KR20020033818A - Biphenyl derivatives used as nhe-3 inhibitors - Google Patents

Abstract

The present invention relates to compounds of formula (I)

(I)

(Wherein,

X, R ¹ , R ² , R ³ , R ⁴ and R ⁵ have the meaning indicated in claim 1. The compounds of the present invention represent inhibitors of subtype 3 sodium / proton exchanger (NHE-3).

Description

Biphenyl derivatives used as NHE-3 inhibitors {BIPHENYL DERIVATIVES USED AS NHE-3 INHIBITORS}

The present invention relates to compounds of formula I as NHE-3 inhibitors:

(Wherein, in the above formula,

R ¹ and R ⁴ are each independently of one another selected from -COA, -CO- [C (R ⁶ ) ₂ ] _n -Ar, -COOA, -OH or by a conventional amino protecting group, NH- _{NH 2, -CO-N = C} (NH 2) 2,

or

(= NH) -NH ₂ , which may be substituted on the nitrogen atom,

R ² , R ³ and R ⁵ are each independently of the other H, A, OR ⁶ , N (R ⁶ ) ₂ , NO ₂ , CN, Hal, NHCOA, NHCOAr, NHSO ₂ A, NHSO ₂ Ar, COOR ⁶ , CON _{^{(R 6) 2, CONHAr,}} 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 (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 ] _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 CO-,

R < ⁶ > is H, A or benzyl,

A is an alkyl having 1 to 20 carbon atoms, in which one or two CH ₂ groups may be replaced by O or S atoms or -CR ⁶ = CR ⁶ - groups and / or one to seven H atoms may be substituted by F ,

Ar is unsubstituted or substituted by ^{A, Ar ', OR 6,} OAr', N (R 6) 2, NO 2, CN, Hal, NHCOA, NHCOAr ', NHSO 2 A, NHSO 2 Ar', COOR 6, CON ( _{^{R 6) 2, CONHAr ',}} COR 6, COAr', S (O) n a or S (O) _n day to Ar' -, phenyl or phenyl or naphthyl which is substituted with three,

Ar 'is unsubstituted or substituted by ^{A, OR 6, N (R} 6) 2, NO 2, CN, Hal, NHCOA, COOR 6, CON (R 6) 2, COR 6 or S (O) days in _n A - , -, trisubstituted phenyl or naphthyl,

Hal is F, Cl, Br or I,

n is 0, 1 or 2,

m is 1 or 2,

and p is 1 or 2)

And salts and solvates thereof.

Other inhibitors of the sodium / proton exchanger subtype 3 are described, for example, in EP 0 825 178.

The present invention was based on the object of finding new compounds with useful properties, in particular those which can be used for the preparation of medicaments.

DE 19819548 discloses that the compounds of formula I and their salts have a potent Xa-inhibiting action and thus are useful for the treatment of thromboembolic disorders such as thrombosis, myocardial infarction, atherosclerosis, inflammation, stroke, angina, restenosis after intervention, It can be used for prevention.

Surprisingly, it has been found that the compounds of formula I and their salts inhibit the sodium / proton exchanger subtype 3 and have excellent resistance.

The compounds of formula I can be used as pharmaceutical active substances in human and veterinary medicine.

The Na ⁺ / H ⁺ exchanger is a family with at least six different variants (NHE-1 to NHE-6), all of which are now all cloned. Subtype NHE-1 is nonspecifically distributed in all tissues of the whole body, while other NHE subtypes are selectively expressed in the kidney or in specific organs such as the luminal wall and the semi-lumenal wall of the small intestine. This distribution is characterized by the specific function provided by the various heterotypes, namely the regulation of intracellular pH and cell volume by the subtype NHE-1 on the one hand and the regulation of the cell volume on the other by the mutant NHE-2 and NHE- Of Na ⁺ absorption and reabsorption. Hyung-Tae NHE-4 was found mainly in the above. The expression of NHE-5 is restricted to the brain and nervous tissue. NHE-6 is a heterodimer that forms a sodium proton exchanger in mitochondria.

Hypothyroidism NHE-3 is expressed, especially in the proximal membranes of proximal tubules; NHE-3 inhibitors therefore exhibit a particularly protective kidney function.

The therapeutic uses of selective inhibitors for NHE-3 variants are varied. NHE-3 inhibitors inhibit or reduce tissue damage and cellular necrosis after pathogenic physiological hypoxia and ischemic events that activate NHE activity, such as during renal ischemia or renal transplantation, kidney removal, transport and reperfusion.

The compounds of formula I are suitable as pharmaceutical active compounds for the treatment of hypertension since they exhibit hypotensive action. They are furthermore suitable as diuretics.

The compounds of formula (I) exhibit anti-ischemic action in combination with NHE inhibitors of their own or with other subtype specificities, and exhibit anti-ischemic action and are useful for the prevention of chronic or acute renal failure as well as pre- and intraoperative, for protection of organs such as kidneys and liver, Atherosclerosis, and vasospasm.

They can also be used for the treatment of stroke, cerebral edema, ischemia of the nervous system, various forms of shock, as well as for improving respiratory motion in conditions such as, for example, central sleep apnea, infant deaths, postoperative hypoxia and other respiratory disorders . Respiratory activity can be further improved by combining it with a carboanhydrase inhibitor.

The compounds of formula (I) exhibit inhibitory effects on cell proliferation, for example fibroblast proliferation and vascular smooth muscle cell proliferation, and thus can be used for the treatment of diseases where cell proliferation is a primary or secondary cause.

The compounds of formula I can be used for late diabetes complications, carcinomatous disorders, fibrotic disorders, endothelial dysfunction, organ hypertrophy and hyperplasias, especially prostatic hyperplasia or enlarged prostate.

They are also suitable as diagnostic methods for identifying and distinguishing certain forms of hypertension, atherosclerosis, diabetes and proliferative disorders.

Since the compounds of formula I also favorably affect the level of serum lipoproteins, they can be used in combination with other medicines for treating, either by themselves or by increased levels of serum lipids.

The present invention relates to compounds of formula I according to claim 1 for the preparation of medicaments for the treatment of ischemic conditions of the thrombosis, heart, peripheral and central nervous system and of stroke, ischemic conditions of the peripheral organs and limbs and shock conditions and their physiologically acceptable Salts and / or solvates thereof.

The present invention also relates to the use of a compound of formula I according to claim 1 and its physiologically acceptable salts and / or solvents for the manufacture of medicaments for use in the preservation and storage of implants for surgical and organ transplantation ≪ / RTI >

The present invention also relates to the use of a compound of formula I according to claim 1 for the manufacture of a medicament for the treatment of diseases in which cell proliferation is a primary or secondary cause, disorders of lipid metabolism or the treatment and prevention of ideal respiratory movement and its physiologically acceptable Salts and / or solvates thereof.

