EP2124955A2

EP2124955A2 - Inhibition of pde2a

Info

Publication number: EP2124955A2
Application number: EP07818641A
Authority: EP
Inventors: Peter Ellinghaus; Andreas Wilmen; Martin Hendrix; Adrian Tersteegen
Original assignee: Bayer Schering Pharma AG
Current assignee: Bayer Pharma AG
Priority date: 2006-10-14
Filing date: 2007-10-02
Publication date: 2009-12-02
Also published as: CA2666355A1; WO2008043461A3; JP2010506561A; US20100035882A1; DE102006048693A1; WO2008043461A2

Abstract

The invention relates to the use of PDE2A inhibitors for producing a medicament for the treatment and/or prophylaxis of coronary heart diseases, particularly stable or instable angina pectoris, acute myocardial infarct, myocardial prophylaxis, heart insufficiency, hypertension and conditions resulting from atherosclerosis, vascular and renal diseases, particularly kidney failure, inflammatory diseases, erectile disorders and the prevention of sudden cardiac death.

Description

Inhibition of PDE2A

The invention relates to the use of PDE2A inhibitors for the production of a medicament for the treatment and / or prophylaxis of heart diseases, in particular heart failure and its underlying cardiomyopathies such as dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM), arrhythmogenic right ventricular cardiomyopathy ( ARVCM), myocarditis and in particular hypertrophic cardiomyopathy (HCM). In addition, the treatment of erectile dysfunction, hypertension and prevention of arteriosclerosis with PDE2A inhibitors is the subject of the invention.

Above all, arterial hypertension damages the brain as well as the kidneys and blood vessels. Prior to the introduction of antihypertensive therapy, arterial hypertension was the leading cause of heart failure and myocardial infarction. Even today, the risk of becoming heart failure and suffering a heart attack is greater even for an antihypertensive hypertensive patient than for a normotensive. Left ventricular hypertrophy is the principal structural mechanism of adaptation of the myocardium to chronic pressure in the course of arterial hypertension. The extent of myocardial hypertrophy increases with the level of blood pressure.

In the early stages of arterial hypertension, the systolic wall tension - the left ventricular afterload - is increased due to the systolic pressure loading on the ventricular wall. The developing left ventricular hypertrophy leads to a normalization of the wall tension due to increase in thickness of the myocardial walls. In this early stage of concentric left ventricular hypertrophy, the left ventricle is able to require a normal cardiac output at a normal cardiac energy expenditure per unit weight myocardium despite hypertensive systolic blood pressure values. Already at this stage there is a disturbance of the diastolic function of the left ventricle.

Chronic compression of the left ventricle leads to abnormal activation of fetal growth factors that alter protein biosynthesis and fetal muscle gene products. Angiotensin II, norepinephrine and other growth hormones have a growth-promoting effect on the myocardium. This results in a further stimulation of the development of myocardial hypertrophy, regardless of the systolic pressure load. Depending on the extent of the growth hormone, a hypotrophic myocardial hypertrophy, which is similar to a hypertrophic cardiomyopathy, may develop. Due to the fact that the adult myocardial tissue is little or no longer capable of cell division due to the blockade of the cell cycle, a pathological hypertrophic reaction of the myocardium results at the molecular level. Cardiac hypertrophy is not a physiological adaptation mechanism to the chronic long-term burden. It is an independent risk factor for cardiac events such as myocardial infarction, heart failure and sudden cardiac death [I].

Therapy methods and agents that prevent cardiac hypertrophy are thus suitable for the treatment of symptoms of the above-mentioned diseases.

The DNA sequence encoding human PDE2A is shown in the Sequence Listing in SEQ ID NO: 1. The amino acid sequence of human PDE2A is shown in SEQ ID NO: 2 of the Sequence Listing.

As shown in Figure 1, it has surprisingly been observed in a cellular hypertrophy model that expression of PDE2A mRNA is increased upon induction of hypertrophy. For this purpose, the rat cardiomyocyte cell line H9c2 (ATCC number: CRL-1446) was supplemented with arginine vasopressin by a hypertrophy stimulus, which may, inter alia, be detected. a. in increased expression of marker genes for cardiac hypertrophy such as ANP (atrial natriuretic peptide) and MYHCB (myosin heavy chain beta-subunit) [2]. Increased expression of the cGMP-hydrolyzing PDE2A can lower the intracellular cGMP level of the cardiomyocytes and thereby suppress the anti-hypertrophic effect of cGMP [3, 4]. It can be deduced from the present observation that the increased expression of PDE2A in hypertrophic H9c2 cells also contributes in vivo to the pathogenesis of cardiac hypertrophy, and inhibition of PDE2A activity by a small molecule drug has a positive effect on cardiac hypertrophy. the cGMP level in the cardiomyocytes is high, and thus the anti-hypertrophic effect of cGMP is retained. [5] To test this hypothesis, H9c2 cells were stimulated with arginine vasopressin and incubated with the PDE2 inhibitor BAY 60-7550 [6]. BAY 60-7550 is the substance 2- (3,4-dimethoxybenzyl) -7- [1- (1-hydroxyethyl) -4-phenylbutyl] -5-methylimidazo [5, lf] [l, 2,4] triazine-4 (3H) -ones having the structural formula:

As shown in Figure 2, the PDE2A inhibitor BAY 60-7550 can suppress the increase in the hypertrophy marker gene MYHCB in a dose-dependent manner. To verify that the incubation of H9c2 cells with the PDE2A inhibitor BAY 60-7550 also increase the intracellular cGMP level and thereby antihypertrophic, the intracellular cGMP content was determined after stimulation of cGMP synthesis by ANP in the presence of the PDE2A inhibitor BAY 60-7550 by EIA. As shown in Figure 3, BAY 60-7550 dose-dependently increases intracellular cGMP levels in H9c2 cells. To verify the in v / fro findings, the antihypertrophic effect of the PDE2 inhibitor BAY 60-7550 in vivo was investigated in a mouse hypertrophy model. For this purpose, 2 mg / kg of isoprenaline was administered subcutaneously once a day to mice of strain C57BL6, and as a positive control in addition to isoprenaline 10 mg / kg of enalapril was administered via the drinking water. BAY 60-7550 was co-administered twice daily with isoprenaline injection at a dose of 10 mg / kg ip. As can be seen from Figure 4, the infusion of isoprenaline increases the animals' heart weight in relation to body weight. Like the positive control enalapril, administration of the PDE2A inhibitor BAY 60-7550 in both dose groups resulted in a significant reduction in the ratio of heart weight to body weight.

