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(54) Title: COMPOUND 2-PHENYL-1- (3,4-DIHYDROXY-5-NITROPHENYL) -1-ETHANONE, PHARMACEUTICAL COMPOSITION, AND, USE OF A COMPOUND.

(30) Unionist Priority: 12/18/19 GB9827996.1 (73) Holder (s): Portela & CA, SA

(72) Inventor (s): David Alexander Learmonth, Jan Benes, Patrício Manuel Vieira Araújo Soares da Silva

COMPOUND 2-PHENYL-1 - (3,4-DIHYDROXY-5 -NITROPHENYL) -1-ETHANONE, PHARMACEUTICAL COMPOSITION, AND, USE OF A COMPOUND Description

The most effective symptomatic treatment of Parkinson's disease involves the administration of LP-3,4-dihydroxyphenylalanine (L-DOPA) the immediate precursor to dopamine. Orally administered, L-DOPA is predominantly metabolized on the periphery by the aromatic L-amino acid decarboxylase (AADC) in dopamine, which can cause serious adverse effects such as emesis, orthostatic hypotension and cardiac arrhythmia. Therefore, L-DOPA is usually administered in combination with a peripheral AADC inhibitor (benserazide or carbidopa). When administered with such inhibitors, very little dopamine is formed on the periphery, but only a small amount of an oral dose of LDOPA reaches the brain because a considerable amount of the drug undergoes methylation to 3-O-methyl-L-DOPA (Mánnistõ, PA , et al., Progress Drug Research, 39: 291 to 350, 1992). The duration of clinical improvement induced by L-DOPA is brief, as a result of the short half-life of L-DOPA which contrasts with the long half-life of 3-O-methyl-L-DOPA. Within a few years of starting L-DOPA therapy with the usual 2 to 4 doses per day, the clinical improvement induced by L-DOPA decreases at the end of each dose cycle, producing the “end of dose” pattern or “Gradual weakening” of motor fluctuations. An intimate relationship has been described between the accumulation of 3-O-methyl-L-DOPA and the development and development of the “gradual weakening” phenomenon (Tohgi, H., et al., Neurosci. Letters, 132: 19 to 22 , 1992). It was predicted that this could result from the inhibition of L-DOPA transport at the blood-brain barrier level by its methylated O2 metabolite (Reches, A. et al., Neurology, 32: 887 and 888, 1982) or simply because there is less L-DOPA available to reach the brain (Nutt, JG, Fellman, JH, Clin. Neuropharmacol., 7: 35 to 49, 1984).

In recent years, the development of new inhibitors of the enzyme catechol-O-methyl transferase (COMT) has been accelerated by the hypothesis that inhibition of this enzyme may provide significant clinical improvements in patients afflicted with Parkinson's disease undergoing treatment with L-DOPA plus a peripheral AADC inhibitor. The rationale for the use of COMT inhibitors is based on its ability to inhibit L-DOPA O-methylation to 3-O-methyl-L-DOPA. Inhibition of COMT decreases the elimination of L-DOPA from the plasma by increasing the plasma half-life (increases the area under the [AUC] curve without changing the LDOPA time in the plasma to reach the peak or maximum concentration). In this way, pharmacokinetic changes can be an advantage over increasing the dose of L-DOPA, which also increases AUC, but additionally raises peak concentrations. In turn, high peak concentrations are related to adverse effects, such as dyskinesia, which occurs immediately when COMT inhibitors are given, but can be prevented by reducing the dose of L-DOPA or by increasing interval times. between doses. The effects of COMT inhibition also differ from those of the controlled-release formulation of L-DOPA which decreases absorption and reduces bioavailability. The pharmacokinetic changes induced by COMT inhibition reduce the daily dose of LDOPA allowing a reduction in each dose or an increase in dose ranges. With repeated doses of L-DOPA every 2-6 hours, in the presence of COMT inhibition, the average concentration of L-DOPA in the plasma is increased and the total concentrations are proportionally increased more than the peak concentrations, despite a reduction in the dose of LDOPA. As could be assumed by the decreased elimination of L-DOPA, the duration of antiparchinsonian action with single doses of L-DOPA is prolonged by COMT inhibition (Nutt, JG, Lancet, 351: 1221 and 1222, 1998).

The most potent and selective COMT inhibitors found so far are very active and do not interact with other enzymes, receptors, ion channels or transporters until very high doses. Some of them have been shown to have beneficial effects both in experimental models of parchinsonism and in patients with Parkinson's disease. Other therapeutic applications of these COMT inhibitors have also been proposed, namely, in the treatment of depression or anxiety, as gastroprotective drugs and as natriuretic and antihypertensive agents.

