The immunophilin-specific ligands as anti-asthma and immunodepressant agents and method of production thereof, pharmaceutically ready form, the drug and its production method

 

(57) Abstract:

Describes new immunophilin-specific ligands of General formula I,

(I)

where R1- the remainder of the methyl ester of the amino acids selected from-NH2-lysine, -Z-NH-lysine, phenylalanine, R2is hydrogen, R butyloxycarbonyl, carboxybenzoyl, the remainder of the 4-piperidinecarboxylic acid, 2-indolinecarboxylic acid4- hydrogen And aromatic group; -D = -CH = C-; X = Oh, Y = C. They possess anti-asthma and immunodepressants action and can be used as starting compounds to obtain drugs. Also describes the method of production thereof, pharmaceutically ready form, the drug and the method of its production. 6 C. and 13 C.p. f-crystals, 1 Il., 3 table.

The invention relates to new immunophilin-specific ligands of the formula I:

< / BR>
Group R1, R2, R3, R4X, Y, A, B and D have the following values:

R1- the remainder of the methyl ester of the amino acids selected from-NH2-lysine, -Z-NH-lysine, phenylalanine,

R2is hydrogen,

R3- butyloxycarbonyl, carboxybenzoyl, the remainder of the 4-piperidinecarboxylic acid, 2-indolinecarboxylic acid,

R4

Cyclosporine A (CsA) and FK 506 - natural compounds isolated from fungi, with immunodepressants properties, inhibit Ca+2dependent transmission signal in some types of cells. In T-cells of both drugs inhibit the transcription of several genes, including the gene for interleukin-2 (IL-2), which is normally activated by stimulation of T-cell receptors (TCR). FK-506 and CsA bind with high affinity to soluble receptor proteins (G. Fischer et al., Nature 337, 476-478, 1989; M. W. Harding et al., Nature 341, 755-760, 1989). FK-506-receptor is called FKBP, CsA-receptor called cyclophilin (Cyp). Both proteins catalyze the isomerization of CIS - and TRANS rotamers of amide linkages in peptides, and in the literature often describes called immunophilins.

The ligand-receptor complexes, CsA-Cyp and, accordingly, FK 506-FKBP, associate calcineurin (CN) and thus inhibit its fosfatazu activity. It is known that the intracellular target for CN is phosphorylated cytoplasmic component of the transcription factor NF-AT, which in the absence in the cell nucleus CN are not active dephosphorylated and therefore cannot form the active Tran the basis of allergic diseases, such as asthma, lies inflammatory reaction caused by T-cells and related mediators. As a means for the treatment of many allergic diseases are still used corticosteroids. CsA and FK 506 has also proven to be an effective therapeutic tool in the case of bronchial asthma and related inflammatory reactions, but only in animal experiments and clinical trials. In animal experiments it was shown blockade of various cytokines, such as IL-2, IL-4 and IL-5, causing the development of inflammatory reactions induced by allergies.

Despite many attempts, so far not managed to find new inhibitors of immunophilins, exceeding the activity of CsA, FK 506 and rapamycin or derivatives thereof, synthesized or isolated from natural sources. However, the high inhibitory properties of CsA, FK 506 and rapamycin significantly impaired because they have a variety of side effects, particularly because of the action on the kidneys and neurotoxicity. The basis of this phenomenon is nespecificnomu interaction, the immunophilin-specific ligands with specific cellular proteins. Consequently, terapeutiche selectivity problematic use of these drugs in the specific case of long-term therapy.

The aim of the invention is the search for new compounds with valuable pharmacological properties and receive them by direct synthesis.

According to the formula of the invention compounds according to formula I represent a completely new class of compounds capable of extremely high specificity to contact the immunophilins and highly effective to inhibit IL-2 proliferation. Join this class and their pharmaceutically acceptable salts have a high affinity for immunophilins, such as CypA, CypB, CypC and FKBP12.

Those compounds according to formula I, which contain asymmetric carbon atom and therefore, as a rule, the form of the racemates, using known methods, for example, in the form of salts with optically active acid, divided into optically active isomers. However, it is possible to use in the first stage of synthesis of optically active starting compound, and in this case, the quality of the final products are obtained the corresponding optically active diastereoisomers connection.

The invention also includes compounds according to the formula is Ecole several asymmetric carbon atoms, diastereoisomeric form.

Depending on the synthesis conditions and starting substances of the compounds according to formula I can be obtained in free form or in the form of salts. The obtained salt by known methods, for example, by the action of acids, alkalis, or by using ion exchangers can be converted into the free base or free acid.

Compounds of formula I in free form with inorganic or organic acids or bases can be converted into the corresponding physiologically compatible acid salt.

