DE10031389A1 - Pyrimidine derivatives and their use for the prophylaxis and therapy of cerebral ischemia - Google Patents

Abstract

The invention relates to 3-substituted 5,6,7,8 tetrahydropyrido[4',3':4,5] thieno [2,3-d] pyrimidine-4(3H)-one derivatives of formula (I), wherein R<1> represents a hydrogen atom or a C1-C4 alkyl group, and to their physiologically compatible salts.

Description

The invention relates to pyrimidine derivatives for prophylaxis and Therapy of cerebral ischemia.

DE 199 00 545.1 describes 3-substituted pyrimidine derivatives of the formula 1

wherein
A represents NH or an oxygen atom,
B represents hydrogen or methyl,
C represents hydrogen, methyl or hydroxy,
D means methyl

means or
D together with E stand for -CH ₂ -CH ₂ -NR ¹ -CH ₂ -, -CH ₂ -NR ¹ -CH ₂ -, -CH ₂ -NR ¹ -CH ₂ -CH ₂ -,
X represents a nitrogen atom,
Y is CH ₂ , CH ₂ -CH ₂ , CH ₂ -CH ₂ -CH ₂ or CH ₂ -CH,
Z represents a nitrogen atom, carbon atom or CH, where the bond between Y and Z can also be a double bond,
n represents the number 2, 3 or 4,
R ^{1 is} a hydrogen atom, a C ₁ -C ₄ alkyl group, an acetyl or benzoyl group, a phenylalkyl-C ₁ -C ₄ radical or phenyl alkoxy-C ₂ -C ₅ radical, the aromatic optionally being replaced by halogen, C ₁ -C ₄ alkyl, trifluoromethyl, hydroxy, C ₁ -C ₄ alkoxy, amino, cyano or nitro groups, a naphthylalkyl C ₁ -C ₃ radical, a phenylalkanone C ₂ - C ₄ radical or a phenyl or pyridylcarbamoylalkyl-C ₂ radical, where the phenyl or pyridyl group can be substituted by halogen, a C ₁ -C ₃ alkyl group, a methoxy group and by a nitro or amino group,
R ^{2 is} optionally mono by halogen atoms, C ₁ -C ₄ alkyl, trifluoromethyl, trifluoromethoxy, hydroxy, C ₁ -C ₄ alkoxy, amino, monomethylamino, dimethylamino, cyano or nitro groups, represents di- or trisubstituted phenyl, pyridyl, pyrimidinyl or pyrazinyl group, if appropriate with a benzene nucleus, optionally substituted by halogen atoms, C ₁ -C ₄ alkyl, hydroxy, trifluoromethyl, C ₁ -C ₄ - Alkoxy, amino, cyano or nitro groups can be mono- or disubstituted and can optionally contain 1 nitrogen atom, or can be fused with a 5- or 6-membered ring which can contain 1 to 2 oxygen atoms, or by a phenyl -C ₁ -C ₂ alkyl or alkoxy group can be substituted, where the phenyl radical can be substituted by halogen, a methyl, trifluoromethyl or methoxy group,
R ³ and R ⁴ independently of one another represent a hydrogen atom or a C ₁ -C ₄ alkyl group.

Because of their 5-HT _1A affinity, these compounds are suitable for the treatment of cerebral ischemia, in particular stroke.

5-HT _1A agonism plays a special role here, as can be seen from the work of SMITHKLINE BEECHAM (EP 345 948), BAYER / TROPON (EP 749 970; De Vry et al., Drugs of the Future 1997, 22 (4) , Pp. 341-349) and SUNTORY (WO 96/24594, WO 99/03847) can be seen.

It has now been found that the 3-substituted 5,6,7,8-tetrahydropyrido- [4 ', 3': 4,5] thieno [2,3-d] pyrimidine contained in the general formula (1) -4 (3H) -one derivatives of the formula I.

wherein
R ^{1 represents} a hydrogen atom or a C ₁ -C ₄ alkyl group,
and their physiologically tolerable salts,
particularly suitable for the production of medicaments for the prophylaxis and therapy of neurodegeneration, brain trauma and cerebral ischemia, particularly in the case of a stroke or the secondary diseases caused by these diseases.

One use according to the invention also relates to neuro protection.

These compounds of formula I can be prepared by using a compound of formula II

in which R ^{1 has} the meaning given above, R ^{3 represents} a C ₁ -C ₃ -alkyl-carboxylic acid ester group and R ^{4 is} C ₁ -C ₃ -alkyl, with ethanolamine of the formula IV

in an inert solvent, preferably alcohols such as. As ethanol, at temperatures between 60 and 120 ° C to the cyclization product V (D = OH)

which is then treated with a halogenating agent, such as. As thionyl chloride or hydrobromic acid, in an organic solvent such as a halogenated hydrocarbon or without solvent at temperatures between room temperature and 100 ° C in the corresponding halogen derivative V (D = Cl, Br) is performed. Finally, the halogen derivative of the formula V (D = Cl, Br) is set with 8- (1-piperazinyl) quinoline of the formula VI

to the end product of formula I according to the invention. This implementation works best in an inert organic solvent, preferably toluene or xylene, in the presence of a base, such as. B. Potassium carbonate or potassium hydroxide, at temperatures between 60 and 150 ° C.

