HRP20040721A2 - Dihydro-thia-phenanthrene-carbonyl-guanidines, method for the production thereof, use thereof as a medicament or diagnostic reagent - Google Patents

Description

The invention relates to dihydro-thia-phenanthrene-carbonyl-guanidines of formula I

[image]

where

R(1) and R(3) independently represent hydrogen, alkyl with 1, 2, 3 or 4 C-atoms, alkoxy with 1, 2, 3 or 4 C-atoms, F, Cl, Br, I, CN, NR(10) R(11), -Op-(CH2)n-(CF2)x-CF3 or -(SOm)p-(CH2)n-(CF2)x-CF3;

R(10) and R(11) are independently hydrogen, alkyl with 1, 2, 3 or 4 carbon atoms or - (CH2)n-(CF2)x-CF3;

m is zero, 1 or 2;

n is zero, 1, 2, 3, 4, 5 or 6;

x and p are independently zero or 1;

R(2) is hydrogen, F, Cl, Br, I, CN, alkyl with 1, 2, 3 or 4 C-atoms, methoxy, cycloalkyl with 3, 4, 5, 6, 7 or 8 C-atoms,

R(4) and R(5) are independently hydrogen, alkyl with 1, 2, 3 or 4 carbon atoms;

R(6), R(7), R(8) and R(9) independently of each other are hydrogen, alkyl with 1, 2, 3 or 4 C-atoms, alkoxy with 1, 2, 3 or 4 C-atoms, F, Cl, Br, I, CN, NR(12)R(13), -Oq-(CH2)r(CF2)s-CF3 or -(SOw)t-(CH2)u-(CF2)v-CF3 ;

R(12) and R(13) are independently hydrogen, alkyl with 1, 2, 3 or 4 carbon atoms;

w is zero, 1 or 2;

r and u are zero, 1, 2, 3, 4, 5 or 6;

q, s, t and v are independently zero or 1; or

R(6) and R(7), or R(7) and R(8), or R(8) and R(9) together with the phenyl ring that carries them form a naphthalene system;

A is -S-, -SO- or -SO2-, as well as their pharmaceutically acceptable salts.

Preference is given to compounds of formula I, in which

R(1) and R(3) are independently hydrogen, methyl, ethyl, methoxy, ethoxy, F, Cl, CN, NR(10)R(11), -Op-(CH2)n-CF3 or -(SOm )p-(CH2)n-CF3;

R(10) and R(11) are independently hydrogen, methyl, ethyl or -CH2-CF3;

m is zero, 1 or 2;

n is zero, 1, 2 or 3;

p are independently zero or 1;

R(2) is hydrogen, F, Cl, CN, alkyl with 1, 2, 3 or 4 C-atoms, methoxy, cycloalkyl with 3, 4, 5 or 6 C-atoms,

R(4) and R(5) are independently hydrogen, methyl or ethyl;

R(6), R(7), R(8) and R(9) are independently hydrogen, alkyl with 1, 2, 3 or 4 C-atoms, methoxy, ethoxy, F, Cl, CN, NR(12 )R(13), -Oq-(CH2)rCF3 or -(SOw)t-(CH2)u-CF3;

R(12) and R(13) are independently hydrogen, methyl or ethyl;

w is zero, 1 or 2;

r and u are zero, 1, 2 or 3;

q and t are independently zero or 1; or

R(6) and R(7), or R(7) and R(8), or R(8) and R(9) together with the phenyl ring that carries them form a naphthalene system;

A is -S-, -SO- or -SO2-, as well as their pharmaceutically acceptable salts.

Particular preference is given to compounds of formula I, in which

R(1) is hydrogen, methyl, ethyl, methoxy, ethoxy, F, Cl, NR(10)R(11), -Op-(CH2)n-CF3 or - (SOm)p-(CH2)n-CF3 ;

R(10) and R(11) are independently hydrogen, methyl, ethyl or -CH2-CF3;

m is zero, 1 or 2;

n and p are independently zero or 1;

R(2) hydrogen, F, Cl, methyl, cycloalkyl with 3, 4, 5 or 6 C-atoms,

R(3), R(4) and R(5) are hydrogen;

R(6), R(7), R(8) and R(9) are independently hydrogen, methyl, methoxy, ethoxy, F, Cl, NR(12)R(13), -Oq-(CH2)rCF3 or -(SOw)t-(CH2)u-CF3;

R(12) and R(13) are independently hydrogen, methyl or ethyl;

w is zero, 1 or 2;

q, r, t and u are independently zero or 1; or

R(6) and R(7), or R(7) and R(8), or R(8) and R(9) together with the phenyl ring that carries them form a naphthalene system; A is -S-, -SO- or -SO2-,

as well as their pharmaceutically acceptable salts.

A very special preference is given to the compounds of formula I, in which

R(1) is hydrogen, methyl, methoxy, ethoxy, Cl, NR(10)R(11), -O-CH2-CF3 or -(SOm)p-(CH2)n-CF3;

R(10) and R(11) are independently hydrogen, methyl, ethyl or -CH2-CF3;

m is zero, 1 or 2;

p is zero or 1;

R(2) is hydrogen, F, Cl or methyl;

R(3), R(4) and R(5) are hydrogen;

R(6), R(7), R(8) and R(9) are independently hydrogen, methyl, methoxy, ethoxy, F, Cl, -O-CH2-CF3 or -(SOw)t-(CH2) u-CF3;

w is zero, 1 or 2;

t and u are independently zero or 1; or

R(6) and R(7), or R(7) and R(8), or R(8) and R(9) together with the phenyl ring that carries them form a naphthalene system;

A is -SO2-, as well as their pharmaceutically acceptable salts.