The biologically active compounds, in the form of free bases or in the form of salts. Compounds of formula I in free form or in salt form can be supplied with physiologically joint salts and bases. The application can be oral, parenteral, intravenous, subcutaneous, or by inhalation.

Further, the invention relates to pharmaceutical ready-made forms with the inclusion of at least one compound according to formula I or their salts with physiologically compatible inorganic or organic acids and/or bases, and in this case, pharmaceutically acceptable carriers capsule, solutions, respectively, capsules, suppositories, plasters, or forms for administration by inhalation.

The dosage of the above-mentioned pharmaceutically ready-made forms depends on the patient and on the form of application. Daily current dose is 0.01-100 mg per kg of body weight.

To prepare compounds of formula I can, for example, by the method of solid-phase synthesis by B. Merrifield, preferably, insoluble polymers, such as swelling in an organic solvent, the polystyrene in the form of spherical granules (for example, copolymer of polystyrene and 1% divinylbenzene), by standard methods of obtaining peptides by the method of solid-phase synthesis.

Compounds of General formula I are synthesized so that the first cover protective groups for functional groups (amino, amino or carboxyl group), and then appropriately modify free third functional group. In this case you can also, if it gives the best result at the first stage to enter the intermediate protective group, after which the second stage to replace the necessary functional group. Suitable protective groups and methods for their introduction are known. Examples of protective gr, 1984; G. Barany and R. B. Merrifield, "The Peptides", Ch. 1, S. 1-285, 1979, Academic Press Inc.

Stepwise synthesis are, for example, so that the first C-terminal amino acid with a protected amino group covalently linked to an insoluble matrix, otscheplaut protective group, the free amino group attached carboxyl group of the next amino acid, is also protected on the amino group. Thus the synthesized peptide attach one amino acid for another in the required sequence, otscheplaut additional functional and protective group, and after joining last amino acids investigate the effectiveness of the immobilized binding of ligand to the receptor (Cyp or FKBP). Stepwise condensation is a normal peptide synthesis from the appropriate protected amino acids. The synthesis can also be carried out on an automatic peptide synthesizer, for example, type Labortec SP 650 (Bachem, Switzerland), using a commercially available protected amino acids.

As examples of compounds according to formula I are the following:

Example 1: N-[1-Boc-piperidin-4-carbonyl]-indolin-2-(R,S) - carboxylic acid-[S-(N--Boc]-lysimetry ether]-amide

Example 2: N-[1-piperidin-4-carb is arbonyl]-indolin-2-(R,S)- carboxylic acid-[S-(N--Boc)-lysimetry ether]-amide

Example 4: N-[indolin-2-(R,S)-carbonyl]-indolin-2-(R,S)-carboxylic acid-[S-(--NH2)-lysimetry ether]-amide

Example 5: N-[1-Boc-indolin-2-(R,S)-carbonyl]-indolin-2-(R,S)- carboxylic acid-(S-(N--Z)-lysimetry ether)-amide

Example 6: 1-Boc-indolin-2-(R,S)-carboxylic acid-(S-(N--Z)-lysimetry ether)-amide

Example 7: 1-Boc-indolin-2-(R,S)-carboxylic acid-(8-phenylalaninamide ether)-amide

Example 8: Methyl ester N-[N'-(4-methoxyphenylacetyl)-piperidin-4-carbonyl] -indolin-2-(R, S)-carboxylic acid (as a preliminary stage for obtaining the amides of General formula (I)

Example 9: Methyl ester of N-(4-methoxyphenylacetyl)-indolin-2-(R,S)-carboxylic acid (as a preliminary stage for obtaining the amides of General formula (I)

Example 10: N-Boc-indoline-2-(R,S)-carboxylic acid-4-piperidyl-amide

Example 11: N-Boc-indoline-2-(R,S)-carboxylic acid-[piperazinone acid-morpholide]-amide

Example 12: N-[1-Boc-piperidin-4-carbonyl] -indolin-2-(R,S)- carboxylic acid-[piperazinone acid-morpholide]-amide

Example 13: N-[N'-(4-methoxyphenylacetyl)-piperidin-4-carbonyl]-indolin-2-(R,S)-carboxylic acid-[S-(N--Z)-lysimetry ether]-amide

In accordance with the present invention the compounds according to the formula may be the UB>, R3, R4, A, B, D, X and Y have the abovementioned meanings, are synthesized so that the indole derivatives of the formula II, where R4, A, B, D, X and Y have values that interact with alcohols III with a hydrocarbon chain C1-C12with the formation of complex indole esters IV, in which R4, A, B, D, X and Y have the above meanings; esters IV injected into reaction with compounds Y, in which R3X and Y have the above values, which resulted in the formation of compound VI in which R3, R4, A, B, D, X and Y have the above meanings; finally, the compounds VI are subjected to saponification to form compounds VII, where R3, R4A, B, D, X and Y have the above meanings, and then connect VII interact with the compounds VIII, where R1and R2have the specified values, with the formation of the desired product I.

