect on the vanilloid TRPV1 receptor [132] and is generally used in test to evaluate 828 the potential effects of compounds at CB1. LY-320135 (73) is worth to be 829 mentioned due to its structurally dissimilarity from SR141716A (70). The 830 compound displays > 70 fold selectivity for CB1 over CB2 receptors (Ki = 141 nM 831 and > 10 µM respectively). It also shows weak binding to both serotonin 5-HT2 and 832 muscarinic receptors. Another notable diarylpyrazole derivative is Surinabant (74, 833 Figure 23) developed by Sanofi-Aventis under the name SR147778. This compound 834

27

displays nanomolar affinity to human CB1 receptors (Ki = 3.5 nM), along with low 835 affinity to human CB2 receptors (Ki = 400 nM) [133]. It has being investigated, in 836 view of potential advantages over Rimonabant (70) [134], as a potential treatment 837 for nicotine addiction to assist smoking cessation. It may also be developed as an 838 anorectic drug to assist with weight loss. CP-945,598, or Otenabant (75, Figure 24), 839 is a diphenyl purine which acts as a potent and highly selective CB1 antagonist. It 840 displays a Ki = 0.7 nM for CB1 receptors and a selectivity >10000 fold over CB2 841 [135]. It was designed by Pfizer for the treatment of obesity, but development for 842 this application has been discontinued following the problems seen during clinical 843 use of the similar drug Rimonabant (70) [136]. 844 845

N

N

NHO

Cl

Cl

Cl

74

N

N

Cl

Cl

N

N

N

NH2

O

NH

75 846

FIGURE 23 Diarylpyrazolic and diphenylpurinic CB1 antagonist/inverse agonist 847 848

849

New Emerging Classes of Cannabinoid Heterocycles 850

851 Albeit a wide range of structures are represented in the synthetic cannabinoid classes 852 and indeed highly potent and selective molecules are already reported, the research 853 in the field seems to not have yet reached its goal. Frontier research in new classes 854 of cannabinoids is continuing apace, in order to find new molecules, more effective, 855 scalable, displaying less side effects, with high bioavailability and a convenient 856 metabolism. 857

Recently, a new class of heterocyclic compounds has been synthesized by J. 858 Meilla-Rapaìn group. These benzimidazole derivatives, called JM series, displayed a 859 high CB1 affinity. JM-6 (76) showed a CB1 Ki = 98.2 nM while its 1-deoxo-6-860 fluorine derivative JM-39 (77) displays an increase in potency of 180 fold, showing 861 a CB1 Ki = 0.53 nM [137]. Structures are shown in Figure 24. 862

All cannabinoid compounds (endocannabinoids, plant cannabinoids and 863 synthetic cannabinoids) are very lipophilic molecules. In fact, this feature represents 864 a limitation in the binding radio assays of new synthetic derivatives. Nowadays 865 efforts are made in the design of “hydrophilic” or less hydrophobic cannabinoids, 866 that do not penetrate the blood-brain barrier, thereby providing the desired 867 pharmacologic activity without the side effects mainly associated with central CBs 868 activation [138]. 869

Since Rimonabant’s withdrawal, several groups are pursuing peripherally 870 selective CB1 antagonists. These compounds are expected to be devoid of 871 undesirable CNS-related effects but to maintain efficacy through antagonism of 872

28

peripherally expressed CB1 receptors. Several analogues of Otenabant (75) have 873 been developed, among these N-{1-[8-(2-chlorophenyl)-9-(4-chlorophenyl)-9H-874 purin-6-yl]piperidin-4-yl}pentanamide (78) (Figure 24) resulted to be a potent, 875 orally absorbed antagonist of the CB1 receptor (>50-fold selective for CB1 over 876 CB2) [139]. 877

Following the same theory, a novel series of aryl alkynylthiophene 878 derivatives has been developed by Hung’s group, leading to the identification of 879 TM8837 (79) as a highly promising CB1 peripheral inverse agonist [140] with 880 excellent potency (IC50 CB1 = 8.5 nM, CB2 = 604.9 nM CB1/CB2 ratio = 71) and 881 poor brain permeability as indicated by a low brain-to-plasma ratio (B/P=1/33). 882 Structures is shown in Figure 24. 883 884

N

N

NH

N

O

Cl

Cl

S

F3C

79

N

N

Cl

Cl

78

N

N

N

HN

Bu

O

76

N

N N

O

N

N NF

77

885 FIGURE 24 Diarylpyrazole, benzimidazole and alkynylthiophene derivatives 886

Some other notable examples in the field are the 4-oxo-1,4-dihydropyridine 887 derivative (80), a selective CB2 receptor agonist (Ki CB2 = 36.5 nM, CB1 > 1000 888 nM) [141] and AKB48 (APINACA, 81) that is a reasonably potent CBs agonist (Ki 889 CB1= 824.0 nM, CB2 = 430.0 nM) and an ingredient of synthetic cannabinoids 890 smoking blends [142]. While the physiological properties of AKB48 (81) compound 891 are not deeply known, in general molecules with adamantyl-carboxamide moieties 892

