HK1097525B - Method for producing (3-oxo-2,3-dihydro-1h-isoindol-1-yl) acetylguanidine derivatives - Google Patents

Description

Process for preparing (3-oxo-2, 3-dihydro-1H-isoindol-1-yl) acetylguanidine derivatives

The present invention relates to a process for producing a (3-oxo-2, 3-dihydro-1H-isoindol-1-yl) acetylguanidine derivative using a 3-hydroxy-2, 3-dihydro-1H-isoindol-1-one derivative or a 3- (2-carbamoylphenyl) acrylate derivative as an intermediate, an optical resolution method, and an intermediate in the process of the present invention.

(3-oxo-2, 3-dihydro-1H-isoindol-1-yl) acetylguanidine derivatives of formula I

Are NHE 1-inhibitors and are described in PCT/EP 03/05279. However, the synthetic methods described in the literature give racemic regioisomeric mixtures, which require expensive and inconvenient separation methods and reduce the yield of the desired compound. To date, it has only been possible to obtain isomers by performing expensive and inconvenient chromatographic separations on chiral supports. However, the yield of the substance is limited by the chromatographic separation method.

Therefore, there is great interest in finding methods for the regioselective preparation of (3-oxo-2, 3-dihydro-1H-isoindol-1-yl) acetylguanidine derivatives and methods for obtaining enantiomers. Improved regioselective preparation of racemic (3-oxo-2, 3-dihydro-1H-isoindol-1-yl) acetylguanidine derivatives was successful via two independent pathways, shown in scheme 1 and scheme 3. Resolution of the racemate the success was achieved by crystallization of the salt of 2, 3-O-acetylated D-or L-tartaric acid shown in scheme 5. If the undesired enantiomer is racemized under mild base catalysis, it is possible to convert the racemate essentially completely into the desired enantiomer. The process enables the simple preparation of enantiomerically enriched or enantiomerically pure (3-oxo-2, 3-dihydro-1H-isoindol-1-yl) acetylguanidine derivatives. The novel process allows simple preparation of large amounts of the compounds of the formula I on an industrial scale.

Accordingly, the present invention relates to a process for the preparation of compounds of formula I:

wherein:

r1 and R2 are each independently of the other hydrogen, F, Cl, trifluoromethoxy, 2, 2, 2-trifluoroethoxy, trifluoromethyl, 2, 2, 2-trifluoroethyl or alkyl having 1, 2, 3 or 4 carbon atoms;

r3 is Alk-R4 or trifluoromethyl;

alk is an alkyl group having 1, 2, 3, or 4 carbon atoms;

r4 is hydrogen, trifluoromethyl or cycloalkyl having 3, 4, 5, 6 or 7 carbon atoms; as shown in scheme 1, the method comprises

Scheme 1

a) Carbamoylating formula IV followed by cyclization to a compound of formula VI,

b) reacting a compound of formula VI with alkoxycarbonylmethylenetriphenylphosphorane with 1-alkoxy-1-trimethylsiloxyethylene or with trialkyl phosphonoacetate to give a compound of formula VII, and

c) reacting a compound of formula VII with guanidine to obtain a compound of formula I and salts thereof,

wherein, in the compounds of formulae IV, VI and VII,

R1-R3 are each as defined for formula I, and

r5 is an alkoxy group having 1, 2, 3 or 4 carbon atoms.

The invention also provides processes for the preparation of compounds of formula I and salts thereof, wherein

R1 and R2 are each independently of the other hydrogen, F, Cl, trifluoromethoxy, 2, 2, 2-trifluoroethoxy, trifluoromethyl, 2, 2, 2-trifluoroethyl or alkyl having 1, 2, 3 or 4 carbon atoms;

r3 is Alk-R4 or trifluoromethyl;

alk is an alkyl group having 1, 2, 3, or 4 carbon atoms;

r4 is hydrogen, trifluoromethyl or cycloalkyl having 3, 4, 5, 6 or 7 carbon atoms;

as shown in scheme 2, the method comprises:

scheme 2

a) Reacting a compound of formula II with an amine of formula III to obtain an amide of formula IV,

b) formylating the amide of formula IV at the ortho position of the amide functionality to give a formylamide of formula V,

c) cyclizing the formylamide of formula V to give the compound of formula VI,

d) reacting a compound of formula VI with alkoxycarbonylmethylenetriphenyl-phosphorane, with 1-alkoxy-1-trimethylsiloxyethylene or with trialkyl phosphonoacetate to give a compound of formula VII, and

e) reacting a compound of formula VII with guanidine to obtain a compound of formula I and a salt thereof,

wherein, in the compounds of formulae II, III, IV, V, VI and VII,

R1-R3 are each as defined for formula I,

r5 is an alkoxy group having 1, 2, 3 or 4 carbon atoms,

x is Cl, Br, OH or an alkoxy group having 1, 2, 3 or 4 carbon atoms.

Typically, the compound of formula II is reacted with the amine of formula III, if appropriate in the presence of an activator, in an inert solvent such as diethyl ether, a hydrocarbon or a halogenated hydrocarbon, for example dichloromethane, at a temperature of from-30 ℃ to the boiling point of the solvent, preferably at room temperature, to give the amide of formula IV.

For example, ortho-formylation may be carried out by initially adding an alkyl metal compound, for example an alkyl lithium compound, preferably t-BuLi, and a complex ligand, preferably TMEDA, in an inert solvent such as diethyl ether or a hydrocarbon, for example THF, at a temperature of from-100 ℃ to 0 ℃, preferably from-80 ℃ to-50 ℃. The amide of formula IV is then added and the deprotonation reaction is carried out at a temperature of-100 ℃ to 0 ℃, preferably-80 ℃ to-50 ℃ for 10 minutes to 10 hours. Next, a formylating agent, preferably DMF, is added and the reaction with the anion is effected at a temperature of-100 ℃ to 40 ℃, preferably-80 ℃ to room temperature. Preferably, after the addition of DMF, the solution is allowed to reach room temperature within 10 minutes to 3 hours, for example within 30 minutes. The amides of the formula V formed are generally cyclized directly as intermediates to the isoindolones of the formula VI.

Reacting an isoindolone of formula VI with (C)₁-C₄) Alkoxycarbonylmethylenetriphenylphosphorane in an inert solvent such as diethyl ether, a hydrocarbon or a halogenated hydrocarbon, for example toluene, at a temperature of from 0 ℃ to the boiling point of the solvent, preferably from 20 ℃ to the boiling point of the solvent, or with tris (C) phosphonoacetic acid₁-C₄) The alkyl ester is reacted in the presence of a base, such as sodium hydride, in an inert solvent such as diethyl ether, a hydrocarbon or a halogenated hydrocarbon, e.g. 1, 2-dimethoxyethane, at a temperature of from 0 ℃ to the boiling point of the solvent, preferably from 20 ℃ to the boiling point of the solvent. Or reacting the isoindolone of formula VI with 1- (C)₁-C₄) Alkoxy-1-trimethylsiloxyethylenes are reacted in the presence of a Lewis acid, such as titanium (IV) chloride or trimethylsilyl trifluoromethanesulfonate, in an inert solvent such as diethyl ether, a hydrocarbon or a halogenated hydrocarbon, for example dichloromethane, at a temperature of from-80 ℃ to the boiling point of the solvent, preferably from-80 ℃ to 20 ℃ (Synth. Commun.1987, 17, 1).

The ester of formula VII may be reacted with guanidine by generally known methods to give the acylguanidine of formula I. The reaction is preferably carried out in a protic or aprotic, polar but inert organic solvent in a manner known to the person skilled in the art. For example, in the methyl ester (formula VII; R5 ═ OCH₃) In the reaction with guanidine, it has been found that the solvent used is methanol, isopropanol or THF at temperatures from 20 ℃ to the boiling of these solvents. For example, most compounds of formula VII are reacted with non-salified guanidine in an aprotic solvent, e.g. an ether such as THF, dimethoxyEthane or dioxane. However, when the reaction of the VII compound with guanidine uses a base such as NaOH, water may also be used as the solvent. In the reaction of a compound of formula VII with a guanidinium salt, e.g., guanidine hydrochloride, the reaction is typically carried out in a base, such as potassium tert-butoxide, sodium methoxide or sodium ethoxide, in an inert solvent, such as dimethylformamide, NMP, 2-propanol, at a temperature of from 20 ℃ to the boiling point of the solvent.

In addition to the carboxylic esters of the formula VII, it is also possible to use other reactive acid derivatives in the reaction with guanidine, for example phosgene, carboxylic thioesters or carboxylic anhydrides. For example, DCC can also be used to activate the carboxylic acid. The activated carboxylic acid derivatives can be prepared in a manner known to the person skilled in the art either directly from the original carboxylic acid esters of the formula VII or from the corresponding carboxylic acids which can be obtained from the esters by customary hydrolysis methods. A series of suitable methods for preparing reactive carboxylic acid derivatives are described in detail in the document j. march, advanced organic chemistry, third edition (John Wiley & Sons, 1985, p.350).

The steps described in schemes 1 and 2 can be carried out continuously or stepwise independently of each other. The treatment of the reaction mixture may be carried out at any step. Work-up and purification of the product, if desired, can be carried out by customary methods such as extraction, pH-separation, chromatography or crystallization and customary drying.

The starting compounds of the formulae II and III are commercially available or can be prepared according to or in analogy to methods described in the literature or familiar to the person skilled in the art.

Also claimed are processes for preparing compounds of formula I:

wherein:

r1 and R2 are each independently of the other hydrogen, F, Cl, trifluoromethoxy, 2, 2, 2-trifluoroethoxy, trifluoromethyl, 2, 2, 2-trifluoroethyl or alkyl having 1, 2, 3 or 4 carbon atoms;

r3 is Alk-R4 or trifluoromethyl;

alk is an alkyl group having 1, 2, 3, or 4 carbon atoms;

r4 is hydrogen, trifluoromethyl or cycloalkyl having 3, 4, 5, 6 or 7 carbon atoms;

as shown in scheme 3, the method comprises:

scheme 3

a) Reacting an amine of formula IX with an alkyl acrylate via a diazonium salt to give a cinnamic acid derivative of formula XI,

b) reacting a compound of formula XI with an amine of formula III and a guanidine to give an acylguanidine of formula I and salts thereof,

wherein, in the compounds of formulae III, IX and XI,

R1-R3 are each as defined for formula I, and

r6 is an alkoxy group having 1, 2, 3 or 4 carbon atoms.

