Medicinal Chemistry of Catechol O-Methyltransferase (COMT

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Medicinal Chemistry of Catechol O-Methyltransferase (COMT) Inhibitors and their Therapeutic Utility Laszlo Erno Kiss, and Patrício Soares-Da-Silva J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500572b • Publication Date (Web): 31 Jul 2014 Downloaded from http://pubs.acs.org on August 20, 2014

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Journal of Medicinal Chemistry

Medicinal Chemistry of Catechol OMethyltransferase (COMT) Inhibitors and their Therapeutic Utility László E. Kiss a,* and Patrício Soares-da-Silva a,b,c a

Department of Research & Development, BIAL - Portela & Cª, S.A., À Avenida da Siderurgia Nacional, 4745-457 S. Mamede do Coronado, Portugal

b

Department of Pharmacology & Therapeutics, Faculty of Medicine, University of Porto, 4200319 Porto, Portugal b

MedInUP - Center for Drug Discovery and Innovative Medicines, University of Porto, 4200319 Porto, Portugal

ABSTRACT

Catechol O-methyltransferase (COMT) is the enzyme responsible for the O-methylation of endogenous neurotransmitters and of xenobiotic substances and hormones incorporating catecholic structures. COMT is a druggable biological target for the treatment of various central and peripheral nervous system disorders, including Parkinson’s disease, depression,

1 ACS Paragon Plus Environment


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schizophrenia and other dopamine deficiency-related diseases. The purpose of this article is fourfold: i) to summarize the physiological role of COMT inhibitors in central and peripheral nervous system disorders; ii) to provide the history and perspective of the medicinal chemistry behind the discovery and development of COMT inhibitors; iii) to discuss how the physicochemical properties of recognized COMT inhibitors are understood to exert influence over their pharmacological properties; and iv) to evaluate the clinical benefits of the most relevant COMT inhibitors.

1. Introduction

In the late 1950s, Marvin Armstrong and coworkers studied the metabolism of norepinephrine in the urine of patients with the adrenaline-forming tumor pheochromocytoma and noted the formation of an O-methylated metabolite (3-methoxy-4-hydroxymandelic acid).1 The endogenous origin of this metabolite had been postulated because its excretion was not affected by dietary changes. Following Armstrong’s work, the group of the 1970 Nobel Prize winner for Physiology or Medicine, Julius Axelrod, reported the normal occurrence of the corresponding 3O-methylated metabolites of dopamine, epinephrine and norepinephrine in mammals.2,

3

The

enzyme, which is responsible for the regioselective O-methylation of one of the hydroxyl groups of catecholamines, was partially purified and characterized from rat liver fractions by the same group and later termed catechol-O-methyltransferase (COMT EC 2.1.1.6).3 Because catecholamines are important neurotransmitters involved in many biological functions, the inhibition of the COMT enzyme became an attractive strategy for manipulating the levels of endogenous neurotransmitters such as dopamine, epinephrine and norepinephrine. Although early COMT inhibitors (often classified as ‘first-generation’ inhibitors) prolonged the


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physiological effect of endogenous neurotransmitters by inhibiting COMT, their efficacy on dopaminergic, adrenergic and noradrenergic mechanisms was limited.4 Furthermore, the role of ‘first-generation’ COMT inhibitors in the inactivation of neurotransmitters at receptor sites was questioned. COMT began to attract increasing attention in the 1970s, when the involvement of the COMT enzyme in the metabolic degradation of the mainstay antiparkinsonian drug L-DOPA (levodopa) was discovered. Parkinson’s disease (PD) is a chronic degenerative neurological disorder predominantly afflicting the aged population. PD is caused by a reduction in the striatal levels of dopamine associated with the gradual degeneration or death of nigral cells in the brain. L-DOPA can be considered a biological precursor of dopamine that can be used as an ‘artificial’ means to manipulate the cerebral levels of this neurotransmitter.5 Despite the beneficial ability of L-DOPA to modulate the cerebral levels of dopamine, one of its major drawbacks is its short in vivo half-life. COMT inhibitors block the unwanted O-methylation of L-DOPA by COMT to 3O-methyl-L-DOPA (3-OMD) in peripheral tissues, thereby prolonging the pharmacological effect of L-DOPA as well as diminishing the therapeutic dose. Over the last thirty years, efforts from several research groups from both academia and the pharmaceutical industry have focused on optimizing L-DOPA therapy with the objective of providing a more continuous and sustained delivery of dopamine to the striatum by targeting COMT. The search for new COMT inhibitors led to the discovery of ‘second-generation’ inhibitors, which were endowed with improved pharmacokinetic profiles compared with those of the ‘first-generation’ predecessors. Clinically relevant COMT inhibitors for the adjunctive treatment of PD are exemplified by nitecapone, (316, OR-462) (Figure 8), entacapone (327, OR-611) (Figure 8), tolcapone (378, Ro40-7592) (Figure 9), nebicapone (399, BIA 3-202) (Figure 9) and opicapone (50c10, BIA 9-1067) (Figure 10).

Although

‘second-generation’

COMT

inhibitors

share

the

same

nitrocatechol



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pharmacophore, subtle differences do exist in the mode of COMT inhibition. These characteristic properties are thought to be of relevance in terms of the downstream clinical efficacy and safety of PD treatment and will be discussed in this perspective article along with the potential therapeutic applications of COMT inhibitors in other dopamine-deficiency related diseases.

2. Catechol-O-methyltransferase (COMT) enzyme Catechol-O-methyltransferase (COMT) is a magnesium-dependent intracellular enzyme that catalyzes the transfer of a methyl group from the common methyl donor S-adenosyl-Lmethionine (AdoMet or SAM) to substrates incorporating a dihydroxybenzene (catechol) motif, resulting in the formation of mono-O-methylated products and S-adenosylhomocysteine (AdoHcy or SAH) (Figure 1).2

Figure 1. COMT catalyzed methylation of simple catechol and pyrogallol substrates The enzymatic O-methylation reaction generally takes place with high regioselectivity. Under in vivo experimental conditions, the COMT-mediated metabolic degradation of catecholic substrates almost exclusively results in the formation of the corresponding meta-O-methylated 4 ACS Paragon Plus Environment

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products.11 In contrast, under in vitro conditions, some COMT substrates have also been shown to undergo O-methylation reactions at the para-position. The extent of the para-O-methylated product is highly dependent on the nature of the substrate and the experimental conditions, such as the pH of the reaction mixture, but in general accounts for ≤ 50% of the total amount of methylated metabolites.12 Catechols bearing a polar side chain (e.g., dopamine) are O-methylated exclusively at the meta position in vitro, whereas substrates that contain a less polar side chain may also undergo O-methylation reactions at the para position. Modeling studies with the 3D protein structure have also confirmed that COMT favors meta-O-methylation over the para-Omethylation reaction. The binding of the para-hydroxyl group of the catechol nucleus to the methyl donor AdoMet forces the side chain to accommodate an unfavorable position, which results in repulsive interactions with the protein residues.13 Vicinal trihydroxy benzenes (e.g., pyrogallol) are methylated on the middle hydroxyl group regardless of the substitution pattern of the middle hydroxyl group (i.e., para or meta) relative to other substituents on the benzene ring.14 COMT is able to methylate only one of the hydroxyl groups, and no dimethylated product is formed. 2.1 Function, substrates and distribution of COMT The main physiological function of COMT is the metabolic inactivation of endogenous catechol neurotransmitters15 and xenobiotic substances.16, 17 Furthermore, the physiological functions of several hormones incorporating a catechol pharmacophore are also terminated by COMT. The COMT physiological substrates include dopamine, epinephrine, norepinephrine, 3,4dihydroxymandelic acid (DOMA), 3,4-dihydroxybenzoic acid (DOPAC), ascorbic acid and catechol estrogens, such as 2-hydroxyestradiol, 4-hydroxyestradiol, 2-hydroxyestrogen, 2hydroxyestron, 2-hydroxyestron and the dihydroxyindolic intermediates of melanin. Several



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drugs incorporating a dihydroxyphenyl moiety are also substrates of COMT, including amino acid decarboxylase (AADC) inhibitors (carbidopa 118 and benserazide 219), dopamine agonists (apomorphine 320, dobutamine 421 and fenoldopam 522), the antihypertensive compound alphamethyl-L-DOPA 623 and bronchodilating agents, such as isoprenaline 724 and rimiterol 825 (Figure 2). Moreover, COMT also plays a key role in the inactivation of exogenous toxic compounds and metabolites bearing catecholic structures. OH

O HO

O

OH HN

HO

NH2

HO NH2

N H

HO H N

OH

OH HO

H 1

2 Benserazide

Carbidopa

N

3 Apomorphine

HO O

OH HO

H N

HO

HO

HO

NH

HO

OH NH2

HO Cl

4 Dobutamine

6

5

alpha-methyl L-DOPA

Fenoldopam

OH HO

H N

OH

NH

OH HO

OH 7

8

Isoprenaline

Rimiterol

Figure 2. Drug substrates of COMT.


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The distribution and cellular localization of COMT have been evaluated in several animal species as well as in humans.26 COMT is considered a ubiquitous enzyme because it can be found in almost all peripheral tissues as well as in the central nervous system (CNS). In peripheral tissues, the COMT enzyme is abundantly expressed in the liver and kidney but is also present in the lung, stomach, spleen, intestines, adrenals, heart, various glands, adipose tissue, the uterus, gonads, muscles and red blood cells (RBCs).15, 27 The COMT enzymatic activity in the liver is three- to fourfold higher than that in other peripheral tissues. The extent of COMT protein and enzymatic activity in the brain is significantly lower than that in peripheral tissues. Different brain areas show quite similar COMT protein distributions. The COMT enzyme has been found to be present in two distinct forms;28 both a soluble form (S-COMT) and a membrane-bound form (MB-COMT) have been cloned and characterized. They are coded by the same gene using two separate promoters.29 The gene encoding the human enzyme is located in chromosome 22, band q11.2.30 Human S-COMT consists of 221 amino acids, whereas human MB-COMT has a 50-residue-long amino-terminal extension that contains the hydrophobic anchor region. The primary amino acid sequences for different mammalian species exhibit high similarity. For instance, human S-COMT is 81% identical with rat S-COMT and displays an 82% homology with the porcine COMT enzyme.31 Although large interspecies differences were found in the relative distribution of S-COMT and MB-COMT, S-COMT is predominant in the periphery, whilst MB-COMT is predominant in the brain.31,

32

The cellular localization of S-

COMT is different from that of MB-COMT. S-COMT is mainly expressed in the cytoplasm, whereas MB-COMT is located on intracellular membranes, such as the rough endoplasmic reticulum, oriented towards the cytoplasm. Both the S-COMT and MB-COMT forms are active towards O-methylation reactions, and no difference in substrate specificity has been reported.



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The primary amino acid sequences of the catalytic sites of S-COMT and MB-COMT are identical, but there is a marked difference in their kinetic behavior. MB-COMT shows a higher substrate affinity (Km) than does S-COMT, which in turn exhibits a much higher capacity (Vmax) than does MB-COMT. These kinetic differences determine the roles of the two isoenzymes. MBCOMT has been considered to be the physiologically more important form of the enzyme because it is able to methylate catecholamines at their physiological concentration.33 S-COMT is important in nonphysiological conditions, such as when the substrate concentration suddenly increases (e.g., during L-DOPA treatment) or when a higher O-methylation reaction rate is needed.34 2.2 Catalytic mechanism and three-dimensional crystal structure of COMT: The methylation of catecholic substrates by the COMT enzyme requires the presence of magnesium ions (II) and the cofactor AdoMet. Early kinetic studies with the partially purified enzyme suggested several different catalytic mechanisms, such as the ping-pong mechanism or the rapid random equilibrium mechanism.35 Later, it was concluded by Woodard and coworkers that the mechanism is sequentially ordered and that the methylation reaction proceeds in a single step via a tight SN2-type transition state in inversion mode without the involvement of a methylated enzyme intermediate.36 Approximately three decades after Axelrod’s pioneering work, the soluble form of recombinant rat COMT in complex with the methyl donor AdoMet, a magnesium (II) ion and a simple tight-binding nitrocatechol, 1937 (3,5-dinitrocatechol, 3,5-DNC) (Table 1), was successfully co-crystallized.13,

38

In the last two decades a number of other crystallographic

structures of human and rat S-COMTs bound to various inhibitors have also been determined and comprehensively reviewed.39 The analysis of the crystal structures revealed that the structural


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fold of human and rat S-COMTs is similar.13, 40 The overall structure of rat S-COMT in complex with 19 and the enlarged view of the catalytic site are shown in Figures 3A and 3B, respectively.

Insert Figure 3

COMT has a single domain, a mixed α/β-protein structure in which the seven-stranded central β-sheet core is surrounded by eight α-helices (Figure 3A). The active site of COMT consists of the AdoMet binding domain and the catalytic region with the magnesium (II) ion. The cofactor AdoMet is located in a more buried cleft, whereas the substrate binding site occupies a shallow groove on the outer surface of the protein (Figure 4). It has been found that AdoMet is the first ligand to bind to the enzyme, followed by the magnesium (II) ion and finally by the substrate.

