NO332538B1 - Dihydropteridinones, method of preparation, use thereof as medicaments and use thereof for the manufacture of pharmaceutical preparations for the treatment of disease - Google Patents

Abstract

The present invention relates to novel dihydropteridinones of general formula (I) wherein groups L and R 1 to R 5 have the meanings given in the claims and description, their isomers, processes for preparing these dihydropteridinones and their use as pharmaceutical preparations.

Description

The present invention relates to new dihydropteridinones with the general formula (I)

where the groups L, R<1>, R<2>, R<3>, R4 and R<5> have the meanings given in the claims and the description, the isomers thereof, methods for the preparation of these dihydropteridinones and their use as drugs.

Background for the invention

Pteridinone derivatives are known from the prior art as active substances with antiproliferative action. WO 01/019825 describes the use of pteridinone derivatives for the treatment of neoplastic and viral diseases. The resistance of many grouse tumors makes it necessary to develop new pharmaceutical preparations for combating tumors.

The purpose of the present invention is to produce new compounds with anti-inflammatory and anti-proliferative action.

Detailed description of the invention

It has surprisingly been found that compounds of the general formula (I), where the groups L and R<1> to R<5> have the meanings given below, act as inhibitors of specific cell cycle kinases. The compounds according to the invention can therefore be used, for example, for the production of preparations to treat diseases which are related to the activity of specific cell cycle kinases and which are characterized by high or abnormal cell proliferation.

The present invention therefore relates to compounds of the general formula (I)

where

R , R which may be the same or different, means hydrogen or optionally substituted d-C 6 alkyl,

or

R<1> and R<2> together mean a 2- to 5-membered alkyl bridge,

R<3> means hydrogen, Ci-Cn-alkyl or phenyl optionally substituted with -OMe or C3-C12-cycloalkyl 1,

R<4> represents a group selected from hydrogen, C1-C6-alkyl or C1-C5-alkyloxy

L means a connecting group selected from C2-C10-alkyl, phenyl, -C2-C4-alkyl-phenyl, -phenyl-C1-C4-alkyl or C3-C12-cycloalkyl,

n means 0 or 1

m means 1 or 2

R<5>means a group selected from optionally Ci-C4-alkyl or tetrahydropyranyl substituted morpholinyl, piperidinyl, piperazinyl, piperazinylcarbonyl, pyrrolidinyl, tropenyl, sulfoxomorpholinyl, sulfonylmorpholinyl, thiomorpholinyl, -NR<8>R<9> and azacycloheptyl,

R<8>, R<9> mean unsubstituted nitrogen substituents on R<5>, which may be the same or different, and are either hydrogen or a group selected from Ci-C6 alkyl,

-Ci-C4-alkyl-phenyl, -Ci-C4-alkyl-C3-C7-cycloalkyl or C1-C4-alkylcarbonyl,

where the substituents on the optionally substituted alkyl groups can be chosen from one or more fluorine atoms,

optionally in the form of the tautomers, racemates, enantiomers, diastereomers and mixtures thereof and optionally the pharmacologically acceptable acid addition salts thereof.

Preferred compounds of formula (I) are those where

R<1> to R4 are as above defined and

L means a connecting group selected from optionally substituted C 2 -C 10 alkyl, phenyl,

-C2-C4-alkyl-phenyl, -phenyl-C1-C4-alkyl or C3-C12-cycloalkyl,

n means 1

m means 1 or 2

R<5>means a group which is bound to L via a nitrogen atom, selected from optionally substituted with C1-C4 alkyl or tetrahydropyranyl morpholinyl, piperidinyl,

R<8->piperazinyl, pyrrolidinyl, tropenyl, sulfoxomorpholinyl, sulfonylmorpholinyl, thiomorpholinyl, -NR<8>R<9>and azacycloheptyl,

R<8>, R<9> mean unsubstituted nitrogen substituents on R<5>, which may be the same or different, and are either hydrogen or a group selected from Ci-C6-alkyl, -Ci-C4-alkyl-phenyl, - C1-C4-alkyl-C3-C7-cycloalkyl or C1-C4-alkylcarbonyl,

optionally in the form of the tautomers, racemates, enantiomers, diastereomers and mixtures thereof and optionally the pharmacologically acceptable acid addition salts thereof.

Also preferred are compounds of formula (I), where

R<1> to R4 are as defined above,

L means a connecting group selected from optionally substituted C 2 -C 10 alkyl, phenyl,

-C2-C4-alkyl-phenyl, -phenyl-C1-C4-alkyl or C3-C12-cycloalkyl,

n means 0 or 1

m means 1 or 2

R<5>means a group which is attached to L via a carbon atom, selected from R<8->piperidinyl, R<8>R<9->piperazinyl, R<8->pyrrolidinyl, R<8->piperazinylcarbonyl, R<8->tropenyl, R<8->morpholinyl

Q

and R -azacycloheptyl,

and

R<8>, R<9> mean unsubstituted nitrogen substituents on R<5>, which may be the same or different, and are either hydrogen or a group selected from Ci-C6-alkyl, -Ci-C4-alkyl-phenyl, - C1-C4-alkyl-C3-C7-cycloalkyl or C1-C4-alkylcarbonyl,

optionally in the form of the tautomers, racemates, enantiomers, diastereomers and mixtures thereof and optionally the pharmacologically acceptable acid addition salts thereof.

Particularly preferred are compounds of formula I where

L, m, n and R<3> to R<9> are as defined above and

R<1>, R<2> which may be the same or different, means a group selected from hydrogen, Me, Et, Pr, or

R<1>and R<2>together form a C2-C4 alkyl bridge,

optionally in the form of the tautomers, racemates, enantiomers, diastereomers and mixtures thereof and optionally the pharmacologically acceptable acid addition salts thereof

Particularly preferred are compounds of formula I, where

R<1>, R<2>, m, n and R<5> to R8 are as above defined and

R<4> represents a group selected from hydrogen, OMe, OEt, O-propargyl and O-butynyl, and

L means a connecting group selected from phenyl, phenylmethyl, cyclohexyl and branched C 1 -C 6 alkyl,

optionally in the form of the tautomers, racemates, enantiomers, diastereomers and mixtures thereof and optionally the pharmacologically acceptable acid addition salts thereof.

The invention further relates to compounds of formula I for use as pharmaceutical preparations.

Of particular importance according to the invention are compounds of formula I for use as pharmaceutical preparations with antiproliferative action.

The invention also relates to the use of a compound of formula I for the production of a pharmaceutical preparation for the treatment and/or prevention of cancer, infections, inflammatory and autoimmune diseases.

The invention also relates to pharmaceutical preparations which contain as active substance one or more compounds of the general formula (I) or the physiologically acceptable salts thereof, optionally combined with conventional excipients and/or carriers. The invention also relates to a method for the preparation of a compound of the general formula (I),

where

R<1->R<5>, m, n and L are as defined above,

characterized by a compound of the general formula (II)

where

R -R is as defined above and A is a leaving group,

is reacted with an optionally substituted compound of the general formula (III),

where

R<4> is as defined above and

R<10> means OH, NH-L-R<5>, -O-methyl, -O-ethyl,

and then possibly the product with the general formula (IV)

where

R<1> to R<4> are as defined above and

R<10> means OH, -NH-L-R<5>, -O-methyl or -O-ethyl,

optionally after prior hydrolysis of the ester group -COR<10>, reacted with an amine of the general formula (V)

where

R<5> is as defined above.

Furthermore, the invention relates to a compound of formula (II),

where

P<J->R<3> is as defined above and A is a leaving group.

The following definitions apply unless otherwise stated in the requirements.

The term alkyl groups, including alkyl groups which are part of other groups, means branched and unbranched alkyl groups with 1 to 12 carbon atoms, preferably 1 - 6, most preferably 1-4 carbon atoms, such as: methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl and dodecyl. Unless otherwise stated, the above terms propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl and dodecyl include all possible isomeric forms. The term propyl includes, for example, the two isomeric groups n-propyl and isopropyl, the term butyl includes n-butyl, iso-butyl, sec-butyl and tert-butyl, the term pentyl includes iso-pentyl and neopentyl.

In the above-mentioned alkyl groups, one or more hydrogen atoms may optionally be replaced by other groups. These alkyl groups can, for example, be substituted with fluorine. All the hydrogen atoms in the alkyl group can optionally also be substituted.

The term alkyl bridge means, unless otherwise stated, branched and unbranched alkyl groups with 1 to 5 carbon atoms, for example methylene, ethylene, propylene, isopropylene, n-butylene, iso-butyl, sec-butyl and tert -butyl bridges. Methylene, ethylene, propylene and butylene bridges are particularly preferred. In the aforementioned alkyl bridges, 1 to 2 C atoms may optionally be replaced by one or more heteroatoms selected from oxygen, nitrogen or sulphur.

The term alkenyl groups (including those which are part of other groups) means

branched and unbranched alkylene groups of 2 to 10 carbon atoms, preferably 2-6 carbon atoms, most preferably 2-3 carbon atoms, provided they have at least one double bond. Examples include: ethenyl, propenyl, butenyl and pentenyl. Unless otherwise stated, the above terms propenyl, butenyl also include all possible isomeric forms. The term butenyl includes, for example, 1-butenyl, 2-butenyl, 3-butenyl, 1-methyl-1-propenyl, 1-methyl-2-propenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl and 1 -ethyl-1-ethenyl.

In the above-mentioned alkenyl groups, unless otherwise stated, one or more hydrogen atoms may optionally be replaced by other groups. These alkyl groups can, for example, be substituted with the halogen atom fluorine. All the hydrogen atoms in the alkenyl group can optionally also be substituted.

The term alkynyl groups (including those which are part of other groups) means branched and unbranched alkynyl groups of 2 to 10 carbon atoms, provided that they have at least one triple bond, for example ethynyl, propargyl, butynyl, pentynyl and hexynyl, preferably ethynyl or propynyl.

In the above-mentioned alkynyl groups, unless otherwise stated, one or more hydrogen atoms may optionally be replaced by other groups. These alkyl groups can, for example, be substituted with fluorine. All the hydrogen atoms in the alkynyl group can optionally also be substituted.

The term aryl means an aromatic ring system with 6 to 14 carbon atoms, preferably 6 or 10 carbon atoms, preferably phenyl, which, unless otherwise indicated, may carry one or more of the following substituents, for example: OH, NO2, CN, OMe, -OCHF2, -OCF3, -NH2, halogen, for example fluorine or chlorine, Ci-Cio-alkyl, preferably C1-C5-alkyl, preferably Ci-C3-alkyl, most preferably methyl or ethyl, -O-Ci-C3- alkyl, preferably -O-methyl or -O-ethyl, -COOH, -COO-d-C4-alkyl, preferably -O-methyl or -O-ethyl, -CONH2.

Examples of heteroaryl groups where up to two carbon atoms have been replaced by one or two nitrogen atoms include pyrrole, pyrazole, imidazole, triazole, pyridine, pyrimidine, where each of the above-mentioned heteroaryl rings can optionally also be fused to a benzene ring, preferably benzimidazole, and provided unless otherwise stated, these heterocyclic groups may carry one or more of the following substituents, for example: F, Cl, Br, OH, OMe, methyl, ethyl, CN, CONH2, NH2, optionally substituted phenyl, optionally substituted heteroaryl, preferably optionally substituted pyridyl.