The invention also relates to processes for preparing compounds of formula I:

wherein:

r1 and R2 are each independently of the other hydrogen, F, Cl, trifluoromethoxy, 2, 2, 2-trifluoroethoxy, trifluoromethyl, 2, 2, 2-trifluoroethyl or alkyl having 1, 2, 3 or 4 carbon atoms;

r3 is Alk-R4 or trifluoromethyl;

alk is an alkyl group having 1, 2, 3, or 4 carbon atoms;

r4 is hydrogen, trifluoromethyl or cycloalkyl having 3, 4, 5, 6 or 7 carbon atoms;

as shown in scheme 4, the method comprises:

scheme 4

a) Converting the nitro compound of formula VIII to an amine of formula IX,

b) converting the amine of formula IX to the diazonium salt of formula X,

c) diazo salt of formula X is reacted with alkyl acrylate to obtain cinnamic acid derivative of formula XI,

d) converting a compound of formula XI to an amide of formula XII, and

e) converting a compound of formula XII to an acylguanidine of formula I, wherein the acylguanidine of formula I is obtained by converting the compound of formula XII to an isoindolone derivative of formula XIII in the presence of a base and subsequently reacting with an activated guanidine (route a), or after forming the isoindolone derivative of formula XIII from the compound of formula XII in the presence of a base, the acylguanidine of formula I is obtained by converting the compound of formula XIII to an ester of formula XIV and subsequently reacting with a guanidine (route B), or the acylguanidine of formula I is obtained by converting the compound of formula XII to an ester of formula XIV in the presence of a strong base and subsequently reacting with a guanidine (route C), or the isoindolone of formula I is obtained by directly reacting the compound of formula XII with a guanidine in the presence of a base while amidifying (guanylation) and cyclizing (route D); and salts thereof, and to the use thereof,

wherein, in the compounds of formulae VIII, IX, X, XI, XIl, XIII and XIV,

R1-R3 are each as defined for formula I, and

r6 and R7 are each independently an alkoxy group having 1, 2, 3 or 4 carbon atoms.

The nitro compound of the formula VIII can be reduced to the aniline of the formula IX by known methods (for example, the method described in "Houben-Weyl, Methodender organischen Chemie", volume XI/1, Nitrogen compound II, Georg Thieme Verlag Stuttgart, 1957, p.360ff). For example, catalytic hydrogenation is preferably carried out using Pd/C, for example using 5% Pd/C or 10% Pd/C, in a solvent, for example an alcohol, preferably ethanol, under a hydrogen pressure of from 1bar to 200bar, preferably from 1bar to 10 bar.

Then, in an inert solvent, preferably ethanol, in an acid whose anion does not replace the diazonium ion itself, for example HBF₄Or HPF₆Preferably HBF₄Or, for example, H₂SO₄In the presence of and in the presence of nitrite, preferably NaNO₂The diazotization of the aniline of formula IX is carried out in the presence of at a temperature of from-30 ℃ to the boiling point of the solvent, preferably at a temperature of from 0 ℃ to 30 ℃.

Preferably on a palladium catalyst, preferably Pd (OAc)₂The diazonium salt of formula X is reacted directly with acrylic acid (C) in the presence of a solvent at a temperature of from 0 ℃ to the boiling point of the solvent, preferably from 45 ℃ to 55 ℃₁-C₄) Alkyl esters, preferably ethyl acrylate, to give cinnamic acid derivatives of formula XI.

The benzoic acid function of the compound of formula XI can be converted to the amide of formula XII by methods known in the art, preferably by acid chloride or with the aid of DCC. The reaction can also be carried out in the manner described, so that the amide of the formula XII is converted directly into the ester of the formula XIV in the reaction mixture, i.e.the reaction to give the ester of the formula XIV from the compound of the formula XI takes place in one step. The reaction can also be carried out under the basic reaction conditions of amide formation or the cyclization can be carried out by addition of a base, for example triethylamine, Hunig's base or potassium tert-butoxide. Other alternatives include the direct conversion of a compound of formula XI to a compound of formula I by sequential amide formation, cyclization and guanylation (guanylation) in the same reaction vessel, in which case the reaction can be carried out without isolation of intermediates.

With respect to the additional conversion of the compound of formula XII to acylguanidines of formula I, there are 4 routes to choose from:

route A: the conversion of the amide of the formula XII is carried out in a solvent such as an alcohol, preferably methanol or ethanol, at a temperature of-30 ℃ to the boiling point of the solvent, preferably at room temperature, with an aqueous alkaline solution, preferably aqueous NaOH solution. Hydrolysis and cyclization of the ester functionality to the isoindolone derivative of formula XIII occurs. The compound of formula XIII is activated for acylation by generally known methods, for example with an acid chloride or with DCC (as depicted in scheme 1) and the acylguanidine of formula I is obtained.

Route B: carboxylic acids of formula XIII are synthesized as described in the a route. Next, standard methods of ester preparation are utilized, preferably in an alcohol such as methanol or ethanol, utilizing SOCl₂For example, methyl or ethyl esters of formula XIV. Next, as shown in scheme 1, the ester of formula XIV is converted to the acylguanidine of formula I.

Route C: the conversion of the amide of formula XII to the methyl or ethyl ester of formula XIV is carried out in a strong alkaline solution, preferably methanolate or tert-butanolate in an alcohol such as methanol or ethanol. The ester of formula XIV is converted to the acylguanidine of formula I as shown in scheme 1.

And (3) pathway D: the amide of the formula XII is converted under the conditions customary for guanidinylation. The solvent used is an inert solvent, such as an ether, a hydrocarbon or a halogenated hydrocarbon, preferably DMF. In general, a guanidinium salt is first reacted with a strong base, preferably KOtBu, to give the free guanidine. This mixture is added to a solution of the compound of formula XII in a solvent such as an alcohol, ether, hydrocarbon or halogenated hydrocarbon, for example DMF, NMP or 2-propanol. During the addition, amidination and cyclization to the isoindolones of formula I occur simultaneously. In another alternative, the compound of formula XI is successively cyclized to the compound of formula XIV and converted directly to the compound of formula I in a solvent such as DMF, NMP or 2-propanol using a catalytic amount of a strong base such as potassium tert-butoxide or sodium methoxide or ethoxide.

Preference is given to the D route, in which the conversion of the benzoic acid derivative of the formula XI is carried out using a one-pot process to give the acylguanidine of the formula I.

The process described in scheme 2 can be carried out continuously or stepwise. The treatment of the reaction mixture may be carried out after any of the steps. Work-up and purification of the product, if desired, can be carried out by customary methods such as extraction, pH-separation, chromatography or crystallization and customary drying.

The starting compounds of the formulae III and VIII are commercially available or can be prepared according to or in analogy to methods described in the literature or known to the person skilled in the art.

The invention also provides compounds of formula XII:

wherein:

r1 and R2 are each independently of the other hydrogen, F, Cl, trifluoromethoxy, 2, 2, 2-trifluoroethoxy, trifluoromethyl, 2, 2, 2-trifluoroethyl or alkyl having 1, 2, 3 or 4 carbon atoms;

r3 is Alk-R4 or trifluoromethyl;

alk is an alkyl group having 1, 2, 3, or 4 carbon atoms;

r4 is hydrogen, trifluoromethyl or cycloalkyl having 3, 4, 5, 6 or 7 carbon atoms;

r6 has alkoxy of 1, 2, 3 or 4 carbon atoms.

Also claimed is the use of a compound of formula XII as a synthetic intermediate.

The compounds of the formula I, enriched in enantiomers or in enantiomerically pure form, can preferably be prepared by novel optical resolution processes, which also form part of the subject matter of the present invention. For this purpose, the racemate of the compound of formula I crystallizes as the 2, 3-O-acetylated D-or L-tartrate salt, in the course of which the crystals or mother liquor are enriched in enantiomers. The free base is then released again from the salt.

Accordingly, the present invention also relates to a process for the separation of compounds of formulae Ia and Ib:

wherein

R1 and R2 are each independently of the other hydrogen, F, Cl, trifluoromethoxy, 2, 2, 2-trifluoroethoxy, trifluoromethyl, 2, 2, 2-trifluoroethyl or alkyl having 1, 2, 3 or 4 carbon atoms;

r3 is Alk-R4 or trifluoromethyl;

alk is an alkyl group having 1, 2, 3, or 4 carbon atoms;

r4 is hydrogen, trifluoromethyl or cycloalkyl having 3, 4, 5, 6 or 7 carbon atoms; as shown in scheme 5, the method comprises:

scheme 5

a) Converting the compound of formula I into a 2, 3-O-acetylated D-or L-tartrate salt and obtaining by crystallization two salts of formula XVa and XVb, respectively, and

b) liberating the two free bases of formula Ia and Ib from the two salts of formula XVa and XVb respectively,

wherein, in the compounds of formulae I, XVa and XVb, R1-R3 are as defined for formulae Ia and Ib,

R^＊is composed of

Or

R8 is alkyl having 1, 2, 3, 4, 5 or 6 carbon atoms or phenyl unsubstituted or substituted with 1, 2 or 3 substituents selected from F, Cl, Br, I, alkyl having 1, 2, 3 or 4 carbon atoms or alkoxy having 1, 2, 3 or 4 carbon atoms.

Also claimed is the above process wherein the undesired enantiomer of formula Ia or Ib is racemized again.

The use of tartaric acid derivatives R in suitable solvents, for example in ethers, for example diethyl ether, diisopropyl ether, dimethoxyethane, tetrahydrofuran or dioxane, in halogenated hydrocarbons, for example dichloromethane, trichloromethane, tetrachloromethane, 1, 2-dichloromethane or trichloroethylene, in alcohols, for example methanol, ethanol, n-propanol, 2-propanol, butanol, in esters, for example ethyl acetate or butyl acetate, in water or in solvent mixtures, preferably in 2-propanol, dimethoxyethane or ethyl acetate, at temperatures from-10 ℃ to the boiling point of the solvent, preferably from 0 ℃ to 40 ℃^＊For example, O ' -dibenzoyl-D-tartaric acid, O ' -dibenzoyl-L-tartaric acid, O ' -bis (4-methylbenzoyl) -D-tartaric acid, O ' -bis (4-methoxybenzoyl) -L-tartaric acid or O, O ' -bis (4-methoxybenzoyl) -D-tartaric acid, preferably the racemate of the compound of formula I is crystallized using O, O ' -dibenzoyl-L-tartaric acid or O, O ' -dibenzoyl-D-tartaric acid. In another variant, two or more mixtures of 2, 3-O-acylated D-or L-tartaric acids of the same configuration but carrying different acyl groups are used for the separation.

A compound of the formula I and a tartaric acid derivative R^＊Salt formation can be carried out on an equivalent basis, i.e.0.5 mol of tartaric acid derivative R containing two carboxylic acid groups per mol of compound of the formula I can be used^＊. However. It is also possible to use less than 0.5mol equivalent of 2, 3-O-acetylated D-or L-tartaric acid, for example from 0.25mol to 0.5mol of tartaric acid derivative R per mol of compound of the formula I^＊In particular from 0.25mol to 0.3mol of tartaric acid derivative R per mol of compound of the formula I^＊Crystallizing the compound of formula I. The desired enantiomer is crystallized in the form of a salt of the formula XVa or XVb and the undesired enantiomer is largely in the form of an enantiomer of the formula Ib or IaRather than being present in the mother liquor as a salt of XVa or XVb. The enantiomeric purity of the salts of formulae XVa and XVb can be increased by repeated crystallization or by stirring the initial crystallization with fresh solvent at elevated temperature and subsequent cooling.