The data show that PDE2A inhibition could also prevent cardiac hypertrophy in humans.

The present invention therefore relates to the use of PDE2A inhibitors for the manufacture of a medicament for the treatment and / or prophylaxis of the following diseases: coronary heart disease, in particular stable and unstable angina, acute myocardial infarction, myocardial infarction, sudden cardiac death, heart failure and hypertension and the Consequences of atherosclerosis, as well as vascular diseases, kidney diseases, and erectile dysfunction.

Antagonists within the meaning of the invention are all substances which bring about an inhibition of the biological activity of PDE2A. Particularly preferred antagonists are nucleic acids including "locked nucleic acids", "peptide nucleic acids" and "Spiegelmers", proteins including antibodies and low molecular weight substances, very particularly preferred antagonists are low molecular weight substances.

The invention relates to:

1. Use of a PDE2A polypeptide or a nucleic acid encoding a PDE2A polypeptide in a test system for the discovery of inhibitors of PDE2A suitable for the treatment and / or prophylaxis of heart failure and its underlying cardiomyopathies. - A -

A nucleic acid encoding a PDE2A polypeptide is a nucleic acid selected from the group consisting of:

a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence disclosed by SEQ ID NO: 2 as well as functional fragments thereof;

b) nucleic acid molecules comprising the sequence shown in SEQ ID NO: 1 as well as functional fragments thereof;

c) nucleic acid molecules whose complementary strand hybridizes with a nucleic acid molecule from a) or b) under stringent conditions and which have the biological function of a PDE2A, wherein a stringent hybridization of nucleic acid molecules in an aqueous solution containing 0.2 × SSC ( Ix standard saline citrate = 150mM NaCl, 15mM trisodium citrate), performed at 68 ^° C (Sambrook et al., 1989); and

d) Nucleic acid molecules which differ from those mentioned under c) due to the degeneration of the genetic code.

A PDE2A polypeptide according to the invention is a polypeptide which is encoded by one of the nucleic acids mentioned under a) - d). In particular, a polypeptide comprising the sequence shown in SEQ ID NO: 1 or comprising a fragment thereof having PDE2A activity is a PDE2A polypeptide.

2. Use according to item 1, which is a cell-free test system.

3. Use according to item 1, which is a test system using whole cells containing a nucleic acid encoding a PDE2A. In this case, the nucleic acid may have been introduced endogenously or recombinantly introduced.

4. Use according to items 1 - 3, wherein a PDE2A activity is measured.

5. Use according to item 4, wherein the cGMP or the GMP level is measured.

6. Use according to items 1 - 3, wherein the expression of PDE2A is measured.

7. Use according to items 1-6, wherein the cardiac insufficiency is cardiomyopathy-induced cardiac insufficiency selected from the group of cardiomyopathies consisting of dilated cardiomyopathy (DCM), the restrictive ones Cardiomyopathy (RCM), arrhythmogenic right ventricular cardiomyopathy (ARVCM), myocarditis and / or hypertrophic cardiomyopathy (HCM).

8. Use of a PDE2A inhibitor, which has been identified by means of one of the methods according to points 1-7, for the manufacture of a medicament for the treatment and / or prophylaxis of cardiac insufficiency.

9. Use of a PDE2A inhibitor which has been identified by means of one of the methods according to items 1-7 for the manufacture of a medicament for the treatment and / or prophylaxis of cardiomyopathy-induced cardiac insufficiency selected from the group of cardiomyopathies consisting of dilated cardiomyopathy ( DCM), restrictive cardiomyopathy (RCM), the arrhythmogenic right ventricular

Cardiomyopathy (ARVCM), myocarditis and / or hypertrophic cardiomyopathy (HCM).

10. Use of a PDE2A-specific antibody, a PDE2A-specific antisense oligonucleotide or a PDE2A-specific siRNA for the production of a medicament for the treatment and / or prophylaxis of cardiac insufficiency.

11. Use of a PDE2A-specific antibody, a PDE2A-specific antisense oligonucleotide or a PDE2A-specific siRNA for the manufacture of a medicament for the treatment and / or prophylaxis of cardiomyopathy-induced heart failure, selected from the group of cardiomyopathies consisting of dilated cardiomyopathy ( DCM), restrictive cardiomyopathy (RCM), arrhythmogenic right ventricular cardiomyopathy (ARVCM), myocarditis and / or hypertrophic cardiomyopathy (HCM). An siRNA is a "short interfering RNA." Those skilled in the art are familiar with methods for providing PDE2A-specific antisense oligonucleotides, antibodies or siRNAs, and suitable antisense oligonucleotides, siRNAs or antibodies ultimately lead to an inhibition of PDE2A

Activity. This can be done by a mechanism that directly involves the PDE2A protein, or at the level of transcription or translation of PDE2A.

12. Use of PDE2A inhibitor for the manufacture of a medicament for the treatment and / or prophylaxis of heart failure.

13. Use according to item 12, wherein the cardiac insufficiency is a cardiomyopathy-induced cardiac insufficiency selected from the group of cardiomyopathies consisting of dilated cardiomyopathy (DCM), the restrictive cardiomyopathy myopathy (RCM), arrhythmogenic right ventricular cardiomyopathy (ARVCM), myocarditis and / or hypertrophic cardiomyopathy (HCM).

14. Use according to item 12 or 13, wherein the PDE2A inhibitor has an IC ₅₀ value of less than 1 μM.

15. Use according to item 12 or 13, wherein the PDE2A inhibitor has an IC ₅₀ value of less than 100 nM.

PDE2A inhibition may e.g. be measured in the PDE2A inhibition test described below.

In this case, preference is given to those PDE2A antagonists which inhibit in the PDE2A inhibition test indicated below with an IC ₅₀ of 1 μM, preferably with an IC ₅₀ of less than 0.1 μM.