Examples of cycloalkyl groups are cycloalkyl groups with 3-12 carbon atoms, for example cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl, preferably cyclopropyl, cyclopentyl or cyclohexyl, where each of the above-mentioned cycloalkyl groups can optionally also carry one or more substituents, for example: OH, NO2, CN, OMe, -OCHF2, -OCF3, -NH2 or halogen, preferably fluorine or chlorine, Ci-Cio-alkyl, preferably Ci-Cs-alkyl, preferably Ci-C3-alkyl, more preferably methyl or ethyl, - O-Ci-Cj-alkyl, preferably -O-methyl or -O-ethyl, -COOH, -COO-Ci-C4-alkyl, preferably -COO-methyl or -COO-ethyl or -CONH 2 . Particularly preferred substituents for cycloalkyl groups are =O, OH, NH2, methyl or F.

Examples of cycloalkenyl groups are cycloalkyl groups with 3-12 carbon atoms which have at least one double bond, for example cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl or cycloheptenyl, preferably cyclopropenyl, cyclopententyl or cyclohexenyl, where each of the above-mentioned cycloalkenyl groups can possibly also carry one or more substituents.

"=0" means an oxygen atom bonded via a double bond.

Unless otherwise described in the definitions, examples of heterocycloalkyl groups include 3- to 12-membered, preferably 5-, 6- or 7-membered, saturated or unsaturated, heterocyclic groups which may contain as heteroatoms nitrogen, oxygen or sulphur, for example tetrahydrofuran, tetrahydrofuranone, γ-butyrolactone, α-pyran, γ-pyran, dioxolane, tetrahydropyran, dioxane, dihydrothiophene, thiolane, dithiolane, pyrroline, pyrrolidine, pyrazoline, pyrazolidine, imidazoline, imidazolidine, tetrazole, piperidine, pyridazine, pyrimidine, pyrazine, piperazine, triazine, tetrazine, morpholine, thiomorpholine, diazepane, oxazine, tetrahydro-oxazinyl, isothiazole, pyrazolidine, preferably morpholine, pyrrolidine, piperidine or piperazine, where the heterocyclic group may optionally carry substituents, for example Cl-C4-alkyl, preferably methyl, ethyl or propyl.

Examples of polycycloalkyl groups are optionally substituted, bi-, tri-, tetra- or pentacyclic cycloalkyl groups, for example pinane, 2,2,2-octane, 2,2,1-heptane or adamantane. Examples of polycycloalkenyl groups are optionally bridged and/or substituted, 8-membered, bi-, tri-, tetra- or pentacyclic cycloalkenyl groups, preferably bicycloalkenyl or tricycloalkenyl groups, if they have at least one double bond, for example norbornene.

Examples of spiroalkyl groups are optionally substituted, spirocyclic Cs-Ci2alkyl groups.

The term halogen usually means fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine, most preferably chlorine.

The leaving group A means either identical or different leaving groups such as -O-methyl, -SCN, chlorine, bromine, iodine, methanesulfonyl, trifluoromethanesulfonyl or p-toluenesulfonyl, preferably chlorine.

The compounds according to the invention can exist in the form of the individual optical isomers, mixtures of the individual enantiomers, diastereomers or racemates, in the form of the tautomers and also in the form of the free bases or the corresponding acid addition salts with pharmacologically acceptable acids - such as, for example, acid addition salts with hydrohalic acids , for example hydrochloric acid or hydrobromic acid, or organic acids, such as oxalic acid, fumaric acid, diglycolic acid or methanesulfonic acid.

The substituent R<1> can mean hydrogen or a group selected from optionally substituted and/or branched C1-C6 alkyl, preferably methyl or ethyl, more preferably methyl or ethyl.

The substituent R<2> can mean hydrogen or a group selected from optionally substituted and/or branched C1-C6 alkyl, preferably methyl or ethyl.

R<1> and R2 together can mean a 2- to 5-membered alkyl bridge, preferably an ethylene, propylene or butylene bridge, more preferably ethylene, propylene.

The substituent R<3> can mean hydrogen or a group selected from optionally substituted and/or branched Ci-Cn alkyl, preferably ethyl, propyl, butyl, pentyl or hexyl, more preferably propyl, butyl, pentyl or hexyl, or optionally substituted phenyl with -OMe or Cj-Cn-cycloalkyl, preferably cyclopentyl or cyclohexyl. It is most preferred that the substituent R<3> means isopropyl, isobutyl, isopentyl, cyclopentyl, phenyl or cyclohexyl.

The substituent R<4> can mean a group selected from hydrogen, C1-C6-alkyl, preferably methyl, ethyl or propyl, C1-C8-alkyloxy, preferably methoxy or ethoxy.

It is most preferred that the substituent R<4> means methoxy, methyl, ethoxy or ethyl.

L can represent a connecting group selected from C2-C10-alkyl, preferably ethyl, propyl, butyl or pentyl, phenyl, -C2-C4-alkyl-phenyl, phenyl -C1-C4-alkyl, preferably -phenyl-methyl or C1-Cn -cycloalkyl, preferably cyclohexyl.

n means 0 or 1

m means 1 or 2, preferably 1.

R<5> can mean a group selected from optionally tetrahydropyranyl substituted morpholinyl, piperidinyl, piperazinyl, piperazinylcarbonyl, pyrrolidinyl, tropenyl, sulfonylmorpholinyl, thiomorpholinyl, -NR<8>R<9> and azacycloheptyl, preferably piperidinyl, morpholinyl, pyrrolidinyl, sulfoxomorpholinyl, piperazinyl, thiomorpholinyl or tropenyl.

The groups R8 and R<9> can be unsubstituted nitrogen substituents at R<5>, they can be identical or different and mean either hydrogen or a group selected from C1-C6 alkyl, preferably methyl, ethyl or propyl, -Ci- C4-alkyl-C3-C10-cycloalkyl, preferably -CH2-cyclopropyl, -C1-C4-alkyl-phenyl, preferably benzyl or C1-C4-alkylcarbonyl.

It is most preferred that the substituent R<8> means methyl, ethyl or propyl.

It is most preferred that the substituent R<9> means methyl, ethyl or propyl.

The compounds according to the invention can be prepared by synthesis method A described below, where the substituents in the general formulas (A1) to (A9) have the meanings indicated above.

Method A

Stage IA

A compound of formula (A1) is reacted with a compound of formula (A2) to obtain a compound of formula (A3) (Diagram IA). This reaction can be carried out according to WO 0043369 or WO 0043372. Compound (A1) can be obtained commercially, for example from City Chemical LLC, 139 Allings Crossing Road, West Haven, CT, 06516, USA. Compound (A2) can be prepared by procedures known from the literature: (a) F. Effenberger, U. Burkhart, J. Willfahrt, Liebigs Ann. Chem. 1986, 314-333; b) T. Fukuyama, C-K. Jow, M. Cheung, Tetrahedron Lett. 1995, 36, 6373-6374; c) R. K. Olsen, J. Org. Chem. 1970, 35, 1912-1915; d) F.E. Dutton, B.H. Byung,

Tetrahedron Easy. 1998, 30, 5313-5316; e) J.M. Ranajuhi, M.M. Joullie Synth.

Commun., 1996, 26,1379-1384./

Diagram IA

In Step IA, 1 equivalent of the compound (Al) and 1 to 1.5 equivalents, preferably 1.1 equivalents, of a base, preferably potassium carbonate, potassium hydrogen carbonate, sodium carbonate or sodium hydrogen carbonate, calcium carbonate, most preferably potassium carbonate, are stirred in a diluent which optionally is mixed with water, for example acetone, tetrahydrofuran, diethyl ether, cyclohexane, petroleum ether or dioxane, preferably cyclohexane or diethyl ether.

At a temperature of 0 to 15 °C, preferably 5 to 10 °C, 1 equivalent of an amino acid of formula (A2) dissolved in an organic solvent, for example acetone, tetrahydrofuran, diethyl ether, cyclohexane or dioxane, is added dropwise . The reaction mixture is heated to a temperature of 18°C to 30°C, preferably about 22°C, with stirring and is then stirred for an additional 10 to 24 hours, preferably about 12 hours. Then the diluent is distilled off, the residue is combined with water and the mixture is extracted two to three times with an organic solvent, such as diethyl ether or ethyl acetate, preferably ethyl acetate. The combined organic extracts are dried and the solvent is distilled off. The residue (compound A3) can be used in Step 2 without prior purification.

Step 2A

The compound obtained in Step IA (A3) is reduced at the nitro group and cyclized to form the compound of formula (A4) (Diagram 2A).

Diagram 2A

[Reduction = reduction]

In Step 2A, 1 equivalent of the nitro compound (A3) is dissolved in an acid, preferably glacial acetic, formic or hydrochloric acid, preferably glacial acetic acid, and heated to 50 to 70°C, preferably about 60°C. Then a reducing agent, for example zinc, tin or iron, preferably iron filings, is added to complete the exothermic reaction, and the mixture is stirred for 0.2 to 2 hours, preferably 0.5 hours, at 100 to 125 °C, preferably at about 117 °C. After cooling to ambient temperature, the iron salt is filtered off and the solvent is distilled off. The residue is taken up in a solvent or a mixture of solvents, for example ethyl acetate or dichloromethane/methanol 9/1 and half-saturated NaCl solution, and is filtered, for example through diatomaceous earth. The organic phase is dried and evaporated. The residue (compound (A4)) can be purified by chromatography or crystallization or can be used as a crude product in Step 3A of the synthesis.

Step 3A

The compound obtained in Step 2A (A4) can be reacted by electrophilic substitution as shown in Diagram 3A, to obtain the compound of formula (A5).

Diagram 3A

In Step 3A, 1 equivalent of the amide of formula (A4) is dissolved in an organic solvent, for example dimethylformamide or dimethylacetamide, preferably dimethylacetamide, and is cooled to approximately -5 to 5°C, preferably 0°C.

Then 0.9 to 1.3 equivalents of sodium hydride and 0.9 to 1.3 equivalents of a methylation reagent, e.g. methyl iodide, added. The reaction mixture is stirred for 0.1 - 3 hours, preferably about 1 hour, at about 0 to 10 °C, preferably at about 5 °C, and can optionally be left for a further 12 hours at this temperature. The reaction mixture is poured into ice water and the precipitate is isolated. The residue (compound (A5)) can be purified by chromatography, preferably over silica gel, or by crystallization, or can be used as a crude product in step 4A of the synthesis.

Step 4A

Amination of the compound (A5) obtained in Step 3A, giving the compound of formula (A9) (Chart 4A), can be carried out using the methods known from the literature for the variants 4,1 A (a) SM.P.V. Boarland, J.F.W. McOmie, J. Chem. Soc. 1951, 1218-1221; b) F. H. S. Curd, F. C. Rose,./. Chem. Soc. 1946, 343-348., 4.2 A (a) Banks, J. Am. Chem. Soc. 1944, 66, 1131 b) Ghosh and Dolly, J. Indian Chem. Soc. 1981, 58, 512-513; c) N. P. Reddy and M. Tanaka, Tetrahedron Lett. 1997, 38, 4807-4810.