The invention further relates to the use of a compound of formula I according to claim 1 and its physiologically acceptable salts and / or solvents for the manufacture of a medicament for the treatment of ischemic kidney, ischemic intestinal disorders or for the prevention of the diseases of acute or chronic renal failure ≪ / RTI >

Methods for identifying substances that inhibit the sodium / proton exchanger subtype 3 are described, for example, in US 5,871,919.

For example, for all radicals of the compound of formula I appearing multiple times, such as R < ⁶ >, the meanings are to be mutually independent.

Hydrates and solvates are understood to mean, for example, semi-, mono- or dihydrates, and the solvates are understood to mean alcohol-added compounds such as, for example, methanol or ethanol.

In the above formulas, A is a straight chain or branched chain alkyl having 1 to 20 carbon atoms, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. A is selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl, also pentyl, 1-, 2- or 3- Or a group selected from the group consisting of 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1-, 2-, 3- or 4-methylpentyl, 1,1-, Ethyl-1-methylpropyl, 1-ethyl-2-methylpropyl, 1,1,2- or 1,2,2-tetramethylbutyl, More preferably is trimethylpropyl, heptyl, octyl, nonyl or decyl.

Furthermore, A is, for example, trifluoromethyl, pentafluoroethyl, allyl or crotyl.

COR ⁶ is acyl, more preferably formyl, acetyl, propionyl, butyryl, pentanoyl or hexanoyl.

COOR ⁶ is preferably methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl or butoxycarbonyl.

Hal is preferably I as well as F, Cl or Br.

R ² , R ³ and R ⁵ are each independently of one another H, fluorine, chlorine, bromine, iodine, hydroxyl, methoxy, ethoxy, propoxy, nitro, amino, methylamino, dimethylamino, Wherein the substituent is selected from the group consisting of amino, acetamido, sulfonamido, methylsulfonamido, phenylsulfonamido, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, methylsulfonyl, ethylsulfonyl, phenylsulfinyl, Or a pharmaceutically acceptable salt thereof, which is selected from the group consisting of a cyano, a carboxyl, a methoxycarbonyl, an ethoxycarbonyl, a carboxymethoxy, a methoxycarbonylmethoxy, a carboxymethyl, a methoxycarbonylmethyl, an aminocarbonylmethoxy, an aminocarbonylmethyl, Methoxy, N-phenylaminocarbonylmethyl, and also acyl or benzoyl.

In particular, R ² and R ⁵ are H.

R ³ is particularly, for example, H, COOA or -OCH ₂ COOR ⁶ (R ⁶ is H or alkyl of 1 to 4 carbon atoms).

R < ⁶ > is H, A or benzyl, but especially H or alkyl 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- ; -CH ₂ O-, -O-CH ₂ - or -CH ₂ -CH ₂ -.

Ar is preferably unsubstituted phenyl or naphthyl such as A, fluorine, chlorine, bromine, iodine, hydroxyl, methoxy, ethoxy, propoxy, butoxy, pentyloxy, Wherein the aryl group is optionally substituted with one or more substituents selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, (4-methylphenyl) sulfone, (4-methylphenyl) sulfone, and the like. Examples of the sulfonamido group include methylamino, ethylamino, formamido, acetamido, propionylamino, butyrylamino, methylsulfonamido, ethylsulfonamido, propylsulfonamido, Amido, carboxymethoxy, carboxyethoxy, methoxycarbonylmethoxy, methoxycarbonylethoxy, hydroxymethoxy, hydroxyethoxy, methoxyethoxy, carboxyl, methoxycarbonyl, ethoxycarbonyl , Cyano, phenylaminocarbonyl, acyl or benzoyl, also biphenyl By mono-, or tri-substituted phenyl or naphthyl are more preferred.

Ar is thus, for example, o-, m- or p-tolyl, o-, m- or p-ethylphenyl, o-, m- or p- O-, m- or p-tertiary butylphenyl, o-, m- or p-hydroxyphenyl, o-, m- or p-nitrophenyl, o-, m- or p- aminophenyl, o -, m- or p- (N-methylamino) phenyl, o-, m- or p-acetamidophenyl, o-, m- or p- methoxyphenyl, o-, m- or p- M- or p- (N, N-dimethylamino) phenyl, o-, m- or p-carboxyphenyl, o-, m- or p- methoxycarbonylphenyl, o-, m- or p- O-, m- or p-formylphenyl, o-, m- or p-acetylphenyl, o-, m- or p-formyl M- or p-bromophenyl, o-, m- or p-chlorophenyl, o-, m- or p-methylsulfonylphenyl, o-, m- or p- o-, m- or p- (phenylsulfonamido) phenyl, o-, m- or p- (methylsulfonamido) phenyl, o-, m- or p-methylthiophenyl Preferred are 2,3-, 2,4-, 2,5-, 2,6-, 3,4- or 3,5-difluorophenyl, 2,3-, 2,4-, 5-, 2,6-, 3,4- or 3,5-dichlorophenyl, 2,3-, 2,4-, 2,5-, 2,6-, 3,4- or 3,5-di Bromophenyl, 2,4- or 2,5-dinitrophenyl, 2,5- or 3,4-dimethoxyphenyl, 3-nitro-4-chlorophenyl, 3- Amino-6-chloro-phenyl, 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, Dichlorophenyl, 3,6-dichloro-4-aminophenyl, 4,5-trichlorophenyl, 2,4,6-trimethoxyphenyl, 2-hydroxy- Fluoro-4-bromophenyl, 3-bromo-6-methoxyphenyl, 3-chloro-6- Methoxyphenyl, 3-chloro-4-acetamidophenyl, 3-fluoro-4-methoxyphenyl, 3- No-6-methylphenyl, more preferably a 3-chloro-4-acetamido-phenyl, or 2,5-dimethyl-4-chlorophenyl.

Ar 'is in particular phenyl or naphthyl, for example o-, m- or p-tolyl, o-, m- or p-ethylphenyl, o-, m- or p- , o-, m- or p-isopropylphenyl, o-, m- or p-tert-butylphenyl, o-, m- or p-hydroxyphenyl, o-, m- or p- m- or p-aminophenyl, o-, m- or p- (N-methylamino) phenyl, o-, m- or p- acetamidophenyl, o-, m- or p- M- or p-methoxy carbonyl phenyl, o-, m- or p- (N, N-diethoxyphenyl, o-, m- or p- O-, m- or p- (N-ethylamino) phenyl, o-, m- or p- O-, m- or p-formylphenyl, o-, m- or p-fluorophenyl, o-, m- or p-bromophenyl, o-, m- or p- -, m- or p-methylsulfonylphenyl.