Physiologically compatible salts of the compounds according to formula I get in a known manner by reaction with inorganic or organic acids, for example hydrochloric, Hydrobromic, phosphoric, sulfuric, acetic, tartaric, citric, fumaric, maleic, lactic, or monowai acids with inorganic or inorganic bases.

Pharmaceutical ready f the content of inorganic fillers or organic acids or bases, and in this case, pharmaceutically acceptable fillers and excipients.

Compounds according to formula I can be used both in free form and in the form of salts with physiologically compatible acids or bases, oral, parenteral, intravenous, subcutaneous injection, or by inhalation.

Any form of drugs, for example, tablets or coated tablets, capsules, solutions and/or capsules, suppositories, plasters, or for administration by inhalation.

Dosage above finished pharmaceutical forms depends on the patient and on the form of application. Daily current dose is 0.01-100 mg per 1 kg of body weight.

According to the invention compounds of formula I have the ability to contact the immunophilins and to inhibit their isomerase activity. Shed-isomerase activity determined according to the standard technique: G. Fischer, H. Bang, A. Schellenberger, Biochim.Biophys. Acta, 791, 87-97, 1984; D. H. Rich et al, J. Med.Chem. 38, 4164-4170, 1995.

In addition to the impact in each case on the peptidyl-CIS-TRANS-isomerase activity of immunophilins, these compounds are extremely specific way inhibit IL-2-the proliferation of mast cells, macrophages and activated T-cells. Coastwe immunosuppressants (R. Y. Calne et al., Br.Med.J. 282, 934-936, 1981), for the treatment of autoimmune diseases (R. H. Wiener et al., Hepatology 7, 1025, Abst. 9, 1987; L. Fry, J. Autoimmun. 5, 213-240, 1992; G. J. Feutren J. Autoimmun. 5, 183-195, 1992; EP 610743), for the treatment of allergic inflammatory reactions (P. Zabel et al, Lancet 343, 1984), asthma (C. Bachert, Atemw. - Lungenkrkh. 20, 59, 1994), insulin-dependent diabetes (Diabetes Mellitus) (C. R. Stiller, Science, 223, 1362-1367, 1984), sepsis, and also be used in combination with known ligands of immunophilins, such as CsA, FK 506 or rapamycin (M. J. Wyvratt, N. H. Sigal, Perspectives in Drug Discovery and Design, Immunosuppression, 2, 1, 1994; WO 92/21313, US 5330993).

Summary of the invention the following examples.

The list of the used abbreviations:

AcOEt - ethyl acetate

Boc-tert-butyloxycarbonyl

(Boc)2O - tert-butyloxycarbonyl-anhydride

CN - calcineurin

CsA - cyclosporine A

Cyp - cyclophilin

DMAP - N,N-dimethylaminopyridine

EA - element analysis

IT is an ester of acetic acid

FKBP - protein-receptor drug FK 506

HPLC - high performance liquid chromatography

i.OPV - in vacuum, generated by an oil pump

Lsg. solution

MeOH - methanol

PPIase - peptidyl-Proline, CIS-TRANS-isomerase

i.RV on a rotary evaporator

i.V. - vacuum

RT - room temperature
emer 1

Synthesis of N-[1-Boc-piperidin-4-carbonyl] -indolin-2-(R, S)-carboxylic acid-[S-(N--Boc]-lysimetry ether]-amide

Stage 1: (R,S)-indoline-2-carboxylic acid, methyl ester, hydrochloride

In a three-neck flask with a volume of 100 ml dissolve 5.3 g (32.5 mmol) of (R,S)-2-indolinecarboxylic acid in 70 ml of absolute methanol and mixed at room temperature from 4.25 g (35,75 mmol) of thionyl chloride. The reaction mixture is yellow heated under reflux for 5 h and after cooling, the solvent is removed in vacuo on a rotary evaporator (i.V. i. RV). After drying in high vacuum (oil pump) (i. OPV) get the crude product as a solid crystalline material, which is washed with diethyl ether and filtered.

Output: 5,4 g (78%)

Stage 2: Boc-piperidine-4-carboxylic acid

B odnogolosy flask of 250 ml dissolve 7 g (54 mmol) of piperidine-4-carboxylic acid in 50 ml of dioxane and 40.5 ml of 2 N NaOH and cooled to 0oC. and Then for 30 min is added dropwise a solution 12,99 g (59,4 mmol) (Boc)2O in 30 ml of dioxane. The reaction mixture was stirred at room temperature for 24 hours of saliva. The dioxane is removed in vacuo on a rotary evaporator and the residue T once with saturated NaCl solution and dried over MgSO4. After removal of solvents in vacuo on a rotary evaporator receive 11,93 g (96%) of white powder.