The most potent COMT inhibitors reported to date, 3,4-dihydroxy-4'-methyl-5-nitrobenzophenone (tolcapone, Australian Patent AU-B-69764/87) and (E) -2-cyano-N, N-diethyl-3- (3,4-dihydroxy-5-nitrophenyl) acrylamide (entacapone, German Patent DE 3740383 Al) have inhibition constants in the low nM range. Tolcapone differs from entacapone in that it is a more potent inhibitor of COMT in the periphery and also in penetrating the brain to inhibit cerebral COMT. It has not been established which of these two inhibitors is most useful in the treatment of Parkinson's disease. Compounds that penetrate the blood-brain barrier can be considered to be more effective, as theoretically they could have additional benefits of decreasing methylation of dopamine to 3 methoxytyramine and to homovanilic acid. Conversely, central inhibition may not be important if the most significant action is to protect L-DOPA from chemical decomposition in the periphery. The distinction may be of practical importance, since the use of COMT inhibitors that are excluded from the brain can avoid the potential unwanted side effects of these agents' CNS.

In this regard, it is of interest to highlight the lack of the anti-parchinsonian action of tolcapone when administered alone (Hauser, RA, et al., Mov. Disord, 1998, 13, 643 to 647) and the relatively frequent observations of increased central dopaminergic stimulus, primarily dyskinesia and confusion, in patients taking L-DOPA associated with tolcapone (Nutt, JG, Lancet, 351: 1221 and 1222, 1998). This suggests that the central effects of COMT inhibition are very small when given alone, but when given with L-DOPA the risk of COMT inhibition of the brain may be associated with the appearance of symptoms related to the increased dopaminergic stimulus that may require interruption of therapy.

Another potential problem with COMT inhibitors concerns its relatively short half-life (tolcapone, 2 hours [Dingemanse, J., et al., Clin. Pharmacol. Ther., 57: 508 to 517, 1997]; entacapone, 0.3 hour [Keranen, T., et al., Eur. J. Clin. Pharmacol; 46: 151 to 157, 1994]). To avoid this problem, it is recommended that both tolcapone and entacapone be administered as often as 3 times a day; because the half-life of entacapone is considerably shorter than that of tolcapone, the recommended dose for entacapone is twice that of tolcapone.

As previously mentioned, the 3,4-dihydroxy-5nitrophenyl group was identified as an active pharmacophor and it was simultaneously found that the presence of a carbonyl group (for example, in tolcapone) or an enone group (for example, in entacapone) conjugated to the pharmacophor of the molecule, in general it intensifies the inhibition of the catalyzed transfer by COMT of the methyl group of the coenzyme S-adenosyl-L-methionine to a substrate containing a catechol functional group. Among many tested compounds that carry a 3,4-dihydroxy-5-nitrobenzoyl group, the corresponding benzophenones have been recognized as the most potent CONT inhibitors with ED₅₀ <1 mg / kg (rat, po) (Borgulya, J., et al.,

Helvetica Chimica Acta 72, 952 to 968, 1989).

The formation of homologues of biologically active compounds known as potentially improved drugs is a well-known principle and is mainly used for the optimization of the activity of structurally non-specific drugs or to obtain changes in the predominant biological action in structurally specific drugs (Korolkovas A., Essentials of Medicinal Chemistry, p. 76, 1988 by J. Wiley & Sons, Inc.). On the other hand, homologation is not generally used nor is it expected to influence a compound's half-life in a predicted manner.

The Applicant surprisingly proved that the next higher homologue of 3,4-dihydroxy-5-nitrobenzophenone, that is, the compound with an additional methylene group between the substituted benzoyl group and the phenyl group is endowed with selective long-term COMT inhibition and that this effect is unique in a series of superior counterparts.

The invention relates to 2-phenyl-1- (3,4-dihydroxy-5-nitrophenyl) 1-ethanones of formula I:

<img file="BR9908084B1_D0001.tif" />

where Ri and R₂ are hydrogen or hydrolyzable groups under physiological conditions, the same or different and significantly optionally substituted lower alkanoyl or aroyl, optionally substituted lower alkyl or arylsulfonyl or optionally substituted lower alkylcarbamoyl or when together significantly lower alkylidene or cycloalkylidene group; R₃, R4 and R₅ are the same or different and are significantly hydrogen, optionally substituted saturated or partially unsaturated lower hydrocarbon residue, hydroxyl, optionally substituted lower alkoxy or aryloxy group, optionally substituted aryl, optionally substituted alkanoyl group or aroyl group, lower alkanoylamino group, dialkanoylamino group optionally substituted lower carboxyl, lower alkyloxycarbonyl or aryloxycarbonyl group, optionally substituted carbamoyl, halogen, nitro, amino, lower alkylamino or lower dialkylamino or cyano group or when together they are significantly aliphatic or heteroaliphatic rings or aromatic or heteroaromatic rings and their pharmaceutically acceptable salts; for the use of compounds for the prevention or treatment of certain pathological conditions in humans and for the preparation of pharmaceutical compositions containing them.

The term "lower" denotes residues with a maximum of 8, preferably a maximum of 4 carbon atoms. The term "alkyl" when alone or in combination with terms such as "alkanoyl, alkoxycarbonyl, alkylidene, cycloalkylidene, alkoxycarbonyloxy, alkylamino" denotes straight or branched saturated hydrocarbon residues. The term halogen denotes fluorine, chlorine, bromine and iodine. The term "aryl" denotes an aromatic carbocyclic group, preferably mono- or bicyclic groups.

