SK3342002A3 - Biphenyl derivatives used as nhe-3 inhibitors - Google Patents

Abstract

The invention relates to compounds of the formula (I), wherein X, R<1>, R<2>, R<3>, R<4> and R<5> have the meaning indicated in claim 1. The inventive compounds represent inhibitors of the subtype 3 sodium/proton exchanger (NHE-3).

Description

Biphenyl derivatives l & hp PPMilhl, 4 FMfl β0Ίΐ /} b £ f / w, 0fpj ^ kfpry h p piwhvjts

Technical field

The invention relates to biphenyl derivatives of the formula I in general

-cr (I) where

R ¹ , R ⁴ independently of one another are -C (= NH) -NH 2, optionally substituted with one -COA, -CO [C (R ⁶ ) 2] _n -Ar, -COOA, -OH or the usual amino protecting group, or represents NH-C (= NH) -NH ₂ , -CO-N = C (NH ₂ ) _2, or a group of the formula

or

N.

N =

CH ₃

R ² , R ³ , R ⁵ independently represent a hydrogen atom, a group

A, OR ^6, N (R ⁶⁾ _2, NO _{2 j} CN, Hal, NHCOA, NHCOAr,

NHSO ₂ A, NHSO ₂ Ar, COOR ⁶ , NOC (R ⁶ ) ₂ , CONHAr, i

COR ⁶ , COAr, S (O) n A, S (O) n Ar, -O- [C (R ⁶ ) 2] m COOR ⁶ , - [C (R ⁶ ) 2] _P -COOR ⁶ , -O- [C ^(R6) 2] ", - CON (R ^a) 2,

- [C (R ⁶ ) 2] p-CON (R ⁶ ) 2, -O- [C (R ⁶ ) 2] m -CONHAr or [C (R ⁶ ) 2] _p -CONHAr;

X is - [C (R ⁶ ) 2] n -, -CR ⁶ = CR ⁶ -, - [C (R ⁶ ) 2] n -O-, O- [C (R ⁶ ) ₂ ] n -, - COO-, -OOC-, -CONR ⁶ - or NR ⁶ COR ⁶ represents a hydrogen atom, a group A or a benzyl group

A represents an alkyl group having 1 to 20 carbon atoms, one or two -CH ₂ groups being optionally replaced by an oxygen or sulfur atom or the groups -CR ⁶ = CR ⁶ - and / or one to seven hydrogen atoms being replaced by a fluorine atom;

Ar represents a phenyl or naphthalyl group unsubstituted or substituted by one, two or three of the groups A, Ar ', OR ⁶ , OAr', N (R ⁶ ) 2, NO 2, CN, Hal, NHCOA, NHCOAr ', NHSO2A, NHSO 2 Ar ', COOR ⁶ , CON (R ⁶ ) 2, CONHAr', COR ⁶ , COAr ', S (O) _n A and S (O) _n Ar';

Ar 1 represents a phenyl or naphthyl group unsubstituted or substituted by one, two or three groups selected from the group consisting of A, OR ⁶ , N (R ⁶ ) 2, NO 2, CN, Hal, NHCOA, COOR ⁶ , CON (R ⁶ ) 2, COR ⁶ and S (O) _n A;

Hal represents a fluorine, chlorine, bromine or iodine atom;

n represents Ο, 1 or 2;

m is 1 or 2;

p is 1 or 2;

and salts and solvates thereof as NHE-3 inhibitors.

BACKGROUND OF THE INVENTION

Other sodium exchanger / proton subtype 3 inhibitors are described, for example, in EP 0 825178.

DE 19819548 discloses that the compounds of formula I and their salts possess Factor Xa inhibiting properties and can therefore be used for the treatment and prevention of thromboembolic diseases such as thrombosis, myocardial infarction, arteriosclerosis, inflammation, apoplexy, angina pectoris, angioplasty restenosis and intermittent curvature.

SUMMARY OF THE INVENTION It is an object of the present invention to provide novel compounds having valuable properties which are particularly useful in the manufacture of medicaments.

SUMMARY OF THE INVENTION

The present invention relates to biphenyl derivatives as described above.

It has surprisingly been found that the compounds of the formula I and their salts, with good tolerability, inhibit the sodium / proton exchangers of subtype 3. The compounds can therefore be used as medicinal active substances in human and veterinary medicine.

It is known that the Na ⁺ / H ⁺ exchanger represents a family with at least 6 different isoforms (NHE-1 to NHE-6), all of which are now all cloned. While the NHE-1 subtype is ubiquitously distributed throughout the body in all tissues, the other NHE subtypes are expressed selectively in specific organs such as the kidney or lumen wall and in the contaluminal small intestine wall. This distribution reflects the specific functions that various isoforms serve to regulate intracellular pH and cell volume by the NHE-1 subtype or for Na ⁺ uptake and re-uptake in the intestine and kidney by the NHE-2 or NHE-3 isoforms. especially in the stomach. Expression of NHE-5 is limited to brain and neuronal tissue. NHE-6 is the isoform that forms the sodium proton exchanger in mitochondria.

The NHE-3 isoform is expressed primarily in the apical membrane of adjacent kidney pelvis; the NHE-3 braking agent therefore performs, inter alia, renal protection.

The therapeutic use of the selective braking agent for the NHE-3 isoforms is multifaceted. NHE-3 inhibits or reduces tissue damage and cell death following pathophysiological hypoxic and ischemic events that lead to NHE activation, such as in renal ischemia or during renal transplant removal, transport and reperfusion.

The compounds of formula I act to lower blood pressure and are useful as medicaments for the treatment of hypertension. They are also suitable as diuretics.

The compounds of formula I act alone or in conjunction with NHE inhibitors of other subtype specificities and are anti-ischemic and can be used in thromboses, atherosclerosis, varicose veins, to protect organs such as kidneys or liver before and during surgery and chronic or acute renal failure.

In addition, they can be used for the treatment of stroke, brain swelling, nervous system ischemia, various forms of shock, breathing improvement, for example in the following conditions: sleep disorder, sudden infant death, postoperative hypoxia and other respiratory disorders.

Combination with a carbonic anhydrase stopper can further improve breath performance.

The compounds of formula I inhibit cell proliferation, for example fibroblast cell proliferation and smooth muscle cell proliferation, and can therefore be used to treat diseases in which cell proliferation is a primary or secondary cause.

The compounds of the formula I can be used against later complications of diabetes, cancer, fibrotic diseases, endothelial dysfunction, hypertrophies, orane hyperplasia, in particular in prostate hypertrophy or hyperplasia.

They are further useful as diagnostics for the determination and differentiation of certain forms of hypertonia, atherosclerosis, diabetes and proliferative diseases.

Since the compounds of formula I, preferably affecting the levels of serum lipoproteins, they may be used for the treatment of raised blood lipid levels, or in combination with other liečivamir · ¹ '-. .

The invention relates to the use of the compounds of formula I and their physiologically acceptable salts and / or solvates for the manufacture of medicaments, for the treatment of thromboses, ischemic conditions of the heart, peripheral or central nervous system and stroke, ischemic conditions of peripheral organs and limbs and shocks.

The invention further relates to the use of the compounds of the formula I and their physiologically acceptable salts and / or solvates for the manufacture of medicaments for use in surgical operations and organ transplants and for the preservation, storage of transplants for surgical operations.

The invention also relates to the use of the compounds of formula I and their physiologically acceptable salts and / or solvates for the manufacture of a medicament for the treatment of diseases in which cell proliferation is a primary or secondary cause, for the treatment or prophylaxis of fat exchange or respiratory disorders.

The invention further relates to the use of the compounds of formula I and their physiologically acceptable salts and / or solvates for the manufacture of a medicament for the treatment of ischemic kidney, ischemic bowel disease or for the prophylaxis of acute or chronic renal diseases.