Diagram 4A

In variant 4.1 A, for example, 1 equivalent of the compound (A5) and 1 to 3 equivalents, preferably about 2 equivalents, of the compound (A6) are heated without a solvent or in an organic solvent such as sulfolane, dimethylformamide, dimethylacetamide, toluene, N-methylpyrrolidone, dimethylsulfoxide or dioxane, preferably sulfolane, for 0.1 to 4 hours, preferably 1 hour, at 100 to 220°C, preferably at about 160°C. After cooling, the product (A9) is crystallized by adding organic solvents or mixtures of solvents, e.g. diethyl ether/methanol, ethyl acetate, methylene chloride or diethyl ether, preferably diethyl ether/methanol 9/1, or is purified by chromatography.

In variant 4.2 A, for example, 1 equivalent of the compound (A5) and 1 to 3 equivalents of the compound (A6) are stirred with acid, for example 1-10 equivalents of 10-38% hydrochloric acid and/or an alcohol, for example ethanol , propanol, butanol, preferably ethanol, at reflux temperature for 1 to 48 hours, preferably about 5 hours.

The precipitated product (A9) is filtered off and possibly washed with water, dried and crystallized from a suitable organic solvent.

In variant 4.3 A, for example, 1 equivalent of the compound (A5) and 1 to 3 equivalents of the compound (A7) are dissolved in a solvent, for example toluene or dioxane, and combined with a phosphine ligand, for example 2,2 '-bis-(diphenylphosphino)-l,r-binaphthyl and a palladium catalyst, for example tris(dibenzylideneacetone)-dipalladium(O) and a base, for example cesium carbonate, and is refluxed for 1-24 h, preferably 17 h. The reaction mixture is purified, for example, over silica gel and the product (A8) is isolated from the solution or obtained by appropriate crystallization.

The product (A8) is dissolved in a suitable solvent, for example dioxane, and mixed with acid, for example semi-concentrated hydrochloric acid, for example in a ratio of solvent to acid of 3:1. Then the mixture is refluxed in 1 - 481, for example 12 h, and the precipitate formed is isolated. If desired, the product (A9) is purified by crystallization.

Step 5A

Diagram 5A

Variant 5.1 A:

1 equivalent of the compound (A9) is for example dissolved with 1 equivalent of an activation reagent, e.g. 0-benzotriazolyl-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), and a base, for example 1.5 equivalents of diisopropylethylamine (DIPEA), in an organic diluent, for example dichloromethane, tetrahydrofuran, dimethylformamide, N -methylpyrrolidone, dimethylacetamide, preferably dichloromethane or dimethylformamide. After addition of 1 equivalent of the amine (A10), the reaction mixture is stirred for 0.1 to 24 hours, preferably about 2 hours at 20°C to 100°C. The product with formula (Al 1) is obtained, for example, by crystallization or chromatographic purification.

The new compounds of the general formula (I) can be synthesized analogously to the following examples of synthesis. However, these examples are intended only as examples of procedures that provide a closer illustration of the invention, without limiting the invention to these examples.

Preparation of some intermediate compounds used to synthesize the examples is also described below.

Preparation of the acids

To synthesize the compounds in Examples 94 and 95, an intermediate

connection Zl

prepared as described below.

50.0 g (0.48 mol) of D-alanine methyl ester x HCl and 49.1 g (0.50 mol) of cyclohexanone were added to 300 ml of dichloromethane and then combined with 41.0 g (0.50 mol) sodium acetate and 159.0 g (0.75 mol) of sodium triacetoxyborohydride. The mixture was stirred overnight and then 300 ml of 10% sodium bicarbonate solution was added. The aqueous phase was extracted with dichloromethane. The combined organic phases were washed with 10% sodium bicarbonate solution, dried over Na2SO4 and evaporated.

Yield: 72.5 g of compound Zla (clear liquid)

72.5 g of compound Zla was added to 500 ml of water, and 76.6 g (0.39 mol) of 2,4-dichloro-5-nitropyrimidine in 500 ml of diethyl ether was added. At a temperature of -5°C, 100 ml of 10% potassium bicarbonate solution was added dropwise. The mixture was stirred for 3 h at -5°C and for an additional 121 at ambient temperature. The organic phase was separated and dried over NazSCv. Upon evaporation, the product crystallized.

Yield: 48.0 g of compound Zlb (yellow crystals)

48.0 g of compound Zlb was dissolved in 350 ml of glacial acetic acid and heated to 60°C. 47.5 g of iron powder was added, whereby the temperature rose to 105°C. The reaction mixture was stirred for three hours at 80°C, and was then filtered hot through cellulose and evaporated. The residue was stirred in water and ethyl acetate, suction filtered and the light gray precipitate was washed with ethyl acetate. The filtrate was washed with dilute ammonia and

water, and the organic phase was dried over Na 2 SC 4 , filtered through activated charcoal and evaporated. A slightly more light gray solid was obtained.

Yield: 29.5 g of compound Zlc (light gray crystals)

32.1 g of compound Zlc was added to 300 ml of dimethylacetamide and combined with 13 ml (0.2 mol) of methyl iodide. At -5°C, 6.4 g (0.16 mol) of sodium as a 60% dispersion in mineral oil was added portionwise. After 2 h, the reaction mixture was poured into 800 ml of ice water. The precipitate formed was suction filtered and washed with petroleum ether.

Yield: 33.0 g of compound Zld (beige crystals)

4.0 g of compound Zld and 2.3 g (15 mmol) of 4-amino-3-methylbenzoic acid were suspended in 50 ml of ethanol and 120 ml of water, were combined with 2 ml of conc. hydrochloric acid and refluxed at 481. The precipitate formed on cooling was suction filtered and washed with water, ethanol and diethyl ether.

Yield: 2.9 g of compound Zl (colorless crystals)

To synthesize the compounds of Example 188 and Example 203, an intermediate compound Z2 was first prepared

prepared as described below.

A solution of 128.2 g (0.83 mol) of D-alanine ethyl ester x HCl and 71.5 g (0.85 mol) of cyclopentanone in 1500 ml of dichloromethane was combined with 70.1 (0.85 mol) of sodium acetate and 265.6 g (1.25 mol) of sodium triacetoxyborohydride. The reaction mixture was stirred for 12 h and then poured into 1.5 L of a 10% sodium bicarbonate solution. The aqueous phase was extracted with dichloromethane. The combined organic phases were dried over Na 2 SC 4 and evaporated.

Yield: 143.4 g of compound Z2a (colorless oil)

66.0 g of compound Z2a was added to 500 ml of water and combined with 85.0 g (0.44 mol) of 2,4-dichloro-5-nitropyrimidine in 500 ml of diethyl ether. At -5°C, 100 ml of 10% potassium hydrogen carbonate solution was added dropwise, and the reaction mixture was stirred for 481 at ambient temperature. The aqueous phase was extracted with diethyl ether, and the combined organic phases were dried over Na2SO4 and evaporated. The dark red solid was stirred with petroleum ether and suction filtered.

Yield: 88.0 g of compound Z2b (yellow crystals)

88.0 g of compound Z2b was dissolved in 1000 ml of glacial acetic acid and was combined at 60°C in portions with 85 g of iron powder, whereby the temperature rose to 110°C. It was stirred for 1 h at 60°C, then hot suction filtration through cellulose and evaporation. The brown solid was stirred with 700 ml of water and suction filtered.

Yield: 53.3 g of compound Z2c (light brown crystals)

53.3 g of compound Z2c was dissolved in 300 ml of dimethylacetamide and combined with 13 ml (0.21 mol) of methyl iodide. At -5°C, 5.0 g (0.21 mol) of sodium hydride as a 60% dispersion in mineral oil was added portionwise. After 12 h, the reaction mixture was poured into 1000 ml of ice water and the precipitate formed was suction filtered.

Yield: 40.0 g of compound Z2d (colorless crystals)

4.0 g of compound Z2d and 2.8 g (16 mmol) of 4-amino-3-chlorobenzoic acid were suspended in 25 ml of ethanol and 60 ml of water, were combined with 3 ml of conc. hydrochloric acid and refluxed for 43 h. The precipitate formed on cooling was suction filtered and washed with water, ethanol and diethyl ether.

Yield: 0.9 g of compound Z2 (colorless crystals)

To synthesize the compounds in Example 19, 21, 22, 23, 45, 55, 58, 116, 128, 131, 133, 134, 136, 138, 177, 217, 231, 239, 46, 184, 166 and 187, an intermediate was first -connection Z3

prepared as described below.

54.0 g (0.52 mol) of D-2-aminobutyric acid was suspended in 540 ml of methanol and slowly combined with 132 g (1.1 mol) of thionyl chloride while cooling with ice. The mixture was refluxed for 1.5 h and then evaporated. The remaining oil was combined with 540 ml of tert-butyl methyl ether and the colorless crystals formed were suction filtered.

Yield: 78.8 g of compound Z3a (colorless crystals)

74.2 g of compound Z3a and 43.5 ml (0.49 mol) of cyclopentanone were dissolved in 800 ml of dichloromethane. After addition of 40.0 g (0.49 mol) of sodium acetate and 150.0 g (0.71 mol) of sodium triacetoxyborohydride at 0 °C, the mixture was stirred for 12 h at ambient temperature and then 500 ml of 20% sodium bicarbonate solution was added . The aqueous phase was extracted with dichloromethane. The combined organic phases were washed with water, dried over MgSCu and evaporated.

Yield: 85.8 g of compound Z3b (light yellow oil)

40.0 g of compound Z3b and 30.0 g (0.22 mol) of potassium carbonate were suspended in 600 ml of acetone and combined with 45.0 g (0.23 mol) of 2,4-dichloro-5-nitropyrimidine in 200 ml acetone while cooling with ice. After 12 h, a further 5.0 g of 2,4-dichloro-5-nitropyrimidine was added and it was stirred for 3 h. The reaction mixture was evaporated, taken up in 800 ml of ethyl acetate and 600 ml of water and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with water, dried over MgSO 4 and evaporated.

Yield: 75.0 g of compound Z3c (brown oil)

100 g of compound Z3c was dissolved in 650 ml of glacial acetic acid, and at 70°C 20 g of iron powder was added in portions. The mixture was stirred in oxygen at 70°C, then for 1.5 h at 100°C and was then filtered hot through diatomaceous earth. The reaction mixture was evaporated, taken up in methanol/dichloromethane, transferred to silica gel and purified with ethyl acetate by Soxhlet extraction. The solvent was removed and the residue was stirred with methanol.

Yield: 30.0 g of compound Z3d (light brown crystals)

25.0 g of compound Z3d and 6.5 ml (0.1 mol) of methyl iodide were added to 250 ml of dimethylacetamide, and at -10°C 3.8 g (0.95 mol) of sodium hydride as a 60% dispersion in mineral oil, added. It was stirred for 20 min at 0 °C, then for 30 min at ambient temperature and finally ice was added. The reaction mixture was evaporated and combined with 300 ml of water. The precipitate formed was suction filtered and washed with petroleum ether.

Yield: 23.0 g of compound Z3e (colorless solid)

6.0 g of compound Z3e and 5.1 g (31 mmol) of 4-amino-3-methoxybenzoic acid were suspended in 90 ml of ethanol and 350 ml of water, were combined with 3.5 ml of conc. hydrochloric acid and refluxed at 481. The reaction mixture was evaporated, the residue was stirred with methanol/diethyl ether and the precipitate formed was suction filtered.

Yield: 6.3 g of compound Z3 (light beige crystals)

To synthesize the compound in Examples 81 and 137, an intermediate compound Z4 was first made

prepared as described below.