For the preparation of pharmaceutical compositions of the compounds of formula I, inert pharmaceutically acceptable carriers are mixed with the active compounds. Pharmaceutically acceptable vehicles can be solid or liquid. Solid form preparations include powders, tablets, dispersible granules and capsules. A solid vehicle can be one or more substances that can also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders or tablet disintegrating agents; they can also be an encapsulating material.

Preferably, the pharmaceutical preparation is in a single dosage form, for example, packaged preparations, the package containing different amounts of preparation such as tablets, capsules and powders packaged in bottles or ampoules.

The dosage can be varied depending on the requirements of the patient, the severity of the disease and the particular compound that is used. For convenience, the total daily dosage can be divided and administered in portions throughout the day. Determining the appropriate dosage for a particular situation is within the skill of those in the medical art.

Reference is now made to the attached drawings where:

Figure 1 is a graph showing COMT brain activity at different times after oral administration of compound B (full squares), entacapone (empty circles) or tolcapone (empty squares).

Figure 2 is a graph showing COMT activity in the liver at different times after oral administration of compound B (full squares), entacapone (empty circles) or tolcapone (empty squares).

Figure 3 is a graph showing the concentration-dependent inhibition of COMT brain activity one hour after oral administration of compound B (filled squares), entacapone (empty circles) or tolcapone (empty squares).

Figure 4 is a graph showing the concentration-dependent inhibition of COMT activity in the liver one hour after oral administration of compound B (filled squares), entacapone (empty circles) or tolcapone (empty squares).

Figure 5 is a graph showing the horizontal activity induced by amphetamine, dependent on concentration, after administration of vehicle (empty columns), tolcapone (full columns), entacapone (shaded columns) and compound B (horizontal dashed columns) .

In Figures 1 and 2 each point represents the average of four to eight experiments per group and the vertical lines the respective SEM.

In Figures 3 and 4 each point represents the average of eight experiments per group and the vertical lines the respective SEM.

In Figures 5 and 6 each column represents the average of eight experiments per group and the vertical lines the respective SEM.

MATERIALS AND METHODS

COMT ACTIVITY TEST

The livers and brains of 60-day-old male Wistar rats weighing 240 to 260 g (Harlan-Interfauna Ibérica, Barcelona, ​​Spain), kept in two per cage under controlled environmental conditions (12-hour light / dark cycle and temperature 24 ° C) were used in all experiments. After decapitation, the organs were immediately removed and homogenized in 5 mM of pH 7.8 phosphate buffer. COMT activity was assessed for its ability to methylate adrenaline into metanephrine. The 0.5 ml aliquots of liver and whole brain homogenates were pre-incubated for 20 minutes with 0.4 ml of phosphate buffer (5 mM); after which, the reaction mixture was incubated for 15 minutes with increased concentrations of epinephrine (0.1 to 2000 μΜ; 0.1 ml) in the presence of a saturated concentration of S-adenosyl-L-methionine, the methyl donor (brain, without μΜ; liver 500 pM); the incubation medium also contained pargyline (100 pM), MgCl₂ (100 pm) and EGTA (1 mM). Pre-incubation and incubation were carried out at 37 ° C under conditions of protection from light with continuous agitation and without oxygenation.

In experiments conducted with the purpose of studying the inhibition effect of COMT inhibitors on the enzyme activity, the reaction mixture was pre-incubated for 20 minutes with increasing concentrations of the test compounds (0.5 to 1,000 nM); incubation was performed in the presence of an adrenaline concentration five times the corresponding Km value as determined in the saturation experiments.

In experiments designed to assess oral bioavailability, half-life and access to the brain, test compounds were administered via the gastric tube to overnight fasted rats. Then, at defined intervals, the animals were killed by decapitation and the livers and brains removed and used to determine COMT activity as described above.

At the end of the incubation period (brain, 15 minutes; liver, 5 minutes) the tubes were transferred to ice and the reaction was stopped by adding 200 μΐ of 2 M perchloric acid. The samples were then centrifuged (200 xg, 4 minutes , 4 ° C) and 500 μΐ aliquots of the supernatant, filtered through 0.22 pm pore size Spin-X filter tubes (Costar) were used for the methanophrine assay.

The methanophrine assay was performed by means of high pressure liquid chromatography with electrochemical detection. The lower limits for methanophrine detection ranged from 350 to 500 fmol (0.5 to 1.0 pmol / mg protein / hour).

The K values_m and V_max for COMT activity, they were calculated from non-linear regression analysis using the GraphPad Prism statistical software package (Motulsky, HG, et al., GraphPad Prisms, GraphPad Prism Software Inc., San Diego, 1994). For the calculation of the IC50 values, the parameters of the equation for inhibiting a site were adjusted for the experimental data. Geometric means are given with 95% confidence limits and arithmetic means are given by SEM Statistical analysis was performed by one-way analysis of variance (ANOVA) using the NewmanKeuls multiple comparison test to compare values.