Insert Figure 4

The major molecular interactions with the protein are shown in Figure 3B. The adenine ring of AdoMet is hydrogen bounded to Ser119 and Gln120 and makes further van der Waals interactions with residues Ile91, Ala118 and Trp143. The methionine fragment of AdoMet forms hydrogen bonds with residues Val42, Ser72 and Asp141.The magnesium (II) ion is located in the center of the catalytic site and has no interaction with the cofactor AdoMet. It is octahedrally coordinated to the oxygen atoms of the side chains of aspartic acid residues Asp141 and Asp169 and asparagine residue Asn170, while another coordination site is occupied by a molecule of water. The fifth and sixth coordinating orbits of the magnesium (II) ion are chelated to each of the two hydroxyl groups belonging to the catechol substrate. In addition to the excellent



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coordination to the protein and the catechol substrate, the magnesium (II) ion lowers the substrate pKa, thereby facilitating the deprotonation of the more acidic hydroxyl group of the catechol nucleus by Lys144; the resultant phenolate anion can be immediately methylated by AdoMet. Further important hydrogen bonding interactions have been identified between the hydroxyls of the catechol ring and the side chains of Glu199 and Lys144. Additional beneficial interactions are provided by the so-called hydrophobic “gatekeeper” residues (Trp38, Trp143, Pro174 and Leu198), which maintain the substrate correctly positioned for methylation by making favorable interactions with the catechol ring. The interaction of the catechol ring with Trp38 was found to be absolutely essential for high binding affinity. Replacement of Trp38 with arginine in pig COMT drastically reduced the binding affinity of the catechol substrate.41 From these observations, several families of inhibitors have been designed and synthesized.39 Overall, the analysis of the crystal structures clearly suggest that COMT can interact well with various types of inhibitors incorporating catechol pharmacophore. Because the catechol ring binds to the enzyme in a groove at the surface of the protein, the side-chain extends out of the catalytic site cavity toward the solvent region and it can be a target for optimization of the pharmacokinetic profile of the inhibitor. 3. Therapeutic applications of COMT inhibitors: COMT inhibitors are used primarily in the treatment of certain central and peripheral nervous system disorders,42 such as Parkinson´s Disease (PD)5, 43, restless leg syndrome44 (RLS), schizophrenia45, mood disorder46, depression,46 cognition improvement,47 the depressive phase of bipolar disorder,48 edema formation state,49 gastrointestinal disturbances50 and other dopamine deficiency-related diseases, such as attention deficit disorder51 (ADD) and attention deficit hyper activity disorder52 (ADHD). The following serves as a brief summary of the most important therapeutic applications.


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3.1 Parkinson’s disease: Parkinson’s disease (PD) is the most prevalent chronic degenerative neurological disorder of the elderly after Alzheimer’s disease (AD). Dopaminergic neurons are gradually destroyed in a specific region of the CNS (mainly in the substantia nigra), resulting in a reduction in brain dopamine levels, which becomes symptomatic over a certain threshold. The classical symptoms of PD are motor-related and commonly include tremor and rigidity as well as mild to severe difficulty in coordinating physical movements. Dopamine is a neurotransmitter that stimulates motor neurons, which are nerve cells that control the muscles. If dopamine production is depleted, the motor system nerves are unable to control movement and coordination. In the later stages of PD, cognitive and behavioral problems may also arise. When clinical symptoms appear, ~ 70-80% of the dopaminergic neurons have already been lost. The etiology of this neuronal destruction is unknown (idiopathic PD), but most likely both genetic and environmental factors contribute.53 PD has an estimated prevalence of 2% in the adult population over 60 years of age. There is currently no cure for PD; however, there are medications that can control the symptoms of the disease. In the early stages of PD, the administration of a dopamine agonist, which mimics the effects of dopamine at the dopaminergic receptors, helps alleviate the symptoms of PD. Alternatively, the administration of a centrally active monoamine oxidase (MAO-B) inhibitor prevents the cerebral breakdown of dopamine by blocking the oxidative deamination of dopamine to 3,4-dihydroxyphenylacetic acid (DOPAC)54, thereby reducing symptoms and improving the quality of life of parkinsonian patients. In certain cases, direct stimulation of the dopaminergic receptors by surgery in the brain (deep brain stimulation) may also be an option for patients afflicted with PD. However, as PD progresses, the symptoms worsen and conventional treatments (dopamine agonists or MAO-B inhibitors) may not provide satisfactory results. The dopamine precursor L-DOPA (levodopa), the most effective



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symptomatic treatment available for PD, has been in clinical practice since the 1960s and remains the gold standard.55 Orally administered L-DOPA is able to compensate for the shortage of cerebral dopamine by transport across the blood-brain barrier (BBB), followed by conversion to dopamine by decarboxylation (Figure 5).

Insert Figure 5

As a result, L-DOPA can be employed clinically as an artificial source of dopamine. Although the majority of PD patients experience considerable improvements in motor function soon after initiating L-DOPA therapy, a major drawback is that this positive clinical response tends to diminish or fluctuate rather quickly over the following years and eventually reaches a stage where undesirable side-effects traceable to the medication, such as motor complications and dyskinesia, are not easily distinguishable from the symptoms of the disease itself. It has also been noted that when L-DOPA is administered orally, ~ 70% of the orally administered dose is extensively degraded to dopamine in the peripheral tissues through enzyme-catalyzed decarboxylation, so that only a limited percentage of the administered L-DOPA remains intact for passage into the brain. As a COMT substrate, a small amount of the administered L-DOPA (~ 10%) is metabolized by the COMT enzyme to 3-O-methyl-L-DOPA (3-OMD), a compound that has not been shown to exhibit any clinical effects (Figure 5). To combat this premature destruction of L-DOPA, a peripheral AADC inhibitor, such as 1 or 2, is administered concomitantly, which reduces the extent of L-DOPA decarboxylation in the periphery, thereby reducing the clinically effective dose of L-DOPA by approximately 70-80%.56 However, due to degradation through the COMT pathway, less than 10% of the orally administered L-DOPA dose


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reaches the brain.57 The plasma concentration of 3-OMD increases due to its long elimination of half-life (t½ ≥ 15 h) and remains high during chronic therapy.58 Moreover, 3-OMD further reduces the bioavailability of L-DOPA by competing with it for transport across the BBB.57 Therefore, the inhibition of COMT should help preserve the integrity of L-DOPA by minimizing the degree of detrimental O-methylation of L-DOPA to 3-OMD by COMT in the periphery. The concomitant administration of a COMT inhibitor and an AADC inhibitor lowers the required oral dose of L-DOPA while retaining its clinical benefit.59 3.2 Psychotic disorders: COMT also represents an important target as a susceptibility gene for cognitive dysfunction in schizophrenia as a result of the unique role of the enzyme in regulating dopaminergic function in the prefrontal cortex. Within the coding sequence of COMT is a common G>A polymorphism that produces a valine-to-methionine (Val/Met) substitution at codons 108 and 158 of S-COMT and MB-COMT, respectively.45,

60

This functional COMT

polymorphism (Val/Met) predicts performance in tasks of prefrontal executive function and the neurophysiological response measured with electroencephalography and functional magnetic resonance imaging in tasks assessing working memory. Individuals with the Val/Val genotype, which encodes for the high-activity enzyme resulting in lower dopamine concentrations in the prefrontal cortex, perform less well and are less efficient physiologically than Met/Met individuals.61, 62 These findings raise the possibility of new pharmacological interventions for the treatment of prefrontal cortex dysfunction and of predicting outcome based on COMT genotype.61 However, recent data suggest a more complex pattern of genetic regulation of COMT function beyond that attributable to the Val/Met locus. Moreover, it is also clear that there is a complex nonlinear relationship between dopamine availability and brain function.



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These two factors, allied to phenotypic complexity within schizophrenia, make it difficult to draw strong conclusions regarding COMT in schizophrenia.62, 63 3.3 Other potential indications: Dopamine plays a dual role in the body, occurring in both neurons and non-neuronal cells. Dopamine is a neurotransmitter in brain dopaminergic neurons, and its central role in the regulation of motor function and behavior is well established. In the peripheral nervous system, dopamine is the first catecholamine in the biosynthesis of neurotransmitters such as norepinephrine and epinephrine. The role of dopamine in the sympathetic nervous system is not limited only to providing a means for the synthesis of the neurotransmitter noradrenaline;64 the amine can also act as a co-transmitter in some circumstances.65 Non-neuronal cells capable of producing dopamine are distributed in the kidney and intestine.66 These renal tubular and intestinal epithelial cells are endowed with a high capacity to synthesize dopamine from its immediate precursor, L-DOPA, where the amine plays a pivotal role as a local hormone with autocrine and paracrine function, regulating water and electrolyte homeostasis.67 The rationale for the usefulness of peripherally selective COMT inhibitors in the treatment of cardiovascular disorders and gastrointestinal medical conditions is based on the considerable O-methylation of dopamine by COMT. In fact, high-sodium diets increased night-time systolic and diastolic blood pressures in wild-type mice, whereas the blood pressures in COMT (-/-) mice remained unaltered.68 Furthermore, in the wild-type mice, the sodium-induced increase in blood pressure was completely normalized by treatment with a peripheral-selective COMT inhibitor.68 Alternatively, some peripherally selective COMT inhibitors may provide protection against angiotensin II-induced renal damage through antioxidative and anti-inflammatory mechanisms rather than through COMT inhibition-induced changes in renal dopaminergic tone.69 In a prospectively followed hypertensive cohort, 174


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patients who suffered a myocardial infarction were identified and compared to 348 controls from the same cohort.70 Patients homozygous for the low activity COMT genotype had a decreased risk of myocardial infarction compared to those with the high activity genotype, with an odds ratio of 0.65 (95% CI 0.44-0.97, p=0.033). The protective effect of the low activity genotype was most evident among older patients (> 58 years of age), with an odds ratio of 0.43 (95% CI 0.230.79, p=0.006). The serum levels of estradiol were increased (p=0.006) in males with the low activity genotype. These data suggest that the low activity COMT genotype is protective against myocardial infarction, and one may speculate that the altered estrogen status could be involved in this effect.70 Other studies have also described the cardiovascular risk effect of the high activity genotype (Val/Val)71, which presents the possibility of using peripherally selective COMT inhibitors in the treatment of cardiovascular disorders. However, there is evidence in the literature that conflicts with these views72, namely, with respect to experimental models of hypertension.73 The inhibition of peripheral COMT, which prevents the mucosal degradation of dopamine, along with various dopamine receptor agonists ameliorates gastroduodenal mucosal damage. This protective effect was suggested to result from the probable electroneutral component of duodenal mucosal bicarbonate secretion stimulated via peripheral dopamine D1 receptors.74 This may also explain the increased incidence of diarrhea in patients receiving COMT inhibitors.50 In addition, some peripheral-selective COMT inhibitors stimulate cAMP-dependent Cl- secretion in the rat colon, and this process is regulated by endogenous prostaglandin and the submucosal enteric nervous system.75 On the other hand, it has also been suggested that the COMT gene 158Met polymorphism might be associated with a reduced risk of developing more severe intestinal metaplasia in Helicobacter pylori-infected older subjects.76



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4. Medicinal Chemistry of COMT inhibitors The medicinal chemistry of COMT inhibitors has been previously reviewed elsewhere.42, 77, 78 The review is structured into different classes of inhibitors exemplified by the most relevant compounds of each class, followed by summary of reported clinical data and concludes with a personal perspective on the current state of the field. 4.1

Competitive inhibitors: Following the characterization of COMT, several structurally

simple, small molecules were reported to inhibit COMT. These early COMT ligands15 included polyhydroxy compounds containing a vicinal-dihydroxy (catechol) or trihydroxybenzene (pyrogallol) motifs (Figure 6 and 7), as described further below. 4.1.1 Simple catechols and related structures: Catechols and simple related derivatives have been shown to inhibit COMT in vitro; representative examples are shown in Figure 6. Compounds 9-15 inhibit COMT in a competitive manner, with inhibition constants in the low micromolar range.4, 15 The binding of 9-15 is fully reversible through the 1,2-dihydroxy moiety. Catechols 9-15 are very good substrates for the COMT enzyme, and thus they easily undergo Omethylation reactions after incubation with liver COMT, resulting in the formation of the corresponding O-methylated metabolites in high quantities.79 The naturally occurring flavonoid 1480 and compound 1581 (U-0521) have been reported to be the most potent COMT inhibitors in vitro from this early catechol class of compounds, with inhibitory constants (Ki) of 8.4 µM and 7.8 µM, respectively. The in vivo efficacy of 9-15 was found to be limited due to the very short half-lives of these molecules.82 For instance, repeated administration of 9 was required to prolong the in vivo COMT inhibitory effect. Furthermore, 9 elicited convulsions in mice after intraperitoneal (ip) administration within a few minutes, which is unrelated to COMT inhibition and may be attributed to the facile penetration of 9 into the brain.83 Compound 15 was reported


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to effectively diminish plasma and brain levels of 3-OMD in different animal species after ip injection, but its target selectivity was questioned because it inhibited other enzymes involved in the catabolism of catecholamines, such as tyrosine hydroxylase.81 Despite its toxicity and unfavorable pharmacokinetic properties, catechol 15 was administered orally to one parkinsonian patent, but no effect on erythrocyte COMT activity was observed.84 O

O OH

HO

HO

OH

OH

OH 11

10

O

NH2 HO OH 13 Dopacetamide

12 Dopamine lutine

Caffeic acid

Protocatechuic acid

O

N H

HO

HO OH

9 Catechol

HO

O

OH

O

OH

HO OH

HO OH 14 Quercetin

15 U-0521

Figure 6. Representative structures of early catechol-based competitive COMT inhibitors. 4.1.2 Pyrogallol and its derivatives: Inhibitor 1685 (pyrogallol) and derivatives 17-1886,

87

,

similarly to 9-15, inhibit the COMT enzyme in a competitive manner because they compete with the substrate for the active site of the enzyme (Figure 7).



