Hydroxypyridinone compounds as inhibitors of catechol-o-methyltransferase

Abstract

The present application relates to N-substituted hydroxypyridinone compounds of Formula (I) which act as inhibitors of catechol-O-methyltransferase. The compounds of Formula (I) herein described are suitable for the treatment of diseases or disorders associated with dopamine deficiency.

Description

hydroxypyridinone compounds as inhibitors of catechol-o-methyltransferase

[0001] This application relates toN-substituted hydroxypyridinone compounds of formula (I) which act as inhibitors of catechol-O-methyltransferase and are suitable for the treatment of diseases or disorders associated with dopamine deficiency.

[0002] The imbalances in dopamine neurotransmission and alterations of brain circuits where dopamine is a key factor are involved in a variety of neurological and neuropsychiatric diseases, from alcohol / drug addiction to schizophrenia, among others. Dopamine is one of the most relevant neurotransmitters, as it seemingly participates directly or indirectly in almost any physiological function occurring in the central nervous system (CNS). A key discovery was the link between dopamine deficiency and Parkinson’s disease (PD).

[0003] Parkinson's disease (PD) is a neurodegenerative disease (ND) of unknown etiology, affecting brain nerve cells responsible for musculoskeletal control and locomotion [1]. PD is the second most common ND, affecting a global estimated 6.3 million people worldwide [2]. Due to the increased life expectancy, this number is expected to increase dramatically in the future and may even double by 2040, imposing severe social and economic burdens on the general population and healthcare systems. PD is characterized by the progressive loss of dopaminergic neurons in thesubstantia nigra[3], which leads to extensive dopamine depletion and contributes to the cardinal motor symptoms of the disease [4]. Moreover, increased iron accumulation has been observed in the brain of PD patients. The deregulation of iron homeostasis has been associated with increased oxidative stress and ferroptosis, leading to synaptic dysfunction and dopamine depletion [5]. The drugs available for the treatment of PD act only symptomatically and aim at restoring the cerebral dopaminergic pool. Levodopa, a biosynthetic dopamine precursor, is the gold standard drug for the clinical management of PD. Due to its extensive metabolism by catechol-O-methyltransferase (COMT) in peripheral tissues after oral administration, levodopa is usually co-administered with COMT inhibitors to prevent its premature decomposition [6]. Currently, three tight-binding nitrocatechol-based COMT inhibitors are used in PD treatments: tolcapone, entacapone, and opicapone. Tolcapone is a potent, centrally active COMT inhibitor. Although the therapeutic use of tolcapone has been associated with potentially fatal liver toxicity, it is still used due to its high efficacy. Entacapone and opicapone are safer alternatives but act only on peripheral COMT. The limitations of nitrocatechol-based COMT inhibitors stress the importance of the discovery of new inhibitors containing moieties that can mimic the nitrocatechol pharmacophore and provide the best balance between bioactivity, blood-brain barrier (BBB) permeability, and safety.

[0004] In this context, heterocycle catechol mimics (HetCAMs) have been developed over the last few years, and recent studies showed their potential as COMT inhibitors [7, 8].

[0005] Although the efficacy of these compounds is well-recognized, their use as putative COMT inhibitors is still in the early stages, and no clinical data concerning theirin vivoefficacy in PD patients is available.

[0006] The application relates to a N-substituted hydroxypyridinone compound of Formula (I), or a pharmaceutically acceptable salt thereof, having the structure:

[0007]

[0008] Formula (I)

[0009] Wherein X is selected from:

[0010] wherein R1=R2=CH3, or R1=R2=H, or R1=CH3 and R2=H, or R1=Cl and R2=H; or

[0011] wherein n is selected from 1, 2, 3, and R3 is independently selected from H or CH3, and R4 is independently selected from H or CH3.

[0012] The application also relates to a method of obtaining a N-substituted hydroxypyridinone compound of Formula (I).General description

[0013] The present application discloses compounds of Formula (I) which are suitable for use as inhibitors of COMT. Consequently, the compounds of Formula (I) herein disclosed are also suitable for the treatment of diseases or disorders associated with dopamine deficiency. These diseases or disorders are neurological or neuropsychiatric diseases or disorders.

[0014] Accordingly, innovative compounds were synthesized. Then, to extend the compound list, new compounds were designed and developed, where the hydroxypyridin-4-one core was preserved, and linkers with different lengths (ethyl, butyl, and hexyl linkers) were introduced between the hydroxypyridinone ring and the unsubstituted aryl moiety.

[0015] The chemical modifications were carefully chosen to select those that can be productive for the subsequent design of a robust list of compounds for the purpose of the present invention. All compounds were screened towards soluble (S-COMT) and membrane-bound COMT (MB-COMT), both isolated from rat brain and liver. The compound with the best COMT inhibition profile followed through to the evaluation of its metal chelation activity, drug-like properties, cytotoxicity, and pharmacokinetic profile.

[0016] For easier understanding of this application, figures are attached in the annex that represent the preferred forms of implementation which nevertheless are not intended to limit the technique disclosed herein.Fig.1

[0017] shows Formula (I).Fig.2

[0018] shows the compounds based on the chemical structure of kojic acid (KA).Fig.3

[0019] shows the synthetic strategy used to obtain compounds9and10. Reaction conditions: (A) methanol, aqueous solution of NaOH 1 M, benzyl chloride, reflux, 7 – 8 h; (B) acetone, Jones reagent 2.5 M, 0 ºC, overnight; (C) diphenyl ether, 250 ºC, 8 – 10 min; optimized: Cu2O, 1,10-phenantthroline, NMP, quinoline, 190 ºC (MW irradiation), 35 min; (D) 3,4-dimethylaniline or 4-chloroaniline, ethanol, HCl 0.38 M, 100 ºC, 1.5 h; (E) anhydrous DCM, BBr3, -80 ºC, 2 h.Fig.4

[0020] a) shows the synthetic strategy used to obtain hydroxypyridinone derivatives 23-33. Reaction conditions: (A) Camphor sulfonic acid, water, reflux, 100 ºC, 48 h. b) shows the synthetic strategy pursued to obtain phenethylamines 15-16 and 20-22. (1A) CH3NO2, AcOH, NH4OAc, rt, 3 days; (1B) LiAlH4, anhydrous THF, argon, reflux, 2 days; (2A) 1. bromoalkanoyl chloride, CH2Cl2 anhydrous, argon atmosphere, 0 °C, 20 min; 2. AlCl3, 0 °C, 3 h; (2B) 1. TFA, CH2Cl2, 0 °C, argon atmosphere, 30 min; 2. Et3SiH, rt, overnight; (2C) phthalimide potassium salt, DMF, reflux, 2 h; (2D) butylamine, ethanol, reflux, 24 h.Fig.5

[0021] shows the saturation curves forbrain MB-COMT (A, B), liver MB-COMT (B, C), and liver S-COMT (E, F) using increasing concentrations of AD (A, C and E) or SAM (B, D and F) as the substrate. Compound9was present in the pre-incubation period. Symbols represent the mean of three independent determinations. Vertical lines show S.E.M..Fig.6

[0022] shows the Ackermann-Potter plot of (A and B) liver S-COMT (0.15-1.5 mg mL-1) using tolcapone (30 and 100 nM) and compound9(3 µM), (C and D) liver MB-COMT (0.2-1.2 mg mL-1) using tolcapone (20 nM) and compound9(50 nM), and (E and F) brain MB-COMT (0.1-1.4 mg mL-1) using tolcapone (3 nM) and compound9(30 nM). Different amounts of protein were incubated in the absence and the presence of the inhibitor. Results are the mean of four determinations per group; vertical lines show S.E.M..Fig.7

[0023] shows(A)UV-vis spectra ofcompound9(40 µM) alone and co-incubated with iron (II) chloride, iron (III) chloride, and copper (II) chloride (20 µM).(B)UV-Vis titration of compound9(40 µM) incubated with iron (II) chloride (0-100 µM).(C)UV-vis titration of compound9(40 µM) incubated with iron (III) chloride (0-100 µM).(D)UV-vis titration of compound9(40 µM) incubated with copper (II) chloride (0-100 µM). Inset: changes in the absorbance at 294 nm with increasing concentrations of Fe2+, Fe3+, or Cu2+derived from the titration; breakpoints were observed at 30 µM. All solutions were prepared in PBS (pH 7.4).Fig.8

[0024] [Fig.8] shows the evaluation of cytotoxicity, by calcein-AM assay (A and C), or by resazurin assay (B and D) of HepG2 cells (A and B) and of SK-N-SH cells (C and D), after incubation with 10, 30 and 50 µM for 24 h, of tolcapone and compound9. Ethanol (70 %, EtOH) was used to eliminate viable cells 15 min before the addition of calcein-AM (A and C) and No cells represent the negative control of the experiment (B and D). Each column represents the mean of nine determinations, vertical lines show S.E.M.. Significantly different values compared with Untreated cells (Control) (*) are shown using a one-way ANOVA test followed by Dunnett's multiple comparisons test.Pvalues lower than 0.05 were considered significant (****p<0.0001vsUntreated). Significantly different values compared with same concentration of Tolcapone (#) are shown using unpaired t-test.Pvalues lower than 0.05 were considered significant (####p<0.0001;##p<0.01;#p<0.05vsTolcapone).Detailed Description of Embodiments

[0025] Now, preferred embodiments of the present application will be described in detail with reference to the annexed drawings. However, they are not intended to limit the scope of this application.

[0026] The present application discloses compounds of Formula (I) which are suitable for use as inhibitors of COMT. Consequently, the compounds of Formula (I) herein disclosed are suitable for the treatment of diseases or disorders associated with dopamine deficiency.

[0027] In one embodiment, the compound of Formula (I) is for use in the treatment of a neurological or a neuropsychiatric disease or disorder associated with dopamine deficiency which are selected from, but not limited to, Parkinson’s Disease, schizophrenia, depression, attention deficit hyperactivity disorder, substance dependency such as opiate, tobacco addiction etc., dementia, Parkinson's disease, Huntington's disease, Pick's disease, Creutzfeldt Jakob disease, perinatal hypoxia, cognitive disorder, or cognitive deficit associated with any of said disorder or disease, among others.

[0028] The compounds of Formula (I), as shown inand, comprise a common core structure which isN-substituted. TheseN-substituted compounds are hydroxypyridinone derivatives. The compounds comprise a different pharmacophore than the actual COMT inhibitors used in therapy, display relevant COMT inhibition and are safe, in the experimental conditions tested, in different cell lines. Some of the compounds are selective for brain COMT and are predicted to cross the BBB.

[0029] The compounds of Formula (I), of pharmaceutically acceptable salts thereof, herein described areN-substituted hydroxypyridinone compounds: compounds 9 and 10, and 23 to 33.

[0030] In the present invention Formula (I) is defined as:

[0031]

[0032] Wherein

[0033] X is selected from:

[0034] wherein R1=R2=CH3, or R1=R2=H, or R1=CH3and R2=H, or R1=Cl and R2=H; or

[0035] wherein n is selected from 1, 2, 3, and R3is independently selected from H or CH3, and R4is independently selected from H or CH3.

[0036] In this Formula (I), the and substituents are attached to the core structure in position X via the N atom.

[0037] In one embodiment, the compounds of Formula (I) are listed as follows:

[0038] Compound 9 is R1=R2=CH3

[0039] Compound 10 is R1=Cl and R2=H

[0040] Compound 23 is R1=R2=H

[0041] Compound 24 is R1=CH3and R2=H

[0042] Compound 25 is n=1, R3=R4=H

[0043] Compound 26 is n=1, R3=CH3and R4=H

[0044] Compound 27 is n=1, R3=R4=CH3

[0045] Compound 28 is n=2, R3=R4=H

[0046] Compound 29 is n=2, R3=CH3and R4=H

[0047] Compound 30 is n=2, R3=R4=CH3

[0048] Compound 31 is n=3, R3=R4=H

[0049] Compound 32 is n=3, R3=CH3and R4=H

[0050] Compound 33 is n=3, R3=R4=CH3.

[0051] The following compounds are intermediate compounds in the synthesis of the Formula (I) compounds described above:

[0052] Compounds 12 is R1=R2=H

[0053] Compounds 13 is R1=CH3and R2=H

[0054] Compounds 14 is n=1, R3=R4=H

[0055] Compounds 15 is n=1, R3=CH3and R4=H

[0056] Compounds 16 is n=1, R3=R4=CH3

[0057] Compounds 17 is n=2, R3=R4=H

[0058] Compounds 18 is n=2, R3=CH3and R4=H

[0059] Compounds 19 is n=2, R3=R4=CH3

[0060] Compounds 20 is n=3, R3=R4=H

[0061] Compounds 21 is n=3, R3=CH3and R4=H

[0062] Compounds 22 is n=3, R3=R4=CH3.

