WO2003000269A2

WO2003000269A2 - Novel use for pde 10a inhibitors

Info

Publication number: WO2003000269A2
Application number: PCT/EP2002/006309
Authority: WO
Inventors: Ulrich Niewöhner; Jens-Kerim ERGÜDEN; Marcus Bauser; Nils Burkhardt; Dietmar Flubacher; Arno Friedl; Irene Gerlach; Volker Hinz; Reinhard Jork; Paul Naab; Thorsten-Oliver Repp; Karl-Heinz Schlemmer; Jürgen Stoltefuss
Original assignee: Bayer Aktiengesellschaft
Priority date: 2001-06-22
Filing date: 2002-06-10
Publication date: 2003-01-03
Also published as: WO2003000269A3; DE10130151A1

Abstract

The invention relates to the use of PDE 10A inhibitors for producing a medicament for the treatment and/or prophylaxis of neurodegenerative diseases, especially Parkinson's disease.

Description

New use for PDE IOA inhibitors

The invention relates to the use of PDE IOA inhibitors for the manufacture of a medicament for the treatment and / or prophylaxis of neurodegenerative

Diseases, especially Parkinson's syndrome.

The cyclic nucleotides cGMP and cAMP are among the most important intracellular messengers. Phosphodiesterases (PDEs) play an important role in regulating the concentrations of cGMP and cAMP. So far, 11 phosphodiesterase isoenzyme groups are known (PDE 1-7: Beavo et al. Mol. Pharmacol. 1994, 399-405; PDE 8-10: Soderling and Beavo Curr. Opin. Cell Biol. 2000, 12, 174- 179; PDE 11: Fawcett et al. Proc. Natl. Acad. Sci. USA 2000, 97, 3702-3707).

The PDE 10A hydrolyzes both cAMP and cGMP. Transcribed PDE 10A was identified primarily in the putamen and caudate nucleus regions of the brain, as well as in thyroid and testicular tissues. Compared to normal tissue, the PDE IOA mRNA is also increasingly expressed in certain tumor tissues, such as tissues of breast, liver, colon and lung tumors.

Parkinson's syndrome is a chronic, progressive disease of the central nervous system. It is caused by the degeneration of dopaminergic neurons in the substantia nigra, which produce and release the neurotransmitter dopamine. The resulting reduction in dopaminergic neurotransmission leads to massive dysfunctions in the extrapyramidal system of motion control. These disorders affect not only the basal ganglia but also other closely linked brain areas. A distinction is made between several forms of Parkinson's syndrome: idiopathic or primary Parkinson's syndrome, symptomatic or secondary Parkinson's syndrome as well as special forms of Parkinson's syndrome such as Parkinson's dementia-ALS complex or Parkinson's plus syndrome (Pschyrembel Klinisches Dictionary, 257th edition, de Gruyter; Berlin, 1994; p.1153, keyword

'Parkinson's syndrome').

The aetiology of idiopathic Parkinson's syndrome is still largely unknown. However, increasing evidence indicates that the cell death of dopaminergic neurons in the substantia nigra due to apoptosis is more mitochondrial

Malfunction occurs. In addition to genetic disorders, increased glutamate levels and / or a deficient supply of neurotrophic factors are also discussed as the cause of the mitochondrial malfunction.

The majority of the currently used therapeutics for Parkinson's syndrome follow a purely symptomatic approach. The goal of these therapies is either the direct substitution of the missing dopamine by a dopamine precursor molecule (L-DOPA), which is metabolized to dopamine in the body, or the stimulation of deficient dopaminergic neurotransmission processes by means of agonists at dopamine receptors or by reducing the dopamine breakdown (MAO -

Inhibitors, COMT inhibitors). However, all current therapies are characterized by strong side effects (e.g. dyskinesias, psychoses, sleep disorders) or long-term loss of effectiveness.

Fujishige (J. Biol. Chem. 1999, 274, 18438-18445) has already speculated about an unspecified connection between PDE 10A and juvenile Parkinsonism.

