**1. Introduction**

The sympathetic nervous system is part of the autonomic nervous system and innervates tissues in almost every organ system. Adrenergic system is important for maintaining the organism homeostasis and mediates the neuronal and hormonal stress response commonly known as the fight-or-flight response.

Central and peripheral adrenergic neurotransmitters are epinephrine and norepinephrine, which act on their specific adrenergic receptors.


**Table 1.** Types and subtypes of adrenergic receptors.

Adrenergic receptors were described for the first time by Ahlquist in 1948, who hypothesized the existence of two different types of receptors, α and β, based on the consideration that adrenaline, noradrenaline, and other pharmacological agonists regulate various physiological functions [1]. This differentiation of receptors was confirmed by the finding that there are antagonists, which selectively block α receptors (e.g., phenoxybenzamine or phentolamine) or β receptors (e.g., propranolol). Every type of adrenergic receptors has different subtypes, which are mentioned in **Table 1** [2–4].

### **1.1. Beta-3 adrenergic receptor discovery and structure**

**1. Introduction**

**Adrenergic receptors types**

**and subtypes**

**α-Adrenergic receptors**

**β-Adrenergic receptors**

known as the fight-or-flight response.

220 Adiposity - Epidemiology and Treatment Modalities

which act on their specific adrenergic receptors.

The sympathetic nervous system is part of the autonomic nervous system and innervates tissues in almost every organ system. Adrenergic system is important for maintaining the organism homeostasis and mediates the neuronal and hormonal stress response commonly

Central and peripheral adrenergic neurotransmitters are epinephrine and norepinephrine,

α1A Heart, blood vessels, smooth muscle, liver, lung, vas deferens, prostate, cerebellum,

α1B Heart, kidney, spleen, lung, blood vessels,

α1D Aorta, coronary artery, platelets, prostate,

α2A Sympathetic neurons, platelets, pancreas, locus coeruleus, brainstem, spinal cord

cortex, hippocampus

cortex, hippocampus

α2c Basal ganglia, cortex, cerebellum,

β1 Heart, kidney, skeletal muscle, cortex, olfactory nucleus, brain stem

β2 Bronchial and gastrointestinal smooth

β3 Adipose tissue, gastrointestinal tract, gallbladder, urinary bladder

Tissue localization and dominant effects (after Goodman & Gillman's 2011, modified).

muscle, blood vessels, heart, lung, skeletal

Adrenergic receptors were described for the first time by Ahlquist in 1948, who hypothesized the existence of two different types of receptors, α and β, based on the consideration that adrenaline, noradrenaline, and other pharmacological agonists regulate various physiological functions [1]. This differentiation of receptors was confirmed by the finding that there are antagonists, which selectively block α receptors (e.g., phenoxybenzamine or phentolamine) or

hippocampus

muscle, cortex

**Table 1.** Types and subtypes of adrenergic receptors.

cortex, brainstem

**Tissue localization Dominant effects**

α2B Liver, kidney, pancreas, blood vessels Mediates α2 vasoconstriction

Contraction of vascular smooth muscle; vasoconstriction of large resistant arterioles in skeletal muscle

Promotes cardiac growth and structure

Vasoconstriction in aorta and coronary

Main inhibitory receptor on sympathetic

Modulates dopamine neurotransmission Inhibits hormone release from adrenal

Positive inotropic and chronotropic

Smooth muscle relaxation, skeletal

Lipolysis, thermogenesis, relaxation of

muscle hypertrophy

artery

neurons

medulla

effects

the bladder

In the early 1980s, Tan S and Curtis-Prior PB proposed the term of beta-3 or beta-hybrid receptor for a new type of beta-adrenergic receptor, based on some studies of four betaadrenergic agonists on isolated rat adipose cells. They observed that lipolytic potency decreased in the order: isoprenaline (beta-1 and beta-2 agonist) > noradrenaline (beta-1 >>> beta-2 agonist) > salbutamol (beta-2 agonist) > prenalterol (beta-1 agonist). They also studied the effects of some beta-antagonists on lipolysis induced by various agonists. Propranolol (nonselective beta-antagonist) was more potent than betaxolol (selective beta-1 antagonist) or ICI 118551 (selective beta-2 antagonist). All results conducted to the idea that lipolysis in adipose tissue is regulated by other adrenergic receptor than the classical ones, beta-1 and beta-2 [5].

In 1989, Emorine et al. first characterized beta-3 receptor by discovering the gene that encodes it [6]. Before that Arch et al. observed that some nonspecific classical beta-receptor agonists, named BRL 26830A, BRL 33725A, and BRL 35135A, had antiobesity actions on obese and diabetic mice [7].