25.0 g (0.19 mol) ethyl-1-aminocyclopropane-1-carboxylate x HC1 and 16.8 g (0.20 mol) cyclopentanone were dissolved in 300 ml dichloromethane and combined with 16.4 g (0.20 mol) sodium acetate and 61.7 g (0.29 mol) of sodium triacetoxyborohydride. It was stirred overnight and the reaction mixture was then poured into 400 ml of 10% sodium bicarbonate solution. The aqueous phase was extracted with dichloromethane. The combined organic phases were dried over Na2SO4 and evaporated.

Yield: 34.5 g of compound Z4a (colorless oil)

42.5 g (0.22 mol) of 2,4-dichloro-5-nitropyrimidine in 350 ml of diethyl ether was added to a mixture of 34.5 g of compound Z4a in 350 ml of water. At -5°C, the mixture was combined with 80 ml of 10% potassium hydrogen carbonate solution and stirred overnight at ambient temperature. The aqueous phase was extracted with diethyl ether. The combined organic phases were dried over Na2SO4 and evaporated.

Yield: 53.8 g of compound Z4b (brown oil)

20.1 g of compound Z4b was dissolved in 200 ml of glacial acetic acid and at 60°C was combined in portions with 19.1 g of iron powder, and after some time the temperature rose to 100°C. The mixture was stirred for 3 h at 60°C, then suction filtered through cellulose and evaporated. The residue was stirred in water and ethyl acetate and the yellow precipitate was suction filtered. The filtrate was washed with dilute ammonia and water, and the organic phase was dried over Na 2 SC* 4 and evaporated. After addition of diethyl ether, more product crystallized out. ;Yield: 4.0 g of compound Z4c (yellow crystals) ;7.8 g of compound Z4c and 2.6 ml (0.04 mol) of methyl iodide were dissolved in 100 ml of dimethylacetamide, and at -5°C 1, 5 g (0.04 mol) of sodium hydride added portionwise as a 60% dispersion in mineral oil. After 2 h, the reaction mixture was poured into ice water and the precipitate formed was suction filtered. ;Yield: 7.5 g of compound Z4d (light brown crystals) ;3.0 g of compound Z4d and 1.9 g (11 mmol) of 4-amino-3-methoxybenzoic acid were suspended in 40 ml of ethanol and 80 ml of water, combined with 2 ml conc. hydrochloric acid and refluxed at 20 L. A further 0.5 g of 4-amino-3-methoxybenzoic acid was added and it was refluxed at 48 L. The precipitate formed on cooling was suction filtered and washed with water, ethanol and diethyl ether. ;Yield: 2.1 g of compound Z4 (colorless crystals), m.p.: 222-223°C ;In order to synthesize the compounds in Example 162, 43, 53, 161, 202, 211, 215 and 212, first an intermediate compound Z5; ; prepared as described below. ;A mixture of 73.4 ml (0.5 mol) ethyl 2-bromoisobutyrate, 87.1 ml (0.75 mol) 3-methyl-l-butylamine, 82.5 g (0.6 mol) sodium iodide and 76.0 g (0.6 mol) of potassium carbonate in 1000 ml of ethyl acetate was refluxed for 3 days. Any salts present were filtered off and the filtrate was evaporated. ;Yield: 97.0 g of compound Z5a (red oil) ;49.0 g (0.25 mol) of 2,4-dichloro-5-nitropyrimidine and 38.3 g (0.28 mol) of potassium carbonate were suspended in 500 ml of acetone and was combined at 0 °C with 93.0 g of compound Z5a in 375 ml of acetone. The reaction mixture was stirred overnight at ambient temperature, filtered and evaporated. The residue was dissolved in ethyl acetate and washed with water, and the organic phase was dried over MgSCu and evaporated. Yield: 102.7 g of compound Z5b (brown oil). ;22.7 g of compound Z5b was dissolved in 350 ml of glacial acetic acid and was combined at 60°C in portions with 17.4 g of iron powder. After the addition was complete, the mixture was refluxed for 0.5 h, filtered hot and evaporated. The residue was taken up in 200 ml of dichloromethane/methanol (9:1) and washed with sodium chloride solution. The organic phase was suction filtered through diatomaceous earth, dried over MgSO4, evaporated and purified by column chromatography (eluent: ethyl acetate/cyclohexane 1:1). ;Yield: 1.9 g of compound Z5c (colorless crystals) ;1.9 g of compound Z5c was dissolved in 32 ml of dimethylacetamide and, while cooling with ice, was combined with 0.3 g (7 mmol) of sodium hydride as a 60% dispersion in mineral oil. After 10 min, 0.5 ml (7 mmol) of methyl iodide was added and it was stirred for 3 h at ambient temperature. The reaction mixture was evaporated and combined with water. The precipitate formed was suction filtered and washed with petroleum ether. ;Yield: 1.6 g of compound Z5d (colorless crystals) ;14.0 g of compound Z5d and 10.0 g (0.06 mol) of 4-amino-3-methoxybenzoic acid were suspended in 200 ml of dioxane and 80 ml of water , was combined with 10 ml conc. hydrochloric acid and refluxed at 401. The precipitate formed on cooling was suction filtered and washed with water, dioxane and diethyl ether. ;Yield: 13.9 g of compound Z5 (colorless crystals) ;To synthesize the compounds in Examples 88, 194, 229 and 89, an intermediate compound Z6 was first ; prepared as described below. ;6.0 g (0.06 mol) of L-2-aminobutyric acid was added to 80 ml of 0.5 M sulfuric acid and was combined at 0 °C with 5.5 g (0.08 mol) of sodium nitrite in 15 ml of water. The reaction mixture was stirred for 22 h at 0°C, combined with ammonium sulfate and filtered. The filtrate was extracted with diethyl ether and the combined organic extracts were dried over MgSO4 and evaporated. ;Yield: 6.0 g of compound Z6a (yellow oil) ;200 ml of methanol was combined under cooling with ice successively with 65.0 ml (0.89 mol) of thionyl chloride and 76.0 g of compound Z6a in 50 ml of methanol. The resulting mixture was stirred for 11 at 0°C and 2 h at ambient temperature, and then methanol and residual thionyl chloride were removed under vacuum at 0°C. ;Yield: 40.0 g of compound Z6b (yellow oil); 30.0 ml (0.17 mol) of trifluoromethanesulfonic anhydride was added to 150 ml of dichloromethane, and under cooling with ice a solution of 20.0 g of compound Z6b and 14.0 ml (0.17 mol) pyridine in 50 ml dichloromethane added over 1 hour. The mixture was stirred for 2 h at ambient temperature, any salts formed were suction filtered and then washed with 100 ml of water. The organic phase was dried over MgSO4 and evaporated. Yield: 42.0 g of compound Z6c (light yellow oil); 42.0 g of compound Z6c in 200 ml of dichloromethane was added dropwise to a solution of 15.5 ml (0, 17 mol) aniline and 24.0 ml (0.17 mol) triethylamine in 400 ml dichloromethane. The mixture was stirred in oxygen at ambient temperature and for a further 2 h at 35°C. The reaction mixture was washed with water, dried over MgSO4 and evaporated. The residue was purified by distillation (95-100°C, 1<*>10"<3>mbar). Yield: 14.0 of compound Z6d (colorless oil)

14.0 g of compound Z6d and 16.0 g (0.1 mol) of potassium carbonate were suspended in 100 ml of acetone and at 10°C were combined with 16.0 g (0.08 mol) of 2,4-dichloro-5 -nitropyrimidine. The mixture was stirred for 41 at 40°C, any salts formed were suction filtered and the filtrate was evaporated. The residue was taken up in 300 ml of ethyl acetate and washed with water. The organic phase was dried over MgSCu and evaporated.

Yield: 31.0 g of compound Z6e (brown oil)

31.0 g of compound Z6e was dissolved in 200 ml of glacial acetic acid and at 60°C was combined in portions with 10 g of iron powder, and after some time the temperature rose to 85°C. The mixture was stirred for an additional 1 hour at 60°C, filtered through diatomaceous earth and evaporated. The residue was stirred with methanol.

Yield: 4.5 g of compound Z6f (brown crystals)

At -20°C, 0.6 g (16 mmol) of sodium hydride as a 60% dispersion in mineral oil was added portionwise to a mixture of 4.5 g of compound Z6f and 1.0 ml (16 mmol) of methyl iodide in 100 ml of dimethylacetamide. After 11, the reaction mixture was combined with 50 ml of water and evaporated. The residue was stirred with 200 ml of water, the precipitate was suction filtered and washed with petroleum ether.

Yield: 4.5 g of compound Z6g (colorless crystals)

A suspension of 1.5 g of compound Z6g and 1.4 g (8 mmol) of methyl 4-amino-3-methoxybenzoate in 30 mL of toluene was combined with 0.4 g (0.6 mmol) of 2,2'- bis-(diphenylphosphino)-l,r-binaphthyl, 0.23 g (0.3 mmol) tris(dibenzylideneacetone)-dipalladium(O) and 7.0 g (21 mmol) cesium carbonate and was refluxed for 17 h. The reaction mixture was transferred to silica gel and purified by column chromatography (eluent: dichloromethane/methanol 9:1).

Yield: 1.7 g of compound Z6h (yellow crystals)

1.7 g of compound Z6h was dissolved in 50 ml of dioxane, combined with 15 ml of semi-conc. hydrochloric acid and refluxed at 121. After cooling, the formed precipitate was suction filtered.

Yield: 1.1 g of compound Z6 (colorless solid)

To synthesize the compounds of Example 26, 20, 32, 56, 101, 112, 209, an intermediate compound Z7 was first

prepared as described below.

50.0 g (0.36 mol) of D-alanine methyl ester x HCl was suspended in 500 mL of dichloromethane and 35 mL of acetone and was combined with 30.0 g (0.37 mol) of sodium acetate and 80.0 g (0. 38 mol) sodium triacetoxyborohydride. The mixture was stirred for 12 h and then poured into 400 ml of 10% sodium bicarbonate solution. The organic phase was dried over Na2SO4 and evaporated.

Yield: 51.0 g of compound Z7a (yellow oil)

A suspension of 51.0 g of compound Z7a in 450 ml of water was combined with 80.0 g (0.41 mol) of 2,4-dichloro-5-nitropyridine in 450 ml of diethyl ether. At -5°C, 100 ml of 10% potassium bicarbonate solution was added dropwise. The reaction mixture was stirred for 3 h, and the organic phase was dried over Na2SO4 and evaporated.

Yield: 74 g of compound Z7b (yellow oil)

18.6 g of compound Z7b was dissolved in 200 ml of glacial acetic acid and was combined at 60°C in portions with 20.0 g of iron powder. The mixture was stirred for 2 h at 60°C and then suction filtered through cellulose. The residue was dissolved in ethyl acetate and washed with water and conc. ammonia. The organic phase was dried over Na 2 SC 4 and evaporated. The residue was crystallized from diethyl ether.

Yield: 9.8 g of compound Z7c (colorless crystals)

17.0 g of compound Z7c and 7 ml (0.1 mol) of methyl iodide were dissolved in 200 ml of dimethylacetamide and combined at -5°C with 4.0 g (0.1 mol) of sodium hydride as a 60% dispersion in mineral oil . The reaction mixture was stirred for 30 min and then poured into 300 ml of ice water. The precipitate formed was suction filtered and stirred with petroleum ether.

