Isoxazoles and crotonamide derivatives and their use as pharmaceuticals and diagnostic agents

专利摘要:
The compound of formula 1 is suitable for the manufacture of a medicament for the treatment of inflammation, carcinoma disease or autoimmune disease. The compounds of formula (4) can be prepared by the preparation of specific antibodies against compounds of formula (1), detection of proteins that specifically bind from cell extracts, serum, blood or synovial fluid, purification of proteins, modification of microtiter plates or chromatographic materials, in particular It is suitable for the preparation of affinity chromatography materials and for use as diagnostic agents. Formula 1 Formula 4 In the above formula, R 1 is a radical of formula (2) or a radical of formula (3). Formula 2 Formula 3
公开号:KR19980070841A
申请号:KR1019980002288
申请日:1998-01-26
公开日:1998-10-26
发明作者:뮐너슈테판；키르쉬바움베른트；쉬바브빌프리트
申请人:야코비 피셔；훽스트아크티엔게젤샤프트；
IPC主号:

专利说明:

Isoxazoles and crotonamide derivatives and their use as pharmaceuticals and diagnostic agents
The present invention relates to novel isoxazole and crotonamide derivatives, their preparation and use as medicaments, and as antigens for the production of antibodies, and the use in diagnostic and purification processes.
Isoxazoles and crotonamide derivatives having anti-inflammatory, immunosuppressive and proliferative effects have already been published (EP 0 013 376; EP 0 217 206; EP 0 527 736). Analytical determination of these compounds in animal and human serum can be used with the aid of conventional chromatographic methods. Disadvantages of this chromatographic method are high cost in terms of equipment, complicated sample preparation steps and low sample throughput.
Immunological measurement and analysis methods can be a fast and reliable alternative to chromatography methods. It is very important to prepare appropriate antibodies in practicing this alternative method.
It is an object of the invention to obtain useful compounds suitable for the preparation of antibodies which can be combined with polymers via modification of isoxazoles and crotonamide derivatives and which can be used as tracers in radioimmunoassays.
It has now been found that compounds of formula (I) having at least one functional group capable of covalently binding to the polymer by itself or via a spacer functional group in the aromatic ring of the aniline residue are suitable for achieving the object of the present invention.
The present invention therefore relates to compounds of formula (1) and / or physiologically acceptable salts thereof and / or optional stereoisomers thereof.
In the above formula,
R ¹ is a radical of formula 2 and a radical of formula 3

ego,
R ² is a) —O— (CH ₂ ) n —CH═CH ₂ ,
b) -O- (CH ₂ ) m-CH ₂ -halogen,
c) a radical of formula 5
(Wherein R ³ is halogen or —NH ₂ and R ⁴ is a hydrogen atom or an amino acid radical), or
d) -NH ₂ ,
n and m are integers of 1, 2 or 3,
Halogen is fluorine, chlorine, bromine or iodine.
Preferred compounds of formula (1) are those in which R ¹ is a radical of formula (2) or a radical of formula (3),
R ² is a) -O- (CH ₂ ) m-CH ₂ -halogen,
b) -O-CH ₂ -CH = CH ₂ or
c) -NH-C (O) -CH (R ³ ) (R ⁴ ),
m is an integer of 2,
Halogen is bromine or iodine,
R ³ is bromine, —NH ₂ — or chlorine,
R ⁴ is a compound which is a hydrogen atom.
Particularly preferred compounds of formula 1 are as follows:
2-cyano-3-hydroxybut-2-enecarboxylic acid (4-allyloxyphenyl) amide,
2-cyano-3-hydroxybut-2-enecarboxylic acid (4- (3-iodopropoxy) phenyl) amide,
2-cyano-3-hydroxybut-2-enecarboxylic acid (4- (2-aminoacetylamino) phenyl) amide,
5-methylisoxazole-4-carboxylic acid (4- (2-aminoacetylamino) phenyl) amide,
2-cyano-3-hydroxybut-2-enecarboxylic acid (4- (2-bromoacetylamino) phenyl) amide or 5-methylisoxazole-4-carboxylic acid (4- (2-bromoacetylamino ) Phenyl) amide.
The radical R ² in formula 1 may be present in the meta-, ortho- or para-position, preferably in the para-position, relative to the phenyl cyclic NH group.
The compound of formula 1 may optionally exist in optical isomers, diastereomers, racemates or mixtures thereof. The term amino acid is understood to mean the stereoisomeric forms of the following compounds, for example the D and L forms. Asparagine, valine, arginine, aspartic acid, glutamine, glutamic acid, tryptophan, β-alanine, lysine, proline, glycine,, -aminobutyrate, Nε-acetyllysine, Nδ-acetylornithine NΥ-acetyldiaminobutyrate, Nα-acetyl Diaminobutyrate, histidine, isoleucine, leucine, methionine, phenylalanine, serine, cysteine, threonine, alanine and tyrosine. L-amino acids are preferred. The amino acid residue Gly is particularly preferred.
Amino acid residues may be derived from the corresponding amino acid. A brief representation of amino acids follows the usual conventional notation. The radical (R ⁴ ) represents the side chain of each amino acid.
Suitable physiologically acceptable salts of compounds of formula 1 are, for example, alkali metal, alkaline earth metal and ammonium salts, including salts of organic ammonium bases and protonated amino acid residues.
The present invention also relates to a) reacting a compound of formula 6 with a compound of formula 7 wherein R ¹ is a radical of formula 2 and R ² is -NH ₂ , -NH-C (O) -CH ₂ -NH-protecting group,- Obtaining a compound of formula 1 which is OH or -NH-C (O) -CH ₂ -halogen;
b) by reacting the compound prepared in step a) with R ⁷ being -OH, with an alkyl halide or dihaloalkane, wherein the alkyl moiety has 2, 3 or 4 carbon atoms To obtain
c) reacting the compound prepared in step a), wherein R ⁷ is -OH, with an unsaturated alkyl halide, wherein the alkyl moiety has 3, 4 or 5 carbon atoms to give the corresponding compound of formula 1 do or,
d) reacting the compound prepared in step a), wherein R ⁷ is -NH _2, with a carboxylic acid halide such as bromoacetyl bromide, where R ² is a radical of formula 5 and R ³ is halogen and R ⁴ is a hydrogen atom To obtain a compound of formula
e) reacting an aromatic diamine such as p-phenylenediamine with a protected amino acid on an amino group to give a compound of formula 7 wherein R ⁷ is a radical of formula 5, R ³ is -NH ₂ and R ⁴ is a protected amino acid Following the reaction as in step a) to yield the corresponding compound of formula
f) removing the protecting group in the compound of formula 1 prepared in step a) or step e), or
g) steps a) to step f) in a compound of Formula 1 prepared by (wherein, R ¹ is, or to switch to a radical of a compound of general formula (I) of the formula (2) is the radical) R ¹ is formula (3),
h) separating the compound of formula 1 prepared in steps a) to g) in free form, or optionally converting the compound to its physiologically acceptable salt, if acidic or basic groups are present, A method for preparing a compound of Formula 1 and / or a physiologically acceptable salt thereof and / or optionally a stereoisomer thereof.

