UNIVERSITA’ DEGLI STUDI DI PISA
FACOLTA’ DI FARMACIA
Corso di Laurea Specialistica in
CHIMICA E TECNOLOGIA FARMACEUTICHE
Tesi di laurea
“DESIGN AND SYNTHESIS OF PYRIDINE-3-CARBOXAMIDE
DERIVATIVES AS CANNABINOID RECEPTOR LIGANDS”
Relatore Candidato
Prof. Clementina Manera Martina Consani
INDEX
INTRODUCTION
PAGE 4-25Description Taxonomia Popular usage
ECS (endocannabinoid system) Cannabinoid Receptor
Endogenous ligands of cannabinoid receptors
Structure of cannabinoid receptors and effects of their activation Biological roles of endocannabinoid system
Role of cannabinoids
Pharmacological and structural classification of exogenous ligands of cannabinoid receptors
INTRODUCTION OF EXPERIMENTAL PART
PAGE 26-42RESULTS AND DISCUSSION
PAGE 43-45EQUIPMENT USED IN SYNTHESIS
PAGE 46
SCHEME OF SYNTHESIS
PAGE 47-54EXPERIMENTAL PART
PAGE 55-774
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Description
Cannabis (Cán-na-bis) is a genus of flowering plants that includes one or more species. The plant is believed to be indigenous to Central Asia, China, and the north-west Himalayas. Cannabis is an annual, dioecious, flowering herb. The leaves are palmately compound, with serrate leaflets. The first pair of leaves usually have a single leaflet, the number gradually increasing up to a maximum of about thirteen leaflets per leaf (usually seven or nine), depending on variety and growing conditions. At the top of a flowering plant, this number again diminishes to a single leaflet per leaf. The lower leaf pairs usually occur in an opposite leaf arrangement and the upper leaf pairs in an alternate arrangement on the main stem of a mature plant.
Fig.1
Cannabis normally has imperfect flowers, with staminate "male" and pistillate "female" flowers occurring on separate plants. It is not unusual, however, for individual plants to bear both male and female flowers. Although monoecious plants are often referred to as "hermaphrodites," true hermaphrodites (which are less common) bear staminate and pistillate structures on individual flowers, whereas monoecious plants bear male and female flowers at different locations on the same
6
plant. Male flowers are normally borne on loose panicles, and female flowers are borne on racemes.
Taxonomia
The taxonomic classification of the genus Cannabis is not unique. The Cannabis genus was first classified using the "modern" system of taxonomic nomenclature by Carolus Linnaeus.He considered the genus to be monotypic, having just a single species that he named Cannabis sativa L.. Subsequent observations have led to the identification of other types of plants of the genus cannabis classified as species or subspecies:
Small and Cronquist distinguish one species (sativa) with two subspecies, each with two varieties:
• Cannabis sativa L.
or ssp. indica (Lam.) E. Small & Cronquist var. indicates, var. kafiristanica Vavilov
or ssp. sativa, var. sativa, var. Spontaneous Vavilov Synonyms = Cannabis chinense; Cannabis indicates Lam. Shultes, instead, divides the genus into three species:
1. Cannabis sativa (sativa = useful; volg. Hemp)
2. Cannabis indica (Indian states =; volg. Or cannabis indica)
3. Cannabis ruderalis (ruderalis = ruderale; volg. Ruderale hemp or Russian or American)
Fig.2 Leaves of Cannabis Sativa (a), Indica (b) and Ruderalis (c).
7
Clarke and Watson (2002) propose that the species Cannabis sativa includes all individuals, except perhaps the varieties used to produce hashish and marijuana in Afghanistan and Pakistan, which should be grouped under the species Cannabis indicates.
In any case, all species, subspecies or varieties may be mentioned among them cross giving rise to a fertile offspring.
Various types of Cannabis have been described, and classified according to the use, quantity and type of active present in them:
-plants cultivated for fiber and seed production, described as low-intoxicant, non-drug, or fiber types
-plants cultivated for drug production, described as high-intoxicant or drug types
-escaped or wild forms of either of the above types.