1H-NMR (DMSO-d6, 270 MHz): 1,25-1,5 (m, 11, Boc, 2-Pip); and 1.8 (m, 2 Pip) and 2.4 (m, 1, H-C4); 2.8 (t, 2, H-C3, H-C5); and 3.8 (d, 2, H-C2, H-C6); 12,25 (s, 1, COOH).

Elemental analysis: calculated for C11H19N1O4(229,1): C 57,62; H 8,29; N 6,11, found: C 57,89; H at 8.36; N 5,86

Stage 3: N-[1-Boc-piperidin-4-carbonyl]-indolin-2-(R,S)-carboxylic acid, methyl ester

4.6 g (22 mmol) of the hydrochloride of(R,S)-indoline-2-carboxylic acid and 7.4 g (32 mmol) of Boc-piperidine-4-carboxylic acid are dissolved in 50 ml of CH2Cl2and added dropwise for 30 min at room temperature to a suspension containing 9,27 g (36 mmol) of 2-chloro-1-methylpyridinium and of 8.06 ml (58 mmol) of triethylamine in 40 ml of CH2Cl2. Then the reaction mixture is heated under reflux for 8 hours the Solvent is removed in vacuo on a rotary evaporator, the residue is treated with 200 ml of IT and the organic phase is washed once with water, twice water polysystem solution of KHSO4twice aqueous 2N NaOH and once with aqueous saturated NaCl solution. The solvent is removed in vacuo on a rotary evaporator and the residue was subjected to purification by chromatography on 400 g of kieselgel motornom evaporator and drying in high vacuum (oil pump) get 4.61 in g (54%) powder light brown color.

So pl. 54-56oC

TLC: CH2Cl2/MeOH, 95:5 Rf= 0,61

1H-NMR (DMSO-d6270 MHz): 1,35-of 1.85 (m, 15, Boc, 6 Pip); 2,7-2,8 (m, 2, H-C3, H-C5); of 3.25 (m, 1, H-C3-lnd); the 3.65 (m, 1, H-C3'-Ind); and 3.8 (s, 3, COOCH3); 3,95 (m, 2, H-C6-Pip); the 5.45 (d, 1, H-C2-Ind); 7,05 (m, 1, Ar); 7,1-7,3 (m, 2, Ar); and 8.1 (d, 1, Ar).

Elemental analysis: calculated for C21H28N2O5(388,47): C 64,92; H 7,27; N 7,21 found: C 65,20; H 7,49; N 7,38.

Mass spectrometry: (ESI+): calculated 388,3 found 389,2

Stage 4: N-[1-Boc-piperidin-4-carbonyl] -indolin-2-(R, S)-carboxylic acid

B odnogolosy flask with a volume of 50 ml dissolve 3.3 g (8,51 mmol) of methyl ester of N-[1-Boc-piperidin-4-carbonyl]-indolin-2- (R,S)-carboxylic acid in 25 ml of MeOH, added 2.14 g (51 mmol) LiOHH2O and stirred at room temperature for 2.5 hours the Solution is acidified polysystem aqueous solution of KHSO4to pH 5 and extracted twice with HER. The organic phase is washed once with saturated NaCl solution, dried over MgSO4and the solvent is removed in vacuo on a rotary evaporator. After drying in high vacuum (oil pump) get to 3.09 g (97%) powder light brown color.

T square 118-119oWITH

TLC: CH2Cl2/MeOH, 95:5 Rf= 0,14

1H-NMR (DMCO-d , Ar); and 8.1 (d, 1, Ar); at 13.0 and 13.3 (s, 1, COOH).

Mass spectrometry: (ESI+): calculated 374,3; found 375,1.

Stage 5: N-[1-Boc-piperidin-4-carbonyl]-indolin-2-(R,S)-carboxylic acid-[S-(N--Boc)-lysimetry ether]-amide.