In the above formula,
R ⁶ is radical OH, Cl or Br,
R ⁷ is 1) -NH ₂ ,
2) —NH—C (O) —CH ₂ —NH—protecting group, wherein the protecting group is an amine protecting group, for example Boc,
3) -NH-C (O) -CH ₂ -halogen or
4) -OH.
In the preparation step according to a), isoxazole-4- in aprotic solvents such as toluene, tetrahydrofuran (THF) or chlorinated hydrocarbons by methods known in the art, for example thionyl chloride or phosphorus oxychloride, Conversion of a carboxylic acid (R ⁶ is —OH) to acid chloride followed by an organic base such as tertiary amine (eg triethyleneamine or N-) in a bipolar aprotic solvent (eg THF or chlorinated hydrocarbon) Ethyl morpholine) can be added and reacted with an optionally substituted aromatic amine using appropriate methods. On the other hand, amide formation can be performed directly from the carboxylic acid with the addition of condensing agents known in peptide chemistry such as dicyclohexylcarbodiimide. Secondary modifications are particularly suitable for aromatic amines with further amino or alcohol radicals.
In the preparation step b) or c), the hydroxy group introduced by the aniline moiety is added to the alkyl halide or dihaloalkane (e.g., 1,3-diiodopropane) with the addition of an organic or inorganic base such as potassium carbonate. ) To give the corresponding substituted non-aliphatic esters, in the latter case (adding an inorganic base), using them as appropriate excess alkylation reagents or solvents to avoid dimerization so that the alkyl halide functional groups in the product To stay longer for the union,
2) Ring opening of the isoxazole moiety (radical of formula 2) under acetone as a solvent in g of a dipolar aprotic solvent (e.g. tetrahydrofuran (THF), acetone or chlorinated hydrocarbon) as a solvent, g) Or functionalize further by reacting with unsaturated alkyl halides (eg aryl or propargyl halides, preferably allyl bromide) under similar reaction conditions for propargyl esters.
In step d) of the preparation method, the amino group is added in a bipolar aprotic solvent (eg THF or chlorinated hydrocarbon) with the addition of an organic base such as a tertiary aromatic amine (eg triethyleneamine or N-ethylmorpholine). React with a carboxylic acid such as bromoacetyl bromide.
In step e) of the preparation, aromatic diamines (e.g. p-) are controlled in a controlled manner using amino acids (e.g. N-Boc-glycine) protected on amino groups, condensing agents (e.g. dicyclohexylcarbodiimide) Phenylenediamine) is converted to monoamide, and aniline is formed as described in step a) of the preparation method and finally the amine protecting groups included together are removed under standard conditions known in peptide chemistry (preparation f). .
In the step of preparation method g), the compounds prepared under a) to f) undergo base-induced ring opening of the isoxazole moiety, which reaction is preferably a water / organic solvent mixture (eg THF / water or ethanol / water). In excess of organic or inorganic bases (eg NaOH), ammonia solution or potassium carbonate.
If the compound of formula 1 is present in the form of diastereomers or enantiomers and is obtained in a selective synthesis as a mixture thereof, the pure stereoisomers are separated by chromatography with an optional chiral support material, or If the racemate compound forms a salt, it can be separated by fractional crystallization of the diastereomeric salt formed with an optically active base or acid as an adjuvant. In principle in the same way, racemic compounds of the formula (1) comprising a basic group such as an amino group can be prepared by optically active acids such as (+)-camphor-10-sulfonic acid, D- and L-tartaric acid, D- And L-lactic acid or (+) and (-)-mandelic acid) can be converted into pure enantiomers.
The invention also provides an effective amount of at least one compound of formula 1 and / or stereoisomeric forms of the compound of formula 1 and / or physiologically acceptable salts of the compound of formula 1, further pharmaceutically suitable and physiologically acceptable excipients. It relates to a medicament comprising an additive and / or an active compound and an adjuvant. The agents according to the invention can be administered intravenously, parenterally, topically, rectally or orally.
The medicaments according to the invention are preferably suitable for the prophylaxis and / or treatment of carcinoma diseases, inflammations, autoimmune diseases.
These include, for example, rheumatic diseases, acute and chronic inflammation of muscles, joints or gastrointestinal tract, allergic airway diseases, psoriasis or autoimmune diseases such as systemic lupus erythema (SLE), type 2 diabetes, myasthenia gravis, Sjogren's syndrome , Dermatitis, scleroderma or complex sclerosis (MS). Carcinoma diseases include, for example, lung cancer, leukemia, Kaposi's sarcoma, ovarian cancer, sarcoma, meningioma, bowel cancer, lymphocyte cancer, brain tumors, breast cancer, gastric cancer, pancreatic cancer, prostate or skin cancer.
The present invention provides a method for preparing a pharmaceutical comprising preparing one or more compounds of Formula 1 in a suitable dosage form using pharmaceutically suitable and physiologically acceptable excipients, and optionally further suitable active compounds, additives or auxiliaries. It is about.
Suitable solid or liquid pharmaceutical formulation forms are, for example, tablets, powders, coated tablets, tablets, (micro) capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions and active compounds. Sustained release formulations and general preparation aids include, for example, excipients, disintegrants, binders, coatings, swelling agents, lubricants, glidants, flavors, sweeteners and solubilizers. Adjuvants that may be mentioned are, for example, animal and animal such as talc, starch, magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, lactoprotein, gelatin, cellulose and derivatives thereof, liver oil, sunflower, peanut or sesame oil. Vegetable oils, polyethylene glycols and sterile water and monohydric or polyhydric alcohols such as glycerol.