[0063] In one embodiment, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is selected from the list comprising:

[0064] Compound 9= 1-(3,4-dimethylphenyl)-3-hydroxypyridin-4(1H)-one;

[0065] Compound 10 = 1-(4-chlorophenyl)-3-hydroxypyridin-4(1H)-one;

[0066] Compound 23 = 3-hydroxy-2-methyl-1-phenylpyridin-4(1H)-one;

[0067] Compound 24 = 3-hydroxy-2-methyl-1-(p-tolyl)pyridin-4(1H)-one;

[0068] Compound 25 = 3-hydroxy-2-methyl-1-phenethylpyridin-4(1H)-one;

[0069] Compound 26 = 3-hydroxy-2-methyl-1-(4-methylphenethyl)pyridin-4(1H)-one;

[0070] Compound 27 = 1-(3,4-dimethylphenethyl)-3-hydroxy-2-methylpyridin-4(1H)-one;

[0071] Compound 28 = 3-hydroxy-2-methyl-1-(4-phenylbutyl)pyridin-4(1H)-one;

[0072] Compound 29 = 3-hydroxy-2-methyl-1-(4-(p-tolyl)butyl)pyridin-4(1H)-one;

[0073] Compound 30 = 1-(4-(3,4-dimethylphenyl)butyl)-3-hydroxy-2-methylpyridin-4(1H)-one;

[0074] Compound 31 = 3-hydroxy-2-methyl-1-(6-phenylhexyl)pyridin-4(1H)-one;

[0075] Compound 32 = 3-hydroxy-2-methyl-1-(6-(p-tolyl)hexyl)pyridin-4(1H)-one;

[0076] Compound 33 = 1-(6-(3,4-dimethylphenyl)hexyl)-3-hydroxy-2-methylpyridin-4(1H)-one.

[0077] In one embodiment, the present application relates to a pharmaceutical composition comprising at least one compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0078] In one embodiment, the present application relates to a pharmaceutical composition at least one compound of Formula (I) or a pharmaceutically acceptable salt thereof, for use in the treatment of a neurological or a neuropsychiatric disease or disorder associated with dopamine deficiency.

[0079] In one embodiment, the present application also relates to a method of producing compounds of formula (I) 9 and 10, comprising the steps of:

[0080] - protection of the hydroxyl group of Kojic acid (compound1), in a concentration between 1 and 3 M, through reacting benzyl chloride in a concentration between 1 and 5 mol in methanol in a concentration between 1 and 10 mL / mmol, with an aqueous solution of NaOH in a concentration between 1 and 2 M, in reflux between 6 and 10 hours to yield 5-(benzyloxy)-2-(hydroxymethyl)-4H-pyran-4-one (compound2);

[0081] - the 2-hydroxymethyl moiety of compound2in a concentration between 1 and 4 M is oxidized into the corresponding carboxylic acid using the Jones reagent at a concentration between 1 and 2.5 M and acetone in a concentration between 1 and 10 mL / mmol, at a temperature between 0ºC and 25ºC, overnight, yielding 5-(benzyloxy)-4-oxo-4H-pyran-2-carboxylic acid (compound3);

[0082] - heat-induced decarboxylation of compound3in a concentration between 1 and 3 M in 1-methyl-2-pyrrolidinone in a concentration between 0.5 and 5 mL / mmol, using Cu2O in a concentration between 0.05 and 1 mol as catalyst, 1,10-phenanthroline as a copper ligand in a concentration between 0.1 and 2 mol, and microwave irradiation at a temperature between 150 ºC and 250 ºC, with reaction time between 15 minutes and 24 hours, yielding C2-unsubstituted intermediate 3-(benzyloxy)-4H-pyran-4-one (compound4);

[0083] - Compound4in a concentration between 1 and 5 M was reacted with 3,4-dimethylaniline or with 4-chloroaniline in a concentration between 1 and 10 molviaMichael addition, under mild acidic conditions provided by EtOH:HCl at a concentration between 0.3 and 3 M with ratio1:1 V / V, at a temperature between 80ºC and 150 ºC, with a reaction time between for 1 hour and 48 hours, affording theN-substituted derivatives 3-(benzyloxy)-1-(3,4-dimethylphenyl)pyridin-4(1H)-one(compound5) or3-(benzyloxy)-1-(4-chlorophenyl)pyridin-4(1H)-one (compound6);

[0084] - deprotection of the hydroxyl group of compounds5or6in a concentration between 1 and 4 M with a boron tribromide (BBr3) solution in dichloromethane in a concentration between 1 and 10 mol, and dichloromethane in a concentration between 1 and 50 mL / mmol, at a temperature between -80ºC and 0ºC, with a reaction time between 1 hour and 48 hours, yielding 1-(3,4-dimethylphenyl)-3-hydroxypyridin-4(1H)-one (compound9) or 1-(4-chlorophenyl)-3-hydroxypyridin-4(1H)-one (compound10),respectively.

[0085] In another embodiment, the application also relates to a method of producing compounds of formula (I)23-33, comprising the steps of:

[0086] - Synthesis of (E)-1,2-dimethyl-4-(2-nitrovinyl)benzene (compound16b) through the reaction of 3,4-dimethylbenzaldehyde (compound16a), in a concentration between 1 and 3 M with nitromethane in a concentration between 1 and 10 mol dissolved in acetic acid in a concentration between 1 and 10 mol, in the presence of ammonium acetate in a concentration between 1 and 10 mol, at a temperature between 10 ºC and 50 ºC, for a reaction time between 24 hours and 72 hours;

[0087] - (E)-1,2-dimethyl-4-(2-nitrovinyl)benzene16bin a concentration between 1 and 3 M was then reduced into 2-(3,4-dimethylphenyl)ethan-1-amine (compound16) with lithium aluminium hydride in a concentration between 1 and 10 mol in anhydrous tetrahydrofuran in a concentration between 1 and 10 mL / mmol, under argon atmosphere and reflux, with a reaction time between 24 hours and 72 hours;

[0088] - Friedel-Crafts acylation between o-xylene19aor toluene21ain a concentration between 1 and 3 M and 4-bromobutyl chloride or 6-bromohexanoyl chloride in a concentration between 1 and 10 M in anhydrous dichloromethane in a concentration between 0.5 and 5 mL / mmol, in the presence of aluminium trichloride in a concentration between 1 and 10 mol, under argon atmosphere, at a temperature between -50ºC and 0ºC, with a reaction time between 1 hour and 48 hours, to afford 4-bromo-1-(3,4-dimethylphenyl)butan-1-one (compound19b); or 6-bromo-1-(p-tolyl)hexan-1-one (compound21b), or 6-bromo-1-(3,4-dimethylphenyl)hexan-1-one (compound22b);

[0089] - Reduction of bromophenylalcan-1-ones19b,or21b, or22bin a concentration between 1 and 3 M into the correspondingbromophenylalcanes with triethylsilane in a concentration between 1 and 10 mol in anhydrous dichloromethane in a concentration between 0.5 and 5 mL / mmol, under acidic conditions provided by trifluoroacetic acid in a concentration between 10 and 100 mol, under argon atmosphere, at a temperature between 0ºC and 25ºC, overnight, the resulting intermediates were then reacted with phthalimide potassium salt in a concentration between 1 and 10 mol in DMF in a concentration between 1.0 and 10 mL / mmol, under argon atmosphere and reflux, with a reaction time between 1 hour and 24 hours, yielding 2-(4-(3,4-dimethylphenyl)butyl)isoindoline-1,3-dione (compound19c), or 2-(6-(p-tolyl)hexyl)isoindoline-1,3-dione (compound21c), or 2-(6-(3,4-dimethylphenyl)hexyl)isoindoline-1,3-dione (compound22c);

[0090] - 1-Bromo-6-phenylhexane (compound20a)in a concentration between 1 and 3 M was reacted with phthalimide potassium salt in a concentration between 1 and 10 mol in DMF in a concentration between 1 and 10 mL / mmol, under argon atmosphere and reflux, with a reaction time between 1 hour and 24 hours, yielding 2-(6-phenylhexyl)isoindoline-1,3-dione (compound20b);

[0091] - The phthalimide group of phenylalkylisoindoline-1,3-diones19c,or20b,or21c,or22cin a concentration between 1 and 3 M was cleaved with butylamine in a concentration between 5 and 100 mol in ethanol in a concentration between 0.5 and 5 mL / mmol, under reflux, with a reaction time between 6 hours and 72 hours, affording 4-(3,4-dimethylphenyl)butan-1-amine (compound19), or 6-phenylhexan-1-amine (compound20), or 6-(p-tolyl)hexan-1-amine (compound21), or6-(3,4-dimethylphenyl)hexan-1-amine (compound22);

[0092] - Single-step one-pot reaction between 3-hydroxy-4H-pyran-4-one11in a concentration between 1 and 3 M and aniline (compound12),orp-toluidine (compound13), or 2-phenylethan-1-amine (compound14), or 2-(p-tolyl)ethan-1-amine (compound15), or 2-(3,4-dimethylphenyl)ethan-1-amine(compound16), or 4-phenylbutan-1-amine (compound17), or 4-(p-tolyl)butan-1-amine (compound18), or 4-(3,4-dimethylphenyl)butan-1-amine (compound19), or 6-phenylhexan-1-amine (compound20), or (p-tolyl)hexan-1-amine (compound21), or 6-(3,4-dimethylphenyl)hexan-1-amine (compound22) in a concentration between 1 and 7 M in water in a concentration between 2 and 20 mL / mmol, under acidic conditions provided by camphor sulfonic acid in a concentration between 0.1 and 2 mol, under reflux, with a reaction time between 24 and 72 hours, yielded 3-hydroxy-2-methyl-1-phenylpyridin-4(1H)-one (compound23), or 3-hydroxy-2-methyl-1-(p-tolyl)pyridin-4(1H)-one (compound24), or 3-hydroxy-2-methyl-1-phenethylpyridin-4(1H)-one (compound25), or 3-hydroxy-2-methyl-1-(4-methylphenethyl)pyridin-4(1H)-one (compound26), or 1-(3,4-dimethylphenethyl)-3-hydroxy-2-methylpyridin-4(1H)-one (compound27), or 3-hydroxy-2-methyl-1-(4-phenylbutyl)pyridin-4(1H)-one (compound28), or 3-hydroxy-2-methyl-1-(4-(p-tolyl)butyl)pyridin-4(1H)-one (compound29), or 1-(4-(3,4-dimethylphenyl)butyl)-3-hydroxy-2-methylpyridin-4(1H)-one (compound30), or 3-hydroxy-2-methyl-1-(6-phenylhexyl)pyridin-4(1H)-one (compound31), or 3-hydroxy-2-methyl-1-(6-(p-tolyl)hexyl)pyridin-4(1H)-one (compound32), or 1-(6-(3,4-dimethylphenyl)hexyl)-3-hydroxy-2-methylpyridin-4(1H)-one (compound33), respectively.

[0093] In the present application, a list of compounds of Formula (I) was developed, in which the hydroxypyridin-4-one derivative9was the most potent COMT inhibitor. The experimental data suggest that compound9has higher selectivity for MB-COMT than S-COMT, as well as greater inhibitory activity for brain MB-COMT than liver MB-COMT. Like tolcapone, the IC50value of compound9towards MB-COMT was within the low nanomolar range. Although tolcapone showed higher COMT inhibitory activity in both the brain and liver MB-COMT than compound9, the latter exhibited higher selectivity towards brain MB-COMT compared to liver S-COMT. Compound9also demonstrated optimal physicochemical properties to act as a CNS-active drug, such as MW, HBD, HBA, RB, tPSA, lipophilicity, and BBB permeability. Unlike tolcapone, which is known to act as a non-competitive inhibitor for the substrate adrenaline (AD), compound9showed a different mechanism of COMT inhibition. Compound9is expected to act as a competitive inhibitor, which was evidenced by increases in COMT affinity for AD (higherKmvalues) without significantly changing theVmaxvalues

[0055] . In addition, the linearity of the curves in the Ackermann-Potter plots in the presence of compound9reflected the non-tight-binding nature recorded with tolcapone. UV-Vis spectrometry demonstrated that compound9interacts with divalent metals, namely iron and copper, which suggests that this compound can act as a chelating agent and possibly regulate ferroptosis in the CNS. Finally, compound9was less cytotoxic than tolcapone in cultured hepatocarcinoma (HepG2) and neuroblastoma (SK-N-SH) cells.Examples

[0094] Experimental section

[0095] In the context of the present application, the term “room temperature” is understood as a temperature between 20 and 25ºC.

[0096] Chemistry

[0097] Reagents and apparatus

[0098] All reagents and solvents were purchased from Sigma-Aldrich (Barcelona, Spain), Fluorochem (Hadfield, UK), and Alfa Aesar (Kandel, Germany) and used without any further purification. All reactions were controlled by thin-layer chromatography (TLC) using precoated silica gel 60 F254 plates acquired from Merck (Darmstadt, Germany). For analytical control, dichloromethane (DCM), ethyl acetate, and DCM / ethyl acetate mobile phases were used in several proportions. The spots were visualized under UV detection (254 and 366 nm) and / or ferric chloride. The crude products were purified by flash column chromatography using silica gel 60 Å (0.040-0.063 mm) (Carlo Erba Reactifs). Following the workup and after extraction, the organic phases were dried over anhydrous sodium sulfate (Na2SO4), filtered, and concentrated.