Cannabis plants produce a unique family of terpeno-phenolic compounds called cannabinoids, which produce the "high" one experiences from smoking marijuana. The two cannabinoids usually produced in greatest abundance are cannabidiol (CBD) and/or ∆9-tetrahydrocannabinol (THC), but only THC is psychoactive.
O H CH3 C H3 CH3 OH H H C H2 O CH3 C H3 CH3 OH H H C H3 Fig.3 cannabidiol (CBD) ∆9-tetrahydrocannabinol (THC)
8
Since the early 1970s, Cannabis plants have been categorized by their chemical phenotype or "chemotype," based on the overall amount of THC produced, and on the ratio of THC to CBD. Although overall cannabinoid production is influenced by environmental factors, the THC/CBD ratio is genetically determined and remains fixed throughout the life of a plant. Non-drug plants produce relatively low levels of THC and high levels of CBD, while drug plants produce high levels of THC and low levels of CBD. When plants of these two chemotypes cross-pollinate, the plants in the first filial (F1) generation have an intermediate chemotype and produce similar
amounts of CBD and THC. Female plants of this chemotype may produce enough THC to be utilized for drug production.
9
Popular usage
The National Institute of Drug Abuse defines drug abuse as "the non-medical use of a substance for psychic effect, dependence, or suicide attempt”. Marijuana (also known as ganja) and hashish are psychoactive products of the plant Cannabis sativa L. subsp. indica (= C. indica Lam.). The herbal form of the drug consists of dried mature inflorescences and subtending leaves of pistillate ("female") plants. The resinous form consists primarily of glandular trichomes collected from the same plant material. It has been reported that commercial hashish is often no more potent than high quality seedless marijuana. 1 However, carefully produced and screened hashish
is up to three times as potent as the highest quality herb.2 The major biologically
active chemical compound in Cannabis is THC. It has psychoactive and physiological effects when consumed, usually by smoking or ingestion. The minimum amount of THC required to have a perceptible psychoactive effect is about 5 mg. A related compound, ∆9-tetrahydrocannabidivarin, also know as THCV, is produced in
appreciable amounts by certain drug strains. This cannabinoid has been described in the popular literature as having shorter-acting, flashier effects than THC, but recent studies suggest that it may actually inhibit the effects of THC. Relatively high levels of THCV are common in African dagga (marijuana), and in hashish from the northwest Himalaya. The nature and intensity of the immediate effects of cannabis consumption vary according to the dose, the species or hybridization of the source plant, the method of consumption, the user's psychical and physical characteristics (such as possible tolerance), and the environment of consumption. This is sometimes referred to as set and setting. Smoking the same cannabis either in a different frame of mind (set) or in a different location (setting) can alter the effects or perception of the effects by the individual. What the user does under the influence can also affect the effects of cannabis. For example, if the user does nothing they will feel relaxed and sleepy, whereas if they engage in intense physical or psychical activity they will feel energized. Effects of cannabis consumption may be loosely classified as cognitive and physical. Anecdotal evidence suggests that drug varieties of Cannabis sativa subsp. sativa tend to produce more of the cognitive or perceptual effects, while C. sativa subsp. indica tends to produce more of the physical effects.
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ECS (endocannabinoid system)
The endocannabinoid system includes cannabinoid receptors, their endogenous ligands, anandamide and 2-arachidonoylglycerol, the anandamide transporter protein, and two enzimes, fatty acid amide hydrolase (FAAH) and monoglyceride lipase (MGL). 3
Fig. 5
Cannabinoid receptors
At present, two cannabinoid receptor types have been unequivocally identified, CB1 and CB2, and both receptors are coupled to G-protein-coupled-seven-transmembrane receptors (GPCRs). However, recent evidence has been presented for existence of a third cannabinoid receptor, with has been detected in the mouse brain.