The protein content in the homogenates was determined by the Bradford process (Bradford, MM, Anal. Biochem., 72: 248 to 254, 1976) with human serum albumin as a standard. The protein content was similar in all samples (approximately 5 mg / 500 μΐ of homogenate). BEHAVIORAL TEST

The experimental design used in the present study was aimed at determining the potentiation of amphetamine-induced hyperactivity in brain dopaminergic systems by COMT inhibitors. For this purpose 128 rats were divided into 16 groups and given the vehicle or one of the three COMT inhibitors tested 6 hours before the behavioral assessment. In all rat groups, behavioral testing started 15 minutes after sc injection vehicle or increasing doses of amphetamine (0.5, 2.0 or 4.0 mg / kg).

On the day of the test, 7 hours before the start of the experiment, the animals were transferred to a dimly lit and soundproofed environment, separated from the environment of the animal colony where the test cages were kept; the temperature and humidity were the same as in the colony environment. Spontaneous locomotor activity was measured using a San Diego Instruments rodent activity monitor (Flex Field model, San Diego Instruments, San Diego, CA) with 48 infrared motion sensors. The bottom structure was 50.5 x 50.5 cm, with 32 photocells (separated by 2.5 cm) located longitudinally 5 cm above the floor. The upper structure was 50.5 x 50.5 cm, with 16 photocells (separated by 2.5 cm) located longitudinally 15 cm from the floor. The test field was an acrylic chamber with internal dimensions of 40 x 40 x 37 cm. The ten-minute recording began immediately after placing the test subject in the center of the chamber. Activity was measured automatically with a personal computer using Flex Field software (San Diego Instruments) that provides user-defined intervals for total outages. Three parameters of normal spontaneous locomotion were recorded: horizontal activity, vertical activity and center time. Stereotypical behavior (intense sniffing, repetitive head and limb movements and licking and biting, as defined by Feldman, RS, Meyer, JS, Quenzer, LF, Principies of Neuropharmacology, 1997, Sinauer Associates, Inc. Publishers, Sunderland, MA) were quantified by an independent observer after being recorded on tape using a video recording system (VP200, HVS Image, Ltd) placed 70 cm above the test field. The animals were accustomed to the test field environment for one hour before the behavioral test.

RESULTS

COMTIN VITRO INHIBITION STUDIES

Incubation of homogenates from liver and whole brain in the presence of increasing concentrations of adrenaline resulted in formation dependent on the concentration of methanophrine, resulting in K values_m (in μΜ) and V_max (in nmol mg protein '¹ H¹) of 0.7 (0.5, 0.9; 95% confidence intervals) and 1.31 ± 0.02 for the brain and 238.5 (128.5; 348.5) and 61.6 ± 3.8 for the liver, respectively. From these kinetic parameters, an adrenaline saturation concentration was chosen for use in inhibition studies (liver, adrenaline = 1000 μΜ; brain, adrenaline = 100 μΜ). The compounds of formulas A through E,

<td>n</td><td>Compound</td>

<td> 0</td><td>THE</td>

<td> 1</td><td>B</td>

<td> 2</td><td>Ç</td>

<td> 3</td><td>D</td>

<td> 4</td><td>AND</td>

<img file="BR9908084B1_D0002.tif" />

plus entacapone and tolcapone (the reference compounds) produced a concentration-dependent decrease in adrenaline O-methylation with IC values₅₀ in the lower nM range for the brain and in the μΜ range for the liver (see Table 1)

TABLE 1

IC VALUES₅₀ (IN nM) FOR THE INHIBITION OF BRAIN AND RAT LIVER COMT.