[0099] Solvents were evaporated under reduced pressure in a Buchi Rotavapor. Compounds9and23-33were further purified by Shimadzu Prominence high-performance liquid chromatography (HPLC), equipped with a Luna C18 column, 150 4.6 mm, 5 µm (Phenomenex, CA, USA) and using a gradient elution system containing acetonitrile:water (20 % / 80 % to 100 % / 0 %, V / V).

[0100] 1H Magnetic Resonance (NMR) spectra were acquired at room temperature and recorded on a Bruker Avance III operating at 400 and 100 MHz, respectively. Tetramethylsilane (TMS) was used as an internal standard, chemical shifts (δ) are expressed in parts per million (ppm), and the coupling constant (J) is given in Hz. Assignments were also made from Distortionless Enhancement by Polarization Transfer (DEPT135, underlined values). The NMR samples were prepared in deuterated methanol (CD3OD-d4), dimethyl sulfoxide (DMSO-d6), or chloroform (CDCl3-d1). Mass spectra (MS) of final compounds were recorded on a Bruker Microtof (Electrospray Ionization - ESI) apparatus and referred in m / z (% relative) of relevant fragments. The purity of the final compounds was verified by preparative HPLC to attain percentages of purity > 98 %.

[0101] Synthesis of the compounds

[0102] Synthesis of 5-(benzyloxy)-2-(hydroxymethyl)-4H-pyran-4-one (compound 2)

[0103] To a solution of kojic acid1(0.50 g, 3.54 mmol) in methanol (5 mL), NaOH 1 M (360 µL) was added, and the mixture was heated to reflux for 1 h. Then, benzyl bromide (410 µL, 3.56 mmol) was added dropwise over 1 h, and the mixture was stirred under reflux for 7 h. The reaction mixture was cooled to room temperature, dissolved in the minimum amount of methanol, and added dropwise to cold water. The solid obtained was collected by filtration, washed with cold water, recrystallized (ethyl acetate), and collected as a white solid. The procedure was adapted from the literature

[0037] .

[0104] Yield: 59 %

[0105] 1H NMR (400 MHz, CDCl3-d1): δ = 4.45 (s, 2H, Ar-CH2-OH), 5.06 (s, 2H, Ar-CH2-O-), 6.50 (s, 1H, H(3)), 7.31–7.41 (m, 5H, H(2’)-H(6’)), 7.51 (s, 1H, H(6)).

[0106] Synthesis of 5-(benzyloxy)-4-oxo-4H-pyran-2-carboxylic acid (compound 3)

[0107] To a solution of 5-(benzyloxy)-2-(hydroxymethyl)-4H-pyran-4-one 2 (1.00 g, 4.06 mmol) in 20 mL of acetone, 5 mL of Jones reagent 2.5 M were added at 0 °C. The reaction was stirred overnight at room temperature. The solid was removed by filtration and washed with acetone, and the filtrate was concentrated. The concentrated residue was poured onto water. The resulting solid was collected by filtration, washed with water, and dried. The product was isolated as a white solid. The procedure was adapted from the literature [38, 39].

[0108] Yield: 73 %

[0109] 1H NMR (400 MHz, DMSO-d6): δ = 4.99 (s, 2H, Ar-CH2-O-), 6.94 (s, 1H, H(3)), 7.34– 7.45 (m, 5H, H(2’)-H(6’)), 8.37 (s, 1H, H(6)).

[0110] Synthesis of 3-(benzyloxy)-4H-pyran-4-one (compound 4)

[0111] 5-(Benzyloxy)-4-oxo-4H-pyran-2-carboxylic acid 3 (0.40 g, 1.63 mmol) was dissolved in diphenyl ether (2.5 mL) and heated to 250 °C in an open flask for 10 min. Upon completion, the crude product was purified by column chromatography (DCM / methanol 99:1 to 9:1), recrystallized (DCM / petroleum ether), and collected as a white solid. The procedure was adapted from the literature

[0040] .

[0112] Yield: 13 %

[0113] Optimization: An oven-dried 10 mL microwave vial was filled with 5-(benzyloxy)-4-oxo-4H-pyran-2-carboxylic acid 3 (0.70 g, 3.00 mmol), Cu2O (21.9 mg, 0.15 mmol). The reaction mixture was made inert, and 1,10-phenanthroline (54.4 mg, 0.30 mmol) and 1-methyl-2-pyrrolidinone (NMP, 1.5 mL) were added via a syringe. The reaction mixture was heated under microwave irradiation at 190 ºC for 35 min. Upon completion, the reaction was diluted in 30 mL of aqueous HCl 5 M and extracted repeatedly with diethyl ether (5 mL portions). The product was obtained by recrystallization (DCM / petroleum ether) as a white solid. The procedure was adapted from the literature

[0041] .

[0114] Yield: 58 %

[0115] 1H NMR (400 MHz, CDCl3-d1): δ = 5.09 (s, 2H, Ar-CH2-O-), 6.43 (d,J= 5.6 Hz, 1H, H(3)), 7.32- 7.41 (m, 5H, H(2’)-H(6’)), 7.55 (d,J= 0.8 Hz, 1H, H(6)), 7.66 (dd,J= 5.6, 0.9 Hz, 1H, H(2)).

[0116] Synthesis of 3-(benzyloxy)-1-(3,4-dimethylphenyl)pyridin-4(1H)-one (compound 5)

[0117] 3-(Benzyloxy)-4H-pyran-4-one 4 (0.12 g, 0.59 mmol) and 3,4-dimethoxyaniline (0.43 g, 3.59 mmol) were dissolved in EtOH:HCl 0.38 M (1:1 V / V, 1 mL) and heated in a microwave oven at 100˚C for 1.5 h. Once the reaction was complete, the solvents were removed under reduced pressure, and the residue was dissolved in DCM (30 mL) and washed with HCl 1M (4 x 10 mL) and brine (20 mL). The organic layer was dried over MgSO4, filtered and concentrated. The crude product was purified by column chromatography (DCM / methanol 99:1 (V / V) to 9:1 (V / V)), and the fractions containing the product were collected and concentrated. The product was isolated as a dark brown oil. The procedure was adapted from the literature [42, 43].

[0118] Yield: 68 %

[0119] 1H NMR (400 MHz, CD3OD-d4): δ = 2.32 (s, 3H, CH3), 2.34 (s, 3H, CH3), 5.15 (s, 2H, Ar-CH2-O-), 6.57 (d, J = 7.3 Hz, 1H, H3), 7.14 (dd, J = 8.1, 2.4 Hz, 1H, H6), 7.20 (d, J = 2.1 Hz, 1H, H4’), 7.41 – 7.27 (m, 4H, H(2’), H(3’), H(5’), H(6’)), 7.50 – 7.46 (m, 2H, H2’’, H5’’), 7.71 (d, J = 2.2 Hz, 1H, H6’’), 7.87 (dd, J = 7.3, 2.2 Hz, 1H, H5).

[0120] Synthesis of 3-(benzyloxy)-1-(4-chlorophenyl)pyridin-4(1H)-one (compound 6)

[0121] The 3-(benzyloxy)-4H-pyran-4-one 4 (0.10 g, 0.49 mmol) and 4-chloroaniline (0.38 g, 2.94 mmol) were dissolved in EtOH:HCl 0.38 M, (1:1 V / V, 1 mL) and heated in a microwave oven at 100 ˚C for 1 h. Upon completion, the solvents were removed under reduced pressure, and the residue was stirred with HCl 1 M (20 mL) for 30 min at room temperature. The mixture was then extracted with DCM (3×10 mL). The combined organic layers were washed with HCl 1 M (2×10 mL), water (2×10 mL), and brine (10 mL), dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (ethyl acetate / methanol 99:1 (V / V) to 9:1 (V / V)), and the fractions containing the product were collected and concentrated. The product was isolated as a yellow solid. The procedure was adapted from the literature [42, 43].

[0122] Yield: 40 %

[0123] 1H NMR (400 MHz, CDCl3-d1): δ = 5.21 (s, 2H, Ar-CH2-O-), 6.57 (d, J = 7.5 Hz, 1H, H2’’), 7.19 – 7.13 (m, 2H, H3’’, H5’’), 7.21 (d, J = 2.4 Hz, 1H, H6’’), 7.40 – 7.29 (m, 3H, H3, H5, H6), 7.48 – 7.41 (m, 5H, H2’-H6’).

[0124] Synthesis of 1-(3,4-dimethylphenyl)-3-hydroxypyridin-4(1H)-one (compound 9)

[0125] To a solution of 3-(benzyloxy)-1-(3,4-dimethylphenyl)pyridin-4(1H)-one 5 (0.12 g, 0.41 mmol) in anhydrous DCM (1.5 mL), under an inert atmosphere and at -80ºC, BBr3 (0.62 mL of a 1 M solution in DCM, 0.62 mmol) was added. The solution was kept at -80 ºC for 2 h and then allowed to reach room temperature overnight. Upon completion, the solution was added to cold water, neutralized with aqueous saturated NaHCO3 solution (10 mL), and stirred at room temperature for 1 h. The solid was filtered and recrystallized (DCM / petroleum ether). The product was isolated as a beige solid. The procedure was adapted from the literature

[0045] .

[0126] Yield: 30 %

[0127] 1H NMR (400 MHz, CDCl3-d1): δ = 2.33 (s, 3H, CH3), 2.34 (s, 3H, CH3), 6.55 (d, J = 6.9 Hz, 1H, H6), 7.08 (dd, J = 8.0, 2.0 Hz, 1H, H6’), 7.13 (d, J = 2.0 Hz, 1H, H2’), 7.22 – 7.28 (m, 1H, H5’), 7.51 (m, 2H, H3, H5).

[0128] Synthesis of 1-(4-chlorophenyl)-3-hydroxypyridin-4(1H)-one (compound 10)

[0129] To a solution of 3-(benzyloxy)-1-(4-chlorophenyl)pyridin-4(1H)-one 6 (0.12 g, 0.41 mmol) in anhydrous DCM (1.5 mL), under an inert atmosphere and at -80ºC, BBr3 (0.62 mL of a 1 M solution in DCM, 0.62 mmol) was added. The solution was kept at -80ºC for 2 h and then allowed to reach room temperature overnight. Upon completion, the solution was added to cold water, neutralized with aqueous saturated NaHCO3 solution (10 mL), and stirred at room temperature for 1 h. The solution was extracted with DCM (3×10 mL), and the combined organic layers were washed with water (2×10 mL) and brine (10 mL), dried over Na2SO4, filtered, and concentrated. The crude product was recrystallized (DCM / petroleum ether) and isolated as a light pink solid. The procedure was adapted from the literature

[0045] .

[0130] Yield: 34 %

[0131] 1H NMR (400 MHz, CDCl3-d1): δ = 6.56 (d, J = 7.3 Hz, 1H, H6), 7.53-7.59 (m, 4H, H2’, H3’, H5’, H6’), 7.77 (d, J = 2.4 Hz, 1H, H3), 7.87 (dd, J = 7.3, 2.4 Hz, 1H, H5).

[0132] General procedure to obtainhydroxypyridinonederivatives (compounds 23-33)

[0133] A mixture of 3-hydroxy-4H-pyran-4-one 11 (1g; 7.93 mmol), the proper amine (12-22) (7.93 mmol) and camphor sulfonic acid (0.79 mmol) in water (20 mL) was heated at 100 °C for 48 h at argon atmosphere. The solution was extracted with DCM (2×20 mL), and the combined organic layers were washed with water (2×20 mL) and brine (10 mL), dried over Na2SO4, filtered, and concentrated. The crude product was purified with preparative HPLC, and the final products were isolated as light-yellow solids. The procedure was adapted from the literature

[0010] .

[0134] Synthesis of 3-hydroxy-1-phenylpyridine-4(1H)-one (compound 23)

[0135] Yield: 9 %

[0136] 1H NMR (400 MHz; CD3OD-d4): δ = 6.56 (d, J = 7.2 Hz, 1H, H6), 7.57 – 7.49 (m, 5H, H2’, H3’, H4’, H5’, H6’), 7.77 (s, 1H, H3), 7.87 (d, J = 7.2 Hz, 1H, H5).

[0137] Synthesis of 3-hydroxy-1-(p-tolyl)pyridin-4(1H)-one (compound 24)

[0138] Yield: 14 %

[0139] 1H NMR (400 MHz; CDCl3-d1): δ = 2.42 (s, 3H, CH3), 6.55 (d, J = 7.6 Hz; 1H, H6), 7.26 (d, J = 3.2 Hz, 2H, H2’, H6’), 7.31 (d, J = 8.4 Hz, 2H, H3’, H5’), 7.49 – 7.51 (m, 2H, H3, H5).