Pharmaceutical preparation is preferably prepared and administered in dosage units, each dosage unit comprising a specific dose of the compound of formula 1 according to the invention as the active ingredient. In the case of solid dosage units such as tablets, capsules, coated tablets or suppositories, the dosage is about 1000 mg or less, preferably about 50 to 300 mg, and in the case of an ampoule injection, about 300 mg or less, preferably about 10 to 100 mg. The daily dose in the treatment of an adult patient weighing about 70 kg in humans and animals—depending on the efficacy of the compound of Formula 1—is about 50-3000 mg of active compound, preferably about 150-1000 mg, for intravenous administration, and When injected within, about 50 to 1000 mg, preferably about 100 to 300 mg. However, under some circumstances higher or lower daily doses are appropriate. The daily dose may be administered once in the form of each dose unit or in several smaller dose units or in several divided doses, especially at time intervals.
The invention also relates to a compound of formula 1 optionally bonded to a polymer or solid via a bridge member.
These are compounds of formula (4).
In the above formula,
R ¹ is a radical of formula (2) or (3)
Formula 2
Formula 3
ego,
L is a) -O-,
b) -NR ⁵ , wherein R ⁵ is a hydrogen atom,
c) -O- (CH ₂ ) _n -CH _2- , where n is an integer 1, 2 or 3,
d) —NH—C (O) —CH— (R ⁴ ) (R ³ ), wherein R ³ is a covalent bond or —NH— and R ⁴ is a hydrogen atom or a radical of an amino acid), or
e) a bridge member selected from the group of bridge members L as defined in a) to d), with spacers which are radicals of the group -NH- (CH ₂ ) _m -S-, where m is an integer from 1 to 12; Is,
X is a polymer or a solid.
The radical LX in formula 4 may be located in the meta-, ortho, or para-position, preferably in the para-position, relative to the phenyl cyclic NH group. The term polymer can be understood to mean a synthetic or natural polymer selected from the group consisting of, for example, polystyrene, polypropylene, polyvinyl chloride, latex, polysaccharides, sepharose, proteins, fats, silicates, hexanes and the like. . Optionally, the polymer may be provided with one or more functional radicals from the group consisting of —OH—, —COOH—, —NH ₂ —, and —CO— to bind to the compound of formula (1). General methods for linking a compound of formula 1 to a solid are described, for example, in BI Application Handbook, Ed. 1994, Figure 4.1, Merk AB, Uppsala, Sweden. The term solid refers to an insoluble object that may be a particle or exist in geometric form (eg, tube, bead or microtiter plate).
Preferred compounds of formula 4 are those wherein the compound of formula 1 is bonded to a solid or liquid. Particularly preferred compounds of formula 4 are 5-methylisoxazole-4-carboxylic acid (4- (2-bromoacetylamino) phenyl) amide or 2-cyano-3-hydroxybut-2-enecarboxylic acid (4- Compounds of formula (1), such as (2-bromoacetylamino) phenyl) amide, are bound to a solid or polymer.
The compounds of formula 4 are also suitable for the discovery of specific-binding proteins in cell extracts, serum, blood or synovial fluid, purification of proteins, modification of microtiter plates or the preparation of chromatography materials, in particular affinity chromatography materials. Suitable proteins for purification are those which directly interact with the compound of formula 1 which is bound to a polymer or solid.
Particularly suitable solids for compounds of formula 4 are ^R BIA core CM5 chips or stationary phases for chromatographic irradiation or separation, and also microtiter plates.
Example 1
5-Methylisoxazole-4-carboxylic acid (4- (2-bromoacetylamino) phenyl) amide
Step a) 5-Methylisoxazole-4-carboxylic acid (4-aminophenyl) amide
10.1 g (0.008 mol) of 5-methylisoxazole-4-carboxylic acid and 8.65 g (0.08 mol) of p-phenylenediamine were dissolved in 300 ml of tetrahydrofuran, and 18.05 g (0.088 mol) of dicyclohexylcarbodiimide was dissolved. Add. The precipitate deposited after 5 hours is filtered off with suction, the organic phase is concentrated and the living is chromatographed on silica gel with ethyl acetate / petroleum ether with the addition of 1% glacial acetic acid and then crystallized from ethyl acetate / petroleum esters. Yield: 6.8 g, acetate salt of melting point 123 ° C to 128 ° C.
Step b) 5-methylisoxazole-4-carboxylic acid (4- (2-bromoacetylamino) phenyl) amide
2.17 g (0.01 mol) of the product of step a) were introduced into 50 ml of tetrahydrofuran together with 1.9 g (0.016 mol) of N-ethylmorpholine in an ice bath, and a solution of 2.4 g (0.012 mol) of bromoacetyl bromide was added dropwise. The mixture is then further stirred for 5 hours at room temperature. After addition of 5 ml of water, acidify to pH 2 with 1N HCl, extract the product with ethyl acetate, wash the organic phase with water, dry and concentrate. The product is crystallized from ethyl acetate / petroleum ether.
Yield: 2.0 g, melting point 179 占 폚.
¹ H-NMR: (DMSO-d ₆ ): 2.7 (s, 3H), 4.05 (s, 2H), 7.5-7.75 (m, 4H), 9.1 (s, 1H), 10.1 and 10.45 (sb, in each case) 1H).
Example 2
2-Cyano-3-hydroxybut-2-encarboxylic acid (4- (2-bromoacetylamino) phenyl) amide
0.5 g (0.0015 mol) of the product of Example 1 is dissolved in 10 ml of tetrahydrofuran and 2 ml of 1N aqueous NaOH is added with ice cooling. The reaction was monitored using TLC and after completion of the reaction (after about 60 to 90 minutes) the product was acidified with 2.25 mL of 1N aqueous hydrochloric acid, precipitated by addition of 100 mL of water, filtered off, washed with water and stirred with stirring. Wash with a small amount of ethyl acetate and dry under reduced pressure.
Yield: 0.28 g, Melting point 210 ℃ or more.
¹ H-NMR: (DMSO-d ₆ ): 2.28 (s, 3H), 4.04 (s, 2H), 7.4-7.7 (m, 4H), 8.5-10 (sb, 1H) 10.4 (sb, 2H)
Example 3
5-Methylisoxazole-4-carboxylic acid (4- (2-aminoacetylamino) phenyl) amide hydrochloride
Step a) 2-3-butoxycarbonylaminoacetylamino-p-phenylenediamine