Further studies have shown that beta-3 receptor is different than beta-1 and beta-2 by some important issues:


The structures of beta-1, beta-2, and beta-3 receptors are similar, being all members of G protein-coupled receptors. Beta-3 receptor is a protein which contains 396 amino acids, found in seven transmembrane segments, with three intracellular and three extracellular loops. The amino-terminal region is extracellular, glycosylated, and with variable length. The carboxylterminal region is intracellular and it does not possess phosphorylation sites, which are present at beta-1 and beta-2 receptors. Essential for interaction with the ligands are the disulfide bond between the second and the third extracellular loops and also four of the seven transmembrane segments. Other two segments are implicated in G-protein stimulation, with adenylate cyclase and second messenger activation [12].

#### **1.2. Metabolic effects of beta-3 adrenergic receptor stimulation**

Some of the most important effects of beta-3 receptor activation are the metabolic ones, especially in the brown adipose tissue. This thermogenic tissue has the role of keeping constant body core temperature of small animals at cold ambient temperatures. Stimulation of beta-3 adrenergic receptors not only activates brown adipose tissue thermogenesis in the short term, but also increases mitochondrial biogenesis and the expression of thermogenin

(UCP1) [13–15]. This protein mediates transport across the internal mitochondrial membrane and interrupts oxidative phosphorylation of the beta oxidation of fatty acids, increasing the use of energy [16]. There have been investigations about the effects of beta-3 adrenergic agonists on thermogenin. The results have shown that beta-3 agonists activated thermogenin, and also other uncoupling proteins, as follows:


The stimulation of thermogenesis by beta-3 adrenergic agonists resulted in a number of experimental studies, which have shown that in animals, these substances lead to weight loss, a selective fat decrease, but without reducing food intake [18].

An *in vitro* study was performed on cells with high levels of beta-3 adrenoreceptors such as the adipocytes of the murine cell line 3T3-F442A. The study demonstrated that insulin and glucocorticoids downregulate beta-3 adrenoreceptor expression through a transcriptional effect. The impairment of beta-3 adrenoceptor gene expression in adipocytes of congenitally obese ob/ob mice could be related to the higher glucocorticoid plasma levels when compared to lean mice [19].

The main studied metabolic actions in preclinical research of beta-3 adrenergic agonists were the reduction of plasma insulin levels, increase glucose tolerance, and reducing body weight in obese diabetic rats. The major implication of beta-3 adrenergic receptor in glucose metabolism and hence in the release of insulin and in obesity has been demonstrated [20, 21].

The development of beta-3 adrenergic agonists was a step forward for the treatment of metabolic diseases by sympathetic activation, because norepinephrine and other relative nonselective derivatives have cardiovascular side effects which limit their use.

#### **1.3. Preclinical studies of beta-3 agonists on carbohydrate and lipid metabolism**

Numerous nonclinical studies have shown that administration of beta-3 agonists decreased glucose and lipids plasma concentrations in diabetic mice derived from genetically modified strains (kk, C57BL/KsJ- db/db) or in rats with experimentally induced diabetes [22, 23].

Several mechanisms of action were highlighted and reported:


The nonclinical research for proving the effects of beta-3 agonists on obesity were conducted on rodent species [28–30] from various strains, both normal and genetically modified to generate predisposition to obesity.

Studies in obese rats treated with the selective beta-3 adrenergic agonists have shown a significant reduction in body weight and reduction of adipose tissue, without food intake being affected by them. It was also demonstrated that an increase of two to three times of the mRNA level and of UCP-type protein, as well as of the guanidine 5'-diphosphate coupled, a relevant index of thermogenesis, in brown and white adipose tissues for the tested rats. In addition, it has been found, after the administration of beta-3 adrenergic agonists, an improvement of glucose tolerance and a decrease of hyperinsulinemia. The researchers suggested two possible mechanisms for defining this aspect, increase of the number of insulin receptors or decrease of glucose transporters in brown and white adipose tissues, which implies an increase in glucose uptake into muscle tissue [31].