<td>Compound</td><td>Brain</td><td>Liver</td>

<td>THE</td><td> 7,1 (3,4,14,8)</td><td> 935 (511,1710)</td>

<td>B</td><td> 3,7(1,7, 8,1)</td><td> 696 (356,1360)</td>

<td>Ç</td><td> 3,1(1,1,8,9)</td><td> 829 (498, 1379)</td>

<td>D</td><td> 2,9(1,4, 6,1)</td><td> 1852 (758, 4522)</td>

<td>AND</td><td> 6,7 (3,5, 12,9)</td><td> 1285 (673, 2454)</td>

<td>Encatope</td><td> 12,8 (4,0,41,3)</td><td> 2320 (741, 7263)</td>

<td>Tolcapone</td><td> 2,2 (0,8, 6,4)</td><td> 927 (551,1561)</td>

The compounds of formulas A through E have been found to be potent inhibitors of COMT in both the brain and liver, the maximum inhibitory effect being achieved within 30 minutes after oral administration (Table 2). Compound A showed a similar inhibitory profile in brain and liver COMT, while Compound E was much more potent in liver COMT than in brain COMT. Similarly, compound B was also much more potent as a peripheral COMT inhibitor than in the brain, compounds with longer carbon chains were less potent in inhibiting brain COMT compared to its effects on liver COMT. This difference may have to do with difficulties in accessing the brain. The compounds with short carbon chains (A, B and C) were not equally potent in inhibiting peripheral and central COMT, but this difference was not as surprising as those observed with compounds with long carbon chains. When looking at the duration of the inhibitory effect with respect to liver COMT, it becomes evident that compound B (two-carbon chain) was a particularly long-acting compound. Notably, the inhibition of liver COMT by this compound within 9 hours after oral administration almost reached 70% inhibition, whereas compounds with shorter and longer carbon chains were not endowed with such a long-acting effect. Tolcapone at 6 hours and 9 hours after administration produced marked inhibition of COMT in the brain and liver. As shown in Figures 1 and 2, nine hours after administration, compound B and tolcapone were equally potent in inhibiting liver COMT, whereas entacapone was almost devoid of COMT inhibitory properties. On the other hand, compound B and entacapone were much less potent than tolcapone in inhibiting brain COMT.

TABLE 2. Percentage of inhibition of COMT activity by compounds from A to E, entacapone (Enta) and tolcapone (TOLC) in rat brain and liver homogenates, determined at 0.5, 1, 3, 6 and 9 hours after its administration through the gastric tube. The results are the means ± SEM of 4 experiments per group.

Brain% Inhibition

Time Course

<td></td><td>0.5 h</td><td>1 h</td><td>3 am</td><td>6 am</td><td>9 am</td>

<td>THE</td><td> 96,3 ± 0,4</td><td> 96,8 ± 0,3</td><td> 97,0 ± 0,3</td><td> 85,8 ± 7,5</td><td> 34,9 ± 6,0</td>

<td>B</td><td> 83,6 ± 1,3</td><td> 80,9 ± 2,7</td><td> 65,0 ±3,9</td><td> 31,5 ±3,2</td><td> 21,9 ±2,7</td>

<td>Ç</td><td> 89,9 ± 0,7</td><td> 86,2 ± 0,5</td><td> 59,8 ±5,8</td><td> 33,4 ± 7,0</td><td> 0,4 ± 5,3</td>

<td>D</td><td> 85,1 ± 1,7</td><td> 69,3 ±5,1</td><td> 33,5 ±4,4</td><td> 26,7 ± 4,0</td><td> 12,2 ±5,8</td>

<td>AND</td><td> 87,4 ± 1,3</td><td> 74,2 ± 4,2</td><td> 25,0 ±3,1</td><td> -5,6 ± 7,5</td><td> -6,7 ± 5,0</td>

<td>So</td><td> 71,7 ±7,0</td><td> 44,8 ± 7,0</td><td> 30,1 ±6,4</td><td> 19,9 ±7,1</td><td> 22,8 ± 3,4</td>

<td>Tolc</td><td> 98,9 ±0,1</td><td> 98,7 ± 0,2</td><td> 97,0 ± 0,5</td><td> 85,8 ±8,2</td><td> 77,5 ± 1,8</td>

<td></td><td>Liver</td><td></td><td colspan="2">% Inhibition</td><td></td>

<td></td><td></td><td colspan="2">Time Course</td><td></td><td></td>

<td></td><td>0.5 h</td><td>1 h</td><td>3 am</td><td>6 am</td><td>9 am</td>

<td>THE</td><td> 99,0 ± 0,2</td><td> 98,7 ± 0,2</td><td> 96,9 ± 2,5</td><td> 80,5 ± 7,3</td><td> 31,7 ±5,5</td>

<td>B</td><td> 98,6 ± 0,4</td><td> 96,7 ± 1,7</td><td> 96,2 ± 0,8</td><td> 75,9 ±4,2</td><td> 69,8 ± 3,6</td>

<td>Ç</td><td> 98,4 ± 0,3</td><td> 97,8 ± 0,2</td><td> 95,0 ±0,7</td><td> 70,8 ± 12,8</td><td> 39,9± 11,1</td>

<td>D</td><td> 97,2 ±0,1</td><td> 95,3 ± 0,8</td><td> 67,5 ± 7,8</td><td> 52,0 ±9,5</td><td> 39,0 ± 13,2</td>

<td>AND</td><td> 99,2 ±0,1</td><td> 98,9 ± 0,3</td><td> 88,1 ±3,9</td><td> 36,0 ±6,3</td><td> -4,0 ± 8,0</td>

<td>So</td><td> 71,7 ±7,0</td><td> 96,2 ± 1,1</td><td> 85,9 ±2,2</td><td> 73,6 ±5,4</td><td> 24,7 ± 7,9</td>

<td>Tolc</td><td> 100,0 ±0,0</td><td> 99,9 ±0,1</td><td> 98,0 ±0,7</td><td> 94,1 ±0,3</td><td> 67,0 ± 4,0</td>

Compounds F through J (seen below were also tested at 6 hours and 9 hours after administration and were found to produce an inhibitory profile similar to that described for compound B (Table 3).