8.65 g (0.08 mol) of p-phenylenediamine was dissolved in 300 ml of anhydrous tetrahydrofuran (THF) together with 15.3 g (0.088 mol) of N-Boc-glycine, and 18.05 g (0.088 mol) of dicyclohexylcarbodiimide Is added in fractions. After 5 hours, the deposited precipitate is suction filtered, the filtrate is concentrated and the product is purified by chromatography on silica gel using ethyl acetate / methanol / acetic acid and crystallized from ethyl acetate / petroleum ether.
Yield: 13.5 g, Melting point: 144 ° C.
Step b) 5-methylisoxazole-4-carbonyl chloride
127.1 g (1.0 mol) of 5-methylisoxazole-4-carbonyl chloride was introduced into 1 L of toluene, 129.8 g (1.1 mol) of thionyl chloride was added dropwise, and the mixture was heated at 80 ° C. for 6 hours. The volume is concentrated to 1/2 under reduced pressure and the toluene solution is used directly for the next reaction.
Step c) 5-methylisoxazole-4-carboxylic acid (4- (2-tert-butoxycarbonylaminoacetylamino) phenyl) amide
13.5 g (0.05 mol) of the product of step a) were initially introduced with 7.6 ml (0.06 mol) of N-ethylmorpholine into 100 ml of THF, and 30 ml (corresponding to 0.06 mol) of step b) were brought to 0 ° C. Drop by The mixture is stirred for an additional 5 hours at room temperature, hydrolyzed with aqueous citric acid and then the product is extracted with ethyl acetate, washed with water, dried over sodium sulphate and concentrated under reduced pressure.
Yield: 12 g, Melting point: 139 ° C.
Step d) 5-methylisoxazole-4-carboxylic acid (4- (2-amino-acetylamino) phenyl) amide hydrochloride
6 g (0.017 mol) of the product of step c) are dissolved in 180 ml of dichloromethane and 18 ml of trifluoroacetic acid is added. The mixture is concentrated by stirring for 1 hour at room temperature, and the product is dissolved in ethanol to add excess ethanolic HCl, and concentrated and stirred to convert the hydrochloride by washing the residue with ethyl acetate. Yield: 2.5 g, Melting point: 210 캜.
¹ H-NMR: (DMSO-d ₆ ): 2.7 (s, 3H), 3.7-3.9 (m, 2H), 7.6 and 7.72 (2H, AA'BB in each case), 8.25 (sb, 2H), 9.3, 10.2 and 10.75 (s, 1H in each case).
Example 4
2-Cyano-3-hydroxybut-2-encarboxylic acid (4- (2-aminoacetylamino) phenyl) amide trifluoroacetate
6 g (0.017 mol) of the product of Example 3, step c) are dissolved in 20 mL of 1N sodium hydroxide solution / 2 mL of ethanol and the mixture is stirred at room temperature until the ring is fully open (about 3 hours). After acidification with aqueous citric acid, the Boc-protected product is obtained in solid form and dried by filtration through a suction filter (melting point 189 ° C., yield 5.5 g). 5 g of this intermediate product are treated with trifluoroacetic acid in dichloromethane similarly to Example 3d). The product is crystallized from ethanol as trifluoroacetate using ethyl acetate and petroleum ether.
Yield: 5.0 g, Melting point: 209 캜.
¹ H-NMR: (DMSO-d ₆ ): 2.15 (s, 3H), 3.7-3.85 (m, 2H), 7.49 (s, 4H), 8.1 (sb, 3H), 10.3 and 11.2 (sb, in each case) 1H).
Example 5
2-Cyano-3-hydroxybut-2-encarboxylic acid (4- (3-iodopropoxy) phenyl) amide
Step a) 5-Methylisoxazole-4-carboxylic acid (4- (3-iodopropoxy) phenyl) amide
6.5 g (0.03 mol) of 5-methylisoxazole-4-carboxylic acid (4-hydroxyphenyl) amide was dissolved in 57 ml (0.5 mol) of 1,3-diiodopropane, and finely divided potassium carbonate with vigorous stirring 26.2 g (0.19 mol) are added. After about 4 hours the reaction is complete. The workup is carried out by filtration, concentration and chromatography on silica gel with ethyl acetate / petroleum ether 1: 1 with addition of 1% glacial acetic acid.
Yield: 6.7 g.
Step b) 2-cyano-3-hydroxybut-2-enecarboxylic acid (4- (3-iodopropoxy) phenyl) amide
5.5 g of the product of step a) were treated in 200 ml of a solution of about 1 N methanolic ammonia until the ring was completely open, the mixture was concentrated, the residue was immersed in dilute acetic acid and ethyl acetate / petroleum ether was added with 1% glacial acetic acid Chromatography using and then crystallize from ethyl acetate / petroleum ether.
Yield: 2.3 g, Melting point: 149 ° C.
¹ H-NMR: (DMSO-d ₆ ): 2.1-2.4 (m, 2H and s, 3H at 2.3 ppm), 3.4 and 4.02 (t, 2H in each case), 6.92 and 7.42 (2H, AA'BB in each case) '), 7.2-8.5 (ssb, 1H), 10.2 (s, 1H).
Example 6
2-Cyano-3-hydroxybut-2-enecarboxylic acid (4-allyloxyphenyl) amide
1.5 g (0.0068 mol) of 5-methylisoxazole-4-carboxylic acid (4-hydroxyphenyl) amide was dissolved in acetone, and 5.8 ml (0.068 mol) of allyl bromide and 9.3 g (0.068 finely divided potassium carbonate) were vigorously stirred. Mol)) and then the mixture is stirred overnight at room temperature. After filtration, the filtrate is concentrated and the residue is recrystallized from ethyl acetate / petroleum ether by immersion in water.
Yield: 0.8 g, Melting point: 162 to 164 ° C.
¹ H-NMR: (DMSO-d ₆ ): 2.3 (s, 3H), 4.45-4.63 (m, 2H), 5.15-5.38 (m, 2H), 5.9-6.2 (m, 1H), 6.95 and 7.42 ( In each case 2H, AA'BB '), 10.0 (sb, 1H), 12.5-14.5 (ssb, 1H).
Pharmacological test
Culture of the cells in the in vitro proliferation test is used to test the activity of the compound of formula (1).
Example 7
Proliferation test
10 L (Seromed, Biochrom, Berlin, FRG) Clicks / RPMI 1640 medium (50:50) in powder form containing L-glutamine and not NaHCO ₃ was dissolved in 9 l of secondary-distilled water, Sterile-filter into bottles.
Wash badge
900 ml of base medium is buffered in 9.5 ml of 7.5% sodium bicarbonate and 5 ml of HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) (Gibco, Eggenstein, FRG).
Badge used
₁₉ mL NaHCO ₃ solution + 900 mL base medium (7.5%; 10 mL HEPES solution and 10 mL L-glutamine solution (200 mM)). The medium used for mitogen-induced lymphocyte proliferation uses a medium containing 1% calf fetal serum inactivated by heat (56 ° C., 30 minutes).
Tumor cell medium
To maintain tumor cells and hybridoma cells, use medium containing 5% FCS is prepared.
Culture medium for cell line
900 ml of use medium containing 10% FCS, 10 ml of NEA solution (non-essential amino acid) (Gibco), 10 ml of sodium pyruvate solution (100 mM, Gibco) and 5 ml of 10 ⁻² M mercaptoethanol to maintain the cell line Mix.
Obtaining and Treating Spleen Cells for Mitogen-Induced Lymphocyte Proliferation