After separation of the two salts of the formulae XVa and XVb and separation of the salt of the formula XVa or XVb from the undesired enantiomer of the formulae Ia or Ib, this is followed, typically by addition of an auxiliary base, for example an amine, for example triethylamine, an inorganic base such as NaHCO₃、Na₂CO₃Or an aqueous solution thereof, from which the enantiomerically enriched compound of formula Ia or Ib is released. It is customary to work in suitable solvents, for example in ethers such as diethyl ether, diisopropyl ether, dimethoxyethane, tetrahydrofuran or dioxane, in halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, 1, 2-dichloroethane or trichloroethane, in alcohols such as methanol, ethanol, n-propanol, 2-propanol or butanol, in esters such as ethyl acetate or butyl acetate, or in water or in solvent mixtures, preferably in ethyl acetate, 2-propanol, dichloromethane or water or mixtures thereof (in which case the reaction mixture may be one or more phases), at temperatures from-10 ℃ to the boiling point of the solvent, preferably from 10 ℃ to 40 ℃. The process can also be carried out, for example, by dissolving the salt in NaHCO₃In aqueous solution, and then extracting the enantiomer of formula Ia or Ib with an organic solvent, such as ethyl acetate.

The enantiomer Ia or Ib, which is in each case undesired, can be converted back into the racemate of formula I by racemization and then by means of a further optical resolution step. In this case, the undesired enantiomer is preferably treated with a small amount of a base, for example KOH, in a solvent such as an alcohol, for example 2-propanol, at a temperature of from 10 ℃ to the boiling point of the solvent, preferably at from 0 ℃ to 40 ℃, the reaction mixture is neutralized and the racemate is isolated after the aqueous extraction treatment. This process can be carried out by suitably selecting the amount of base and the reaction temperature so that the substance does not undergo any chemical change other than racemization in practice.

The invention also provides compounds of formulae XVa and XVb:

wherein

R1 and R2 are each independently of the other hydrogen, F, Cl, trifluoromethoxy, 2, 2, 2-trifluoroethoxy, trifluoromethyl, 2, 2, 2-trifluoroethyl or alkyl having 1, 2, 3 or 4 carbon atoms;

r3 is Alk-R4 or trifluoromethyl;

alk is an alkyl group having 1, 2, 3, or 4 carbon atoms;

r4 is hydrogen, trifluoromethyl or cycloalkyl having 3, 4, 5, 6 or 7 carbon atoms;

R^＊is composed of

Or

R8 is alkyl having 1, 2, 3, 4, 5 or 6 carbon atoms or phenyl unsubstituted or substituted with 1, 2 or 3 substituents selected from F, Cl, Br, I, alkyl having 1, 2, 3 or 4 carbon atoms or alkoxy having 1, 2, 3 or 4 carbon atoms.

When the compounds described above, for example compounds of the formulae I, Ia, Ib, VII, XIII, XIV, XVa or XVb, contain one or more asymmetric centers, they may each, independently of one another, have the S or R configuration, unless otherwise indicated. Unless more precisely defined, the compounds may exist in the form of optical isomers, diastereomers, racemates or mixtures thereof. When there is a double bond, either the E or Z configuration may be present unless otherwise indicated. The present invention encompasses all tautomeric forms of the above compounds, for example, compounds of formulae I, Ia, Ib, XVa and XVb.

The alkyl group may be linear or branched. This applies when they carry substituents or are present as substituents of other groups, for example fluoroalkyl or alkoxy. Examples of alkyl groups are methyl, ethyl, n-propyl, ethylpropyl (═ 1-methylethyl), n-butyl, isobutyl (═ 2-methylpropyl), sec-butyl (═ 1-methylpropyl), tert-butyl (═ 1, 1-dimethylethyl), n-pentyl, isopentyl, tert-pentyl, neopentyl and hexyl. Preferred alkyl groups are methyl, ethyl, n-propyl and isopropyl, more preferably methyl or ethyl. In alkyl groups, one or more, for example 1, 2, 3, 4 or 5, hydrogen atoms may be substituted by fluorine atoms. Examples of the fluoroalkyl group are a trifluoromethyl group, a 2, 2, 2-trifluoroethyl group and a pentafluoroethyl group, and a trifluoromethyl group or a 2, 2, 2-trifluoroethyl group is preferable. Substituted alkyl groups may be substituted at any position.

Examples of cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl.

The phenyl radicals may be unsubstituted or mono-or polysubstituted, for example mono-, di-or trisubstituted, by identical or different radicals. When the phenyl group is substituted, it preferably has one or two substituents which may be the same or different. In the mono-substituted phenyl group, the substituent may be at the 2-position, 3-position or 4-position. The disubstituted phenyl groups may be substituted at the 2, 3-position, 2, 4-position, 2, 5-position, 2, 6-position, 3, 4-position or 3, 5-position. In the trisubstituted phenyl group, the substituent may be in the 2, 3, 4-position, 2, 3, 5-position, 2, 4, 6-position, 2, 3, 6-position or 3, 4, 5-position.

In the process of the present invention, the above-mentioned compounds, for example the compounds of formulae I, Ia and Ib, can be used in the form of their salts and/or isolated in the form of their salts. The salts may be obtained from other salts by conventional methods, for example by reaction with an acid or base in a solvent, or by anion exchange or cation exchange. For example, acid addition salts of compounds of formulae I, Ia and Ib which may be used are halides, especially the hydrochloride, hydrobromide, lactate, sulphate, citrate, tartrate, acetate, phosphate, methanesulphonate, benzenesulphonate, p-toluenesulphonate, adipate, fumarate, gluconate, glutamate, glycerophosphate, maleate, benzoate, oxalate and pamoate salts and trifluoroacetate salts. In the case of the preparation of the active ingredients, it is preferred to prepare physiologically tolerable salts and pharmaceutically acceptable salts. Examples include salts of the compounds of formulae I, Ia and Ib with fumaric acid, in particular salts containing 1mol of fumaric acid per mol of the compounds of formulae I, Ia and Ib, and thus may be the hydrogen fumarate and the hemifumarate salts. Advantageous properties such as crystallinity, stability, in particular low hygroscopicity, low racemization tendency and good solubility are characteristic of the compounds, in particular, for example, the (S) -N- {2- [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl ] acetyl } guanidine hydrogen fumarate hydrate of the formula XVI, all tautomeric forms also forming part of the subject matter of the present invention.

When the compounds contain acid groups, they may form salts with bases, for example alkali metal salts, preferably sodium or potassium salts or ammonium salts, for example salts with ammonia or organic amines or amino acids. The compounds containing both basic groups and acidic groups may also be present in the form of zwitterions.

A particular embodiment of the invention relates to compounds wherein R1 and R2 are not both hydrogen, in particular to compounds wherein R1 is hydrogen and R2 is fluoro, chloro or trifluoromethyl, in particular trifluoromethyl. In compounds wherein R1 is hydrogen, the R2 substituent is preferably para to the C ═ O-group in the phenyl ring in the isoindolone system.

The Alk group is preferably an alkyl group having 1, 2 or 3 carbon atoms, in particular having 1 or 2 carbon atoms, more in particular having 1 carbon atom. R4 is preferably trifluoromethyl or cycloalkyl having 3, 5 or 6 carbon atoms, especially 3 carbon atoms, more preferably trifluoromethyl. A particular embodiment of the invention relates to compounds wherein R3 is trifluoromethyl or 2, 2, 2-trifluoroethyl, in particular 2, 2, 2-trifluoroethyl.

A particular embodiment of the invention relates to the preparation of N- {2- [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl ] acetyl } guanidine and its corresponding isomeric forms and salts thereof.

X is preferably chlorine or methoxy, in particular chlorine. R5 is preferably methoxy or ethoxy, especially ethoxy. R6 is preferably methoxy or ethoxy, especially ethoxy. R7 is preferably methoxy or ethoxy, especially ethoxy.

In a particular embodiment of the invention, R8 is phenyl, in particular unsubstituted phenyl, unsubstituted or substituted with 1, 2 or 3 substituents selected from F, Cl, alkyl having 1, 2, 3 or 4 carbon atoms or alkoxy having 1, 2, 3 or 4 carbon atoms.

Compounds of formulae I, Ia, Ib, XVa and XVb and their pharmaceutically acceptable salts are substituted acylguanidines which inhibit cellular sodium proton antiporters (Na +/H + -exchangers, NHE), particularly the NHE-1 subtype.

Due to their NHE inhibiting properties, the compounds of formulae I, Ia, Ib, XVa and XVb and/or their pharmaceutically acceptable salts are suitable for the prevention and treatment of diseases caused by active or activated NHE and diseases secondary to NHE-related damage.

For example, the compounds of formulae I, Ia, Ib, XVa and XVb can also be used by using low doses for the treatment and prevention of diseases which only require partial inhibition of NHE.

Since NHE inhibitors act primarily through their effect on the regulation of the pH of the cell, they may in general preferably be combined with other compounds that regulate the pH in the cell, suitable combinations being dehydratase inhibitors, systemic inhibitors of the transport of bicarbonate ions, such as sodium bicarbonate co-transporter (NBC) inhibitors or sodium-dependent chloride-bicarbonate exchanger (NCBE) inhibitors, and NHE inhibitors having an inhibitory effect on other NHE subtypes, since it is possible by them to enhance or modulate the pharmacologically relevant pH-regulating effect of the NHE inhibitors described herein.

The use of the compounds of formula I, Ia, Ib, XVa, XVb or XVI according to the invention relates to the prevention and treatment of acute and chronic diseases in animals and humans, for example the NHE inhibitors according to the invention are suitable for the treatment of diseases caused by ischemia and reperfusion.

The compounds described herein are suitable as antiarrhythmic drugs due to their pharmacological properties. Owing to their cardioprotective component, NHE inhibitors are particularly suitable for the prophylaxis of infarctions and for the treatment of angina pectoris, in which case they also preventively inhibit or greatly reduce the pathophysiological processes when ischemia-induced damage occurs, in particular when ischemia-induced arrhythmias have occurred. Due to their protective effect on pathological hypoxic and ischemic situations, the compounds of the formulae I, Ia, Ib, XVa, XVb and XVI and/or their pharmaceutically acceptable salts used according to the invention can be used as medicaments for the treatment of all acute or chronic ischemia-induced injuries or primary or secondary diseases induced thereby, because of the inhibition of the cellular Na +/H + exchange mechanism.

The invention also relates to the use thereof as a medicament for surgical interventions. Thus, the compounds may be used during organ transplantation, either before or during the removal to protect the donor organ or to protect the removed organ, for example during treatment therewith or during its storage in a physiological bath and during transplantation to a recipient.

The compounds of the invention are also valuable pharmaceuticals having a protective effect on the heart and peripheral organs and vessels during surgical interventions in angioplasty.

When bypass grafting is performed, for example, the compounds of the invention may also be used in coronary bypass grafting and in Coronary Artery Bypass Grafts (CABG).

Since the compounds of formula I according to the invention have an action against ischemia-induced damage, they can be used even for resuscitation after cardiac arrest.

The compounds of the present invention are useful as agents for treating life-threatening arrhythmia disorders. Terminating ventricular fibrillation and restoring physiological sinus rhythm.

Since NHE1 inhibitors of human tissues and organs, particularly the heart, are effective not only against damage caused by ischemia and reperfusion, but also against the cytotoxic effects of drugs used in cancer therapy and autoimmune disease therapy, it is appropriate to administer them in combination with NHE inhibitors to inhibit the cytotoxic side effects, particularly the cardiotoxic side effects, of the compounds. The reduction in cytotoxic effects, particularly cardiotoxicity, of the combination of agents with NHE1 inhibitors makes it possible to increase the dose of cytotoxic therapeutic agents and/or prolong the treatment with these agents. The therapeutic advantages of this cytotoxic treatment can be significantly increased by the combination with NHE inhibitors.

