WO1992011013A1

WO1992011013A1 - 5-cyanomethylene-10-(piperazine-1-yl)-dibenzo[a,d]cycloheptenes for relieving migraine

Info

Publication number: WO1992011013A1
Application number: PCT/EP1991/002379
Authority: WO
Inventors: Gerd Steiner; Alfred Bach; Liliane Unger; Manfred Raschack
Original assignee: Basf Aktiengesellschaft
Priority date: 1990-12-22
Filing date: 1991-12-12
Publication date: 1992-07-09
Also published as: DE4041463A1

Abstract

The description relates to the use of 5-cyanomethylene-10-(piperazine-1-yl)-dibenzo[a,d]cycloheptenes of formula (I), in which R?1, R2 and R3¿ have the significance given in the specification, for relieving migraine.

Description

5-cyanomethylene-10- (piperazin-1-yl) -dibenzo [a, d] cycloheptene to combat migraines

description

The present invention relates to a new use of 5-cyanomethylene-10- (piperazin-1-yl) dibenzo [a, d] cycloheptenes.

5-cyanomethylene-10- (piperazin-1-yl) -dibenzo [a, d] cycloheptenes are already known from EP-PS 35,711. Neuroleptic, sedative, hypnotic and tranquilizing effects have been described for these compounds.

It has now been found that 5-cyanomethylene-10- (piperazin-1-yl) dibenzo- [a, d] cycloheptenes of the formula I

in the

R ^{1 is} a hydrogen atom or a C _1-3 alkyl radical and

R ² and R ^{3 are} hydrogen atoms or one of the radicals R ² and R ^{3 can} also represent a fluorine or chlorine atom, and their salts with physiologically compatible acids also have a good action against migraines.

The new effect is particularly pronounced with (E), (Z) -5-cyanomethylene-2-chloro-10- (4-methylpiperazin-1-yl) -dibenzo [a, d] cyclo-heptene and especially with (Z) Isomers (A). The preparation of the compounds I is described in EP-PS 35 711. The same applies to the separation of the compounds I into the cis and trans isomers and for the preparation of galenical application forms with the compounds I.

The action of the compounds I against migraines results from their high affinity for 5-hydroxytryptamine (5-HT _1D ) receptors, which is considerably stronger than that of sumatriptan (Merck Index, 11th edition 1989: No. 8979).

The effect was shown by the following tests:

1) 5-HT _1D receptor test with cerebral cortex from bovine cattle brain was immediately placed in 0.32 M sucrose solution (4 ° C) and transported in ice. The frontal cortex was homogenized with about 5 volumes of sucrose solution at 0 ° C in a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 3000 × g for 15 min, the supernatant was collected and centrifuged at 40,000 × g (10 min, 4 ° C.). The pellet was washed twice with resuspension and recentrifugation with buffer (50 mM Tris-HCl, pH 7.4) and briefly homogenized. The homogenate was portioned and taken up in liquid nitrogen. The protein was determined using the BCA protein assay (Pierce, Rockford,

Illinois, USA).

The homogenate was diluted with incubation buffer (50 mM Tris-HCl, 10 μM pargylin, 4 mM CaCl ₂ ; 0.1% ascorbic acid, pH 7.7).

The test batches (1 ml) were composed of: 600 μg homogenate, 3 nM - ³ H-serotonin (NEN, Dreeich; specific radioactivity 23.2 Ci / mmol). To cover the 5-HT _1A and 5-HT _1C receptors, 100 nM 8-hydroxydipropylaminotetralin and 10 nM mianserin were added to the batches. The non-specific binding was determined with 1 μM serotonin; it was 65% of the total binding. 2) Vector construction for the transient expression of the

5HT _1D- like receptor

The cDNA coding for the 5HT _1D- like receptor was isolated from the vector mp18 with the aid of the restriction enzymes HindIII and EcoRI and was commercially available from the

Company Luvitrogen available BstXI linkers with the sequence

5 'C T T A G A G C A C A - 3'

G A A T C T C

Mistake. Then the modified DNA sequence of the SHT _1D- like receptor was also used by the company

Luvitrogen integrates commercially available cloning vector pCDM8. For this purpose, the vector was restricted with the enzyme BstXI and the so-called stuffer fragment was separated from the vector by gel electrophoresis. After the gel elution for the stuffer fragment shortened pCDMδ vector was ligated provided with BstXI linkers encoding the 5HT1 _D receptor -like DNA in the pre-treated as described above pCDM8. The ligation took place at 12 ° C and lasted 14 h. The methods used are described in "Current Protocols in Molecular Biology" Volumes I and II, published by Greene Publishing Associates and Wiley Interscience ISBN 0-471-50338-X.