29

display high affinity for peripheral CB2 but greatly reduced affinity for central CB1 893 [143]. 894

As AKB48 (81), the chemical structure of MMB2201(82) [144] recalls the 895 similar structures in synthetic cannabinoids of the indazole-3-carboxamide group 896 published by Pfizer, e.g. AB-FUBINACA (83) (Ki values CB1 = 0.9 nM, CB2 = 897 23.2 nM) [145]. Albeit found in the illegal circulation, the physiological and 898 toxicological properties of MMB2201 (82) are not yet known, and no binding data 899 are available up to date. Structures are shown in Figure 25. 900

901

80

N

N O O

81

N

N

NH

O

O

N

NH

O

F

O

82

NH2

N

N

NH

O

O

83 F

902

FIGURE 25 New heterocyclic cannabinoids 903

Allosteric Modulators of Cannabinoid Receptors 904

905 Cannabinoids allosteric modulators are substances which indirectly influence the 906 effects of an agonist or inverse agonist at cannabinoid receptors, CB1 and/or CB2, 907 by binding to a site distinct from the orthosteric agonist binding site. Basically, the 908 effect of an allosteric ligand is to change the affinity of an orthosteric ligand for the 909 receptor, and vice versa [146]. Allosteric modulators of cannabinoid receptors can 910 be divided into two classes: endogenous and exogenous. To the first class belong 911 Lipoxin A4 (84) (Figure 26) [147], along with a family of peptide endocannabinoids 912 (PepCans) [148]. Among the studied pepcans, named as pepcan-12 to pepcan-23 913 referring to peptide length, pepcan-12, also known as the α-haemoglobin-derived 914 dodecapeptide RVD-haemopressin (RVDPVNFKLLSH), resulted to be a negative 915 allosteric modulator of CB1 receptor, exhibiting potent negative allosteric 916 modulation of the orthosteric agonist-induced cAMP accumulation, [

35S]GTPγS 917

binding, and CB1 receptor internalization. Pepcans are the first endogenous 918 allosteric modulators identified for CB1 receptors. 919

30

Lipoxins are members of the family of bioactive products generated from 920 arachidonic acid (AA). Lipoxin A4 (84) is reported to be an endogenous allosteric 921 enhancer of CB1 receptor, increasing the affinity of the endogenous cannabinoid 922 AEA (1) for CB1. Among the synthetic ones, indole-2-carboxamides named 923 Organon Compounds, are reported to be allosteric modulators of CB1 receptors. 5-924 chloro-3-ethyl-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-carboxamide (85, Org 925 27569) was the first molecule of a new class of indole-2-carboxamides that exhibits 926 allostery of CB1, along with its analogues Org 27759 (86) and Org 29647 (87) 927 [149]. In an equilibrium binding assay the Org compounds significantly increased 928 the binding of the CB1 agonist [

3H] CP55,940, indicating a positive cooperative 929

allosteric effect. In a subsequent study on the SAR of this class of molecules was 930 showed that the presence of the carboxamide functionality was required in order to 931 obtain a stimulatory effect. The maximum stimulatory activity on CB1 was exerted 932 by carboxamides (88) (EC50 = 50 nM) and (89) (EC50 = 90 nM) [150] bearing a 933 dimethylamino or piperidinyl group, respectively, at position 4 of the phenethyl 934 moiety and a chlorine atom at position 5 of the indole. Structures are shown in 935 Figure 26. No natural exogenous allosteric modulator of cannabinoids receptors 936 have been reported up to date. 937

84

O

OH

OH

OH

OH

NH

F

NH

O

NH

F

NH

O

NH

F

NH

O

N

N

86

85

87

NH

Cl

NH

O

NH

Cl

NH

O

N N

8988

938 FIGURE 27 Structures of allosteric modulators of cannabinoid receptors 939

940

THE THERAPEUTIC POTENTIAL OF CANNABINOIDS 941

942

In the recent years the Endocannabinoid System has emerged as an important 943 physiological system and an interesting target for new medicines. Its receptors and 944 endogenous ligands play a modulatory role in many functions including immune 945 response, food intake, cognition, emotion, perception, behavioural reinforcement, 946 motor co-ordination, body temperature, wake/sleep cycle, bone formation and 947