In case of suitable substitution, the compounds of formula I can exist in stereoisomeric forms. If the compounds of formula I have one or more centers of asymmetry, they can independently have an S configuration or an R configuration. The invention includes all possible stereoisomers, eg enantiomers or diastereomers, and mixtures of two or more stereoisomeric forms, eg enantiomers and/or diastereomers in any ratio. The invention therefore includes enantiomers, for example, in enantiomerically pure form, as well as left- and right-handed antipodes, and also in the form of a mixture of two enantiomers in different proportions or in the form of a racemate. The production of individual stereoisomers can be carried out, if desired, by separating the mixture using usual procedures or, for example, by stereoselective synthesis. If there are displaceable hydrogen atoms, the proposed invention also includes all tautomeric forms of the compounds of formula I.

Said alkyl radicals can have a straight chain or can be branched.

The invention further relates to the process for the production of compound I, which is characterized in that the compound of formula II

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where

R(1) to R(9) as well as A have the stated meanings, a

L is a leaving group that can be easily nucleophilically substituted, it reacts with guanidine.

Activated acid derivatives of the formula II, in which L represents alkoxy, preferably a methoxy group, a phenoxy group, a phenylthio-, methylthio-, 2-pyridylthio group, a heterocycle with nitrogen, preferably 1-imidazolyl, are primarily obtained in a known manner from basic carboxylic acid chlorides (formula II, L = Cl), which in turn can be easily produced, also in a known manner, from basic carboxylic acids (formula II, L = OH), for example with thionyl chloride.

In addition to chlorides of carboxylic acids of formula II (L = Cl), further activated acid derivatives of formula II can be obtained in a known manner from derivatives of basic benzoic acid (formula II, L = OH), as methyl ester of formula II with L = OCH3, by treatment with with gaseous HCl in methanol, the imidazolide of formula II by treatment with carbonyldiimidazole [L = 1-imidazolyl/ Staab, Angew. Chem. Int. Ed. English 1, 351-367 (1962)], mixed anhydrides II with Cl-COOC2H5 or tosyl chloride in the presence of triethylamine in an inert solvent, as well as by activating benzoic acid with dicyclohexylcarbodiimide (DCC) or with O-[(cyano(ethoxycarbonyl)- methylene)amino]-1,1,3,3-tetramethyluronium tetrafluoroborate ("TOTU") [Proceedings of the 21st European Peptide Symposium, Peptides 1990, published by E. Giralt and D. Andreu, Escom, Leiden, 1991] . A number of suitable procedures for the production of activated carboxylic acid derivatives of formula II are set forth with reference to J. March, Advanced Organic Chemistry, Third Edition (John Wiley & Sons, 1985), p. 350.

The reaction of activated carboxylic acid derivatives of formula II with guanidine is carried out in a known manner in a protic or aprotic polar but inert organic solvent. At the same time, when converting the methyl ester of benzoic acid (II, L = OMe) with guanidine, methanol, isopropanol or THF at 20°C up to the boiling point of that solvent proved to be good solvents. In most reactions of compounds II with salt-free guanidine, it is useful to work in aprotic inert solvents such as THF, dimethoxyethane, dioxane. However, in addition to using a base, such as for example NaOH as a solvent for the conversion of compound II with guanidine, water can also be used as a solvent.

If L represents Cl, it is useful to work with the addition of an acid-binding agent, eg in the form of excess guanidine to bind the hydrohalic acid.

For the construction of the structure of dihydro-thia-phenanthrene-carboxylic acid, it is useful to start with an appropriately substituted benzylsulfanylene, phenylmethanesulfinylene or phenylmethanesulfonylene. They undergo intramolecular aryl-aryl coupling, which is known in principle (see Chem. Rev. 95 (7), 2457 (1995) or "Metal-catalyzed Cross-coupling Reactions", Diederich, Francois; Stang, Peter J.; publisher for Germany (1998): Wiley-VCH, Weinheim, Germany, 517) or Tetrahedron (1998), 54 (3/4), 263). It is advantageous to associate the boric acid with a suitable aryl halide, such as an aryl chloride, an aryl bromide, an aryl iodide, or with a suitable aryl ester such as an aryl mesylate or an aryl trifluoromethanesulfonate. At the same time, the functional group of boric acid can be introduced both on benzylic and also on benzoic acid as a second participant in the reaction. Also particularly advantageous is the use of bis(pinacolato)diboron, as described in Tetrahedron Lett. (1997), 38 (22), 3841-3844. Formula III shows such a starting material, in which R(1) to R(9), as well as A and L have the meanings indicated, and X and Y are in each case halogen or -O-SO2CH3 or -O-SO2CF3. A favorable metal catalyst is palladium, and it is particularly favorable in the form of its complex Pd(dppf)2. The reaction is carried out in a dipolar aprotic solvent, preferably in DMF or DMA, at a temperature between 0°C and the boiling point of the solvent, preferably at a temperature between 40°C and 120°C.