[0140] Synthesis of 3-hydroxy-1-phenylethylpyridin-4(1H)-one (compound 25)

[0141] Yield: 8 %

[0142] 1H NMR (400 MHz; CD3OD-d4): δ = 3.08 (t, J = 6.9 Hz, 2H, Ar-CH2), 4.20 (t, J = 6.9 Hz, 2H, N-CH2), 6.30 (d, J = 7.0 Hz, 1H, H6), 7.08 – 7.16 (m, 2H, H2’, H6’), 7.17 – 7.31 (m, 3H, H3’,H4’,H5’), 7.36 (dd, J = 7.0, 2.3 Hz; 1H; H3), 7.43 (d, J = 2.3 Hz, 1H, H5).

[0143] Synthesis of 3-hydroxy-1-(4-methylphenylethyl)pyridin-4(1H)-one (compound 26)

[0144] Yield: 8 %

[0145] 1H NMR (400 MHz; CD3OD-d4): δ = 2.28 (s, 3H, CH3), 3.03 (t, J = 6.8 Hz, 2H, Ar-CH2), 4.16 (t, J = 6.8 Hz, 2H, N-CH2), 6.31 (d, J = 7.0 Hz, 1H, H6), 6.99 (d, J = 8.0 Hz, 2H, H2’, H6’), 7.07 (d, J = 7.9 Hz, 2H, H3’, H5’), 7.35 (d, J = 7.0, 2.0 Hz, 1H, H3), 7.42 (d, J = 2.1 Hz, 1H, H5).

[0146] Synthesis of 1-(3,4-dimethylphenylethyl)-3-hydroxypyridin-4(1H)-one (compound 27)

[0147] Yield: 5 %

[0148] 1H NMR (400 MHz; CD3OD-d4): δ = 2.19 (d, J = 3.2 Hz, 6H, 2xCH3), 2.99 (t, J = 6.8 Hz, 2H, Ar-CH2), 4.15 (t, J = 7.0 Hz, 2H, N-CH2), 6.31 (d, J = 7.0 Hz, 1H, H6), 6.80 (dd, J = 7.7, 1.9 Hz, 1H, H6’), 6.86 (d, J = 1.9 Hz, 1H, H2’), 7.00 (d, J = 7.6 Hz, 1H, H5’), 7.34 (1H, dd, J = 7.0, 2.2 Hz, 1H, H5), 7.41 (d, J = 2.2 Hz, 1H, H3).

[0149] Synthesis of 3-hydroxy-1-(4-phenylbutyl)pyridin-4(1H)-one (compound 28)

[0150] Yield: 7 %

[0151] 1H NMR (400 MHz; CD3OD-d4): δ = 1.61 – 1.65 (m, 2H, N-CH2-CH2), 1.78 – 1.86 (m, 2H, Ar-CH2-CH2), 2.65 (t, J = 7.5 Hz, 2H, Ar-CH2-CH2), 3.98 (t, J = 7.2 Hz, 2H, N-CH2-CH2), 6.43 (d, J = 7.0 Hz, 1H, H6), 7.16 (d, J = 7.2 Hz, 3H, H3’, H4’, H5’), 7.23 – 7.26 (m, 2H, H2’,H6’), 7.53 (d, J = 2.2 Hz, 1H, H3), 7.59 (dd, J = 7.0, 2.2 Hz, 1H, H5).

[0152] Synthesis of 3-hydroxy-1-(4-(p-tolyl)butyl)pyridin-4(1H)-one (compound 29)

[0153] Yield: 8 %

[0154] 1H NMR (400 MHz; CD3OD-d4): δ = 1.56 – 1.60 (m, 2H, N-CH2CH2), 1.77 – 1.80 (m, 2H, CH2CH2-Ar), 2.27 (s, 3H, CH3), 2.59 (t, 2H, J = 7.5 Hz, CH2-Ar), 3.95 (t, J = 7.2 Hz, 2H, N-CH2CH2), 6.41 (d, J = 7.0 Hz, 1H, H6), 7.01 – 7.05 (m, 4H, H(2´), H(3´), H(5´), H(6´)), 7.49 (d, 1H, J = 2.2 Hz, H3), 7.56 (dd, 1H, J = 7.0, 2.2 Hz, H5).

[0155] Synthesis of 1-(4-(3,4-dimethylphenyl)butyl)-3-hydroxypyridin-4(1H)-one (compound 30)

[0156] Yield: 8 %

[0157] 1H NMR (400 MHz; CD3OD-d4): δ = 1.54 – 1.61 (m, 2H, N-CH2CH2), 1.75 – 1.83 (m, 2H, Ar-CH2CH2), 2.20 (d, 6H, J = 4.1 Hz, 2xCH3), 2.55 (t, J = 7.4 Hz, 2H, CH2Ar’), 3.95 (t, J = 7.2 Hz, 2H, N-CH2), 6.41 (d, J = 7.0 Hz, 1H, H6), 6.85 (dd, J = 7.6, 1.9 Hz, 1H, H6’), 6.91 (d J = 1.8 Hz, 1H, H2’), 7.00 (d, J = 7.7 Hz, 1H, H5’), 7.49 (d, J = 2.2 Hz, 1H, H3), 7.56 (dd, J = 7.1; 2.2 Hz, 1H, H5).

[0158] Synthesis of 3-hydroxy-1-(6-phenylhexyl)pyridin-4(1H)-one (compound 31)

[0159] Yield: 7 %

[0160] 1H NMR (400 MHz; CD3OD-d4): δ = 1.27 – 1.41 (m, 4H, N-CH2-CH2-CH2-CH2), 1.57 – 1.66 (m, 2H, N-CH2-CH2), 1.74 – 1.82 (m, 2H, Ar-CH2-CH2), 2.58 (t, J = 7.6 Hz, 2H, Ar-CH2-CH2), 3.93 (t, J = 7.2 Hz, 2H, N-CH2-CH2), 6.42 (d, J = 7.0 Hz, 1H, H6), 7.10-7.18 (m, 3H, H3’, H4’, H5’), 7.21 – 7.25 (m, 2H, H2’,H6’), 7.52 (d, J = 2.2 Hz, 1H, H3), 7.57 (dd, J = 7.0, 2.1 Hz, 1H, H5).

[0161] Synthesis of 3-hydroxy-1-(6-(p-tolyl)hexyl)pyridin-4(1H)-one (compound 32)

[0162] Yield: 6 %

[0163] 1H NMR (400 MHz; CD3OD-d4): δ = 1.29 – 1.37 (m, 4H, N-CH2-CH2-CH2-CH2), 1.56 – 1.63 (m, 2H, N-CH2-CH2), 1.75 – 1.82 (m, 2H, Ar-CH2-CH2), 2.27 (s, 3H, CH3), 2.54 (t, J = 7.5 Hz, 2H, Ar-CH2-CH2), 3.94 (t, J = 7.2 Hz, 2H, N-CH2-CH2), 6.42 (d, J = 7.1 Hz, 1H, H6), 6.98 – 7.08 (m, 4H, H2’, H3’, H5’, H6’), 7.52 (d, J = 2.2 Hz, 1H, H3), 7.57 (dd, J = 7.1, 2.2 Hz, 1H, H5).

[0164] Synthesis of 1-(6-(3,4-dimethylphenyl)hexyl)-3-hydroxypyridin-4(1H)-one (compound 33)

[0165] Yield: 8 %

[0166] 1H NMR (400 MHz; CD3OD-d4): δ = 1.21 – 1.38 (m, 4H, N-CH2-CH2-CH2-CH2), 1.52 – 1.63 (m, 2H, N-CH2-CH2), 1.72 – 1.72 (m, 2H, Ar-CH2-CH2), 2.19 (d, J = 5.3 Hz, 6H, 2xCH3), 2.50 (t, J = 7.5 Hz, 2H, Ar-CH2-CH2), 3.91 (t, J = 7.1 Hz, 2H, N-CH2-CH2), 6.42 (d, J = 7.0 Hz, 1H, H6), 6.84 (dd, J = 7.6, 1.9 Hz, 1H, H6’), 6.90 (d, J = 1.8 Hz, 1H, H2’), 6.97 (d, J = 7.6 Hz, 1H, H5’), 7.51 (d, J = 2.1 Hz, 1H, H3), 7.55 (dd, J = 6.9, 1.8 Hz, 1H, H5).

[0167] Evaluation of catechol O-methyltransferase inhibitory activity

[0168] Animals

[0169] Male Wistar rats obtained from Harlan (Barcelona, Spain) were kept 8 per cage, under controlled environmental conditions (12 h light / dark cycle and room temperature of 22 ±1 °C) with food and tap water allowed ad libitum. Twenty minutes before sacrifice, animals were anesthetized with sodium pentobarbital (60 mg kg-1) administered intraperitoneally. Before sample collection, anesthetized animals were euthanized by decapitation. All animal procedures were approved by the local Ethics Committee for the welfare of experimental animals and performed following national legislation.

[0170] Isolation of MB and S-COMT fractions

[0171] For the isolation of membrane-bound (MB) or soluble (S) forms of COMT, we used the method described by Nissien et al.

[0046] . Briefly, rat liver and brain were thawed at room temperature and homogenized 1:4 (w / v) and 1:2 (w / v) in 5 mM sodium phosphate buffer pH 7.8, respectively, using a Polytron homogenizer (Heidolph). The homogenates were centrifuged at 15,000xg for 20 min at 4 ºC, and the supernatants at 100,000xg for 60 min at 4 ºC. The high-speed supernatants were used for the determination of the S-COMT activity. The microsomal fraction was washed twice (100,000xg for 60 min at 4 ºC) in sodium phosphate buffer and used to determine the activity of MB-COMT.

[0172] COMT activity assay

[0173] The COMT inhibitory activity of tolcapone and novel compounds was evaluated using a method described previously by Vieira-Coelho et al.

[0047] . Briefly, 2 mg mL-1 of liver MB-, S-COMT, or brain MB-COMT were preincubated with increasing concentrations of the inhibitor (liver S-COMT 0,001-100 µM; liver MB-COMT 0,00003-10 µM; brain MB-COMT 0.003 nM -30 µM) for 20 min. The reaction started with the addition of adrenaline, AD, (liver S-COMT: 1000 µM for 5 min; liver MB-COMT: 10 µM for 5 min; brain MB-COMT: 10 µM for 15 min). After the incubation period, the reaction was stopped with the addition of 50 µL of PCA 2M. The assay of MN was carried out using HPLC-ED. IC50 values were obtained by fitting the experimental data to the equation variable slope (four parameters) using Prism 9 (GraphPad Software, San Diego, CA, USA).

[0174] Tight-binding inhibition and Ackermann-Potter analysis

[0175] To evaluate the tight-binding nature of the inhibitors, we used the method described by Borges et al.

[0048] . Liver S-COMT (0.15-0.45 mg mL-1), liver MB-COMT (0.2-1.2 mg mL-1) or brain MB-COMT (0.1-2 mg mL-1) at different concentrations were incubated with a saturating concentration of SAM (500 µM for the liver and 100 µM for the brain). The test compounds (liver S-COMT, tolcapone 30 nM and 100 nM and compound 9 3 µM; liver MB-COMT, tolcapone 20 nM and compound 9 50 nM; brain MB-COMT, tolcapone 3 nM and compound 9 30 nM) were pre-incubated with COMT isoforms for 20 min, and the reaction was started with the addition of AD (liver S-COMT: 1000 µM for 5 min; liver MB-COMT: 10 µM for 5 min; brain MB-COMT: 10 µM for 15 min). The reaction was stopped with the addition of 50 µL of PCA 2 M. The MN assay was performed using HPLC-ED. Results were fitted to the Ackermann-Potter equation for tight-binding inhibitors

[0049] .

[0176] Iron and copper chelating activity

[0177] The chelating properties were studied using a UV-Vis spectrophotometric assay. Stock solutions of metal ions (10 mM) were freshly prepared in ethanol (iron (II) chloride, iron (III) chloride, copper (II) chloride, or magnesium (II) chloride). A stock solution of compound 9 (10 mM) was prepared in DMSO. The absorption spectra of each compound (final concentration: 40 µM), in the absence or the presence of iron (II) chloride, iron (III) chloride, copper (II) chloride, or magnesium (II) chloride (final concentration: 20 µM), were recorded after 30 min of incubation in a multiplate reader (BioTek Epoch 2 microplate spectrophotometer from BioTek Instruments, Winooski, VT, USA). The analysis was performed at room temperature in phosphate-buffer saline (PBS) (1X, pH 7.4) between 200-550 nm, with a reading step of 2 nm.

[0178] UV−Vis titration experiments were performed for the determination of the stoichiometry of a complex between the ligand and Cu(II), Fe(II), or Fe(III) following the molar ratio method

[0016] . A solution of 9 (final concentration: 40 μM) was co-incubated for 30 min with increasing concentrations of iron (II) chloride, iron (III) chloride, or copper (II) chloride (final concentration: 0-100 μM). The absorption spectra were recorded in PBS. Blank wells were run with PBS and ethanol. The procedure was adapted from the literature [50, 51].

[0179] Assessment of drug-like properties

[0180] The calculation of several molecular descriptors, such as molecular weight (MW), topological polar surface area (tPSA), number of hydrogen bond donors and acceptors (HBD and HBA, respectively), and number of rotatable bond count (RB), was performed using SwissADME (http: / swissadme.ch / index.php (accessed 08 February 2023)).