2 g (5,35 mmol) N-[1-Boc-piperidin-4-carbonyl]-indolin-2- (R,S)-carboxylic acid and 1.59 g (to 5.35 mmol) of the hydrochloride of the methyl ester of N-Boc-lysine was dissolved in 20 ml of CH2Cl2and added dropwise for 30 min at room temperature to a suspension containing 2,81 g (11 mmol) of 2-chloro-1-methylpyridinium and of 1.62 g (16 mmol) of triethylamine in 30 ml of CH2Cl2. Then the reaction mixture is heated under reflux for 8 hours the Solvent is removed in vacuo on a rotary evaporator, the residue is treated with 200 ml of IT and the organic phase is washed once with water, twice polysystem aqueous solution of KHSO4twice with an aqueous solution of 2N NaOH and once with saturated aqueous NaCl. The solvent is removed in vacuo on a rotary evaporator and the residue was subjected to purification by chromatography on 400 g of kieselgel using as eluent CH2Cl2/MeOH, 95:5. After removal of solvents in vacuo on a rotary evaporator and drying in high vacuum (oil pump) the eOH, 95:5 Rf= 0,48

Infrared spectrometry (FT-IR) (KBr): 3365 SL.(N-H): 2976 SL. (C-H); 1744 cf. (C= O); 1684 S. (CONH); 1540 SL. (C-O); 1407 cf (C-H); 1170 C. (C-O); 755 cf. (C=C).

1H-NMR (DMSO-d6, 270 MHz): 1,25-1,9 (m, 28, 18 Boc+3 CH2-Lys+4 Pip); 2,7-3,05 (m, 5, -CH2-Lys + H-C3-lnd + 2 Pip); 3,55 to 3.7 (m, 3, COOMe); 3,9-4,1 (m, 2, Pip); 4,15-4,3 (m, 1, H-C3-Ind); further 5.15 (m, 1, H-C2-Ind); to 6.8 (m, 1, Ar-Ind); 7,0 (m, 1, Ar-Ind); 7,1-7,3 (m, 2, Ar-Ind+ -NHCO); to 8.1 (d, 1, Ar-Ind); 8,7-8,9 (dd, 1, NHCO-Boc).

Mass spectrometry: (ESI+): calculated 616,4 found 617,5.

HPLC: two peaks with retention times of 24.25 and 24,63 minutes

Elemental analysis: calculated for C32H48N4O8(616,4): C 62,34; H 7,47; N is 9.09 found: C 62,08; H to 7.67; N 8,86.

Example 2

Synthesis of N-[1-piperidyl-4-carbonyl] -indolin-2-(R, S)-carboxylic acid- [S-(-NH2-lysimetry ether]-amide

In odnogolosy flask with a volume of 25 ml was dissolved 500 mg (0,812 mmol) N-[1-Boc-piperidin-4-carbonyl] -indolin-2-(R, S)-carboxylic acid-[S-(N--Boc)-lysimetry ether]-amide 2.8 ml of CH2Cl2. To the resulting solution was added 15 equivalents (0,0122 mol, with 0.93 ml) triperoxonane acid and stirred for 2 h at room temperature. To the solution add 10 ml of diethyl ether, in this fall a white precipitate, which is filtered off and washed 6 times with diethyl ether. After drying in: 164-165oWITH

TLC (reverse phase): CH3CN/H2O, 1:1, 1% TFU, Rf= 0,61.

IR spectroscopy (KBr): 3435 SL. (N-H); 3049 SL. (C-H); 1740 SL. (C=O); 1676 c. (CONH); 1420 cf. (C-H); 1205 Wed, 1135 S. (C-O).

1H-NMR (DMSO-d6, 270 MHz): 1,2-2,05 (m, 10, 3CH2, -Lys + 4Pip); 2,7-3,15 (m, 5, -CH2- Lys + H-C3-Ind + 2Pip); 3,55 to 3.7 (m, 3, COOMe); 4,1 - of 4.25 (m, 1, C3-Ind); further 5.15 (d, 1, H-C2-lnd); to 6.95 (m, 1, Ar-Ind); 7,1-7,3 (m, 2, Ar-Ind); 7,7-a 7.85 (s, 3, NH3+); and 8.1 (d, 1, Ar-Ind); 8,7-8,9 (m, 2, NH2+).

Mass spectrometry: (ESI+): calculated 418,2 found 417,3 and 209,1 to m/2.

HPLC: 2 peaks with retention times 11,54 and 12,65 minutes

Example 3

Synthesis of N-[1-Boc-indolin-2-(R, S)-carbonyl] -indolin-2-(R,S)- carboxylic acid-[S-(N--Boc)-lysimetry ether]-amide

Stage 1: Boc-(R,S)-indoline-2-carboxylic acid

B odnogolosy flask of 250 ml dissolve 5 g (30,8 mmol) of (R,S)-indoline-2-carboxylic acid in 30 ml of dioxane and 23 ml of 2N NaOH and cooled to 0oC. To the resulting solution was added dropwise over 30 min a solution of 7,39 g (33,9 mol) and (Boc)2O in 20 ml of dioxane and stirred at room temperature for 24 hours of saliva. The dioxane is removed in vacuo on a rotary evaporator, the residue is treated with saturated solution of KHSO4and extracted twice. The organic phase is about a rotary evaporator and drying in high vacuum (oil pump) get 7,76 g (96%) of a brown powder.