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O

O

HO

HO

HO OH

OH

HO

OBu

HO

HO OH

OH

16

17

18

Pyrogallol

Gallic acid

GPA 1714

Figure 7. Representative structures of early pyrogallol-based competitive COMT inhibitors. The parent pyrogallol compound 16 is one of the most potent competitive COMT inhibitors in vitro within the trihydroxy benzene series, with a Ki value of 13 µM. The higher potency of 16 over the parent catechol compound 9 (Ki = 30 µM) may be attributed to the presence of the extra hydroxyl group on the catechol nucleus, which provides beneficial van der Waals interactions with the protein in the catalytic pocket. Nevertheless, 16 displayed a similar in vivo pharmacological profile to that of 9, including limited efficacy and high toxicity. Compound 16 at a dose of 200 mg/kg was found to be short-acting in rats, and a repeated dose was required to sustain effective inhibition. Although 16 easily penetrated mice brains at a repeated dose of 50 mg/kg every 30 min, its efficacy on cerebral levels of catecholamines was insufficient. Inhibitor 17 (Gallic acid) is a markedly less potent inhibitor than 16, with a Ki value of 70 µM. Although 16 and 17 share the same 1,2,3-trihydroxybenzene pharmacophore, they have quite different physicochemical properties, which make 17 more toxic than 16. To test a simple ester of 17, namely, compound 18 (GPA 1714), as a COMT inhibitor appeared to be rational because esters of 17 have been used as safe food additives due to their antioxidant properties. A few clinical trials were performed with the N-butyl ester 18, which showed some beneficial effects at a daily dose of 750 mg in patients afflicted with Huntington’s chorea and spasmodic troticollis.88 Compound 18 has been reported to reduce the dose of L-DOPA by an average of 29% in patients


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with parkinsonism, but failed to show any advantage over AADC inhibitors administered concomitantly with L-DOPA.88 Furthermore, in patients afflicted with chronic schizophrenia, compound 18 failed to provide a beneficial therapeutic effect at a daily dose of 5 g.89 Due to their unfavorable pharmacokinetic properties and questionable efficacies against cerebral levels of catecholamines, no compounds from the trihydroxy benzene series have advanced into clinical development. 4.2 Tight-binding inhibitors In the mid-1980s, two different industrial research groups, at Orion Corp. and Hoffman-la Roche Ltd., simultaneously discovered that di-substituted catechols bearing strongly electronwithdrawing groups (EWGs) (such as nitro or cyano) at the ortho-position to one of the hydroxyl groups were more potent inhibitors of the COMT enzyme than the previously studied simple catechols 9-15 and pyrogallols 16-18.37,

90

The new derivatives were shown to alter L-DOPA

metabolism in vivo and were found to be longer-acting inhibitors than 9-18. Moreover, they did not interfere with other enzymes involved in the metabolism of catecholamines, including tyrosine hydroxylase, dopamine-β-hydroxylase (DβH) and MAO. In contrast with the earlier competitive inhibitors 9-18, the new type of inhibitors did not undergo extensive O-methylation by COMT, which suggests that they bind to the active site of the enzyme more tightly, but conversely are poorer substrates. The presence of an EWG markedly lowers the pKa values of the adjacent hydroxyl group, thereby decreasing the reactivity of the resulting phenolate anion towards O-methylation through delocalization of the negative charge.

Table 1. Representative examples of early ‘second-generation’ COMT inhibitors and IC50 values



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a

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Compound

R1

R2

R3

IC50 (nM)

19

-NO2

-H

-NO2

12a

20

-H

-NO2

-NO2

442b

21

-CN

-H

-NO2

88b

22

-CHO

-H

-NO2

162b

23

-COOMe

-H

-NO2

361b

24

-CONH2

-H

-NO2

1924b

25

-NO2

-H

-CHO

24a

26

-CF3

-H

-CHO

2300a

27

-SO2CH3

-H

-CHO

20000a

IC50 determined in rat brain. bIC50 determined in rat liver. In the in vitro screening of different compound collections, 19 was identified as the most

potent hit (Table 1).90 Compound 19 had an ED50 of 1.3 mg/kg at 1 h post-dose in rats, and concomitant administration of 50 mg/kg of 19 with L-DOPA and benserazide (100 and 50 mg/kg p.o., respectively) almost completely suppressed the formation of 3-OMD.37 The acute toxicity of 19 was found to be in the range of 312-625 mg/kg after single oral administration to mice. Despite the outstanding in vivo pharmacological profile of 19 over catechols 8-15 and pyrogallols 16-18, it was concluded that compound 19 was not suitable for clinical development due to safety and toxicological concerns.37 Preliminary structure-activity relationship (SAR) studies on structure 19 revealed that the position of the EWG is important, and the best result was obtained with compounds substituted with EWGs at the ortho-position relative to one of the 20 ACS Paragon Plus Environment

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hydroxyl groups of the catechol nucleus (cf. 19 and 2037). Replacement of the nitro group with other EWGs, such as cyano (2137), aldehyde (2237), carboxylic acid ester (2337) or carboxyamide (2437) groups, drastically reduced COMT inhibition in vitro. Attempts to replace the nitro group of 2590 with trifluoromethyl or mesyl groups gave the practically inactive compounds 2690 and 2790, respectively. The nitro group located at the furthest place from the catechol hydroxyls in structure 19 has inspired scientists to try to replace it with other groups to modulate the pharmacokinetic properties of 19. The nitrocatechol moiety (1,2-dihydroxy 3-nitrobenzene) has been a standard structural feature on most subsequent COMT inhibitor designs because it exhibits greatly improved in vitro potency over the ‘first-generation’ inhibitors 9-18. Efforts by academic research groups and pharmaceutical companies have led to the identification of several new classes of ‘second-generation’ (tight-binding) COMT inhibitors with improved pharmacokinetic profiles over 19 and related structures. Several of these research groups have developed compounds that have reached clinical trials, as described further below. 4.2.1 Meta-nitrated catechols 4.2.1.1 Vinylic derivatives: A series of vinylic compounds disclosed by Orion Corp. and exemplified by 28-32 showed high in vitro potency (IC50 < 25 nM) in rat brain tissues more or less independently of the nature of the substituents on the vinyl group (Figure 8).90, 91



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Figure 8. Representative examples of vinylic meta nitrated COMT inhibitors and IC50 values in rat brain homogenates. Inhibitors 28-32 exhibited a greater than 1,000-fold increased specificity for COMT versus tyrosine hydroxylase and a greater than 10,000-fold increased specificity versus DβH, MAO-A and MAO-B enzymes. Compounds 28-30 were found to significantly reduce the plasma levels of 3-OMD in rats by 89%, 26% and 49%, respectively, 5 h after oral administration at a dose of 30 mg/kg. The pharmacodynamic interaction of the dicyano derivative 28 with L-DOPA was also assessed. Compound 28 provided a two-fold increase in the plasma levels of L-DOPA 3 h postadministration, which was steadily sustained over the next 2 h. Compounds 31 and 32, which are structurally closely related to 28-30, were selected as clinical candidates of Orion for the adjunctive treatment of PD. Inhibitor 31 has been reported to be a tight-binding inhibitor of COMT, with IC50 values of 18 and 307 nM in rat brain and liver, respectively. The Ki value of 31 was calculated to be 23 nM in rat liver.92, 93 COMT inhibition by 31 was strictly confined to the periphery, and 31 was found to 22 ACS Paragon Plus Environment

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be devoid of acute toxicity risk (LD50 > 2500 mg/kg). The beneficial effect of 31 on the striatal levels of L-DOPA, dopamine, DOPAC and 3-OMD in rat lasted for 5 h after the oral administration of 31 plus L-DOPA and an AADC inhibitor and were found to be dose-dependent (3-30 mg).94 Compound 31 demonstrated relatively modest in vivo efficacy in cynomolgus monkeys after intravenous (iv) administration of L-DOPA/AADC inhibitor 1 boluses, but when administered to rats at an oral dose of 30 mg/kg concomitantly with L- DOPA and AADC inhibitor 1, inhibited the formation of 3-OMD in serum by 80%.95 Compound 32 is a fully reversible potent inhibitor of COMT with IC50 values of 10 and 160 nM in rat duodenum and liver, respectively.96 Inhibitor 32 achieved nearly complete inhibition of duodenal COMT for 1-3 h after an oral dose of 30 mg/kg in rats. Compound 32 is a longer-acting inhibitor than is 31, sustaining significant COMT inhibition up to 6 h post-administration in rats. Compound 32 was found to be a purely peripheral COMT inhibitor at doses of up to 30 mg/kg. Furthermore, 32 demonstrated a marked effect on rat plasma 3-OMD and L-DOPA pharmacokinetics in a dose-dependent manner (3-30 mg/kg).94, 96 Although 32 is the most widely prescribed antiparkinsonian COMT inhibitor, it has limitations in clinical practice. Due to its low to moderate bioavailability (F = 29-46%) and relatively short-acting COMT inhibitory profile, its dosages are elevated (200 mg) and must be repeated up to eight times per day.97 The reason for the limited exposure of 32 may be related to its inadequate physicochemical properties. Compound 32 exhibits very low aqueous solubility at low pH (80 µg/mL at pH ≤ 5), which affects its rate of absorption from the acidic environment in the stomach.98, 99 At the higher pH values (pH ≥ 5) found in the small intestine, 32 exists in the ionized form, which limits its passage through the intestinal wall. The partition coefficient (Log P) of the ionized form of 32 has been determined to be -0.4 in a mixture of 1,2-dichloroethane-water.98 The low lipophilicity



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of 32 at physiological pH can also explain its limited passage across the BBB. In addition to its low aqueous solubility and lipophilicity, the enzymatic stability of 32 is also limited. Compound 32 was found to be extensively metabolized through glucuronidation of its hydroxyls, which resulted in short half-lives in different animal species as well as in humans.100 The therapeutic usefulness of vinylic structures, such as compounds 28-32, has been demonstrated primarily in the adjunctive treatment of PD, but other potential therapeutic applications have also been claimed by Orion in several patent applications. In a divisional application of Orion’s initial patent application91, peripherally selective COMT inhibitors bearing nitrocatechol motifs (Table 1 and Figure 8) were indicated to be useful in the treatment of hypertension and heart failure, while depression could be treated with CNS-active COMT inhibitors, though no experimental data were reported.101 The effect of COMT inhibitors on the treatment of ulcers and lesions in the gastro-intestinal tract has also been reported.102 Compound 19 and 31 significantly reduced both the incidence and the severity of cysteamine-induced duodenal ulcers in rats. In another experiment, the administration of absolute ethanol to rats resulted in severe gastric damage consisting of grossly hemorrhagic and necrotic lesions. An oral dose of 30 mg/kg 31 and 19 caused 37% and 77% reductions in the areas of the lesions, respectively. In a separate patent application, Orion reported the anticancer effects of 19 and 32 on the MCF-7 human breast cancer cell line.103 Both 19 and 32 markedly decreased the cell growth of MCF-7 cells at low micromolar concentrations. Compound 31 has been shown to have an effect on the prevention of diabetic vascular dysfunctions, such as nephropathy, retinopathy and neuropathy.104 The effect of 31 on the glomerular filtration rate (GFR) and albumin excretion rate (AER) was studied in diabetic rats. Inhibitor 31 at a dose of 30 mg/kg was found to normalize both GFR and AER when compared to non-inhibitor 31 treated animals. In 2001,