Yield: 14.8 g of compound Z7d (beige crystals)

0.9 g of compound Z7d and 1.5 g (9 mmol) of 4-amino-3-methoxybenzoic acid were heated to 210°C for 30 min. After cooling, the residue was stirred with ethyl acetate and the precipitate obtained was suction filtered.

Yield: 1.2 g of compound Z7 (gray crystals)

The following acids were prepared, for example, analogously to the methods in the described synthesis:

Synthesis of the amino components L-R5

The following amines were obtained as follows:

1,1-dimethyl-2-dimethylamino-1-γ-1-ethylamine and 1,1-dimethyl-2-piperidin-1-yl-ethylamine

The compounds were prepared according to the following references: a) S. Schuetz et al., Arzneimittel-Forschung 1971, 21, 739-763 b) V. M. Belikov et al, Tetrahedron 1970, 26, 1199-1216. c) E.B. Butler and McMillan, J. Amer. Chem. Soc. 1950, 72.2978.

Other amines were prepared as follows, in a modified manner in relation to the literature mentioned above.

1,1-dimethyl-2-morpholin-1-yl-ethylamine

8.7 mL of morpholine and 9.3 mL of 2-nitropropane were prepared while cooling with ice, and 7.5 mL of formaldehyde (37%) and 4 mL of a 0.5 mol/L NaOH solution were slowly added dropwise (< 10°C). The mixture was then stirred for 11 at 25°C and 11 at 50°C. The solution was treated with water and ether, and the aqueous phase was extracted 3x with ether. The combined org. phases were dried over Na 2 SC 4 and combined with HCl in dioxane (4 mol/l), and the precipitate formed was filtered with suction. Yield: 21.7 white powder.

5 g of the white powder was dissolved in 80 ml of methanol and, with the addition of 2 g of RaNi, was treated with hydrogen at 35°C and 50 psi for 40 minutes. This gave 3.6 g of 1,1-dimethyl-2-morpholin-1-yl-ethylamine.

The following amines were prepared by this method:

1,1-Dimethyl-N-methylpiperazin-1-yl-ethylamine

1, 1 - dimethyl- 2- pyrrolidin- 1 - yl- ethylamine Synthesis of 1, 3- dimorpholin- 2- amino- propane 5 g 1,3 dimorpholin-2-nitropropane acquired from Messrs. Aldrich, was dissolved in 80 mL of methanol and treated with hydrogen for 5.5 h at 30°C and 50 psi while adding 2 g of RaNi. This gave 4.2 g of 1,3-dimorpholine-2-amino-propane.

4-aminobenzylmorpholine

The preparation of this amine is described in the following reference: S. Mitsuru et al, J. Med. Chem. 2000, 43, 2049-2063

4-amino-1-tetrahydro-4H-pyran-4-vl-piperidine

20 g (100 mmol) of 4-tert-butyloxycarbonylaminopiperidine was dissolved in 250 ml of CH2Cl2 and stirred for 12 h at RT with 10 g (100 mmol) of tetrahydro-4H-pyran-4-one and 42 g (200 mmol) of NaBH( OAc)3. Then water and potassium carbonate were added, the org. the phase was separated and dried and the solvent was removed under vacuum. The residue was dissolved in 200 ml of CH 2 Cl 2 and stirred at 121 at RT with 100 ml of trifluoroacetic acid. The solvent was removed under vacuum and the residue was taken up in CHCl 3 and re-evaporated, then taken up in acetone and the hydrochloride precipitated with ethereal HCl. Yield: 14.3 g (56%).

Cis- and trans- 4- morpholino- cvclohexylamine

Dibenzvl- 4- morpholino- cyclohexylamine

3.9 g (30 mmol) of 4-dibenzylcyclohexanone was dissolved in 100 ml of CH 2 Cl 2 and stirred for 12 h at RT with 3.9 g (45 mmol) of morpholine and 9.5 g (45 mmol) of NaBH(OAc) 3 . Then water and potassium carbonate were added, the org. the phase was separated and dried and the solvent was removed under vacuum. The residue was purified through a silica gel column (about 20 ml silica gel; about 500 ml ethyl acetate 90/methanol 10 + 1% conc. ammonia). Relevant fractions were evaporated under vacuum. Yield: 6.6 g (60%) cis isomer and 2 g (18%) trans isomer.

Alternatively, trans-dibenzyl-4-morpholino-cyclohexylamine can be prepared by the following method: 33 g (112 mmol) of 4-dibenzylcyclohexanone was dissolved in 300 ml of MeOH, was combined with 17.4 g (250 mmol) of hydroxylamine hydrochloride and stirred for 41 at 60°C. The solvent was evaporated under vacuum, combined with 500 ml of water and 50 g of potassium carbonate and extracted twice with 300 ml of dichloromethane. The org. the phase was dried, evaporated under vacuum, and the residue was crystallized from petroleum ether, dissolved in 1.5 L of EtOH and heated to 70°C. 166 g of sodium was added in portions and the mixture was refluxed until the sodium had dissolved. The solvent was removed under vacuum, the residue was combined with 100 ml of water and extracted twice with 400 ml of ether. The org. phase was washed with water, dried, evaporated under vacuum and the trans isomer was isolated using a column (approx. 1.5 L silica gel; approx. 2 L ethyl acetate 80/methanol 20 + 2% conc. ammonia). Yield: 12.6 g (41.2%).

6.8 g (23 mmol) of trans-1-amino-4-dibenzylaminocyclohexane was dissolved in 90 ml of DMF and stirred for 8 h at 100°C with 5 ml (42 mmol) of 2,2'-dichloroethyl ether and 5 g of potassium carbonate. After cooling, 30 ml of water was added, and the precipitated crystals were suction filtered and purified through a short column (about 20 ml of silica gel, about 100 ml of ethyl acetate). The residue was crystallized from methanol and conc. HC1 as the dihydrochloride. Yield: 7.3 g (72.4%).

Trans-4-morpholino-cyclohexylamine

7.2 g (16.4 mmol) of trans-dibenzyl-4-morpholino-cyclohexylamine was dissolved in 100 ml of MeOH and hydrogenated on 1.4 g of Pd/C (10%) at 30-50°C. The solvent was removed under vacuum and the residue was crystallized from ethanol and conc. HC1. Yield: 3.9 g (93%); m.p. 312°C.

The cis-isomer can be prepared analogously.

Cis- and trans-4-piperidino-cyclohexylamine

Trans-dibenzyl-4-piperidino- cvclohexvlamin

2.0 g (6.8 mmol) of trans-1-amino-4-dibenzylaminocyclohexane (see Example 2) was dissolved in 50 ml of DMF and stirred for 48 h at RT with 1.6 g (7 mmol) of 1,5- dibromopentane and 2 g of potassium carbonate. The mixture was cooled, combined with water, extracted twice with 100 ml of dichloromethane and dried, and the solvent was removed under vacuum. The residue was purified through a column (about 100 ml silica gel, about 500 ml ethyl acetate 80/methanol 20 + 1% conc. ammonia). The desired fractions were evaporated under vacuum and crystallized from petroleum ether. Yield: 1.2 g (49%).

Trans-4-piperidino-cyclohexylamine

1.7 g (4.8 mmol) of trans-dibenzyl-4-piperidino-cyclohexylamine was dissolved in 35 ml of MeOH and hydrogenated on 350 mg of Pd/C (10%) at 20°C. The solvent was removed under vacuum and the residue was crystallized from ethanol and conc. HC1. Yield: 1.1 g (78%).

The cis-isomer can be prepared analogously.

Cis- and trans- 4-(4-phenyl-piperazine-l-vl)- cvclohexvlamine

4.1 g (25.3 mmol) of 4-dibenzylcyclohexanone was dissolved in 50 ml of dichloromethane and stirred for 12 h at RT with 7.4 g (25.3 mmol) of N-phenylpiperazine and 7.4 g (35 mmol) of NaBH (OAc)3. Water and potassium carbonate were then added, and the org. the phase was separated and dried, and the solvent was removed under vacuum. The residue was purified over a silica gel column (ethyl acetate 80/methanol 20 + 0.5% conc. ammonia). Yield: 1.7 g (15.8%) cis isomer and 0.27 (2.5%) trans isomer.

Trans-4-(4-phenyl-piperazine-1-vD-cyclohexylamine

270 mg (0.61 mmol) of trans-dibenzyl-[4-(4-phenyl-piperazin-1-yl)-cyclohexyl]-amine was dissolved in 5 mL of MeOH and hydrogenated on 40 mg of Pd/C (10%) at 20-30 °C. The solvent was removed under vacuum and the residue was crystallized from ethanol and conc. HC1. Yield: 110 mg (69%).

The cis-isomer can be prepared analogously.

Cis- and trans-4-(4- cyclopropylmethyl-piperazine-1-vl')- cyclohexylamine

9.8 g (33.4 mmol) of 4-dibenzylcyclohexanone was dissolved in 100 ml of dichloromethane and stirred for 12 h at RT with 5.6 g (40 mmol) of N-cyclopropylmethylpiperazine and 8.5 g (40 mmol) of NaBH(OAc )3. Water and potassium carbonate were then added, and the org. the phase was separated and dried, and the solvent was removed under vacuum. The residue was purified over a silica gel column (approximately 50 ml silica gel, approximately 3 L ethyl acetate 95/ methanol 5 + 0.25% conc. ammonia. Relevant fractions were evaporated under vacuum. The fastest eluting cis compound was crystallized from ethyl acetate. Trans - compound was crystallized from ethanol + conc. HCl Yield: 8.5 g (61%) cis-isomer and 2.2 (13%) trans-isomer.

Cis- 4-( 4- cyclopropylmethyl- piperazine- 1 - vD- cyclohexylamine

8.5 g (20 mmol) of cis-dibenzyl-[4-(4-cyclopropylmethyl-piperazin-1-yl)-cyclohexyl]-amine was dissolved in 170 ml of MeOH and hydrogenated on 1.7 g of Pd/C (10% ) at 30-50 °C. The solvent was removed under vacuum and the residue was crystallized from ethanol and conc. HC1. Yield: 4.4 g (91%).

The trans isomer can be prepared analogously.

Synthesis of the examples

Example 152

0.15 g of compound Z10, 0.14 g of TBTU, 0.13 ml of DIPEA were dissolved in dichloromethane and stirred for 20 minutes at 25°C. Then 90 µl of 1-(3-aminopropyl)-4-methylpiperazine was added and it was stirred for a further 2 hours at 25°C. The solution was then diluted with dichloromethane and extracted with water. The product was precipitated by addition of

petroleum ether, ether and ethyl acetate to the organic phase. Yield: 0.16 g beige solid.

Example 164

0.10 g of compound Z10, 0.1 g of TBTU, 0.08 ml of DIPEA were dissolved in 4 ml of dichloromethane and stirred for 20 minutes at 25°C. Then 44 µl of dimethylaminopropylamine was added and it was stirred for a further 2 hours at 25°C. The solution was then diluted with dichloromethane and extracted with water. The product was precipitated by adding petroleum ether, ether and acetone to the organic phase. Yield: 0.08 g yellow solid.

Example 242

0.15 g of compound Z10, 0.14 g of TBTU, 0.13 ml of DIPEA were dissolved in 5 ml of dichloromethane and stirred for 20 minutes at 25°C. Then 75 µl of 1-(2-aminoethyl)-piperidine was added and it was stirred for a further 2 hours at 25°C. The solution was then diluted with dichloromethane and extracted with water. The product was precipitated by adding petroleum ether, ether and ethyl acetate to the organic phase Yield: 0.14 g yellow solid.