[image]

Derivatives of the general formula III are conveniently prepared from 3-mercapto-benzoic acid derivatives or from 3-sulfino-benzoic acid derivatives of the general formula IV with activated benzyl derivatives of the general formula V:

[image]

Moreover, group Z is a leaving group that can be easily nucleophilically substituted, such as chlorine, bromine, iodine, mesylate, tosylate or trifluoromethanesulfonate. Derivatives IV and V are reacted in a suitable solvent, such as DMF, THF or acetonitrile, with the use of an auxiliary base such as triethylamine or DIPEA, at a temperature between -20°C and the boiling point of the solvent, preferably at a temperature between 0°C and 40° C.

Aroylguanidines I are generally weak bases and they can bind acids to form salts. Suitable acid addition salts include all pharmacologically tolerable acids, for example halides, especially hydrochlorides, lactates, sulfates, citrates, tartrates, acetates, phosphates, methyl sulfonates, p-toluenesulfonates.

Compounds I are substituted acylguanidines.

Compared to known compounds, the compounds according to the invention are distinguished by an exceptionally high effect in inhibiting the Na/H exchanger.

They also do not have any unwanted and unfavorable salidiuretic properties like the known compounds, but they have very good antiarrhythmic properties, which are important, for example, for the treatment of diseases that occur when there is a lack of oxygen. Due to their pharmacological properties, these compounds are particularly suitable as antiarrhythmic drugs with a cardioprotective component for the prophylaxis of heart attacks and for the treatment of heart attacks, as well as for the treatment of angina pectoris, whereby they can also preventively inhibit or greatly reduce pathophysiological processes in the occurrence of injuries caused by ischemia, especially in cardiac arrhythmias caused by ischemia. Due to their protective action against pathological hypoxic and ischemic conditions, due to the inhibition of the cellular Na+/H+ exchange mechanism, the compounds of formula I according to the invention can be used as drugs for the treatment of all acute or chronic injuries caused by ischemia or diseases caused by it primarily or secondarily. This refers to their use as drugs for surgical procedures, e.g. in organ transplants, where these compounds can be used to protect organs in the donor, before and during organ retrieval, to protect extracted organs, for example during processing or their disposal in baths of physiological fluids, as well as during transfer into the recipient's organism. These compounds are also valuable drugs with a protective effect during angioplasty operations, for example on the heart, as well as on peripheral blood vessels. In accordance with their protective action against ischemia-induced injuries, these compounds are also suitable as drugs for the treatment of ischemia of the nervous system, especially of the SNS, where they are suitable, for example, for the treatment of stroke or cerebral edema. In addition, the compounds of formula I according to the invention are also suitable for the treatment of forms of shock, such as for example allergic, cardiogenic, hypovolemic and bacterial shock.

The compounds of the formula I according to the invention are further characterized by a strong inhibitory effect on cell proliferation, for example the proliferation of fibroblast cells and on the proliferation of smooth muscle cells of blood vessels. Therefore, the compounds of formula I come into consideration as valuable therapeutic agents for diseases in which cell proliferation is a primary or secondary cause, and therefore they can be used as antiatherosclerotics, agents against diabetic late complications, cancer diseases, fibrotic diseases such as pulmonary fibrosis, liver fibrosis or kidney fibrosis, hypertrophy and hyperplasia of organs, especially hyperplasia or hypertrophy of the prostate.

The compounds according to the invention are effective inhibitors of cellular sodium-proton antiporters (Na+/H+-exchangers) which are also elevated in numerous diseases (essential hypertension, atherosclerosis, diabetes, etc.) in such cells that are easily accessible for measurements, such as for example erythrocytes, platelets or leukocytes.

The compounds according to the invention are therefore suitable as a distinguished and simple scientific tool, for example, in their use as diagnostics for determining and distinguishing certain forms of hypertension, but also atherosclerosis, diabetes, proliferative diseases, etc. Furthermore, the compounds of formula I are suitable for preventive therapy to prevent the genesis of high blood pressure, for example essential hypertension.

In addition, compounds of formula I have been found to exhibit a beneficial effect on serum lipoproteins. It is generally recognized that for the development of arteriosclerotic changes in blood vessels, especially coronary diseases, excessive levels of fat in the blood, so-called hyperlipoproteinemia, are an important risk factor. Therefore, for the prophylaxis and regression of atherosclerotic changes, the reduction of elevated serum lipoproteins is extremely important. In addition to the total reduction of cholesterol in the serum, the reduction in the share of specific atherogenic lipid fractions of that total cholesterol, especially low density lipoproteins (e. low density lipoproteins, LDL) and very low density lipoproteins (e. very low density lipoproteins, VLDL), is particularly significant, because these lipid fractions represent an atherogenic risk factor. In contrast, high-density lipoproteins have been attributed a protective function against coronary diseases. Accordingly, hypolipidemics must be able to reduce not only total cholesterol, but especially VLDL and LDL cholesterol fractions in serum. The compounds of formula I have been found to exhibit valuable, therapeutically applicable properties in affecting the amount of lipids in the serum. Thus, they significantly lower the elevated concentration of LDL and VLDL in the serum, which is observed, for example, with increased dietary intake of food rich in cholesterol and lipids or with pathological changes in metabolism, for example with genetically determined hyperlipidemia. They can therefore be used for prophylaxis and for the regression of atherosclerotic changes, whereby they exclude the causal risk factor. This includes not only primary hyperlipidemias, but also certain secondary hyperlipidemias, such as those that occur in diabetes. Furthermore, the compounds of formula I lead to a clear reduction of infarcts caused by metabolic anomalies, and in particular to a significant reduction in the size of such infarcts and their degree of severity. Furthermore, compounds of formula I lead to effective protection against endothelial injuries caused by metabolic anomalies. With this protection of blood vessels against endothelial dysfunction syndrome, the compounds of formula I are a valuable drug for the prevention and treatment of coronary spasms of blood vessels, atherogenesis and atherosclerosis, left atrial hypertrophy and dilated cardiomyopathy, and thrombotic diseases.