WO 01/29199 discloses a cyclic nucleotide phosphodiesterase, designated 22045 and homologous to PDE 10A. According to WO 01/29199, modulators can be used to determine the concentration or activity of this PDE Diseases are examined. Brain diseases, including Parkinsonism, are listed under a variety of diseases (p.19, Z.32).

WO 01/24781 (p.33) suggests the treatment of neuronal dysfunctions, such as, for example, Huntington's disease, by upregulating PDE

10A activity before. Another treatable neuronal disease is called Parkinson's disease. However, pharmacologically, the upregulation of PDE 10A activity corresponds to the opposite of PDE 10A inhibition.

Unexpectedly, however, selective PDE IOA inhibitors work in animal models for neurodegenerative diseases, in particular Parkinson's syndrome.

It is also shown for the first time that selective PDE IOA inhibitors work in animal models for Parkinson's syndrome.

The present invention therefore relates to the use of PDE IOA inhibitors for the production of a medicament for the treatment and / or neurodegenerative diseases, in particular of Parkinson's syndrome, in particular of idiopathic Parkinson's syndrome.

Preferred PDE 10A inhibitors are those which inhibit PDE 10A with an IC ₅₀ of less than 1 μM, preferably less than 0.1 μM, in the test given below.

The PDE IOA inhibitors according to the invention are preferably also selective towards other PDEs, particularly preferably towards PDE IC, 2A, 3B, 4B, 5A and 7B. Very particularly preferred compounds of the invention inhibit PDE 10A at least stronger than the other PDEs by a factor of 10, ie the IC ₅₀ - value is for PDE 10A by at least a factor of 10 lower than the value recorded for the other PDEs IC ₅ o value. The IC ₅₀ values for the PDEs are measured according to the conditions specified below. PDE IOA inhibitors with the property profile described above can be identified with these assays.

Inhibition of PDE 10A

PDE 10A (WO 01/29199, Fig. 1A) is recombinantly expressed in full length in Sf9 insect cells (Invitrogen, Carlsbad, CA) using the Bac-to-Bac ™ baculovirus expression system from Life Technologies (Gaithersburg, MD). 48 hours after

Infection, the cells are harvested and in 20 mL (per IL culture) lysis buffer (50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1 raM MgCl ₂ , 1.5 raM EDTA, 10% glycerol plus 20 μL protease inhibitor cocktail set III [ CalBiochem, La Jolla, CA USA]) suspended. The cells are treated with ultrasound at 4 ° C. for 1 minute and then centrifuged for 30 minutes at 4 ° C. at 10,000 rpm. The

Supernatant (PDE IO preparation) is collected and stored at -20 ° C.

To determine their in vitro effect on PDE 10A, the test substances are dissolved in 100% DMSO and serially diluted. Typically, dilution series from 200 μM to 1.6 μM are prepared (resulting final concentrations in

Test: 4 μM to 0.032 μM). 2 μL of the diluted substance solutions are placed in the wells of microtiter plates (Isoplate; Wallac Inc., Atlanta, GA). Then 50 μL of a dilution of the PDE10A preparation described above are added. The dilution of the PDE IO preparation is chosen such that less than 70% of the substrate is converted during the later incubation (typical dilution: 1: 10000; dilution buffer: 50 mM Tris / HCl pH 7.5, 8.3 mM MgCl ₂ , 1.7 mM EDTA , 0.2% BSA). The substrate, [5 ', 8- ³ H] -cAMP (. 1 uCi / ul; Amersham Pharmacia Biotech, Piscataway, NJ) is 1: 2000 with assay buffer (50 mM Tris / HCl pH 7.5, 8.3 mM MgCl _2, 1.7 mM EDTA) diluted to a concentration of 0.0005 μCi / μL. By adding 50 μL

(0.025 μCi) of the diluted substrate, the enzyme reaction is finally started. The test batches are incubated for 60 min at room temperature and the reaction is stopped by adding 25 μL of a suspension with 18 mg / ml Yttrium ScintiUation Proximity Beads (Amersham Pharmacia Biotech., Piscataway, NJ.). The microtiter plates are sealed with a film and left to stand for 60 min at room temperature. The plates are then measured in a Microbeta scintillation counter (Wallac Inc., Atlanta, GA) for 30 s per well. IC ₅₀ values are determined on the basis of the graphical plot of the substance concentration against the percentage inhibition.