Accordingly, the present invention relates more particularly to the use of said compounds of formula I, wherein said one or more radicals have one of these preferred meanings. Some preferred groups of compounds correspond to formula (I) and the radicals not specifically indicated have the meanings indicated in formula (I)

Ia)

R ¹ and R ⁴ are independently -C (= NH) -NH ₂ , which may be monosubstituted independently by OH or -CO-N═C (CH ₂ ) ₂ ;

Ib)

R ² , R ⁵ is H;

Ic)

R ¹ and R ⁴ are each independently -C (= NH) -NH ₂ which may be monosubstituted by OH or -CO-N═C (NH ₂ ) ₂ ,

R ² , R ⁵ is H,

R ³ is H or COOR ⁶ ;

Id)

R ¹ and R ⁴ are each independently -C (= NH) -NH ₂ which may be monosubstituted by OH or -CO-N═C (NH ₂ ) ₂ ,

R ² , R ⁵ is H,

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

Ie)

X is -CH ₂ -O- or -O-CH ₂ -, and;

If

R ¹ and R ⁴ are each independently -C (= NH) -NH ₂ which may be monosubstituted by OH or -CO-N═C (NH ₂ ) ₂ ,

R ² , R ⁵ is H,

R ³ is H or COOR ⁶ ,

X is -CH ₂ -O- or -O-CH ₂ -, and;

Ig)

R ¹ and R ⁴ are each independently -C (= NH) -NH ₂ which may be monosubstituted by OH or -CO-N═C (NH ₂ ) ₂ ,

R ² , R ⁵ is H,

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

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

Ih)

R ¹ and R ⁴ are each independently -C (= NH) -NH ₂ which may be monosubstituted by OH or -CO-N═C (NH ₂ ) ₂ ,

R ² , R ⁵ is H,

R ³ is ^{H, 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,

A is alkyl having 1 to 4 carbon atoms;

Ii)

R ¹ and R ⁴ are each independently -C (= NH) -NH ₂ which may be monosubstituted by OH or -CO-N═C (NH ₂ ) ₂ ,

R ² , R ⁵ is H,

R ³ is H, COOR ^6, and ^{_{-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,

A is alkyl having 1 to 4 carbon atoms

Can be expressed by the sub-expressions Ia to Ii.

Alternatively, the compounds of formula (I) and the starting materials for their preparation can be prepared by methods known to those skilled in the art, such as those described in the literature (for example, standard guidelines such as Houben-Weyl, Methoden der organischen Chemie [Methods of Organic Chemistry], George-Thieme-Verlag, Stuttgart) Is prepared by a method known per se, i. E., Is suitable for the reaction described above and is prepared under known reaction conditions. Although not mentioned in detail herein, are also usable in variants known per se.

If desired, the starting material may be reacted immediately without separation from the mixture by forming in situ to form the compound of formula (I).

Compounds of formula (I) are preferably obtained by treating them with a solubilizing or hydrodemic cleaving agent to liberate a compound of formula (I) from one of its functional derivatives.

Preferred starting materials for solvolysis or hydrolysis are those corresponding to formula I but containing corresponding protected amino and / or hydroxyl groups instead of one or more free amino and / or hydroxyl groups, preferably nitrogen (R 'is an amino protecting group) instead of the HN group, and / or a hydroxyl protecting group instead of the hydrogen atom of the hydroxyl group Have, for example, those corresponding to formula I, but with -COOR " (where R " is a hydroxyl protecting group) instead of the -COOH group. A preferred starting material is also an oxadiazole derivative which can be converted to the corresponding amido compound.

Introduction of the oxadiazole group is carried out, for example, by reaction of a cyano compound with hydroxylamine and reaction with phosgene, dialkyl carbonate, chloroformic acid ester, N, N'-carbonyldiimidazole or acetic anhydride do.

Much-identical or other protected amino and / or hydroxyl groups may be present in the molecule of the starting material. If the protecting groups present are different, in many cases they can be selectively removed.

The term " amino protecting group " refers to a group which is generally known and is suitable for protecting (or blocking) amino groups from chemical reactions but which can easily be removed after the desired chemical reaction takes place at other positions in the molecule. Typical groups of this type are in particular unsubstituted or substituted acyl, aryl, aralkoxymethyl or aralkyl groups. Since the amino protecting group is removed after the desired reaction (or reaction sequence), its size and nature are not critical; However, it is preferably 1 to 20 carbon atoms, especially 1 to 8 carbon atoms. The expression "acylase" is interpreted as the broadest concept related to this manufacturing process. It includes acyl groups derived from aliphatic, aromatic aliphatic, aromatic or heterocyclic carboxylic acids or sulfonic acids and, in particular, alkoxycarbonyl, aryloxycarbonyl and especially aralkoxycarbonyl groups. Examples of acyl groups of this type include alkanoyls such as acetyl, propionyl, butyryl; Aralkanoyls such as phenylacetyl; Aroyl, such as benzoyl or toluyl; Aryloxyalkanoyls such as POA; Alkoxycarbonyls such as methoxycarbonyl, ethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, BOC (tert-butyloxycarbonyl), 2-iodoethoxycarbonyl; Aralkyloxycarbonyls such as CBZ (carbobenzoxy), 4-methoxybenzyloxycarbonyl, FMOC; There are arylsulfonyls such as Mtr. Preferred amino protecting groups are BOC and Mtr, CBZ, Fmoc, benzyl and acetyl.

The expression " hydroxyl protecting group " is likewise generally known and relates to groups which are suitable for protecting the hydroxyl group from chemical reactions but which are easily removable after the desired chemical reaction takes place at other positions in the molecule. Typical groups of this type are the unsubstituted or substituted aryl, aralkyl, or acyl groups, and also alkyl groups. The nature and size of the hydroxyl protecting group is not critical, since the desired chemical reaction or reaction sequence is removed again, but groups of 1 to 20 carbon atoms, especially 1 to 10 carbon atoms, are preferred. Examples of hydroxyl protecting groups are in particular benzyl, p-nitrobenzoyl, p-toluenesulfonyl, tert-butyl and acetyl, benzyl and tert-butyl are particularly preferred.

Depending on the protecting group used, for example, strong acids can be used, conveniently TFA or perchloric acid, and also with other strong inorganic acids such as hydrochloric acid or sulfuric acid, strong organic carboxylic acids such as trichloroacetic acid or sulfonic acids such as benzene- or p- , The compound of formula (I) is liberated from a functional derivative of a compound of formula (I). Additional inert solvents may be present but are not always necessary. Suitable inert solvents include, for example, carboxylic acids such as acetic acid, ethers such as tetrahydrofuran or dioxane, amides such as DMF, halogenated hydrocarbons such as dichloromethane, alcohols such as methanol, ethanol or isopropanol, It is also preferable that it is water. In addition, mixtures of the above-mentioned solvents are possible. TFA is preferably used in excess in the absence of other solvents in the form of a 9: 1 mixture of acetic acid and 70% perchloric acid. The reaction temperature for the decomposition is conveniently from about 0 to about 50 [deg.], And the reaction is preferably carried out at from 15 to 30 [deg.] (Room temperature).