The said compounds can therefore be usefully used for the production of a drug for the treatment of hypercholesterolemia; for the production of a drug for the prevention of atherogenesis; for the production of a drug for the prevention and treatment of atherosclerosis, for the production of a drug for the prevention and treatment of diseases caused by an increased amount of cholesterol, for the production of a drug for the prevention and treatment of diseases caused by endothelial dysfunction, for the production of a drug for the prevention and treatment of hypertension caused by atherosclerosis, for the production of a drug for the prevention and treatment of thrombosis caused by atherosclerosis, for the production of a drug for the prevention and treatment of ischemic injuries caused by hypercholesterolemia and endothelial dysfunction and postischemic reperfusion injuries, for the production of a drug for the prevention and treatment of cardiac hypertrophy caused by hypercholesterolemia and endothelial dysfunction, cardiomyopathy and congestive heart failure (CHF), for the production of a drug for the prevention and treatment of coronary spasms of blood vessels caused by hypercholesterolemia and endothelial dysfunction and myocardial infarction, for the production of a drug for the treatment of women's complaints in combinations with blood pressure lowering agents, primarily with ACE (e. Angiotensin Converting Enzyme, ACE) inhibitors and angiotensin receptor antagonists. The combination of an NHE inhibitor of formula I with an active substance that lowers the amount of fat in the blood, primarily with an HMG-CoA reductase inhibitor (e.g. lovastatin or pravastatin), whereby the latter is attributed a hypolipidemic effect and thus enhances the hypolipidemic properties of NHE inhibitors of formula I, has been shown is like a favorable combination with enhanced action and reduced use of active substances.

In addition, the administration of sodium-proton exchange inhibitors of the formula I as a new drug for reducing the increased amount of fat in the blood, as well as combinations of sodium-proton exchange inhibitors with blood pressure-lowering and/or hypolipidemic drugs, is required.

In addition, the administration of sodium-proton exchange inhibitors of formula I as well as combinations of sodium-proton exchange inhibitors with blood pressure-lowering drugs, especially with ACE inhibitors (for example ramipril) as well as with angiotensin receptor antagonists (for example losartan) as a new drug is required for the treatment of CHF.

At the same time, the drugs containing compound I can be given orally, parenterally, intravenously, rectally or by inhalation, whereby the favorable method of application depends in each case on the presenting picture of the disease. Here, compounds I can be used alone or together with galenic excipients, both in veterinary medicine and in human medicine.

Based on his professional knowledge, the expert will know which excipients are suitable for the desired formulation of the medicine. In addition to solvents, gel forming agents, suppository bases, tablet excipients, other carriers of active substances can also be used, for example antioxidants, dispersants, emulsifiers, antifoaming agents, flavor correctors, preservatives, dissolution aids or colored.

For the forms to be administered, the orally active compounds are mixed with excipients suitable for this purpose, such as carriers, stabilizers or inert excipients, and are brought into forms suitable for administration by conventional methods, such as tablets, dragees, capsules, aqueous , alcoholic or oily solutions. Gum arabic, magnesium oxide, magnesium carbonate, potassium phosphate, milk sugar, glucose or starch, especially corn starch, can be used as inert carriers. At the same time, the preparation can be in the form of dry or wet granules. Suitable oil carriers or solvents are, for example, vegetable or animal oils, such as sunflower oil or cod liver oil.

For subcutaneous or intravenous administration, the active compounds are optionally brought into solution, suspension or emulsion with conventional substances such as solubilizing agents, emulsifiers or other auxiliary substances. Suitable solvents include water, saline solution or alcohols, for example ethanol, propanol, glycerin, and in addition also sugar solutions such as glucose or mannitol solutions, or also a mixture of the various mentioned solvents.

As pharmaceutical formulations for administration in the form of aerosols or sprays, solutions, suspensions or emulsions of the active substance of formula I in a pharmaceutically acceptable solvent, such as in particular ethanol or water, or a mixture of such solvents, are suitable.

If necessary, the formulation may also contain other pharmaceutical auxiliary substances such as surfactants, emulsifiers and stabilizers as well as propellant gas. Such a preparation contains an active substance usually in a concentration of approx. 0.1 to 10, especially from approx. 0.3 to 3 wt. %.

The dosage of the given active substance of formula I and the frequency of administration depend on the strength of the active substance and the duration of action of the compounds used; in addition, it also depends on the type and severity of the treated disease as well as on the sex, age, weight and individual reaction of the treated mammal.

On average, the daily dose of the compound of formula I for a patient with a severe condition is approx. 75 kg is at least 0.001 mg/kg, preferably 0.01 mg/kg, up to a maximum of 10 mg/kg, preferably up to a maximum of 1 mg/kg of body weight. In the case of an acute outbreak of the disease, possibly immediately after surviving a heart attack, even higher and above all more frequent dosages may be required/ eg up to 4 individual doses per day. Especially with i.v. application, possibly in patients with a heart attack in intensive care, may require up to 200 mg per day per kg of body weight.