Mice are killed by cervical dislocation and the spleen is removed under sterile conditions. From the sterile sieve with a mesh butterfly of 80 mesh, the spleen is cut out using a rod piston of plastic syringe (10 ml) and carefully filtered into a Petri dish containing the medium of use. To remove erythrocytes from the spleen cell suspension, the mixture is incubated for about 1 minute at room temperature with occasional shaking in a thermogenic 0.17 M ammonium chloride solution. Red blood cells dissolve during this process, but the life and reactivity of lymphocytes is not affected. After centrifugation (7 min / 340 g), the lysate is discarded and the cells are washed twice and then immersed in each test medium.
Mitogen-Induced Lymphocyte Proliferation
Purified 5 × 10 ⁵ splenocytes of female NMRI mice are pipetted with various mitogens and formulations (compounds of Formula 1) into flat titer microtiter plates with 200 μl of test medium per pupil. The following are the concentrations of mitogen and preparation used.
Concanavalin A (Serva): 0.5- 0.25- 0.12 μg / ml
Lipopolysaccharide (Calbiochem): 1.0- 0.5-0.1 μg / ml
Phytohemagglutinin (Gibco): 0.5- 0.25- 0.12% stock
Folkweed mitogen (Gibco) compound 1 or 2: 50, 25, 10, 7.5, 5, 2.5, 1,
0.5, 0.1 μmol
Positive control is defined as the group that adds mitogen and no preparation. Negative controls are cells in culture medium with the addition of the preparation and no mitogen. Each concentration of mitogen is tested four times with all formulation concentrations. After 48 hours of incubation at 37 ° C./5% CO ₂ , a 25 μl / tritium-thymidine (Amersham) cavity (9.25 × 10 ³ μs) with ²⁵ μCi / cavity activity is added to the cells. In addition, it is incubated for 16 hours under the same conditions. To assess the test batch, cells are harvested through filter paper using Flow Laboratories and thymidine not included in the cells is collected in a separate wash bottle. After drying the filter paper, it was perforated and added to the flash vessel with 2 ml of the flashing agent (Rotiszint 22, Roth) and cooled at 4 ° C. for an additional 2 hours. The amount of radioactivity contained in the cells is measured using a beta-counter (Packard, Tricarb-460c).
Preparation of Tumor Cells and Cell Lines for Proliferation Testing
Tumor cells or cell lines used in this experiment are taken from stocks on exponential growth, washed twice with wash media and suspended in appropriate media.
Conduct and evaluation of proliferation test
Proliferation tests are performed on original microtiter plates. Compounds of formula (1) and interleukin are lysed at each 50 μl / cavity of appropriate medium and the cell number (5 × 10 ⁵ ) is adjusted to 100 μl / cavity to a final volume of 200 μl / cavity. In all tests, the figures are measured four times in total. Cells without preparations and growth factors are defined as negative controls, cells with growth factors and no preparations are counted as values of positive controls. The value of the negative control is subtracted from all measured values and the difference between the positive control and the negative control minus 100%.
Microtiter plates are incubated for 72 hours at 37 ° C./5% CO ₂ and the growth rate is measured corresponding to the growth of mitogen-induced lymphocytes. Cell lines are obtained from strain collection (American Type Culture Collection (ATCC)).
Concentration at which 50% inhibition occurs Compound of Formula 1 according to the embodimentCell lineA20.2J (μM)EL 4 (μM)K562 (μM) One0.80.850 215.06.020 550.025.0Do not measure
Example 8
Compound according to Example 2 ^RHow to bind to BIA Core CM5 matrix
The starting material was the Pharmacia Biosensor CM5 chip with a carboxymethyldextran surface (BIApplication Handbook, Ed. 1994, Merk AB, Uppsala, Sweden). All binding steps are performed at a flow rate of 5 μl / min. All steps are performed at 25 ° C. using brine (HBS) buffered with Hepes pH4 as the working buffer.
Step 1: 35 μl 1: 1 mixture of 0.05M NHS (N-hydroxysuccinimide) and 0.2M EDC (N-ethyl-N ′-(dimethylaminopropyl) carbodiimide) to activate the chip surface.
Step 2: 35 μl of 40 mM cystamine dihydrochloride in 35 μl of 0.1 M sodium borate buffer at pH 8.5.
Step 3: 35 μl of ethanolamine hydrochloride at pH 8.5 to saturate the empty matrix structure.
Step 4: 35 μl of 0.1 M dithiothreitol in 0.1 M sodium borate buffer at pH 8.5.
Step 5: 35 μl (100 mg / μl) of the solution containing the compound according to Example 2 in 0.1 M sodium borate buffer at pH 7.5.
Example 9
Used for analysis ^RBIAcore method
The analysis is essentially described in Current Biology, Vol. 3, No. 12, 1993, 913-915p.
System: ^R BIAcore 2000, Pharmacia Biosensor AB Uppsala, Sweden with corresponding software)
Chip: CM5 Sensor Chip, Pharmacia Biosensor AB
Sheath: according to Example 8
work
Buffer: HBS buffer, certified BIA, Pharmacia Biosensor AB
Flow rate: 10 μl / min
Injection: 5 min binding phase per 50 ml of sample to be analyzed
washing
Time: 180 seconds, three minutes separate phase
Regeneration: 2x15 seconds using 0.05% sodium dodecyl sulfate
A) Binding of Various Serum Albumin
shapeResonance unit Small Human Rat Mouse Donkey Sheep5029410611518480331
The term resonance unit is a quantitative unit that is proportional to the amount of protein bound.
B) Combination of Various Dehydrogenases
enzymeResonance unit Glyceraldehyde Phosphate DH (25 nM) Glyceraldehyde Phosphate DH (50 nM) Glyceraldehyde Phosphate DH (100 nM) Glyceraldehyde Phosphate DH (250 nM) Dihydroorotate DH (50 nM) Dihydroorotate DH (100 nM) Dihydro Orotate DH (250nM) pyruvate kinase (50nM) pyruvate kinase (100nM) pyruvate kinase (250nM)69148297603203425106485189505
DH refers to the enzyme dehydrogenase.
The relative binding strength of the investigated dehydrogenases is compared at each defined protein concentration. At a protein concentration of 1 μM, the binding of dihydroorotate DH is strongest, followed by lactate DH, glyceraldehyde phosphate DH and pyruvate.
Example 10
Affinity Chromatography
The compound according to example 1 is bound to the support matrix via its reactive bromine group. The support used is Fractogel ^R EMD-SH (Merck KGaA, Darmstadt). Covalently bind according to the standard protocol described in DE 43 10 964. The gel obtained is charged into a Superformance ^R (1 cm × 5 cm) column (Merck KGaA) and connected to a high pressure liquid chromatography unit (HPLC).