The compounds of formula I according to the invention can either by recrystallization from the usual organic solutions agents, preferably from a lower alcohol such as ethanol crystallized or purified by column chromatography.

The free 3-substituted 5,6,7,8-tetrahydropyrido [4 ', 3': 4,5] - thieno [2,3-d] pyrimidine-4 (3H) -one derivatives of the formula I can be found in usually in the acid addition salts of a solution with the stoichiometric amount of the corresponding acid. pharmaceutical compatible acids are, for example, hydrochloric acid, phosphoric acid, Sulfuric acid, methanesulfonic acid, amidosulfonic acid, maleic acid, Fumaric acid, oxalic acid, tartaric acid or citric acid.

The compounds of the present invention have a surprisingly high affinity for the 5-HT _1A receptor, as shown by binding studies with cloned human 5-HT _1A receptors.

The following test setup was used to determine the Receptor binding affinity:

5-HT _1A binding assay with membranes of HEK293 cells expressing 5-HT _1A receptor

Culture of 5-HT _1A receptor expressing HEK293 cells 5-HT _1A expressing HEK293 cells are in RPMI / Glutamax medium (RPMI 1640, 25 mM Hepes, 2 mM Glutamax, 10% FCS, 2 mM Glutamine, penicillin / streptomycin (100 IU / ml each), Geneticin G-418 sulfates 400 mg / l, NaHCO ₃ 1.2 g / l) in culture bottles (TripleFlasks T-175) in a 5% CO ₂ atmosphere at 37 ° C in. After confluence is reached, the medium is removed and the bottles are filled with 15 ml of sterile PBS (phosphate buffered cream). The cells are incubated for 10 minutes (incubator, 37 ° C) with a trypsin solution (0.05% trypsin, 0.0004% EDTA, 0.02% EGTA, 2.682 mM KCL, 1.47 mM KH ₂ PO ₄ , 6.46 mM NaHPO ₄ , 136.89 mM NaCl). The detachment of the cells is promoted by tapping the bottom of the bottle. After transferring them into 50 ml tubes (Greiner), the cells are centrifuged at 250 × g at room temperature. The supernatant is discarded and the cells are resuspended in 10 ml of medium. The cells are redistributed to culture bottles and cultured for a further 5 to 6 days until the membranes are prepared.

Preparation of the membranes of 5-HT _1A receptor-expressing HEK293 cells

The supernatants of the cells are removed and the culture bottles are filled with PBS. The cells are then incubated for 10 minutes with a trypsin solution (for composition, see above). The detachment of the cells is promoted by tapping the bottom of the bottle. The cell suspension is removed and the remaining cells are also taken up in PBS by washing the culture twice with PBS. The collected cell suspension is distributed into 150 ml Falcon tubes and centrifuged for 10 minutes at 250 × g at 4 ° C. The supernatants are discarded and the cells in the pellet are resuspended in PBS. 20 μl of the cell suspension are removed and the cell density is determined. The cells are centrifuged again at 250 × g (4 ° C.) for 10 minutes, the supernatant is discarded and the cells in the pellet in 50 mM Tris-HCl pH 7.4 (1 ml / 10 ⁸ cells) using an Ultra-Turrax ( 30 sec) homogenized. The homogenate is distributed into cryotubes (1 ml / Kry tube) and stored in liquid nitrogen until use in the binding assay.

5-HT _1A binding assay

The frozen membranes are thawed at 37 ° C, at 48000. g (20 minutes), centrifuged and resuspended in binding buffer (50 mM Tris-HCl pH 7.4, 5 mM CaCl ₂ ). An incubation batch contains membrane material of 50 mg / sample, 0.15 µmol (= 0.15 nM) 3H-8-OH-DPAT as well as the substances to be tested in a total of 1 ml binding buffer. The non-specific binding is determined in the presence of 10 ^-5 M 5-carboxamidotryptamine. After incubation at 22 ° C. for 90 minutes, the bound and free ligand is separated from one another by filtration through CF / B filters and subsequent washing with 5 to 9 ml of ice-cold binding buffer. The GF / B filters are treated with 0.3% polyethyleneimine for at least 2 hours before use. After filtration, the filters are mixed with 3 to 4 ml Packard Ultima Gold XR and the radioactivity is determined by liquid scintillation counting in the Packard Tricarb.

Evaluation of the data from the 5-HT _1A binding assay

The displacement curves are analyzed by nonlinear regression using a modified version of the "Ligand" program by Munson & Rodbard (Anal. Biochem., 107, 220 (1980)). The value for the theoretical non-specific binding is estimated as the theoretical radioligand binding with an infinitisimally high ligand concentration. The measured values for the non-specific binding are treated as data points of the displacement curve, which correspond to measuring points at an infinitisimally high ligand concentration. When testing less than 4 concentrations of a substance or when the radioligand is specifically displaced <25% (at all concentrations tested), an IC ₅₀ value is estimated using the Hill equation and the K _i value according to the equation by Cheng and Prusoff (Biochem. Pharmacol. 22, 3099 (1973)).