In addition, NHE1 inhibitors may be used when the heart is damaged to overproduce thyroid hormone, thyrotoxicosis, or when exogenous thyroid hormones are administered. Thus, the compounds of formulae I, Ia, Ib, XVa, XVb and XVI and/or pharmaceutically acceptable salts thereof are suitable for use in the treatment of drugs that ameliorate cardiotoxicity.

The compounds according to the invention are also suitable, on account of their protective action against damage caused by ischemia, as medicaments for the treatment of ischemia of the nervous system, in particular of the central nervous system, for example in the treatment of stroke or cerebral edema.

NHE inhibitors are also useful for the treatment and prevention of diseases and disorders which are caused by excessive excitation of the central nervous system, in particular for the treatment of epilepsy, centrally induced clonic and tonic spasms, psychological depression, anxiety and psychosis. In these cases, it is possible to use the NHE inhibitors described herein alone or in combination with other substances with anti-epileptic or antipsychotic activity or carbonic anhydrase inhibitors, such as acetazolamide, as well as with other NHE inhibitors or sodium-dependent chloride-bicarbonate exchanger (NCBE).

In addition, NHE inhibitors are equally useful in the treatment of shock type diseases such as anaphylactic, cardiogenic, hypovolemic and bacterial shock.

The compounds of formulae I, Ia, Ib, XVa, XVb and XVI and/or their pharmaceutically acceptable salts are likewise useful for the prophylaxis and treatment of thrombotic disorders, since they are themselves capable of inhibiting platelet aggregation as NHE inhibitors. In addition, they are able to inhibit or prevent excessive release of inflammatory and coagulation mediating factors, in particular von Willebrand factor and prothrombin selectin, following ischemia or reperfusion. Thus, the pathogenic effects of important prothrombin factors can be reduced and eliminated. Therefore, the NHE inhibitor of the present invention may be combined with other anticoagulant and/or thrombolytic active ingredients, for example, recombinant or native tissue plasminogen activators, streptokinase, urokinase, acetylsalicylic acid, thrombin antagonists, factor Xa antagonists, drugs having fibrinolytic activity, thromboxane receptor antagonists, phosphodiesterase inhibitors, factor vila antagonists, clopidogrel, ticlopidine, and the like. The NHE inhibitors of the invention are particularly useful in combination with NCBE inhibitors and/or with carbonic anhydrase inhibitors such as, for example, with acetazolamide.

In addition, NHE inhibitors have a strong inhibitory effect on cell proliferation, such as fibroblast proliferation and vascular smooth muscle cell proliferation. Therefore, the compounds of formulae I, Ia, Ib, XVa, XVb and XVI and/or pharmaceutically acceptable salts thereof are suitable for use as valuable therapeutic agents for diseases in which cell proliferation is manifested as a major or minor cause, and as anti-atherosclerotic drugs, therapeutic agents for chronic renal failure or cancer.

NHE inhibitors may have an inhibitory effect on cell migration. Therefore, the compounds of formulae I, Ia, Ib, XVa, XVb and XVI and/or pharmaceutically acceptable salts thereof are suitable for use as valuable therapeutic agents for diseases in which cell migration is a major or minor cause, for example, cancer with a significant tendency to metastasize.

NHE inhibitors may also alleviate or prevent fibrotic diseases. Therefore, they are suitable as excellent therapeutic agents for the treatment of cardiac and pulmonary fibrosis, hepatic fibrosis, renal fibrosis and other fibrotic diseases. They are useful in the treatment of organ hypertrophy and hyperplasia, for example in the treatment of cardiac and prostate hypertrophy and hyperplasia. They are therefore suitable for the prophylaxis and treatment of heart failure (congestive heart failure — CHF) and for the treatment and prevention of prostate hyperplasia and hypertrophy.

Due to the marked increase in NHE in patients with essential hypertension, the compounds of formulae I, Ia, Ib, XVa, XVb and XVI and/or their pharmaceutically acceptable salts are suitable for the prophylaxis and treatment of hypertension and cardiovascular diseases. In these cases, they can be used alone or in combination with suitable combination products and formulated combinations for the treatment of hypertension and cardiovascular diseases. Thus, for example, they may be used in combination with one or more diuretics having a thiazide-like action, loop diuretics, aldosterone and pseudo-aldosterone antagonists such as hydrochlorothiazide, indapamide, polythiazide, furosemide, piretanide, torasemide, bumetanide, amiloride, triamterene, spironolactone or eplerone. The NHE inhibitors of the invention may also be used in combination with calcium channel antagonists such as verapamil, diltiazemAmlodipine or nifedipine, and ACE inhibitors such as ramipril, enalapril, lisinopril, fosinopril or captopril. Other advantageous co-agents may also be beta blockers such as metoprolol, salbutamol and the like, angiotensin receptor and its sub-receptor antagonists such as losartan, irbesartan, valsartan, omatrara, gemopatrilat, endothelin antagonists, renin inhibitors, adenosine receptor agonists, potassium channel inhibitors and activators such as glibenclamide, glimepiride, diazoxide, cromakelim, minoxidil and its derivatives, activators of the mitochondrial ATP-sensitive potassium channel (mitok (ATP) channel), kv1.5 inhibitors and the like.

NHE1 inhibitors have been found to have significant anti-inflammatory effects and are therefore useful as anti-inflammatory agents. In this connection, the inhibitory effect on the release of inflammatory mediators is very pronounced. Thus, the compounds can be used for the prevention or treatment of chronic and acute inflammatory diseases, either alone or in combination with anti-inflammatory agents. The effective combination is a steroidal or non-steroidal anti-inflammatory drug. The compounds of the invention are also useful in the treatment of protozoan diseases such as malaria and coccidiosis in poultry.

It was also found that NHE inhibitors showed beneficial effects on serum lipoproteins. It is generally accepted that blood lipid levels (when too high, called hyperlipoproteinemia) are a major risk factor for the development of arteriosclerotic vascular lesions, in particular coronary heart disease. Thus, reducing elevated serum lipoproteins is important for the prevention and regression of atherosclerotic coronary lesions. In addition to lowering total serum cholesterol, it is particularly important to reduce the proportion of certain atherogenic lipid moieties, in particular Low Density Lipoprotein (LDL) and Very Low Density Lipoprotein (VLDL), in said total cholesterol, since these lipid moieties constitute risk factors for atherogenesis. In contrast, high density lipoproteins have a protective function against coronary heart disease. Thus, hypolipidemic agents should be able to lower not only total cholesterol, but also the VLDL and LDL serum cholesterol moieties in particular. It has now been found that NHE1 inhibitors show valuable therapeutically useful properties in relation to influencing serum lipid levels. Thus, it was observed that they can significantly reduce the elevated serum concentrations of LDL and VLDL, for example, as a result of increased intake of a cholesterol-and lipid-rich diet, or in the case of pathological metabolic changes, such as hereditary hyperlipidemia. Therefore, they can be used to prevent and resolve atherosclerotic lesions by eliminating the causative risk factors. This document includes not only primary hyperlipidemia, but also certain secondary hyperlipidemia that occurs, for example, hyperlipidemia associated with diabetes. In addition, NHE inhibitors significantly reduce infarcts caused by metabolic abnormalities, and in particular, significantly reduce the size and severity of induced infarcts.

Thus, the compounds of formulae I, Ia, Ib, XVa, XVb and XVI are advantageously used for the manufacture of a medicament for the treatment of hypercholesterolemia; for the manufacture of a medicament for the prevention of atherogenesis; for the production of a medicament for the prophylaxis and treatment of atherosclerosis; for the production of a medicament for the prophylaxis and treatment of diseases caused by high cholesterol levels; for the production of a medicament for the prophylaxis and treatment of diseases caused by endothelial dysfunction; for the production of a medicament for the prophylaxis and treatment of hypertension caused by atherosclerosis; for the production of a medicament for the prophylaxis and treatment of atherosclerosis-induced thrombosis; for the production of a medicament for the prevention and treatment of ischemic injury and post-ischemic reperfusion injury caused by hypercholesterolemia and endothelial dysfunction; for the production of a medicament for the prophylaxis and treatment of cardiac hypertrophy and cardiomyopathy and Congestive Heart Failure (CHF) caused by hypercholesterolemia and endothelial dysfunction; for the production of a medicament for the prevention and treatment of coronary vasospasm and myocardial infarction caused by hypercholesterolemia and endothelial dysfunction; for use in combination with a hypotensive agent, preferably an Angiotensin Converting Enzyme (ACE) inhibitor and an angiotensin receptor antagonist, for the manufacture of a medicament for the treatment of said disease. The combination of an NHE inhibitor with a lipid-lowering active ingredient, preferably with an HMG-CoA reductase inhibitor (e.g. lovastatin or pravastatin), has been shown to be advantageous in enhancing the effect and reducing the use of the active ingredient, the latter component producing a lipid-lowering effect and thus increasing the lipid-lowering properties of the NHE inhibitor.

Therefore, NHE inhibitors produce potent protection against endothelial injury of various origins. This protective effect on the blood vessels against endothelial dysfunction syndrome makes the compounds of formulae I, Ia, Ib, XVa, XVb and XVI and/or their pharmaceutically acceptable salts valuable medicaments for the prophylaxis and treatment of coronary vasospasm, peripheral vascular diseases, in particular intermittent claudication, atherogenesis and atherosclerosis, left ventricular hypertrophy and dilated cardiomyopathy and thrombotic disorders.

In addition, NHE inhibitors have been found to be suitable for the treatment of non-insulin dependent diabetes mellitus (NIDDM), being limited to insulin resistance. In this connection, it is advantageous for enhancing the antidiabetic activity and quality of the compounds of the invention for the combined use with biguanides (e.g. metformin), antidiabetic sulfonylureas (e.g. glibenclamide, glimepiride, tolbutamide, etc.), glucosidase inhibitors, PPAR agonists (e.g. rosiglitazone, pioglitazone, etc.), insulin products in different administration forms, DB4 inhibitors, insulin sensitizers or meglitinide.

In addition to the acute antidiabetic effect, NHE inhibitors block the development of diabetic late complications, and thus are useful as agents for the prevention and treatment of diabetic late damage such as diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy and other diseases caused by diabetes. In this case, they are advantageously combined with the antidiabetic drugs described above in the treatment of NIDDM. In this connection, combination with a beneficial dosage form of insulin should be of particular importance.

NHE inhibitors, in addition to exhibiting a protective effect against acute ischemic events and subsequent also acute emergency reperfusion events, also exhibit a direct therapeutic effect against diseases and conditions of the whole mammalian organism associated with the manifestation of a chronic progressive aging process, also occurring independently in acute hypoperfusion conditions and in normal cases non-ischemic diseases. Treatment with NHE inhibitors of pathological, age-related symptoms such as diseases, labor loss and death which can be improved during long-term aging is mainly diseases and disorders which are caused by age-related changes in vital organs and their function and become increasingly important in aging organisms.