Preferably, the PDE2A inhibitors according to the invention can not pass the blood / brain barrier and act systemically and not centrally.

The present invention also provides the use of compounds of the general formula (I)

Wonn

R ^{1 is} phenyl, naphthyl, quinolinyl or isoquinolinyl, which are up to three times identical or different with radicals selected from the group consisting of (C 1 -C ₄ ) -alkyl, (C 1 -C ₄ ) -alkoxy, halogen, cyano, -NHCOR ⁸ , -NHSO ₂ R ⁹ , -SO ₂ NR ¹⁰ R ¹¹ , -SO ₂ R ¹² , and -NR ¹³ R ¹⁴ may be substituted, means

wherein

R ⁸ , R ¹⁰ , R ", R ¹³ and R ^{14 are} independently hydrogen or (C _r C ₄ ) alkyl, and

R ⁹ and R ¹² are independently (C _r _C4) alkyl, or

R ¹⁰ and R ¹¹ together with the adjacent nitrogen atom azetidin-1-yl, pyrrol-1-yl, piperid-1-yl, azepin-1-yl, 4-methyl-piperazin-l-yl or morpholine Form -1-yl radical,

or

R ¹³ and R ¹⁴ together with the adjacent nitrogen atom azetidin-1-yl, pyrrol-1-yl, piperid-1-yl, azepin-1-yl, 4-methyl-piperazin-l-yl or morpholine Form -1-yl radical,

R ² and R ³ independently of one another denote hydrogen or fluorine,

R ⁴ (CC.) - Alkyl.

R ^{5 is} (C ₁ -C ₃ ) -alkyl,

R ^{6 is} hydrogen or methyl,

R ⁷ is phenyl, thiophenyl, furanyl, up to three times identically or differently by radicals selected from the group consisting of may be substituted (Ci-C _t) alkyl, (Ci-C ₄₎ -alkoxy, halogen and cyano, or ( C ₅ -C ₈ ) -cycloalkyl,

L is carbonyl or hydroxymethanediyl, and

M is (C ₂ -C ₅ ) -alkanediyl, (C ₂ -C ₅ ) -alkendiyl or (C ₂ -C ₅ ) -alkanediyl,

and their physiologically acceptable salts for the manufacture of a medicament for the treatment and / or prophylaxis of cardiac insufficiency.

In the context of the invention, C ₁ -C 4 -alkyl and (C ₁ -C ₁₎ -valent are a straight-chain or branched alkyl radical having 1 to 4 or 1 to 3 carbon atoms, for example: methyl, ethyl, n-propyl, isopropyl, isobutyl, , t-butyl, preferred are methyl and ethyl.

(In the context of the invention, Cr-CO-alkanediyl represents a straight-chain or branched alkanediyl radical having 2 to 5 carbon atoms, for example ethylene, propan-1,3-diyl, propan-1,2-diyl, propane-2,2- Diyl, butane-l, 3-diyl, butane-2,4-diyl, pentane-2,4-diyl. A straight-chain (C ₂ -C ₅ ) -alkane-l, ω-diyl radical is preferred called ethylene, propane-1,3-diyl, butane-1,4-diyl, pentane-l, 5-diyl. Particularly preferred are propane-1,3-diyl and butane-1,4-diyl. (C2-dV) -Alkendiyl in the context of the invention represents a straight-chain or branched alkenediyl radical having 2 to 5 carbon atoms. Examples which may be mentioned ethene-l, 2-diyl, ethene-1,1-diyl, propene-1,1-diyl, propene-1,2-diyl, prop-2-en-l, 3-diyl, propene-3 , 3-diyl, propene-2,3-diyl, but-2-en-1, 4-diyl, pent-2-en-1, 4-diyl. Preference is given to a straight-chain (C ₂ -C ₅ ) -alkene-1, (o-diyl radical. Examples which may be mentioned are ethene-1,2-diyl, prop-2-en-1, 3-diyl, but-2-one en-1, 4-diyl, but-3-en-1, 4-diyl, pent-2-en-1, 5-diyl, pent-4-en-1, 5-diyl Particularly preferred are prop-2 -en-l, 3-diyl, but-2-en-l, 4-diyl and but-3-en-l, 4-diyl.

In the context of the invention, (Cϊ-Cs) alkanediyl represents a straight-chain or branched alkynediyl radical having 2 to 5 carbon atoms, for example ethin-1, 2-diyl, ethyn-1,1-diyl, prop-2-yn 1, 3 - diyl, prop-2-yn-1, 1-diyl, but-2-yn-1, 4-diyl, pent-2-yn-1, 4-diyl, Preferred is a straight-chain (C ₂ -C ₅ ) - Alkene-l, ω-diyl radical. Examples which may be mentioned ethyne-1,2-diyl, prop-2-yn-l, 3-diyl, but-2-yn-l, 4-diyl, but-3-yn 1-l, 4-diyl, pent-2-yn-l, 5-diyl, pent-4-yn-l, 5-diyl. Particular preference is given to prop-2-yn-1, 3-diyl, but-2-ynyl. l, 4-diyl and but-3-yn-l, 4-diyl.

In the context of the invention, (C ₁ -Q) -alkoxy is a straight-chain or branched alkoxy radical having 1 to 4 carbon atoms. Examples which may be mentioned are: methoxy, ethoxy, n-propoxy, isopropoxy, t-butoxy, n-pentoxy and n-hexoxy. Preferred are methoxy and ethoxy.

(Cs-CgVcycloalkyl in the context of the invention represents cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl. Preference may be given to cyclopentyl, cyclohexyl or cycloheptyl.

Halogen is in the context of the invention in general for fluorine, chlorine, bromine and iodine. Preference is given to fluorine, chlorine and bromine. Particularly preferred are fluorine and chlorine.

As salts, physiologically acceptable salts of the compounds according to the invention are preferred in the context of the invention.

Physiologically acceptable salts of the compounds according to the invention may be acid addition salts of the substances according to the invention with mineral acids, carboxylic acids or sulphonic acids. Particularly preferred are e.g. Salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, propionic acid, lactic acid, tartaric acid, citric acid, fumaric acid, maleic acid or benzoic acid.