<img file="BR9908084B1_D0003.tif" />

The potency of compound B, tolcapone and entacapone in inhibiting COMT in the brain and liver was evaluated in experiments in which rats were given increasing doses of the compounds under test (from 0.3 to 30 mg / kg). In these experiments, the rats were killed 1 hour after administration of the compounds (at_max) and COMT activity determined as described above, the results obtained are shown in Figures 3 and 4 and indicate that compound B and tolcapone were equally potent in inhibiting liver COMT with ED₅₀0.7 ± 1.1 and 0.5 ± 0.1 mg / kg, respectively; entacapone was slightly less potent with an ED value₅o 1.9 ± 0.2 mg / kg. However, compound B was less potent than tolcapone in inhibiting brain COMT with ED₅o's of 5.3 ± 1.1 and 1.6 ± 0.1 mg / kg, respectively. At the highest dose tested (30 mg / kg), entacapone failed to reach the 50% inhibition level.

TABLE 3. Percentage of inhibition of COMT activity by compounds from F to J in rat brain and liver homogenates, 10 determined at 6 and 9 hours after its administration through the gastric tube. The results are the means ± SEM of 4 experiments per group.

<td></td><td colspan="2">Liver</td><td colspan="2">Brain</td>

<td></td><td>6 am</td><td>9 am</td><td>6 am</td><td>9 am</td>

<td>F</td><td> 70,2 ± 3,3</td><td> 37,7 ± 4,2</td><td> 10,3 ±5,1</td><td> 0,4 ±7,1</td>

<td>G</td><td> 77,8 ±4,5</td><td> 51,2 ±3,7</td><td> 28,2 ±5,1</td><td> 27,1 ±5,0</td>

<td>H</td><td> 82,8 ± 2,2</td><td> 45,8 ± 10,3</td><td> 17,1 ±4,0</td><td> 6,8 ±2,7</td>

<td>I</td><td> 74,0 ± 4,6</td><td> 46,3 ± 10,6</td><td> 33,0 ±2,0</td><td> 24,6 ± 6,6</td>

<td>J</td><td> 68,6 ± 4,2</td><td> 57,1 ±8,1</td><td> 12,1 ±3,2</td><td> 25,8 ±2,1</td>

BEHAVIORAL TEST

Amphetamine is a potent psychostimulant that, depending on the dose administered, produces locomotor behavior and increased stereotypic activities. A single low dose of amphetamine administered to rats leads to a characteristic pattern of response that consists of increased locomotor activity, rearing, mild sniffing and head shaking. Gradually increasing the dose of amphetamine results in a decrease in locomotion and in rearing which are replaced by focused stereotypes (repetitive behaviors, apparently without purpose, performed in a relatively invariable manner) confined to a small area of ​​the cage floor (Feldman, RS, Meyer JS, Quenzer, L.

F., Principies of Neuropharmacology, 1997, Sinauer Associates, Inc. Publishers, Sunderland, MA). The brain dopaminergic system has traditionally been crucial to amphetamine's ability to stimulate locomotor activity and stereotypical behaviors. With respect to the substrates of the action of amphetamine, there is evidence that the stimulus of dopaminergic activity in the accumulating nucleus is responsible for the locomotor activity induced by amphetamine, whereas the stimulus of dopaminergic activity in the caudate drupa lump is linked with focused stereotypes produced for high doses of amphetamine.

As predicted, low doses of amphetamine (0.5 and 2.0 mg / kg, sc) were observed to produce dose-dependent increases in horizontal activity and rearing, with no evidence of stereotyped behavior (Figures 5 and 6).

In contrast, a high dose of amphetamine (4.0 mg / kg, sc) was observed to produce no additional increase in locomotor activity, however it resulted in the appearance of stereotypes that lasted 250 seconds during the 600 second observation period. Tolcapone (30 mg / kg, po) administered 6 hours before amphetamine inoculation was observed to significantly increase locomotor activity in rats treated with 0.5 and 2.0 mg / kg of amphetamine. In contrast, in rats administered 4.0 mg / kg of amphetamine, tolcapone produced a marked decrease in locomotor activity and increased the duration of stereotyped behavior twice. Rats treated with entacapone (30 mg / kg, po) or compound B six hours before inoculation of amphetamine showed the same patterns of locomotor activity and stereotyped behavior as their corresponding controls.

CONCLUSION

The compounds of Formula I are very potent catechol-O-methyltransferase (COMT) inhibitors and have potentially valuable pharmaceutical properties in the treatment of some disorders of the central and peripheral nervous system where inhibition of catecholamine O-methylation can be of therapeutic benefit, such as Parkinson's disease and parchinsonian disorders, gastrointestinal disorders, edema and hypertension. The possibility for the use of a COMT inhibitor of lasting action with limited access to the brain, such as compound B opens new perspectives in said therapies, improving selectively and prolonging COMT inhibition. It is particularly important when thinking about treating patients afflicted with Parkinson's disease and taking L-DOPA plus a peripheral AADC inhibitor. Due to the possibility that COMT inhibitors that have easy access to the brain may cause excessive dopaminergic stimulation, that is, by inducing dyskinesia and mental confusion in patients treated with L-DOPA, the use of a substance such as compound B is intended to be devoid of such effects while still having the benefits of a long-acting substance.