The BOC, OBut, and Myr groups can be desirably removed, for example, using about 3-5N HCl in dioxane at 15-30 [deg.] Or using TFA in dichloromethane; The Fmoc group can be desirably removed using a 5-50% solution of dimethylamine, diethylamine or piperidine in DMF at 15-30 [deg.].

For example, by treating with a catalyst in the presence of a catalyst (for example, a noble metal catalyst such as palladium, conveniently a noble metal catalyst such as carbon or a wet Raney nickel, for example, with added acetic acid) Hydrolytically removable protecting groups (e. G., A glass of amzdinocyte from CBZ, benzyl or oxadiazole derivatives thereof) can be removed. Suitable solvents in this context are those indicated above, in particular alcohols such as, for example, methanol or ethanol, or amides such as DMF. In general, hydrocracking is carried out at a temperature of from about 0 to 100 degrees and at a pressure of from about 1 to 200 bar, preferably at a temperature of from 20 to 30 degrees and at a pressure of from 1 to 10 bar. Hydrogenolysis of the CBZ group is easily carried out, for example, on 5-10% Pd / carbon in methanol at 20-30 ° using ammonium formate (instead of hydrogen) or Pd / carbon in methanol / DMF.

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

The cyano group is converted, for example, to an amidino group by reduction of N-hydroxyamidine with hydrogen in the presence of a catalyst such as Pd / carbon or Raney nickel after reaction with, for example, hydroxylamine.

It is also possible to add ammonia to the nitrile of the formula I (R ¹ = CN) in order to prepare an amidine of the formula I (R ¹ = -C (= NH) -NH ₂ ). The addition is carried out in a manner known per se by known methods a) conversion of the nitrile using H ₂ S to a thioamide converted to the corresponding S-alkylimidothioester using an alkylating agent such as CH ₃ I, further comprising: a part is switched to the step, b) imidoesters corresponding nitrile by under HCl present using an alcohol such as ethanol to produce the amidine by reaction with NH ₃ and is treated with ammonia, or c) lithium-bis nitrile ( Trimethylsilyl) amide and then hydrolyzing the product.

The preparation of cyano compounds is carried out by methods known per se.

Compounds of formula (I) wherein R ¹ and R ⁴ are -CON (═NH) -NH ₂ can be preferably obtained from the corresponding alkoxycarbonyl compounds by reacting with guanidine.

For example, O an amino group acylated, or by reducing the nitro group to an amino group (e. G., In an inert solvent such as methanol or ethanol Raney by hydrogenation over nickel or Pd / carbon) at least one radical R ^1, R ^2, R ^3, It is also possible to convert the compounds of formula I into other compounds of formula I by converting R ⁴ and / or R ⁵ into one or more radicals R ¹ , R ² , R ³ , R ⁴ and / or R ⁵ .

The ester can be hydrolyzed, for example, using NaOH or KOH in water, water / THF or water / dioxane at a temperature of 0-100 °, or using acetic acid.

Acylation of the free amino group in the customary manner using an acid chloride or anhydride or conveniently in the presence of a base such as triethylamine or pyridine in an inert solvent such as dichloromethane or THF conveniently at temperatures of -60 to + It is also possible to alkylate them using unsubstituted or substituted alkyl halides under the conditions described in US Pat.

In general, the reaction is carried out in an inert solvent in the presence of acid binders, preferably alkali metal or alkaline earth metal hydroxides, carbonates or bicarbonates, or other salts of weak acids of alkali or alkaline earth metals, preferably potassium, sodium, calcium or cesium .

It is preferable to add an organic base such as triethylamine, dimethylaniline, pyridine or quinoline or an excess of an amine component of the formula (II) or an alkylated derivative of the formula (III). Depending on the conditions used, the reaction time is from about 14 to about 14 days, and the reaction temperature is from about 0 to 150, typically 20 to 130.

Suitable inert solvents include, for example, hydrocarbons such as hexane, petroleum ether, benzene, toluene or xylene; Chlorinated hydrocarbons such as trichlorethylene, 1,2-dichloroethane, 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 esters such as ethylene glycol monomethyl or monoethyl ether (methyl glycol or ethyl glycol), ethylene glycol dimethyl ether (diglyme); Ketones such as acetone or butanone; Amides such as acetamide, dimethylacetamide, N-methylpyrrolidone (NMP) or dimethylformamide (DMF); Nitriles such as acetonitrile; Sulfoxides such as dimethylsulfoxide (DMSO); Carbon disulfide; Carboxylic acids such as formic acid or acetic acid; Nitro compounds such as nitromethane or nitrobenzene; Esters such as ethyl acetate or mixtures of the solvents mentioned.

The base of formula (I) can be converted to the corresponding acid addition salt using an acid, for example, by reacting an acid with an equivalent amount of an acid in an inert solvent such as ethanol, followed by evaporation. Suitable acids for this reaction are those that produce particularly physiologically acceptable salts. Therefore, as the inorganic acid, for example, a hydrohalogenic acid such as sulfuric acid, nitric acid, hydrochloric acid or hydrobromic acid, phosphoric acid such as orthophosphoric acid, and sulfamic acid may be used, and organic acids such as aliphatic, alicyclic, aromatic aliphatic, aromatic or hetero ring Examples of the mono- or polybasic carboxylic acid, sulfonic acid or sulfuric acid include formic acid, acetic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimetic acid, fumaric acid, maleic acid, lactic acid, , N-butanol mono-and disulfonic acid, and lauryl sulfuric acid can be used in the present invention. Examples of the organic acid include, but are not limited to, hydrochloric acid, hydrobromic acid, hydrobromic acid, hydrobromic acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, have. Salts with physiologically unacceptable acids, such as, for example, peak rates, can also be used for the separation and / or purification of compounds of formula (I).

On the other hand, the compounds of formula (I) can be converted to the corresponding metal salts, in particular alkali metal or alkaline earth metal salts, or to the corresponding ammonium salts using bases (for example sodium hydroxide or potassium hydroxide or sodium or potassium carbonate).

Physiologically acceptable organic bases such as ethanolamine may also be used.

The present invention also relates to the use of a compound of formula I as an NHE-3 inhibitor and / or a physiologically acceptable salt thereof for the preparation of pharmaceutical preparations, in particular for the preparation of pharmaceutical preparations via non-chemical routes. In this regard, they can be made into a suitable formulation together with one or more solid, liquid and / or semi-liquid vehicles or excipients and, if appropriate, one or more other active compounds.

The present invention also relates to pharmaceutical preparations comprising one or more of the NHE-3 inhibitors of formula I and / or one of its physiologically acceptable salts and solvates.