Obtaining Soluble Cell Protein
Incubate for 48 hours until strain RAW264.7 (ATCC strain) is confluent, wash three times with cold 4 ° C. PBS buffer to remove culture medium, transfer cells to PBS buffer and ca. 10 Centrifuge at 200 g for minutes. Cell processing is performed at 4 ° C. The cell pellet is resuspended in Buffer A consisting of 20 mM Tris base, 2 mM MgCl ₂ × 6H ₂ O and 1 mM Dithiothreitol (DTT). Various protease inhibitor mixtures are added to prevent proteolytic degradation by cell-endogenous proteases. The mixture is then homogenized in a glass bowl. The suspension is centrifuged at 22,000 g at 4 ° C. for about 30 minutes to yield soluble protein. The supernatant is immediately reused in affinity chromatography. An aliquot of the supernatant is retained as reference material and the remainder is injected into the affinity column.
HPLC
Affinity chromatography instruments from Kontron Instruments (Milan, ITALY) are used. The system consists of an autosampler 465, HPLC pumps 422 and 422S, high-pressure mixing valve M800, Vesta motor valve and bipolar vacuum tube array detector 440. Data recording and analysis is performed using the data system 450-MT2 / DAD series. The cell supernatant is diluted to a volume of 30 ml and injected at a flow rate of 1 ml / min. Fractions are collected as soon as chromatography is complete and stored at 4 ° C.
The following buffer was used for gradient elution of proteins:
Components of PBS Buffer:
NaCl 8.00 g / ℓ
KCl 0.20 g / ℓ
KH ₂ PO ₄ 0.20 g / ℓ
Na ₂ HPO 4 × 7H ₂ 0 2.16 g / ℓ
Composition of the buffer used in Table 2:
Buffer: Composition:
1 PBS
2 PBS + 0.5M NaCl
3 PBS + 150 mM A 77 1726 (N- (4-trifluoromethylphenyl) -2-cyano-3-hydro
Sodium salt of oxycrotonamide)
4 H ₂ O
5 6M elements
6 60% acetonitrile + 0.1% trifluoroacetic acid (TFA)
After injecting the protein solution, the gradient according to Table 2 begins with a flow rate of 3 ml / min.
MinutesgradientFraction 0-10H ₂ O (4)One 10-15Concentration gradient with PBS (1)2 15-20PBS (1) 20-35Concentration gradient with PBS + 0.5M NaCl (2)3 35-40PBS + 0.5 M NaCl (2) 40-50Concentration gradient with PBS (2) 50-55PBS (1)4 55-70Concentration gradient with PBS + preparation (3)5 70-75PBS + Products (3) 75-90H ₂ O (4)6 90-100Element7 100-110H ₂ O (4)8 110-120Acetonitrile (6)9
The numbers in parentheses relate to the buffer used.
Fractions are collected and dialyzed against H ₂ O at 4 ° C. for 48 h (exclusion limit 6000-8000 Da of the used dialysis membrane) and then lyophilized.
Protein measurement
After freezing, the protein is dissolved in 1 ml of 1% sodium dodecyl sulfate (SDS) and then diluted with 0.5% SDS by addition of water. An aliquot of this solution is taken and used to measure protein using a BCA test (Pierce). Protein was evaporated (UNIVASPO 150H, UniEquip Power Heater, Martinsried, Germany), then 10 g of sucrose, 9 ml of 0.25M Tris / 1M glycine solution, 7.8 ml of 10% SDS, 2.5 ml of bromophenol blue (0.1%), β- Dissolve in an appropriate volume of sample buffer consisting of 4 ml of mercaptoethanol and 1.7 ml of water.
SDS-PAGE
The following solutions are prepared when preparing SDS gels having a polyacrylamide gradient of 10 to 17%.
A: B:
5 ml of acrylamide (30%, 0.5%) 8.5 ml of acrylamide (30%, 0.5%)
3.75 ml of Tris (3M, pH 8.8) 3.75 ml of Tris (3M, pH 8.8)
6.12ml water glycerol 3.275ml
SDS 0.15ml SDS 0.15ml
Polymerization is initiated by adding 100 μl of 10% APS and 12.5 μl of TEMED. Collection gels are prepared from 3 ml of acrylamide (10%, 0.5%), 1.36 ml of water, 1.45 ml of 0.5 M Tris pH 6.8, 60 µl of 10% SDS, 106 µl of 10% APS and 9 µl of TEMED.
When preparing an assay gel, 100 μg of preparation gel, about 10 μg of protein per track is filled, and when preparing a preparative gel, about 100 μg protein per track.
The molecular weight is measured in comparison to the protein standard mixture.
Sequence analysis
Protein fractions showing bands of concentrated protein in the assay gel are separated according to production scale and the separated proteins are fixed by blotting with PVDF membrane (Millipore). Proteins are stained with coomassie and visualized, followed by direct Edman degradation. After electrophoresis on a Coomassie-stained gel, the N-terminally blocked protein is carefully cleaved and washed with water until neutral. Small pieces of gel are superimposed on two steel sieves, 100 μm and 32 μm, high, compacted and homogenized. Ointment-like materials are dried in UNIVAPO 150 H (UniEquip Power Heater, Martinsried, Germany) to reduce residual moisture. The obtained protein was incubated for 7 hours at 37 ° C. with the enzyme endoprotein Lysd C (Boehringer Mannheim) (enzyme to protein ratio of about 1:10) in a buffer consisting of 25 mM Tris HCl and 1 mM EDTA at pH 8.5. Cut to
The obtained peptide is eluted twice at 37 ° C. for 4 hours using 1 ml of 60% acetonitrile and 0.1% TFA, respectively. The combined eluate is evaporated in UNIVAPO and the peptide obtained is separated by reverse-phase chromatography. Chromatography was performed for 70 minutes on a LiChroCART ^R 125-2 Superspher ^R 60 column using a first gradient (1% / min) of A (0.1% TFA in H ₂ O) to B (0.1% TFA in acetonitrile). To perform.
Amino acid sequencing of the purified peptides is performed using a 477 A gas-phase sequencer (Applied Biosystems) according to the Edman degradation principle. Identification of PTH-AA is carried out at 269 nm using an amino acid analyzer of 120 A phenylthiohydantoin.
Western Blot Analysis
After transferring the protein from the SDS gel to the PVDF membrane (Millipore), incubated with 2.5% of chicken albumin for 2 hours in TBS consisting of 45 g NaCl, 5 L Tris base 30.3 g pH 7.4 to block the unencapsulated area.
The first antibody is diluted in titration ratio using blocking buffer and incubated for 1 hour. The blot is then washed three times for 5 minutes with TBS and 0.1% Tween 20 and incubated with POD-labeled second antibody for 1 hour. The washing procedure is repeated and the bound antibody is visualized with a solution of 60 mg 4-chloro-1-naphthol, 20 mL methanol, 100 mL TBS and 200 μl hydrogen peroxide (30%).