(UCP1) [13–15]. This protein mediates transport across the internal mitochondrial membrane and interrupts oxidative phosphorylation of the beta oxidation of fatty acids, increasing the use of energy [16]. There have been investigations about the effects of beta-3 adrenergic agonists on thermogenin. The results have shown that beta-3 agonists activated thermogenin,

**•** UCP3 that is found in skeletal muscle and has an important role in basal thermogenesis [17]. The stimulation of thermogenesis by beta-3 adrenergic agonists resulted in a number of experimental studies, which have shown that in animals, these substances lead to weight loss,

An *in vitro* study was performed on cells with high levels of beta-3 adrenoreceptors such as the adipocytes of the murine cell line 3T3-F442A. The study demonstrated that insulin and glucocorticoids downregulate beta-3 adrenoreceptor expression through a transcriptional effect. The impairment of beta-3 adrenoceptor gene expression in adipocytes of congenitally obese ob/ob mice could be related to the higher glucocorticoid plasma levels when compared

The main studied metabolic actions in preclinical research of beta-3 adrenergic agonists were the reduction of plasma insulin levels, increase glucose tolerance, and reducing body weight in obese diabetic rats. The major implication of beta-3 adrenergic receptor in glucose metabolism and hence in the release of insulin and in obesity has been demonstrated [20, 21].

The development of beta-3 adrenergic agonists was a step forward for the treatment of metabolic diseases by sympathetic activation, because norepinephrine and other relative

Numerous nonclinical studies have shown that administration of beta-3 agonists decreased glucose and lipids plasma concentrations in diabetic mice derived from genetically modified strains (kk, C57BL/KsJ- db/db) or in rats with experimentally induced diabetes [22, 23].

**–** decrease in glucose release from the liver, increase noninsulin-dependent uptake of

**–** increase of glucose tolerance at doses lower than those that stimulate lipolysis in adipose

The nonclinical research for proving the effects of beta-3 agonists on obesity were conducted on rodent species [28–30] from various strains, both normal and genetically modified to

glucose from white and brown adipose tissue and skeletal muscles [26], and

tissue, without affecting the amount of food intake or body weight [27].

nonselective derivatives have cardiovascular side effects which limit their use.

**–** improving insulin resistance and increased tissue response to insulin [24],

Several mechanisms of action were highlighted and reported:

**–** increasing insulin secretion in beta cells of the pancreas [25],

generate predisposition to obesity.

**1.3. Preclinical studies of beta-3 agonists on carbohydrate and lipid metabolism**

and also other uncoupling proteins, as follows:

a selective fat decrease, but without reducing food intake [18].

**•** UCP2 that is found in many tissues.

222 Adiposity - Epidemiology and Treatment Modalities

to lean mice [19].

Lorente Ferrer et al. investigated the effect of beta-3 receptors agonists on thermogenesis in deep adipose tissue. In general, these agonists increase energy consumption but their effects are quickly counteracted by glucocorticoids. Thus, their potential for long-term treatment of obesity is reduced. Since the metabolic effects of beta-3 receptor agonists (β3A) overlap only partially with those of oleoyl-estrone (OE) (loss of appetite, weight change, loss of body fat), the possibility of combining them in the energy balance in order to accelerate the decrease of fat deposits was studied. Rats receiving OE or OE + β3A significantly reduced weight compared with the control group, the maximum reduction corresponding to the group which received the combination [32].

The effect of beta-3 adrenergic receptor agonists was investigated on two strains of rats with different genetic predisposition to obesity: male rats aged 8 weeks Osborne Mendel (OM) strain and S5B/P1 (S5B) strain. Animals were treated with beta-3 adrenergic agonist CL316243 after they have been adapted to either a high fat diet (56% fat-based energy) or low fat (10% fatbased energy), but both equivalent diets in terms of protein content (24% based on protein energy). The animals were fed *ad libitum* and were injected with CL316243 in three doses: 0.1; 0.3; and 3 mg/kg at the beginning of the night. Food intake was measured at 1, 3, and 24 hours after injection. The results showed that CL316243 significantly reduced food intake for all measurements, in both types of rats. Inhibition of food intake was still higher in S5B-type mice. CL316243 significantly decreased serum leptin and serum glucose at both types of rats, especially at S5B. In OM rats, beta-3 adrenergic agonist increased serum insulin levels, while in S5B rats fed with a low-fat diet, the level of serum insulin decreased. In another experiment, CL316243 was administered to rats kept fasting overnight. It was observed after 30 minutes a significant reduction in insulin levels in both types, more pronounced in S5B. The glucose level in OM rats decreased after 30 and 60 minutes, while in rats S5B a decrease was observed only after 30 minutes from the administration. Experiments have shown that beta-3 agonist CL316243 has a much more obvious effect on rat strain resistant to obesity induced by highfat diet [33].

Another study used a transgenic model of mice, lacking beta-3 adrenergic receptors. CL316243 blocked the activation of adenylate cyclase and lipolysis when it was administered to these mice. A modest growth of fat tissues especially in females was observed. These mice showed an increase in the level of mRNA for beta-1 receptor, but not for receptor beta-2. This showed a functional compensation between the genes for beta-1 and beta-3 receptors. Finally, a sharp increase of insulin levels and lipolysis after administration of CL316243 in normal mice was noted; effects were not found after administration of beta-3 adrenergic agonists in beta-3 receptor-deficient mice [34].