These agents can be used as medicaments in human or veterinary drugs. Suitable vehicles include organic or inorganic materials that are not reacted with the novel compounds and are suitable for intestinal (e.g. oral) or parenteral administration or topical application, examples of which include water, vegetable oils, benzyl alcohol, alkyl Hydrocarbons such as glycerol, glycerol triacetate, gelatin, lactose or starch, magnesium stearate, talc and petrolatum. In particular, tablets, pills, preparations, capsules, powders, granules, syrups, juices or drops are used for oral administration; Suppositories are used for rectal administration; Solutions, preferably oily or aqueous solutions, and also suspensions, emulsions or implants are used for parenteral administration; Ointments, creams, and powders are used for topical applications. The novel compounds can also be lyophilized and the lyophiles obtained can be used, for example, in the preparation of injectables. The formulations can be sterilized and / or contain excipients such as lubricants, preservatives, stabilizers and / or wetting agents, emulsifiers, salts affecting osmotic pressure, buffer substances, coloring agents, flavoring agents and / Compounds (e. G., One or more vitamins).

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

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

In this regard, it is preferred that the substance according to the invention is administered in a dosage of approximately 0.1 to 100 mg, in particular 1 to 10 mg, per dosage unit. The daily dosage is preferably about 0.001 to 10 mg per kg of body weight. It will be understood, however, that the specific dose for each patient will depend upon a wide variety of factors, such as the efficacy of the particular compound employed, age, weight, general condition of health, sex, diet, time and route of administration, excretion rate, As shown in FIG. Oral administration is preferred.

All temperatures above and below are indicated in degrees Celsius. In the following examples, " normal reaction finishing " means: water is added, if necessary, and the pH of the mixture adjusted to 2 to 10, if necessary, depending on the composition of the final product, extracted with ethyl acetate or dichloromethane, Separate, dry with sodium sulfate, evaporate, and chromatograph the residue on silica gel and / or crystallize to purify.

Mass spectrometry (MS):

EI (electron impact ionization) M ⁺

FAB (accelerated atomic impact) (M + H) ⁺

[Example 1]

A solution of 2.06 g of 3-bromobenzonitrile and 1.50 g of 3-tolyl boric acid in 50 ml of dimethoxyethane was added to a solution of 247 mg of palladium (II) acetate, 335 mg of tri-O-tolylphosphine, 20 ml of water and 954 mg of Treated with anhydrous sodium carbonate and heated at 100 < 0 > C for 18 hours with stirring. The mixture was reaction quenched by conventional methods and the residue was chromatographed on a silica gel column using petroleum ether / ethyl acetate 9: 1 to give 3'-methylbiphenyl-3-carbonitrile as a colorless solid ("A" EI 193 is obtained.

A solution of 1.17 g of "A" in 10 ml of carbon tetrachloride is treated with 1.09 g of N-bromosuccinimide (NBS) and 60 mg of azobisisobutyronitrile and heated at 70 ° C. for 18 hours. The reaction was worked up in a conventional manner and the residue was chromatographed on a silica gel column using petroleum ether / ethyl acetate 9: 1 to give 3'-bromomethylbiphenyl-3-carbonitrile as a colorless solid ("B") .

A solution of 500 mg of " B " and 238 mg of 3-hydroxybenzonitrile in 10 ml of acetonitrile is treated with 652 mg of cesium carbonate and stirred at room temperature for 40 hours. After a typical reaction finish, the residue is chromatographed on a reverse-phase column using 65:35 acetonitrile / water. 3'- (3-cyanophenoxymethyl) biphenyl-3-carbonitrile ("C") as a colorless solid, FAB 311.

90 mg of " C " and 125 mg of hydroxylammonium chloride solution in 10 ml of ethanol are treated with 1.2 g of polymer-bound dimethylaminopyridine (DMAP) and stirred at room temperature for 18 hours. The polymer was filtered and the solvent removed to give N-hydroxy-3'- [3- (N-hydroxycarbamimidoyl) phenoxymethyl] biphenyl-3-carboximidine ("D") as a colorless solid, FAB 377 < / RTI >

76 mg of " D " solution in 10 ml of methanol are treated with 100 mg of water-wet Raney nickel and 30 mg of acetic acid and hydrogenated at room temperature and normal pressure for 18 hours. The catalyst was filtered off and 3 ' (3-carbamimidoyl phenoxymethyl) to remove the solvent-3-carboxamide, pyrimidine, acetate, EI 327 (M ⁺ -NH _3), 310 (M ⁺ - NH 2 ₃ ).

Analogously, the following compounds are obtained.

3'- (3-carbamimidoyl phenoxymethyl) biphenyl-4-carboximidine, diacetate, FAB 345;

3'- (4-carbamimidoyl phenoxymethyl) biphenyl-4-carboxinidine, diacetate, FAB 345;

3'- (4-carbamimidoyl phenoxymethyl) biphenyl-3-carboxymidine, diacetate FAB 345;

4'- (4-carbamimidoyl phenoxymethyl) biphenyl-4-carboximidine,

4'- (4-carbamimidoyl phenoxymethyl) biphenyl-3-carboximidine,

4'- (3-carbamimidoyl phenoxymethyl) biphenyl-3-carboximidine and

4'- (3-carbamimidoyl phenoxymethyl) biphenyl-4-carboximidine.

[Example 2]

Similar to Example 1, the compound 3'-methoxybiphenyl-3-carbonitrile is obtained by reacting 3-bromobenzonitrile with 3-methoxyphenylboric acid.

Subsequently, the ether is decomposed using aluminum triiodide in acetonitrile and reacted with 3-bromomethylbenzonitrile to give 3'- (3-cyanobenzyloxy) biphenyl-3-carbonitrile.

Reaction with hydroxylamine and reduction with hydrogen under Ra Ni catalysis gives 3'- (3-carbamimidoylbenzoyloxy) biphenyl-3-carboxymidine.

Analogously, the following compounds are obtained.

4'- (4-carbamimidoylbenzyloxy) biphenyl-4-carboxymidine, diacetate, FAB 345;

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

[Example 3]

Similar to Example 1, 3-cyanophenylboric acid and methyl 3-bromo-5-methylbenzoate were reacted to obtain methyl 3'-cyano-5-methylbiphenyl-3-carboxylate. Brominated using NBS and reacted with 3-hydroxybenzonitrile to give methyl 3'-cyano-5- (3-cyanophenoxymethyl) biphenyl-3-carboxylate.

Reacted with hydroxylamine and reduced with H ₂ / Ra ₂ Ni to obtain the compound methyl 3'-carbamimidoyl-5- (3-carbamimidoyl phenoxymethyl) biphenyl-3-carboxylate.

Water-soluble NaOH is used to hydrolyze the ester, from which 3'-carbamimidoyl-5- (3-carbamimidoyl phenoxymethyl) biphenyl-3-carboxylic acid is obtained.

Chromatography on a reverse-phase column using an acetonitrile / water / TFA mixture provided 3'-carbamimidoyl-5- (3-carbamimidoyl phenoxymethyl) biphenyl-3-carboxylic acid, bistrifluoroacetate .