11 Fig.6
The CB1 receptor (CB1R) subtype is mainly located in the central nervous system, with the highest density in the cerebellum, the basal ganglia, the substantia nigra pars compacta, and some regions of globus pallidus. CB1 is also present in peripheral organs such as adrenal glands, bone marrow, lung, adipose tissue, liver, muscle, gastrointestinal tract, testis, and uterus. 4
The CB2 recptor (CB2R) was originally identified from macrophages present in the spleen, and it is expressed primarily in cells associated with the immune system, like spleen, thymus, and tonsils.5 Although absent from the CNS in normal conditions,
CB2 receptor might be induced in brain microglia cells in response to different
damaging conditions associated with local inflammatory events,6 and recent studies
using human neutrophils indicate that the CB2 receptor may suppress neutrophil
migration during inflammation. It is also located in retina,7 skin8 and some malignant
12
Endogenous ligands of cannabinoid receptors
The individuation of endogenous ligands for these receptors has been yielded after cannabinoid receptors discovery. The endogenous ligand of the CB1 receptor is anandamide whereas the endogenous ligand of the CB2 is 2-arachidonylglycerol (2-AG).
Fig.7
They are synthesized from membrane-derived phospholipids whose biologic effects are mediated through coupling with the specific, widely expressed ECS receptors located presynaptically.10 The synthesis of anandamide is Ca2+-dependent and is
produced locally by the phospholipase D-mediated cleavage of the membrane precursor called N-arachidonoyl- phosphatidylethanolamine (NArPE). While the synthesis of 2-arachidonylglycerol is produced by the DAG lipase(diacylglycerol lipase; DAG-α and DAG-β) cleavage of the membrane precursor. Because endocannabinoids are lipophilic compounds derived from membrane phospholipids, they do not need to be stored in synaptic vesicles like other neurotransmitters11. In
the brain, they are produced by neurons at their sites of action and act on demand, generating a transient, rapid effect before being hydrolyzed and inactivated by FAAH (fatty acid amine hydrolase), which breaks the amide bond and releases arachidonic acid and ethanolamine. 11, 10 Because of their lipophilic nature and the mechanism of
their synthesis and release, endocannabinoids are considered as local neuromodulators.12
13 Fig.8
In particular, in the brain, the biosynthetic and degradative enzymes for 2-AG are localized, with respect to CB1receptors, in away that post-synaptic neurons, which express the DAGL-α in dendritic spines and somatodendritic compartments, by producing and releasing this endocannabinoid, can control the activity of the complementary pre-synaptic neurons, where the CB1 receptor is often expressed.13
This “retrograde” modulatory action is terminated by MAGL (monoacylglycerol lipase) expressed on the same pre-synaptic terminal. CB1 activation, then, by reducing the activity of voltage-activated Ca2+ channels and enhancing that of
inwardly rectifying K+channels, can inhibit the release of neurotransmitters14,15. This
paracrine signalling mechanism represents a “circuitbreaking” mechanism13 and,
hence, can re-establish an excessive activity of the post-synaptic neurons, such as during certain pathological neurological conditions.13,16,17 The general strategy of
14
Structure of cannabinoid receptors and effects of their
activation
The CB1 and CB2 receptors are two G-protein-coupled-seven-transmembrane receptors (GPCRs). The G protein (Gi/Go) associated to cannabinoid receptors is sensible to pertussis toxin and it is constituted by the α and βγ subunits. The affinity of CB1 and CB2 for Gi or Go proteins may be different as revealed by several studies on cannabinoid ligand binding or regulation of [35 s] GTPγS binding. Whereas, activation of both of them display a high affinity for Gi, agonist stimulation of CB1 also result in a high-affinity saturable interaction with G0 but CB2 receptor do not interact efficiently with G0. It has been reported that the affinity of CB1 receptor for G0 is ten fold higher than that of the CB2.19 It has been described that the
juxtamembrane C-terminal region of CB1 receptors (amino acids 401-417) and the second and third intracellular loops are critical for Gi/o protein coupling and that the distal C-terminal tail domain profoundly modulates both the magnitude and kinetics of signal transduction.20 In the CB2 receptor it has been described the existence of
two cysteins, C313 and C320, that are located in this C-terminal region, that may play important roles for receptor-G protein coupling and receptor desensitization. Also, the third transmembrane domain in the CB2, particularly the Asp-Arg-Tyr motif, may be crucial for interacting with G proteins.