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Aho and Linden reported analgesic effects for COMT inhibitors.105 The relationship between COMT inhibition and analgesic effects has remained unclear. However, inhibitors 19 and 31-32 displayed significant reductions in the pain thresholds of different pharmacological models, such as in the Randall-Selitto test and the acetic acid-induced writhing test. Interestingly, the same COMT inhibitors (19 and 31-32) had no effect in a hot plate test, which might be attributed to their peripheral mechanism of action. Orion published two patent applications on the use of COMT inhibitors in combination with L-DOPA and AADC inhibitors for the symptomatic treatment of patients afflicted with restless legs syndrome (RLS)106 and for the delay of the progression of motor dysfunction in patients afflicted with PD.107 It has been reported that 32 potentiates the turning behavior induced by L-DOPA and AADC inhibitor 1 in rats with unilateral nigral lesions.108 4.2.1.2 Acetylated and carbamoylated derivatives: Acetylated and carbamoylated series of compounds were first identified by Hoffmann la Roche Ltd., aimed at the development of an orally bioavailable, reversible-binding and CNS-active COMT inhibitor for the treatment of PD. Representative examples 33-36 are shown in Figure 9.37, 109



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Figure 9. Representative examples of acetylated and carbamoylated meta-nitro COMT inhibitors and their IC50 values in rat liver homogenates. Among structures 33-35, no significant influence of different side chain substituents on in vitro activity has been observed. Despite good in vitro COMT enzymatic activity, compounds 33-35 showed moderate in vivo efficacy and exhibited considerable toxicity. The ED50 values of 33-35 have been determined in rats at 4 h post-dose to be 6.7, 5.1 and 3.4 mg/kg, respectively. The optimization of the acetyl substituent of 35 led to the identification of benzoyl derivative 36, which was slightly more potent in vitro than was the parent compound 35, though it was 26 ACS Paragon Plus Environment

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endowed with markedly enhanced in vivo efficacy over 33-35. This ortho-fluoro analogue 36 (Ro41-0960) was found to be very potent, with an oral ED50 of 0.28 mg/kg in rats at 1 h postdose. Moreover, compound 36 (3-100 mg, po, 2 h) blocked the formation of 3-OMD and homovanillic acid (HVA) in rats in a dose-dependent manner. Concomitant administration of 50 mg/kg 36 with L-DOPA and benserazide (100 and 50 mg/kg po, respectively) almost completely suppressed the formation of 3-OMD (9% of control).37 Despite the favorable pharmacological and toxicological profile of 36, Roche has not considered the clinical development of COMT inhibitor 36. However, from the same class of compounds, a structurally very close analogue to 36, inhibitor 37, was chosen for clinical evaluation for the adjunctive treatment of PD.110 Compound 37 was characterized as a tight-binding and reversible inhibitor of the COMT enzyme and found to be more potent under in vitro experimental conditions than was 31 or 32 against rat liver and brain COMT, with IC50 values of 36 and 2.2 nM, respectively.110 Furthermore, 37 displayed longer duration of action in different animal species and appeared to be more potent than were compounds 31 and 32.111 Compound 37 showed full inhibition in the heart and kidney when dosed to rats (100 mg/kg) at 0.5 h post-dose, and the liver COMT was inhibited by over 90% at the same time point. Significant inhibition (≥ 50%) was maintained up to 8 h (in liver) and 11 h (heart and kidney) after dosing.112 The COMT enzymatic activity was fully recovered at 16 h post-dose in all tissues. 37 increased the extracellular levels of L-DOPA, dopamine and DOPAC in the rat striatum in a dose-dependent manner (3-30 mg), while the levels of 3-OMD were markedly decreased when 37 was co-administered with L-DOPA and DDC inhibitors. Although 37 shares with 31 and 32 the same nitrocatechol pharmacophore, it displays a different pharmacokinetic profile, which was demonstrated by its cerebral inhibition effect.113 The high permeation of 37 across the BBB is believed to be due to the presence of the



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lipophilic 4’-methylbenzoyl side chain. Compound 37 has been shown to be an equipotent inhibitor of both brain and peripheral COMT, with a cerebral ID50 of 26-28 mg/kg in rats.112 The L-DOPA induced increase in HVA output was attenuated by 37, which further confirms the occurrence of central COMT inhibition.113 The beneficial effect of central COMT inhibition has been demonstrated in several pharmacological models. Inhibitor 37 administered alone has demonstrated antidepressant activity in an animal model of the anhedonic state, but an L-DOPA boost was needed to show a more marked antidepressant effect in two other rat models of depression (the forced swimming test and learned helplessness paradigm).114, 115 Compound 37 has also been shown to increase the exploratory activity of rats after oral administration at a dose of 10 mg/kg and was found to potentiate the hyperactivity and stereotypy induced by amphetamine and nomifensine in rats.116 The acute toxicity of 37 was found to be low. The oral LD50 values in rat were higher than 2 g/kg.110, 117 The oral bioavailability of 37 was demonstrated to be superior (F = 60%) to that of 32. Compounds 32 and 37 have similar pKa values and poor aqueous solubility profiles. Experimental Log P values for both the neutral (3.17) and ionized (0.2) forms of 37 were found to be higher than that of 32 (2.16 and -0.4, respectively), which supports its enhanced intestinal absorption and higher permeation across the BBB over 32. During clinical trials, 37 altered liver enzyme activity in 1-3% of subjects.118 Shortly after the launch, the marketing authorization of 37 was suspended due to liver toxicity concerns highlighted by several post-launch cases of fatal fulminant hepatitis.119 Several possible causes have been postulated for the toxicity of 37. In humans, compound 37 is almost completely metabolized prior to excretion, with less than 1% of an orally administered dose of 37 excreted unchanged.120 Although the reduction of the nitro group to the corresponding catecholic aniline accounted for 8% of the excretion of the


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administered dose of 37, this metabolic pathway could be responsible for forming reactive intermediates arising from the oxidation of the amino metabolite of 37. It has also been shown that these reactive intermediates form glutathione adducts in vitro upon incubation with human liver microsomes and may be capable of forming covalent adducts with hepatic proteins, resulting in damage to liver tissue.121 Another possible mechanism of toxicity involves the uncoupling of oxidative phosphorylation, which has been observed in vitro and in vivo.122, 123 Furthermore, 37 has been shown to be toxic to human neuroblastoma SH-SY5Y cells and caused a profound reduction in ATP synthesis with or without a functional respiratory chain.124 Limitations in the clinical use of 32 and 37 have prompted the discovery of novel COMT inhibitors with improved efficacies (more efficacious than 32), peripheral selectivities (more selective than 37) and safety profiles (safer than 37). To this end, the analogue-based design approach has been taken at BIAL - Portela & Cª, S.A. to identify new compounds that selectively inhibit COMT in peripheral tissues. A homologous series (n=1-4) of nitrocatechol structures 38 have been disclosed, which were derived from the CNS active 37 (Figure 9).125 It had been speculated that insertion of an aliphatic spacer (n = 1-4) between the carbonyl group and the phenyl ring might result in COMT inhibitors approximately equipotent to the parent 37 but with improved peripheral selectivity. Compounds with different spacer lengths (n=1-4) were shown to inhibit in vitro both rat brain and liver COMT with IC50 values ranging from 3.7 nM to 12.8 nM and from 696 nM to 1285 nM, respectively. It has also been shown that compounds with the general formula 38 (n= 1-4) achieved maximal inhibitory effects within 30 min after their oral administration to rats at a dose of 30 mg/kg. The parent benzophenone derivative (n=0) showed a similar inhibitory profile against brain and liver COMT, whereas compounds with longer carbon chains (n=1-4) were less potent at inhibiting brain COMT in comparison with their inhibitory



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effect on liver COMT. Inhibitor 39126 was found to exhibit the longest duration action and was endowed with enhanced peripheral selectivity over 37. Furthermore, 39 and 37 were equally potent inhibitors of liver COMT in the rat, with ED50 values of 0.7 mg/kg at 1 h post-dose. Inhibition by 39 had a shorter duration than did that by 37 but a longer duration than did that by 32. Compound 39 displayed weaker brain COMT inhibition than did 37, with ED50 values of 5.3 mg/kg and 1.6 mg/kg, respectively. Substitution on the phenyl ring of core structure 38 had no effect on the inhibitory profiles of the parent unsubstituted structures, such as 39.126 Incubation of 39 with liver microsomes resulted in the formation of the corresponding meta- and para-Omethylated metabolites The crystal structures of COMT were used to study the molecular interactions between 39 and rat S-COMT by unrestrained flexible-docking simulations.127 It has been found that the catechol ring of 39 can bind in two different orientations to the active site of COMT, which explains the lack of regioselectivity of O-methylation seen in vitro. In contrast, under in vivo conditions 39 has been shown to undergo O-methylation exclusively at the metaposition relative to the phenylacetyl side-chain, as a result of the preferential cytochrome P450mediated O-demethylation of the para-O-methyl regioisomer.127 The functional relevance of brain COMT inhibition by 39 was evaluated based on the potentiation of amphetamine-induced hyperactivity. Amphetamine is known to be a potent psychostimulant that, depending on the dose administered, increases locomotion and induces stereotypies. Peripherally selective COMT inhibitors are expected not to alter amphetamineinduced locomotion and stereotypies. Indeed, rats treated (30 mg/kg) with 32 or 39 6 h before amphetamine challenge (4 mg/kg) displayed the same pattern of locomotion and stereotyped behavior as did the corresponding controls. On the basis of the overall pharmacological profile, 39 entered clinical evaluation as an adjunct to L-DOPA therapy for PD. The clinical


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development of 39 was discontinued because its safety profile was not considered sufficiently improved over that of 37. Further efforts to modify 37 led to the identification of a distinct series of compounds incorporating heteroatom-containing side chains, such as alkyl-phenols, thiophenols and disubstituted amines (Figure 9).128 Compounds from phenoxy and thiophenoxy series 40 were found to exhibit slightly improved in vitro (in SK-N-SH cells, 100 nM) COMT inhibition (8594%) over 32 (77%) but were less efficacious than was 37 (97%).129 The length of the hydrocarbon spacer (n=1-4) had no effect on the in vitro COMT inhibition. In mice at a dose of 30 mg/kg, compounds from the same class 40 were shown to be potent liver COMT inhibitors (70-78% inhibition at 1 h) and were reasonably selective for the periphery (20-39% central inhibition at 1 h). In the alkylamino series 41,128 compounds containing N-phenyl piperazine side chains were equipotent to 37. Substitution with less bulky groups, such as N,N-dimethyl, morpholine and alkyl-piperazine, led to reductions in COMT inhibition. Thus, systematic modification of N-phenyl piperazine side chains (n = 2) led to the identification of inhibitor 42129 (BIA 3-335) (Figure 9), which was found to be a long-acting peripheral COMT inhibitor retaining 74% inhibition at 6 h post-dose (30 mg/kg, mice), while 32 provided only 26% inhibition in liver tissues. Furthermore, 42 displayed better peripheral selectivity (14% cerebral inhibition at 1 h) compared to 37 (99% brain inhibition at 1 h). The poor BBB penetration of 42 may be attributed to the presence of two piperazinyl nitrogen atoms beyond the customary nitrocatechol motif along with the solvent accessible carbonyl function, which altogether represent an extended hydrophilic surface. Moreover, the hydrogen-bond acceptor atoms have a further negative impact on BBB permeation. In conjunction with 3D structures of COMT in complex with other inhibitors, it has been found that nitrocatechol ring of 42 binds to the



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substrate binding site and the bulky side-chain extends out of the catalytic pocket toward the solvent region without providing beneficial interaction with the protein.130 4.2.1.3 Heterocyclyl nitrocatechol derivatives: Researchers at Hofmann la-Roche Ltd. disclosed the first structurally varied series of COMT inhibitors bearing heterocyclic rings at the meta-position relative to the nitro group.37

O

O

O

HO

HO

N

H N

N HO

N

HO

HO

HO

NO2

NO2

NO2 43

44

N

NO2

Cl

S HO

45 IC50 = 23nM

IC50 = 23nM

IC50 = 29nM

N

HO

NH2 HO

HO

HO N N

HO

NO2

OH

N NH 47

46

48

IC50 = 39nM NO2

NO2

HO

HO N N

O

HO

O

HO

50a: R1-R4 = H 50b: R1 = CF3; R2-R4 = H N

N R1

R3

N 49

50c: R1 = R3 = Cl; R2 = R4 = Me opicapone (BIA 9-1067)