List of abbreviations

DIPEA diisopropylethylamine

DMA N,N-dimethylacetamide

DME 1,2-dimethoxyethane

DMF N,N-dimethylformamide

EE ethyl acetate (EtOAc)

eq. equivalent

MeOH methanol

Pd(dppf)2 (1:1) complex [1,1'-bis-(diphenylphosphino)-ferrocene]palladium(II) chloride-methylene chloride

RT room temperature

Srap smelter (Italian)

THF tetrahydrofuran

Experimental part

General working procedure for the synthesis of dihydro-thia-phenanthrene-carbonyl-guanidine

Level 1)

4-Bromo-5-chlorosulfonyl-2-methyl-benzoic acid methyl ester

12 g of 4-bromo-5-chlorosulfonyl-2-methyl-benzoic acid (J. Med. Chem. 1997, 40, 2017) is boiled with 20 ml of thionyl chloride for 8 hours with exclusion of moisture and under reflux. Excess thionyl chloride is removed in vacuo on a rotary evaporator, the residue is taken up with approx. 50 ml of dry toluene and evaporate once more. The crude acid chloride is dissolved in 25 ml of anhydrous toluene, 1.7 ml of MeOH is added and stirred for 2 h at 50°C. Then another 1.7 ml of MeOH was added and stirred for another 4 h at 50°C. The reaction mixture was diluted with 200 ml of EE and washed with 100 ml of saturated aqueous NaHCO3 solution. It is dried over Na2SO4 and the solvents are removed in vacuo. 11.0 g of pale yellow oil is obtained, which is used without further purification.

Level 2)

2-bromo-5-methoxycarbonyl-4-methyl-benzenesulfinic acid

Dissolve 550 mg of Na2SO3 in 2 ml of water and add dropwise to a solution of 337 mg of 4-bromo-5-chlorosulfonyl-2-methyl-benzoic acid methyl ester in 2 ml of DME at 70°C. During addition, the solution is weakly acid (pH = 5). It is stirred for another 2.5 hours at 70°C, allowed to cool and adjusted to pH = 1-2 with an aqueous solution of HCl. It is diluted with 50 ml of EE and washed with 50 ml of saturated aqueous NaCl solution. It is dried over Na2SO4 and the solvents are removed in vacuo. 228 mg of pale yellow oil is obtained, which is used without further purification.

Level 3)

4-bromo-5-(2-bromo-phenylethanesulfonyl)-2-methyl-benzoic acid methyl ester

150 mg (0.51 mmol) of 2-bromo-5-methoxycarbonyl-4-methyl-benzenesulfinic acid (step 2) was dissolved in 1.5 ml of DMF. To this, 128 mg (0.51 mmol) of 2-bromobenzyl bromide dissolved in 0.5 ml of DMF and 0.1 ml (0.56 mmol) of DIPEA are added and allowed to stir for 16 hours at room temperature with moisture excluded. The reaction solution is filtered, diluted with 20 ml of EE, washed with 20 ml of 1N hydrochloric acid and then with 20 ml of 5% NaCl solution. The organic phase is passed through a drying cartridge (anhydrous sodium sulfate), and the cartridge is washed with 5 ml of EE. The filtrate is evaporated. The crude product is purified by preparative HPLC.

In accordance with the general reaction equation, the following products were obtained analogously:

[image]

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The benzyl bromides used, if not obtained commercially, were produced from the corresponding methylaroates by radical bromination with N-bromosuccinimide or from benzyl alcohol by reaction with aqueous HBr or with methanesulfonic acid/triethylamine chloride, and then with tetrabutylammonium bromide.

The crude products are mixed with acetonitrile/water = 9:1 (1 ml), possibly with the addition of 0.2 ml of DMF, suction through the cartridge and washed with acetonitrile/water = 9:1 (0.5 ml). The secreted solid substance was dried in a vacuum drying chamber at 50°C, and Purities according to HPLC/MS were >80%. The mother liquor is purified using preparative HPLC, because it still contains a large part of the product.

Level 4)

6-methyl-9,9-dioxo-9,10-dihydro-9-thia-phenanthrene-7-carboxylic acid methyl ester

68 mg (0.266 mmol) of bis(pinacolato)diboron, 71 mg (0.725 mmol) of potassium acetate and 9 mg (0.012 mmol) of Pd(dppf) 2 were placed in 2 ml of DMA. 112 mg (0.242 mmol) of 4-bromo-5-(2-bromo-phenylmethane-sulfonyl)-2-methyl-benzoic acid methyl ester (grade 3) dissolved in 4 ml of DMA was added and stirred overnight at 80 °C under protective gas. The reaction solution is filtered through silica gel and washed with 20 ml of EE. The organic phase is washed with water and 5% NaCl solution, dried over anhydrous sodium sulfate and evaporated in a vacuum. The crude product is purified by preparative HPLC.