The effects due to activation of CB1 and CB2 receptors may be mediated or not by interaction with G proteins.21 Transduction mechanisms of CB1 receptor involve
inhibition of cAMP production through inhibition of adenylate cyclase, inhibition of calcium influx, activation of potassium channels, and activation of the mitogen-activated protein kinase (MAPkinase ) that induce cellular proliferation. The mammalian MAPK family consists of three subfamilies with multiple members: the extracellular signal-regulated kinases (ERK), the Jun amino-terminal kinases/stress-activated kinases (JNK/SAPK), and the p38 MAPKs. While ERK is involved in regulation of cell division and growth, the other two subfamilies are activated by stress signals and inflammatory cytokines and have been related with cellular death and immune disorders.
15
Fig.9 Signal transduction activated by CB1 receptors
Two main mechanisms have been identified for the transduction machanisms of CB2 receptor: inhibition of adenylate cyclase and stimulation of mitogen activated protein Kinase (MAP kinase). Furthermore unlike CB1 receptor, the CB2 receptor does not have effect on ion channels. In addition, the activation of CB2 receptors has
been also linked to the stimulation of additional intracellular pathwaysincluding the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which has beenassociated with pro-survival effects, and the de novo synthesis of the sphingolipid messenger ceramide, which has been linked with the pro-apoptotic effects of cannabinoids.
16
17
Biological roles of endocannabinoids system
The endo-cannabinoid system is implicated in two major biological roles: modulation of neurotransmitter release and modulation of immune functions. Cannabinoids has been demonstrated to inhibit the evoked release of Ach, GABA, NA, DA, 5-HT, Glu, Gly, d-Asp, and CCK.22 The effect of cannabinoids on the function of immune cell is
still unclear and requires elucidation but they produce a deleterious effect on the immune response causing the impairment of macrophage functions, perturbation of immunoglobulin production and down-regulation of immune cells activity. It has been evidenced that THC can suppress the human immune response in a host of leukocyte subsets and alveolar macrophages. The degranulation of mast cells induced by substance P is fully abrogated by the endogenous ligands at cannabinoid receptors. The mechanism by which CB2 ligands modulate mast cell activation is by generating
NO and PGE2. It is involved in the controlling vascular homeostasis23 and synaptic
transmission.24 NO can be induced by proinflammatory factors under pathological
conditions. NO production is stimulated by anandamide in human monocytes. This stimulation could explain some cannabinoids effects like vasodilatation and neurotransmission release inhibition. Cannabinoids could be used as therapeutic agents in NO-mediated inflammation leading to neurodegeneration. Modulation of iNOS induction by cannabinoids required NO production. In addition AEA and exogenous cannabinoids induce arachidonic acid mobilization and activation of the enzymes of arachidonic acid cascade in many cells. It is important to note that AEA degradation into cells by fatty acid amide hydrolase (FAAH) yield arachidonic acid that could mediate some biological actions of endocannabinoids like vasorelaxation but, in this case, the effect is independent of cannabinoid receptors25 (Figure 11).
18
Fig.11
Three enzymes are involved in the release of arachidonic acid from membrane phospholipid domain: phospholipase D (PLD), phospholipase C (PLC) and phospholipase A2 (PLA2). The three enzymes have been shown to be activated by
cannabinoids. PLD causes phospholipid hydrolysis yielding arachidonate containing diacylglicerols generation that, after hydrolysis by DAG lipases, may release arachidonic acid. The primary pathway is represented by activation of PLA2 which
liberate arachidonic acid from phospholipids. The main mechanism of cytosolic PLA2
activation is phosphorylation by MAP kinase. The release of arachidonic acid induced by THC in hippocampal neurones can be blocked by the CB1 receptor antagonist
SR141716 but not by pre-treatment with pertussis toxin, suggesting an involvement of cannabinoid receptor by a mechanism independent of Gi coupling. It has been recently shown that cannabinoids may modulate arachidonate metabolic enzymes. In human platelets THC inhibits COX-2 activity blocking the synthesis of its pro-inflammatory metabolites with the redistribution of products towards lipoxygenase pathway.26 In human neuroglioma cells THC and methanandamide stimulated
COX-2 mRNA expression and subsequent PGECOX-2 synthesis via a non cannabinoid receptor-mediated mechanism.