It is possible to identify proteins obtained in the NaCl (3) and A771726b (5) fractions (see Table 2) using amino acid-length peptides of 7-14. For 22 kDa protein, the measured amino acid sequence is designated as two proteins, MSP23 and KNEF-A. The basis for this is the high sequence homology of the two proteins, 93%. The results of the sequence analysis of affinity chromatography are summarized in Table 3.
FractionproteinAmino acid sequenceMolecular Weight [Da] NaCl (3)Lactate DH M Chain (LDH-A)SADTLWGIQK36489 Cyclophylline ATAENFRALSTGEK17971 A 771726 B (5)HSP90-β (HSP84)EQVANSAFVERVRK83185 HSP70 (HSP73)EIAEAYLGK70871 Pyruvate kinase M ₂ GPEIRTGLIK57755 EF1-αSTTTGHLIYK50164 ActinEITALAPSTMKRGILTLK41876 GAPDHVIPELNGK35679 Maleate DHITPFEEKVVE--FV35596 Lactate DHNLRRVHP36367 Phosphoglycerate MutasePMQFLGDEETVRK28635 MSP23 / NKEF-AAEAVMPDGQFK22176
Fraction numbers according to Table 2 are shown in parentheses in the column fractions.
Description of the identified protein
The 18 kDa protein concentrated in fraction (3) is identified as cyclophilin A. Cyclophilin A is a type of peptidylpropyl cis / trans isomerase, which is involved in the mechanism of action of many immunosuppressive agents such as, for example, cyclosporin A.
Lactate dehydrogenase, an enzyme of anaerobic glycolysis catalyzing the reaction of converting pyruvate to lactate, is further identified in fraction (3). Lactate dehydrogenase has at least 5 different isoenzyme forms in animal tissues. The predominant isoenzyme in skeletal muscle contains four A chains, and the predominant isoenzyme in heart comprises four B chains. In this case, peptides from the A chain of lactate dehydrogenase are identified.
Two proteins belonging to the heat shock proteins are identified in fraction (5). The heat shock protein HSP70, which is predominant in eukaryotes, is a member of the multigene family. Heat shock protein HSP90 is a cytoplasmic protein, which is always expressed constantly under normal conditions. It consists of two separate gene products, HSP84 and HSP86, which have 86% sequence homology with each other. It is known that HSP90 complexes with steroid hormone receptors and interferes with the transcription of certain genes.
In fraction (5), additionally three proteins of glycolysis were found, namely glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase M ₂ and phosphoglycerate mutase. These three enzymes are derived from the lower phase of glycolysis and exist as polyenzyme complexes under inherent conditions.
Badly concentrated also in fraction (5) was identified as Elongation Factor (EF-1α). EF-1α is an important translation factor in eukaryotes. This can be compared to the prokaryotic length growth factor EF-Tu and carries aminoacyl-tRNA from the cytoplasm to the corresponding receptor site of ribosomes in the course of GTP-dependent protein biosynthesis.
Actin can be further detected in fraction (5). Actin is present in large amounts in almost all parts of eukaryotes. It is a major member of muscle tissue and cell constitution. Actin polygenes encode four or more muscle actin forms and also encode two cytoplasmic actin forms (β- and murine-actin). The actin presented herein is a murine-chain in the form of cytoplasmic actin.
Malate dehydrogenase, an enzyme of the citric acid cycle and an enzyme of the maleate-aspartate shuttle, is also identified. Maleate dehydrogenase is present in the isoform of the cytoplasm and mitochondria in animal tissues. The two isozymes play an important role in the malate-aspartate shuttle between the cytoplasm and the mitochondria with aspartate aminotransferase.
The 22 kDa protein of fraction 5 is assigned to two proteins based on the sequenced peptide fragments: macrophage 23 kDa stress protein (MSP23) and natural killer known as osteoblast-specific factor 3 (OSF-3). Natural killer cell enchancing factor-A (NKEF-A). Activated macrophages produce reactive oxygen compounds such as H ₂ O ₂ and O ₂ . Since they must withstand the oxidative stress, they must have an effective defense system to protect themselves from the reactive compounds. NKEF is a cytoplasmic protein derived from human erythrocytes and increases the activity of natural killer cells. These are lymphocytes and recognize and destroy numerous tumor cells after cytokine stimulation. Natural killer cell enhancers have a molecular weight of 44 kDa and consist of two equally sized subunits with 88% mutual sequence homology.
Example 11
Comparative Affinity Chromatography of Unstimulated, LPS-stimulated and LPS-stimulated, Leflunomide-treated RAW 264.7 Cellular Extracts
Stimulation of macrophage line RAW 264.7 by lipopolysaccharide is used as an in vitro model of the inflammatory process. Lipopolysaccharide (LPS) is an essential component of the outer membrane of Gram-negative bacteria, which is recognized by immune cells of almost all organisms. In particular, macrophages are activated by LPS stimulation to synthesize numerous cytokines such as TNF-α, IL-1 and IL-6. Potential changes in the protein pattern accompanying this stimulus should be prevented by simultaneous administration of leflunomide, an immunomodulatory agent.
RAW 264.7 cells are incubated for 24 hours with 10 ng LPS per ml culture medium. In the case of LPS-stimulated, leflunomide-treated cells, they are incubated simultaneously with 60 μM of A 77 1726B for 24 hours. The cells are treated and examined for cytoplasmic extracts using affinity chromatography with a Fractogel ^R column derived according to Example 2. The formulation fractions of these three batches are then applied to the analytical gradient gels and each is compared with each other.
The increase in the band near 35 kDa, already identified by malate dehydrogenase by sequencing, is particularly clearly defined. The strength of these bands is markedly increased when compared with other protein bands that tend to weaken in culture with LPS and A 77 1726B.
The compounds of the present invention are suitable for the production of a medicament for the treatment of inflammatory reactions, carcinoma diseases or autoimmune diseases.