[0181] Chromatographic Hydrophobicity Index

[0182] Chromatographic hydrophobicity indexes (CHIs) at pH 2.3 were determined using an experimental protocol described elsewhere [26, 52, 53]. A calibration curve was obtained using a mixture of the following reference compounds: theophylline, paracetamol, caffeine, benzimidazole, colchicine, carbamazepine, indole, propiophenone, butyrophenone and valerophenone. The results were expressed as mean values ± standard error mean (SEM) from three independent experiments.

[0183] Evaluation of in vitro blood-brain barrier permeability

[0184] Materials

[0185] The blood-brain barrier (BBB) permeability was estimated using the BBB parallel artificial membrane permeability assay (PAMPA). Reference compounds (verapamil, quinidine, propranolol, lidocaine, progesterone, corticosterone, and theophylline), deep well plates, 96-well PAMPA sandwich plates, BBB-1 lipid solution, PRISMA HT and Brain Sink Buffer (BSB) were purchased from pION Inc. (Billerica, MA, USA). Each 96-well PAMPA sandwich plate was composed of an acceptor microplate, containing a polyvinylidene fluoride (PVDF) filter membrane (pore size 0.45 µm), and a donor plate, with each well containing rims 3 mm above the filter. Polytetrafluoroethylene (PTFE) hydrophilic syringe filters (diameter 13 mm, pore size 0.45 µm) were acquired from ALWSCI (Zhejiang, China). Greiner UV- star 96 well plates were purchase from Sigma Aldrich (St. Louis, MO, USA)

[0186] Parallel artificial membrane permeability assay

[0187] The PAMPA-BBB experiments were conducted according to the manufacturer’s instructions with minor modifications. Briefly, stock solutions of reference and test compounds were prepared in DMSO, filtered, and diluted in PRISMA HT buffer on a deep well plate (final concentrations: reference compounds (50-1250 µM); test compounds (100-500 µM); % DMSO = 5 %). Blank (150 µL of PRISMA HT buffer) and reference plates (150 µL of test solutions in triplicate) were prepared in UV-transparent microplates, and the UV-vis spectra were recorded in a microplate reader (BioTek Epoch 2 microplate spectrophotometer) using the PAMPA Explorer software Version 3.8 (pION Inc). The donor plate was filled with 200 µL of the test solutions. After coating the filter surface of the acceptor plate with 5 µL of BBB-1 lipid formulation, 200 µL of BSB were added. The acceptor filter plate was carefully placed over the donor plate to form a sandwich, which was left undisturbed for 4 h at 25 °C in a humidified chamber. After incubation, 150 µL of each well of the acceptor plate were transferred to a UV-transparent microplate, and the UV-vis spectra were recorded. The same procedure was performed with the donor plate. Based on the UV-vis spectra obtained with blank, reference, donor, and acceptor plates, -log Pe values were calculated at pH 7.4 by the PAMPA Explorer software version 3.8. The results are expressed as mean -log Pe ±SD of three independent experiments. Assay validation was performed by comparing the experimental -log Pe values with the reference values CNS± designations were assigned by comparison with compounds that were identified in previous reports

[0026] : CNS+: - log Pe < 4.6, high permeability (i.e., can enter the CNS); CNS-:- log Pe ≥ 6.8, low permeability (i.e., excluded from the CNS); CNS+ / -, 5.0 ≤ - log Pe < 6.8, uncertain permeability.

[0188] In vitro toxicology

[0189] Cell lines and culture conditions

[0190] Human neuroblastoma SK-N-SH (ATCC® HTB-11™) cells were routinely cultured into 75 cm2 flasks and maintained in MEM supplemented with 10 % heat-inactivated FBS (v / v), 1 % antibiotic-antimycotic solution (v / v). Cells were maintained at 37 °C in a humidified, 5 % CO2−95 % air atmosphere, and the culture medium was changed every 2-3 days. The cultures were passed once a week by trypsinization (0.25 % trypsin / 1 mM EDTA). Human hepatocarcinoma HepG2 cells (ATCC HB-8065) were cultured in MEM (Sigma Chemical Company, St. Louis, MO, USA), containing 10 % FBS (v / v) (Sigma) and 1 % antibiotic-antimycotic solution (Gibco™). Both cell lines were maintained at 37 °C in a humidified atmosphere containing 5 % CO2−95 % air atmosphere and the culture medium was changed every 2-3 days. Every week and after reaching the confluence, the cells were dissociated by trypsinization (0.25 % trypsin / 1 mM EDTA).

[0191] Evaluation of cytotoxicity profile

[0192] Calcein-AM assay was performed as previously described by Pedrosa et al.

[0054] . After a 24 h incubation period with the test compounds, the cell culture medium was removed, and cells were washed twice with HBSS. Then, calcein-AM (2 μM) in HBSS was added, and cells were incubated at room temperature for 30 min. Fluorescence was recorded at the selected time point on a fluorescence microplate reader (Spectramax® Gemini™, Molecular Devices) set to 485 nm excitation and 530 nm emission. To determine minimum staining for calcein-AM, wells were treated with ethanol 15 min before calcein-AM addition.

[0193] Resazurin assay was performed as previously described by

[0053] . After the incubation period with the test compounds (24 h), 10 µM of the resazurin solution (0.1 mg mL-1) was added to each well. Then cells were incubated at 37 °C for 3 h (until intracellular pink resorufin products were visible). The fluorescence of resorufin was measured on the fluorescence microplate reader (Spectramax® Gemini™, Molecular Devices) set to 560 nm excitation and 590 nm emission. The wells without cells (No cells) and with only the medium represent the negative control of the assay.

[0194] Statistical analysis

[0195] Vmax and Km values for S- and MB-COMT were calculated by nonlinear regression analysis using Prism 9 (GraphPad Software, San Diego, CA, USA). IC50 values for COMT inhibition studies were calculated using variable slope (four parameters). Geometric means are given with 95 % confidence intervals and arithmetic means are given with S.E.M.. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test and unpaired t-test. A P value lower than 0.05 was considered significantly different.

[0196] Results

[0197] Kojic acid (KA)-based 9 and 10 were synthesized following the synthetic routes depicted in. The first step included the protection of the hydroxyl group of KA (1) through the reaction with benzyl chloride in methanol and aqueous solution of NaOH 1 M to yield compound 2 (, step A). Then, the 2-hydroxymethyl moiety of compound 2 was oxidized into the corresponding carboxylic acid using the Jones reagent, leading to the obtainment of intermediate 3 (, step B). A subsequent heat-induced decarboxylation of compound 3 in diphenyl ether afforded the C2-unsubstituted intermediate 4 (, step C). This reaction was further optimized using microwave (MW) irradiation and Cu2O as catalyst; the reaction yield was improved from 13 to 58 %. Compound 4 was then reacted with the suitable primary amine, under mild acidic conditions, via Michael addition, affording the N-substituted derivatives 5-6 (, step D). Finally, the deprotection of the hydroxyl group of intermediates 5-6 with boron tribromide (BBr3) solution afforded the compounds 9 and 10 in moderate yields (≥ 30 %) (, step E).

[0198] Hydroxypyridinone derivatives 23-33 were synthesized in a single-step one-pot reaction using 3-hydroxy-4H-pyran-4-one and the primary arylamines as starting materials (a). The synthesis involved a nucleophilic substitution reaction between the α,β-unsaturated function of 3-hydroxy-4H-pyran-4-one (11) and phenylalkylamines 12-22, followed by a Michael-type addition reaction [10, 11]. Camphorsulfonic acid was used as an acidic catalyst. Compounds 23-33 were obtained in low yields after 48 h of heating (1-17 %). The deprotected 3-hydroxyl group of the pyran-4-one underwent side reactions, which resulted in the formation of undesirable by-products and decreased the overall yields.

[0199] Since phenylalkylamines 15-16 and 20-22 () were not obtained commercially, their synthesis was also described inb.

[0200] Catechol O-methyltransferase inhibition studies

[0201] Inhibitory activity in rat liver S- and MB-COMT and in brain MB-COMT

[0202] Compounds 9-10 and 23-33 were tested against rat liver S- and MB-COMT and brain MB-COMT, using a fixed amount of protein (2 mg mL-1), in the presence of saturating concentrations of adrenaline (AD). Tolcapone was used as a reference inhibitor. The IC50 values (mean ± S.E.M.) obtained for liver and brain COMT isoforms are given in Table 1. Results are the mean of three determinations for each point.

[0203] Table 1.COMT inhibition for compounds9-10and23-33and the reference inhibitor tolcapone.IC50(nM)CompoundRat LiverS-COMTRat LiverMB-COMTRat BrainMB-COMTSI (1)bSI (2)c9620±1981±824±325.83.41013968±7491328±28219±1663.86.1232640±516380±49101±4426.13.8249665±3490430±35633±7615.30.725n.d.an.d.an.da____26n.d.an.d.an.d.a____27n.d.an.d.an.d.a____28n.d.an.d.an.d.a____294806±547330±6191±1725.21.7304764±432311±10137±634.82.331n.d.an.d.an.d.a____32n.d.an.d.an.da____33n.d.an.d.an.da____Tolcapone67±420±74±116.85.0

[0204] aCompounds did not reach 50 % inhibition at the highest compound concentration tested (30 µM).

[0205] bSI (1): selectivity index = IC50(rat liver S-COMT) / IC50(rat brain MB-COMT).

[0206] cSI (2): selectivity index = IC50(rat liver MB-COMT) / IC50(rat brain MB-COMT).

[0207] TheN-substituted hydroxypyridinones9and10were able to inhibit all COMT isoforms. In general, MB-COMT isoforms were more potently inhibited than liver S-COMT. Tolcapone,9and10inhibited liver MB-COMT (20 nM, 81 nM and 1328 nM, respectively) with higher potency than liver S-COMT (67 nM, 620 nM and 13968 nM, respectively). Moreover, the IC50values of tolcapone,9and10towards brain MB-COMT (4 nM, 24 nM and 219 nM, respectively) were lower than those towards liver MB-COMT, showing higher inhibition potency towards the former.

[0208] Compound9exhibited lower IC50values towards all COMT isoforms than compound10. These results indicate that, compared to the 4-Cl group, the presence of 3,4-dimethyl substituents at the aromatic ring improves the compound’s inhibitory activity. Although compound9displayed higher IC50values against liver and brain COMT isoforms than tolcapone, compound9presented a higher selectivity towards brain MB-COMT compared to liver S-COMT than tolcapone, (Compound9: SIb= 25.8; Tolcapone: SIb= 16.8).

[0209] With the aim of improving the COMT inhibition properties of compound9,N-substituted hydroxypyridinone componds were synthesized (compounds23-33,). Two chemical modifications were performed on the chemical structure of compound9to attain structural diversity. The first was the incorporation of alkyl linkers with different lengths (ethyl, butyl, hexyl) between the hydroxypyridinone and the benzenic rings. The second was the modification of the substitution pattern of the benzenic ring (phenyl, 4-methylphenyl, or 3,4-dimethylphenyl). Unfortunately, none of the structural modifications resulted in improved COMT inhibition potency. In fact, from all the tested compounds, only compounds23, 24, 29and30displayed significant inhibitory activities towards rat brain and liver COMT isoforms, although the IC50values obtained were significantly higher than those of compound9 (Table 1).

[0210] In summary, compound9stands out as the best non-nitrocatechol COMT inhibitor of the series and was selected for the subsequent studies.

[0211] Kinetic parameters of compound 9

[0212] To elucidate the mechanism of COMT inhibition by compound 9, saturation curves were performed with increasing concentrations of AD or with increasing concentrations of the co-substrate S-adenosyl-L-methionine (SAM) in the absence and the presence of compound 9. Saturation curves for brain MB-COMT, liver MB-COMT and S-COMT are depicted in. Tables 2 and 3 show the corresponding kinetic parameters in the absence and the presence of compound 9.

[0213] Table 2.Kinetic parameters (Kmin µM andVmaxin nmol.mg prot-1.h-1) of brain MB-COMT and liver MB-, S-COMT activity after incubation with increasing concentrations of AD (0.05-50 µM – MB-COMT; 10-2500 µM – S-COMT), and with fixed concentrations of SAM (brain MB-COMT, 100 µM; liver MB-, S-COMT 500 µM), or with fixed concentrations of AD (10 µM for brain MB-COMT and liver MB-COMT, 1000 µM for liver S-COMT) and with increasing concentrations of SAM (5-1000 µM – brain MB-COMT; 1-1000 µM - liver MB-COMT; 1-500 µM - liver S-COMT) in the absence (untreated) or the presence of compound9.Increasing concentrations of ADuntreatedcompound 9compound 9 concentrationBrain MB-COMTVmax4.0 (3.8 to 4.3)3.9 (3.6 to 4.2)30 nMKm1.5 (1.1 to 1.9)5.3 (4.1 to 6.6)Liver MB-COMTVmax41.4 (38.0 to 44.7)33.47 (29.57 to 37.36)150 nMKm2.3 (1.4 to 3.2)15.4 (11.2 to 19.7)Liver S-COMTVmax89.6 (84.1 to 95.3)22.8 (18.4 to 28.7)2 µMKm136.7 (111.4 to 167.2)245.0 (112.2 to 509.2)Increasing concentrations of SAMuntreatedcompound 9compound 9 concentrationBrain MB-COMTVmax3.3 (3.2 to 3.4)2.2 (2.1 to 2.3)30 nMKm4.0 (3.1 to 5.1)1.6 (0.8 to 2.6)Liver MB-COMTVmax34.2 (32.1 to 36.4)10.5 (9.6 to 11.46)150 nMKm6.3 (4.8 to 8.4)1.5 (0.8 to 2.4)Liver S-COMTVmax103.9 (98.0 to 110.2)42.0 (37.5 to 47.1)1 µMKm52.0 (42.9 to 62.9)18.95 (11.6 to 30.1)

[0214] Results are the mean of three determinations per group.