R2

O

R4


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Figure 10. Representative examples of heterocyclyl meta-nitro COMT inhibitors and their IC50 values in rat brain homogenates. Representative examples 43-4637 are shown in Figure 10. In vitro COMT inhibition by compounds 43-46 ranged from 23 nM to 39 nM in rat brain tissues. Compounds 43-46 exhibited low toxicity in mice (LD50 > 5000 mg/kg), but their in vivo efficacy was found to be weaker than that of 37, most likely due to poor gastrointestinal absorption.37 For instance, the benzoxazinone derivative 43 had an oral ED50 of 32 mg/kg in rats 4 h post-administration, which corresponds to a more than ten-fold weaker efficacy than that of 37. Applying the prodrug approach to compounds 43-46 resulted in modest improvements in the in vivo potency.37 In another effort to identify novel COMT inhibitors endowed with enhanced in vivo potency over 43-46, researchers at BIAL - Portela & Cª, S.A. have reported the optimization of a screening hit 47131, identified by preliminary in vitro screening of different compound banks (Figure 10). The trihydroxy benzene ring of 47 was replaced with the customary nitrocatechol subunit, leading to the identification of potent COMT inhibitors in vitro, such as 48131 (100% inhibition at 3 µM), with limited in vivo inhibitory ability in mice liver (39% inhibition at 30 mg/kg, 6 h post-dose). In a subsequent optimization phase, the replacement of the pyrazole ring with oxadiazole rings resulted in compound 49131, which was found to be equipotent to 37 in mice (93% liver inhibition at 30 mg/kg, 6 h post-dose). Oxadiazole 49 showed only a three-fold preference for peripheral COMT inhibition in the rat 3 h post-administration (3 mg/kg). Furthermore, in an in vitro toxicity assay (mouse neuroblastoma cell line at 30 µM), the viable cell count was reduced to 43% after 24 h of exposure to compound 49, suggesting that 49 might be toxic. In a subsequent phase of SAR aimed at reducing toxicity risk and improving tissue selectivity, the replacement of the phenyl ring with heterocyclic rings led to the identification of



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a series of meta-pyridyl-N-oxides exemplified by 50a-c.131 Notably, the ortho- and para-pyridine N-oxide derivatives were generally found to be less potent than were their meta–pyridyl-N-oxide regioisomers. Although the prototype compound 50a displayed modest inhibition (33%) in mice at a dose of 3 mg/kg (3 h post-dose), it showed very little alteration in the cell viability count (93% of Control) after 24 h of exposure to a mouse neuroblastoma cell line at a concentration of 30 µM. Further work on 50a demonstrated that the pyridyl N-oxide ring could be substituted with small lipophilic groups (CF3 50b; or methyl and halogen 50c), which restored its high potency (95-99% inhibition) and reduced its toxicity in a mouse neuroblastoma cell line (87-96% viable cells after 24 h exposure) compared with 37 (27% viable cells). Based on the presented data, it was not obvious what type of molecular properties might be responsible for the lower toxicity risk of the pyridine N-oxide compounds. Nevertheless, the authors were convinced that the lower lipophilicity (log P) and higher hydrophilic surface area were the key molecular descriptors of the toxicity risk of COMT inhibitors such as 50a-c.131 The best compound from the pyridine N-oxide series was 50c, which is under clinical evaluation for the adjunctive treatment of PD.10 In a time course experiment (rat, po, 3 mg/kg), 50c achieved the maximum inhibitory (99%) effect within 3 h of oral administration, which was continued by 80-90% inhibition up to 9 h post-administration, followed by a gradual return to lower levels over the next 15 h. Furthermore, 50c was shown to modulate the plasma levels of L-DOPA (101% increase) and 3OMD (43-58% of decrease) at a dose of 3 mg/kg. Compound 50c was designed as a hydrophilic 1,2,4-oxadiazole analogue with a pyridine N-oxide residue at position 3 to provide high COMT inhibitory potency and prevent cell toxicity.131 Inhibitor 50c is endowed with an exceptionally high binding affinity (sub-picomolar Kd)132 that translates into a slow complex dissociation rate


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constant and a long duration of action in vivo.133 In liver and brain homogenates from rats administered 50c, 37 and 32 by a gastric tube, compound 50c showed a stronger and more sustained COMT inhibitory effect than did the COMT inhibitors 37 and 32. One hour after administration, the COMT inhibition was 99% for 50c versus 82% for 37 and 68% for 32. Nine hours after administration, 32 showed no COMT inhibition, and 37 produced a minimal inhibitory effect (16%), whereas 50c continued to inhibit COMT activity by 91%.133 Chronic administration of 50c to cynomolgus monkeys resulted in increased L-DOPA bioavailability, both peripheral and central, with a concomitant reduction in 3-OMD formation.134 Compound 50c increased the L-DOPA exposure (from 2031 to 3352 ng.h/ml) in the dorsal striatum dialysate without changing the L-DOPA Cmax values and reduced the exposure to 3-OMD (from 315 to 105 ng.h/ml) with marked decreases in the 3-OMD Cmax values (from 29.7 to 7.6 ng/ml). Similar findings were observed in the substantia nigra and prefrontal cortex. Inhibitor 50c increased the L-DOPA systemic exposure (from 267,627 to 506,858 ng.h/ml) without changing the L-DOPA Cmax values and reduced the exposure to 3-OMD (from 417,304 to 66,544 ng.h/ml) with marked decreases in the 3-OMD Cmax values (from 1,909 to 427 ng/ml). These changes were accompanied by ~80-85% reductions in the erythrocyte COMT activity.134 4.2.1.4 Diphenyl sulfone scaffolds: Takehiro and collaborators at Kissei Pharmaceuticals reported a new family of COMT inhibitors characterized by the general formula 51, shown in Figure 11, where the nitrocatechol pharmacophore is further functionalized at the 5-position with a benzene-sulfonyl group.135 A number of compounds have been synthesized and shown to inhibit the COMT enzyme in vitro at low nanomolar concentrations. In particular, compound 52135 was shown to be endowed with an IC50 of 6.2 nM.



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Figure 11 COMT inhibitors developed by Kissei Pharmaceuticals. A selected series of compounds have been subjected to in vitro cellular toxicity tests in a rat hepatocyte cell line. The EC50 values (50% of viable cells) were determined for each compound, and the results were compared to those for 37 and 32. It was found that some of the disclosed derivatives (such as compound 52) displayed lower toxicity than did either 37 (EC50=34.3 µM) or enatacapone 32 (EC50= 111 µM). 4.2.2 Ortho-nitrated catechols: In the early 1990s, Pérez and coworkers disclosed the first example of “ortho-nitrated” catechols, exemplified by 53, where the nitro group is located at the ortho position relative to the formyl group (Figure 12).136 Compound 53 exhibited an IC50 value of 1.5 µM against pig liver COMT, which was slightly superior to that of the corresponding 3,4-dihydroxy-5-nitro regioisomer (IC50 = 2.0 µM). Further studies by the same group led to the discovery of 54 (QO IIR2), which incorporated a vinylic side chain at the ortho position.137 Compound 54 was found to be a selective, reversible, tight-binding COMT inhibitor with an apparent inhibition constant (Kapp) of 200 nM, which is 3.5-fold lower than that obtained for 31.138 Compound 54 modulated the striatal levels of 3-OMD in a dose-dependent fashion after concomitant ip administration with L-DOPA (7.5-60 mg/kg) and AADC inhibitor 1 (50 mg/kg).139


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Figure 12. Representative examples of ortho-nitrated COMT BIAL - Portela & Cª, S.A., have disclosed a systematic comparative study of regioisomerically nitrated derivatives of common meta-nitrated COMT inhibitors, such as 39 and 42.140 General structures and representative examples of this work are shown in Figure 12. The alkylaryl 55140 and alkylamino 56140 compounds were shown to maintain good in vitro potency in comparison to their meta-nitrated derivatives (39 and 42), whereas the ring-constrained analogues 57140 exhibited weaker COMT inhibitory potency than did the corresponding open chain derivatives in human neuroblastoma SK-N-SH cells at a concentration of 100 nM. Conversely, the ortho-nitro analogues 55 and 56 failed to provide enhanced in vivo inhibition (rat, 30 mg/kg, po) over their meta-nitrated derivatives 39 and 42. Generally, compounds from series 55 and 56 displayed much shorter peripheral durations of inhibition than did their meta-nitro regioisomers 39 and 42. Additionally, examples of 55 and 56 were endowed with little or no peripheral selectivity. The best inhibitor from the “ortho-nitrated” series was compound 58140 (BIA 8-176), which subsequently advanced into the preclinical phase. Benzophenone analogue 58 inhibited rat brain and liver COMT with IC50 values of 3 and 130 nM, respectively. Compound 58 rapidly (tmax = 37 ACS Paragon Plus Environment


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0.5 h) achieved the maximum inhibitory effect in rat liver at a dose of 30 mg/kg, and thereafter the inhibitory ability of 58 had fallen by 50% at 3 h post-dose. The profile of central inhibition by 58 was similar to that of liver inhibition. After the rapid onset (tmax = 0.5 h) of cerebral COMT inhibition (>90%) by 58, the inhibitory effect relatively quickly decreased over the next 2.5 h. The shorter-acting effect of ortho-nitrated catechols over the corresponding meta-nitrated compounds may be attributed to their reduced metabolic stability. In vitro metabolic studies with 58 provided information on its binding orientation. Compound 58 is predominantly methylated at the ortho- rather than the meta-position (7.5:1). The preferred ortho binding mode was confirmed by molecular modeling studies and X-ray crystallography.141 The ortho biding mode is thermodynamically more favorable than the meta orientation. Furthermore, in meta orientation, the nitro group encounters steric conflicts with a hydrophobic side chain of Leu198 and is strongly destabilized by repulsive electrostatic interactions with the negatively charged Glu199.142 In contrast, in ortho binding mode, benzophenone 58 makes favorable van der Waals interactions contacts with Trp143. The development of 58 was discontinued due to metabolic stability concerns. 4.2.3 Trisubstituted catechol derivatives: In a patent application, Orion has claimed that trisubstituted catechol derivatives inhibit the COMT enzyme (Figure 13).143


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CN

O

CN Ki = 190nM 61

60

NO2 O

R N O N

HO

HO HO

IC50 = 30nM

59

HO

NO2

HO

CN

62a R = Ph-COIC50 = 9.4 nM EC50 = 683 µM 62b R = H

HO HO

O O S

CN OH

OH

IC50 = 15nM

O2N

O

HO

HO HO

NO2

HO

O N H

NO2 O

N H

Cl IC50 = 8 nM EC50 > 300 µM 63

HO CN IC50 = 6.2 nM EC50 > 300 µM 64

Figure 13. Representative examples of tri-substituted catechol structures of COMT inhibitors The most interesting reported inhibitors from this work are compounds 59143 and 60143, with IC50 values of 15 nM and 30 nM, respectively, representing improvements over their mono-nitro analogues. In rats, at a dose of 10 mg/kg, compounds 59 and 60 displayed plasma concentrations 0.5 h post-dose that were markedly higher than those for 31. Both compounds 59 and 60 exhibited better bioavailability than did 31, but the dicyano derivative 59 exhibited a shorter plasma half-life than did the dinitro analogue 60, which may be attributed to differences in their metabolic stabilities. The compounds have been proposed for use in PD and hypertension. In a very recent patent application, Orion has disclosed a series of isophthalonitrile similar to 59 exemplified by compound 61 (Figure 13).144 Compound 61 inhibited human recombinant S-



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COMT with a Ki value of 190 nM. Additionally, 61 demonstrated an improved effect on LDOPA pharmacokinetics over 32. The structure of the clinical candidate from this work has not been disclosed to date. In 2009, Kissei pharmaceuticals published a novel series of trisubstituted oxadiazolylnitrocatechol derivatives, such as 62a.145 The synthesized compounds are structurally quite similar to a previously disclosed oxadiazole compound, 62b,109 which can be found in Roche’s patent application.109 Compound 62a is a potent inhibitor of human COMT, with an IC50 of 9.4 nM. Moreover, 62a was found to possess apparently lower toxicity compared with that of 62b in the rat hepatocyte cell line. The pharmacodynamic interaction of oxadiazole 62a and the comparator 62b with L-DOPA was assessed in rats. Compounds 62a-b were administered at a dose of 3 mg/kg, and then L-DOPA (5 mg/kg) was co-administered with AADC inhibitor 1 (30 mg/kg) at either 4 h or 6 h post-dose. After 2 h, the L-DOPA was quantified. Compound 62a caused a two-fold increase in the plasma levels of L-DOPA 4 h post-administration, which was steadily sustained over the next 2 h. In contrast, 62b provided only a 40% increase in the plasma levels of L-DOPA, and thereafter the L-DOPA levels quickly returned to the baseline at 6 h postadministration. Finally, the potentiation of the efficacy of L-DOPA in unilateral 6hydroxydopamine-lesioned hemiparkinsonian rats was assessed. Compounds 62a-b were orally administered (10 mg/kg) with concomitant oral administration of L-DOPA (5 mg/kg) and AADC inhibitor 2 (30 mg/kg). Three weeks after the lesion, the potency was measured as the number of contralateral turnings. Oxadiazole 62a displayed remarkable potentiation of the drug effects when compared with control animals treated only with L-DOPA and inhibitor 2. It is unknown whether these new analogues, such as 62a, have any advantage over BIAL’s oxadyazolyl COMT inhibitors, such as 50c.131


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Hiroaki and colleagues at Kissei Pharmaceuticals characterized trisubstituted nitrovanillic amide derivatives as inhibitors of the COMT enzyme, represented by compound 63 (Figure 13).146 Compound 63 was shown to be equipotent to 32 (IC50 = 10 nM) and displayed improved in vitro efficacy over 37 (IC50 = 36 nM). Additionally, in the rat hepatocyte cell line, compound 63 presented a lower toxicity risk than did 32 and 37. Further efforts to identify novel COMT inhibitors at Kissei Pharmaceuticals led to the discovery of novel tri-substituted COMT inhibitors (Figure 13).147 A representative structure of this work is compound 64.147 which was found to be potent in vitro, with an IC50 value of 6.2 nM. Compound 64 is devoid of toxicity in the rat hepatocyte cell line. The in vivo COMT inhibitory profile of 64 was evaluated in rat liver and brain homogenates at a dose of 10 mg/kg. 64 was found to achieve a 100% inhibitory effect in the liver 1 h after oral administration and thereafter sustained constant inhibition of COMT over the next 4 h. Conversely, 32 and 37 displayed shorter durations in peripheral inhibition. The extent of central COMT inhibition by nitrocatechol 64 was markedly lower than that of 37 and remained below the 40% level. Compound 64 was tested for its potentiation of the efficacy of L-DOPA in unilateral 6hdroxydopamine lesioned hemiparkinsonian rats. The authors claimed that compound 64 presented similar potentiation drug effects to those of 37. 4.2.4 Fused nitrocatechol derivatives: In the early 2000s, Orion’s interest turned to fused bicyclic nitrocatechol structures (Figure 14).