However, salts with customary bases can also be mentioned as salts, for example alkali metal salts (for example sodium or potassium salts), alkaline earth salts (for example calcium or magnesium salts) or ammonium salts derived from ammonia or organic amines such as, for example, Example, diethylamine, triethylamine, ethyldiisopropylamine, procaine, dibenzylamine, N-methylmorpholine, dihydroabietylamine, 1-ephenamine or methyl-piperidine.

The compounds of the invention may exist in stereoisomeric forms that behave either as image and mirror image (enantiomers) or that do not behave as image and mirror image (diastereomers). The invention relates to both the enantiomers or diastereomers or their respective mixtures. The racemic forms can be separated as well as the diastereomers in a known manner in the stereoisomerically uniform components.

The use of compounds of general formula (I), wherein R ¹ is phenyl, whose meta and / or para positions up to three times identically or differently by radicals selected from the group consisting of (C _r _C4) alkyl is preferably (C _r C ₄₎ -alkoxy and -SO ₂ NR ¹⁰ R ¹¹ are substituted, group, and R ^2, R ^3, R ^4, R ^5, R ^6, R ^_7, R ^10, R ^11, L and M are have the abovementioned meaning for the preparation of a medicament for the treatment and / or prophylaxis of cardiac insufficiency.

The meta and para positions of the phenyl ring are to be understood as meaning those positions which are meta or para to the CR ² R ³ group. These positions can be illustrated by the following structural formula (Ic):

Particularly preferred is the use of compounds of the general formula (Ic) in which the para and a meta position of the phenyl radical are substituted, and the second meta position is unsubstituted for the manufacture of a medicament for the treatment and / or prophylaxis of heart failure.

Also preferred is the use of compounds of general formula (I) wherein R ^{7 is} phenyl and R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , L and M have the abovementioned meaning for the preparation of a medicament for the treatment and / or prophylaxis of heart failure.

Very particular preference is given to the use of compounds of the general formula (I)

in which R ¹ phenyl, its meta and / or para positions up to three times the same or different with radicals selected from the group consisting of (Ci-C ₄ ) alkyl, (C _r C ₄ ) alkoxy and -SO ₂ NR ¹⁰ R "are substituted, naphthyl or quinolinyl,

wherein R ¹⁰ and R ¹¹ are independently hydrogen or (C _r C ₄₎ alkyl,

R ¹ and R ^{2 are} hydrogen,

R ⁴ is methyl or ethyl,

R ^{5 is} methyl,

R ^{6 is} hydrogen or methyl,

L is carbonyl or hydroxymethanediyl, and

M is straight-chain (C ₂ -C ₅ ) -alkane-1, α) -diyl, straight-chain (C ₂ -C ₅ ) -alkene-1, ω-diyl or straight-chain (C ₂ -C ₅ ) -alkyne-1, ω -diyl means for the manufacture of a medicament for the treatment and / or prophylaxis of cardiac insufficiency.

Likewise preferred is the use of the substance 2- (3,4-dimethoxybenzyl) -7- [1- (1-hydroxyethyl) -4-phenylbutyl] -5-methylimidazo [5, lf] [l, 2,4] triazine-4 (3H) -ones having the structural formula:

for the manufacture of a medicament for the treatment and / or prophylaxis of cardiac insufficiency.

Also preferred is the above-described use of the structural formulas disclosed above, wherein cardiac insufficiency is cardiomyopathy-induced cardiac insufficiency selected from the group of cardiomyopathies consisting of dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM), arrhythmogenic cardiomyopathy right ventricular cardiomyopathy (ARVCM), myocarditis and / or hypertrophic cardiomyopathy (HCM). The compound described above, their action as PDE2 inhibitors and processes for their preparation are known from WO 02/050078 Al.

Description of the figures:

Fig. 1: Comparison of the relative expression of PDE2A RNA in H9c2 cells after 72 h incubation with 1 micromole of arginine vasopressin. Shown is the expression of PDE2A relative to L32 ribosomal protein (rE) in H9c2 rat cardiomyocytes. The results are tabulated (mean of the relative expression rE from a triplicate, mean of the Ct values for PDE2A and L32).

Ct r-PDE2A Ct r-L32 rE MW STABW

Control

29.35 17.35 160.90 159.73 12.15

Control

29.47 17.35 147.03

Control

29,48 17,01 171,25

Vasopressin lμM

30.89 16.31 584.07 613.64 228.15

Vasopressin l μM

32.38 16.23 401.71

Vasopressin l μM

31.14 16.55 855.13

Fig. 2: Relative expression of the hypertrophic marker MYHCB in H9C2 rat cells after stimulation by vasopression ± PDE2A inhibitor BAY 60-7550.

Shown is the expression of ANP and MYHCB relative to L32 ribosomal protein in

H9C2 cells after 72 h incubation with vasopressin (0.1 micromol, 1 micromal, 10 micromol). It has been shown that the simultaneous presence of the PDE2A inhibitor BAY 60-7550 dose-dependently suppressed the induction of the hypertrophic marker MYHCB by vasopressin. Following are the Results listed in tabular form. (Mean of the relative expression rE from a duplicate determination).

Ct r-MYHCB Ct r-L32 rE MW STABW

Control

34.99 16.07 0.53 Control 34.77 15.69 0.47 0.50 0.03

Vasopressin 1 μM 30.01 16.29 19.43 Vasopressin 1 μM

2,20 30,21 16,12 15,03 17,23

Vasopressin 1 μM + 0.1 μM BAY 60-7550 31.97 17.14 9.00

Vasopressin 1 μM + 0.1 μM BAY

3.44 60-7550 30.46 16.45 15.89 12.44

Vasopressin 1 μM + 1 μM BAY 60-7550 32.76 17.16 5.28

Vasopressin 1 μM + 1 μM BAY 60-7550 31.12 3.26

16.68 11.79 8.54

Vasopressin 1 μM + 10 μM BAY 60-7550 32.72 16.91 4.56

Vasopressin 1 μM + 10 μM BAY 60-7550 33.75 0.78

17.34 3.01 3.79

Fig. 3: Change in intracellular cGMP concentration in H9c2 rat cardiomyocytes after stimulation with ANP in the presence of different concentrations of the PDE2A inhibitor BAY 60-7550. Shown is the cGMP content in the cells after 15 min incubation with ANP and the indicated dosages of the PDE2A inhibitor BAY 60-7550. It can be seen that the PDE2A inhibitor BAY 60-7550 increases dose-dependently and synergistically the intracellular cGMP level in the H9c2 cells.