Example 1 inhibited under these conditions PDE 10A with an IC ₅ o value of

35nm.

Inhibition of PDEs 1-5 and 7

Recombinant PDE IC (GenBank / EMBL Accession Number: NM_005020), PDE

2A (Rosman et al. Gene 1997 191, 89-95), PDE 3B (Miki et al. Genomics 1996 36, 476-485), PDE 4B (Böiger et al. Mol. Cell. Biol. 1993 13, 6558-6571 ), PDE 5A (GenBank / EMBL Accession Number: AJ004865) and PDE 7B (Hetman et al. Proc. Natl. Acad. Sei. USA 2000 97, 472-476) are converted into Sf9 cells with the aid of the pFASTBAC baculovirus expression system (GibcoBRL) expressed.

The in vitro effect of test substances on recombinant PDE 3B, PDE 4B, and PDE 7B is determined according to the test protocol described above for PDE 10A. To determine a corresponding effect on recombinant PDE IC, PDE2A and PDE5A, the protocol is adapted as follows: With PDE IC, additional Calmodulin 10 ^"7 M and CaCl ₂ 3mM are added to the reaction mixture. In the test, PDE 2A is added by adding cGMP 1 μM stimulated and tested with a BSA concentration of 0.01%. For PDE 5A is used as a substrate [8- ³ H] -cGMP (Amersham Pharmacia Biotech., Piscataway, NJ). Example 1 inhibits the PDE IC, 2A, 3B, 4B, 5A and 7B with IC ₅₀ values of 2μM,>10μM,> 4μM, 2.5μM, 10μM and 3.8μM.

The suitability of the compounds according to the invention for the treatment of Parkinson's syndrome can be shown in the following animal models:

6-Hydroxydopamine (6-OH-DA) lesion in the rat

The clinical picture of Parkinson's syndrome can be simulated to a large extent by injecting the neurotoxin 6-OH-DA intracerebrally into rats.

30 minutes before the lesion, Pargyline (50 mg / kg ip) and Desmethylimipramine HC1 (25 mg / kg ip) were administered to the animals to prevent the metabolism of 6-hydroxydopamine or to absorb 6-hydroxydopamine in noradrenergic structures to prevent. The lesion of the nigrostriatal neurotransmission is then performed under anesthesia by giving the test animals a single stereotactic injection of 8 μg 6-OH-DA. The coordinates of the injection according to König and Klippel are: 2.4 mm anterior, 1.49 mm lateral, -2.7 mm ventral. In the verum group, the animals were treated with test substance one day after the operation until the end of the experiment 28 days after the operation.

The motor failures of the lesionized rats were quantified using the following tests as described in the relevant literature:

a) Staircase test (motor skills test of the front extremity): Barneoud et al: Effects of complete and partial lesions of the dopaminergic mesotelencephalic system on skilled forelimb use in the rat. Neuroscience 1995, 67, 837-848.

b) Accelerating Rotarod Test: Spooren et al .: Effects of the prototypical mGlu ₅ receptor antagonist 2-methyl-6- (phenylethynyl) -pyridine on rotarod, locomotor activity and rotational responses in unilateral 6-OHDA-lesioned Board. Eur. J. Pharmacol. 2000, 406, 403-410. c) Tensile force measurement of the forelimbs: Dunnet et al .: A laterised grip strength test to evaluate unilateral nigrostriatal lesions in rats. Neurosci. Leu. 1998, 246 ^" , 1-4.

Example 1 improved the motor skills of the front extremities in the staircase test in a dose range of 0.3 to 3.0 mg / kg bid p.o. In a comparable dose range, the other test parameters, namely balance test and tensile force measurement, were also positively influenced.