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Figure 14. Fused nitrocatechol structures of COMT inhibitors COMT inhibitors in which the pharmacophore is an appropriately substituted coumarin ring system were published in 2002.148 The invention is represented by the 5-nitro analogue 65148 and 8-nitro derivative 66.148 Compounds 65 and 66 inhibited COMT in the low nanomolar range with IC50 values of 10 nM and 35 nM, respectively. The in vivo COMT activity data for the coumarin compounds were not disclosed in the patent application. The second scaffold is based upon naphthalene and isoquinoline derivatives (Figure 14).149 The application is represented by compounds 67149 and 68.149 The napthyl derivative 67 exhibited an IC50 of 15 nM, while isoquinolinyl example 68 was shown to have a much weaker COMT inhibitory effect (IC50 = 140 nM) than the structurally similar napthyl analogues, such as 67. Another “bicyclic” patent application by Orion focused on benzofused five-membered heterocycles with the preferred structures represented by compounds 69 and 70 (Figure 14).150 Examples 69 and 70 exhibited


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impressive inhibitory constants with Ki values of 0.5 nM and 2.0 nM, respectively. Compounds 69 and 70 were evaluated in vitro for their ability to uncouple oxidative phosphorylation, which appears to be one of the most important mechanisms of the cell toxicity caused by some nitrocatechol derivatives, such as 37.122, 123 The presented data confirmed that compounds 69 and 70 did not uncouple oxidative phosphorylation. Furthermore, compounds 69 and 70 were demonstrated to be endowed with enhanced in vitro uridine-5’-diphosphglucuronic acid (UGDPA) metabolic stability in human liver microsomes over 32. However, in a related publication, benzothiophene 69 was reported to possess poor oral bioavailability and high systemic clearance.151 The exact structure of the clinical candidate for the treatment of PD has not been revealed publicly, but it was later reported that the phase I clinical trial was discontinued due to the conclusion that the clinical candidate pharmacological properties would not outperform those of 32 or 37. 4.3 Miscellaneous inhibitors In addition to the catechol- and pyrogallol-based inhibitors discussed in sections 4.1 and 4.2, several

structurally

‘atypical’

molecules,

which

do

not

share

the

dihydroxy-

or

trihydroxybenzene ring, have been reported to show COMT inhibitory activity and are discussed further below. 4.3.1 Tropolone-based inhibitors: Compound 71 (tropolone) and its alkylated derivatives 72-73 inhibit COMT in a competitive fashion, with Ki values of 27.0, 8.9 and 10.1 µM, respectively (Figure 15).152 Although tropolones 71-73 do not incorporate a catechol motif in their structure, they are able to interact with COMT through α-keto-enol tautomerism.



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Figure 15. Tropolone-based inhibitors The binding mode of 71-73 is reversible, and the strength of the interaction with COMT is relatively weak, which results in short acting in vivo effects in different animal species, such as in rats.153 Compound 71 exhibited high cerebral concentrations 0.5 h after ip administration in rats at a dose of 100 mg/kg. The concentration was markedly reduced in all tissues over the next 2-3 h. Tropolones 71-73 exerted modest effects on catecholamine levels when administered ip to rats at doses of 40-100 mg/kg. Due to the considerable toxicity of 71-73, no clinical development was initiated with tropolone derivatives. 4.3.2 Hydroxy-pyridones, -pyrimidones, -quinolinones and -pyrones: Hydroxy-pyridones, pyrimidones, -quinolinones and -pyrones interact with the COMT enzyme in a similar fashion as do tropolones 71-73, as they can be considered isosteric compounds of catechol.154 The early prototype compounds 74-76154 share similar pharmacological properties with pyrogallols 16-18 and catechol derivatives 9-15, such as moderate efficacy in vitro, a lack of selectivity and noticeable toxicity (Figure 16). These characteristics precluded the successful clinical development of any of the early pyridone, quinoline and pyrone ligands as COMT inhibitors. Despite the unfavorable pharmacodynamic and pharmacokinetic properties of the early ligands 74-76, the optimization of pyridone 74 by scientists at Merck led to the discovery of a novel series of hydroxyl-pyridones and pyrimidones, represented by 77155 and 78156 (Figure 16). Pyrimidones 77 and 78 were shown to be potent inhibitors of human MB-COMT, with IC50 44 ACS Paragon Plus Environment

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values of 8 and 31 nM, respectively. A structurally similar pyrimidone analogue, 79,157 exhibited lower potency in the same in vitro assay (IC50 = 93 nM). O

OH OH

O N

OH

O N H 76

75

74

Cl N

O

O

F N

HN

OH

F3C

O

OH N

N

O

N

N O OH

77

78

IC50 = 8 nM

IC50 = 31 nM

79 IC50 = 93 nM

Figure 16. Pyrimidone, quinoline and pyrone based inhibitors The compounds disclosed in the patent applications have been claimed to be useful for the symptomatic treatment of certain psychiatric disorders, such as schizophrenia, depression, bipolar disorders and other dopamine deficiency-related diseases. 4.3.3 Bifunctional inhibitors: Inhibitors bearing two catecholic structures in the same molecule have been synthesized and evaluated for their in vitro COMT inhibitory ability. Representative examples are shown in Figure 17.



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Figure 17. Representative examples of bifunctional COMT inhibitors In an early series, the two carboxyamide subunits were separated by different length alkyl spacers.158 The authors concluded that no evidence could be found for a direct correlation between potency and bifunctional nature, although the length of the hydrocarbon spacer influenced the in vitro potency. The most potent catechol compound was 80a158 (n = 3), which displayed a competitive inhibition pattern with a Ki of 0.3 µM. In the trihydroxy series, the most interesting compound, 80b,158 also had an n-propyl spacer. Compound 80b displayed a Ki of 6.6 µM, and the inhibition mode was found to be uncompetitive. Derivatives with either longer or shorter hydrocarbon chains displayed marked reductions in activity (80c158, Ki = 136 µM; 80d158 , Ki = 26.7 µM). Follow-up work on bifunctional inhibitors led to a novel series of asymmetrical phenol-nitrocatechol type inhibitors exemplified by structures 81a-c (Figure 17).159 Compounds 81a-c were found to exhibit higher in vitro potency than did their first generation analogues, with Ki values of 0.64, 0.77 and 0.97 µM, respectively. The in vivo evaluation of bifunctional inhibitors has not been reported in the literature to date. 4.3.4 Bisubstrate inhibitors: The rationale for designing bisubstrate inhibitors was to target simultaneously the catechol and the AdoMet binding sites in the catalytic site of the enzyme. Bisubstrate COMT inhibitors represented by general structure 82 (Figure 18) were expected to have an advantage over single-substrate-based compounds. The first bisubstrate inhibitor, 82a,


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was developed by structure-based design using the crystal structure of the quaternary complex between the COMT enzyme, the cofactor AdoMet, 19 and the magnesium (II) ion.13,

160

The

inhibitor 82a displayed a modest potency with an IC50 of 2000 nM. Later, Lerner and coworkers designed two additional analogues (guided by computer modeling studies) with shorter (82b) and conformationally more rigid (82c) linkers between the adenine and catechol moieties.161,

162

Bisubstrate inhibitor 82b displayed a 10-fold greater inhibitory potency than did compound 82a. The less flexible derivative 82c was the most potent bisubstrate inhibitor to date, with an IC50 of 9 nM.161 Kinetic analysis with compound 82c displayed competitive inhibition mechanism with respect to the co-substrate and AdoMet binding site, and more complex inhibition mechanism with respect to the catecholic binding site. Compound 82c was successfully co-crystallized with rat S-COMT.161 The crystal structure reveals that inhibitor 82a binds to both the AdoMet and the catechol binding sites. Binding of the bisubstrate inhibitor 82a to COMT causes little structural changes of the enzyme compared with the protein in complex with a simple nitrocatechol substrate such as 19 and the co-factor AdoMet.



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NH2 N O

HO

O

X

N O

N

N

N H

HO

HO

X=

OH

82b

82a

82c

IC50 = 199 nM

IC50 = 2000 nM

IC50 = 9 nM

NO2 82 R N

NH2 N O

O

HO

N H

HO

HO

N

HO

N N

O

O N H

HO

X

N

N

Y

OH

N N

R 83

N

F

N NH 85

84a X = Y = OH, R = NH2 84b X = Y = H, R = NH2 84c X= F, Y = OH, R = NH-CH3 84d X = Y = OH, R = CH3

Figure 18. Bisubstrate inhibitors of the COMT enzyme and structure of AdoMet competitive fragment. Scientists at Hoffman la-Roche Ltd. synthesized and characterized analogues of 82c. The SAR analyses of the Markush structure 83 revealed that its potency could be attributed to the nature of the R group.

163

Incorporation of electron donating substituents (e.g., R = -iPr) resulted in low

micromolar potency inhibitors. In contrast, the presence of EWGs (R1 = -CN, CF3, -(CO)CF3, Cl, aryl or heteroaryl) was found to be beneficial for in vitro COMT inhibition. The 4-fluoro phenyl analogue 84a showed excellent potency, with an IC50 value of 21 nM.164 No experimental in vivo data for bisubstrate inhibitors have been published to date. The compounds have been proposed for the treatment of various medical conditions, such as PD, depression and


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schizophrenia and for cognition improvement. Another attempt to identify important interactions between COMT and bisubstrate inhibitors involved the modification of the ribose and the adenine moieties. Replacement of the hydroxyls of the ribose ring with hydrogen in 84a gave derivative 84b which was less active than the parent compound 84a.165 Further work on inhibitor 84b demonstrated that in vitro potency could be restored by inclusion of fluorine (84c).

165

In

another study attempts were made to modify the adenine moiety of 84a using structure-based design.166 The nitrogen atom of the adenine ring at position 1 is crucial for high binding affinity due to hydrogen bonding to the backbone NH of Ser119. The 6-methylpurine derivative 84d was found to be the most potent adenine modified analogue.166 In a very recent report scientists at Takeda Pharmaceutical Company Ltd. have identified a series of AdoMet competitive fragments, which are structurally unrelated to AdoMet. Successful cocrystallization of 85 with mouse COMT confirmed its binding to the AdoMet pocket.167 Compound 85 exhibits a Ki value of 16 μM and can serve as starting point for development of novel bisubstrate inhibitors. 4.4 Prodrugs and Codrugs: The most clinically relevant COMT inhibitors bear nitrocatechol rings, which greatly determine the overall physicochemical properties of the inhibitor, such as its aqueous solubility and polarity. Inadequate physicochemical properties in nitrocatechol COMT inhibitors may result in limited oral bioavailability. As discussed in section 4.2.1.1, 32 has limited absorption due to certain molecular properties.98, 99 To tackle this problem, several water soluble prodrugs of 32 have been synthesized to improve the oral bioavailability of 32. These prodrug derivatives include phosphate ester 86168 and various O-substituted esters 87a-i169 as well as N-alkyl 88a-c170 and N,N-dialkyl carbamate esters 89a-c170 (Figure 19). These prodrugs are expected to release the parent drug 32 in systemic circulation.