权利要求:
Claims (10)
[1" claim-type="Currently amended] A compound of Formula 1 and / or a physiologically acceptable salt thereof and / or any stereoisomeric form thereof.
Formula 1

In the above formula,
R ¹ is a radical of the formulas (2) and (3)
Formula 2

Formula 3

ego,
R ² is a) —O— (CH ₂ ) _n —CH═CH ₂ ,
b) -O- (CH ₂ ) _m -CH ₂ -halogen,
c) a radical of formula 5
Formula 5

(Wherein R ³ is halogen or NH ₂ and R ⁴ is a hydrogen atom or a radical of an amino acid), or
d) NH ₂ ,
n and m are integers of 1, 2 or 3,
Halogen is fluorine, chlorine, bromine or iodine.
[2" claim-type="Currently amended] The compound of claim 1, wherein R ¹ is a radical of Formula 2 or 3,
R ² is a) -O- (CH ₂ ) _m -CH ₂ -halogen,
b) -O-CH ₂ -CH = CH ₂ or
c) -NH-C (O) -CH (R ³ ) (R ⁴ ),
m is an integer of 2,
Halogen is bromine or iodine,
R ³ is bromine, —NH ₂ or chlorine,
R ⁴ is a hydrogen atom.
[3" claim-type="Currently amended] 3. The compound of formula 1 according to claim 1, wherein the radical R ² in formula 1 is present in the meta-, ortho- or para-position, preferably in the para-position, relative to the NH group of the phenyl ring.
[4" claim-type="Currently amended] The method of claim 1,
2-cyano-3-hydroxybut-2-enecarboxylic acid (4-allyloxyphenyl) amide,
2-cyano-3-hydroxybut-2-enecarboxylic acid (4- (3-iodopropoxy) -phenyl) amide,
2-cyano-3-hydroxybut-2-enecarboxylic acid (4- (2-amino-acetylamino) phenyl) amide trifluoroacetate,
5-methylisoxazole-4-carboxylic acid (4- (2-aminoacetylamino) -phenyl) amide hydrochloride,
2-cyano-3-hydroxybut-2-enecarboxylic acid (4- (2-bromo-acetylamino) phenyl) amide or
5-Methylisoxazole-4-carboxylic acid (4- (2-bromoacetylamino) -phenyl) amide.
[5" claim-type="Currently amended] a) Reacting a compound of formula 6 with a compound of formula 7 wherein R ¹ is a radical of formula 2 and R ² is -NH ₂ , -NH-C (O) -CH ₂ -NH-protecting group, -OH or -NH To obtain a compound of Formula 1 wherein -C (O) -CH ₂ -halogen;
b) by reacting the compound prepared in step a) with R ⁷ being -OH, with an alkyl halide or dihaloalkane, wherein the alkyl moiety has 2, 3 or 4 carbon atoms To obtain
c) reacting the compound prepared in step a), wherein R ⁷ is -OH, with an unsaturated alkyl halide, wherein the alkyl moiety has 3, 4 or 5 carbon atoms to give the corresponding compound of formula 1 do or,
d) reacting the compound prepared in step a), wherein R ⁷ is -NH _2, with a carboxylic acid halide such as bromoacetyl bromide, where R ² is a radical of formula 5 and R ³ is halogen and R ⁴ is a hydrogen atom To obtain a compound of formula
e) reacting an aromatic diamine such as p-phenylenediamine with a protected amino acid on an amino group to give a compound of formula 7 wherein R ⁷ is a radical of formula 5, R ³ is -NH ₂ and R ⁴ is a protected amino acid Following the reaction as in step a) to yield the corresponding compound of formula
f) removing the protecting group in the compound of formula 1 prepared in step a) or step e), or
g) steps a) to step f) in a compound of Formula 1 prepared by (wherein, R ¹ is, or to switch to a radical of a compound of general formula (I) of the formula (2) is the radical) R ¹ is formula (3),
h) separating the compound of formula 1 prepared in step a) to g) in free form, or optionally converting the compound to its physiologically acceptable salt, if acidic or basic groups are present, or
i) separation of the compound of formula (I), prepared by steps a) to h) in diastereomeric or enantiomeric form due to its chemical structure, into the pure stereoisomer by chromatography on any chiral support material Or isolating a racemic compound of formula (1) capable of forming a salt using any active base or acid such as an adjuvant by fractional crystallization of the formed diastereomeric salts, or (+)-camphor-10-sul Racemates of Formula 1 containing basic groups such as amino groups using any active acids such as phonic acid, D- and L-tartaric acid, D- and L-lactic acid or (+)-and (-)-mandelic acid A process for preparing a compound of formula 1 according to any one of claims 1 to 4 comprising separating the compound into pure enantiomers.
Formula 6

Formula 7

In the above formula,
R ⁶ is radical OH, Cl or Br,
R ⁷ is 1) -NH ₂ ,
2) —NH—C (O) —CH ₂ —NH—protecting group, wherein the protecting group is an amine protecting group, in particular Boc,
3) -NH-C (O) -CH ₂ -halogen or
4) -OH.
[6" claim-type="Currently amended] A medicament comprising the compound of formula 1 according to any one of claims 1 to 4 and a pharmaceutically acceptable excipient.
[7" claim-type="Currently amended] Use of a compound of formula 1 according to any one of claims 1 to 4 for the manufacture of a medicament for the treatment of cancer disease, inflammation or autoimmune disease.
[8" claim-type="Currently amended] Compound of formula 4.
Formula 4

In the above formula,
R ¹ is a radical of formula (2) or (3)
Formula 2

Formula 3

ego,
L is a) -O-,
b) -NR ^5- , wherein R ⁵ is a hydrogen atom,
c) -O- (CH ₂ ) _n -CH _2- , where n is an integer 1, 2 or 3,
d) —NH—C (O) —CH— (R ⁴ ) (R ³ ), wherein R ³ is a covalent bond or —NH— and R ⁴ is a hydrogen atom or a radical of an amino acid), or
e) a bridge member selected from the group of bridge members L as defined in a) to d), with spacers which are radicals of the group -NH- (CH ₂ ) _m -S-, where m is an integer from 1 to 12; Is,
X is a polymer or a solid.
[9" claim-type="Currently amended] The process of formula 4 according to claim 8 for the purpose of finding specific-binding proteins from cell extracts, blood or synovial fluid, purifying proteins, modifying microtiter plates or preparing chromatography materials, in particular affinity chromatography materials. Use of the compound.
[10" claim-type="Currently amended] Use of a compound of formula 4 according to claim 8 in diagnosis.

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引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1997-01-28|Priority to DE19702988.4

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1997-01-28|Priority to DE19702988A

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1998-01-26|Application filed by 야코비 피셔, 훽스트아크티엔게젤샤프트

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1998-10-26|Publication of KR19980070841A

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2006-10-13|Application granted

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2006-10-13|Publication of KR100572808B1

优先权:

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申请号 | 申请日 | 专利标题

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DE19702988.4|1997-01-28|

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DE19702988A|DE19702988A1|1997-01-28|1997-01-28|Isoxazole and crotonic acid amide derivatives and their use as pharmaceuticals and diagnostics|