[0215] For untreated samples, the maximum velocity (Vmax,nmol.mg prot-1.h-1) and the Michaelis constant (Km, µM)values for S-COMT were significantly higher MB-COMT (Table 2). Therefore, S-COMT exhibits higher metabolic capacity and lower substrate affinity than MB-COMT. Moreover,Vmaxvalues for brain MB-COMT were lower than those of liver MB-COMT (Table 2). Therefore, the hepatic isoform has a higher metabolic capacity than the brain isoform. Concerning theKmvalues, brain MB-COMT presented lowerKmvalues than liver MB-COMT (Table 2). The lowerKmvalues for brain MB-COMT suggest that the brain isoform has higher affinity for substrates than the hepatic isoform. These results are in line with previous reports

[0012] .

[0216] The incubation of COMT isoforms with increasing concentrations of AD and with compound 9 led to non-significant decreases in Vmax values compared to the untreated groups (Table 2). In contrast, the presence of compound 9 contributed to significant increases in Km values, compared to the untreated group (Table 2). In other words, compound 9 reduced the maximal rate of O-methylation of AD and the COMT affinity for AD. Thus, one can conclude that compound 9 acts as a competitive inhibitor for the real substrate, AD, since the binding of compound 9 to COMT significantly changes the Km values while maintaining Vmax values. Furthermore, compound 9 may bind to the enzyme’s active site and prevent the binding of the catechol substrate.

[0217] Compared to the untreated group, brain MB-COMT activity after incubation with increasing concentrations of SAM in the presence of compound 9 led to decreases in Vmax and Km values (Table 2). These results indicate that compound 9 acts as an uncompetitive inhibitor against the co-substrate, SAM, meaning that compound 9 only interacts with COMT / SAM complex and not with the free enzyme. In other words, compound 9 does not prevent SAM from binding to the enzyme. However, when the inhibitor is bound to the COMT / SAM complex, the enzyme cannot perform its catalytic activity.

[0218] Tight-binding inhibition and Ackermann-Potter analysis

[0219] Nitrocatechol COMT inhibitors bind to the enzyme’s active site in a “tight-binding inhibition” manner. Tight-binding inhibitors are characterized by their high inhibition potency, slow association and dissociation from the enzyme, and their titrating properties (stoichiometric relationship between enzyme and inhibitor)

[0013] . To assess if compound 9 acts as a tight-binding COMT inhibitor, the enzymes’ maximum velocities were measured using different protein concentrations, in the absence or presence of different compound concentrations (Ackermann-Potter plot). Tolcapone was used as a reference tight-binding inhibitor. The results obtained for liver S-COMT, liver MB-COMT, and brain MB-COMT are depicted in.

[0220] The plots of Vmax (pmol mL-1 min-1) against different protein concentrations (liver S-COMT and MB-COMT and brain MB-COMT) at different tolcapone concentrations were characterized by asymptotic concave curves (A, 6C, and 6 E). This behaviour is characteristic of a tight-binding inhibitor. However, using different concentrations of compound 9, the curves of velocity vs protein concentration were linear, and, unlike tolcapone, the inhibition of compound 9 is independent of the enzyme concentration. These results indicate that compound 9 does not act as a tight-binding COMT inhibitor (B, 6 D, and 6 F).

[0221] Evaluation of chelating properties towards transition metals

[0222] The metal chelating capacity of compound 9 was investigated by UV-Vis spectrometry [14, 15]. Full scan spectra of 40 μM solutions of compound 9 in phosphate buffer saline (PBS 1X, pH 7.4), in the absence or presence of 20 µM of iron(II) chloride, iron(III) chloride, or copper(II) chloride, were recorded(A). Compound 9 showed a maximum absorption sharp peak at 294 nm. The co-incubation of compound 9 with iron(II) chloride, iron(III) chloride, and copper(II) chloride resulted in a decrease of the absorbance at 294 nm, suggesting the formation of iron and copper complexes with compound 9. The Mg(II) chelating properties of compound 9 were also evaluated with magnesium(II) chloride. However, only a small shift of the maximum absorbance peak was observed, suggesting that compound 9 was unable to form a stable complex with Mg(II) (data not shown).

[0223] Titration analysis of compound 9 was carried out using the molar ratio method

[0016] with a fixed concentration (40 μM) of the ligand and increasing concentrations of iron(II) chloride (B), iron(III) chloride (C) and copper(II) chloride (D) (0-100 µM). The results showed the formation of complexes with 1.3:1 Fe2+ / ligand, 1.3:1 Fe3+ / ligand, and 1.3:1 Cu2+ / ligand stoichiometry.

[0224] Overall, the data obtained indicate that, in addition to selective brain MB-COMT inhibition, compound 9 may act as an iron and copper chelating agent.

[0225] Evaluation of drug-like properties

[0226] The evaluation of the physicochemical properties of compounds in the early stages of drug development is of utmost importance. They broadly influence the absorption, distribution, metabolism, excretion, and toxicity (ADME-Tox) profiles of compounds, namely their solubility, permeability, and metabolic clearance

[0017] . In the development of CNS drug candidates, drug delivery across the blood-brain barrier (BBB) is one of the main obstacles because this highly selective semipermeable barrier restricts the access of most therapeutic agents to the brain

[0018] . Small lipophilic drugs may cross the BBB by passive diffusion, which is strongly dependent on physicochemical properties such as molecular weight (MW), number of hydrogen bond donors (HBD), number of hydrogen bond acceptors (HBA), polar surface area (tPSA), and number of rotatable bonds (RB)

[0019] .

[0227] In this context, the physicochemical properties of compound 9 and reference inhibitors tolcapone (CNS-permeant COMT inhibitor) and entacapone (peripheral COMT inhibitor) were assessed (Table 3) [20, 21]. Most of the predicted parameters fell within the optimal values for CNS-active drugs (MW < 450 g.mol-1; HBD < 3; HBA < 7; RB< 8)

[0020] . The estimated tPSA value of compound 9 was in accordance with the general drug-likeness requirements of CNS drugs (<60 Å2)

[0022] . The same was not observed for tolcapone and entacapone, which exhibit tPSA values above the recommended limits.

[0228] Table 3.Physicochemical properties and calculated CHI PH 2.3 and CHI LogP values of compound9, deferiprone,and reference COMT inhibitors tolcapone and entacapone.

[0229] Compound 9tolcaponeentacaponeCNS+drugs

[0018] MW[a]215.25273.24305.29< 450RB[a]136< 8HBD[a]122< 3HBA[a]256< 7tPSA (Å2)[a]42.23103.35130.38< 60tR / min8.17610.3389.482__CHI pH 2.3[b]41.0270.1356.40__CHI Log P[c]1.192.922.271.5 – 2.7-Log Pe ± SD[d]4.8 ± 0.405.2 ± 0.446.8 ± 0.42< 5.0CNS+ / -predictionCNS+CNS+ / -CNS-CNS+

[0230] [a] Properties predicted using SwissADME (http: / swissadme.ch / index.php (accessed 08 February 2023)).

[0231] [b] CHI values calculated by the equation: CHI = 15.378tr – 106.01 obtained in linear correlation.

[0232] [c] CHI LogP represents the partition coefficient of a compound between octanol and water and was back-calculated from the CHI values of compounds using the following equation: CHI LogP = 0.047 CHI + 0.36 HBD – 1.10

[0023] .

[0233] [d]; CNS+:- log Pe < 5.0, high permeability (i.e., can enter the CNS); CNS-:- log Pe > 6.8, low permeability (i.e., excluded from the CNS); CNS+ / -, 5.0 < - log Pe < 6.8, uncertain permeability. Data are represented as means ± standard deviation (SD) from three independent experiments (n = 3).

[0234] MW: molecular weight; RB: number of rotatable bonds; HBD: number of hydrogen donors; HBA: number of hydrogen acceptors; tPSA: topological polar surface area.

[0235] The evaluation of a compound’s lipophilicity is crucial in the early stages of drug discovery. Lipophilicity affects the ability of small molecules to interact with physiological membranes and, consequently, their permeability and toxicity

[0024] . The chromatographic hydrophobic index (CHI) is a parameter directly correlated with the compound’s hydrophobicity. The CHI values at pH 2.3 of compound 9, tolcapone, and entacapone were determined from the retention times (tR) obtained by reverse phase HPLC [25-27]. The CHI values were then used to calculate the compounds’ CHI LogP. The results obtained are presented in Table 3. Compound 9 presented lower CHI than tolcapone and entacapone indicating lower hydrophobicity. The CHI LogP value determined for compound 9 was close to the suggested limit of CNS+ drugs (LogP: 1.5-2.7; compound 9: CHI logP = 1.19).

[0236] Altogether, the data obtained suggest that, in general, compound 9 presents suitable physicochemical properties to act as a CNS drug.

[0237] Evaluation of the blood-brain barrier permeability

[0238] To obtain experimental data concerning the BBB permeability of compound 9, tolcapone, and entacapone, the parallel artificial membrane permeation (PAMPA-BBB) assay was performed, following the method previously described by Di et al.

[0028] . In this assay, the in vitro permeability (- log Pe) of compounds through an artificial lipid membrane was determined to predict their ability to cross the BBB by passive diffusion

[0029] . The assay validation was performed by plotting the experimental permeability (log Pe (exp.)) vs the reported values (log Pe (rep.)) for commercial drugs. The good correlation attained (log Pe (exp) = 0.9013 log Pe (rep.) – 1.059, R2 = 0.9690) allowed to establish the ranges of permeability as follows: CNS+:- log Pe < 5.0 for compounds with high permeability (i.e., can enter the CNS); CNS-:- log Pe > 6.8, for compounds with low permeability (i.e., excluded from the CNS); CNS+ / -: 5.0 < - log Pe < 6.8, for compounds with uncertain permeability. The results obtained are displayed in Table 3.

[0239] The data obtained for tolcapone (CNS+ / -) and entacapone (CNS-) were in accordance with the literature [30-32]. Compound 9 exhibited a -log Pe value of 4.8, suggesting that it is predicted to cross the BBB by passive diffusion. Therefore, the results obtained experimentally in the PAMPA-BBB assay for 9 were in line with the conclusions drawn from the estimation of the compound’s physicochemical properties.

[0240] Evaluation of cytotoxicity profile

[0241] The cytotoxicity of tolcapone and compound 9 was assessed in human hepatocarcinoma HepG2 cells ([Fig.8] .A and 8 B) and human neuroblastoma SK-N-SH cells ([Fig.8] C and 8 D). After cell treatment with the test compounds (tolcapone and compound 9) at three concentrations (10, 30, and 50 µM) for 24 h, calcein fluorescence and resorufin fluorescence were evaluated by calcein-AM and resazurin reduction assays, respectively. These assays are a direct measure of cell metabolic capacity or cell membrane integrity (calcein-AM test), or a measure of cell death (resazurin test) [33-34]. The results are presented as mean calcein fluorescence (% of control) ± S.E.M. ([Fig.8] .A and 8 C) or as mean resorufin fluorescence (% of control) ± S.E.M. ([Fig.8] B and 8 D).

[0242] Results showed that tolcapone and compound 9 reduced cell viability in a concentration-dependent manner ([Fig.8]). Cellular viability assays using the hepatocarcinoma HepG2 cells showed that tolcapone induced significant decreases in cell viability at all the concentrations tested: calcein fluorescence values ranged 59 – 85 % ([Fig.8] .A), and resorufin fluorescence ranged between 68 – 83 % in Hep G2 cells ([Fig.8] B). These results are in line with results by other authors, as tolcapone is known for its hepatotoxicity, which could be due to the formation of toxic reactive metabolites and protein adducts, oxidative stress and uncoupling of the mitochondrial respiratory chain

[0035] . In contrast, compound 9 at 10 µM did not show significant decreases in calcein fluorescence (93 %) ([Fig.8] .A) and resorufin fluorescence (102 %) ([Fig.8] B) compared to untreated cells (100 %). Significant decreases in cell viability were only observed when cells were incubated with higher concentrations of compound 9. For instance, compound 9 at 30 µM and 50 µM induced mild, but significant, decreases in calcein fluorescence ([Fig.8] .A, 82 % and 74 %, respectively). The reduction in calcein fluorescence with compound 9 may be facing some decrease in cellular metabolic activity or a lower cell membrane uptake of calcein-AM. However, the resorufin fluorescence in the presence of compound 9 was similar to that in untreated cells at all concentrations ([Fig.8] B), which means that cells are viable though facing some decrease in cellular metabolic activity. Furthermore, when comparing both compounds in the same concentrations, compound 9, in all conditions, induced significantly less cytotoxicity than tolcapone ([Fig.8] .A and 8 B).