Fig. 4: Change in heart weight compared to body weight (HW: BW) by subcutaneous isoprenaline (2 mg / kg / d) and the influence of enalapril as a positive control 810 mg / kg / d in drinking water) or the PDE2A inhibitor BAY 60-7550. Shown is the quotient of heart weight: body weight depending on the treatment. It turns out that the PDE2A inhibitor BAY 60-7550 given in two independent animal groups at 10 mg / kg / d (ip) for 5 days can suppress isoprenaline-induced weight gain almost as well as the positive control enalapril.

5 shows the cDNA sequence of the human PDE2A (Accession No. NM_002599, SEQ ID NO: 1).

Figure 6: Figure 6 shows the amino acid sequence of human PDE2A (Accession No. NP_002590, SEQ ID NO: 2).

Studies of PDE2A expression and MYHCB expression in H9c2 rat cells.

The relative expression of PDE2A in H9c2 rat cells is determined by quantifying the mRNA using the real-time polymerase chain reaction [7]. Compared to classical PCR, real-time PCR offers the advantage of more accurate quantification by introducing an additional, fluorescently labeled oligonucleotide. This so-called probe contains the fluorescent dye FAM (6-carboy-fluorescein) at the 5 'end and the fluorescence quencher TAMRA (6-carboxy-tetramethylrhodamine) at the 3' end. During the polymerase chain reaction, in the TaqMan PCR, the fluorescence dye FAM is cleaved off the probe by the 5'-exonuclease activity of the Taq polymerase, thereby obtaining the previously quenched fluorescence signal. The threshold value [treshold cyle (Ct value)] records the number of cycles at which the fluorescence intensity is approximately 10 standard deviations above the background fluorescence.

From the H9c2 rat cardiomyocytes, the total RNA is isolated from a 6-well (approximately 4 × 10 ⁵ cells) using an RNeasy kit (Qiagen Hilden). 1 μg of total RNA per tissue is removed for 15 minutes at room temperature to remove contaminations with genomic DNA with 1 unit of DNase I (Invitrogen). Dnase I is inactivated by adding 1 μl of EDTA (25 mM) followed by heating to 65 ° C. (10 min).

Subsequently, in the same reaction batch, the cDNA synthesis is carried out according to the instructions for the "SUPERSCRIPT-π RT cDNA synthesis kit" (Invitrogen) and the reaction volume is made up to 200 μl with distilled water.For the PCR, 5 μl each of the diluted cDNA Solution 7.5 μl mixture of primer and probe and 12.5 μl TaqMan reaction solution (qPCR master mix, Eurogentec Co.) The final concentration of the primers is 300 nM, that of the probe 150 nM "Forward" and "reverse" primers for the rat PDE2A are: 5'-CCAAATCAGGGACCTCATATTCCO '(SEQ ID NO: 3) and 5'-GGTGTCCCACAAGTTCACCAT-3' (SEQ ID NO: 4), the sequence of the fluorescent Probe 5'-6FAM-AACAACTCGCTGGATTTCCTGGA-TAMRA-S '(SEQ ID NO: 5) "Forward" and "reverse" primers for r-MYHCB is: 5'-TGGAGAACGACAAGCAGCAG-S '(SEQ ID NO: 6) or 5'-CCTGGCGTTGAGTGCATTTA-S' (SEQ ID NO: 7), the sequence the fluorescent probe 5'-6FAM-TGGATG AGCGACTC AAAAAG AAGG ACTTTG-T AMRA-3 '(SEQ ID NO: 8).

The PCR is carried out on an ABI Prism SDS 7700 instrument (Applied Biosystems) in accordance with the manufacturer's instructions. 40 cycles are carried out. The Ct value (see above) obtained for each gene in the respective cDNA corresponds to the cycle in which the fluorescence intensity of the released probe is about 10 standard deviations above the background signal. The lower the Ct value, the sooner the duplication begins, ie the more mRNA is contained in the original sample. In order to compensate for possible variations in the cDNA synthesis, the expression of a so-called "housekeeping gene" is analyzed in all the samples examined, which should always be expressed equally independently of the treatment of the cells L32 ribosomal protein is used The sequence of the "forward" or "reverse" primer for rat L32 is 5'-GAAAGAGCAGCACAGCTGGC-S '(SEQ ID NO: 9), and 5'-TCATTCTCTTCGCTGCGTAGC-3' (SEQ ID NO: 10), the sequence of the probe 5'-6FAM-TCAGAGTCACCAATCCCAACGCCA-T AMRA-3 '(SEQ ID NO: 11) The evaluation of the data is carried out as follows: For each RNA, the dCt value is calculated. Value is the difference between the Ct value for the candidate gene (ie: MYHCB or PDE2A) and the Ct value of the household gene in the respective tissue, from which value a relative expression rE is calculated according to the following formula: r _{E = 2} (18- dC) _

Determination of cGMP content in H9c2 cells after preincubation with the PDE2A inhibitor BAY 60-7550

The determination of the intracellular cGMP content in H9c2 cells was carried out using the Biotrak (EIA) immunoassay from Amersham (catalog No. RPN 226) in accordance with the manufacturer's protocol. For this purpose 105 H9c2 cells / well are seeded overnight in 12 well plates and after washing with 1 × PBS (1 ml) for 15 min at room temperature with 800 μl medium without FCS and the indicated concentrations of the PDE2A inhibitor BAY 60- 7550 and ANP incubated. The supernatants are discarded and the cells are mixed with 500 ml ice-cold 70% ethanol. After 2 minutes shaking at room temperature (150 u / min), the plates are frozen at -20 ⁰ C overnight and transferred to the lysed cells after thawing in Eppendorf vessels. After evaporation of the ethanol in a Speed-Vac (3 h at 35 ° C), the samples are reconstituted in 200 μl assay buffer and worked up as indicated in the kit description. The measurement of Fluorescence is done at 450/570 in a Tecan Spectrafluor photometer. The OD values obtained are converted into fmol / well using the standard calibration curve according to the kit instructions.