Analogously, the following compounds are obtained.

Methyl 4'-carbamimidoyl-4- (4-carbamimidoyl phenoxymethyl) biphenyl-3-carboxylate, FAB 403;

Methyl 4'-carbamimidoyl-4- (3-carbamimidoyl phenoxymethyl) biphenyl-2-carboxylate, FAB 403;

Methyl 3'-carbamimidoyl-4- (4-carbamimidoyl phenoxymethyl) biphenyl-2-carboxylate, FAB 403;

Methyl 3'-carbamimidoyl-4- (3-carbamimidoyl phenoxymethyl) biphenyl-2-carboxylate, FAB 403;

Methyl 4'-carbamimidoyl-5- (3-carbamimidoyl phenoxymethyl) biphenyl-4-carboxylate, FAB 403;

Methyl 3'-carbamimidoyl-5- (3-carbamimidoyl phenoxymethyl) biphenyl-4-carboxylate, FAB 403.

[Example 4]

Similar to Example 1, 3'-methylbiphenyl-3-carboxylate is obtained by reacting methyl 3-bromobenzoate with 3-tolyl boric acid. Bromo using NBS and reacting with methyl 3-hydroxybenzoate to obtain methyl 3 '-( 3-methoxycarbonylphenoxymethyl) -biphenyl-3-carboxylate therefrom. To obtain N- [3'- (3-guanidinocarbonylphenoxymethyl) biphenyl-3-carbonyl] guanidine from this by reacting with guanidine hydrochloride in a methanolic sodium methoxide solution.

The compound N- [4'- (4-guanidinocarbonylphenoxymethyl) biphenyl-4-carbonyl] guanidine is similarly obtained.

[Example 5]

A solution of 7.0 g of 3-bromo-5-methylphenol and 5.97 g of methyl bromoacetate and 13 g of cesium carbonate in 100 ml of acetonitrile is stirred at room temperature overnight. After a typical finishing operation, 9.70 g of methyl (3-bromo-5-methylphenoxy) acetate (" AB ") is obtained.

A suspension of 2.0 g of "AB", 100 mg of tetrakis- (triphenylphosphine) palladium and 0.85 g of sodium carbonate in 50 ml of toluene is heated until boiling. A solution of 2.94 g of 3-cyanophenylboric acid in 30 ml of methanol is then added dropwise and the mixture is heated under reflux for 14 hours. The reaction is finished in a conventional manner to obtain 2.17 g of methyl (3'-cyano-5-methylbiphenyl-3-yloxy) acetate ("AC").

A solution of 1.2 g of " AC " and 0.765 g of NBS in 50 ml of carbon tetrachloride is irradiated at room temperature using UV or the like. After a typical reaction finish, 1.54 g of methyl (3'-cyano-5-bromomethylbiphenyl-3-yloxy) acetate ("AD") is obtained.

A solution of 185 mg of " AD ", 63.1 mg of 4-hydroxybenzonitrile and 172.7 mg of cesium carbonate in 10 ml of acetonitrile is stirred at room temperature for 4 days. After a typical reaction finish, methyl [3'-cyano-5- (4-cyanophenoxymethyl) biphenyl-3-yloxy] acetate ("AE") is obtained.

A solution of 60 mg of " AE ", 69.5 mg of hydroxylammonium chloride and 101 mg of triethylamine in 10 ml of ethanol is heated under reflux for 14 hours. The solvent is removed and the residue is taken up in water. The solid is separated and 70 mg of methyl [3'-N-hydroxyamidino-5- (4-N-hydroxyamidinophenoxymethyl) biphenyl-3- yloxy] acetate ("AF" . Methyl [3'-amidino-5- (4-amidinophenoxymethyl) biphenyl-3-yloxy] acetate, FAB 433 is obtained by reduction with H ₂ / Raney nickel.

Analogously, the following compounds are obtained.

Methyl [4'-amidino-5- (4-amidinophenoxymethyl) -biphenyl-3-yloxy] acetate, FAB 433

Methyl [3'-amidino-5- (3-amidinophenoxymethyl) -biphenyl-3-yloxy] acetate, FAB 433

Methyl [4'-amidino-5- (3-amidinophenoxymethyl) -biphenyl-3-yloxy] acetate, FAB 433.

If methyl bromoacetate is replaced by tert-butyl bromoacetate in the first step, trifluoroacetic acid can be used to decompose the tertiary-butyl ester obtained in the final stage, and the corresponding carboxylic acid

[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 triethylphosphite are mixed and heated slowly to 150 °. The mixture is stirred at 150 ° for 6 hours and after the usual reaction finish, 6.05 g of diethyl (3'-cyanobiphenyl-3-ylmethyl) phosphonate ("BA") is obtained.

150 mg of sodium hydride is added to a solution of 1.0 g of "BA" and 3-cyanobenzaldehyde in 20 ml of ethylene glycol dimethyl ether under ice-cooling under nitrogen. The mixture is then stirred for 4 hours and reaction is carried out in the usual manner to give 0.93 g of 3'- [2- (3-cyanophenyl) vinyl] biphenyl-3-carbonitrile ("BB"). The compound 3'- [2- (3-amidinophenyl) ethyl] biphenyl-3-carboxymidine, FAB 343, is obtained analogously to Example 1, after reaction with hydroxylammonium chloride and hydrogenation using Raney nickel .

Compound 3'- [2- (4-amidinophenyl) ethyl] biphenyl-3-carboxidin, FAB 343 is similarly obtained.

[Example 7]

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

For example, according to the following reaction scheme:

Similarly, the compound

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

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

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

.

Pharmacological test

Methods used to characterize compounds of formula I as NHE-3 inhibitors are described below.

The compounds of formula (I) were characterized in terms of their selectivity to NHE-I to NHE-3. Three heterozygotes were stably expressed in mouse fibroblast cells. The inhibitory action of the compounds was assessed by confirming EIPA-sensitive ²² Na ⁺ uptake into cells after intracellular acidemia.

In order to characterize the Na ⁺ / H ⁺ exchange inhibitor in relation to its dendritic selection, we have shown that EIPA-sensitive ²² Na ⁺ uptake into cells after intracellular acidosis is stable in mouse fibroblast cells (see method section) NHE-1, MHE-2, and NHE-3, which are expressed in the NHE-expressing cells.

Materials and methods

LAP1 cell line expressing other NHE heterozygotes

LAP1 cell lines expressing NHE-1, -2 and -3 (mouse fibroblast cell line) were obtained from Professor J. Pouyssegur (Nice, France). Transfection was performed by the procedure of Franchi et al. (1989). Cells were cultured in DMEM (Dulbecco ' s modified Eable medium) with 10% inactive FCS (fetal calf serum). To select NHE-expressing cells, the so-called " acid destruction procedure " of Sardet et al. (1989) was used. Cells were first transferred to NH ₄ Cl-containing bicarbonate and sodiumbuminate buffer by washing with unchallenged acid, no NH ₄ Cl and Na sodium buffer and incubated with a bicarbonate NaCl-containing buffer. Only cells functionally expressing NHE were able to survive in their exposed cellular acidification.