TLC: CH2Cl2/MeOH, 95:5 + 1% NEt3Rf= 0,91

1H-NMR (DMSO-d6, 270 MHz): 1,4-1,7 (s, 9, Boc); 3,1 (m, 1, H-C3); 3,5 (m, 1, H-C3') and 4.9 (m, 1, H-C2); 7,0 (m, 1, Ar); 7,1-7,3 (m, 2, Ar); 7,5-7,9 (m, 1, Ar); 11,5 (m, 1, COOH).

Elemental analysis: calculated for C14H17N1O4(263,2) C 63,88; H 6,46; N, 5,32, found: C 64,05; H 6,53; N 5,41.

Stage 2: Methyl ester of N-[Boc-indolin-2-(R,S)-carbonyl]-indolin-2-(R, S-carboxylic acid

5 g (is 0.023 mol) of the hydrochloride of the methyl ester of (R,S)-indoline-2-carboxylic acid and 12.11 g (46 mmol) of Boc-indoline-2(R,S)-carboxylic acid are dissolved in 40 ml of CH2Cl2and the resulting solution was added dropwise for 30 min at room temperature to a suspension containing 12,92 g (51 mmol) of 2-chloro-1-methylpyridinium and 10,23 ml (74 mmol) of triethylamine in 40 ml of CH2Cl2. Then the reaction mixture is heated under reflux for 8 hours the Solvent is removed in vacuo on a rotary evaporator and the residue is treated with 200 ml of HER, the organic phase is washed once with water, twice polysystem aqueous solution of KHSO4twice aqueous 2N NaOH and once with saturated aqueous NaCl. The solvent is removed in vacuo on a rotary evaporator and the residue was subjected to purification by chromatography on 400 g of Kizel rotary evaporator and drying in high vacuum (oil pump) receive free 5.01 g (51%) of the powder is dark brown.

So pl.: 86oC

TLC: CH2Cl2/MeOH, 95:5 Rf= 0.67 and 0.7 to

IR spectrometry: (KBr): 3448 SL. (N-N); 2976 SL. (C-N); 1751 S., 1707 c. (C = O); 1680 c.(CONH): 1485 c. (C-H): 1168 cp. (C-O): 1020 cp. (C-O); 752 c. (C=C).

Mass spectrometry: (ESI+) calculated 422,4 found 423,3.

Elemental analysis: calculated for C24H26N2O5(422,4): C 68,25; H 6,16; N 6,64 found: C 67,96; H 6,17; N 6,4.

Stage 3: N-[1-Vos-indolin-2-(R,S)-carbonyl]-indolin-2-(R,S)-carboxylic acid

In odnogolosy flask with a volume of 50 ml dissolve 2,84 g (6,77 mmol) of methyl ester of N-[1-Vos-indolin-2-(R,S)-carbonyl]-indolin-2-(R,S)-carboxylic acid in 20 ml of MeOH. To the resulting solution was added 1,71 g (41 mmol) LiOHH2O and stirred at room temperature for 2.5 h Then the solution is acidified polysystem solution of KHSO4pH 5 and extracted twice. The organic phase is washed once with saturated NaCl solution, dried over MgSO4and remove the solvent in vacuo on a rotary evaporator. After drying in high vacuum (oil pump) get a 2.71 g (98%) of the powder is dark brown.

So pl.: 118-119oC

TLC: CH2Cl2/MeOH, 95:5 Rf= 0,14

Mass spectrometry: (ESI+) calculated 408,2 found 409,]-amide

2 g (4.9 mmol) of N-[1-Boc-indolin-2-(R,S)-carbonyl]-indolin-2- (R,S)-carboxylic acid and 1.45 g (4.9 mmol) of the hydrochloride of the methyl ester of N-Boc-S-lysine was dissolved in 20 ml of CH2Cl2and added dropwise at room temperature within 30 min to a suspension containing of 2.51 g (9.8 mmol) of 2-chloro-1-methylpyridinium and 2,04 ml (14.7 mmol) of triethylamine in 30 ml of CH2Cl2. Then heated under reflux for 8 hours the Solvent is removed in vacuo on a rotary evaporator, the residue is treated with 200 ml of IT and the organic phase is washed once with water, twice polysystem aqueous solution of KHSO4twice with an aqueous solution of 2N NaOH and once with aqueous saturated NaCl solution. The solvent is removed in vacuo on a rotary evaporator and the residue was subjected to purification by chromatography on 400 g of kieselgel using as eluent CH2Cl2/MeOH, 95:5. After removal of solvent in vacuo on a rotary evaporator and drying in high vacuum (oil pump) gain of 2.21 g (69%) of brown powder.