Methods for identifying substances that inhibit the sodium / proton exchanger subtype 3 are described, for example, in U.S. Patent No. 5,871,919.

All symbols that appear several times in the general formulas, for example R ⁶ , have independent meanings.

The term hydrates and solvates refers to hemihydrates, monohydrates or dihydrates, and solvates are, for example, to addition salts. alcohols, e.g. m ethanol. or with ethanol.

A represents a straight-chain or branched alkyl group having 1 to 20, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. A is preferably methyl, furthermore ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl, furthermore also pentyl, 1-, 2- or 3-methylbutyl, 1,1-, 1,2- or 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1-, 2-, 3 or 4-methylpentyl, 1,1-, 1,2-, 1,3-, 2,2-, 2,3- or 3, 3-dimethylbutyl, 1- or 2-ethylbutyl, 1-ethyl-1-methylpropyl, 1-ethyl-2-methylpropyl, 1,1,2- or 1,2,2-trimethylpropyl, heptyl, octyl, nonyl or decyl. A is furthermore, for example, trifluoromethyl, pentafluoroethyl, allyl or crotyl.

The symbol COR ⁶ represents an acyl, preferably a formyl, acetyl, propionyl, furthermore also a butyryl, pentanoyl or hexanoyl group.

The symbol COOR ⁶ represents a methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl or butoxycarbonyl group.

Hal is preferably fluorine, chlorine or bromine, but also iodine.

R @ ² , R @ ³ and R @ ⁵ independently of one another are preferably hydrogen, fluorine, bromine or iodine, <RTIgt; 5, </RTI> hydroxy, methoxy, ethoxy-S-group, propoxy, nitro, amino, methylamino, dimethylamino, ethylamino, diethylamino, acetamido, sulfonamido, methylsulfonamido, phenylsulfonamido, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl. methylsulfonyl,. ethylsulfonyl, phenylsulphinyl, phenylsulphonyl, cyano, carboxyl, methoxycarbonyl, ethoxycarbonyl, carboxymethoxy, methoxycarbonylmethoxy, carboxymethyl, methoxycarbonylmethyl, aminocarbonylmethoxy, aminocarbonylmethyl, N-fenylaminokarbonylmetoxy, N-fenylaminokarbonylmetylovú, furthermore an acyl or benzoyl group, wherein R ² and

In particular, R represents a hydrogen atom and R represents in particular, for example, a hydrogen atom, -COOA or OCH ₂ COOR ⁶ , wherein R ⁶ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

R @ ⁶ represents a hydrogen atom, a group A or a benzyl group, in particular a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

X is preferably, for example, -CH ₂ -, -CH = CH-, -CH ₂ O-, -O-CH ₂ -, -COO-, -OOC-, -CONH- or NHCO-, especially representing -CH ₂ O-, O-CH ₂ -or-CH ₂ -CH ₂ -.

Ar is preferably unsubstituted phenyl or naphthyl, further preferably phenyl or naphthyl, monosubstituted, disubstituted or trisubstituted from the group consisting of A, fluorine, chlorine, bromine and iodine. ..hydroxyskupinu, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, benzyloxy, phenethyloxy, methylthio, ethylthio, methylsulfinyl group, ethylsulfinyl, methylsulfonyl, ethylsulfonyl, phenylsulphinyl, fenylsulfonvlovú, nitro, amino, methylamino, ethylamino, dimethylamino, diethylamino, formamido , acetamido, propionylamino, butyrylamino, methylsulfonamido, ethylsulfonamido, propylsulfonamido, butylsulfonamido, phenylsulfonamido (4-methylphenyl), sulfonamido, carboxymethoxy, carboxymethoxy, carbonylmethoxy, methoxycarbonylethoxy, hydroxymethoxy, hydroxyethoxy, methoxyethoxycarboxy, methoxycarbonyl, ethoxycarbonyl, cyano, phenylaminocarbonyl, acyl and benzoyl, and furthermore is biphenyl.

Thus, Ar is preferably, for example, o-, m- or p-tolyl, o-, m- or p-ethylphenyl, o-, m- or p-propylphenyl, o-, m- or p-isopropylphenyl, o- , male or p-butylphenyl, o-, m- or p-hydroxyphenyl, o-, male- or p-nitrophenyl, o-, m- or p-aminophenyl, o-, male- or p- (N-methylamino) phenyl, o-, m- 'or pacetamidophenyl, o-, m- or p-methoxyphenyl, o-, m- or p-ethoxyphenyl, o-, m- or p-carboxyphenyl, o-, m- or p-methoxycarbonylphenyl, o-, m - or p- (N, N-dimethylamino) phenyl, o-, m- or p- (N-ethylamino) phenyl, o-, m- or p- (N, N-diethylamino) phenyl, o-, m- or pacetylphenyl , o-, m- or p-formylphenyl, o-, m- or fluorophenyl, o-, m- or p-bromophenyl, o-, m- or p-chlorophenyl, o-, m- or p-mephthylphenylphenyl, o-, male- or p- (phenylsulfonamido) phenyl, o-, m- or pmethylsulfonamido) phenyl, o-, m- or p-methylthiophenyl, further preferably a 2,3- group; 2,4; 2,5-; 2,6; 3,4- or 3,5-difluorophenyl, 2,3-; 2,4; 2,5-; 2,6; 3,4- or 3,5-dichlorophenyl, 2,3-; 2,4; 2,5-; 2,6; 3,4- or 3,5-dibromophenyl, 2,4- or 2,5-dinitrophenyl, 2,5- or 3,4-dimethoxyphenyl, 3-nitro-4-chlorophenyl, 3-amino-4-chloro, 2-amino- 3-chloro, 2-amino-4-chloro, 2-amino-5-chloro or 2-amino-6-chlorophenyl, 2-nitro-, 4-N, N-dimethylamino- or 3-nitro-, 4-N , N-dimethylaminophenyl, 2,3-diaminophenyl, 2,3,4-, 2,3,5-, 2,3,6-, 2,4,6- or 3,4,5-trichlorophenyl, 2,4 6-trimethoxyphenyl, 2-hydroxy-3,5-dichlorophenyl, iodophenyl, 3,6-dichloro-4-aminophenyl, 4-fluoro-32-fluoro-4-bromophenyl,

3-bromo-6-methoxyphenyl,

3-chloro-4-acetamido phenyl,

3-amino-6-methylphenyl, chlorophenyl, bromophenyl, methoxyphenyl, methoxyphenyl,

2,5-difluoro-43-chloro-63-fluoro-43-chloro-4-acetamidophenyl or 2,5-dimethyl-4-chlorophenyl.

Ar 'is preferably, for example, phenyl or naphthyl, further preferably, for example, o-, m- or ptolyl, o-, m- or p-ethylphenyl. o-, m- or p-propylphenyl, o-, m- or p-isopropylphenyl, o-, m- or p-tert-butylphenyl, o-, m- or p-hydroxyphenyl, o-, male- or p-nitrophenyl, o- , m- or p-aminophenyl; o-, male- or p- (N-methylamino) phenyl, o-, m- or pacetamidophenyl, o-, m- or p-methoxyphenyl, o-, m- or p-ethoxyphenyl, o-, m- or p-carboxyphenyl , o-, m- or p-methoxycarbonylphenyl, o-, m- or p- (N, N-dimethylamino) phenyl, o-, m- or p- (N, N-diethylamino) phenyl _with o-, m- or p-acetylphenyl , o-, m, or p-formylphenyl, o-, m- or p-fluorophenyl, o-, male- or p-bromophenyl, O-, m- or p-chlorophenyl or O-, m- or p-methylsulfonylphenyl groups .