Characterization of NHE inhibitors in relation to selectivity

Following the procedure described in the literature (Counillon et al (1993) and Scholz et al (1995)), using the mouse fibrous asei strain expressing NHE-I, NHE-2 and NHE-3, The selectivity was tested. The cells were intracellularly acidified by incubation in a NH ₄ Cl pre-pulse procedure followed by a bicarbonate ²² Na ⁺ -containing buffer. Because of intracellular acidification, NHE was activated and sodium was absorbed into the cells. The effect of the test compound was indicated by the inhibition of EIPA (ethylisopropylamiloride) - sensitive ²² Na ⁺ uptake.

Cells expressing NHE-1, NHE-2 and NHE-3 were inoculated at a density of 5 to 7.5 × 10 ⁴ cells / tank and transferred to a 24-minute microtiter plate and cultured for 24 to 48 hours to confluent. The medium was aspirated and the cells were incubated in NH ₄ Cl buffer (50 mM NH ₄ Cl, 70 mM Choline chloride, 15 mM MOPS, pH 7.4) for 60 minutes at 37 ° C. Remove the buffer and then the cell was quickly choline chloride wash buffer covered twice with (120mM choline chloride, 15mM PIPES / tris, 0.1mM elegant _{bar, 1mM MgCl 2, 2mM CaCl 2} , pH7.4) layer; Cells were incubated in this buffer for 6 minutes. After the incubation time, the culture buffer was aspirated. To remove extracellular radioactivity, cells were washed 4 times rapidly with ice-cold PBS (phosphate-buffered saline solution). Then, 0.3 ml of 0.1 N NaOH / group was added to dissolve the cells. The cell-fragment-containing solution was transferred to a scintillation tube. Each bath was further washed twice with 0.3 ml of 0.1 N NaOH and the wash solution was added to the corresponding scintillation tube in this manner. The tube containing the cell lysate was mixed with the scintillation cocktail and the radioactivity absorbed into the cells was confirmed by the detection of beta radiation.

In rabbit red blood cells ²² Na ⁺ Inhibition of absorption

In acidified rabbit red blood cells²²Na⁺By observing the absorption of ions Na⁺/ H⁺Exchange activity. Rabbit red blood cells are Na⁺/ H⁺(Escobales & Fugueroa, 1991: Morgan & Canessa, 1990). In acidified red blood cells²²Na⁺The EIPA-sensitive part of the absorption Na⁺/ H⁺-Dependence²²Na⁺Absorption.

Cell Manufacturing

The production of erythrocytes and the internal acidification of erythrocyte microparticles were performed similar to the methods of Morgan and Canessa (1990).

Blood was obtained from rabbits (for example, New Zealand white). Blood was collected in a 50 ml Falcon centrifuge tube containing 5 ml of sodium heparin solution (250 U / ml). Blood and heparin solution were mixed well. Red blood cells were recovered by centrifugation at 2000 x g at 4 < 0 >C; Plasma and buffy coats were removed. The remaining solution was filtered with a 200 탆 gauge. The filtrate was again with the washing buffer _{(140mM KCl, 0.15mM MgCl 2,} 10mM TRIS / MOPS, pH 7.4) were suspended in the original volume. Red blood cells were collected again by centrifugation (2000 x g, 4 캜). The washing procedure was repeated twice.

Intracellular acidification

And for the cells in acidified precipitate collected 5ml of red blood cells using acidified buffer _{(170 mM KCl, 0.15mM MgCl 2} , 0.1 mM elegant bar, 10mM glucose, 10mM sucrose as agarose, 20mM tris / Mes, pH 6.2 ) in 45ml , And suspended again. The suspension of erythrocytes was incubated at 37 占 폚 for 10 minutes (with occasional mixing). To fix the internal pH, it was treated with 200 μM and 1 mM or less of DIDS or DIAMOX (acetazolamide). The mixture was further incubated at 37 DEG C for 30 minutes.

The red blood cells were then recovered by centrifugation (2000 x g, 4 min at 4 ° C); They were resuspended in ice-cold non-buffered wash solution (170 mM KCl, 40 mM sucrose, 0.15 mM MgCl ₂ ) and washed 4 times with it.

²² Na ⁺ Culture and measurement of absorption

The culture was carried out in a giant coarse tube strip in the form of 8 x 12 cells. 200㎕ incubation buffer solution (160mM KCl in, ²² NaCl (37 MBq / _{tank), 10mM NaCl, 0.15mM MgCl 2} , 0.1mM elegant bar, 10mM glucose, 40mM sucrose as agarose, 10mM tris / MPOS, pH 8.0, 0.5mM Diamox, 1% DMSO) and 20 microliters of (pre-warmed) acidified solution of erythrocytes. The test material was firstly dissolved using 100% DMSO and the solution was diluted to the corresponding concentration using culture buffer. And incubated at 37 ° C for about 5 minutes (preliminary experiments showed that the uptake rate of ²² Na under these culture conditions was linear during the 5 min incubation period). The culture was stopped by adding 800 μl of ice-cold stop solution (112 mM MgCl ₂ , 0.1 mM Grabine). The tube was temporarily stored on ice. The tubes were then covered with parafilm and centrifuged at 2000 × g and 4 ° C. for 7 minutes to collect red blood cells. Four tubes adjacent to each other can be sucked simultaneously; The spacer ring at the other end of the tip aspirated the supernatant with a suction device made in Germany to prevent the tip from being too deeply immersed in the tube and the pellet of red blood cells being drawn. All ²² Na-containing supernatant and wash solutions were stored and discarded as radioactive waste.

The red blood cells were washed three times with 900 의 of ice-cold stop solution (i. E., Repeating the suspension / centrifugation step described above). After the last wash, the red cell pellet was mixed with 200 의 of water. The tube was then sonicated for 2 x 30 minutes. The giant coarse tube strips were then disassembled and each tube inverted into individual scintillation tubes; Hemolyzed solutions of erythrocytes gently shaken into the scintillation vials. Each vial was treated with 3 ml of cilantation emulsion Aquasafe 300 PS, capped and mixed well.

The radioactivity absorbed into erythrocytes was measured in a scintillation counter by measuring the beta decomposition.