MPTP mouse - model

The neuroprotective in vivo effect of the compounds according to the invention was shown in a mouse model for Parkinson's syndrome, the so-called MPTP model. MPTP (= l-methyl-4-phenyl-l, 2,3,6-tetrahydropyridine) is a neurotoxin that causes the degeneration of dopaminergic neurons in the substantia nigra characteristic of Parkinson syndrome in humans and animals and the motor symptoms typical of Parkinsonism.

The mice were given 3 mg / kg MPTP i.p. on 3 consecutive days. applied (method modified according to Bezard E. et al., Kinetics of nigral degeneration in a chronic model of MPTP-treated mice, Neurosci. Lett. 1997, 234, 47-50). The experimental animals then show a reduced number of dopaminergic neurons in the substantia nigra pars compacta.

The extent of cell damage is quantified by a histological examination on the tenth day after MPTP injection. For this purpose, dopaminergic neurons are made immunohistochemically visible as cells that contain the enzyme tyrosine hydroxylase, which is essential in dopamine metabolism, and finally quantified using a computer program (Nelson EL et al., Midbrain dopaminergic neurons in the mouse: Computer assisted mapping, J. Comp. Neurol. 1996, 369, 361-371).

Mice that used Example 1 in a dose of 3 mg / kg bid po for 10 days from the first day of MPTP intoxication. had significantly more dopaminergic neurons in the substantia nigra pars compacta than control animals that only received MPTP.

The active compounds can be converted in a known manner into the customary formulations, such as tablets, dragées, pills, granules, aerosols, syrups, emulsions, suspensions and solutions, using inert, non-toxic, pharmaceutically suitable excipients or solvents. Here, the therapeutically active compound should in each case be present in a concentration of about 0.5 to 90% by weight of the total mixture, i.e. in amounts sufficient to achieve the dosage range indicated.

The formulations are prepared, for example, by stretching the active ingredients with solvents and / or carriers, optionally using emulsifiers and / or dispersants, e.g. in the case of the use of water as a diluent, organic solvents can optionally be used as auxiliary solvents.

The application is carried out in the usual way, preferably orally, transdermally or parenterally, in particular perlingually or intravenously. However, it can also be done by inhalation through the mouth or nose, for example with the aid of a spray, or topically via the

Skin.

In general, it has proven to be advantageous to administer amounts of approximately 0.001 to 10 mg / kg, preferably approximately 0.005 to 3 mg / kg of body weight in the case of oral use in order to achieve effective results. Nevertheless, it may be necessary to deviate from the amounts mentioned, depending on the body weight or the type of application route, on the individual behavior towards the medication, the type of its formulation and the time or interval at which the administration takes place , In some cases it may be sufficient to make do with less than the aforementioned minimum quantity, while in other cases the above upper limit must be exceeded. In the case of application of larger quantities, it may be advisable to distribute them in several individual doses over the day.

2- (3, 4-Dimethoxyphenyl) -5, 7-dimethyl-4- (3, 4,5-trimethoxyphenoxy) imidazo [5, 1-fj- [1,2,4] triazine (Example 1) was as follows manufactured:

a) 3, 4-Dimethoxybenzenecarboximidamide hydrochloride

21.4 g (400 mmol) of ammonium chloride are suspended in 200 ml of anhydrous toluene in a three-necked flask equipped with a thermometer, condenser, dropping funnel and mechanical stirrer under an argon atmosphere and cooled to 0 ° C. 400 mmol of trimethyl aluminum (200 ml of 2 M solution in hexane) are added dropwise, and the mixture is stirred at room temperature until no more gas evolution is observed (approx. 1.5 h). A solution of 33.6 g (200 mmol) of 3,4-dimethoxybenzonitrile in 100 ml of dry toluene is added dropwise and the reaction mixture is stirred at 80 ° C. for 18 h.