The invention thus relates in particular to compounds of the formula I in which at least one of the symbols has the above-mentioned preferred meaning. Some preferred groups of compounds of formula (I) are the following compounds of formulas (Ia) to (Ii), wherein in particular the not shown symbols are of formula (I), wherein in the formula (Ia) R ¹ , R ⁴ independently represent -C (= NH). -NH ₂ optionally substituted with one hydroxyl group or is -CO-N = C (NH ₂ ) 2;

1b) R ² , R ^{5 are} hydrogen;

1c) R ¹ , R ⁴ independently of one another -C (= NH) -NH ₂ optionally substituted with one hydroxyl group or is -CO-N = C (NH ₂ ) ₂ ;

R ² , R ^{5 are} hydrogen and

R ³ is hydrogen or COOR ⁶ ;

1d) R ¹ , R ⁴ independently of one another -C (= NH) -NH ₂ optionally substituted with one hedroxyl group or is -CO-N = C (NH ₂ ) ₂ ;

R ² , R ^{5 are} hydrogen and

R ³ is hydrogen, COOR ⁶ or -O (CH ₂ ) COOR ⁶ ;

le) X is -CH ₂ -O- or -O-CH ₂ -;

1f) R ¹ , R ⁴ independently of one another -C (= NH) -NH ₂ optionally substituted with one hydroxyl group or is -CO-N = C (NH ₂ ) _2;

R ² , R ^{5 are} hydrogen and

R ³ is hydrogen, COOR ⁶ ;

X is -CH ₂ -O- or -O-CH ₂ -;

1g) R ¹ , R ⁴ independently of one another -C (= NH) -NH ₂ optionally substituted with one hydroxyl group or is -CO-N = C (NH ₂ ) ₂ ;

R ² , R ^{5 are} hydrogen and

R ³ is hydrogen, COOR ⁶ or -O (CH ₂ ) COOR ⁶ ;

x -CH 2 -O-, -O-CH ₂ - or -CH ₂ -CH ₂ ;

1h) R ¹ , R ⁴ independently of one another -C (= NH) -NH ₂ optionally substituted with one hedroxyl group or is -CO-N = C (NH ₂ ) ₂ ;

R ² , R ^{5 are} hydrogen and

R ³ is hydrogen, COOR ⁶ , -O-CH ₂ -COOR ⁶ ,

-CH ₂ -COOR ⁶ , -O-CH 2 -CON (R ⁶ ) 2, CH ₂ -CON (R ⁶ ) ₂ , O-CH ₂ -CONHAr or -CH ₂ -CONHAr;

X is -CH ₂ -O-, -O-CH ₂ - or -CH ₂ -CH ₂ ;

R ⁶ is H or A akupinu;

A is C1-C4alkyl; and li) R ¹ , R ⁴ independently of one another -C (= NH) -NH ₂ optionally substituted with one hydroxyl group, or -CO-N = C (NH ₂ ) _2;

R ² , R ^{5 are} hydrogen;

R ³ is H, COOR ^6, -O-CH 2 COOR ^6, -CH 2 -COOR ^6, -O-CH 2 CON (R ⁶⁾ 2, or CH ₂ CON (R ⁶⁾ _2;

X is -CH ₂ -O-, -O-CH ₂ - or -CH ₂ -CH ₂ ;

R ⁶ is H or A;

And an alkyl group having 1 to 4 carbon atoms.

The compounds of formula I and the starting materials for their preparation are prepared by known methods described in the literature (for example, in standard publications such as Houben-Weyl, Methoden der organischen Chemie, Georg Thieme Verlag, Stuttgart) under reaction conditions which are said reactions are known and suitable. It is also possible to use known variants not described here in greater detail.

Alternatively, the starting materials may be formed in situ, i.e. they are not isolated from the reaction mixture, but are immediately used to prepare compounds of formula I.

The compounds of formula (I) may be prepared by being liberated from their functional derivatives by solvolysis, in particular by hydrolysis or hydrogenolysis agents.

The starting materials for solvolysis or hydrogenolysis are compounds which otherwise correspond to the above formulas, but instead have free amino or hydroxyl groups corresponding to protected amino or hydroxy groups, preferably compounds which, instead of a hydrogen atom which is bonded to a nitrogen atom, have corresponding amino or hydroxy groups. have an amino protecting group, especially compounds having an R'-N group instead of the HN group, wherein R 'is an amino protecting group and / or compounds having a hydroxyl protecting group instead of a hydroxyl atom in a hydroxyl group, for example they correspond to the compounds of the general formula I, but instead of the group -COOH they have a COOR group, where R represents a hydroxyl protecting group.

Preferred starting materials are also oxadiazole derivatives which can be transferred to the corresponding amidone compounds.

The introduction of the oxadiazole moiety is possible, for example, by reaction of the cyano compound with hydroxylamine and reaction with phosgene, dialkyl carbonate, chloroformic acid ester, Ν'-carbonyldiimidazole or acetic anhydride.

Several of the same or different amino and / or hydroxyl protecting groups may be present in the molecule of the starting material. As long as they are protecting groups. they may be selectively cleaved in many cases.

The term &quot; amino protecting group &quot; is generally known and refers to groups which are suitable for protecting (to block) the amino group from chemical reactions but which are readily removable when the desired reaction elsewhere in the molecule is carried out. Typically, these groups are unsubstituted or substituted acyl, aryl, aralkoxymethyl or aralkyl groups. Since the amino protecting groups are removed after the desired reaction (or reaction sequence), their type and size are not critical. Preferred are, however, groups having 1 to 20, and in particular 1 to 8, carbon atoms. The term "acyl group" is always meant here in the broadest sense. It includes acyl groups derived from aliphatic, araliphatic, aromatic or heterocyclic carboxylic or sulfonic acids, in particular alkoxycarbonyl, aryloxycarbonyl groups.

Examples of such acyl groups include alkanoyl groups such as acetyl, propionyl, butyryl;

aralkanoyl such as phenylacetyl; aroyl such as benzoyl or toluyl; aryloxyalkanoyl such as phenoxyacetyl; alkoxycarbonyl such as methoxycarbonyl, ethoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl iodoethoxycarbonyl; aralkyloxycarbonyl benzyloxycarbonyl (CBZ), 4-methoxybenzyloxycarbonyl, 9-fluorinatedethoxycarbonyl (FMOC) and arylsulfonyl as Mtr. Preferred amino protecting groups are BOC and Mtr, CBZ, Fmoe, benzyl and acetyl.

2,2,2-trichloro (BOC), 2 as a group

The term &quot; hydroxy protecting group &quot; is generally the same as those which are suitable for protecting (blocking) a hydroxy group from chemical reactions but which are readily removable when the desired reaction is carried out elsewhere in the molecule. Typical of such groups are the above-mentioned unsubstituted or substituted aryl, aralkyl or acyl groups as well as alkyl groups. Since the hydroxyl protecting groups are removed after the desired reaction (or reaction sequence), their type and size are not critical. Preferred are, however, groups having 1 to 20 and especially 1 to 10 carbon atoms. Examples of hydroxyl protecting groups are benzyl, p-nitrobenzoyl, p-toluenesulfonyl, tert-butyl and acetyl, with benzyl and tert-butyl being particularly preferred.

Depending on the protecting group used, the release of the compounds of formula I from their functional derivatives is successful, for example with strong acids, preferably trifluoroacetic acid or perchloric acid, but also with other strong inorganic acids such as hydrochloric or sulfuric acid, strong organic carboxylic acids such as trichloroacetic acid acids such as benzenesulfonic acid or toluenesulfonic acid. The presence of an additional inert solvent is possible, but not always necessary. Suitable inert solvents are, for example, organic carboxylic acids such as acid, acetic, ethers such as tetrahydrofuran or dioxane, amides such as dimethylformamide (DMF), halogenated hydrocarbons such as dichloromethane, alcohols such as methanol, ethanol or isopropanol and also Water. Mixtures of these solvents may also be contemplated. Trifluoroacetic acid is preferably used in excess without addition of other solvents, perchloric acid in the form of a 9: 1 mixture of acetic acid and 70% orthobutyric acid. The reaction temperature is suitably about 0 to about 50 ° C, preferably 15 to 30 ° C (room temperature).