Diseases associated with age-related functional impairment or with symptoms of age-related organ loss are, for example, inappropriate responses and responsiveness of blood vessels to contraction and relaxation. NHE inhibitors can significantly eliminate or slow this age-related decline in vascular responsiveness to systolic and diastolic stimuli, which are essential processes of the cardiovascular system and also essential processes of life and health. An important function and measure of preservation of vascular responsiveness is to prevent or delay the progression of age-related endothelial dysfunction, which is most significantly abrogated by NHE inhibitors. Therefore, NHE inhibitors are well suited for the treatment and prevention of the progression of age-related endothelial dysfunction, in particular intermittent claudication.

Another parameter that manifests as an aging process is the deterioration of the contractile force of the heart and the adaptability of the heart to the desired cardiac pumping capacity. This decline in cardiac function due to aging is in most cases associated with cardiac dysfunction, particularly dysfunction caused by the deposition of connective tissue in myocardial tissue. Deposition of connective tissue manifests itself as increased heart weight, enlarged heart and limited cardiac function. Surprisingly, this aging of the heart organs can be almost completely suppressed. Therefore, NHE inhibitors are very suitable for the treatment and prevention of heart failure, Congestive Heart Failure (CHF).

The proliferation inhibition not only can treat the cancer which already occurs, but also has the prevention and very obvious effect of delaying the age-related cancer incidence rate through an NHE inhibitor. Of particular note is the discovery that disease of all organs resulting from aging, not just certain types of cancer, is inhibited or very significantly delayed. Therefore, NHE inhibitors are very suitable for the treatment, in particular prevention, of age-related cancers.

It has been found that NHE inhibitors not only delay the incidence of age-related diseases in all organs investigated, including heart, blood vessels, liver, etc., very significantly over time and beyond normal statistics, but also in the incidence of cancer in elderly people. Furthermore, it also surprisingly extends life to a degree not reached by other drugs or any natural products. This unique action of NHE inhibitors allows to combine these NHE inhibitors with other active principles, methods, substances and natural products used in geriatric medicine and based on different mechanisms of action, in addition to the use of the active principle alone in humans and animals. The types of active ingredients used in the medical treatment of elderly are: in particular vitamins and substances with antioxidant activity. Because of the correlation between caloric load or food intake and the aging process, it is possible to coordinate eating methods, for example with appetite suppressionAnd (4) matching the preparations. Also contemplated are hypotensive agents such as ACE inhibitors, angiotensin receptor antagonists, diuretics, Ca²⁺Antagonists, etc., or in combination with metabolism-normalizing agents such as cholesterol-lowering agents. Therefore, NHE inhibitors are particularly useful for preventing age-related tissue changes and prolonging life while maintaining a high quality of life.

The compounds of the invention are potent inhibitors of cellular sodium-proton antiporters (Na/H exchangers) which are also increased in cells such as erythrocytes, platelets or leukocytes, as readily measured in a variety of diseases (essential hypertension, atherosclerosis, diabetes, etc.). The compounds of the invention are therefore suitable as good and simple scientific tools as diagnostic agents for e.g. identifying and distinguishing between different types of hypertension and atherosclerosis, diabetes and complications of the latter stages of diabetes, proliferative diseases, etc.

Also claimed is a human, veterinary or plant protection medicament comprising an effective amount of one or more compounds of formulae XVa, XVb and XVI, alone or in combination with other active pharmaceutical ingredients or drugs, and/or pharmaceutically acceptable salts thereof, together with pharmaceutically acceptable carriers and additives. Thus, for example, medicaments containing compounds of formulae I, Ia, Ib, XVa, XVb and XVI and/or pharmaceutically acceptable salts thereof may be administered orally, parenterally, intravenously, rectally or by inhalation, the preferred method of administration depending on the nature of the disease. The compounds of formulae I, Ia, Ib, XVa, XVb and XVI may be used alone or in combination with a pharmaceutically acceptable excipient in veterinary or human medicine. The medicament will generally contain from 0.01mg to 1g per dosage unit of a compound of formulae I, Ia, Ib, XVa, XVb and XVI and/or a pharmaceutically acceptable salt thereof.

Excipients suitable for use in the intended pharmaceutical formulation are well known to the skilled artisan based on their expertise. In addition to solvents, gel formers, suppository bases, tablet excipients and other active ingredient carriers, for example, antioxidants, dispersants, emulsifiers, antifoams, fragrances, preservatives, solubilizers or colorants can be used.

In pharmaceutical preparations for oral administration, the active compounds are mixed with additives suitable for this purpose, such as carriers, stabilizers or inert diluents, and converted by customary methods into suitable dosage forms, such as tablets, coated tablets, hard gelatin capsules, aqueous, alcoholic or oily solutions. Examples of inert carriers which can be used are gum arabic, magnesium oxide, magnesium carbonate, potassium phosphate, lactose, glucose or starch, in particular corn starch. The formulations can also be prepared using dry granules or wet granules. Examples of suitable oily carriers or solvents are vegetable or animal oils, such as sunflower oil or cod liver oil.

In the preparation for subcutaneous, intramuscular or intravenous administration, the active compounds used are, if desired, formulated as solutions, suspensions or solvents with substances conventionally used for this purpose, such as solubilizers, emulsifiers or other excipients. Examples of suitable solvents are: water, physiological saline or alcohols, e.g. ethanol, propanol, glycerol, and sugar solutions such as glucose or mannitol solutions, or mixtures of the solvents.

For example, suitable pharmaceutical formulations for administration as aerosols or sprays are solutions, suspensions or emulsions of the active ingredients of the formulae I, Ia, Ib, XVa, XVb and XVI and/or their pharmaceutically acceptable salts in a pharmaceutically acceptable solvent, in particular ethanol or water, or a mixture of such solvents. If desired, the formulations may also contain other pharmaceutically acceptable excipients such as surfactants, emulsifiers and stabilizers, and propellant gases. For example, the formulations will generally contain the active ingredient in a concentration of about 0.1 to about 10%, particularly about 0.3 to about 3%, by weight.

The dosage and frequency of administration of the active ingredients of formulae I, Ia, Ib, XVa, XVb and XVI depend on the intensity and duration of action of the compounds used; but also on the nature and severity of the disease to be treated, on the sex, age, weight and individual responsiveness of the mammal to be treated.

For a patient having a body weight of about 75kg, the average daily dosage of a compound of formulae I, Ia, Ib, XVa, XVb and XVI and/or a pharmaceutically acceptable salt thereof is at least 0.001mg/kg, preferably 0.01mg/kg, and at most 10mg/kg, preferably 1mg/kg body weight. For acute onset diseases, for example immediately after a myocardial infarction, it may also be necessary to administer higher doses, in particular more frequently, for example up to 4 single doses per day. Particularly when administered intravenously, for example in patients with myocardial infarction in the care unit, it may be necessary to administer up to 700 mg/day, and the compounds of the invention may be administered by infusion.

Abbreviation table:

DCC dicyclohexylcarbodiimide

DIP diisopropyl ether

TLC thin layer chromatography

DMF N, N-dimethylformamide

EA Ethyl acetate

eq. equivalent

Et₃N-Triethylamine

Et₂O Ether

EtOH ethanol

h hours

HEP n-heptane

HOAc acetic acid

KOtBu 2-methyl-2-propanoic acid potassium salt

MeOH methanol

min for

mp melting point

MTB tert-butyl methyl ether

NMP 1-methylpyrrolidin-2-one

Pd(OAc)₂Palladium acetate (II)

RT Room temperature

rt Retention time

tBu tert-butyl

THF tetrahydrofuran

TMEDA N, N, N ', N' -tetramethyl-ethane-1, 2-diamine

The retention times given below refer to retention times obtained by HPLC analysis with the following parameters:

the method A comprises the following steps:

stationary phase: waters Symmetry C8(5 μ) 3.9X 150mm

Mobile phase: isocratic CH₃CN/0.1％ CF₃CO₂35:65 parts of H aqueous solution; λ 220 nm;

1ml/min。

the method B comprises the following steps:

stationary phase: waters Symmetry C8(5 μ) 3.9X 150mm

Mobile phase: isocratic CH₃CN/0.1％ CF₃CO₂40:60 parts of H aqueous solution; λ 230 nm; 1 ml/min.

The method C comprises the following steps:

stationary phase: waters Symmetry C8(5 μ) 3.9X 150mm

Mobile phase: isocratic CH₃CN/0.1％ CF₃CO₂50:50 of H aqueous solution; λ 220 nm;

1ml/min。

example 1

a) N- (2, 2, 2-trifluoroethyl) -4-trifluoromethylbenzamide

5.0g (24mmol) of 4-trifluoromethyl-benzoyl chloride and 5.0ml (36mmol) of triethylamine are dissolved in 50ml of CH₂Cl₂2.4g (24mmol) of 2, 2, 2-trifluoroethylamine are slowly added dropwise at room temperature. The mixture was stirred at room temperature for 4h, then the volatile components were removed under reduced pressure. The residue was dissolved in 100ml of MTB and initially saturated with 30ml of Na₂CO₃Washed with an aqueous solution and then with 30ml of saturated NaHSO₄And (4) washing with an aqueous solution. With MgSO₄Drying gave 6.1g (94%) of a colourless resin which crystallised on standing; mp: 117 deg.C)⁺

R_f(DIP)＝0.50 MS(EI)：271(M+1)⁺

b) (R, S) -3-hydroxy-2- (2, 2, 2-trifluoroethyl) -5-trifluoromethyl-2, 3-dihydroisoindol-1-one

0.37ml (2.4mmol) of TMEDA and 1.4ml (2.3mmol) of a solution of 1.5M t-BuLi in N-pentane are dissolved at-75 ℃ in 2ml of THF (anhydrous) and 0.30g (1.1mmol) of N- (2, 2, 2-trifluoroethyl) -4-trifluoromethylbenzene are added dropwise at-75 ℃Solution of formamide in 2ml of THF. The mixture was stirred at-75 ℃ for 3h, then 0.43ml (5.5mmol) DMF was added dropwise and the mixture was warmed to room temperature over 30 min. The reaction mixture was poured into 100ml of saturated NaHCO₃The aqueous solution was extracted 3 times with 30ml of ethyl acetate each time. With MgSO₄Dried and the solvent removed under reduced pressure. Chromatography on silica gel using DIP. In addition to 110mg of the mixture of starting materials, 80mg of (R, S) -3-hydroxy-2- (2, 2, 2-trifluoroethyl) -5-trifluoromethyl-2, 3-dihydroisoindol-1-one are obtained. The mixture was separated again by reverse phase HPLC (conditions see below) and further 40mg of (R, S) -3-hydroxy-2- (2, 2, 2-trifluoroethyl) -5-trifluoromethyl-2, 3-dihydroisoindol-1-one was obtained; the total yield is 30%.