In accordance with the general reaction equation, the following products were obtained analogously:

[image]

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Grade 5)

Dihydro-thia-phenanthrene-carbonyl-guanidine, general prescription

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53 mg (0.5 mmol) of potassium tert-butylate are suspended in 2 ml of dry DMF. 50 mg (0.55 mmol) of guanidine hydrochloride was added to this and the suspension was stirred for 30 minutes at room temperature with exclusion of moisture. Then 0.1 mmol of methyl ester from step 4 dissolved in 1 ml of DMF is added and allowed to stir overnight at room temperature. The precipitated salt is filtered off and the filtrate is purified directly by preparative HPLC (Merck Supersphere RP 18e column material, acetonitrile/water gradient with 0.1% formic acid as buffer). The resulting product was characterized using analytical HPLC/MS on an Agilent Series 1100 instrument.

Mass determination was performed with positive ionization. Method A:

column: MERCK LiChroCart 55-2

packaging: PuroSpher STAR RP 18

flow rate: 0.75 ml/min

temperature: 40°C

Gradient:

solvent A: acetonitrile/water (90:10) + 0/5% formic acid,

solvent B: acetonitrile/water (10:90) + 0.5% formic acid.

[image]

Method B:

column: YMC J'Sphere ODS H80

packing: 4 μ

flow rate: 1.0 ml/min

temperature: 30°C

Gradient:

solvent A: water + 0.05% trifluoroacetic acid

solvent B: acetonitrile

[image]

The title compounds of examples 1-11 were synthesized according to the general procedure for the synthesis of dihydro-thia-phenanthrene-carbonyl-guanidine:

Example 1

N-(6-methyl-9,9-dioxo-9,10-dihydro-9-thia-phenanthrene-7-carbonyl)-guanidinium formate

[image]

MS (ES): 330 (M+1)4, retention time 2.384 min (220 nm, procedure A)

Example 2

N-(2-methyl-S,5-dioxo-5,6-dihydro-5-thia-benzo[c]phenanthrene-3-carbonyl)-guanidinium formate

[image]

MS (ES) : 380(M+1)+, retention time 1.793 min (220 nm, procedure B}

Example 3

N-(3,6-dimethyl-9,9-dioxo-9,10-dihydro-9-thia-phenanthrene-7-carbonyl)-guanidinium formate

[image]

MS () : 344 (M+1) f retention time 2.411 min (220 nm, method A)

Example 4

N-(2-chloro-6-methyl-9,9-dioxo-9,10-dihydro-9-thia-phenanthrene-7-carbonyl)-guanidinium formate

[image]

MS (ES): 364 (M+1)+, retention time 2,390 min (220 nm, method A)

Example 5

N-(3-fluoro-6-methyl-9,9-dioxo-9,10-dihydro-9-thia-phenanthrene-7-carbonyl)-guanidinium formate

[image]

MS ES) : 348 (M+1)+, retention time 2.215 min (220 nm, procedure A)

Example 6

N-(1-fluoro-6-methyl-9,9-dioxo-9,10-dihydro-9-thia-phenanthrene-7-carbonyl)-guanidinium formate

[image]

MS (ES) : 348 (M+1)+, retention time 2.218 min (220 nm, procedure A)

Example 7

N-(3-chloro-6-methyl-9,9-dioxo-9,10-dihydro-9-thia-phenanthrene-7-carbonyl)-guanidinium formate

[image]

MS (ES): 364 (M+1)+, retention time 2.394 min (220 nm, procedure A)

Example 8

N-(2-methoxy-6-methyl-9,9-dioxo-9,10-dihydro-9-thia-phenanthrene-7-carbonyl)-guanidinium formate

[image]

MS (ES) : 360 (M+1)+, retention time 2.271 min (220 nm, procedure A)

Example 9

N-(1-chloro-6-methyl-9,9-dioxo-9,10-dihydro-9-thia-phenanthrene-7-carbonyl)-guanidinium formate

[image]

MS (ES) : 364 (M+1)+, retention time 2.358 min (220 nm, procedure A)

Example 10

N-(4, 6-dimethyl-9,9-dioxo-9,10-dihydro-9-thia-phenanthrene-7-carbonyl)-guanidinium formate

[image]

MS (ES) : 344 (M+1)+, retention time 2.302 min (220 nm, method A)

Example 11

N-(2, 6-dimethyl-9,9-dioxo-9,10-dihydro-9-thia-phenanthrene-7-carbonyl)guanidinium formate

[image]

MS (ES) : 344(M+1)+, retention time 2.671 min (220 nm, procedure A)

Procedure for NHE inhibition according to Jansen

The IC50 value [nM] of NHE-1 inhibition was determined as follows:

FLIPR assay for determination of NHE-1 inhibitors using measurement of pH increase in transfected cell lines expressing human NHE-1

The experiment was carried out in FLIPR (Fluorescent imaging plate reader) microtiter plates with 96 wells, with black walls and a transparent bottom. Transfected cell lines, expressing different subtypes of HNE (parental cell line LAP-1 [obtained from Prof. Pouyssegur, Nizza] results in mutagenesis and then selection of no endogenous NHE activity), were seeded a day earlier at a density of approx. 25,000 cells per well. [Growth medium for transfected cells (Iscove + 10% fetal calf serum) additionally contains G418 as a selection antibiotic, to determine the presence of transfected sequences].

The experiment itself begins with the removal of the growth medium and the addition of 100 μl/well of loading buffer (5 µM BCECF-AM[2',7'-bis-(carboxyethyl)-5-(i-6)-carboxyfluorescein, acetoxymethyl ester] in 20 mM NH4Cl, 115 mM choline chloride, 1 mM MgCl2, 1 mM CaCl2/5 mM KCl, 20 mM HEPES, 5 mM glucose; pH 7.4 [adjusted with KOH]). The cells are then incubated for 20 minutes at 37°C. This incubation leads to loading of the cells with a fluorescent dye, whose fluorescence intensity depends on the pHi, and with NH4Cl, which leads to a slight alkalinization of the cells.