For example, the BOC, OBut, and Mtr groups may be conveniently cleaved with trifluoroacetic acid in dichloromethane or about 3-5% hydrochloric acid in dioxane at 15 to 30 ° C, FMOC with a 5 to 50% solution of dimethylamine, dimethylamine or piperidine in dimethylformamide at 15 to 30 ° C. - - ^:

Hydrogenolytically removable protecting groups (e.g., CBZ or benzyl, (or amidino release from its oxadiazole derivative)) can be cleaved, for example, by treatment with hydrogen in the presence of a catalyst (e.g., a noble metal catalyst such as palladium, suitably supported on carbon or Raney nickel wetted with additives such as acetic acid) The solvents mentioned above are suitable, in particular, for example, alcohols such as methanol or ethanol or amides such as dimethylformamide Hydrogenolysis is generally carried out at a temperature of about 0 to 100 ° C at a pressure of about 0.1 The hydrogenolysis of the CBZ group performs well, for example, on 5 to 10% palladium-on-carbon in methanol or ammonium formate (instead of hydrogen) in the presence of palladium-on-carbon. coal in methanol / dimethylformamide system at temperature 20-30 ° C.

Compounds of formula I wherein R ¹ and R ⁴ are -C (= NH) -NH ₂ may be obtained advantageously from the corresponding cyano compounds.

The conversion of the cyano group to the amidino group takes place

By reaction with, for example, hydroxylamine and subsequent reduction of N-hydroxyamidine with hydrogen in the presence of a catalyst such as palladium on carbon or Raney nickel.

To prepare an amidine of formula I (R ¹ = C (= NH) -NH ₂ ), ammonia can also be added to the nitrile of formula I (P 2 = CN). This reaction is preferably carried out in several stages, in a manner known per se

(a) the nitrile is reacted with hydrogen sulphide to give a thioamide which is converted to an S-alkylimidothioester with an alkylating agent, for example methyl iodide, which is reacted with ammonia (NH 3) to give an amidine;

(b) the nitrile is converted to the corresponding imidoester with the aid of an alcohol such as ethanol in the presence of hydrochloric acid and the latter is reacted with ammonia; or

c) the nitrile is reacted with lithium bis (trimethylsilyl) amide and the product is subsequently hydrolyzed.

The cyano compounds are prepared in a known manner.

Compounds of formula I wherein R ¹ and R ⁴ are -CON (= NH) -NH ₂ may be advantageously obtained from the corresponding alkoxycarbonyl compounds by reaction with guanidine.

In addition, it is possible to convert a compound of the formula I to another compound of the formula I in such a way that one or more groups R ¹ , R ² , R ³ , R ⁴ and / or R ^{5 are} changed to one or more others. , R ^{@ 1} , R ^{@ 2} , R ^{@ 3} , R ^{@ 4} and / or R ^{@ 5 by,} for example, acylating or reducing nitro groups to amino groups (e.g., by hydrogenation in the presence of Raney nickel or palladium on carbon in an inert solvent such as is methanol or ethanol).

Esters can be saponified, for example, with acetic acid or sodium hydroxide or potassium hydroxide in water, in a water-tetrahydrofuran or water-dioxane system at a temperature of 0 to 100 ° C.

In addition, the free amino groups can be acylated in a known manner with an acid chloride or anhydride or alkylated with an unsubstituted or substituted alkyl halide, preferably in an inert solvent such as dichloromethane or tetrahydrofuran and / or in the presence of a base such as triethylamine or pyridine at -60 to 30 C.

The reaction is generally carried out in an inert solvent in the presence of an acid binding agent, for example an alkali metal or alkaline earth metal hydroxide, carbonate or bicarbonate or other alkali metal or alkaline earth metal salt of a weak acid, preferably such as potassium, sodium, calcium or cesium. The addition of an organic base such as triethylamine, domethylaniline, pyridine or quinoline is also favorable, or an excess of an amino component or an alkylating agent may be beneficial. The reaction time is, depending on the reaction conditions, a few minutes to 14 days, the reaction temperature being about 0 to 150 ° C, typically 20 to 130 ° C.

Suitable inert solvents are, for example, hydrocarbons such as hexane, petroleum ether, benzene, toluene or xylene; chlorinated hydrocarbons such as trichlorethylene, N, T, 2-dichloroethane or carbon tetrachloride, chloroform or dichloromethane; alcohols such as methanol, ethanol, isopropanol, n-propanol, n-butanol or tert-butanol; ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran (THF) or dioxane; glycol ethers such as ethylene glycol monomethyl ether or ethylene glycol monoethyl ether (methyl glycol or ethyl glycol), ethylene glycol dimethyl ether (diglyme); ketones such as acetone or butanol; amides such as acetamide, dimethylacetamide, N-methylpyrrolidone (NMP), dimethylformamide (DMF); nitriles such as acetonitrile; sulfoxides such as dimethyl sulfoxide (DMSO); carbon disulphide; organic carboxylic acids such as formic or acetic acid; nitro compounds such as nitromethane or nitrobenzene; esters such as ethyl acetate; or mixtures of these solvents.

The base of formula (I) may be converted into an appropriate acid addition salt by acid, for example by reacting an equivalent amount of base and acid in an inert solvent such as ethanol and subsequently evaporating the solvent. Suitable acids for this reaction are, in particular, those which give physiologically acceptable salts. Inorganic acids such as sulfuric acid, nitric hydrohalic acids such as hydrochloric or hydrobromic acids, phosphoric acids such as orthophosphoric acid, sulfamic acid and organic acids, in particular aliphatic, alicyclic, araliphatic, aromatic or heterocyclic mono- or polybasic carboxylic, sulfonic or sulfuric acids may be used. formic, acetic, propionic, pivalic, diethylacetic, malonic, succinic, pimelic, fumaric, maleic, lactic, tartaric, malic, citric, gluconic, ascorbic, nicotinic, isonicotinic, methanesulfonic, ethanesulfonic, ethanesulfonic, ethanesulfonic, ethanesulfonic, ethanesulfonic, ethanesulfonic, ethanesulfonic, ethanesulfonic, ethanesulfonic, ethanesulfonic, ethanesulfonic p-toluenesulfonic, naphthalene-monosulfonic and naphthalenedisulfonic and lauryl sulfuric acids. Salts with physiological or non-citric acids, for example picrates, can be used to isolate and / or purify the compounds of formula I.

On the other hand, the compounds of formula I by reaction with a base (e.g. with hydroxide or sodium or potassium carbonate) can be converted into their physiologically acceptable metal salts, in particular alkali metal or alkaline earth metal salts or the corresponding ammonium salts. Physiologically acceptable organic bases such as ethanolamine can also be used.

The present invention also provides the use of the compounds of formula I and / or their physiologically acceptable salts as ΝΗΕ-3-inhibitors for the manufacture of pharmaceutical compositions, in particular by a non-chemical route. For this purpose, they can be converted into a suitable dosage form with at least one solid and / or liquid or semi-liquid carrier or excipient and optionally in admixture with one or more other active substances.

The invention therefore also relates to pharmaceutical compositions comprising at least one ΝΗΕ-3-inhibitor of the formula Ia / or a physiologically acceptable salt or solvate thereof.