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Figure 19. Prodrugs of COMT inhibitors 32 and 69. The phosphate ester 86 was endowed with greatly improved aqueous solubility (30 mg/mL) over 32 in a low pH range (1.2-7.4), which represents the full pH scale of the gastrointestinal


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track. The chemical stability of 86 was shown to be high, and no significant degradation was observed in a buffered solution at physiological pH and temperature (t½ = 2227 h). In human plasma, phosphate ester 86 was found to also be stable. After incubation with rabbit liver homogenate, 86 underwent slow enzymatic hydrolysis (over 1 h) to the parent drug 32 (E isomer) and its Z geometric isomer (E/Z ratio 1:6). Its overly high stability towards enzymatic degradation may be related to the steric hindrance and the strong electron withdrawing effect of the nitro group, which in turn limit the activity of phosphatase enzymes. Further efforts to enhance the oral absorption of 32 involved the preparation and characterization of a series of Oacyl, O-acyloxyacyl, and O-alkyloxycarbonyl esters as well as an O-acyloxy alkyl ether represented by the general structure 87 (Figure 19). It has been postulated that compounds 87a-i might be endowed with higher lipophilicity and, in certain cases, with better aqueous solubility over the parent drug 32. Although all reported examples of 87a-i were found to have higher apparent partition coefficients (Log Papp = 1.23-4.0) than that of 32 (Log Papp = 0.65), the di-Osubstituted esters 87b-f had insufficient solubility. The prodrugs having branched substituents on the catechol hydroxyls, 87b and 87g-h, displayed greater stability towards chemical hydrolysis than did the straight-alkyl chain analogue 87c. Compounds 87b and 87g-h were shown to be more stable at lower pH values than at neutral pH. The mono substituted esters 87a and 87i exhibited comparable or superior aqueous solubility (1.62 mg/mL and 6.99 mg/mL, respectively) at pH 7.4 than did 32 (1.75 mg/mL). Prodrug 87i underwent rapid enzymatic hydrolysis at physiological pH (t½ = 0.7 h), but its chemical stability was insufficient due to intramolecular hydrolysis involving the free vicinal hydroxyl group of the catechol nucleus. On the basis of its overall physicochemical properties, the best compound of the above series was the mono-Opivaloyl ester 87a, which failed to provide better oral absorption (F = 7%) over 32 (F = 10.4%)



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in rats. The lower absolute oral bioavailability of 87a can be attributed to its lower aqueous solubility at low pH values because absorption is likely to take place in the upper part of the gastrointestinal tract, where the environment is more acidic. Further work designing potential new prodrugs resulted in a series of N-alkyl and N,N-dialkyl carbamates (Figure 19).170 Prodrugs 88a-c and 89a were shown to present reasonable chemical stabilities in phosphate buffer (pH = 7.4) with t½ values in the range of 14.9-20.7 h. Furthermore, their in vitro enzymatic hydrolysis was rapid (t½ = 0.8-2.7) enough to fulfill the prodrug criteria. Unexpectedly, N,N-dialkyl carbamates 89a-c failed to release 32 under in vivo conditions, presumably due to the involvement of other metabolic pathways besides the targeted hydrolytic cleavage. The mono Nalkyl carbamates 88a-c were endowed with good to excellent aqueous solubility (≥40, 1.94 and 2.46 mg/mL, respectively), and the log Papp values of 88b-c were shown to be five- and fourfold higher, respectively, than that of 32. When prodrugs 88a and 88c were administered at doses equivalent to 5.7 mg/kg of 32, lower area under the plasma concentration-time curve (AUC0-2h) values were obtained (318 and 261 ngh/mL, respectively) than for the parent compound 32 (644 ngh/mL). Prodrugs of another investigational drug candidate molecule, benzothiophene 69, have also been disclosed in the literature (Figure 19). The carboxylate group of 69 was esterified with different alcohols to give the corresponding alkyl esters (90a-d and 91a), acyloxyalkyl esters (92a-d), aminoacyloxy alkyl ester (92e) and cyclic carbonate ester (91b).151 The prepared lipophilic esters (90a-d, 91a-b and 92a-d) were expected to counterbalance the very polar nature of 69, while the aminoacyloxy alkyl ester 92e was aimed for use as a substrate for dipeptide intestinal transporters.151 Simple alkyl (90a-d) and alkylaryl esters (91a) were shown to be stable in human serum, but in rat liver homogenate were unstable (t½ = 19.8-130.9 min.). Diesters 92a-


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d, designed to increase recognition by esterases, were also evaluated. Prodrugs 92a-d were prone to quantitative hydrolysis in human serum with t½ values ranging from 5.1 to 32.3 h. Furthermore, 92a-d underwent rapid hydrolytic degradation to the parent compound 69 in rat liver homogenate with t½ values of less than a minute. Compounds 92a, 92d-e and 91b were selected for in vivo evaluation in rats at a concentration of 10 µM/kg, but they failed to provide marked improvement over the parent compound 69. Only prodrugs 91b and 92a caused a twofold increase in the plasma concentration of benzothiophene 69. Another attempt to improve the bioavailability of 32 involved the preparation of a codrug product of 32 and L-DOPA, where the two different entities (L-DOPA and enatacapone 32) were linked together to form a single molecule (Figure 20).171 The codrug approach was expected to be an effective way to simplify the administration regime of L-DOPA and 32 in addition to facilitating their in vivo delivery to the site of action.

Figure 20. Structure of codrug of 32 Due to chemical stability problems, the most convenient way to link L-DOPA and 32 is through a carbamate linker between the furthest hydroxyl group from the nitro group in 32 and the amino group of L-DOPA. Codrug 93 was found to be quite stable towards chemical hydrolysis at a wide range of pH values (t½ = 12.1 h, pH = 1.2; t½ = 1.4 h, pH = 5.0; t½ = 1.1 h, pH = 7.4).171 Nevertheless, 93 underwent rapid enzymatic hydrolysis in rabbit liver homogenates at physiological pH (t½ = 7 min) and released the parent drugs (i.e., 32 and L-DOPA), 53 ACS Paragon Plus Environment


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demonstrating the bioreversibility of 93. No in vivo experiments with codrug 93 have been reported thus far in the literature. Codrugs of other common COMT inhibitors have also not been published. 5. COMT inhibitors in clinical trials Until the late 1980s, early pyrogallol and catechol-based competitive inhibitors demonstrated poor in vivo efficacy, which precluded their clinical development. The tight-binding COMT inhibitors (i.e., nitrocatechols) were more promising than their competitive pyrogallol and catechol predecessors. The first potent and selective COMT inhibitor in clinical trials was 31, followed by 32 and 37 and later by 39 and 50c. Their major clinical pharmacology properties are summarized below. 5.1 Nitecapone: Compound 31 was progressed into Phase I clinical trials by Orion in 1987 for the adjunctive treatment of PD.6, 172 Twelve healthy volunteers were enrolled in a Phase I study with 31 and received either 100 mg of L-DOPA and 25 mg of AADC inhibitor 1 alone or the two drugs simultaneously in increasing single doses (10-100 mg) with 31. Compound 31 was rapidly absorbed, with a median tmax of 0.5 h. The plasma peak concentrations and the AUC values increased in a dose-dependent manner. Clinical candidate 31 exhibited a very short elimination half-life (T½ = 30-45 min). Inhibitor 31 was found to partially inhibit S-COMT in erythrocytes 30 min after drug intake in a dose-dependent manner and had no inhibitory effect on cerebral COMT. Compound 31 slightly increased the relative bioavailability of L-DOPA. Doseproportional decreases in 3-OMD and HVA and significant increases in DOPAC were observed. Inhibitor 31 was found to be well tolerated in a dose range of 10-100 mg, and no side effects related to the use of the compound were reported except for the discoloration of urine, which is a common effect of polyhydroxy nitrobenzenes. Although the Phase I clinical studies of 31


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showed improvements in L-DOPA pharmacokinetics, it eventually was replaced by 32, which was judged to be a more efficacious clinical candidate. However, 31 subsequently advanced into Phase I clinical trials for the treatment of gastrointestinal lesions, but the development subsequently was discontinued in Phase II due to a lack of efficacy. 5.2 Entacapone: Orion successfully completed Phase I-III clinical trials with 32 in the late 1990s, and 32 has been approved for the adjunctive treatment of PD. In humans, Compound 32 is rapidly absorbed after oral administration, with a tmax of 1 h. Food or low gastric activity does not affect 32’s pharmacokinetics. Clinical candidate 32 reversibly and dose-dependently inhibits erythrocyte S-COMT.97 Following an oral dose of 800 mg of 32, 82% COMT inhibition was reported. Compound 32 displays a linear pharmacokinetic profile within the dose range of 5 to 800 mg. It has also been reported that 32 is extensively metabolized, with >1% of the orally administered dose excreted in unchanged form.100 Compound 32 was shown to be endowed with high selectivity in terms of peripheral COMT inhibition. Due to the limited bioavailability (F = 29-46%)97 and short duration of action of 3297, the dosages administered to PD patients are high and repeated up to eight times daily; its clinical effectiveness in the treatment of PD has been questioned. 5.3 Tolcapone: In the late 1990s, the clinical candidate of Hoffman la-Roche Ltd. for PD was 37. In a Phase I study, the effect of single doses of 37 (5-800 mg) on the metabolism of L-DOPA (100 mg plus 25 mg of an AADC inhibitor) was studied. In healthy volunteers, 37 was rapidly absorbed after single oral doses ranging from 5-800 mg, with tmax ranges of 0.9-2 h.8 The maximum inhibitory effect of 37 was achieved within 2 h. The elimination of 37 was also rapid, with a t½ of 2.0-2.3 h. In clinical trials, 37 showed much greater bioavailability than did 32. Oral doses of 100-200 mg of 37 caused 80% COMT inhibition in RBCs. The COMT enzymatic



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activity was recovered after 13-23 h, depending on the administered dose. The inhibitory activity of 37 was found to be higher and more prolonged than that of either 31 or 32. Inhibitor 37 increased (up to 200 mg) the AUC of L-DOPA in a dose-dependent manner without markedly changing its Cmax or tmax values. In contrast, the AUC of 3-OMD was shown to be significantly decreased in a dose-dependent fashion. Doses higher than 200 mg of 37 had no beneficial effect on L-DOPA pharmacokinetics because L-DOPA absorption was inhibited by 37, most likely due to competition for intestinal carriers. In PD patients, the effect of single doses of 37 increased the duration of the ON time (symptoms of PD are controlled) by 70%. Due to the toxicity reasons discussed in section 4.2.1.2, 37 can only be used in patients afflicted with PD who are unresponsive to other treatments and only with regular monitoring of liver function, which is expensive and uncomfortable for patients.173 5.4 Nebicapone: When oral single doses of 39 were administered to humans, the maximum plasma concentrations (Cmax=2.7-5.9 mg/ml) were reached within 0.5-2 h, depending on the given dose (50-200 mg), followed by rapid elimination (t½=2.0-2.4 h).174 Clinical candidate 39 was shown to display a linear pharmacokinetic profile over the dose range of 50 to 200 mg. In a single dose study with 39 administered together with L-DOPA and a peripherally selective AADC inhibitor, the AUC of L-DOPA increased by 39-80%, while the AUC of 3-OMD markedly decreased (by 38-62%) depending on the given dose of 39 (50, 100, 200 and 400 mg).175 Inhibition of human RBCs by 39 was shown to occur in the range of 57-84%. 39 demonstrated a similar inhibitory profile to that of 37. Oral doses of 100 and 200 mg of 39 caused 69 and 81% COMT inhibition, respectively. The peripheral COMT inhibition by 39 was maintained up to 18 h post-administration. An oral dose of 75 mg of 39 and 200 mg of 32 exhibited very similar levels of COMT inhibition, but the inhibitory effect of 39 lasted longer


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than did that of 32.9 The metabolic and toxicological profile of 39 reflects quite well the profile of 37. Unfortunately, the clinical development of was discontinued due to safety concerns. 5.5 Opicapone: In 2010, BIAL - Portela & Cª, S.A disclosed that 50c was a clinical candidate for the adjunctive treatment of PD. In a study that evaluated the tolerability, pharmacokinetics (including the effect of food) and pharmacodynamics (effect on COMT activity) following single oral doses of 50c (10 to 1,200 mg) in young healthy male volunteers, was well-tolerated and presented dose-proportional kinetics. Clinical candidate 50c demonstrated marked and sustained inhibition of erythrocyte S-COMT activity. Based on the observation that the half-life of COMT inhibition was independent of the dose and that it reflected an underlying kinetic process that was consistent with the koff value of the COMT-inhibitor 50c complex, it was proposed that the sustained COMT inhibition, which continued even after the clearance of the circulating drug, was due to the long residence time of the reversible complex formed between COMT and 50c.10 In another study to assess the tolerability, pharmacokinetics and inhibitory effect on erythrocyte S-COMT activity following repeated doses of 50c, the drug was tested in a randomized, placebocontrolled, double-blind study that enrolled healthy male subjects who received either a once daily placebo or 50c at a dose of 5, 10, 20 or 30 mg for 8 days.176 Compound 50c was well tolerated, and its systemic exposure increased in an approximately dose-proportional manner, with an apparent terminal half-life of 1.0 to 1.4 hours. Sulfation was the main metabolic pathway, and the 50c metabolites recovered in urine accounted for less than 3% of the amount of 50c administered, suggesting that bile was likely the main route of excretion. The maximum SCOMT inhibition (Emax) ranged from 69.9% to 98.0% following the last dose of 50c. The induced S-COMT inhibition showed a half-life in excess of 100 h, which was dose-independent and much longer than the plasma drug exposure. Such a half-life reflects a putative underlying