[The previous dye step BCECF-AM, which is not fluorescent, passes through the membranes as an ester. Intracellularly, esterases release the own dye BCECF, which does not pass through membranes.]

After this 20-minute incubation, the loading buffer, containing NH4Cl and free BCECF-AM, was removed by washing three times in a cell washer (Liquid Columbus) in each case with 400 μl of wash buffer (133.8 mM choline chloride , 4.7 mM KCl, 1.25 mM MgCl2, 1.25 mM CaCl2/ 0.97 mM K2HPO4, 0.23 mM KH2PO4, 5 mM HEPES, 5 mM glucose; pH 7.4 [adjusted with KOH]). The volume remaining in the wells is 90 μl (possibly 50-125 μl). This washing procedure removes free BCECF-AM and this removal of external NH4+ ions results in intracellular acidification (pHi approx. 6.3-6.4).

Since the balance of intracellular NH4+ with NH3 and H+ is disturbed with the removal of extracellular NH4 and then with the immediate passage of NH3 through the cell membrane, the flushing process leads to the retention of intracellular H+, which is the cause of intracellular acidification. This can eventually lead to cell death, if it persists long enough.

At this point, it is important that the wash buffer is sodium-free (<1 mM), as extracellular sodium ions would lead to immediate pHi repair with the activity of the cloned NHE iso-forms.

It is also important that all the buffers used (loading buffer, washing buffer, collection buffer) do not contain HCO3- ions, because the presence of bicarbonate leads to the activation of the disturbed bicarbonate-dependent pH regulation system found in the parental cell lines LAP-1 .

Then the microtiter plates are transferred to FLIPR (up to 20 minutes after acidification). In FLIPR, an intracellular fluorescent dye is excited with light of a wavelength of 488 nm, which is obtained from an argon laser, and the measurement parameters (laser power, illumination time, and shadowing in the CCD camera installed in FLIPR are selected so that the average fluorescence signal per well lies between 30,000 and 35,000 relative fluorescence units.

An individual measurement in FLIPR starts with taking two images each using a computer program with a CCD camera. After ten seconds, the recovery of intracellular pH values was adjusted with the addition of 90 μl of recovery buffer (133.8 mM NaCl, 4.7 mM KCl, 1.25 mM MgCl2, 1.25 mM CaCl2, 0.97 mM K2HPO4, 0 ,23 mM KH2PO4, 10 mM HEPES, 5 mM glucose; pH 7.4 [adjusted with NaOH]) using a 96-well pipetting device built into the FLIPR.

Positive controls (100% NHE activity) are wells to which pure recovery buffer has been added. Negative controls (0% NHE activity) contain wash buffer. Recovery buffer with twice the concentration of the test substance was added to all other wells. Measurement in FLIPR ends after 60 measurement points (two minutes).

The raw data was transferred to the ActivityBase program. With this program, NHE activities are first calculated for each concentration of the test substance and from this the IC50 values for the substances are calculated. Since the course of pHi recovery is not linear during the entire experiment, but it eventually decreases due to the decrease of NHE activity at higher pHi values, it is important to choose the part where the increase in fluorescence of the positive control is linear to display the measurement results.

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Claims (17)