The compositions of the invention may be used as medicaments in human and veterinary medicine. Suitable carriers are inorganic and inorganic substances which are suitable for enteral (e.g. oral) or parenteral or topical administration and which do not react with the compounds of formula I, such as water, vegetable oils, benzyl alcohols, alkylene glycols, polyethylene glycols, glycerine acetate gelatin, carbohydrates such as lactose or starches, magnesium stearate, talc and petrolatum. Especially suitable for oral use are tablets, pills, dragees, capsules, powders, granules, syrups, juices or drops, for rectal administration of lace, for parenteral administration of solutions, in particular oily or aqueous solutions, further suspensions, emulsions or implants, for topical use ointments, creams or powders and for transdermal use of the patch. The compounds of the invention may also be lyophilized and the resulting lyophilisates used, for example, for the preparation of injectables. The compositions may be sterilized and / or may contain adjuvants such as glidants, preservatives, stabilizers and / or wetting agents, emulsifiers, salts for affecting the osmotic pressure, buffers, coloring agents, flavoring agents and / or one or more more active ingredients such as vitamins.

Suitable pharmaceutical compositions for administration in the form of aerosols or sprays are, for example, solutions, suspensions or emulsions of the active compound of the formula I in a pharmaceutically acceptable solvent.

The compounds of formula I and their physiologically acceptable salts and solvates can be used for the treatment and / or prophylaxis of the aforementioned diseases or disease states.

The compounds of the formula I according to the invention are generally used in doses of approximately 0.1 to 100 mg, in particular 1 to 10 mg, per dosage unit. The daily dose is preferably about 0.001 to 10 mg / kg body weight. The dose for each individual depends on a variety of factors, such as the efficacy of the particular compound used, age, body weight, general health, sex, diet, time and route of administration, the rate of excretion, the combination of drugs and the severity of the particular. ochorennia. Oral administration is preferred.

The invention is illustrated, but not limited, by the following examples. Temperatures are always given in degrees Celsius. In the following examples, the expression "usual processing" means:

optionally, water is added, optionally according to the constitution of the end product. the pH is adjusted to 2 to 10, the reaction mixture is extracted with ethyl acetate or dichloromethane, separation, drying of the organic phase with sodium sulfate, evaporation and purification by silica gel chromatography and / or crystallization are carried out.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

A solution of 2.06 g of 3-bromobenzonitrile and 1.50 g of 3-tolylboronic acid in 50 ml of dimethoxyethane was mixed with 247 mg of palladium acetate, 335 mg of tri-o-tolylphosphine, 20 ml of water and 954 mg of anhydrous sodium carbonate. of stirring at 100 ° C for 18 hours. After working up in the usual manner, the residue is chromatographed on silica gel with petroether / ethyl acetate 9: 1 as eluent to give 3'-methylbiphenyl-3-carbonitrile as a colorless solid ("A"), Eli 93.

A solution of 1.17 g of "A" in 10 ml of carbon tetrachloride is mixed with 1.09 g of N-bromosuccinimide (NBS) and 60 mg of azobisisobutyronitrile and held at 70 ° C for 18 hours. After working up in the usual way, the residue is chromatographed on silica gel with petroleum ether / ethyl acetate 9: 1 as eluent to give 3'-bromomethylbiphenyl-3-carbonitrile as a colorless solid ("B").

A solution of 500 mg &quot; B &quot; and 238 mg of 3-hydroxybenzonitrile and 10 ml of acetonitrile was mixed with 652 mg of cesium carbonate and the mixture was stirred at room temperature for 40 hours. After working up in the usual way, the residue is subjected to reverse phase chromatography with a 65:35 acetonitrile / water system as eluent. 3- (3-cyanophenoxymethyl) biphenyl-3-carbonintril ("C") is obtained as a colorless solid, FAB 311.

A solution of 90 mg of "C" and 125 mg of polymer-bound hydroxylammonium pyridine (DMAP) was stirred at room temperature for 18 hours. After filtration, the solvent is removed and recovered

N-hydroxy-3 '- [(3-carbamimidoyl) phenoxymethyl] biphenyl-3-carboxamidine ("D") as a colorless solid, FAB 377.

A solution of 76 mg of "D" in 10 ml of methanol is mixed with 10 mg of water-moistened Raney nickel and 30 mg of acetic acid and hydrogenated at room temperature at ambient pressure for ¹ hour. Filtration and removal of the solvent gave 3 '- (3-carbamimidoylphenoxymethyl) biphenyl-3-carboxamidine acetate, E1 327 (M ⁺ -NH 3), 310 (M ⁺ -NH 3).

H N

NH 2

Similarly, the following compounds are obtained:

3 '- (3-carbamimidoylphenoxymethyl) biphenyl-4-carboxamidine diacetate, FAB 345 3' - (4-carbamimidoylphenoxymethyl) biphenyl-4-carboxamidine diacetate, FAB 345 3 '- (4-carbamimidoylphenoxymethyl) biphenyl-3-carboxamidine diacetate,

4 '- (4-carbamimidoylphenoxymethyl) biphenyl-4-carboxamidine

4 '- (4-carbamimidoylphenoxymethyl) biphenyl-3-carboxamidine

4 '- (3-carbamimidoylphenoxymethyl) biphenyl-3-carboxamidine a

4 '- (3-carbamimidoylphenoxymethyl) biphenyl-4-carboxamidine.

Example 2

Similar to Example 1, 3'-methoxybiphenyl-3-carbonitrile is obtained by reacting 3-bromobenzonitrile with 3-methoxyphenylboronic acid. Subsequent ether cleavage with aluminum iodide in acetonitrile and treatment with β-bromomethylbenzonitrile affords 3 '- (3-cyanobenzyloxy) biphenyl-3-carbonitrile. Reaction with hydroxylamine and reduction with hydrogen in the presence of Raney nickel catalyst provides 3 '- (3-carbamimidoylbenzyloxy) biphenyl-3-carboxamidine

HN

NH

Similarly, it is obtained:

4 '- (4-carbamimidoylbenzyloxy) biphenyl-4-carboxamidine diacetate, FAB 345, 4' - (3-carbamimidoylbenzyloxy) biphenyl-4-carboxamidine diacetate, FAB 345.

Example 3

Similar to Example 1, 3'-cyano-5-methylbiphenyl-3-carboxylic acid methyl ester is obtained by reacting 3-cyanophenylboronic acid with 3-bromo-5-methylbenzoate methyl ester. Bromination using

NBS and treatment with 3-hydroxybenzonitrile gives 3'-cyano-5- (3-cyanophenoxymethyl) biphenyl-3-carboxylic acid. Treatment with hydroxylamine and reduction with hydrogen in the presence of Raney nickel gives 3'-carbamimidoyl-5- (3-carbamimidoyl-phenoxymethyl) -biphenyl-3-carboxylic acid methyl ester. Saponification of the ester with aqueous sodium hydroxide solution affords 3'-carbamimidoyl-5- (3-carbamimidoylphenoxymethyl) biphenyl-3-carboxylic acid.

Reverse column chromatography using acetonitrile / water / trifluoroacetic acid as eluent gave 3'-carbamimidoyl-5- (3-carbamimidoyl-phenoxymethyl) -biphenyl-3-carboxylic acid bistrifluoroacetate.

Similarly, the following compounds are obtained:

4'-carbamimidoyl-4- (4-carbamimidoyl-phenoxymethyl) -biphenyl-3-carboxylic acid methyl ester, FAB 403, 4'-carbamimidoyl-4- (3-carbamimidoyl-phenoxymethyl) -biphenyl-2-carboxylic acid methyl ester, FAB 403,

3'-Carbamimidoyl-4- (4-carbamimidoyl-phenoxymethyl) -biphenyl-2-carboxylic acid methyl ester, FAB 403, 3'-Carbamimidoyl-4- (3-carbamimidoyl-phenoxymethyl) -biphenyl-2-carboxylic acid methyl ester, FAB 403, methyl ester 4 1'-carbamimidoyl-5- (3-carbamimidoylphenoxymethyl) biphenyl-4-carboxylic acid, FAB 403, 3'-carbamimidoyl-5- (3-carbamimidoylphenoxymethyl) biphenyl-4-carboxylic acid methyl ester, FAB 403.