[0243] Cellular viability assays were also done in human neuroblastoma SK-N-SH cells ([Fig.8] C and 8 D) as reported previously for tolcapone

[0036] . Using SK-N-SH cells, as in Hep G2 cells, tolcapone induced higher decreases in cell viability than compound 9 at all the concentrations tested: calcein fluorescence values ranged between 32 – 74 % in SK-N-SH cells ([Fig.8] C), and resorufin fluorescence ranged between 37 – 67 % ([Fig.8] D). Additionally, compound 9 only at 30 µM and 50 µM induced significant decreases in calcein fluorescence ([Fig.8] C, 74 % and 53 %, respectively) and resorufin fluorescence ([Fig.8] D, 74 % and 68 %, respectively).

[0244] Overall, the cytotoxic assays suggest that tolcapone is more cytotoxic than compound9in neuroblastoma and hepatocarcinoma cells. Moreover, compound9is, in general, well tolerated at lower concentrations (≤10 µM).

[0245] 1. Balestrino, R. and A. Schapira, Parkinson disease. Eur. J. Neurol., 2020. 27(1): p. 27-42.

[0246] 2. Chen, R. et al., Prevalence, incidence, and mortality of PD. Neurology, 2001. 57(9): p. 1679.

[0247] 3. Choudhury, S. et al., Altered neural cell junctions and ion-channels leading to disrupted neuron communication in Parkinson’s disease. Parkinsons Dis., 2022. 8(1): p. 66.

[0248] 4. Charvin, D. et al., Therapeutic strategies for Parkinson disease: beyond dopaminergic drugs. Nat. Rev. Drug Discov., 2018. 17(11): p. 804-822.

[0249] 5. Lin, K. et al. Iron Brain Menace: The Involvement of Ferroptosis in Parkinson Disease. Cells, 2022. 11(2023): p. 3829.

[0250] 6. Harrison-Jones, G. et al., Opicapone versus entacapone: Head-to-head retrospective data-based comparison of healthcare resource utilization in people with Parkinson's disease new to catechol-O-methyltransferase (COMT) inhibitor treatment. Eur. J. Neurol., 2023. 30(10): p. 3132-3141.

[0251] 7. Harrison, S. et al., Synthesis and Evaluation of Heterocyclic Catechol Mimics as Inhibitors of Catechol-O-methyltransferase (COMT). ACS Med. Chem. Lett., 2015. 6(3): p. 318-323.

[0252] 8. Silva, T. et al., Liver says no: the ongoing search for safe catechol O-methyltransferase inhibitors to replace tolcapone. Drug Discov. Today, 2020. 25(10): p. 1846-1854.

[0253] 9. Sheng, R. et al., Novel 1-Phenyl-3-hydroxy-4-pyridinone Derivatives as Multifunctional Agents for the Therapy of Alzheimer’s Disease. ACS Chem. Neurosci., 2016. 7(1): p. 69-81.

[0254] 10. Petrović Peroković, V. et al., Synthesis of 3-Hydroxy-1-(p-methoxyphenyl)-2-methylpyridine-4-one and Spectrophotometric Extraction Studies on its Complexation of Vanadium(V). Croatica Chem. Acta, 2014. 87(2): p. 103-109.

[0255] 11. Zhang, Z. et al., Physical and structural studies of N-carboxymethyl- and N-(p-methoxyphenyl)-3-hydroxy-2-methyl-4-pyridinone. Canadian J. Chem., 1992. 70(3): p. 763-770.

[0256] 12. Bonifácio, M. et al., Catechol-O-methyltransferase and its inhibitors in Parkinson's disease. CNS Drug Rev., 2007. 13(3), p. 352–379.

[0257] 13. Cha, S., Tight-binding inhibitors—I: Kinetic behavior. Biochem. Pharmacol., 1975. 24(23), p. 2177-2185.

[0258] 14. Sharma, A. et al., Bifunctional Compounds for Controlling Metal-Mediated Aggregation of the Aβ42 Peptide. J. Am. Chem. Soc., 2012. 134(15): p. 6625-6636.

[0259] 15. Geng, J. et al., Liberation of Copper from Amyloid Plaques: Making a Risk Factor Useful for Alzheimer’s Disease Treatment. J. Med. Chem., 2012. 55(21): p. 9146-9155.

[0260] 16. Marcus, Y., On the Use of the Molar Ratio Method for Determining Association Stoichiometry. Israel J. Chem., 1967. 5(4): p. 143-149.

[0261] 17. Leeson, P. et al., Target-Based Evaluation of “Drug-Like” Properties and Ligand Efficiencies. J. Med. Chem., 2021. 64(11): p. 7210-7230.

[0262] 18. Cornelissen, F. et al., Explaining Blood–Brain Barrier Permeability of Small Molecules by Integrated Analysis of Different Transport Mechanisms. J. Med. Chem., 2023.

[0263] 19. Gosselet, F. et al., Central nervous system delivery of molecules across the blood-brain barrier. Neurochem. Int., 2021. 144: p. 104952.

[0264] 20. Pajouhesh, H. et al., Medicinal Chemical Properties of Successful Central Nervous System Drugs. NeuroRx, 2005. 2(4): p. 541-553.

[0265] 21. Waring, M.J., Defining optimum lipophilicity and molecular weight ranges for drug candidates—Molecular weight dependent lower logD limits based on permeability. Bioorg. Med. Chem. Lett., 2009. 19(10): p. 2844-2851.

[0266] 22. Clark, D., Rapid calculation of polar molecular surface area and its application to the prediction of transport phenomena. 2. Prediction of blood-brain barrier penetration. J. Pharmaceut. Sci., 88(8): p. 815-821.

[0267] 23. Valko, K. et al., Rapid method for the estimation of octanol / water partition coefficient (log P(oct)) from gradient RP-HPLC retention and a hydrogen bond acidity term (zetaalpha(2)(H)). Curr. Med. Chem., 2001. 8(9): p. 1137-46.

[0268] 24. Arnott, J. et al., The influence of lipophilicity in drug discovery and design. Expert Opin. Drug Discov., 2012. 7(10): p. 863-75.

[0269] 25. Valkó, K. et al., Chromatographic Hydrophobicity Index by Fast-Gradient RP-HPLC:  A High-Throughput Alternative to log P / log D. Anal. Chem., 1997. 69(11): p. 2022-9.

[0270] 26. Camurri, G. et al., High-Throughput Liquid Chromatography / Mass Spectrometry Method for the Determination of the Chromatographic Hydrophobicity Index. Anal. Chem., 2001. 73(15): p. 3716-3722.

[0271] 27. Valko, K. et al., Rapid-gradient HPLC method for measuring drug interactions with immobilized artificial membrane: comparison with other lipophilicity measures. J. Pharm. Sci., 2000. 89(8): p. 1085-96.

[0272] 28. Di, L. et al., High throughput artificial membrane permeability assay for blood-brain barrier. Eur. J. Med. Chem., 2003. 38(3): p. 223-232.

[0273] 29. Di, L. et al., High throughput artificial membrane permeability assay for blood–brain barrier. Eur. J. Med. Chem., 2003. 38(3): p. 223-232.

[0274] 30. Lerner, C. et al., Design of Potent and Druglike Nonphenolic Inhibitors for Catechol O-Methyltransferase Derived from a Fragment Screening Approach Targeting the S-Adenosyl-l-methionine Pocket. J. Med. Chem., 2016. 59(22): p. 10163-10175.

[0275] 31. Chavarria, D. et al., Boosting caffeic acid performance as antioxidant and monoamine oxidase B / catechol-O-methyltransferase inhibitor. Eur. J. Med. Chem., 2022. 243: p. 114740.

[0276] 32. Hider, R. et al., The potential application of iron chelators for the treatment of neurodegenerative diseases. Metallomics, 2011. 3(3): p. 239-249.

[0277] 33. Uggeri, J et al., Calcein-AM is a detector of intracellular oxidative activity. Histochem. Cell Biol., 2000.122(5), p. 499–505.

[0278] 34. Riss, T. et al., Cell Viability Assays Assay Guidance Manual. Assay Guid. Man., 2004, p. 1–23.

[0279] 35. Pinto, M et al., Cellular and Mitochondrial Toxicity of Tolcapone, Entacapone, and New Nitrocatechol Derivatives. ACS Pharmacol & Transsl. Sci., 2024. 7(5), p. 1637–1649.

[0280] 36. Pinheiro, S. et al., Pharmacodynamic evaluation of novel Catechol-O-methyltransferase inhibitors. Eur. J. Pharmacol., 2019. 847, p. 53–60.

[0281] 37. Rai, B. et al., Synthesis, Physicochemical Properties, and Evaluation of N-Substituted-2-alkyl-3-hydroxy-4(1H)-pyridinones. J. Med. Chem., 1998. 41(18): p. 3347-3359.

[0282] 38. Ellis, B. et al., Synthesis, physicochemical properties, and biological evaluation of hydroxypyranones and hydroxypyridinones: novel bidentate ligands for cell-labeling. J. Med. Chem., 1996. 39 19: p. 3659-70.

[0283] 39. Harding, K et al., Selective oxidation of allylic alcohols with chromic acid. J. Org. Chem., 1975. 40(11): p. 1664-1665.

[0284] 40. Zhao, Z. et al., Synthesis and optimization of N-heterocyclic pyridinones as catechol-O-methyltransferase (COMT) inhibitors. Bioorg. Med. Chem. Lett., 2016. 26(12): p. 2952-2956.

[0285] 41. Goossen, L. et al., Microwave-Assisted Cu-Catalyzed Protodecarboxylation of Aromatic Carboxylic Acids. J. Org. Chem., 2009. 74(6): p. 2620-2623.

[0286] 42. Silva, A.M.G., et al., Microwave-assisted synthesis of 3-hydroxy-4-pyridinone / naphthalene conjugates. Structural characterization and selection of a fluorescent ion sensor. Tetrahedron, 2010. 66(44): p. 8544-8550.

[0287] 43. Agrawal, A. et al., Chelator fragment libraries for targeting metalloproteinases. Chem. Med. Chem., 2010. 5(2): p. 195-199.

[0288] 44. Bi, X. et al., POCl3 promoted metal-free synthesis of tertiary amides by coupling of carboxylic acids and N,N-disubstituted formamides. Phosphorus, Sulfur, and Silicon and the Related Elements, 2019. 194(3): p. 236-240.

[0289] 45. Teixeira, J. et al., Development of a Mitochondriotropic Antioxidant Based on Caffeic Acid: Proof of Concept on Cellular and Mitochondrial Oxidative Stress Models. J. Med. Chem., 2017. 60(16): p. 7084-7098.

[0290] 46. Nissinen, E., et al., Catechol-O-methyltransferase activity in human and rat small intestine. Life Sci., 1988. 42(25): p. 2609-2614.

[0291] 47. Vieira-Coelho, M. et al., Effects of tolcapone upon soluble and membrane-bound brain and liver catechol-O-methyltransferase. Brain Res., 1999. 821(1): p. 69-78.

[0292] 48. Borges, N. et al., Studies on the tight-binding nature of tolcapone inhibition of soluble and membrane-bound rat brain catechol-O-methyltransferase. J. Pharmacol. Exp. Ther., 1997. 282(2): p. 812-7.

[0293] 49. Cha, S., Tight-binding inhibitors—I: Kinetic behavior. Biochem. Pharmacol., 1975. 24(23): p. 2177-2185.

[0294] 50. Xie, S. et al., Synthesis and evaluation of selegiline derivatives as monoamine oxidase inhibitor, antioxidant and metal chelator against Alzheimer's disease. Bioorg. Med. Chem., 2015. 23(13): p. 3722-9.

[0295] 51. Silva, T., et al., Repurposing nitrocatechols: 5-Nitro-α-cyanocarboxamide derivatives of caffeic acid and caffeic acid phenethyl ester effectively inhibit aggregation of tau-derived hexapeptide AcPHF6. Eur. J. Med. Chem., 2019. 167: p. 146-152.

[0296] 52. Valko, K., et al., Fast gradient HPLC method to determine compounds binding to human serum albumin. Relationships with octanol / water and immobilized artificial membrane lipophilicity. J. Pharm. Sci., 2003. 92(11): p. 2236-48.

[0297] 53. Benfeito, S., et al., Modulating Cytotoxicity with Lego-like Chemistry: Upgrading Mitochondriotropic Antioxidants with Prototypical Cationic Carrier Bricks. J. Med. Chem., 2023. 66(3): p. 1835-1851.