Test of PDE2A inhibition

PDE2A Assays Formats for identifying PDE2A inhibitors are known to those of skill in the art. An example of a possible PDE2A activity test system format is described below.

Human PDE2A (GenBank / EMBL Accession Number: NM_002599, Rosman et al., Gene 1997, 191, 89-95) is expressed in Sf9 insect cells using the Bac-to-Bac ™ baculovirus expression system. 48 h post-infection, cells are harvested and placed in lysis buffer (20 mL / L culture, 50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1 mM MgC12, 1.5 mM EDTA, 10% glycerol, 20 μL Protease Inhibitor Cocktail Set in [CalBiochem, La Jolla, CA USA]). The cells are disrupted at 4 ° C for using ultrasound and then centrifuged for 30 min at 4 ⁰ C at 15,000. The supernatant (PDE2A preparation) was collected and stored at -8O ⁰ C.

The test substances are dissolved in 100% DMSO and serially diluted to determine their in vitro effect on PDE 2A. Typically, dilution series from 200 μM to

1.6 μM (resulting final concentrations in the assay: 4 μM to 0.032 μM). 2 μL each of the diluted substance solutions are placed in the wells of microtiter plates (Isoplate, Wallac Inc., Atlanta, GA). Subsequently, 50 μL of a dilution of the PDE2A preparation described above are added. The dilution of the PDE2A preparation is chosen such that during the later incubation less than 70% of the substrate is reacted (typical dilution: 1: 200,000; dilution buffer: 50 mM Tris / HCl pH 7.5, 8.3 mM MgC12;

1.7 mM EDTA, 0.2% BSA). The substrate, [5 ', 8-3H] adenosine 3', 5'-cyclic phosphate (1 μCi / μL; Amersham Pharmacia Biotech., Piscataway, NJ), is assayed 1: 2000 with assay buffer (50 mM Tris / HCl pH 7 5, 8.3 mM MgC12, 1.7 mM EDTA) to a concentration of 0.0005 μCi / μL and cGMP (1 μM final concentration in the assay), which serves to stimulate PDE2. By adding 50 μL (0.025 μCi) of this substrate solution, the enzyme reaction is finally started. The test mixtures are incubated for 60 min at room temperature and the reaction is stopped by addition of 25 μL of a suspension of 18 mg / mL Yttrium Scintillation Proximity Beads (Amersham Pharmacia Biotech., Piscataway, NJ). The microtiter plates are sealed with a foil and left for 60 min at room temperature. The plates are then measured for 30 seconds per well in a Microbeta scintillation counter (Wallac Inc., Atlanta, GA). IC50 values are determined by plotting the concentration of the substance versus percent inhibition. Inhibition of PDEs 1, 3, 4, 5, 7, 8, 9, 10 and 11

Recombinant human PDE3B (GenBank / EMBL Accession Number: NM_000922, Miki et al., Genomics 1996 36, 476-485), PDE4B (GenBank / EMBL Accession Number: NM_002600, Obernolte et al., Gene, 1993, 129, 239-247), PDE7B (GenBank / EMBL Accession Number: NM_018945, Hetman et al, Proc Natl Acad, U.S.A. 2000, 97, 472-476), PDE8A (GenBank / EMBL Accession Number: AF_056490, Fisher et al., Biochem. Biophys. Res. Commun. 1998 246, 570-577), PDE9A (GenBank / EMBL Accession Number: NM_002606, Fisher et al., J. Biol. Chem. 1998 273, 15559-15564), PDE10A (GenBank / EMBL Accession Number: NM_06661, Fujishige et J. Biol. Chem., 1999 274, 18438-45, PDEI IA (GenBank / EMBL Accession Number: NM_016953, Fawcett et al., Proc. Natl. Acad., 2000, 97, 3702-3707) were prepared using the pF ASTBAC Bovine PDE1 was obtained from Sigma-Aldrich (P 9529) PDE5 was isolated from human platelets by sonication followed by centrifugation and column chromatography Chromatography of the supernatant on Mono Q 10/10 (linear NaCl gradient, elution with 0.2-0.3 M NaCl in 20 mM Hepes pH 7.2, 2 mM MgC12).

The in vitro activity of test substances on recombinant PDE3B, PDE4B, PDE7B, PDE8A, PDE10A and PDEI IA is determined according to the assay protocol described above for PDE2A, with the exception that the cGMP used for the stimulation of PDE2A is not added to the assay. For the determination of a corresponding effect on PDE1, PDE5 and PDE9A, the protocol is additionally modified as follows: For PDE1, calmodulin 10-7 M and CaC12 3 mM are additionally added to the reaction mixture. For PDE5 and PDE9A, substrate [8-3H] cGMP (1 μCi / μL; Amersham Pharmacia Biotech., Piscataway, NJ) is used in the dilution indicated above. To stop the PDE9A reaction, 25 μl of a PDE9A inhibitor dissolved in assay buffer (e.g., BAY 73-6691, 5 μM final concentration) is added just before adding the Yttrium Scintillation Proximity Bead suspension.

Inhibitors of PDE2A may also attack at the level of transcription or translation of PDE2A. Test systems for finding corresponding inhibitors are well known to the person skilled in the art.

Test of PDE2A inhibitors for anti-hypertrophic action in vivo:

To test the antihypertensive effect of the PDE2A inhibitor BAY 60-7550, the so-called mouse isoprenaline model is used []. Here 8 mice (strain C57bl / 6) per dose group isoprenaline at a dose of 2 mg / kg / d administered subcutaneously for 5 days while the control group as a vehicle control received a saline solution. As a positive control was added in addition to isoprenaline, a group of the ACE inhibitor enalapril is administered via the drinking water at a dose of 10 mg / kg / d during two additional groups in addition to isoprenaline the PDE2A inhibitor BAY 60-7550 intraperitoneally at a dose of 10 mg / kg / d was administered. As a measure of the degree of cardiac hypertrophy, the quotient of heart weight / body weight (HW: BW) is determined after 5 days and the effect of the substances is set in relation to the effect of isoprenaline.