Example 4 '•'••• Xr ....

Similar to Example 1, 3'-methylbiphenyl-3-carboxylic acid methyl ester is obtained by reacting 3-bromobenzoic acid methyl ester with 3-tolylboronic acid. NBS bromination and treatment with 3-hydroxybenzoic acid methyl ester gives 3 '- (3-methoxycarbonylphenoxymethyl) biphenyl-3-carboxylic acid methyl ester. Reaction with guanidine hydrochloride in methanolic sodium methane gives N- [3 '- (3guanidinocarbonylphenoxymethyl) biphenyl-3-carbonyl] guanidine.

ABOUT.

Ή ₂ Ν ^ Ν nh ₂

Similarly, methyl 1-biphenyl-4-carbonyl] guanidine.

Example 5

A solution of 7.0 g of 3-bromo-5-methylphenol and 5.97 g of bromoacetate methyl ester and 13 g of cesium carbonate in 100 ml of acetonitrile is stirred overnight at room temperature. The usual work-up yielded 9.70 g of (3-bromo-5-methyl-phenoxy) -acetic acid methyl ester ("AB"). Suspension 2,0 g "AB",

A, bk. NH, gives N- [4 '- (4-guanidinocarbonylphenoxy) 28

100 mg of tetrakis (triphenylphosphine) palladium and 0.85 g of sodium carbonate in 50 ml of toluene are heated to boiling. A solution of 2.94 g of 3-cyanophenoxyboronic acid in 30 ml of methanol is added dropwise and the mixture is refluxed for 14 hours. The usual work-up gave 2.17 g of (3'-cyano-5-methyl-biphenyl-3-yloxy) -acetic acid methyl ester ("AC").

A solution of 1.2 g of 'AC' and 0.765 g of NBS in 50 ml of carbon tetrachloride is irradiated with UV light at room temperature. The usual work-up gave 1.54 g of (3'-cyano-5-bromomethyl-biphenyl-3-yloxy) -acetic acid methyl ester ("AD").

Solution 185 mg 'AD', 63,1 mg 4-hydroxybenzonitrile a

172.7 mg of cesium carbonate in 10 ml of acetonitrile was stirred at room temperature for 4 days. Customary work-up gave [3'-cyano-5- (4-cyanophenoxymethyl) biphenyl-3-yloxy] acetic acid methyl ester ("AE").

Solution 60 mg 'AE', 69,5 mg hydroxylammonium chloride a

101 mg of triethylamine in 10 ml of methanol are kept at reflux for 14 hours. After removal of the solvent, the mixture is taken up in water. After separation, 70 mg of [3'-N-hydroxyamidine-5- (4-N-hydroxyamidinophenoxymethyl) -biphenyl-3-yloxy] -acetic acid methyl ester ("AF") is obtained.

Reduction with H ₂ / Raney nickel gives the methyl ester in [3'-amidino-5- (4-amidinophenoxymethyl) biphenyl-3yloxy] acetic acid, FAB 433.

Similarly, the following compounds are obtained:

[4'-amidino-5- (4-amidinophenoxymethyl) biphenyl-3yloxy] acetic acid methyl ester, FAB 433 [3'-amidino-5- (3-amidinophenoxymethyl) biphenyl-3yloxy] acetic acid methyl ester, FAB 433 [4'-amidino-5-methyl ester - (3-amidinophenoxymethyl) biphenyl-3-yloxy] -acetic acid, FAB 433

If, in the first step, the methyl bromoacetate is replaced with tert-butyl bromoacetate, the tert-butyl esters obtained in the last step can be cleaved with trifluoroacetic acid to give the corresponding carboxylic acids:

[3'-amidino-5- (4-amidinophenoxymethyl) biphenyl-3-yloxy] acetic acid, bistrifluoroacetate FAB 419, [4'-amidino-5- (4-amidinophenoxymethyl) biphenyl-3-yloxy] acetic acid, [3'-amidino-5- (3-amidinophenoxymethyl) biphenyl-3-yloxy] acetic acid, [4'-amidino-5- (3-amidinophenoxymethyl) biphenyl-3-yloxy] acetic acid.

Example 6

A solution of 5.0 g of 3'-bromomethylbiphenyl-3-carbonitrile and 5 ml of triethylphosphine was combined and heated slowly to 150 ° C. Stir for 6 hours at 150 ° C and work-up to give 6.05 g of (3'-cyanobiphenyl-3-ylmethyl) phosphonic acid diethyl ester ("BA").

150 mg of sodium hydride are added to a solution of 1.0 g of "BA" and 3-cyanobenzaldehyde in 20 ml of ethylene glycoidimethyl ether under ice-cooling and under nitrogen. The mixture was stirred for 4 hours and worked up in the usual manner to give 0.93 g of 3 '- [2- (3-cyanophenyl) vinyl] biphenyl-3-carbonitrile ("BB").

Hydrogenation of 360 mg of "BB" in the presence of 5% palladium on carbon in methanol gave 360 mg of 3 '- [2- (3-cyanophenyl) ethyl] biphenyl-3-carbonitrile ("BC").

Reaction with hydroxyammonium chloride and hydrogenation in the presence of Raney nickel gave, similar to Example 1, 3 '- [2- (3-amidinophenyl) ethyl] biphenyl-3-carboxamidine, FAB 343.

Similarly, 3 '- [2- (4-amidinophenyl) ethyl] biphenyl-3-carboxamidine, FAB 343, is obtained.

Example 7

N- [3 '- (4-guanidinocarbonylbenzyloxy) biphenyl-3-carbonyl] guanidine, dihydrochloride, FAB 431

is obtained, for example, according to the following reaction scheme

Br +

(HO) jB

Pd (PPh ₃ ) ₃ / Na, CO ₃

- 1-chloro-1-methylpyridinium iodide N-methylpyrrolidone

N-ethyldiisopropylamine (Mukaryama)

Similarly, the following compounds are obtained:

N- [3 '- (3-guanidinocarbonylbenzyloxy) biphenyl-3-carbonyl] guanidine dihydrochloride, FAB 431

N- [3 '- (4-guanidinok, arbonylbenzyloxy) biphenyl-4-carbonyl] -, guanidine, dihydrochloride, FAB 431

N- [3 '- (3-guanidinocarbonylbenzyloxy) biphenyl-4-carbonyl] guanidine, dihydrochloride, FAB 431.

Pharmacological tests

The methodology used to characterize the compounds of formula I as NHE-3 inhibitors is described.

The compounds of formula I are characterized with respect to selectivity over the isoforms NHE-1 to NHE-3. These three isoforms are stably expressed in mouse phybroblast cell lines. The braking effect of the compounds is assessed by determining EIPA-sensitive ²² Na ⁺ uptake into cells after intracellular acidosis. To characterize Na ⁺ / H ⁺ -change. inhibiting compounds with respect to their isomorphic selectivity are investigated for compounds inhibiting NHE-isoforms NHE-1 to NHE-3 that have been stably expressed in the mouse phybroblast cell line (see method) by determining EIPAsensitive for uptake into cells after intracellular acidosis.

Material and method

LAP-1 cell lines that express various isoforms

NHE

LAP-1-cell lines that express isoform NHE-1, -2 and -3 (mouse phybroblast cell line) were obtained from Professor J. Pouyssegur (Nice, France). Transfection is performed as described by Franchi et al. (Proc. Natl. Acad. Sci. USA 83: 9388-9392, 1986). The cells are cultured in Dulbecco's modified Eagle (DMEM) medium with 10% inactivated germ-line calf serum (FKS). The so-called "acid-killing" method of Sardet et al. (Cell 56, 271-280, 1989). Cells are first removed in bicarbonate-containing ammonium chloride and sodium-free buffer by washing the bicarbonate, ammonium chloride and sodium-free buffer for 30 minutes, and incubating sodium chloride-containing bicarbonate-free buffer. Only cells that functionally express NHE can survive in the intracellular acidification to which they are exposed.