The following results (K _i values) are obtained:

The following examples serve to illustrate the invention:

A Preparation of the starting materials of the formula II, V and VI

The starting materials of formula II and V are in the DE 196 36 769.7.

a) tert-Butyl4- (8-quinolinyl) -1-piperazine carboxylate

A solution of 9.0 g of 8-chloroquinoline (55.0 mmol), 10.2 g of tert-butyl-1-piperazine carboxylate (55.0 mmol), 0.66 g of 2- (di- (tert-butyl ) -phosphino) -1,1'-biphenyl (2.2 mmol) and 8.23 g of sodium tert-butoxide (85.6 mmol) in 300 ml of anhydrous toluene was mixed with 0.25 g of palladium (II) acetate (1.1 mmol) were added and the mixture was heated under reflux for 10 h under nitrogen. The reaction mixture was cooled and the solvent was removed. The residue obtained was taken up in ethyl acetate and extracted with saturated aqueous sodium chloride solution and dried over sodium sulfate. After removal of the solvent, 17.6 g of a crude product were obtained, which was purified by flash column chromatography (silica gel; heptane / ethyl acetate, 3/1). The main fraction obtained 13.3 g (77%) of the title compound: ¹ H-NMR (CDCl ₃ , 270 MHz) d = 1.5 (s, 9H), 3.35 (t, 4H), 3.8 (t, 4H), 7.17 (m, 1H), 7.4 (m, 1H), 7.45 (m, 2H), 8.15 (dd, 1H), 8.9 (m, 1H).

b) 8- (1-piperazinyl) quinoline

A mixture of 13.28 g tert-butyl 4- (8-quinolinyl) -1-piperazine carboxylate (42.38 mmol), 13.0 g trifluoroacetic acid (169.5 mmol) and 9.2 ml anisole ( 84.8 mmol) was heated to 80 ° C. with stirring for 3 h. The volatile constituents were then distilled off in vacuo, the residue obtained was taken up in dichloromethane, washed with saturated aqueous sodium bicarbonate solution and dried over sodium sulfate. After the solvent had been stripped off, 7.16 g of the slightly contaminated title compound were obtained, which was reacted further untreated: ¹ H-NMR (CDCl ₃ , 400 MHz) d = 2.0 (br, 1H), 3.25 (m, 4H), 3.4 (m, 4H), 7.15 (m, 1H), 7.4 (m, 1H), 7.45 (m, 2H), 8.1 (dd, 1H), 8.9 (m, 1H).

B Production of the end products example 1 7-methyl-3- [2- (4- (8-quinolinyl) -1-piperazinyl) ethyl] - 5,6,7,8-tetrahydropyrido [4 ', 3': 4.5] thieno [2,3-d] pyrimidine 4 (3H) -one

A mixture of 2.66 g of 3- (2-chloroethyl) -7-methyl-5,6,7,8-th trahydropyrido [4 ', 3': 4,5] thieno [2,3-d] pyrimidine- 4 (3H) -one (9.38 mmol), 1.0 g of 8- (1-piperazinyl) quinoline (4.69 mmol), 0.135 g of lithium hydroxide (5.63 mmol) and 0.35 g of sodium iodide (2nd , 35 mmol) was mixed with 25 ml of butanol and heated to 80 ° C. with stirring for 8 h. The reaction mixture was freed from the solvent, the residue was taken up in dichloromethane and washed with saturated aqueous sodium chloride solution and dried over sodium sulfate. After distilling off the solvent, a crude product was obtained, which was purified by medium pressure liquid chromatography (silica gel; MeOH in CH ₂ Cl ₂ , 0 to 100%). The main fraction obtained was 1.63 g of the slightly contaminated product, which was dissolved in dichloromethane and converted into a hydrochloride by adding 1 M ethereal HCl. The solid was filtered off, dichloromethane was added and the mixture was washed with 2 M sodium hydroxide solution. The organic phase was dried over sodium sulfate and concentrated. 1.07 g (50%) of the title compound were obtained: ¹ H-NMR (CDCl ₃ , 400 MHz) d = 2.5 (s, 3H), 2.8 (t, 2H), 2.85 (t, 2H), 2.9 (m , 4H), 3.4 (br, 4H), 3.65 (s, 2H), 4.15 (m, 2H), 7.15 (m, 1H), 7.4 (m, 1H), 7.45 (m, 2H), 8.0 (s, 1H), 8.1 (dd, 1H), 8.9 (m, 1H); LC-MS: m / z = 461.15 [MH] ⁺ .

Claims (3)

1. 3-substituted 5,6,7,8-tetrahydropyrido [4 ', 3': 4,5] -thieno [2,3-d] pyrimidin-4 (3H) -one derivatives of the formula I.
wherein
R ^{1 represents} a hydrogen atom or a C ₁ -C ₄ alkyl group
and their physiologically tolerable salts. 2. Use of compounds according to claim 1 for the preparation of medication. 3. Use according to claim 2 for prophylaxis and therapy of neurodegeneration, brain trauma and cerebral ischemia, especially stroke, or those caused by these diseases resulting sequelae.