PDE2A inhibitor formulations

The PDE2A inhibitors may be converted in a known manner into the usual formulations, such as tablets, dragees, pills, granules, aerosols, syrups, emulsions, suspensions and solutions, using inert, nontoxic, pharmaceutically suitable excipients or solvents. Here, the therapeutically active compound should be present in each case in a concentration of 0.5 to 90 wt .-% of the total mixture, i. in amounts sufficient to achieve the stated dosage margin.

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 using water as the diluent, organic solvents may optionally be used as auxiliary solvents.

The application is carried out in a customary manner, preferably orally, transdermally, intravenously or parenterally, in particular orally or intravenously. But it can also be done by inhalation through the mouth or nose, for example by means of a spray, or topically on the skin.

In general, it has been found to be beneficial to administer levels of from about 0.001 to 10 mg / kg, preferably about 0.005 to 3 mg / kg of body weight when administered orally to achieve effective results.

Nevertheless, it may be necessary to deviate from the stated amounts, depending on the body weight or the type of application route, from the individual

Behavior towards the drug, the nature of its formulation and the timing or

Interval at which administration takes place. Thus, in some cases it may be sufficient to manage with less than the aforementioned minimum amount, while in other cases, the said upper limit must be exceeded. In the case of the application of larger quantities, it may be advisable to distribute these in several single doses throughout the day. literature

1st Scherer, CR, Dissertation Univ. Frankfurt, 2002.

2. Brostrom MA, Reilly BA, Wilson FJ, Brostrom CO. Vasopressin-induced hypertrophy in H9c2 heart-derived myocytes. Int J Biochem Cell Biol. 2000 Sep; 32 (9): 993-1006.

3. Calderone A, Thaik CM, Takahashi N, Chang Dl, Colucci M, Nitric Oxide, Atrial Natriuretic Peptides, and Cyclic GMP inhibit the growth-promoting effects of norepinephrine in cardiac myocytes and fibroblasts. J. Clin Invest. 1998 Feb 15; 101 (4): 812-8.

4. Booz GW, putting the brakes on cardiac hypertrophy: exploiting the NO cGMP counter-regulatory system. Hypertension. 2005 Mar; 45 (3): 341-6.

5. Mendelsohn ME, Nat. Med. 11, 2005, 115-116

6. Boess FG, Hendrix M, van der Staay FJ, Erb C, Schreiber R, van Staveren W, de Vente J, Prickaerts J, Blokland A, Koenig G. Inhibition of phosphodiesterase 2 increases neuronal cGMP, synaptic plasticity and memory performance, Neuropharmacology. 2004 Dec; 47 (7): 1081-92

7. Heid CA, Stevens J, Livak KJ, Williams PM., Real time quantitative PCR. Genome Res 6 (1996), 986-994.

8. Hassan MA, Ketat AF., Sildenafil citrate increases myocardial cGMP content in rat heart, decreases its hypertrophic response to isoproterenol and decreases myocardial leak of creatine kinase and troponin T, BMC Pharmacol. 2005 Apr 6; 5 (1): 10.

Abbreviations

ANP atrial natriuretic peptide

AVP: arginine vasopressin

BW: Body weight Ct: Threshold (threshold cycle)

HW: heart weight

MYHCB: myosin heavy chain, beta subunit (myosin heavy chain beta subunit)

PBS Phosphate-buffered saline rE: relative expression SD: standard deviation

Claims

claims

1. Use of a PDE2 A polypeptide or a nucleic acid encoding a PDE2A polypeptide in a test system for the discovery of inhibitors of PDE2A suitable for the treatment and / or prophylaxis of heart failure and its underlying cardiomyopathies, as well as coronary heart disease, in particular stable and unstable angina pectoris, acute myocardial infarction, sudden cardiac death, hypertension, the consequences of atherosclerosis, vascular diseases, kidney disease, and / or erectile dysfunction.

2. Use according to claim 1, wherein it is a cell-free test system.

3. Use according to claim 1, which is a test system using whole cells containing a nucleic acid encoding a PDE2A.

4. Use according to claims 1-3, wherein a PDE2A activity is measured.

5. Use according to claim 4, wherein the cGMP or the GMP level is measured.

6. Use according to claims 1-3, wherein the expression of the PDE2A is measured.

7. Use according to claims 1-6, wherein the cardiac insufficiency is cardiomyopathy-induced cardiac insufficiency selected from the group of cardiomyopathies consisting of dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM), arrhythmogenic right ventricular cardiomyopathy (ARVCM), myocarditis and / or hypertrophic cardiomyopathy (HCM).

8. Use of a PDE2A inhibitor, which has been identified by means of one of the methods according to claims 1-7, for the manufacture of a medicament for the treatment and / or prophylaxis of cardiac insufficiency, as well as of coronary heart diseases, in particular stable and unstable angina pectoris, of acute myocardial infarction, sudden cardiac death, hypertension, the consequences of atherosclerosis, vascular disease, kidney disease, and / or erectile dysfunction.

9. Use of a PDE2A inhibitor, which has been identified by means of one of the methods according to claims 1-7, for the manufacture of a medicament for the treatment and / or prophylaxis of cardiomyopathy-induced cardiac insufficiency selected from the group of cardiomyopathies consisting of the dilatative Cardiomyopathy (DCM), restrictive cardiomyopathy (RCM), arrhythmogenic right ventricular cardiomyopathy (ARVCM), myocarditis and / or hypertrophic cardiomyopathy (HCM).

10. Use of a PDE2A-specific antibody, a PDE2A-specific antisense oligonucleotide or a PDE2A-specific siRNA for the preparation of a medicament for the treatment and / or prophylaxis of heart failure, as well as of coronary

Cardiac disorders, in particular stable and unstable angina pectoris, acute myocardial infarction, sudden cardiac death, hypertension, the consequences of atherosclerosis, as well as vascular diseases, diseases of the kidney, and / or erectile dysfunction.