19
Role of cannabinoids
The discovery of psychoactive principle of Cannabis sativa, delta 9-tetrahydrocannabinol initiated research into the physiological role of cannabinoids. The cannabinoids produce euphoria, alterations in cognition and analgesia, have anticonvulsant properties and affect temperature regulation, sleep and appetite. However, cannabinoids also possess immunomodulatory activity and anti-inflammatory properties. Many diseases of the CNS involve inflammation, and cause an up regulation of cytochines and other inflammatory mediators in the CNS. Alzheimer’s disease (AD), Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS) are among the best examples of neurodegenerative disorders of the CNS associated with intense inflammation, whereas multiple sclerosis (MS) and HIV-associated dementia are inflammatory disorders of the CNS that lead to diffuse neuronal damage.
Therefore cannabinoids may be potential therapeutic agents in neurological diseases. Recent studies revealed mainly role of cannabinoids in:
- Cognition: the most prominent among the various consequences of CB1 receptor activation by exogenous agonist are disruptive effects on working memory, i.e. on processes necessary to learn and react to new information that differs from session to session.
- Emotionally: blockade of CB1 receptors by SR141716A caused an increase in anxiety-related behaviour22, 23, 24. In contrast, lower doses of SR141716A had no effects25. Data obtained in mice were more inconsistent. Administration of SR141716A either decreased26 or increased anxiety-related behavior, depending on the genetic background of animals and the test situation.
- Multiple sclerosis (MS): is the most important chronic inflammatory demyelinanting disorder of the CNS. The cannabis and cannabinoid agonists may be effective in ameliorating symptomatology of MS, especially spasticity and pain. The benefits of cannabinoids agonist the symptomatology associated with chronic
20
inflammatory demyelinanting pathologies, may be exert at multiple levels: by improving motor function, by limiting neuroinflammation, by promoting remyelination.
- Immune modulation: the cannabinoid CB2 receptor is expressed abundantly in various types of inflammatory cells in particular in B cells. The CB2 receptors has been associated with most of immunomodulatory activity of cannabinoids, but also CB1 may be linked to cannabinoid-mediated alterations of immune cell reactivity. Cannabinoids exhibit immunosuppressive properties.
- Appetite and energy regulation: the ECS is postulated to connect the physical and emotional responses to stress with appetite and energy regulation (general stress-recovery system). Stimulation of the ECS may possibly occur as a consequence of obesity, leading to increased levels of endocannabinoids, which disrupt the feedback mechanism involved in energy balance. The ECS affects energy balance, glucose homeostasis, and lipogenesis because of the cannabinoids receptors are located in the adipose tissue, the liver, the pancreas and the skeletal muscle (Figure 11).
- Obesity: activation of the ECS increases food intake and promotes weight gain. The blockade of the CB1 receptor reduces body weight in animals through central and peripheral action.
21
Pharmacological and structural classification of ligands of
cannabinoid receptors
On a Pharmacological point of view, the cannabinoid ligands might be divided into three major categories:18
I. Dual CB1/CB2 agonist: compounds which are not, or poorly, selective for one cannabinoid receptor subtype such as classical cannabinoids ∆9
-tetrahydrocannabinol (THC) and related compounds, nonclassical cannabinoids as CP-55,940 and derivatives, and aminoalkylindoles as WIN-55,512-2. These compounds have been, and still are, very useful in pharmacological studies on the function of cannabinoid receptors, but are unlikely to generate new therapeutic drugs.
Fig.13
II. Compounds which are selective for the cannabinoid CB1 receptor subtype. These compounds could be:
- Selective CB1 agonists, such as arachidonoylchloroethanolamide and arachidonoyl-cyclopropylamide (ACEA). Such compounds have been very
CP-55,940 THC
22
useful in both in vitro and in vivo studies to distinguish the effects of CB1 receptor activation from those associated to CB2 receptor.