So pl.: 78-80oC

TLC: CH2Cl2/MeOH, 95:5 Rf= 0,51

IR spectrometry: (KBr) 3504 SL. (N-H); 2975 SL. (C-H); 1749 S., c 1690. (CONH, C=O); 1490 c.(C-H); 1407 cp. (C-H); 1170 c. (C-O); 757 cf. (C-C).

, H-C3-Ind); of 3.5-3.7 (m, 3, COOMe); 4,2-4,3 (m, 1, H-C3-Ind); 4,7-4,9 (m, 1, H-C2-Ind); of 5.0-5.5 (m, 1, H-C2-Ind); 6,7-6,8 (m, 1, Ar-Ind); 6,85 of 7.3 (m, 6, Ar-Ind); 7,7-8,9 (m, 3, NHCO, Ar-Ind, -NHCO).

Mass spectrometry: (ESI+) calculated 650,2 found 651,4.

HPLC: 4 peaks with retention times 24,82 min, and 29.9 min, 30,3 min, and 31.2 minutes

Example 4

Synthesis of N-[indolin-2-(R, S)-carbonyl] -indolin-2-(R,S)-carboxylic acid-[S-(-NH2)-lysimetry ether]-amide

In odnogolosy flask with a volume of 25 ml was dissolved 500 mg (0,812 mmol) N-[1-Boc-indolin-2-(R, S)-carbonyl] -indolin-2-(R, S)-carboxylic acid-[S-(N--Boc)-lysimetry ether] -amide 2.8 ml of CH2Cl2. To the resulting solution was added 15 equivalents (0,0122 mol, with 0.93 ml) triperoxonane acid and stirred for two hours at room temperature. To the solution add 10 ml of diethyl ether, forms a white precipitate, which is filtered off and washed 6 times with diethyl ether. After drying in high vacuum (oil pump) receive 513 mg (98%) of white powder.

So pl.: 164-165oC

TLC (reverse-phase): CH3CN/H2OH, 1:1, 1% TFU, Rf= 0,61

IR-spectroscopy: (KBr) 3435 SL. (N-H); 3049 SL. (C-H); 1740 SL. (C=O); 1676 c. (CONH); 1420 cf. (C-H); 1205 Wed, 1135 S. (C-O).

1H-NMR (DMSO-d6, 270 MHz): 1,2-2,05 (m, 10, 3 CH23+); and 8.1 (d, 1, Ar-Ind); 8,7-8,9 (m, 2, NH3+).

Mass spectrometry: (ESI+) calculated 418,2 found 417,3 and 209,1 to m/2.

HPLC: 2 peaks with retention times 11,54 min and 12,65 minutes

Example 5

Synthesis of N-[1-Boc-indolin-2-(R, S)-carbonyl] -indolin-2-(R, S)-carboxylic acid-(S-(N--Z)-lysimetry ether)-amide

2.5 g (6,13 mmol) N-[1-Boc-indolin-2-(R,S)-carbonyl]-indolin-2-(R,S)-carboxylic acid and 2.03 g (6,13 mmol) of the hydrochloride of the methyl ester of N-Z-lysine was dissolved in 20 ml of CH2Cl2and add at room temperature for 30 min to a suspension containing 2.35 g (9.2 mmol) of 2-chloro-1-methylpyridinium and 2.13 ml (15 mmol) of triethylamine in 30 ml of CH2Cl2. The mixture is then refluxed for 8 hours the Solvent is removed in vacuo on a rotary evaporator, the residue is treated with 200 ml of IT and the organic phase is washed once with water, twice polysystem solution of KHSO4twice with a solution of 2N NaOH and once with saturated NaCl solution. The solvent is removed in vacuo on a rotary evaporator and the residue was subjected to purification by chromatography on 400 g of kieselgel using as eluent CH2Cl2/MeOH, 95:5. After removal of solvent in vacuo on a rotary sewage color.

So pl.: 68oWITH

TLC: CH2Cl2/MeOH, 95:5 Rf= 0,48.

IR spectrometry: (KBr) 3329 SL. (N-H); 2935 SL. (C-H); 1701 S. (C=O); 1485 S. (C-H); 1260 cf. (C-O); 1149 cf., 1020 cf. (C-O); 753 cf. (C=C).

Mass spectrometry: (ESI+) calculated 684,5 found 685,4

Elemental analysis: (calculated for C38H44N4O8(684,5): C 66,67; H to 6.43; N 8,19, found: C 64,15; H 6,5; N 7,88.