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rate constant that is comparable to the estimated dissociation rate constant of the COMT- 50c complex.176 The S-COMT inhibitory effect by 50c described in these studies was much stronger than that reported for 37 and 32 in healthy subjects. Emax was reported as, respectively, 72% and 80% for 100 mg and 200 mg 378 and 65% for 200 mg 32.97 Whereas the S-COMT activity returned to the baseline approximately 18 h after 37

8

and 8 h after 32

97

administration, the

inhibitory effect of 50c on S-COMT activity lasted longer: 24 h after the last dose of 50c (which is the expected dosing interval for 50c in clinical use), the S-COMT activity was still depressed by 42.8%, 52.4%, 56.8% and 64.9% in the 5 mg, 10 mg, 20 mg and 30 mg 50c groups, respectively. Another recent study compared the L-DOPA pharmacokinetic profile throughout a day driven by the COMT inhibition either following repeated doses of 50c or concomitant administration with 32.177 This was a randomized, double-blind, gender-balanced, parallel-group study in 4 groups of 20 healthy subjects each. Four (4) subjects in each group received placebo during the entire study. Sixteen (16) subjects in one group received placebo once-daily for 11 days and, on Day 12, 200 mg concomitantly with each L-DOPA/carbidopa dose (three times separated by a 5 hour interval). Sixteen (16) subjects in each of the remaining three groups received, respectively, 25, 50 and 75 mg 50c once-daily for 11 days and, on Day 12, placebo concomitantly with each L-DOPA/carbidopa dose. L-DOPA Cmin for each L-DOPA/carbidopa dose and for the mean of all L-DOPA/carbidopa doses increased substantially with all active treatments (32 and 50c) when compared to the control group (placebo) with values ranging from 1.7-fold (200 mg 32) to 3.3-fold (75 mg 50c). No statistical difference was found for L-DOPA peak of systemic exposure (as assessed by Cmax) between all active treatments and placebo. A significant increase in the L-DOPA extent of systemic exposure (as assessed by AUC) occurred with all 50c treatments in relation to placebo. No statistical difference was found for L-DOPA


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AUC when 32 was compared to placebo. Point estimates (PE) of the geometric mean ratios (GMR) 32/placebo was 102.97% and the 90%CI was 76.16-139.21%. When compared to 32, both 50 and 75 mg 50c presented a statistical difference for the L-DOPA AUC increase (PE 50c/32 [90%CI] = 148.23% [105.88-207.50%]) with 50 mg 50c and 173.74% [124.11-243.22%] with 75 mg 50c). All active treatments significantly inhibited both peak (as assessed by Emax) and extent (as assessed by AUEC) of the COMT activity in relation to placebo. When compared to 32, all 50c treatments significantly decreased the extent (AUEC) of the COMT activity due to a long-lasting and sustained effect. It was concluded that 50c when compared to 32, provides a superior response upon the bioavailability of L-DOPA associated to more pronounced, longlasting and sustained COMT inhibition. Overall, the results clearly suggest that 50c has a unique pharmacodynamic profile adequate for a once-daily regimen, which in the treatment of PD patients could represent an advantage over 32 and 37, which have a short S-COMT inhibitory effect and require multiple daily doses. 50c is currently under phase III clinical trials to demonstrate the efficacy and safety of 50c once-daily treatment for patients with PD and end-ofdose motor fluctuations in comparison to a placebo178 or a placebo and 32.179 6. Conclusions and future directions: The historical development of COMT inhibitors and the corresponding patent landscape has been reviewed. Various structurally diverse COMT inhibitors have been reported and optimized to produce potent and selective inhibitors. The majority of disclosed inhibitors share catechol, pyrogallol or nitrocatechol pharmacophores. The ‘first-generation’ competitive inhibitors showed relatively weak in vitro COMT inhibitory activity and exhibited weak in vivo efficacy and poor target selectivity, which precluded their further clinical use. The development of clinically relevant COMT inhibitors, such as 31 (Orion), 32 (Orion), 37 (Hoffman-la Roche)



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and 39 (BIAL) was based on a nitrocatechol scaffold (‘secobd-generation’ inhibitors), which is one of the most potent pharmacophores for the COMT enzyme. The presence of a nitro group at the ortho-position relative to the hydroxyl group of the catechol nucleus markedly alters the physicochemical properties of the molecule. Generally, nitrocatechol structured compounds are poor substrates for the COMT enzyme and known as tight-binding inhibitors, but their binding to the COMT enzyme is fully reversible. Moreover, they are highly selective inhibitors of COMT versus the other enzymes involved in catecholamine metabolism. From an efficacy point of view, it is important to incorporate a second EWG group into the molecule in addition to the nitro group to further enhance in vitro activity. Medicinal chemistry problems, such as extensive metabolism through glucuronidation of the phenolic hydroxyls (e.g. benzothiophene 69), limited oral bioavailability (e.g. 32) and liver toxicity (e.g. 37) sometimes have been associated with the presence of the nitocatechol ring. In the last ten years, several modifications have been made to nitrocatechol COMT inhibitors, which were intended to improve their pharmacokinetic profile. Replacement of the nitro group with two other EWGs (e.g., cyano) resulted in highly potent trisubstituted COMT inhibitors in vitro, but some exhibited shorter in vivo half-lives than their disubstituted nitrocatechol analogues. Other approaches include prodrugs and codrugs of known COMT inhibitors (e.g., 32) as well as novel aromatic nitrocatechols and novel heterocyclic nitrocatechols (e.g., 50c). The design of new COMT inhibitors has been facilitated by a number of available X-ray structures of COMT in complex with various inhibitors. The catalytic site of COMT is very tight and located at the outer surface of the protein. Therefore, the side-chain of the inhibitor has limited role in the interaction with COMT, but conversely it can be used for optimization of the pharmacokinetic profile of the inhibitor.


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Over the last 50 years, COMT has become an attractive target for the treatment of various peripheral and CNS disorders. The sole clinical application of COMT inhibitors thus far is their co-administration with an AADC inhibitor plus L-DOPA (a biological precursor of dopamine) for the symptomatic treatment of PD. The first orally bioavailable COMT inhibitors, such as 37 from Hoffman-la Roche and 32 from Orion, were found to alter the in vivo metabolism of LDOPA (i.e., they prolonged its half-life) in different animal species and initially showed high promise in clinical studies. However, their daily clinical use has revealed several disadvantages, such as short in vivo half-lives and low oral bioavailability (32) as well as selectivity and liver toxicity problems (37). After the withdrawal of from the market, 32 has become the sole COMT inhibitor available in the clinic for use as an adjunct to the L-DOPA treatment of PD patients. Although other nitrocatechol-based inhibitors, such as 39, 42 and 58 exhibit improved pharmacokinetic properties (i.e., selectivity and duration of action) over 32 and 37, none of them have advanced to clinical trials. Structurally related nitrocatechol COMT inhibitors have been developed by Kissei Pharmaceuticals. The information included in the patent applications about these compounds is limited, but from a structural point of view, the disclosed compounds may show metabolic instability (e.g., ortho-nitrated derivatives), poor aqueous solubility and high lipophilicity. Heterocyclic COMT inhibitors appear to be the most attractive scaffolds from the nitrocatechol group. In contrast with phenyl-substituted nitrocatechols (e.g., 37), the large structural variations in heterocyclic rings provide increased opportunities to tune the physicochemical properties of the molecule, resulting in inhibitors with enhanced pharmacokinetic profiles that maintain excellent pharmacodynamic interactions with COMT. Several classes of condensed and non-fused heterocyclic COMT inhibitors have been designed and claimed in the patent literature. One of the main challenges with such structures is to



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mitigate the potential metabolism of the heterocyclic ring by appropriate substitution and to balance both the lipophilicity and aqueous solubility of the molecule in order to improve the negative impact of the nitrocatechol nucleus on intestinal absorption. The most advanced COMT inhibitor from the heterocyclic class is 50c, which is being evaluated in Phase III clinical trials as an adjunct to L-DOPA therapy in patients with PD. Inhibitor 50c has shown high efficacy (i.e., very long acting) and an excellent safety profile in clinical studies and is expected to be an alternative to other COMT inhibitors (e.g., 32 or 37) in the adjunctive L-DOPA therapy of PD. In our opinion, bi-substrate molecules are not promising clinical candidates as COMT inhibitors. They exhibit excessive molecular weights and are very polar, which may limit their oral absorption. In principle, the codrug approach could be an alternative to the simple consecutive administration of L-DOPA and a COMT inhibitor. However, due to potential pharmacokinetic interactions, L-DOPA and COMT inhibitors are not necessarily administered at the same time, which may limit the clinical usefulness of codrugs. Finally, COMT inhibitors for other possible therapeutic applications (listed in the introduction of this review) have yet to reach the clinical phase. Recently published structures by Merck (hydroxy-pyridones and hydroxyl-pyrimidones) have been claimed to be useful in the treatment of psychotic disorders such as schizophrenia; however, the disclosed pharmacological data are not sufficient to evaluate their potential clinical efficacy.

Author information *Tel: +351- 22-9866100. Fax: 351- 22-9866192. E-mail: [email protected].

Acknowledgement


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Zsuzsa Sárkány is thanked for the generation of Figures 3 and 4.

Abbreviations used COMT, catechol O-methyltransferase; S-COMT, soluble form of COMT; MB-COMT, membrane bound form of COMT; DβH, dopamine β-hydroxylase; MAO, mono-amine oxidase; PD, Parkinson’s disease; ADD, attention deficit disorders; ADHD, attention deficit hyper activity disorder; RLS, restless leg syndrome; AD, Alzheimer’s disease; Val/Met, valine-tomethionine; CNS, central nervous system; GFR, glomerular filtration rate; AER, albumin excretion rate; L-DOPA or levodopa, L-3,4-dihydroxy phenyl alanine; 3-OMD, 3-O-methyl-LDOPA: DOMA, 3,4-dihydroxymandelic acid; DOPAC, 3,4-dihydroxybenzoic acid; 3-MT, 3hydroxy-4-methoxytrianine; HVA, homovanillic acid; 3,5-DNC, 3,5-dinitro catechol; SAM or AdoMet, S-adenosyl-L-methionine; AdHcy or SAH, S-adenosyl-L-homocysteine; AADC, amino acid decarboxylase; RBC, red blood cell; iv, intravenous; Log P, partition coefficient; UGDPA. uridine-5’-diphosphglucuronic acid; BBB, blood-brain barrier; EWG, electron-withdrawing group; SAR, structure-activity relationship; AUC, area under the plasma concentration-time curve: PE, point estimates; GMR, geometric mean ratios.

Biographies

László E. Kiss received his M.Sc. in 1994 and his Ph.D. in organic chemistry from Eötvös Loránd University, Budapest, Hungary with Professor József Rábai. From 1998-2002, he worked as a combinatorial chemist and Project Leader at Comgenex Inc. in Budapest, Hungary. In 2002,



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he joined BIAL – Portela & Cª, S.A., S. Mamede do Coronado, Portugal as a medicinal chemist and became the leader of the Medicinal Chemistry Group of BIAL in 2011, working on several lead generation and optimization programs. His research interests focus on cardiovascular and CNS disorders.

Patrício Soares-da-Silva received his medical degree in 1981 and his Ph.D. in Medicine (Physiology and Pharmacology) in 1988 at the University of Porto (Porto, Portugal). Since 1996, he has been a full professor of Clinical Pharmacology & Therapeutics at the Faculty of Medicine of the University of Porto (Portugal) and heads the Department of Research and Development at BIAL – Portela & Cª, S.A. (S. Mamede do Coronado, Portugal). His main research interests concern pharmacology, toxicology and the clinical pharmacology of new molecular entities on cardiovascular and CNS disorders.

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Table of Contents Graphic



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Figure 3. Rat S-COMT in complex with the methyl donor AdoMet, the Mg2+ ion and ligand 19. A) Schematic representation of the three-dimensional structure of the protein; B) Enlarged view of the catalytic site. Figures drawn using program PyMOL 40x17mm (300 x 300 DPI)

ACS Paragon Plus Environment

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Figure 4. Molecular surface of COMT shown in green with the methyl donor AdoMet and ligand 19 represented in sticks. The Mg2+ ion is depicted in magenta. Figure drawn using program PyMOL 59x38mm (300 x 300 DPI)



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Figure 5. Metabolism and active transport of L-DOPA 225x121mm (96 x 96 DPI)


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Medicinal Chemistry of Catechol O-Methyltransferase (COMT

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