The invention disclosed herein is exemplified by the following preparation examples, which should not be construed as limiting the scope of the disclosure. Alternative paths and analogous structures may be evident to those skilled in the art.

Example 1 1 - (3,4-dihydroxy-5-nitrophenyl) -2-phenyl-1-ethanone

A solution of 20 g (82.64 mmol) of O-benzylvaniline in 200 ml of dry tetrahydrofuran was added slowly to a stirred solution of benzyl magnesium chloride (103.30 mmol) in 150 ml of diethyl ether at 10 ° C for 20 minutes and the reaction mixture was then boiled for 10 minutes, cooled, quenched with a mixture of ice and diluted hydrochloric acid and evaporated under reduced pressure. The residue was dissolved in dichloromethane, the solution washed with brine, dried with sodium sulfate and the solvent was evaporated under reduced pressure leaving a crystalline residue which was recrystallized from diethyl ether and petroleum ether. 1- (4-Benzyloxy-3-methoxyphenyl) -2-phenyl-1-ethanol was obtained as white crystals, mp 97-98 ° C.

A solution of 10 g (30 mmoles) of the above secondary alcohol in 90 ml of dichloromethane and 30 ml of diethyl ether was cooled to 0 ° C and 7.5 g of Celite® was added at once with stirring, followed by 9 g (90 mmoles) of chromium trioxide. The reaction mixture was stirred overnight at room temperature, filtered and the filtrate was evaporated under reduced pressure. The crystalline residue was recrystallized from a mixture of dichloromethane and diethyl ether, producing 1- (4-benzyloxy-3-methoxyphenyl) -2-phenyl-1-ethanone as white crystals, mp 134 to 135 ° C.

A solution of 5.9 g, (17.8 mmoles) of the above ketone in a mixture of dichloromethane (60 ml) and 30% hydrobromic acid in acetic acid (27 ml) was stirred for 1.5 hours at room temperature and then the dichloromethane was evaporated under reduced pressure and the reaction mixture was poured over 200 ml of a mixture of ice and water. The formed precipitate was filtered off and dried in vacuo to provide 1- (4-hydroxy 3-methoxyphenyl) -2-phenyl-1-ethanone as beige crystals, mp 107 at 108 ° C.

To a solution of 3.87 g (16 mmoles) of the above intermediate in 40 ml of acetic acid was added 1.4 ml (17.6 mmoles) of 12.6 M nitric acid under cooling to 10 ° C and the mixture of The reaction was stirred for 30 minutes at room temperature and then poured over a mixture of ice and water. The formed precipitate was filtered off, washed with water and dried to give 1- (4-hydroxy-3-methoxy-5-nitrophenyl) -2-phenyl-1-ethanone as a yellow powder, mp 129 at 130 ° C.

The above nitroderivate (3.769, 13 mmoles) was boiled with a mixture of azeotropic hydrobromic acid (37 ml) and 30% HBr in acetic acid (18 ml) for 16 hours and the cooled reaction mixture was poured over an ice mixture and water. The formed precipitate was filtered off, washed carefully with water and recrystallized from acetic acid to give the desired product as yellow crystals, mp 181 to 182 ° C. Examples 2 to 12

By applying the technique described above and related procedures known to those skilled in the art and using appropriate metallorganic reagents the following compounds were prepared:

- (3,4-dihydroxy-5-nitrophenyl) -2- (4-hydroxyphenyl) -1 -ethanone 1 - (3,4-dihydroxy-5-nitrophenyl) -2- (2-methylphenyl) -1 -ethanone 1 - (3,4-dihydroxy-5-nitrophenyl) -2- (3-methylphenyl) -1 -ethanone

1- (3,4-dihydroxy-5-nitrophenyl) -2- (4-methylphenyl) -l-ethanone 1 - (3,4-dihydroxy-5-nitrophenyl) -2- (4-butylphenyl) -1 -ethanone 1 - (3,4-dihydroxy-5-nitrophenyl) -2- (3,4-dimethylphenyl) -1-ethanone 1 - (3,4-dihydroxy-5-nitrophenyl) -2- (3,4-dimethoxyphenyl) -1-ethanone l- (3,4-dihydroxy-5-nitrophenyl) -2- (4-butyloxyphenyl) -l-ethanone 1 - (3,4-dihydroxy-5-nitrophenyl) -2- (l-methyl- 5-indolyl) -l -ethanone 1 - (3,4-dihydroxy-5-nitrophenyl) -2- (3,4-methylenedioxyphenyl) -1 -ethanone 1- (3,4-dihydroxy-5-nitrophenyl) -2- (2,4,6-trimethylphenyl) -l-ethanone