In another model of knockout mice lacking functional beta-3 adrenoreceptors, there were no responses for food intake and insulin secretion in white and brown adipocytes after administration of beta-3 adrenergic agonist CL316243, indicating the implication of beta-3 receptors in these metabolic effects [35].

An increase in insulin levels during "fasted/fed" transition in rats has been demonstrated, associated with a decrease in the mRNA level of beta-3 adrenergic receptor and a decrease of the response in brown and white adipose tissues. It was concluded that there is a close relationship between the food intake, plasma levels of insulin, and beta-3 adrenergic receptors. Downregulation of the beta-3 receptors could be a possible mechanism by which insulin determines lipid storage and prevents lipid mobilization after food intake [36].

In another study, CL316243 was administered in obese diabetic KKAy mice for 2 weeks. The results showed a decrease of serum levels of glucose, insulin, triglyceride, free fatty acid, and tumor necrosis factor-alpha (TNF-alpha) and an increase of adiponectin. The beta-3 adrenergic receptor agonist recovered the mRNA expressions of adiponectin, adiponectin receptors, and beta-3 adrenoreceptor, which were reduced in epididymal white adipose tissue in KKAy mice. Also, CL316243 suppressed the overexpressed mRNA level of TNF-alpha in both epididymal white and brown adipose tissues. It was concluded that the normalization of adiponectin, adiponectin receptors and TNF-alpha could contribute at the amelioration of obesity-induced insulin resistance [28].

In the study conducted on nonobese/nondiabetic Sprague-Dawley rats, the selective beta-3 agonist CL 312243 increased food intake, metabolic rate, and body temperature after 7 days of treatment. The author also showed a decrease in intra-abdominal and subepithelial fat, a hepatic glucose level independent of variations in body weight, an increase in interscapular fat, and in total glucose, which stimulates the production of insulin. According to the results, only white and brown adipose tissues have been affected. A more important role of adipose tissue in glucose uptake underlining the potential role of beta-3 adrenergic agonist drugs for the treatment of obesity and insulin-resistant diabetes was suggested [8].

By a critical analysis of the published data in nonclinical research, it was concluded that beta-3 adrenergic receptor activation in the experiment-induced diabetes and obesity [37, 38] determines an increase of glucose tolerance and lipolysis activation in adipose tissue [22]. It seems that the effects are dose dependent and the selectivity of actions for glucose metabolism occurs at lower doses than those used for influencing lipid metabolism.

Comparing the effects of beta-3 agonists in rats and humans, Arch and Wilson stated that these compounds, with remarkable effects on rodents, have not convinced in clinical studies because of limited efficacy or serious side effects. The explanations mentioned by the authors included low pharmacokinetic properties and a low biotransformation to active compounds. A possible more important distinction between rats and humans is the different structure of beta-3 receptors, leading to lower efficacy of compounds in humans than in rats. In addition, it seems that the number of beta-3 receptors is lower than beta-1 and beta-2 receptors in the tissues that mediate thermogenesis in humans [39]. This is one of the reasons why the clinical studies conducted on beta-3 selective agonists had some contradictory results.

Mirabegron, a currently approved drug for the treatment of overactive bladder, was recently studied in humans for its effects on brown adipose tissue. This drug has several advantages over other members of its class, including a higher bioavailability and a higher *in vitro* affinity for the human beta-3 adrenoreceptor. Mirabegron was orally administered in the dose of 200 mg to 12 healthy male subjects with detectable brown adipose tissue. The results showed that all treated subjects had a higher brown adipose tissue metabolic activity, measured with 18Ffluorodeoxyglucose (18F-FDG) using positron emission tomography (PET) combined with computed tomography (CT). These are promising results for a possible future use of beta3 agonists for metabolic disease [40].

Based on all these preclinical and clinical considerations new chemical entities with betaphenylethylamine nucleus, substituted in various positions on the nucleus or side chain, with potential action on diabetes and/or obesity were synthesized [41, 42]. Chemists led synthesis in order to obtain derivatives with increased beta-3 receptor selectivity. The compounds were conventionally named A1-βPhEA–A13-βPhEA (**Figure 1**).


**Figure 1.** General structure of the newly synthesized compounds. X = H, alkoxy, halogen, dihalogen; Y = 4-carbopropoxy-phenoxy, 4-carbomethoxymethylene-phenoxy, 4-carbomethoxyethylene-phenoxy.