31

various aspects of hormonal control. In disease it may act as part of the 948 physiological response or as a component of the underlying pathology. The potential 949 of cannabinoid medicines in the following indications has extensively been 950 reviewed: symptomatic relief in multiple sclerosis, chronic neuropathic pain, 951 intractable nausea and vomiting, loss of appetite and weight in the context of cancer 952 or AIDS, psychosis, epilepsy, addiction, and metabolic disorders [151] as well as in 953 glaucoma, Gilles de Tourette syndrome, epilepsy, Parkinson’s disease, dystonia 954 [152]. Recently, a frontier Cannabinoid-Based treatment for Alzheimer’s Disease 955 has been suggested. It is well established that, considering the numerous complex 956 mechanisms involved in the progression of Alzheimer’s Disease, treatments 957 targeting a single causal offer limited benefit. Cannabinoids exhibit pleiotropic 958 activity, targeting in parallel several processes that play key roles in Alzheimer’s 959 Disease, including Aβ and tau aberrant processing, neuroinflammation, 960 excitotoxicity, mitochondrial dysfunction, and oxidative stress. Cannabinoids 961 treatment displays an improvement of patients’ behavioral disturbances [153]. Due 962 to their pro-apoptotic effect and to the correlation that exists between chronic 963 inflammation and neoplasia, interest in developing cannabinoids as cancer therapies 964 has increased [154]. 965

Despite all the beneficial abovementioned effects, cannabinoids drugs hardly 966 reached the market. Up to date only few synthetic cannabinoid have been approved 967 for use as medicine (Nabilone (31), Dronabinol (32)) and, at least in one case, a 968 cannabinoid drug was withdrawn due to the risks outweighing the benefits 969 (Rimonabant (70)). The search for the “cannabinoid golden molecule” is set to 970 continue. 971

972

CONCLUDING REMARKS 973

974

Cannabinoids are an interesting group of natural metabolites that exert their actions 975 by binding to specific receptors: the CB1 found primarily, but not exclusively, in the 976 CNS and CB2 that are mainly, but not exclusively, found in peripheral tissues and in 977 particular in the immune system. CB1 receptors appear to be responsible for the 978 euphoric and anticonvulsive effects of Cannabis, while CB2 receptors are able to 979 modulate the immune cells activity. Nowadays, the term endocannabinoid system 980 indicates the whole signalling system that comprises cannabinoid receptors, 981 endogenous ligands and enzymes for ligand biosynthesis and inactivation. 982

The modulation of the endocannabinoid system is an appealing therapeutic 983 option in a wide range of disparate diseases and pathological conditions, ranging 984 from movement disorders such as Parkinson’s and Huntington’s disease, multiple 985 sclerosis and spinal cord injury, neuropathic pain, to cancer, atherosclerosis, 986 obesity/metabolic dysfunctions, to name just a few. 987

Plant-based cannabinoids (phytocannabinoids) and chemically (natural and 988 synthetic) related compounds, have been found to exert significant analgesic effects 989 in various chronic pain conditions. Due to their important biological activities, the 990 search for non-psychotropic cannabinoids is still active. Cannabinoid receptors can 991 be engaged directly by agonists or antagonists, or indirectly by manipulating 992 endocannabinoid metabolism. The aim for the future is to better understand the 993 pharmacology of endocannabinoids, of cannabinoid receptors, of cannabinoid 994

32

receptor allosteric sites in view to develop novel therapeutic approaches in a number 995 of diseases for which current treatments do not fully address the patients’ need. 996

997

ABBREVIATIONS 998

999 2-AG 2-Acylglycerol 1000 AA Arachidonic Acid 1001

ABHD α−β Hydrolase 1002 ACEA Arachidonoyl-2’-chloroethylamide 1003 ACPA N-Arachidonoylcyclopropylamine 1004 AEA Anandamide 1005 AMP Adenosine Mono Phosphate 1006 CB1 Cannabinoid Receptor Type 1 1007 CB2 Cannabinoid Receptor Type 2 1008 CBC Cannabichromene 1009 CBD Cannabidiol 1010 CBG Cannabigerol 1011 CBN Cannabinol 1012 COX Cyclooxygenase 1013 CSN Central Nervous System 1014 DAG Diacylglycerol 1015 DIM Diindolylmethane 1016 EC Endocannabinoid 1017 EC50 Half Maximal Effective Concentration 1018 FAAH Fatty Acid Amide Hydrolase 1019 FU Official Italian Pharmacopoeia 1020 GlyRs Glycine Receptors 1021

HHC (-)-9-Nor-9β-hydroxyhexahydrocannabinol 1022 IUPAC International Union of Pure and Applied Chemistry 1023 IC50 Half Maximal Inhibitory Concentration 1024 Ki Inhibitory (or affinity) Constant 1025 MAGL MonoAcylGlicerol Lipase 1026 NADA N-Arachidonoyl Dopamine 1027 NAE N-Acylethanolamine 1028 NSAID Non Steroidal Anti Inflammatory Drug 1029 OAE O-Arachidonoyl-ethanolamine 1030 OLDA N-Oleoyl-dopamine 1031 PepCans Peptide Cannabinoids 1032 SAR Structrure Activity Relationship 1033 THC Tetrahydrocannabinol 1034 TRPV1 Transient Receptor Potential Vanilloid Type 1 1035 1036

33

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