HPLC: gradient, run time 20min

Eluent: 0.1% CF₃CO₂Aqueous H, acetonitrile (Chromasolv);

flow rate: 30ml/min

Column: waters Xterra^TM MS C₁₈ 5μm，30×100mm

Gradient: 0-2.5min 10% acetonitrile

3.0min 25% acetonitrile

14.0min 75% acetonitrile

15.0min 95% acetonitrile

17.5min 10% acetonitrile

R_f(DIP)＝0.50 MS(EI)：299(M+1)⁺

c) (RS) - [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl ] -acetic acid ethyl ester

Ethyl (diethoxyphosphoryl) acetate (135mg, 0.6mmol) was dissolved in anhydrous dimethoxyethane (10ml) under an argon atmosphere. To this solution was added 17.6mg NaH (60% oil solution) at room temperature and stirred at room temperature for 10 min. Then, a solution of 120mg (0.04mmol) of (RS) -3-hydroxy-2- (2, 2, 2-trifluoroethyl) -5-trifluoromethyl-2, 3-dihydroisoindol-1-one in anhydrous dimethoxyethane (5ml) was added, and the mixture was stirred under reflux for 2 hours, and the reaction solution was allowed to cool; the reaction solution was then poured into 50ml of 5% sodium bicarbonate solution and extracted twice with 20ml of ethyl acetate each time; the organic phase is MgSO₄Dried and concentrated under reduced pressure, and the residue was purified by chromatography on silica gel using DIP as eluent. This gave 90mg (61%) (RS) - [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl]Ethyl acetate as a colorless oil, crystallized from heptane to give a beige solid.

R_f(DIP)＝0.31

The NMR spectrum was the same as for the material prepared in example 4.

d) (R, S) -N- {2- [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-H-isoindol-1-yl ] acetyl } guanidine

Ethyl (RS) - [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl ] acetate can be reacted with guanidine as described in example 2 g).

Example 2

a) 2-nitro-4-trifluoromethylbenzoic acid

11.97g of 4-trifluoromethylbenzoic acid (63mmol) were slowly added in portions to 48ml of HNO at room temperature₃(100%). The mixture was then heated to reflux for 1h, then cooled to room temperature and poured onto approximately 600g of ice. The mixture was stirred for 1h, then the precipitate was filtered off and washed with 1l of water. The filtrate was diluted with 300ml of CH₂Cl₂Extracting, combining the organic phase and the precipitate, and adding Na₂SO₄And (5) drying. The solvent was removed under reduced pressure and the residue was recrystallized by dissolving in 1l of DIP at 68 ℃, adding 2l of n-heptane at this temperature and finally slowly cooling the solution to room temperature. The crystalline product is washed with 1l of n-heptane and dried under reduced pressure to give 7.1g of product (48%), mp136 ℃ -138 ℃.

b) 2-amino-4-trifluoromethylbenzoic acid

250g of 2-nitro-4-trifluoromethylbenzoic acid (1.06mol) were dissolved in 1l of EtOH and 7.5g of Pd/C (5%) were added and the mixture was hydrogenated under 1-2.5bar of hydrogen. During the hydrogen absorption, the temperature was temporarily raised from 10 ℃ to 104 ℃. After 2h, the hydrogen uptake was complete. Subsequently, the catalyst was filtered off and the solvent was removed under reduced pressure to give 215g (99%) of a pale yellow solid, mp 174-.

c)2- ((E) -2-ethoxycarbonylvinyl) -4-trifluoromethylbenzoic acid

520mg NaNO₂(7.6mmol) was dissolved in 2ml of water and added dropwise at 0 ℃ to 1.3g of 2-amino-4-trifluoromethylbenzoic acid (6.5mmol) in 2.6ml of 48% HBF₄Aqueous solution and 30ml ethanol. Then theThe mixture was stirred at 0 ℃ for 10 minutes and then warmed to room temperature. Then 0.3ml of 48% HBF was added₄Aqueous solution, 30ml ethanol, 0.9g ethyl acrylate (9.0mmol) and 26.9mg Pd (OAc)₂(0.12 mmol). The mixture was then stirred at 50-60 ℃ for 1 h. The solvent was then removed under reduced pressure and the residue was taken up in 25ml of ethyl acetate and washed first with 25ml of 1N aqueous HCl solution and then with 25ml of aqueous NaCl solution. Na for organic phase₂SO₄Dried and the solvent removed under reduced pressure. The residue is suspended in 25ml of heptane and the precipitated product is filtered off. Yield 1.3g (69%) of a light brown solid. The analytical samples were purified by crystallization from heptane/ethyl acetate.

The NMR spectrum was the same as for the material prepared in example 3 a.

d) (E) -3- [2- (2, 2, 2-Trifluoroethylcarbamoyl) -5-trifluoromethyl-phenyl ] acrylic acid ethyl ester

1.3g of 2- (2-ethoxycarbonylvinyl) -4-trifluoromethylbenzoic acid (4.5mmol) and 453mg of 2, 2, 2-trifluoroethylamine (4.5mmol) are dissolved in 5ml of DMF and 0.93g of DCC are added. The mixture was stirred at room temperature for 4 h. The urea by-product was removed by filtration and then the solvent was removed under reduced pressure. The residue was recrystallized from DIP to give 1.6g (96%) of white crystals.

The NMR spectrum was the same as for the material prepared in example 3 b.

e) (RS) - [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl ] -acetic acid

2.2g of (E) -3- [2- (2, 2, 2-trifluoroethylcarbamoyl) -5-trifluoromethyl-phenyl]Ethyl acrylate (5.9mmol) was dissolved in 10To ml of methanol was added 1.5ml of 5M aqueous NaOH solution (7.5 mmol). The mixture was stirred at room temperature for 18h and then adjusted to pH 7 with aqueous HCl. The solvent was removed under reduced pressure and the residue was suspended in 10ml of water. The suspension is brought to pH 2 with 2N aqueous HCl and extracted 3 times with 10ml of ethyl acetate. With Na₂SO₄Dried and the solvent removed under reduced pressure. The residue was crystallized from diethyl ether/DIP, mp: 202 ℃ and 204 ℃.

Yield: 1.8g (89%).

¹H-NMR(400MHz，CDCl₃)：δ＝3.07(dd，J₁＝17Hz，J₂＝6Hz，1H)，3.23(dd，J₁＝17Hz，J₂＝5Hz，1H)，4.27(m，1H)，4.58(m，1H)，5.08(t，J＝5Hz，1H)，7.91(d，J＝8Hz，1H)，7.96(d，J＝8Hz，1H)，8.12(s，1H)，12.50(bs，1H)ppm。

And (3) combustion analysis: c₁₃H₉F₆NO₃(341.2): calcd for C45.76, H2.66, N4.10; found C45.71, H2.43, N4.11.

f) (RS) - [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl ] -acetic acid ethyl ester

2.6ml of SOCl₂(35mmol) are dissolved in 20ml of ethanol and 3.4g of (R, S) - [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl are added at-10 ℃]Acetic acid (10 mmol). The mixture was stirred at room temperature for 18h and then the volatile components were removed under reduced pressure. The residue was chromatographed on silica gel with 3:1 HEP/EA. Yield: 3.0g (81%) of a colorless oil, crystallized from heptane to give a beige solid.

The NMR spectrum was the same as that of the material prepared in example 4.

g) (RS) -N- {2- [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-H-isoindol-1-yl ] acetyl } guanidine

Guanidine hydrochloride (11.5g, 120mmol) was dissolved in NMP (45ml) and KOtBu (11.2g, 100mmol) was added with stirring, the mixture was stirred at room temperature for 1.5h and filtered. The filtrate was added dropwise to (RS) - [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl group with stirring at room temperature]A solution of ethyl acetate (7.38g, 20mmol) in NMP (12ml) was stirred at room temperature for a further 60 min. Ice water (270ml) was then added, the mixture was adjusted to pH 7 with 2N HCl, ethyl acetate (60ml) was added and then by addition of NaHCO₃The pH of the aqueous solution is adjusted to 8-8.5. The mixture was stirred well at room temperature for 1h, the precipitate formed was filtered off with suction and washed with water. This gives 7.06g (83%) (R, S) -N- {2- [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-H-isoindol-1-yl]Acetyl guanidine, containing 0.5 equivalents of ethyl acetate as pale yellow crystals, mp.160-161 ℃, gradually heated, ethyl acetate escaping at about 90 ℃.

R_f(Ethyl acetate/methanol) ═ 0.45

¹H-NMR(400MHz，CDCl₃)：δ＝2.54(dd，J₁＝8Hz，J₂＝16Hz，1H)，3.09(dd，J₁＝4Hz，J₂＝16Hz，1H)，4.25(m，1H)，4.64(m，1H)，5.18(m，1H)，6.65(bs，2H)，7.75(bs，2H)，7.88(d，J＝8Hz，1H)，7.95(d，J＝8Hz，1H)，8.02(s，1H)ppm。

C₁₄H₁₂F₆N₄O₂1/2C₄H₈O₂(426.33): calcd for C45.08, H3.78, N13.14; found C45.07, H3.79, N13.01.

Example 3

a)2- ((E) -2-ethoxycarbonylvinyl) -4-trifluoromethylbenzoic acid (variant of example 2 c)

To a solution of 339g 2-amino-4-trifluoromethylbenzoic acid (1.65mol) in 6.8l EtOH (anhydrous) was added 658ml 48-50% HBF at room temperature₄An aqueous solution. The temperature rose from 21 ℃ to 26 ℃. The mixture was then cooled to 0 ℃ and 125g NaNO was added dropwise over 17 minutes at 0 ℃ to 5 ℃₂500ml of an aqueous solution. The initially yellowish solution first becomes an orange-red suspension and finally a yellowish suspension. The progress of the reaction was monitored by HPLC (method B; 2-amino-4-trifluoromethylbenzoic acid, retention time ═ 6.4 min; 2-carboxy-5-trifluoromethylbenzene diazonium salt intermediate ═ 1.1 min). Conversion to 2-carboxy-5-trifluoromethylbenzene diazonium salt within 30 minutes>99% complete. Then, the mixture was added to 231g of ethyl acrylate (2.31mol), 11.1g of Pd (OAc)₂(49mmol) and 6.8l ethanol (anhydrous) and the mixture is heated to 49-51 ℃. It was observed that the uniform release of nitrogen increased with increasing temperature. The reaction was monitored by HPLC (method B; 2- ((E) -2-ethoxycarbonylvinyl) -4-trifluoromethylbenzoic acid retention time ═ 16.4 min). After 45 minutes, the degree of conversion exceeded 99%. Then, the mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was dissolved in 3l of ethyl acetate and filtered. The filtrate was then washed 3 times with 2.1l each of aqueous HCl and then with 1l of saturated aqueous NaCl solution. With Na₂SO₄Drying and removal of the solvent under reduced pressure gave 449g of a light brown solid. Yield was 83% when impurities (4-trifluoromethylbenzoic acid; 6.3%) and solvent residue (EA; 4%) were considered. The analytical sample was purified by crystallization from heptane/ethyl acetate. And Mp: 132 ℃ and 133 DEG C

¹H-NMR(400MHz，CDCl₃)：δ＝1.36(t，J＝7Hz，3H)，4.31(q，J＝7Hz，2H)，6.41(d，J＝16Hz，1H)，7.72(d，J＝8Hz，1H)，7.86(s，1H)，8.21(d，J＝8Hz，1H)，8.51(d，J＝16Hz，1H)，8.5-9.5(bs，1H)ppm。

And (3) combustion analysis: c₁₃H₁₁F₃O₄(288.23): calcd for C54.17, H3.85; found C54.24, H3.74.