[0298] 54. Pedrosa, R. and P. Soares-da-Silva, Oxidative and non-oxidative mechanisms of neuronal cell death and apoptosis by L-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine. British J. Pharmacol., 2002. 137(8): p. 1305-1313.

[0299] 55. Borges, N., Vieira-Coelho, M. A., Parada, A., & Soares-da-Silva, P., Studies on the tight-binding nature of tolcapone inhibition of soluble and membrane-bound rat brain catechol-O-methyltransferase. J. Pharmacol. Exp.Ther., 1997. 282(2), p. 812–817.

[0300] This description is of course not in any way restricted to the forms of implementation presented herein and any person with an average knowledge of the area can provide many possibilities for modification thereof without departing from the general idea as defined by the claims. The preferred forms of implementation described above can obviously be combined with each other. The following claims further define the preferred forms of implementation.

Claims

A N-substituted hydroxypyridinone compound of Formula (I), or a pharmaceutically acceptable salt thereof, having the structure:Formula (I)Wherein X is selected from:wherein R1=R2=CH3, or R1=R2=H, or R1=CH3 and R2=H, or R1=Cl and R2=H; orwherein n is selected from 1, 2, 3, and R3 is independently selected from H or CH3, and R4 is independently selected from H or CH3.A compound according to the previous claim, wherein the compound is selected from the list comprising: 1-(3,4-dimethylphenyl)-3-hydroxypyridin-4(1H)-one (compound 9); 1-(4-chlorophenyl)-3-hydroxypyridin-4(1H)-one (compound 10); 3-hydroxy-2-methyl-1-phenylpyridin-4(1H)-one (compound 23); 3-hydroxy-2-methyl-1-(p-tolyl)pyridin-4(1H)-one (compound 24); 3-hydroxy-2-methyl-1-phenethylpyridin-4(1H)-one (compound 25); 3-hydroxy-2-methyl-1-(4-methylphenethyl)pyridin-4(1H)-one (compound 26); 1-(3,4-dimethylphenethyl)-3-hydroxy-2-methylpyridin-4(1H)-one (compound 27); 3-hydroxy-2-methyl-1-(4-phenylbutyl)pyridin-4(1H)-one (compound 28); 3-hydroxy-2-methyl-1-(4-(p-tolyl)butyl)pyridin-4(1H)-one (compound 29); 1-(4-(3,4-dimethylphenyl)butyl)-3-hydroxy-2-methylpyridin-4(1H)-one (compound 30); 3-hydroxy-2-methyl-1-(6-phenylhexyl)pyridin-4(1H)-one (compound 31); 3-hydroxy-2-methyl-1-(6-(p-tolyl)hexyl)pyridin-4(1H)-one (compound 32); 1-(6-(3,4-dimethylphenyl)hexyl)-3-hydroxy-2-methylpyridin-4(1H)-one (compound 33).A compound according to any of the previous claims for use in the treatment of a neurological or a neuropsychiatric disease or disorder associated with dopamine deficiency.A compound according to the previous claim, wherein the neurological or neuropsychiatric disease or disorder is selected from, Parkinson’s Disease, schizophrenia, depression, attention deficit hyperactivity disorder, substance dependency such as opiate, tobacco addiction etc., dementia, Parkinson's disease, Huntington's disease, Pick's disease, Creutzfeldt Jakob disease, perinatal hypoxia, cognitive disorder, or cognitive deficit associated with any of said disorder or disease.A pharmaceutical composition comprising at least one compound of Formula (I), or a pharmaceutically acceptable salt thereof, described in any of claims 1 to 2.Pharmaceutical composition according to the previous claim, for the use in the treatment of a neurological or a neuropsychiatric disease or disorder associated with dopamine deficiency.Pharmaceutical composition according to the previous claim, wherein the neurological or neuropsychiatric disease or disorder is selected from, Parkinson’s Disease, schizophrenia, depression, attention deficit hyperactivity disorder, substance dependency such as opiate, tobacco addiction etc., dementia, Parkinson's disease, Huntington's disease, Pick's disease, Creutzfeldt Jakob disease, perinatal hypoxia, cognitive disorder, or cognitive deficit associated with any of said disorder or disease.Method of obtaining a N-substituted hydroxypyridinone compound of Formula (I) selected from compounds 9 or 10 described in claim 2, comprising the steps of:protection of the hydroxyl group of Kojic acid in a concentration between 1 and 3 M, through reacting benzyl chloride in a concentration between 1 and 5 mol in methanol in a concentration between 1 and 10 mL / mmol, with an aqueous solution of NaOH in a concentration between 1 and 2 M, in reflux between 6 and 10 hours to yield 5-(benzyloxy)-2-(hydroxymethyl)-4H-pyran-4-one;the 2-hydroxymethyl moiety of 5-(benzyloxy)-2-(hydroxymethyl)-4H-pyran-4-one in a concentration between 1 and 4 M is oxidized into the corresponding carboxylic acid using the Jones reagent at a concentration between 1 and 2.5 M and acetone in a concentration between 1 and 10 mL / mmol, at a temperature between 0ºC and 25ºC, overnight, yielding 5-(benzyloxy)-4-oxo-4H-pyran-2-carboxylic acid;heat-induced decarboxylation of 5-(benzyloxy)-4-oxo-4H-pyran-2-carboxylic acid in a concentration between 1 and 3 M in 1-methyl-2-pyrrolidinone in a concentration between 0.5 and 5 mL / mmol, using Cu2O in a concentration between 0.05 and 1 mol as catalyst, 1,10-phenanthroline as a copper ligand in a concentration between 0.1 and 2 mol, and microwave irradiation at a temperature between 150 ºC and 250 ºC, with reaction time between 15 minutes and 24 hours, yielding C2-unsubstituted intermediate 3-(benzyloxy)-4H-pyran-4-one;3-(benzyloxy)-4H-pyran-4-one in a concentration between 1 and 5 M was reacted with 3,4-dimethylaniline or with 4-chloroaniline in a concentration between 1 and 10 mol via Michael addition, under mild acidic conditions provided by EtOH:HCl at a concentration between 0.3 and 3 M with ratio1:1 V / V, at a temperature between 80ºC and 150 ºC, with a reaction time between for 1 hour and 48 hours, affording the N-substituted derivatives 3-(benzyloxy)-1-(3,4-dimethylphenyl)pyridin-4(1H)-one or 3-(benzyloxy)-1-(4-chlorophenyl)pyridin-4(1H)-one;deprotection of the hydroxyl group of 3-(benzyloxy)-1-(3,4-dimethylphenyl)pyridin-4(1H)-one or 3-(benzyloxy)-1-(4-chlorophenyl)pyridin-4(1H)-one in a concentration between 1 and 4 M with a boron tribromide solution in dichloromethane in a concentration between 1 and 10 mol and dichloromethane in a concentration between 1 and 50 mL / mmol, at a temperature between -80ºC and 0ºC, with a reaction time between 1 hour and 48 hours, yielding 1-(3,4-dimethylphenyl)-3-hydroxypyridin-4(1H)-one or 1-(4-chlorophenyl)-3-hydroxypyridin-4(1H)-one, respectively.Method of obtaining a N-substituted hydroxypyridinone compound of Formula (I) selected from compounds 23 to 33 described in claim 2, comprising the steps of:Synthesis of (E)-1,2-dimethyl-4-(2-nitrovinyl)benzene through the reaction of 3,4-dimethylbenzaldehyde in a concentration between 1 and 3 M, with nitromethane in a concentration between 1 and 10 mol dissolved in acetic acid in a concentration between 1 and 10 mol, in the presence of ammonium acetate in a concentration between 1 and 10 mol, at a temperature between 10 ºC and 50 ºC, for a reaction time between 24 hours and 72 hours;(E)-1,2-dimethyl-4-(2-nitrovinyl)benzene in a concentration between 1 and 3 M was then reduced into 2-(3,4-dimethylphenyl)ethan-1-amine with lithium aluminium hydride in a concentration between 1 and 10 mol in anhydrous tetrahydrofuran in a concentration between 1 and 10 mL / mmol, under argon atmosphere and reflux, with a reaction time between 24 hours and 72 hours;Friedel-Crafts acylation between o-xylene or toluene in a concentration between 1 and 3 M and 4-bromobutyl chloride or 6-bromohexanoyl chloride in a concentration between 1 and 10 M in anhydrous dichloromethane in a concentration between 0.5 and 5 mL / mmol, in the presence of aluminium trichloride in a concentration between 1 and 10 mol, under argon atmosphere, at a temperature between -50ºC and 0ºC, with a reaction time between 1 hour and 48 hours, to afford 4-bromo-1-(3,4-dimethylphenyl)butan-1-one, or 6-bromo-1-(p-tolyl)hexan-1-one, or 6-bromo-1-(3,4-dimethylphenyl)hexan-1-one;Reduction of bromophenylalcan-1-ones 4-bromo-1-(3,4-dimethylphenyl)butan-1-one, or 6-bromo-1-(p-tolyl)hexan-1-one, or 6-bromo-1-(3,4-dimethylphenyl)hexan-1-one in a concentration between 1 and 3 M into the corresponding bromophenylalcanes with triethylsilane in a concentration between 1 and 10 mol in anhydrous dichloromethane in a concentration between 0.5 and 5 mL / mmol, under acidic conditions provided by trifluoroacetic acid in a concentration between 10 and 100 mol, under argon atmosphere, at a temperature between 0ºC and 25ºC, overnight, the resulting intermediates were then reacted with phthalimide potassium salt in a concentration between 1 and 10 mol in DMF in a concentration between 1.0 and 10 mL / mmol, under argon atmosphere and reflux, with a reaction time between 1 hour and 24 hours, yielding 2-(4-(3,4-dimethylphenyl)butyl)isoindoline-1,3-dione, or 2-(6-(p-tolyl)hexyl)isoindoline-1,3-dione, or 2-(6-(3,4-dimethylphenyl)hexyl)isoindoline-1,3-dione;1-Bromo-6-phenylhexane in a concentration between 1 and 3 M was reacted with phthalimide potassium salt in a concentration between 1 and 10 mol in DMF in a concentration between 1 and 10 mL / mmol, under argon atmosphere and reflux, with a reaction time between 1 hour and 24 hours, yielding 2-(6-phenylhexyl)isoindoline-1,3-dione;The phthalimide group of phenylalkylisoindoline-1,3-diones 2-(4-(3,4-dimethylphenyl)butyl)isoindoline-1,3-dione, or 2-(6-phenylhexyl)isoindoline-1,3-dione, or 2-(6-(p-tolyl)hexyl)isoindoline-1,3-dione, or 2-(6-(3,4-dimethylphenyl)hexyl)isoindoline-1,3-dione in a concentration between 1 and 3 M was cleaved with butylamine in a concentration between 5 and 100 mol in ethanol in a concentration between 0.5 and 5 mL / mmol, under reflux, with a reaction time between 6 hours and 72 hours, affording 4-(3,4-dimethylphenyl)butan-1-amine, or 6-phenylhexan-1-amine, or 6-(p-tolyl)hexan-1-amine, or 6-(3,4-dimethylphenyl)hexan-1-amine;Single-step one-pot reaction between 3-hydroxy-4H-pyran-4-one in a concentration between 1 and 3 M and aniline, or p-toluidine, or 2-phenylethan-1-amine, or 2-(p-tolyl)ethan-1-amine, or 2-(3,4-dimethylphenyl)ethan-1-amine, or 4-phenylbutan-1-amine, or 4-(p-tolyl)butan-1-amine, or 4-(3,4-dimethylphenyl)butan-1-amine, or 6-phenylhexan-1-amine, or (p-tolyl)hexan-1-amine, or 6-(3,4-dimethylphenyl)hexan-1-amine in a concentration between 1 and 7 M in water in a concentration between 2 and 20 mL / mmol, under acidic conditions provided by camphor sulfonic acid in a concentration between 0.1 and 2 mol, under reflux, with a reaction time between 24 and 72 hours, yielded 3-hydroxy-2-methyl-1-phenylpyridin-4(1H)-one, or 3-hydroxy-2-methyl-1-(p-tolyl)pyridin-4(1H)-one, or 3-hydroxy-2-methyl-1-phenethylpyridin-4(1H)-one, or 3-hydroxy-2-methyl-1-(4-methylphenethyl)pyridin-4(1H)-one, or 1-(3,4-dimethylphenethyl)-3-hydroxy-2-methylpyridin-4(1H)-one, or 3-hydroxy-2-methyl-1-(4-phenylbutyl)pyridin-4(1H)-one, or 3-hydroxy-2-methyl-1-(4-(p-tolyl)butyl)pyridin-4(1H)-one, or 1-(4-(3,4-dimethylphenyl)butyl)-3-hydroxy-2-methylpyridin-4(1H)-one, or 3-hydroxy-2-methyl-1-(6-phenylhexyl)pyridin-4(1H)-one, or 3-hydroxy-2-methyl-1-(6-(p-tolyl)hexyl)pyridin-4(1H)-one, or 1-(6-(3,4-dimethylphenyl)hexyl)-3-hydroxy-2-methylpyridin-4(1H)-one, respectively.

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