After cooling, the mixture is added dropwise at -10 ° C with 60 ml of methanol and then stirred vigorously at RT for 90 min. The mixture is filtered off with suction and the residue is washed with methanol (5 × 200 ml). The filtrate is concentrated, the residue is washed with methanol / diethyl ether mixture and diethyl ether and the solid obtained (yield: 28.2 g) is dried. The washing phases are concentrated, taken up in ethanol and decolorized with activated carbon. The activated carbon is filtered off and the filtrate is concentrated. The residue obtained is mixed with diethyl ether and suction filtered. Another 11.2 g of product are obtained.

Total yield 92% of theory Th.

Η NMR (200 MHz, DMSO-d ₆ ): δ = 3.85 (s, 3H), 3.86 (s, 3H), 7.17 (d, 1H), 7.45 (d, 1H), 7.47-7.53 (m, 1H ). b) Ethyl 3- (acetylamino) -2-oxobutanoate

N-acetyl-alanine (4.92 g, 37.5 mmol), 9.10 ml pyridine and 150 mg DMAP are dissolved in 200 ml THF and the solution is brought to a boil. 8.6 ml (10.5 g, 75 mmol) of ethyl oxalyl chloride are added dropwise at the boiling point, and after the addition has ended, the mixture is stirred at the boiling point for a further 3 h. After cooling, the reaction mixture is poured onto 600 ml of ice water, extracted with ethyl acetate (4 x 150 ml), the combined organic phases are saturated with 200 ml. Washed NaCl solution, dried over sodium sulfate and concentrated. The material obtained is further reacted without delay in ethanol.

c) N- {1- [3- (3,4-Dimethoxyphenyl) -5-oxo-4,5-dihydro-1,2,4-triazin-6-yl] ethyl} acetamide

3,4-Dimethoxybenzenecarboximidamide hydrochloride (5.42 g, 25 mmol) is placed in 100 ml of ethanol. 1.34 ml of hydrazine hydrate (1.34 g, 27.5 mmol) are added and the mixture is stirred at 45 ° C. for 3 h. After this time, ethyl 3- (acetylamino) -2-oxobutanoate in 50 ml of ethanol is added and the reaction mixture is stirred for 6 hours at 80 ° C. bath temperature and then for 12 hours at room temperature. The mixture is concentrated and the residue is purified by flash chromatography (mobile phase gradient dichloromethane / methanol 40: 1 to 20: 1).

Yield: 2.85 g (35% of theory), amorphous solid. Mp .: 218 ° C

1H-NMR (300 MHz, DMSO-d ₆ ): δ = 1.35 (d, 3H), 1.84 (s, 3H), 3.84 (s, 3H), 3.85 (s, 3H), 5.00 (quint, 1H), 7.16 (d, 1H), 7.59-7.77 (m, 2H), 8.24 (d, 1H), 13.93 (s, 1H).

d) 2- (3, 4-Dimethoxyphenyl) -5, 7-dimethylimidazo [5, 1 -f] [1,2,4] triazin-4 (3H) -one

N- {l- [3- (3,4-Dimethoxyphenyl) -5-oxo-4,5-dihydro-l, 2,4-triazin-6-yl] ethyl} acetamide (2.60 g, 8.13 mmol) is placed in 100 ml of 1,2-dichloroethane and the solution with

0.19 ml (2.04 mmol) of phosphoryl chloride were added. The mixture is stirred at the boil for 24 h. After cooling, the precipitate is filtered off with suction, the residue is washed with water (2 × 50 ml) and diethyl ether (50 ml) and dried.

Yield: 1.90 g (77% of theory) 1H-NMR (300 MHz, DMSO-ds): δ = 2.55 (s, 3H), 2.64 (s, 3H), 3.84 (s, 3H), 3.86 (s, 3H), 7.13 (d, 1H), 7.58 -7.62 (m, 1H), 7.64-7.71 (m, 1H).

e) 2- (3, 4-dimethoxyphenyl) -5, 7-dimethyl-4- (lH-l, 2,4-triazol-l -yl) imidazo [5, 1- f] [l, 2,4] triazine