b) (E) -3- [2- (2, 2, 2-Trifluoroethylcarbamoyl) -5-trifluoromethyl-phenyl ] acrylic acid ethyl ester

315g of oxalyl chloride (2.48mol) are added to 650g of 2- ((E) -2-ethoxycarbonylvinyl) -4-trifluoromethylbenzoic acid (2.25mol), 33ml of DMF and 7.81CH over 24 minutes at a temperature of 15 to 18 DEG C₂Cl₂In the mixture of (1). The mixture was stirred at room temperature for 1h, then cooled to 5 ℃ and 285g Et were added over 27min at a temperature of 5 ℃ to 10 ℃₃N ((2.81 mol.) the mixture is stirred at 5 ℃ for 10min, then 279g of 2, 2, 2-trifluoroethylamine (2, 81 mol.) are added over 27min at a temperature of from 9 ℃ to 20 ℃, the mixture is stirred at room temperature for 10min, during which a thick precipitate precipitates, and in order to change the stirrability of the mixture, 1lCH is additionally added₂Cl₂. The reaction was monitored by HPLC (method C; 2- ((E) -2-ethoxycarbonylvinyl) -4-trifluoromethylbenzoic acid with a retention time of 5.9 min; (E) -3- [2- (2, 2, 2-trifluoroethylcarbamoyl) -5-trifluoromethylphenyl]Ethyl acrylate rt ═ 13.2 min). After stirring at room temperature for a further 50min, the reaction was complete. The volatile constituents of the reaction mixture are then removed under reduced pressure, the residue is dissolved in 12l of ethyl acetate and washed 3 times with 2.5l of water each time and then with saturated NaHCO₃The aqueous solution was washed twice, 2.51 each time, and finally washed with 1.51 saturated aqueous NaCl solution. With MgSO₄Drying and removal of the solvent under reduced pressure gave 802g of (E) -3- [2- (2, 2, 2-trifluoroethylcarbamoyl) -5-trifluoromethylphenyl]Ethyl acrylate as a brown solid. This crude material was combined with another batch of crude material (177g) and dissolved in 3l of acetic acid at 60-70 deg.CTo ethyl ester, 14l of n-heptane were added portionwise in 1l at this temperature, and the mixture was then heated to 80 ℃ and stirred at this temperature for 1.5 b. The mixture was then added to 5.6l of n-heptane at 70 ℃ and the mixture was then cooled to room temperature over 5h with stirring. The product is then filtered, washed with 3l of n-heptane and air-dried, yielding 689g of (E) -3- [2- (2, 2, 2-trifluoroethylcarbamoyl) -5-trifluoromethyl-phenyl]Ethyl acrylate (67%) gave a light brown solid. And Mp: 161.5-162 ℃.

¹H-NMR(400MHz，CDCl₃)：δ＝1.33(t，J＝7Hz，3H)，4.05(m，2H)，4.26(q，J＝7Hz，2H)，6.19(bs，1H)，6.46(d，J＝16Hz，1H)，7.63(d，J＝8Hz，1H)，7.68(d，J＝8Hz，1H)，7.87(s，1H)，7.90(d，J＝16Hz，1H)ppm。

And (3) combustion analysis: c₁₅H₁₃F₆NO₃(369.27): calcd for C48.79, H3.55, N3.79; found C48.93, H3.51, N3.92.

c) (R, S) -N- {2- [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-H-isoindol-1-yl ] acetyl } guanidine

386g of (E) -3- [2- (2, 2, 2-trifluoroethylcarbamoyl) -5-trifluoromethyl-phenyl]Ethyl acrylate (1.05mol) was suspended in 600ml of DMF and added slowly in portions to 4.7g of KOtBu (42mmol) at a temperature of from 5 ℃ to 15 ℃. The cyclization of the isoindolones was monitored by TLC (HEP/EA ═ 2: 1; ethyl acrylate: Rf ═ 0.32; isoindolones: Rf ═ 0.41). After 1 hour, the reaction was complete. 587g of KOtBu are simultaneously suspended in 2.2l of DMF and 600g of guanidine hydrochloride are added at a temperature of from 20 ℃ to 25 ℃. The mixture was stirred at 25 ℃ for 1h, and then the KCl was filtered off. The filtrate containing the liberated guanidine is then added to the reaction mixture containing the isoindolone and stirred at room temperature for 2 h. By HPLC (method B; Bobo)230nm and 254nm long; isoindolones: the retention time is 15.1 min; acyl guanidine: retention time ═ 2.9min) the conversion to acylguanidine was monitored. The reaction mixture is subsequently poured into 14l of ice water, brought to a pH of 8.5 to 9.0 with aqueous HCl and extracted 4 times with 3l of ethyl acetate. The mixture was then washed 3 times with 3l each of saturated aqueous NaCl solution and with Na₂SO₄Drying and removing the solvent under reduced pressure. 329g (82%) of a brown solid are obtained. Combining the product with 3 other batches of products prepared by the same preparation method; the total amount was 842 g. 842g (2.2mol) of this product are reacted at 30 ℃ in 2l of ethyl acetate and 5l of Et₂And soaking and cooking in O for 2 h. The solid is then filtered off with Et₂O-washed twice, 2l each time and dried under reduced pressure. 693g (82% recovery) of an almost white solid was obtained. The compound crystallized from 2-propanol was a clathrate containing 0.5 equivalent of 2-propanol.

The NMR spectrum was identical to the S-enantiomer prepared in example 5 b.

Example 4

Preparation of ethyl (RS) - (2- (2, 2, 2-trifluoroethyl) -3-oxo-6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl) acetate from 2- ((E) -2-ethoxycarbonylvinyl) -4-trifluoromethylbenzoic acid by a one-pot reaction

At room temperature, adding SOCl₂(1.98g, 27.2mmol) was added to a suspension of 2- ((E) -2-ethoxycarbonylvinyl) -4-trifluoromethylbenzoic acid (2.9g, 10.1mmol) in toluene (30 ml). The mixture was stirred at room temperature for 5min and then heated to 105 deg.C (bath temperature) over 30 min. The gas venting was started at about 70 ℃. The mixture was stirred at 105 ℃ for 3h, then cooled to room temperature, filtered by suction through a layer of Kieselgur (2.5 × 0.5cm) and washed with toluene, and the filtrate was concentrated by evaporation under reduced pressure. The acid chloride (3.34g) was obtained as a red brown oil. 2, 2, 2-trifluoroethylamine (1.2g, 12.1mmol) and triethylamine (2.58g, 25.3mmol) were dissolved in dichloromethane at 5 deg.C(15ml), an acid chloride dissolved in methylene chloride (20ml) was added dropwise at a rate such that the temperature was kept at 5 ℃ to 10 ℃ under ice-cooling. The ice bath was then removed and the excess trifluoroethylamine and part of the dichloromethane were distilled under a general vacuum. The mixture was then heated to boiling under reflux for 10 h. After cooling, the mixture was diluted with dichloromethane (50ml) and extracted 2 times with 2N aqueous HCl (50ml each time), the combined organic phases were washed with water (100ml) and Na₂SO₄Dried and concentrated under reduced pressure. Ethyl (RS) - (2- (2, 2, 2-trifluoroethyl) -3-oxo-6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl) acetate (3.51g, 94%) was obtained as a dark brown oil which was purified by crystallization from n-heptane. And Mp: 54.5-55.5 ℃.

¹H-NMR(400MHz，CDCl₃)：δ＝1.15(t，J＝7Hz，3H)，2.85(dd，J1＝6Hz，J₂＝16Hz，1H)，3.01(dd，J₁＝5Hz，J₂＝16Hz)，1H)，3.83(m，1H)，4.12(q，J＝7Hz，2H)，4.73(m，1H)，5.17(t，J＝6Hz，1H)，7.80(m，2H)，8.01(d，J＝8Hz，1H)ppm。

And (3) combustion analysis: c₁₅H₁₃F₆NO₃(369.27): calcd for C48.79, H3.55, N3.79; found C48.54, H3.49, N3.79.

Example 5

a) (S) -N- {2- [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl ] acetyl } guanidine, O, O' -dibenzoyl-L-tartrate

First a solid of (RS) -N- {2- [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl ] acetyl } guanidine (inclusion compound with ethyl acetate, content 87.06%, 44g, 100mmol by NMR) and O, O' -dibenzoyl-L-tartaric acid (11.2g, 31mmol) was added and 2-propanol (500ml) was added dropwise with stirring. The solid was initially dissolved well and a white solid precipitated out. After 30min, the mixture was heated to 70 ℃. Again an almost clear solution was obtained. It was left to cool to room temperature over 4h and then stirred at this temperature overnight. The mixture was then stirred at 10 ℃ for 4h and subsequently filtered with suction. The residue was washed twice with 2-propanol (100ml each) and dried. 28.05g of (S) -N- {2- [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl ] acetyl } guanidine, O, O' -dibenzoyl-L-tartrate (based on the (S) -enantiomer, yield 74%) with an enantiomeric purity of 82% ee were obtained as colorless crystals determined by HPLC (Chiracel OD/21, 250X 4.6mm, 50:5: 2N-heptane/ethanol/methanol, 1ml/min, 30 ℃). First 20g (14.6mmol) of these crystals were added and 2-propanol (400ml) was added dropwise. The mixture was heated to 80 ℃ with stirring and then allowed to cool gradually to room temperature. The mixture is stirred at this temperature for a further 2h and then filtered off with suction, and the residue is washed twice with 2-propanol (50ml each) and dried. 16.3g (based on (S) -enantiomer in 100%) of (S) -N- {2- [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl ] acetyl } guanidine, O, O' -dibenzoyl-L-tartrate with an enantiomeric purity > 97% ee, determined by HPLC (conditions above), were obtained as colorless crystals, mp: 192 ℃ and 193 ℃.

And (3) combustion analysis: c₁₄H₁₂F₆N₄O₂·1/2C₁₈H₁₄O₈(561.43): calcd for C49.21, H3.41, N9.98; found C49.17, H3.30, N9.97.

b) (S) -N- {2- [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl ] acetyl } guanidine

Mixing (S) -N- {2- [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl]Acetyl } guanidine, O, O' -dibenzoyl-L-tartrate (113mg, 0.20mmol) was dissolved in a mixture of water (1ml) and ethyl acetate (5ml) and NaHCO was added₃(50mg) in water (7.5 ml). The mixture was stirred at room temperature for 16h and then extracted 3 times with ethyl acetate (5ml each). The combined organic phases are treated with NaHCO₃(50mg) in water (20ml) was shaken once and then with purified water (20ml) and Na₂SO₄Dried and concentrated by evaporation under reduced pressure. This gave 75mg (97%) (S) -N- {2- [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl]Acetyl guanidine. The product was crystallized from 2-propanol to give a clathrate containing 0.5 equivalents of 2-propanol, mp: 80-82 ℃. This material can be recrystallized from ethyl acetate and crystallized with 0.5 equivalents of ethyl acetate, mp.: 121.5-122 ℃.

The enantiomeric purity, determined by HPLC (Chiracel OD/21, 250X 4.6mm, n-heptane/2-propanol 4:1.1ml/min, 30 ℃), was > 97%.