Example 13 1 - (3,4-dihydroxy-5-nitrophenyl) -2- (2-methylphenyl) -1 -ethanone

To a mixture of guaiacol (1.24 g, 10 mmol), otolylacetic acid (1.50 g, 10 mmol) and ZnCl₂ (5 g, 36.7 mmoles) POC1 was added₃ (15 ml, 161 mmoles) and the resulting suspension was stirred and heated to 80 ° C for 1.5 hours. The reaction mixture was cooled and poured over ice and water and the resulting suspension was stirred at room temperature for 1 hour and then extracted with ethyl acetate. The organic layer was separated, washed with brine and dried over sodium sulfate. The volatiles were evaporated under reduced pressure and the residue was dissolved in diethyl ether. The solution was extracted twice with 50 ml of 2N aqueous NaOH solution and the combined aqueous layers were combined and acidified with hydrochloric acid to pH = 2. The emulsion formed was extracted with ethyl acetate and the organic layer was washed with brine, dried and the solvent was evaporated under reduced pressure. The residue was subjected to chromatography on a silica gel column with a mixture of petroleum ether and ethyl acetate to give l- (4-hydroxy-3-methoxyphenyl) -2- (2-methylphenyl) -l-ethanone as yellowish-white crystals, mp 79 to 81 ° C.

To a solution of 4.01 g (16 mmoles) of the above intermediate in 40 ml of acetic acid, 1.4 ml (17.6 mmoles) of 12.6 M nitric acid were added under cooling to 10 ° C and the mixture The reaction mixture was stirred for 30 minutes at room temperature and then poured into a mixture of ice and water. The formed precipitate was filtered off, washed with water and dried to give 1- (4-hydroxy-3-methoxy-5-nitrophenyl) -2- (2-methylphenyl) -lethanone as a yellow powder mp 150 to 151 ° C .

The above nitroderivate (3.91 g, 13 mmoles) was boiled with a mixture of azeotropic hydrobromic acid (37 ml) and 30% HBr in acetic acid (18 ml) for 16 hours and the cooled reaction mixture was poured over a mixture of ice and water. The formed precipitate was filtered off, washed carefully with water and recrystallized from acetic acid to give the desired product as yellow crystals, mp 128 to 129 ° C. Examples 14 to 21

By applying the technique described above and related procedures known to those skilled in the art and using appropriately substituted phenylacetic acids, the following compounds have been prepared:

- (3,4-dihydroxy-5-nitrophenyl) -2- (4-carboxyphenyl) -1 -ethanone 1 - (3,4-dihydroxy-5-nitrophenyl) -2- (2-nitrophenyl) -1 -ethanone 1 - (3,4-dihydroxy-5-nitrophenyl) -2- (4-biphenyl) -1-ethanone 1 - (3,4-dihydroxy-5-nitrophenyl) -2- (3-cyanophenyl) -1-ethanone 1 - (3,4-dihydroxy-5-nitrophenyl) -2- (1-naphthyl) -1 -ethanone l- (3,4-dihydroxy-5-nitrophenyl) -2- (2-naphthyl) -l-ethanone l - (3,4-dihydroxy-5-nitrophenyl) -2- (2-chlorophenyl) -l-ethanone 1- (3,4-dihydroxy-5-nitro phenyl) -2- (4-chlorophenyl) -l-ethanone

Example 22 1- (3,4-diacetoxy-5-nitrophenyl) -2-phenyl-1-ethanone

A suspension of 9.20 g (33.6 mmoles) of 1- (3,4-dihydroxy-5nitrophenyl) -2-phenyl-1-ethanone in 90 ml of dichloromethane was treated with 7.85 g (100 mmoles) of acetyl chloride, 7.51 g (95 mmol) of pyridine and a catalytic amount of 4-dimethylaminopyridine. After 1 hour of stirring at room temperature, the solution formed was washed successively with ice-cooled 0.2 N hydrochloric acid, 1% aqueous sodium bicarbonate solution and brine. The dried solution (Na₂ONLY₄) was evaporated under reduced pressure and the residue recrystallized from a mixture of diethyl ether and petroleum ether giving the desired product as yellow crystals, mp 94 to 95 ° C.

Examples 23 to 27

By applying the technique described above and related procedures known to those skilled in the art and using 1 (3,4-dihydroxy-5-nitrophenyl) -2-phenyl-1-ethanones and appropriately substituted acid halides or anhydrides, following compounds were prepared:

- (3,4-dimethoxymethyloxy-5-nitrophenyl) -2-phenyl-1-ethanone l- (3,4-dibutyryloxy-5-nitrophenyl) -2-phenyl-l-ethanone 1 - (3,4-di- (4-tolylsulfonyloxy) -5-nitrophenyl) -2-phenyl-1-ethanone 1- (3,4-dibutyryloxycarbonyloxy-5-nitrophenyl) -2-phenyl-1-ethanone 1 - (3,4-diacetoxy-5- nitrophenyl) -2- (4-acetoxyphenyl) -1 -ethanone