1. Dihydro-thia-phenanthrene-carbonyl-guanidines of formula I [image] indicated by that R(1) and R(3) independently represent hydrogen, alkyl with 1, 2, 3 or 4 C-atoms, alkoxy with 1, 2, 3 or 4 C-atoms, F, Cl, Br, I, CN, NR(10) R(11), -Op-(CH2)n-(CF2)x-CF3 or -(SOm)p-(CH2)n-(CF2)x-CF3; R(10) and R(11) are independently hydrogen, alkyl with 1, 2, 3 or 4 carbon atoms or -(CH2)n-(CF2)x-CF3; m is zero, 1 or 2; n is zero, 1, 2, 3, 4, 5 or 6; x and p are independently zero or 1; R(2) is hydrogen, F, Cl, Br, I, CN, alkyl with 1, 2, 3 or 4 C-atoms, methoxy, cycloalkyl with 3, 4, 5, 6, 7 or 8 C-atoms, R(4) and R(5) are independently hydrogen, alkyl with 1, 2, 3 or 4 carbon atoms; R(6), R(7), R(8) and R(9) independently of each other are hydrogen, alkyl with 1, 2, 3 or 4 C-atoms, alkoxy with 1, 2, 3 or 4 C-atoms, F, Cl, Br, I, CN, NR(12)R(13), -Oq-(CH2)r(CF2)s-CF3 or -(SOw)t-(CH2)u-(CF2)v-CF3 ; R(12) and R(13) are independently hydrogen, alkyl with 1, 2, 3 or 4 carbon atoms; w is zero, 1 or 2; r and u are zero, 1, 2, 3, 4, 5 or 6; q, s, t and v are independently zero or 1; or R(6) and R(7), or R(7) and R(8), or R(8) and R(9) together with the phenyl ring that carries them form a naphthalene system; A is -S-, -SO- or -SO2-, as well as their pharmaceutically acceptable salts. 2. Compounds of formula I according to claim 1, characterized in that in them R(1) and R(3) are independently hydrogen, methyl, ethyl, methoxy, ethoxy, F, Cl, CN, NR(10)R(11), -Op-(CH2)n-CF3 or -(SOm )p-(CH2)n-CF3; R(10) and R(11) are independently hydrogen, methyl, ethyl or -CH2-CF3; m is zero, 1 or 2; n is zero, 1, 2 or 3; p are independently zero or 1; R(2) is hydrogen, F, Cl, CN, alkyl with 1, 2, 3 or 4 C-atoms, methoxy, cycloalkyl with 3, 4, 5 or 6 C-atoms, R(4) and R(5) are independently hydrogen, methyl or ethyl; R(6), R(7), R(8) and R(9) are independently hydrogen, alkyl with 1, 2, 3 or 4 C-atoms, methoxy, ethoxy, F, Cl, CN, NR(12 ) R(13), -Oq-(CH2)rCF3 or -(SOw)t-(CH2)u-CF3; R(12) and R(13) are independently hydrogen, methyl or ethyl; w is zero, 1 or 2; r and u are zero, 1, 2 or 3; q and t are independently zero or 1; or R(6) and R(7), or R(7) and R(8), or R(8) and R(9) together with the phenyl ring that carries them form a naphthalene system; A is -S-, -SO- or -SO2-, as well as their pharmaceutically acceptable salts. 3. Compounds of formula I according to claims 1 or 2, characterized in that in them R(1) is hydrogen, methyl, ethyl, methoxy, ethoxy, F, Cl, NR(10)R(11), -Op-(CH2)n-CF3 or -(SOm)p-(CH2)n-CF3 ; R(10) and R(11) are independently hydrogen, methyl, ethyl or -CH2-CF3; m is zero, 1 or 2; n and p are independently zero or 1; R(2) hydrogen, F, Cl, methyl, cycloalkyl with 3, 4, 5 or 6 C-atoms, R(3), R(4) and R(5) are hydrogen; R(6), R(7), R(8) and R(9) are independently hydrogen, methyl, methoxy, ethoxy, F, Cl, NR(12)R(13), -Oq-(CH2)rCF3 or -(SOw)t-(CH2)u-CF3; R(12) and R(13) are independently hydrogen, methyl or ethyl; w is zero, 1 or 2; q, r, t and u are independently zero or 1; or R(6) and R(7), or R(7) and R(8), or R(8) and R(9) together with the phenyl ring that carries them form a naphthalene system; A is -S-, -SO- or -S02-, as well as their pharmaceutically acceptable salts. 4. Compounds of formula I according to claims 1 to 3, characterized in that in them R(1) is hydrogen, methyl, methoxy, ethoxy, Cl, NR(10)R(11), -O-CH2-CF3 or - (SOm)p-(CH2)n-CF3; R(10) and R(11) are independently hydrogen, methyl, ethyl or -CH2-CF3; m is zero, 1 or 2; p is zero or 1; R(2) is hydrogen, F, Cl or methyl; R(3), R(4) and R(5) are hydrogen; R(6), R(7), R(8) and R(9) are independently hydrogen, methyl, methoxy, ethoxy, F, Cl, -O-CH2-CF3 or -(SOw)t-(CH2) u-CF3; w is zero, 1 or 2; t and u are independently zero or 1; or R(6) and R(7), or R(7) and R(8), or R(8) and R(9) together with the phenyl ring that carries them form a naphthalene system; A is -S02-, as well as their pharmaceutically acceptable salts. 5. Use of the compound according to claim 1, characterized in that it is used for the production of a drug for the treatment or prophylaxis of diseases caused by ischemic conditions. 6. A method for the treatment and prophylaxis of diseases caused by ischemic conditions, characterized in that an effective amount of compound I according to claim 1 is mixed with common additives and given in a form suitable for application, 7. Use of compound I according to claim 1, characterized in that it is used for the production of a drug for the treatment or prophylaxis of heart attacks and arrhythmias. 8. Use of compound I according to claim 1, characterized in that it is used for the production of a medicine for the treatment or prophylaxis of angina pectoris. 9. Use of the compound according to claim 1, characterized in that it is used for the production of a drug for the treatment or prophylaxis of ischemic heart conditions. 10. Use of compound I according to claim 1, characterized in that it is used for the production of a drug for the treatment or prophylaxis of ischemic conditions of the peripheral and central nervous system and stroke. 11. Use of compound I according to claim 1, characterized in that it is used for the production of a drug for the treatment or prophylaxis of ischemic conditions of peripheral organs and limbs. 12. Use of compound I according to claim 1, characterized in that it is used for the production of a medicine for the treatment of shock conditions. 13. Use of compound I according to claim 1, characterized in that it is used for the production of a medicine used in surgical operations and organ transplants. 14. The use of compound I according to claim 1, characterized in that it is used for the production of a medicine for preservation and disposal of grafts for surgical procedures. 15. The use of compound 1 according to claim 1, characterized in that it is used for the production of a drug for the treatment of diseases in which cell proliferation is the primary or secondary cause. 16. Use of compound I according to claim 1, characterized in that it is used for the production of a drug for the treatment or prophylaxis of fat metabolism disorders. 17. Medicine, characterized in that it contains an effective amount of compound I according to one or more claims 1 to 4.