Example 188

0.1 g of compound Z2, 0.09 g of TBTU, 0.05 ml of DIPEA were dissolved in 15 ml of dichloromethane and stirred for 20 minutes at 25°C. Then 33 mg of 1-methyl-4-aminopiperidine was added and the mixture was stirred for a further 3 hours at 25°C. The solution was extracted with 20 ml of water and then evaporated under vacuum. The product was crystallized using ether. Yield: 0.047 g of white crystals.

Example 203

0.1 g of compound Z2, 0.09 g of TBTU, 0.5 ml of DIPEA were dissolved in 15 ml of dichloromethane and stirred for 30 minutes at 25°C. Then 50 mg of 4-amino-1-benzylpiperidine was added

and the mixture was stirred for an additional 3 hours at 25°C. The solution was extracted with 20 ml of water and then evaporated under vacuum. The residue was then chromatographed over silica gel and the isolated product was crystallized with ether. Yield: 0.015 g of white crystals.

Example 94

0.17 g of compound Zl, 0.19 g of TBTU, 0.11 ml of DIPEA were dissolved in 50 ml of dichloromethane and stirred for 30 minutes at 25°C. Then 63 mg of 1-methyl-4-aminopiperidine was added and the mixture was stirred for a further 17 hours at 25°C. 50 ml of water and 1 g of potassium carbonate were added to the solution and the organic phase was separated using a phase separation column and then evaporated under vacuum. The product was then purified by silica gel chromatography and the purified product was crystallized with ether. Yield: 0.1 g of white crystals.

Example 95

0.17 g of compound Zl, 0.19 g of TBTU, 0.11 ml of DIPEA were dissolved in 50 ml of dichloromethane and stirred for 30 minutes at 25°C. Then 77 mg of exo-3-p-aminotropane was added and the mixture was stirred for a further 17 hours at 25°C. 50 ml of water and 1 g of potassium carbonate were added to the solution and the organic phase was separated using a phase separation cartridge and then evaporated under vacuum. The product was then purified by silica gel chromatography, and the purified product was crystallized with ether. Yield: 0.03 g of white crystals.

Example 46

0.15 g of compound Z3, 0.12 g of TBTU, 0.12 ml of DIPEA were dissolved in 5 ml of dichloromethane and stirred for 30 minutes at 25°C. Then 50 mg of 1-methyl-4-aminopiperidine was added, and the mixture was stirred for a further 2.5 hours at 25°C. The solution was then extracted with water and evaporated. The residue was dissolved in hot ethyl acetate and crystallized from ether and petroleum ether. Yield: 0.025 g of white crystals. Melting point: 203°C as the base.

Example 80

0.2 g of compound Z8, 0.2 g of TBTU, 0.1 ml of DIPEA were dissolved in 10 ml of dichloromethane and stirred for 30 minutes at 25 °C. Then 100 mg of 1-methyl-4-aminopiperidine was added, and the mixture was stirred for a further 17 hours at 25°C. The solution was then extracted with a dilute potassium carbonate solution and evaporated. The residue was crystallized using ether. Yield: 0.12 g of white crystals.

Example 190

0.2 g of compound Z8, 0.2 g of TBTU, 0.3 ml of DIPEA were dissolved in 5 ml of dichloromethane and stirred in lt at 25°C. Then 0.13 g of 4-amino-1-benzylpiperidine was added, and the mixture was stirred for a further 1 hour at 25°C. The solution was then diluted with 10 ml of methylene chloride and extracted with 20 ml of water. The product was then purified over silica gel and crystallized from ethyl acetate and ether. Yield: 0.23 g of compound Z8. 0.23 g of the benzylamine Z8 was dissolved in 10 ml of methanol, combined with 50 mg of Pd/C and hydrogenated under 3 bar for 3 h at 25°C. On addition of petroleum ether and ethyl acetate, white crystals were formed. These were chromatographed over silica gel and crystallized from ethyl acetate and ether. Yield: 0.075 g of white crystals.

Example 196

0.1 g of compound Z10, 0.09 g of TBTU, 0.3 ml of DIPEA were dissolved in 4 ml of dichloromethane and stirred for 20 minutes at 25°C. Then 67 mg of xx amine was added and it was stirred for a further 2 hours at 25°C. The solution was then diluted with dichloromethane and extracted with water. It was then chromatographed over silica gel and the residue was dissolved in acetone, combined with ethereal HCl and the precipitate formed was isolated. Yield: 0.09 g pale yellow solid.

Example 166

0.1 g of compound Z10, 0.11 g of TBTU, 0.14 ml of DIPEA were dissolved in 2 ml of dimethylformamide and stirred for 3 h at 50°C. Then 55 mg of 4-morpholino-methylphenylamine was added. The reaction mixture was then cooled to

ambient temperature during 17 h. Then the dimethylformamide was removed under vacuum, and the residue was taken up in dichloromethane and extracted with water. It was then chromatographed over silica gel and the product was crystallized from ethyl acetate and ether. Yield: 0.06 g of yellowish crystals.

Example 81

0.2 g of compound Z4, 0.2 g of TBTU, 0.1 ml of DIPEA were dissolved in 10 ml of dichloromethane and stirred for 30 minutes at 25°C. Then 0.1 g of 1-methyl-4-aminopiperidine was added and the mixture was stirred for a further 17 hours at 25°C. The solution was then extracted with aqueous potassium carbonate solution and evaporated. The product was crystallized using ether. Yield: 0.16 g of white crystals.

Example 162

0.1 g of compound Z5, 0.07 g of TBTU, 0.15 ml of DIPEA were dissolved in 5 ml of dichloromethane and stirred for 20 minutes at 25°C. Then 0.04 g of 1-methyl-4-aminopiperidine was added and the mixture was stirred for a further 2 hours at 25°C. The solution was then diluted with 15 ml of dichloromethane and extracted with 20 ml of water. The residue was dissolved in MeOH and acetone, combined with 1 mL ethereal HCl and evaporated. A crystalline product was formed using ether, ethyl acetate and a little MeOH. Yield: 0.1 g of white crystals.

Example 88

0.1 g of compound Z6, 0.12 g of TBTU, 0.12 ml of DIPEA were dissolved in 10 ml of dichloromethane and stirred for 30 minutes at 25°C. Then 0.04 g of 1-methyl-4-aminopiperidine was added and the mixture was stirred for a further 2 hours at 25°C. The solution was then diluted with 10 ml of dichloromethane and extracted with 10 ml of water. A crystalline product was formed using ether, ethyl acetate and petroleum ether. Yield: 0.6 g of white crystals.

Example 89

0.1 g of compound Z6, 0.08 g of TBTU, 0.08 ml of DIPEA were dissolved in 10 ml of dichloromethane and stirred for 30 minutes at 25°C. Then 37 (µl g of N,N-dimethylneopentanediamine was added and the mixture was stirred for a further 2 hours at 25°C. The solution was then diluted with 10 ml of dichloromethane and extracted with 10 ml of water. The product was then chromatographed over silica gel and crystallized from ethyl acetate , ether and petroleum ether Yield: 0.005 g white crystals.

Example 26

0.15 g of compound Z7, 0.16 g of TBTU, 1 ml of DIPEA were dissolved in 5 ml of dichloromethane and stirred for 30 minutes at 25°C. Then 0.1 g of 4-morpholinocyclohexylamine was added and the mixture was stirred for a further 17 hours at 25°C. The residue was then combined with 10 ml of 10% potassium carbonate solution, and the precipitate was isolated and washed with water. It was then dissolved in dichloromethane and evaporated again. The product was crystallized from ethyl acetate. Yield: 0.1 g of white crystals.

Example 9

150 mg of compound Z9 and 93 mg of amine were dissolved in 5 ml of dichloromethane and stirred with 160 mg of TBTU and 1 ml of DIPEA for 12 h at RT. The solvent was removed under

vacuum, and the residue was combined with 10 ml of 10% potassium carbonate solution. The precipitate was suction filtered, washed with water, taken up in dichloromethane and dried, and the solvent was removed under vacuum. The residue was crystallized from ethyl acetate. Yield: 82.0 mg; sm.p. 253°C (as base).

Example 16

150 mg of compound Z8 and 73 mg of trans-4-piperidino-cyclohexylamine were dissolved in 5 ml of dichloromethane and stirred with 160 mg (0.50 mmol) of TBTU and 1 ml of DIPEA for 12 h at RT. The solvent was removed under vacuum and the residue was combined with 10 ml of 10% potassium carbonate solution. The precipitate was suction filtered, washed with water, taken up in

dichloromethane and dried, and the solvent removed under vacuum. The residue was crystallized from ethyl acetate. Yield: 87.0 mg; sm.p. 237 °C (as base).

Example 37

100 mg of compound Z9 and 42 mg of 3-amino-1-ethyl-pyrrolidine were dissolved in 10 ml of dichloromethane and stirred with 90 mg of TBTU and 0.5 ml of DIPEA for 12 h at RT. The solvent was removed under vacuum and the residue was combined with 10 ml of 10% potassium carbonate solution. The precipitate was suction filtered, washed with water, taken up in dichloromethane and dried, and the solvent was removed under vacuum. The residue was crystallized from ethyl acetate/petroleum ether. Yield: 24.0 mg.

Example 120

100 mg of compound Zl 1 and 73 mg of 4-amino-1-tetrahydro-4H-pyran-4-yl-piperidine were dissolved in 10 ml of dichloromethane and stirred with 90 mg of TBTU and 0.5 ml of DIPEA for 1 h at RT. The solvent was removed under vacuum and the residue was combined with 10 ml of 10% potassium carbonate solution. The precipitate was suction filtered, washed with water, taken up in dichloromethane and dried, and the solvent was removed under vacuum. The residue was crystallized from ethyl acetate/petroleum ether. Yield: 89 mg.

Example 212

150 mg of compound Z5 and 150 mg of trans-4-(4-cyclopropylmethyl-piperazin-1-yl)-cyclohexylamine (as the hydrochloride) were dissolved in 5 ml of dichloromethane and stirred with 160 mg of TBTU and 2 ml of DIPEA for 21 at RT. The solvent was removed under vacuum and the residue was combined with 10 ml of 10% potassium carbonate solution. The precipitate was suction filtered, washed with water, taken up in dichloromethane and dried, and the solvent removed under vacuum. The residue was purified over a column (20 ml silica gel, 300 ml ethyl acetate 90/methanol 10 + 2% conc. ammonia). Relevant fractions were evaporated under vacuum and crystallized from ethyl acetate. Yield: 140 mg; sm.p. 187 °C (as base).

Example 232

390 mg of compound Zl 1 and 240 mg of trans-4-(4-t-butyloxycarbonyl-piperazin-1-yl)-cyclohexylamine were dissolved in 2.5 ml of NMP and stirred with 482 mg of TBTU and 1 ml of triethylamine for 21 at RT . Then 100 ml of water and 200 mg of potassium carbonate were added, and the precipitate was suction filtered, washed with water and purified through a silica gel column. Relevant fractions were evaporated under vacuum, dissolved in 2 ml of dichloromethane, combined with 2 ml of trifluoroacetic acid and stirred for 2 h at RT, combined with an additional 100 ml of water and 200 mg of potassium carbonate, and the precipitate was suction filtered and washed with water. The precipitate was then purified through a silica gel column. Relevant fractions were evaporated under vacuum and the residue was crystallized from ethanol and conc. hydrochloric acid. Yield: 95 mg; sm.p. 291 °C.