Arachidonoylchloroethanolamide Fig.14
-Selective antagonists/inverse agonists for CB1 receptors, such as SR141716A (rimonabant), SR147778 (surinabant), AM251, AM281, MK-0363 (Taranabant), LY320135, CP-945598 and AVE1625. Some of these compounds have already found clinical use as anti-obesity agents as well as against metabolic disorders such as dyslipidemia and type 2 diabetes, although their use in these pathologies has been discontinued due to their psychiatric side effects (namely anxiety and depression). Other possible uses might be against steatosis and steatohepatitis, nicotine and alcohol abuse, relapse of heroin and cocaine abuse, hypotension, cardiopathies, encephalopathy and liver fibrosis in cirrhosis, Parkinson’s and Alzheimer’s disease, schizophrenia and osteoporosis. Efforts are ongoing to develop non-brain-permeant CB1 receptor antagonists/inverse agonists, which should be devoid of the central side effects of rimonabant and taranabant and still useful against some metabolic disorders. Fig.15 SR141716A (rimonabant) AM251
23
- Allosteric modulators of CB1 receptors, including Org27596, and PSNCBAM-1 . These compounds enhance the affinity of CB1 receptor agonists but reduce their efficacy, and might, therefore, find application in the same pathological conditions as CB1 antagonists/inverse agonists.
Fig.16
III. Compounds that are selective for the CB2 cannabinoid receptor subtype. These compounds could be:
- Selective CB2 agonists, such as HU-308, JWH-015, JWH-133 and AM1241. Such compounds have been very useful in both in vitro and in vivo studies to distinguish the effects of CB2 receptors from those of associated to CB1
receptors. They might also represent important templates for the development of non-psychotropic anti-inflammatory and analgesic drugs.
Fig.17
Org29647 PSNCBAM-1
24
-Selective CB2 antagonist/inverse agonists, i.e. SR144528, AM630 and JTE907.
Fig.18
From a structural point of view,22 however, the ligands of the cannabinoid receptors
may be divided into:
a) like classic cannabinoid, as ∆9-THC
b) non-classic cannabinoid as CP-55940
c) aminoalkylindole derivatives, such as WIN-55212-2
d) compounds that are supposed to interact in the aminoalkilindole binding site as Oxoquinolines and Oxonaphthyridines27.
Aminoalkylindole derivatives are structurally dissimilar from the other classes, and are hypothesized to interact in a binding site different from that of the other cannabinoid receptors agonists. This class of ligands appeared to be quite interesting due not only to their particular molecular structure but also to the selectivity properties. Infact WIN-55212-2 has selectivity for the CB2 receptor, relative to the CB1 receptor. However it has not only very high affinity for the CB2 receptor ( Ki CB2R=3.3 nM), but it has affinity for the CB1 receptor ( Ki CB1R=62.3 nM).
25
In a patent of Japan Tobacco Inc.28 2-oxoquinoline structures were studied. These
compounds act selectively at cannabinoid receptor, especially at the CB2R, reported. The most selective and active compounds is 43, which behaves as an inverse agonist in vitro and possesses anti-inflammatory properties in vivo.
N R4 O C H3 R3 R2 O O R1
More recently our research group reported a new series of 1,8-naphthyridin-4(1H)-on-3-carboxamide and quinolin-4(1H)-1,8-naphthyridin-4(1H)-on-3-carboxamide derivatives of general structure designed below.29,30
X N R2 R1 O NH O R3
These compounds generally exhibited a remarkable CB2 affinity, with a Ki value in the nanomolar range. This affinity was accompanied by an high selectivity with respect to CB1 receptor. Moreover, the [35S]GTPγ binding assay and functional
studies on human basophils indicated that these derivatives behaved as CB1 and CB2 receptor agonist. Finally reduced the growth of DLD-1 (D-lactate dehydrogenase-1) cells in a mouse model of colon cancer inducing apoptosis through ceramide de novo synthesis. Our date unveiled, foe the first time, that TNF-α (tumor necrosis factor-α) acts as a link between cannabinoid receptor activation and ceramide production.31
Compound 43
R1=HN[benzo[d][1,3]dioxol-4-yl]; R2=H; R3=OC5H11; R4=H