0.53 ml (879 mg, 5.67 mmol) of phosphoryl chloride become under argon a solution of 568 mg (1.89 mmol) of N- {l- [3- (3,4-dimethoxyphenyl) -5-oxo-4 , 5-di-hydro-l, 2,4-triazin-6-yl] ethyl} acetamide was added dropwise in 80 ml of dry pyridine at 0 ° C. and the mixture was stirred for 20 min. A solution of 3.33 g (47 mmol) of 1,2,4-triazole in 80 ml of dry pyridine is then added at 0 ° C. and the mixture is stirred at RT for 16 h after the addition has ended. The dark red reaction mixture is concentrated, the residue is mixed with 150 ml of ice water, and the

Mixture extracted with dichloromethane (3 x 100 ml). The combined organic phases are dried (sodium sulfate) and the solvent is removed in vacuo. The residue is purified by flash chromatography (mobile phase dichloromethane / methanol 40: 1). 238 mg (36% of theory) of product are obtained.

MS (ESI): 352 [M + H] ⁺

1H-NMR (400 MHz, CDC1 ₃ ): δ = 2.81 (s, 3H), 2.87 (s, 3H), 3.98 (s, 3H), 4.02 (s, 3H), 6.99 (d, 1H), 7.88 ( d, 1H), 8.00 (q, 1H), 8.26 (s, 1H), 9.36 (s, 1H). Mp .: 220 ° C f) 2- (3, 4-Dimethoxyphenyl) -5, 7-dimethyl-4- (3, 4, 5-trimethoxyphenoxy) imidazo [5, 1- f] - [1,2,4] triazine

A solution of 208 mg (1.85 mmol) of potassium tert-butoxide, 682 mg (3.70 mmol) of 3,4,5-trimethoxyphenol and 650 mg (1.85 mmol) of 2- (3,4-dimethoxyphenyl) -5,7-dimethyl-4- (lH-l, 2,4-triazol-l-yl) imidazo [5, l-fJ [l, 2,4] triazine in 120 ml pyridine are at boiling for 16 h touched. After cooling, the solvent is removed and the residue is taken up in 200 ml of dichloromethane. It is washed with 2N hydrochloric acid (3 x 50 ml) and sat. Sodium chloride soln. (50 ml), dries over sodium sulfate and removes the solvent in vacuo. It is first cleaned by flash chromatography (mobile phase gradient dichloromethane-dichloromethane / methanol 20: 1), then by HPLC, and dried in a high vacuum.

Yield: 525 mg (61% of theory)

Mp: 184 ° C;

MS (DCI): 467 [M + H] ⁺ ;

1H-NMR (200 MHz, DMSO-d ₆ ): δ = 2.61 (s, 3H), 2.66 (s, 3H), 3.69 (s, 3H), 3.71 (s, 3H), 3.78 (s, 9H), 6.84 (s, 2H), 7.06 (d, 1H), 7.59-7.68 (m, 2H).

Claims

claims

1. Use of PDE IOA inhibitors for the manufacture of a medicament for the treatment and / or prophylaxis of neurodegenerative diseases.

2. Use according to claim 1, wherein the neurodegenerative disease is Parkinson's syndrome.

3. Use according to claim 1 or 2, wherein the PDE IOA inhibitor has an IC ₅ o value of less than 1 micron.

4. Use according to claim 1 or 2, wherein the PDE IOA inhibitor has an IC ₅₀ value of less than 1 OOnM.

5. Use according to one of claims 1 to 4, wherein the PDE 10A-

Inhibitor is selective for other phosphodiesterases.

6. Use according to any one of claims 1 to 4, wherein the PDE 10A inhibitor is selective for PDE IC, 2A, 3B, 4B, 5A and 7B.

7. Use according to claim 5 or 6, wherein the PDE 10A inhibitor inhibits the PDE 10A more potent than the other phosphodiesterases by at least a factor of 10.

8. Use of PDE IOA inhibitors according to one of claims 1 to 7 for

Manufacture of a medicament for the treatment and / or prophylaxis of idiopathic Parkinson's syndrome.