Example 213

60 mg of the compound from Example 232 was dissolved in 10 ml of ethyl acetate and stirred with 1 ml of acetic anhydride and 1 ml of triethylamine for 30 min at RT. The solvent was removed under vacuum, the residue combined with water and ammonia, and the precipitated crystals were suction filtered and washed with water and a little cold acetone. Yield: 40 mg; sm.p. 248 °C.

Example 218

1.2 g of compound Z9 and 0.5 g of 1,4-dioxaspiro[4,5]dec-8-ylamine were dissolved in 20 ml of dichloromethane and stirred with 1.28 g of TBTU and 4 ml of triethylamine for 12 h at RT. Then 50 ml of water and 0.5 g of potassium carbonate were added, and the org. the phase was separated, dried and evaporated under vacuum. The residue was crystallized from ethyl acetate, combined with 25 ml of 1 N hydrochloric acid and 20 ml of methanol and stirred for 30 min at 50°C. The methanol was removed under vacuum and the precipitate was suction filtered, washed with water and dried.

The residue was taken up in 20 ml of dichloromethane, stirred with 0.5 g of thiomorpholine and 0.5 g of NaBH(OAc) 3 for 12 h at RT. Water and potassium carbonate were then added, and the org. the phase was separated and dried, and the solvent was removed under vacuum. The residue was purified over a silica gel column. Relevant fractions were evaporated under vacuum and the hydrochloride was precipitated with ethereal HCl. Yield: 86 mg trans-isomer; amorphous powder.

Example 187

200 mg of compound Z3 in 5 ml of dichloromethane was combined with 0.1 ml of diisopropylethylamine and 180 mg of TBTU and stirred for 30 min. Then 191 mg of 4-(4-methyl-piperazin-1-yl)-phenylamine was added and the mixture was stirred overnight. The reaction mixture was combined with water and the aqueous phase was extracted with dichloromethane. The combined organic phases were dried over Na2SO4 and evaporated. The residue was purified by column chromatography (eluent: dichloromethane/methanol 100:7).

Yield: 128 mg (light yellow crystals)

The compounds with formula (I) given in Table 1 are, among other things, obtained analogously to the method described above. Abbreviations Xi, X2, X3, X4 and X5

which is used in Table 1, means in each case a link to a position in the general formula shown in Table 1, instead of the corresponding groups R<1>,R<2>,R<3>,R<4>and L-R <5.>

It has been found that the compounds of the general formula (I) are characterized by their wide range of applications in the therapeutic field. In particular, those applications are mentioned where the inhibition of specific cell cycle kinases, especially the inhibitory effect on the proliferation of cultured human tumor cells, but also the proliferation of other cells, such as endothelial cells, plays a role.

It can be demonstrated by FACS analysis that the inhibition of proliferation caused by the compounds according to the invention is mediated by arrest of the cells, especially in the G2/M phase, in the cell cycle. The cells are stopped, regardless of the cells used, for a specific period of time in this phase of the cell cycle before programmed cell death is initiated. Arrest in the G2/M phase of the cell cycle is triggered, for example, by inhibition of specific cell cycle kinases. Studies with model organisms such as Schizosaccharomyces pombe or Xenopus, or studies with human cells, have shown that the transition from the G2 phase to mitosis is regulated by the CDK1/cyclin B kinase (Nurse, 1990). This kinase, which is also known as "mitosis-promoting factor" (MPF), phosphorylates and consequently regulates several proteins, such as nuclear lamins, kinesin-like motor proteins, condensins and Golgi matrix proteins, which play an important role in the breakdown of the nuclear hyster, in centrosome separation, in the formation of the mitotic spindle apparatus, chromosome condensation and breakdown of the Golgi apparatus (Nigg. E. , 2001). A murine cell line with a temperature-sensitive CDK1 kinase mutant shows rapid degradation of the CDK1 kinase and subsequent arrest in the G2/M phase after a temperature increase (Th'ng et al., 1990). Treatment of human tumor cells with inhibitors of CDK1/cyclin B, such as e.g. butyrolactone, also leads to arrest in the G2/M phase and subsequent apoptosis (Nishio, et al. 1996). Another kinase involved in the G2 and mitosis phase is polo-like kinase 1 (Plk1), which is responsible for the maturation of the centrosomes, for the activation of the phosphatase Cdc25C, as well as for the activation of the anaphase-promoting complex (Glover et al, 1998, Qian, et al, 2001). Injection of Plkl antibodies leads to G2 arrest in non-transformed cells, while tumor cells are arrested in the mitosis phase (Lan and Nigg, 1996). In addition, it has been described that the protein kinase aurora B has an essential function during the transition to mitosis. Aurora B phosphorylates histone H3 at Serl 1 and thereby initiates chromosome condensation (Hsu, J.Y. et al., 2000). However, specific cell cycle arrest in the G2/M phase can also be triggered, e.g. of inhibition of specific phosphatases such as e.g. Cdc25C

(Russell and Nurse, 1986). Yeast with a defective cdc25 gene arrests in the G2 phase, while overexpression of cdc25 leads to an early transition to the mitosis phase (Russell and Nurse, 1987). Arrest in G2/M phase can, however, also be triggered by inhibition of certain motor proteins, "so-capped" kinesins such as e.g. Eg5 (Mayer et al, 1999), or by agents that stabilize or destabilize microtubules (eg, colchicine, taxol, etoposide, vinblastine, vincristine) (Schiff and Horwitz, 1980).

On the basis of their biological properties, the compounds of the general formula I according to the invention, their isomers and physiologically acceptable salts, are suitable for the production of preparations for the treatment of diseases characterized by high or abnormal cell proliferation.

Such diseases include, for example: viral infections (eg HIV and Kaposi's sarcoma); inflammatory and autoimmune diseases (eg, colitis, arthritis, Alzheimer's disease, glomerulonephritis and wound healing); bacterial, fungal and/or parasitic infections; leukemias, lymphomas and solid tumors; skin diseases (eg psoriasis); bone diseases; cardiovascular diseases (eg restenosis and hypertrophy). They are also suitable for protecting proliferating cells (eg, hair, gut, blood and progenitor cells) against DNA damage caused by radiation, UV treatment and/or cytostatic treatment (Davis et al., 2001).

The new compounds can be used for the prevention, short-term or long-term treatment of the above-mentioned diseases, also in combination with other active substances used for the same indications, e.g. cytostatics.

The activity of the compounds according to the invention was determined by the cytotoxicity test with cultured human tumor cells and/or by a FACS analysis, for example with HeLaS3 cells. In both test methods, the compounds showed good to very good activity, i.e. for example an EC 50 value in the HeLaS3 cytotoxicity test lower than 5 µmol, usually lower than 1 µmol.

Measurement of cytotoxicity towards cultured human tumor cells

To measure cytotoxicity against cultured human tumor cells, cells from the cervical cancer-inducing cell line HeLaS3 (purchased from American Type

Culture Collection (ATCC)) grown in Ham's F12 Medium (Life Technologies) and 10% fetal calf serum (Life Technologies) and harvested in logarithmic growth. The HeLaS3 cells were then placed in 96-well plates (Costar) at a density of 1000 cells per well. well, and were incubated overnight in an incubator (at 37°C and 5% CO2), where 6 wells on each plate were filled with medium only (3 wells for control medium, 3 wells for incubation with reduced AlamarBlue). The active substances were added to the cells in different concentrations (dissolved in DMSO; final concentration: 1%) (in each case as a triplicate measurement). After 72 h of incubation, 20 µl of AlamarBlue (AccuMed International) was added to each well, and the cells were incubated for an additional 7 h. As a control, 20 µl of reduced AlamarBlue (AlamarBlue reagent that had been autoclaved for 30 min) was added to 3 wells. After 7 h of incubation, the color change of the AlamarBlue reagent in the individual wells was determined in a Perkin Eimer fluorescence spectrophotometer (excitation 530 nm, emission 590 nm, slits 15, integration time 0.1). Amount of converted AlamarBlue reagent represents the metabolic activity of the cell. The relative cell activity was calculated as a percentage of control (HeLa S3 cells without inhibitor), and the concentration of active substance that inhibits the cell activity by 50% (IC<50>) was determined. The values were calculated from the mean of three individual measurements with correction for the control value (medium control).

FACS analysis

Propidium iodide (PI) binds stoichiometrically to double-stranded DNA and is therefore suitable for determining the percentage of cells in G1, S and G2/M phase of the cell cycle, based on the DNA content of the cell. Cells in G0 and G1 phase have diploid DNA content (2N), while cells in G2 or mitosis have 4N DNA content.

For PI labeling, 0.4 million HeLaS3 cells were seeded, for example, in a 75 cm<2>cell culture flask, and after 241 either 1% DMSO was added as a control or the substance was added in different concentrations (in 1% DMSO). The cells were incubated for 241 with the substance or with DMSO, before the cells were washed with 2 x PBS and detached with trypsin/EDTA. The cells were centrifuged (1000 rpm, 5 min, 4°C), and the cell pellet was washed 2x with PBS, before the cells were suspended in 0.1 ml of PBS. The cells were then fixed with 80% ethanol for 16 hours at 4°C, or alternatively for 2 hours at -20°C. The fixed cells (10<6> cells) were centrifuged (1000 rpm, 5 min, 4°C), washed with PBS and then centrifuged again. The cell pellet was suspended in 2 ml of Triton X-100 in 0.25% PBS, and incubated for 5 min on ice, before 5 ml of PBS was added and the mixture was centrifuged again. The cell pellet was suspended in 350 µl PI stain solution (0.1 mg/ml Raze A, 10 µg/ml presidium iodide in 1x PBS). The cells were incubated for 20 min in the dark with staining buffer before being transferred to sample measurement containers for FACS scanning. The DNA measurement was performed in a Becton Dickinson FACS Analyzer with an argon laser (500 mW, emission 488 nm), and the DNA Cell Quest Program (BD). Logarithmic PI fluorescence was determined with a bandpass filter (BP 585/42). The cell populations in the individual phases of the cell cycle were quantified with the ModFit LT program of Becton Dickinson.

The compounds with the general formula (I) can be used alone or combined with other active substances according to the invention, possibly also together with other pharmacologically active substances. Suitable preparations include, for example, tablets, capsules, suppositories, solutions, especially solutions for injection (s.c., i.v., i.m.) and infusion, syrups, emulsions or dispersible powders. The amount of pharmaceutically active compound should in all cases be in the range from 0.1 - 90% by weight, preferably 0.5 - 50% by weight of the total preparation, i.e. in amounts sufficient to achieve the dose range stated below. The specified doses can be given several times a day if necessary.

Suitable tablets can be obtained, for example, by mixing the active substance(s) with known excipients, for example inert diluents such as calcium carbonate, calcium phosphate or lactose, disintegrants such as corn starch or alginic acid, binders such as starch or gelatin, smoothing agents such as magnesium stearate or talc and/or sustained release agents such as carboxymethyl cellulose, cellulose acetate phthalate or polyvinyl acetate. The tablets can also comprise several layers.