11. Use of a PDE2A-specific antibody, a PDE2A-specific antisense oligonucleotide or a PDE2A-specific siRNA for the manufacture of a medicament for the treatment and / or prophylaxis of cardiomyopathy-induced heart failure, selected from the group of cardiomyopathies consisting of dilated cardiomyopathy ( DCM), restrictive cardiomyopathy (RCM), arrhythmogenic right ventricular cardiomyopathy (ARVCM), myocarditis and / or hypertrophic cardiomyopathy (HCM).

12. Use of a PDE2A inhibitor for the manufacture of a medicament for the treatment and / or prophylaxis of heart failure and coronary heart disease, in particular stable and unstable angina pectoris, acute myocardial infarction, sudden cardiac death, hypertension, the consequences of atherosclerosis, and from

Vascular disorders, kidney disease, and / or erectile dysfunction.

13. Use according to claim 12, wherein the heart failure is cardiomyopathy-induced cardiac insufficiency selected from the group of cardiomyopathies consisting of dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM), arrhythmogenic right ventricular cardiomyopathy

(ARVCM), myocarditis and / or hypertrophic cardiomyopathy (HCM).

14. Use of a compound of general formula (I),

wherein

R ¹ is phenyl, naphthyl, quinolinyl or isoquinolinyl, up to three times identically or differently by radicals selected from the group consisting of (dC ₄₎ -alkyl, (C _r _C4) -alkoxy, halogen, cyano -NHCOR ^8, -NHSO ₂ R ⁹ , -SO ₂ NR ¹⁰ R ¹¹ , -SO ₂ R ¹² , and -NR ¹³ R ¹⁴ may be substituted, means

wherein

R ⁹ and R ^{12 are} independently of one another (C 1 -C ₄ ) -alkyl,

or

R ² and R ³ independently of one another denote hydrogen or fluorine,

R ⁴ (CC.) - Alkyl.

R ^{5 is} (C ₁ -C ₃ ) -alkyl,

R ^{6 is} hydrogen or methyl, R ⁽⁷ phenyl, thiophenyl, furanyl, which may be substituted consisting of (C _r _C4) alkyl, (Ci-C ₄₎ -alkoxy, halogen and cyano up to three times identically or differently by radicals selected from the group, or C ₅ -C ₈ ) -cycloalkyl,

L is carbonyl or hydroxybutylmethane, and

and their physiologically acceptable salts

for the manufacture of a medicament for the treatment and / or prophylaxis of cardiac insufficiency, as well as coronary heart diseases, in particular stable and unstable angina pectoris, acute myocardial infarction, sudden cardiac death, hypertension, the consequences of atherosclerosis, as well as vascular diseases, disorders of the Kidney, and / or erectile dysfunction.

Use of compounds as defined in claim 14 wherein R ^{1 is} phenyl, its meta and / or para positions being up to three times the same or different with radicals selected from the group consisting of (C _r C ₄ ) alkyl, (C _r C ₄ ) alkoxy and -SO ₂ NR ¹⁰ R "are substituted, and wherein R ¹⁰ and R ^{11 have} the meaning given in claim 1, for the preparation of a medicament for the treatment and / or prophylaxis of heart failure, as well as coronary heart disease , in particular stable and unstable angina pectoris, acute myocardial infarction, sudden cardiac death, hypertension, the consequences of atherosclerosis, as well as vascular diseases, kidney diseases, and / or erectile dysfunction.

16. Use of compounds we defined in claim 14 or 15, wherein R ^{7 is} phenyl, for the manufacture of a medicament for the treatment and / or prophylaxis of heart failure, as well as coronary heart disease, especially stable and unstable angina, acute myocardial infarction, the sudden Cardiac death, hypertension, the consequences of atherosclerosis, as well as vascular diseases, of

Kidney disease, and / or erectile dysfunction.

17. Use of compounds as defined in claim 14,

in which

R ^{1 is} phenyl whose meta and / or para positions are up to three times identical or different with radicals selected from the group consisting of (C 1 -C ₄ ) -alkyl, (C ¹ -C ₄ ) -alkoxy and -SO ₂ NR ¹⁰ R ¹ 'are substituted, naphthyl or quinolinyl,

in which R ¹⁰ and R ¹¹ independently of one another are hydrogen or (C 1 -C ₄ ) -alkyl,

R ¹ and R ² are hydrogen,

R ^{4 is} methyl or ethyl,

R ^{5 is} methyl,

R ^{6 is} hydrogen or methyl,

L is carbonyl or hydroxybutylmethane, and

M straight-chain (C ₂ -C ₅ ) -alkan-1, ω-diyl, straight-chain (C ₂ -C ₅ ) -alkene-1, ω-diyl or straight-chain (C ₂ -C ₅ ) -alkyne-1, ω- diyl means

for the manufacture of a medicament for the treatment and / or prophylaxis of cardiac insufficiency and coronary heart disease, in particular stable and unstable angina pectoris, acute myocardial infarction, sudden cardiac death, hypertension, the consequences of atherosclerosis, as well as vascular diseases, diseases of the Kidney, and / or erectile dysfunction.

18. Use of compounds of the general formula (II),

wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , L and M have the meaning given in claim 1,

and their salts for the manufacture of a medicament for the treatment and / or prophylaxis of cardiac insufficiency, as well as coronary heart disease, in particular stable and unstable angina, acute myocardial infarction, sudden cardiac death, hypertension, the consequences of atherosclerosis, as well as vascular diseases, diseases kidney, and / or erectile dysfunction.

19. Use of the substance 2- (3,4-dimethoxybenzyl) -7- [1- (1-hydroxyethyl) -4-phenylbutyl] -5-methylimidazo [5, lf] [l, 2,4] triazine-4 ( 3H) -one with the structural formula:

for the manufacture of a medicament for the treatment and / or prophylaxis of cardiac insufficiency and coronary heart disease, in particular stable and unstable angina pectoris, acute myocardial infarction, sudden cardiac death, hypertension, the consequences of atherosclerosis, as well as vascular diseases, kidney diseases, and / or erectile dysfunction.

20. Use according to claims 14 - 19, wherein the heart failure is cardiomyopathy-induced cardiac insufficiency selected from the group of cardiomyopathies consisting of dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM), arrhythmogenic right ventricular cardiomyopathy ( ARVCM), myocarditis and / or hypertrophic cardiomyopathy (HCM).