¹H-NMR(400MHz，CDCl₃)：δ＝2.54(dd，J₁＝8Hz，J₂＝16Hz，1H)，3.09(dd，J₁＝4Hz，J₂＝16Hz，1H)，4.25(m，1H)，4.64(m，1H)，5.18(m，1H)，6.65(bs，2H)，7.75(bs，2H)，7.88(d，J＝8Hz，1H)，7.95(d，J＝8Hz，1H)，8.02(s，1H)ppm。

Combustion analysis (inclusion compound containing 0.5 equivalent of 2-propanol): c₁₄H₁₂F₆N₄O₂·1/2C₃H₈O (412.32): calcd for C45.15, H3.91, N13.59; found C45.23, H4.27, N13.10.

Example 6

(RS) -N-2- [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl ] acetyl } guanidine preparation by racemization of (R) -N- {2- [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl ] acetyl } guanidine

Mixing (R) -N- {2- [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl]Acetyl } guanidine (clathrate with 0.5 eq 2-propanol (M ═ 412.3), 43g, 104 mmol; prepared by concentrating (S) -N- {2- [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl) from example 5a)]Acetyl guanidine, O, O' -dibenzoyl-L-tartrate and the use of NaHCO as described in example 5b)₃Treatment) was dissolved in 2-propanol (1.8) and a solution of KOH (85%, 660mg, 10mmol) in 2-propanol (400ml) was added with stirring at room temperature. The mixture was stirred at room temperature for 24h, then acidified with glacial acetic acid (720mg, 1.5ml) and concentrated by evaporation at a bath temperature of about 40 ℃ under reduced pressure, the residue partitioned between water (500ml) and ethyl acetate (400 ml). The aqueous phase was extracted twice with ethyl acetate (300 ml each) and the combined organic phases were combined with NaHCO₃(10g) The solution of (2) in water (500ml) was shaken and then again with pure water. Na for organic phase₂SO₄Dried and concentrated by evaporation under reduced pressure at a bath temperature of about 40 ℃. 39.8g of (RS) -N- {2- [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl-having a content of 87% by NMR are obtained]Acetyl } guanidine, inclusion complex containing 0.5 equivalents of ethyl acetate, as pale yellow crystals, yield: 87 percent. And Mp: 164-166 ℃ ethyl acetate escaped at about 100 ℃ when gradually heated.

The enantiomeric ratio was 49:51 as determined by HPLC (Chiracel OD/21, 250X 4.6mm, n-heptane/2-propanol 4:1.1ml/min, 30 ℃).

Example 7

(S) -N- {2- [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl ] acetyl } guanidine hydrogen fumarate hydrate

(S) -N- {2- [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl ] acetyl } guanidine [ inclusion compound containing 0.5 equivalent of 2-propanol, (M ═ 412.3), 110g, 266mmol ] was dissolved in dimethoxyethane (2l) and mixed with fumaric acid solution (0.5M dimethoxyethane/water 9:1, 512ml), and the resulting clear solution was concentrated by evaporation under reduced pressure. The residue was dissolved in dichloromethane (2l), and the mixture was concentrated again by evaporation under reduced pressure. The residue was suspended in water (1.5l), filtered off with suction at room temperature and dried overnight at room temperature. This gave 125.9g (95%) of (S) -N- {2- [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl ] acetyl } guanidine hydrochloride hydrate as colorless crystals at mp.210 ℃.

Measurement of NHE inhibition

Inhibitory concentration IC of NHE-1 inhibition was determined as follows₅₀。

In the FLIPR assay, the pH was determined in transfected cell lines expressing human NHE-1_iRecovery of (b) to determine NHE inhibitory Activity IC₅₀。

The assay was performed on a FLIPR (fluorescent imaging plate reader) equipped with a 96-well microtiter plate with clear bottom and black walls on the periphery. The day before, transfected cell lines expressing various subtypes of NHE were seeded at a density of-25000 cells/well (the parent cell line LAP-1 showed no endogenous NHE activity as a result of mutagenesis and subsequent selection). The growth medium of the transfected cells (Iscove + 10% fetal calf serum) also contains G418 as a selection antibiotic to ensure the presence of the transfection sequence.

By removing the growth medium and adding 100. mu.l of loading buffer (5. mu.M BCECF-AM [2 ', 7' -bis (carboxyethyl) -5- (and 6-) -carboxyfluorescein acetoxymethyl ester) to each well]NH at 20mM₄Cl, 115mM choline chloride, 1mM MgCl₂1mM of CaCl₂5mM KCl, 20mM HEPES and 5mM glucose; pH 7.4[ adjusted with KOH]) The test was started. Then, the cells were cultured at 37 ℃ for 20 minutes. The incubation allows for a fluorescent dye (the fluorescence intensity of which depends on pH)_i) And NH to slightly basify the cells₄Cl was loaded in the cells.

The non-fluorescent dye precursor BCECF-AM is an ester that is capable of passing through the cell membrane. In cells, esterases release the authentic BCECF dye which cannot cross the cell membrane.

After 20 minutes of incubation, the loading buffer was removed by 3 washes in a cell washing apparatus (Tecan Columbus) containing NH₄Cl and free BCECF-AM 400. mu.l of wash buffer (133.8mM choline chloride, 4.7mM KCl, 1.25mM MgCl) was used for each wash₂1.25mM CaCl₂0.97mM K₂HPO₄0.23mM KH₂PO₄5mM HEPES and 5mM glucose; pH 7.4[ adjusted with KOH]). The residual volume remaining in the wells was 90. mu.l (possibly between 50 and 125. mu.l). This washing step removes free BCECF-AM and results in intracellular acidification (pH)_i6.3-6.4) due to removal of external NH₄ ⁺Caused by ions.

Due to the removal of extracellular NH₄ ⁺And NH₃Immediately thereafter, cross the cell membrane to make the NH inside the cell₄ ⁺And NH₃And H⁺The balance between them is disrupted and the washing process results in intracellular H⁺This is retained, which is responsible for intracellular acidification. This acidification, if sustained for a sufficient period of time, can ultimately lead to cell death. The washing buffer is sodium-free (<1mM) which would otherwise result in a pH due to the activity of the cloned NHE isoform_iIs immediately increased.

All buffers used (loading buffer, washing buffer and regeneration buffer) did not contain any HCO₃ ^-Ions are also important, otherwise the presence of bicarbonate can lead to interference with bicarbonate dependent pH_iThe regulation system is activated and the regulation system is activated,the system is comprised in the LAP-1 parental cell line.

The microtiter plates containing the acidified cells (20 min after acidification) were then transferred to a FLIPR. In the FLIPR, intracellular fluorochromes are activated with 488nm wavelength light generated by an argon laser, and the measurement parameters (laser power, illumination time and aperture of a CDD camera mounted on the FLIPR) are chosen such that the mean value of the fluorescence signal per well is between the relative fluorescence units of 30000 and 35000.

Measurements were taken every 2 seconds with a CCD camera under software control. After 10 seconds, 90. mu.l of regeneration buffer (133.8mM NaCl, 4.7mM KCl, 1.25mM MgCl) was added using a 96-well pipette device mounted in the FLIPR₂1.25mM CaCl₂0.97mM K₂HPO₄0.23mM KH₂PO₄10mM HEPES and 5mM glucose; pH 7.4[ adjusted with NaOH]) The intracellular pH starts to increase. Some wells with pure regeneration buffer added served as positive controls (100% NHE activity). Negative control (0% NHE activity) contained wash buffer. Regeneration buffer with two test substance concentrations was added to all other wells. After 60 measurements, the measurement in FLIPR was terminated (2 min).

Raw data is input to the ActivityBase program. The program first calculates the NHE activity at each test substance concentration to obtain the IC of each substance₅₀The value is obtained. Since the pH was maintained throughout the experiment_iThe regeneration process is not linear and decreases somewhat at the end point due to the high pH_iLower NHE activity was decreased, so it was important to select the portion where the positive control fluorescence increase was linear to evaluate the assay.

Substance(s)	NHE 1-inhibited IC[nM]
		(S) -N- {2- [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2,3-dihydro-1H-isoindol-1-yl]Acetyl guanidine	0.3

Claims (4)

1. A process for preparing a compound of formula I:

wherein

R1 and R2 are each independently of the other hydrogen, F, Cl, trifluoromethoxy, 2, 2, 2-trifluoroethoxy, trifluoromethyl, 2, 2, 2-trifluoroethyl or alkyl having 1, 2, 3 or 4 carbon atoms;

r3 is Alk-R4 or trifluoromethyl;

alk is an alkyl group having 1, 2, 3, or 4 carbon atoms;

r4 is hydrogen, trifluoromethyl or cycloalkyl having 3, 4, 5, 6 or 7 carbon atoms;

the method comprises the following steps:

a) carbamoylating formula IV followed by cyclization to a compound of formula VI,

b) reacting a compound of formula VI with alkoxycarbonylmethylenetriphenyl-phosphorane, with 1-alkoxy-1-trimethylsiloxyethylene or with trialkyl phosphonoacetate to give a compound of formula VII, and

c) reacting a compound of formula VII with guanidine to give a compound of formula I, optionally converted into a salt, wherein, in the compounds of formulae IV, VI and VII,

R1-R3 are each as defined for formula I, and

r5 is an alkoxy group having 1, 2, 3 or 4 carbon atoms.

2. A process for the preparation of a compound of formula I or a salt thereof as claimed in claim 1 wherein

R1 and R2 are each independently of the other hydrogen, F, Cl, trifluoromethoxy, 2, 2, 2-trifluoroethoxy, trifluoromethyl, 2, 2, 2-trifluoroethyl or alkyl having 1, 2, 3 or 4 carbon atoms;

r3 is Alk-R4 or trifluoromethyl;

alk is an alkyl group having 1, 2, 3, or 4 carbon atoms;

r4 is hydrogen, trifluoromethyl or cycloalkyl having 3, 4, 5, 6 or 7 carbon atoms;

wherein

a) Reacting a compound of formula II with an amine of formula III to obtain an amide of formula IV,

b) formylating the amide of formula IV at the ortho position of the amide functionality to give a formylamide of formula V,

c) cyclizing the formylamide of formula V to give the compound of formula VI,

d) reacting a compound of formula VI with alkoxycarbonylmethylenetriphenylphosphorane, with 1-alkoxy-1-trimethylsiloxyethylene or with a trialkyl phosphonoacetate to give a compound of formula VII, and

e) reacting a compound of formula VII with guanidine to give a compound of formula I, optionally converted into a salt, wherein, in the compounds of formulae II, III, IV, V, VI and VII,

R1-R3 are each as defined for formula I,

r5 is an alkoxy group having 1, 2, 3 or 4 carbon atoms,

x is Cl, Br, OH or an alkoxy group having 1, 2, 3 or 4 carbon atoms.

3. The process of claim 1 or 2, wherein the steps are carried out continuously or batchwise, independently of one another.

4. A method according to claim 3 wherein the compound of formula I is defined as N- {2- [ 3-oxo-2- (2, 2, 2-trifluoroethyl) -6-trifluoromethyl-2, 3-dihydro-1H-isoindol-1-yl ] acetyl } guanidine, or a pharmaceutically acceptable salt thereof.