Accordingly, coated tablets can be prepared by coating cores prepared analogously to the tablets with substances commonly used for

tablet coating, for example collidon or shellac, gum arabic, talc, titanium dioxide or sugar. To achieve delayed release or prevent incompatibility, the core may also consist of multiple layers. In a similar way, the tablet coating can consist of several layers to achieve delayed release, possibly by using the excipients mentioned above for the tablets.

Syrups or elixirs containing the active substances or combinations thereof according to the invention may additionally contain a sweetener such as saccharin, cyclamate, glycerol or sugar, and a flavor enhancer, e.g. a flavoring such as vanillin or orange extract. They may also contain suspension adjuvants or thickeners such as sodium carboxymethyl cellulose, wetting agents such as condensation products of fatty alcohols with ethylene oxide, or preservatives such as p-hydroxybenzoates.

Solutions for injection and infusion are prepared in the usual way, e.g. by adding preservatives such as p-hydroxybenzoates, or stabilizers such as alkali metal salts of ethylenediaminetetraacetic acid, optionally by using emulsifiers and/or dispersing agents, and if water is used as a diluent, organic solvents can optionally be used as solubilizers or additional solvents, and transferred for injection bottles or ampoules or infusion bottles.

Capsules containing one or more active substances or combinations of active substances can, for example, be prepared by mixing the active substances with inert carriers such as lactose or sorbitol, and filling gelatin capsules.

Suitable suppositories can be prepared, for example, by mixing with carriers suitable for this purpose, such as neutral fatty substances or polyethylene glycol or derivatives thereof.

Suitable excipients can be, for example, water, pharmaceutically acceptable organic solvents, such as paraffins (e.g. petroleum fractions), oils of vegetable origin (e.g. peanut or sesame oil), mono- or polyfunctional alcohols (e.g. ethanol or glycerol), carriers such as e.g. natural mineral powders (e.g. kaolin, clay, talc, lime), synthetic mineral powders (e.g. highly dispersed silica and silicates), sugars (e.g. glucose, lactose and dextrose), emulsifiers (e.g. lignin, used sulphite liquor, methyl cellulose, starch and polyvinylpyrrolidone) and smoothing agents (e.g. magnesium stearate, talc, stearic acid and sodium lauryl sulphate).

The preparations are administered in the usual way, preferably orally or transdermally, particularly preferably orally. When the tablets are administered orally, they can of course contain additives, such as e.g. sodium citrate, calcium carbonate and dicalcium phosphate, together with various additives, such as starch, preferably potato starch, gelatin and the like, in addition to the above carriers. Smoothing agents such as magnesium stearate, sodium lauryl sulfate and talc can also be used to prepare tablets. In the case of aqueous suspensions, the active substances can be combined with various flavor enhancers or dyes, in addition to the above-mentioned auxiliaries.

For parenteral use, solutions of the active substances can be prepared using suitable liquid carrier materials.

The dose for intravenous use is 1 - 1000 mg per hour, preferably between 5 - 500 mg per hour.

However, it may be necessary to deviate from the specified amounts, depending on body weight or method of administration, the individual response to the medication, the type of formulation used and the time or intervals at which it is administered. In some cases it may therefore be sufficient to use less than the minimum quantity specified above, while in other cases the specified upper limit will be exceeded. When a large quantity is administered, it may be advisable to spread it into several single doses during the day.

The formulation examples that follow illustrate the present invention without limiting its scope:

Examples of pharmaceutical formulations

The finely ground active substance, lactose and some of the corn starch are mixed together. The mixture is sieved and then moistened with a solution of polyvinylpyrrolidone in water, kneaded, wet granulated and dried. The granules, the rest of the cornstarch and magnesium stearate are sieved and mixed together. The mixture is compressed to form tablets of suitable shape and size.

The finely ground active substance, some of the corn starch, lactose, microcrystalline cellulose and polyvinylpyrrolidone are mixed together, and the mixture is sieved and worked with the rest of the corn starch and water to form a granule which is dried and sieved. The sodium carboxymethyl starch and magnesium stearate are added and mixed, and the mixture is compressed to form tablets of suitable size.

C) Ampoule solution

The active substance is dissolved in water at natural pH or possibly at pH 5.5 to 6.5 and sodium chloride is added to make the solution isotonic. The solution obtained is filtered to remove pyrogens, and the filtrate is transferred under aseptic conditions to ampoules which are then sterilized and sealed by fusion. The ampoules contain 5 mg, 25 mg and 50 mg of active substance.

Claims (14)

1) Compound with the general formula (I), where R , R which may be the same or different, means hydrogen or optionally substituted Ci-Ce alkyl, or R<1> and R<2> together mean a 2- to 5-membered alkyl bridge, R<3> means hydrogen, Ci-Cn-alkyl or phenyl optionally substituted with -OMe or C3-Ci2-cycloalkyl, R<4> denotes a group selected from hydrogen, C1-C6-alkyl or C1-C8-alkyloxy L means a connecting group selected from C2-C10-alkyl, phenyl, -C2-C4-alkyl-phenyl, -phenyl-C1-C4-alkyl or C3-C12-cycloalkyl, n means 0 or 1 m means 1 or 2 R<5>means a group selected from optionally Ci-C4-alkyl or tetrahydropyranyl substituted morpholinyl, piperidinyl, piperazinyl, piperazinylcarbonyl, pyrrolidinyl, tropenyl, sulfoxomorpholinyl, sulfonylmorpholinyl, thiomorpholinyl, -NR<8>R<9> and azacycloheptyl, R<8>, R<9> mean unsubstituted nitrogen substituents on R<5>, which may be the same or different, and are either hydrogen or a group selected from Ci-C6-alkyl, -Ci-C4-alkyl-phenyl, - C1-C4-alkyl-C3-C7-cycloalkyl or C1-C4-alkylcarbonyl, where the substituents on the optionally substituted alkyl groups can be chosen from one or more fluorine atoms, optionally in the form of the tautomers, racemates, enantiomers, diastereomers and mixtures thereof and optionally the pharmacologically acceptable acid addition salts thereof. 2) Connection according to claim 1, where R<1> to R<4> are as defined above and L means a connecting group selected from optionally substituted C2-C10-alkyl, phenyl, -C2-C4-alkyl-phenyl, -phenyl-C1-C4-alkyl or C3-C12-cycloalkyl, n means 1 m means 1 or 2 R<5>means a group which is bound to L via a nitrogen atom, selected from optionally substituted with Ci-C4 alkyl or tetrahydropyranyl morpholinyl, piperidinyl, R<8->piperazinyl, pyrrolidinyl, tropenyl, sulfoxomorpholinyl, sulfonylmorpholinyl, thiomorpholinyl, - NR<8>R<9>and azacycloheptyl, R<8>, R<9> mean unsubstituted nitrogen substituents on R<5>, which may be the same or different, and are either hydrogen or a group selected from Ci-C6-alkyl, -Ci-C4-alkyl-phenyl, - C1-C4-alkyl-C3-C7-cycloalkyl or C1-C4-alkylcarbonyl, optionally in the form of the tautomers, racemates, enantiomers, diastereomers and mixtures thereof and optionally the pharmacologically acceptable acid addition salts thereof. 3) Connection according to claim 1, where R<1> to R<4> are as defined above, L means a connecting group selected from optionally substituted C2-C10-alkyl, phenyl, -C2-C4-alkyl-phenyl, -phenyl-C1-C4-alkyl or C3-C12-cycloalkyl, n means 0 or 1 m means 1 or 2 R<5>means a group which is attached to L via a carbon atom, selected from R<8->piperidinyl, R<8>R<9->piperazinyl, R<8->pyrrolidinyl, R<8->piperazinylcarbonyl, R<8->tropenyl, R<8->morpholinyl and R<8->azacycloheptyl, and R<8>, R<9> mean unsubstituted nitrogen substituents on R<5>, which may be the same or different, and are either hydrogen or a group selected from Ci-C6-alkyl, -Ci-C4-alkyl-phenyl, - C1-C4-alkyl-C3-C7-cycloalkyl or C1-C4-alkylcarbonyl, optionally in the form of the tautomers, racemates, enantiomers, diastereomers and mixtures thereof and optionally the pharmacologically acceptable acid addition salts thereof. 4) Compound according to one of claims 1 to 3, where L, m, n and R<3> to R<9> are as defined above and R<1>, R<2> which may be the same or different, means a group selected from hydrogen, Me, Et, Pr, or R1 and R<2> together form a C2-C4 alkyl bridge, optionally in the form of the tautomers, racemates, enantiomers, diastereomers and mixtures thereof and optionally the pharmacologically acceptable acid addition salts thereof. 5) Compound according to one of claims 1 to 4, where R<1>, R2, m, n and R<5> to R<8> are as defined above and R<4> means a group selected from hydrogen, OMe, OEt, O-propargyl and O-butynyl, and L means a connecting group selected from phenyl, phenylmethyl, cyclohexyl and branched Ci-Ce alkyl, optionally in the form of the tautomers, racemates, enantiomers, diastereomers and mixtures thereof and optionally the pharmacologically acceptable acid addition salts thereof. 6) The following compounds with the general formula (I) according to one of claims 1 to 5: where the abbreviations Xi, X2, X3, X4 and X5 used in the table each mean a bond to a position in the general formula shown below the table, instead of the corresponding groups R1, R2, R3, R4 and L-R5. 7) The following compound with the general formula (I) according to one of claims 1 to 6: where the abbreviations Xi, X2, X3, X4 and X5 used in the table each mean a bond to a position in the general formula shown below the table, instead of the corresponding groups R<1>, R<2>, R<3>, R<4> and L-R<5>. 8) The following compounds with the general formula (I) according to one of claims 1 to 5: where the abbreviations Xi, X2, X3, X4 and X5 used in the table each mean a bond to a position in the general formula shown below the table, instead of the corresponding groups R<1>,<R2,><R3,>R<4>and L-R <5>. 9) Compound of formula I according to one of claims 1 to 8 for use as a pharmaceutical preparation. 10) Compound of formula I according to one of claims 1 to 8 for use as a pharmaceutical preparation with an antiproliferative effect. 11) Use of a compound of formula I according to one of claims 1 to 8 for the production of a pharmaceutical preparation for the treatment and/or prevention of cancer, infections, inflammatory and autoimmune diseases. 12) Pharmaceutical preparation, containing as active substance one or more compounds with the general formula (I) according to one of claims 1 to 8 or the physiologically acceptable salts thereof, optionally combined with conventional additives and/or carriers. 13) Process for the preparation of a compound of the general formula (I), where R<1->R<5>, m, n and L have the meanings given in claims 1 to 8, characterized in that a compound with the general formula (II) where R!<->R<3> has the meanings given in claims 1 to 4 and A is a leaving group, is reacted with an optionally substituted compound of the general formula (III), where R<4> has the meanings given in claims 1 to 5 and R<10> means OH, NH-L-R5, -O-methyl, -O-ethyl, and then possibly the product with the general formula (IV) where R<1> to R<4> have the meanings given in claims 1 to 5 and R<10> means OH, -NH-L-R<5>, -O-methyl or -O-ethyl, optionally after prior hydrolysis of the ester group - COR<10>, reacted with an amine of the general formula (V) where R<5> has the meanings given in claims 1 to 5. 14) Compound of formula (II), where R1-R3 have the meanings given in claims 1 to 5 and A is a leaving group