Measurements per material concentration were performed three times. The mean of counting measurements in the presence of 10 μM EIPA was subtracted from each value to include Na ⁺ / H ⁺ -independent ²² Na ⁺ uptake into erythrocytes. The mean of the remaining coefficients in the absence of material was used as a 100% control; The mean value in the presence of the test compound was expressed as a percentage of this control value. The percent absorbance data is presented in a semi-log plot; IC50 values were obtained by fitting the values to a nonlinear curve using the formula f (x) = 100 / (1+ (IC50 / x) ^** n).

references:

Counillon et al. (1993) Mol. Pharmacol. 44: 1041-1045

Escobales and Figueroa (1991) J. Membrane Biol. 120, 41-49

Franchi et al. (1986) Proc. Natl. Acad. Sci. USA 83: 9388-9392

Morgan and Canessa (1990) J. Membrane Biol. 118, 193-214

Sardet et al. (1989) Cell 56: 271-280

Scholz et al. (1995) Cardiovasc.Res. 29: 260-268

Test result

One.

Code EMD 221960

IC50 (NHE-3) = 1-2 [mu] M

2.

Diacetate;

Code EMD 221963

IC50 (NHE-3) = 1 [mu] M

3.

Diacetate;

Code EMD 246326

IC50 (NHE-3) = 3 [mu] M

4.

Diacetate;

Code EMD 246327

IC50 (NHE-3) = 3-4 [mu] M

The following examples relate to pharmaceutical preparations.

Example A: Vial for injection

A solution prepared by dissolving 100 g of the NHE-3 inhibitor of formula (I) and 5 g of disodium hydrogen phosphate in 3 l of secondary distilled water was adjusted to pH 6.5 with 2N hydrochloric acid, sterilized by filtration and dispensed into the injection vial, Dry, and sterilized. An injection vial containing 5 mg of the active compound is obtained.

Example B: Suppositories

A mixture of 20 g of the NHE-3 inhibitor of formula (I), together with 100 g of soybean lecithin and 1400 g of cocoa butter, is poured into a mold and allowed to cool. A suppository containing 20 mg of the active compound is obtained.

Example C: Solution

1 g of the NHE-3 inhibitor of formula (I), 9.38 g of NaH ₂ PO ₄ .2H ₂ O, 28.48 g of Na ₂ HPO ₄ .12H ₂ O and 0.1 g of benzalkonium chloride are dissolved in 940 ml of distilled water. The solution is adjusted to pH 6.8, adjusted to 1 L, and sterilized by irradiation to prepare a liquid agent. This liquid can be used in eye drops form.

Example D: ointment preparation

500 mg of the NHE-3 inhibitor of formula I is mixed with 99.5 g of petrolatum under sterile conditions to prepare an ointment.

Example E: Purification

A mixture of 1 kg of the NHE-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 is compressed in a conventional manner to prepare tablets, each tablet containing 10 mg of the active compound.

Example F:

Similar to Example E, the tablets are squeezed, followed by sieving with a conventional method using sucrose, potato starch, talc, tragacanth and a coloring agent to prepare a preparation.

Example G: Capsules

2 kg of the NHE-3 inhibitor of formula I are filled into hard gelatine capsules in the usual manner so that each capsule contains 20 mg of the active compound.

Example H: Ampoules

A solution obtained by dissolving 1 kg of the NHE-3 inhibitor of formula (I) in 60 l of secondary distilled water is sterilized and filtered, packed in ampoules, lyophilized under sterilization conditions, and sterilized. An ampoule containing 10 mg of the active compound is obtained.

Claims (7)

A compound of formula I as an NHE-3 inhibitor: (I) (Wherein, R ^1, R ⁴ are each independently of each other by ^{-COA, -CO- [C (R 6} ) 2] n -Ar, COOA, -OH or a conventional amino-protecting group, NH-C (= NH) -NH 2, - _{CO-N = C (NH 2} ) 2, or (= NH) -NH ₂ , which may be monosubstituted by one or more R ² , R ³ and R ⁵ are each independently of the other H, A, OR ⁶ , N (R ⁶ ) ₂ , NO ₂ , CN, Hal, NHCOA, NHCOAr, NHSO ₂ A, NHSO ₂ Ar, COOR ⁶ , CON _{^{(R 6) 2, CONHAr,}} 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 (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 ] _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 CO-, R < ⁶ > is H, A or benzyl, A is alkyl having 1 to 20 carbon atoms, one or two CH ₂ groups may be replaced by O or S or -CR ⁶ = CR ⁶ - groups and / or 1 to 7 hydrogen atoms may be replaced by F, Ar is unsubstituted or substituted with ^{A, Ar', OR 6, OAr'} , N (R 6) 2, NO 2, CN, Hal, NHCOA, NHCOAr', NHSO 2 A, NHSO 2 Ar', COOR 6, CON (R ⁶⁾ _2, CONHAr', COR ^6, COAr', S (O) _n a or S (O) _n day by Ar' -, phenyl or a trisubstituted phenyl or naphthyl which, Ar' is unsubstituted or substituted by ^{A, OR 6, N (R} 6) 2, NO 2, CN, Hal, NHCOA, COOR 6, CON (R 6) 2, COR 6, S (O) _n day by A -, < / RTI > e. G., Phenyl or naphthyl, Hal is F, Cl, Br or I, n is 0, 1 or 2, m is 1 or 2, and p is 1 or 2) And salts and solvates thereof. A compound according to claim 1 as an NHE-3 inhibitor: a) 3'- (3-carbamimidoyl phenoxymethyl) biphenyl-3-carboximidine; b) 3'- (3-carbamimidoylbenzyloxy) biphenyl-3-carboximidine; c) 3'-carbamimidoyl-5- (3-carbamimidoyl phenoxymethyl) biphenyl-3-carboxylic acid; d) N- [3'- (3-guanidinocarbonylphenoxymethyl) biphenyl-3-carbonyl] guanidine; e) Methyl [3'-amidino-5- (4-amidinophenoxymethyl) biphenyl-3-yloxy] acetate; f) [3'-Amidino-5- (4-amidinophenoxymethyl) biphenyl-3-yloxy] acetic acid; And salts and solvates thereof. Claims 1. Compounds of formula I according to claim 1 for the preparation of medicaments for the treatment of ischemic conditions of peripheral organs and limbs and for the treatment of shock conditions of thrombosis, heart, peripheral and central nervous system and stroke and their physiologically acceptable The use of salts and / or solvates. Use of a compound of formula I according to claim 1 and its physiologically acceptable salts and / or solvates for the preparation of medicaments for the preservation and storage of implants for surgical and organ transplantation and for surgical evaluation . The use of a compound of the formula I according to claim 1 for the manufacture of a medicament for the treatment of diseases in which cell proliferation is a primary or secondary cause, disorders of lipid metabolism or the treatment or prevention of an ideal respiratory movement and its physiologically acceptable salts and / Or the use of a solvent compound. Use of a compound of formula I according to claim 1 and its physiologically acceptable salts and / or solvates for the manufacture of a medicament for the treatment of ischemic renal, ischemic intestinal disorders or for the prevention of acute or chronic renal impairment. Characterized in that it comprises one of the at least one NHE-3 inhibitor according to claim 1 and / or one of its physiologically acceptable salts and / or solvates.