Characterization of NHE-braking compounds with respect to their isoform selectivity

Using the aforementioned mouse phybroblast lines expressing the isoforms NHE-1, NHE-2 and NHE-3, the compounds were tested according to the method of Counillon et al. (Mol. Pharmacol. 44: 1041-1045, 1993) and Scholz et al. (Cardiovasc. Res. 29, atr. 260-268, 1995) with respect to selectivity to isoforms. The cells were acidified intra-cellularly with NH ₄ Cl-prepulse method and subsequently acidified by incubation in a Na-free bicarbonate buffer. Based on intrabunkic acidification, NHE is activated and sodium is absorbed by the cells. The action of the test compound is expressed as inhibition of EIPA (ethyl isopropylamiloride) by sensitive absorption of ²² Na ⁺ .

Cells that express NHE-1, NHE-2 and NHE-3 are seeded at a density of 5 to 7.5 x 10 ⁴ cells / well in 24-well microtiter plates and cultured until sintered for 24 to 48 hours. The medium is aspirated and the cells are incubated for 60 minutes at 37 ° C in ammonium chloride buffer (50 M NH ₄ Cl, 70 mM choline chloride, 15 mM MOPS, pH 7.0). The buffer is then removed and the cells are rapidly overlaid twice with choline chloride wash buffer (120 mM choline chloride, 15 mM PIPEST / Tris, 0.1 mM Ouabain, 1 mM, MgCl 2, 2 mM CaCl 2, pH 7.4); in this buffer, the cells are incubated for 6 minutes. After the incubation period, the incubation buffer is aspirated. To remove extracellular radioactivity, cells are washed four times with ice-cold phosphate buffered saline (PBS). The cells are then solubilized by the addition of 0.3 ml of 0.1 N sodium hydroxide solution per well. Solutions containing cell fragments are transferred to scintillation tubes. Each well was washed two more times with 0.3 ml of 0.1 N sodium hydroxide solution and the wash solutions were also added to scintillation tubes. Tubes containing the cell lysate were combined with a scintillation cocktail and the radioactivity absorbed by the cells was determined by irradiation determination.

Braking of ²² Na ⁺ absorption in rabbit erythrocytes

The Na ⁺ / H ⁺ exchanger activity is determined by observing the absorption of ²² Na ⁺ ions into acidified rabbit erythrocytes. Rabbit erythrocytes have been shown to be useful in investigating Na ⁺ / H ⁺ exchanger activity (Escobales & Fugueroa, J. Membrane Biol. 120: 41-9, 1991; Morgan & Canessa, J. Membrane Biol. 118, pp. 193-214). 1990). The EIPA-sensitive fraction of ²² Na ⁺ absorption in acidified erythrocytes is considered to be ²² Na ⁺ absorption as Na ⁺ / H ⁺ dependent.

Preparation of cells

The preparation of the red blood cells and the inert acidification of the red blood cells is carried out substantially according to the method described by Morgan and Canessa (J. Membrane Biol. 118, pp. 193-214, 1990).

Blood is collected from a rabbit (e.g., New Zealand White). Fill into 50 ml Falcon centrifuge tubes containing 5 ml of sodium heparin solution (250 U / ml). The blood is mixed. with heparin solution. Red blood cells are obtained by centrifugation at 2000 xg at 4 ° C. Plasma and leukocyte cuff are removed. Filter the remaining solution through 200 µm gauze. The filtrate was suspended in the wash buffer to the original volume (140 mM KCl, 0.15 mM MgCl ₂ , 10 mM TRIS / MOPS, pH 7.4). Red blood cells are recovered by centrifugation (2000 xg, 4 ° C). Washing is repeated twice.

Intrabunkové acidification

For intra-cellular acidification, 5 ml of settled collected red blood cells are suspended again with 45 ml of acidification buffer (170 mM KCl, 0.15 mM MgCl ₂ , 0.1 mM Ouabain, 10 mM glucose, 10 mM sucrose, 20 mM TRIS / Mes, pH 6.2). The red blood cell suspension is incubated for 10 minutes at 37 ° C (with occasional stirring). To fix the internal pH, 200 µm and 1 mM DIDS or DIAMOX (acetazolamide) are mixed. Incubate for 30 minutes at 37 ° C.

The red blood cells are then collected by centrifugation (4 minutes at 2000 xg, 4 ° C); they are resuspended in ice-cold wash solution (170 mM KCl, 40 mM sucrose, 0.15 MgCl ₂ ) and washed four times.

Incubation and measurement of ²² Na ⁺ absorption

Incubation is performed. 8 x 12 Masrowell-Tube strips are incubated by mixing 200 μΐ incubation buffer (160 mM KCl, ²² NaCl (37 MBq / well), 0.15 mM MgCl ₂ , 0.1 mM Ouabain, 10 mM glucose, 40 mM sucrose, 10 mM TRIS / MOPS, pH 8.0, 0.5 mM Diamox, 1% DMSO) with 20 μΐ (preheated) acidified red blood cell solution. Test compounds are first dissolved in 100% DMSO and diluted with incubation buffer to appropriate concentrations. Incubate for 5 minutes at 37 ° C. (Preliminary experiments have shown that under these incubation conditions the Na absorption rate is linear over the 5 minute incubation period.) Incubation is terminated by the addition of 800 µL ice stop solution (112 mM MgCl ₂ , 0.1 mM Ouabain). Place the tubes briefly on ice. The tubes are sealed with paraffin and the red blood cells are obtained by a 7-min centrifugation at 2000 × g, ⁴ C. The supernatant was aspirated own apparatus to be aspirated simultaneously from the four tubes. Spacer rings at the far ends of the tips prevent the tips from being too deeply dipped into the tubes and the pellets of red blood cells being sucked off. All supernatants containing both Na and wash solutions are stored and radioactive waste discarded.

The red blood cells are washed three times with 900 μΐ of ice-cold stop solution by repeating the suspension and centrifugation steps described above. After the final wash, the pellet of red blood cells is mixed with 200 μΐ of water. Finally, the tubes are sonicated for 2x30 minutes. The Macrowell-Tube strips are disassembled and each tube is placed upside down in its own scintillation tube, and the hemolyzed red blood cell solution is emptied into the scintillation vials by lightly tapping. Each vial is mixed with 3 ml of Aquasafe 300 PS scintillation fluid and the vials are capped and mixed well.

Radioactivity absorbed by red blood cells is determined in a scintillation counter by monitoring β-decay. The determination is repeated three times for each compound concentration. The mean of counting in the presence of 10 μΜ EIPA is subtracted from each value to avoid the absorption of ²² Na ^{+ not} increasing Na ⁺ / H ⁺ into the erythrocytes. The mean value of remaining readings in the absence of test compound was used as a 100% control; the mean value in the test conditions is expressed as a percentage of this control value. Percent absorption data are plotted on a semi-logarithmic plot. IC ₅₀ values are obtained by adapting the values to a non-linear curve using the equation f (x) = 100 (1 + (IC ₅ o / x) ** n).

literature:

Counillon et al., Mol. Pharmacol. 44, p. 1041-1045, 1993;

Escobales and Fugueroa, J. Membrane Biol. 120, p. 41-9, 1991;

Franchi et al., Proc. Natl. Acad. Sci. USA 83, p. 9388 to 9392, 1986;

Morgan and Canessa, J. Membrane Biol. 118, p. 193-214, 1990;

Sardet et al., Cell 56, p. 271-280, 1989;

Scholz et al., Cardiovasc. Res. 29, atr. 260-268, 1995.