3) Transient expression of the cloned 5HT _1D -like

Receptor in 293 cells

If not specifically described, the methods for cell culture can be found in "Cell and Tissue Culture" by Lindl and Bauer (Gustav Fischer Verlag).

293 cells were cultivated under standard conditions in a 10 cm cell culture dish up to a cell number of 7 to 8 × 106 cells. After trypsinization, the cells were diluted 1: 3 in MEM medium (Gibco 041 to 1090 M), which contained 2.2 g / l NaHCO ₃ , and sown again in 10 cm petri dishes. The cells were then cultured at 37 ° C. for 40 to 48 h. The DNA to be transfected was prepared as follows: 20 μg of the DNA solution (1 mg / ml), purified via

CsCl-Grandient, 437 ul H ₂ O were added, then 62.5 ul ₂ M CaCl _{2 were} added and finally

500 ul BBS. This order must be strictly observed. Formed within 10 min at room temperature

Ca ⁺⁺ precipitates.

The solution was placed on a 10 cm cell culture dish containing the 293 cells cultured as described above. After thorough mixing, the cells were cultured in a 3% CO ₂ incubator at 37 ° C. for 15 to 20 h. 5 ml of serum-free medium were then carefully added. After removing all of the medium and repeating the washing process with 5 ml of serum-free medium, 10 ml of complete medium were added to the cells. After 48 h of incubation in a 5% CO ₂ incubator, the cells could be used for pharmacological and electrophysiological studies. 2.5 ml of cold PBS was added to the transfected and cultured cells. After 5 minutes of incubation at room temperature, a further 5 ml of PBS were added and the cells were carefully washed down from the surface of the culture dish. The cell suspension was transferred to a centrifuge tube and centrifuged at approximately 1200 g for 10 minutes. After carefully removing the supernatant, the cells were used for receptor tests.

4) Receptor test with a rat 5-HT _1D- like transiently expressed receptor clone

Human embryonic kidney cells from Example 3 were taken up in 50 mM Tris-HCl, pH 7.4 and homogenized with ultrasound (Sinifer 20 sec, 90% at output 3, 0 ° C.). The homogenate was diluted with incubation buffer (50 μM Tris-HCl, 10 μM pargyline, 4 μM CaCl ₂ , 0.1% ascorbic acid, pH 7.7). After the homogenate had been incubated (30 min at 25 ° C.), the batches were filtered through glass fiber filters (Whatman GF / F) and washed with ice-cold buffer (50 μM Tris-HCl, pH 7.4). The radioactivity retained on the filters was determined by means of liquid scintillation measurement.

The test batch of 1 ml was composed of 35 μg of the homogenate obtained, 3 nM ³ Η-serotonin (NEN, Dreeich; specific radioactivity 23.2 Ci / mmol). Non-specific binding was carried out with serotonin and was 15% of the total binding.

The competition curves were evaluated using iterative nonlinear regression analysis based on the LIGAND program (Munson and Rodbard: Anal. Biochem. 107, 220, (1980).

Results

Ki (nM)

Substance 5-HT _1D 5-HT _1D -like

Cerebral cattle receptors

A 30 6.5

Sumatriptan 50 11 The methods used are described in C. Waeber et al .:

5-HT _1D receptors in guinea-pig and pigeon brain radioligand binding and biochemical studies, Naunyn-Schmiedeberg's

Arch. Pharmacol. 340: 479-485 (1989). For the new indication, the compounds I in one

Dosage of 10-200 mg per patient per day with oral administration or 1-100 mg per patient per day with parenteral (intravenous, intramuscular) administration.

Claims

1. Use of 5-cyanomethylene-10- (piperazin-1-yl) -dibenzo- [a, d] cycloheptenes of the formula I

in the

R ^{1 is} a hydrogen atom or a C _1-3 alkyl radical and R ² and R ^{3 are} hydrogen atoms or one of the radicals R ² and R ^{3 can} also represent a fluorine or chlorine atom, and their salts with physiologically tolerable acids for combating migraines .

2. Use of (E), (Z) -5-cyanomethylene-2-chloro-10- (4-methylpiperazinl-yl) -dibenzo [a, d] cycloheptene and its salts with physiologically acceptable acids to combat migraines.

3. Use of (Z) -5-cyanomethylene-2-chloro-10- (4-methylpiperazinl-yl) -dibenzo [a, d] cycloheptene and its salts with physiologically acceptable acids to combat migraines.