Test results

First

EMD Code 221960

IC50 (NHE-3) = 1-2 μΜ

Second

EMD Code 221963

IC 50 (NHE-3) = 1 μΜ

Third

NH

H ₂ N

NH.

diacetate;

EMD Code 246326

IC 50 (NHE-3) = 3 µΜ

4th

H ₂ N

NH

diacetate;

EMD Code 246327

IC50 (NHE-3) = 3-4 μΜ

The following examples illustrate pharmaceutical compositions.

Example A

Injection ampoules

A solution of 100 g of the NHE inhibitor of formula I and 5 g of disodium hydrogen phosphate in 3 liters of double-distilled water is adjusted to pH 6.5 with 2N hydrochloric acid, sterile filtered and filled into injection vials, lyophilized under sterile conditions and sterile. apluky close. Each vial contains 5 mg of active ingredient.

Example B

lace

A mixture of 20 g of an NHE inhibitor of the formula I is melted with 100 g of soya lecithin and 1400 g of cocoa butter, poured, poured into molds and allowed to cool. Each suppository contains 20 mg of active ingredient.

Example C

solution

Prepare a solution of 1 g of the -Ε-3-inhibitor of the general formula I, 9.38 g of sodium dihydrogen phosphate dihydrate, 28.48 g of 12-water disodium hydrogen phosphate and 0.1 g of benzalkonium chloride in 940 ml of double-distilled water. The pH of the solution is adjusted to 6.8, made up to 1 liter and sterilized by irradiation. This solution can be used as eye drops.

Example D

ointment

500 mg of the ΝΗΕ-3-inhibitor of formula I are mixed with 99.5 g of petroleum jelly under aseptic conditions.

Example E

tablets

Tablets are conventionally compressed from a mixture of 1 kg of a ΝΗΕ-3-inhibitor of formula I, 4 kg of lactose, 1.2 kg of potato starch, 0.2 kg of talc and 0.1 kg of magnesium stearate such that each tablet contains 10 mg of the active substance.

Example F

dragee

Similar to Example E, tablets are compressed, which are then usually coated with sucrose, potato starch, talc, tragacanth and colorant.

Example G

capsules

In a known manner, 2 kg of the ΝΗΕ-3-inhibitor of formula I are filled into hard gelatin capsules such that each capsule contains 20 mg of the active ingredient.

Example H

ampoule

A solution of 1 kg of the ΝΗΕ-3-inhibitor of formula I in 60 liters of double-distilled water is sterile filtered and filled into ampoules, lyophilized under sterile conditions and the ampoules sealed. Each ampoule contains 10 mg of active ingredient.

Industrial usability

Biphenyl derivatives and pharmaceutically acceptable salts and solvates thereof are suitable as NHE-3 inhibitors for the manufacture of pharmaceutical compositions for the treatment of thromboses, ischemic conditions of the heart, peripheral and central nervous system and stroke, peripheral organs and limb ischemic conditions and for the treatment of shock conditions.

Claims (7)

PATENT CLAIMS A biphenyl derivative of the general formula I R (d) where R ¹ , R ⁴ where R ¹ , R ⁴ independently of one another are -C (= NH) -NH 2, optionally substituted with one -COA, -CO [C (R ⁶ ) ₂ ] _n -Ar, -COOA, -OH or the usual amino-protecting group, or represents NH-C (= NH) -NH ₂ , -CO-N = C (NH ₂ ) _2, or a group of the formula or CH are independently of one another, H, A, ^OR6, N ^(R6) 2, NO2, CN, Hal, NHCOA, NHCOAr, NHSO2, NHSO2Ar, COOR ^6, CON (R ⁶⁾ 2, CONHAr, COR ^6, COAr, S (O) nA, S (O) nAr, -O- [C (R ⁶ ) 2] _m COOR ⁶ , - [C (R ⁶ ) 2] p-COOR ⁶ , -O- [C (R) ⁶ ) 2] _m -CON (R ⁶ ) 2, - [C (R ⁶ ) 2] _P -CON (R ⁶ ) 2, -O- [C (R ⁶ ) 2] _m -CONHAr or [C (R ⁶ ) 2] ⁶ ) ₂ ] _p -CONHAr; X is - [C (R ⁶ ) 2] n -, -CR ⁶ = CR ⁶ -, - [C (R ⁶ ) 2] _n -O-, O- [C (R ⁶ ) ₂ ] _n -, - C00-, -00C-, -CONR ⁶ - or NR ⁶ COR ⁶ represents a hydrogen atom, a group A or a benzyl group A represents an alkyl group having 1 to 20 carbon atoms, one or two -CH ₂ groups being optionally replaced by an oxygen or sulfur atom or the groups -CR ⁶ = CR ⁶ - and / or one to seven hydrogen atoms being replaced by a fluorine atom; Ar ______ represents a phenyl or naphthalyl group unsubstituted or substituted by one, two or three of A, Ar ', OR ⁶ , OAr', N (R ⁶ ) 2, NO 2, CN, Hal, NHCOA, NHCOAr ', NHSO2A, NHSO 2 Ar ', COOR ⁶ , CON (R ⁶ ) 2, CONHAr', COR ⁶ , COAr ', S (O) _n A and S (O) _n Ar'; Ar 1 represents a phenyl or naphthyl group unsubstituted or substituted by one, two or three of A, OR ⁶ , N (R ⁶ ) 2, NO 2, CN, Hal, NHCOA, COOR ⁶ , CON (R ⁶ ) 2 , COR ⁶ and S (O) _n A; Hal represents a fluorine, chlorine, bromine or iodine atom; n is 0, 1 or 2; m is 1 or 2; represents 1 or 2; and salts and solvates thereof as NHE-3 inhibitors. A biphenyl derivative according to claim 1 of formula I selected from the group consisting of a) 3 '- (3-carbamimidoylphenoxymethyl) biphenyl-3-carboxamidine; (b) 3 '- (3-carbamimidoylbenzyloxy) biphenyl-3-carboxamidine, c) 3'-carbamimidoyl-5- (3-carbamimidoylphenoxymethyl) biphenyl-3-carboxylic acid, (d) N- [3 '- (3-guanidinocarbonylphenoxymethyl) biphenyl-3-carbonyl] guanidine; (e) [3'-amidino-5- (4-amidinophenoxymethyl) -biphenyl-3-yloxy] -acetic acid methyl ester, f) [3'-amidino-5- (4-amidinophenoxymethyl) biphenyl-3-yloxy] acetic acid. Use of compounds according to claim 1 of the general formula I and their physiologically acceptable salts and solvates for the manufacture of medicaments for the treatment of thromboses, ischemic conditions of the heart, peripheral and central nervous system and stroke, peripheral organs and limb ischemic conditions and for the treatment of shock conditions. Use of the compounds according to claim 1 of the general formula I and their physiologically acceptable salts and solvates for the manufacture of medicaments for use in surgical operations and organ transplantation and for the preservation and preservation of transplants for surgical purposes. Use of the compounds according to claim 1 of the formula I and their physiologically acceptable salts and solvates for the manufacture of a medicament for the treatment of diseases in which the primary or secondary cause of cell proliferation, for the treatment or prophylaxis of fat exchange disorders or impaired respiratory disorders. Use of the compounds according to claim 1 of formula I and their physiologically acceptable salts and solvates for the manufacture of a medicament for the treatment of ischemic kidney disease, ischemic bowel disease or for the prophylaxis of acute or chronic kidney disease. A pharmaceutical composition comprising, as an active ingredient, at least one NHE-3 inhibitor according to claim 1 of the formula I or a physiologically acceptable salt or solvate thereof.