Example 6

Synthesis of 1-Boc-indolin-2-(R,S)-carboxylic acid-(S-(N--Z)- lysimetry ether)-amide

6,36 g (0,0242 mol) of 1-Boc-indolin-2-(R,S)-carboxylic acid and 8.0 g (or 24.2 mmol) of the hydrochloride of the methyl ester of N-Z-lysine are dissolved in 70 ml of CH2Cl2and added dropwise for 30 min at room temperature to a suspension containing 9,27 g (36,3 mmol) of 2-chloro-1-methylpyridinium and 8.41 ml (60,4 mmol) of triethylamine in 60 ml of CH2Cl2. Then the reaction mixture is heated under reflux for 8 hours the Solvent is removed in vacuo on a rotary evaporator, the residue is treated with 200 ml of IT and the organic phase is washed once with water, twice polysystem aqueous solution of KHSO4twice with an aqueous solution of 2N NaOH and once with saturated aqueous NaCl. The solvent is removed in vacuo on a rotary evaporator and the residue was subjected to purification by x is rastvoritelei in vacuum on a rotary evaporator and drying in high vacuum (oil pump) get 10,91 g (84%) powder light brown color.

TLC: CH2Cl2/MeOH 95:5, Rf= 0,74

1H-NMR (DMSO-d6, 270 MHz): 1,3-1,75 m, 15, 9 Boc + 6 CH2- Lys); of 2.8-3.0 (m, 3, CH2- Lys + H-C3-Ind); 3,4-3,55 (m, 1, H-C3'-Ind); the 3.65 (s, 3, SOON3), 4,2 (m, 1, H-C-Lys); and 4.8 (m, 1, H-C2-Ind); 5,0 (s, 2, CH2Z); 6,85 (m, 1, Ar-Ind); to 7.15 (t, 2, Ar-Ind); of 7.2 to 7.4 (m, 5, Ph-Z), and 7.7 (m, 1, NHCO); and 8.4 (m, 1, Ar-Ind).

Elemental analysis: calculated for C29H37N3O7(539,4): C 64,56; H 6,86; N 7,79 found: C 64,61; H 7,06; N to 7.67.

Mass spectrometry: (ESI+) calculated: 539,4 found: 540,3.

Example 7

Synthesis of 1-Boc-indolin-2-(R,S)-carboxylic acid-(3-phenylalaninamide ether)-amide

Stage 1: the Hydrochloride of the methyl ester of S-phenylalanine

B odnogolosy flask of 100 ml was added dropwise to 5.3 ml (to 72.6 mmol) of thionyl chloride for 30 min at room temperature to a suspension containing 8.0 g (48,4 mmol) of S-phenylalanine in 50 ml of MeOH. The mixture is then heated under reflux for 3 hours, the Methanol and the excess thionyl chloride is distilled off, first on the water-jet pump, and then on a rotary evaporator. The residue is dissolved in 50 ml of MeOH and mixed with 800 ml of diethyl ether. Roll a white precipitate, which is filtered off.

Get to 7.93 g (75%) of white powder.

1H-NMR (DMSO-d6, 270 MHz): 3,0-3,2 (m, 2, CH2); 3,6 illuminatingly ether)-amide

3.5 g (16.2 mmol) of the hydrochloride of the methyl ester of S-phenylalanine and 4,27 g (0,0162 mmol) of 1-Boc-indolin-2-(R,S)-carboxylic acid are dissolved in 70 ml of CH2Cl2and added dropwise for 30 min at room temperature to a suspension containing 6,21 g (a 24.3 mmol) of 2-chloro-1-methylpyridinium and 5.32 ml (40,5 mmol) of triethylamine in 60 ml of CH2Cl2. Then the reaction mixture is heated under reflux for 8 hours the Solvent is removed in vacuo on a rotary evaporator, the residue is treated with 200 ml of IT and the organic phase is washed once with water, twice polysystem aqueous solution of KHSO4twice with an aqueous solution of 2N NaOH and once with saturated aqueous NaCl. The solvent is removed in vacuo on a rotary evaporator and the residue was subjected to purification by chromatography on 400 g of kieselgel using as eluent CH2Cl2/MeOH, 95:5. After removal of solvents in vacuo on a rotary evaporator and drying in high vacuum (oil pump) get 7,71 g (62%) of powder light yellow color.

TLC: CH2Cl2/MeOH, 95:5 Rf= 0,87

1H-NMR (DMSO-d6, 270 MHz): 1.2 to 1.5 (m, 9, Boc); 2,3-a 2.45 (m, 0,5, H-C3-Ind); 2.8 to 3.5 (m, 3,5, C3-Ind + CH2); the 3.65 (d, 3, COOMe); 4,4-the 4.65 (m, 1, C2-Ind); and 4.8 (m, 1, H-C); 6,5(424,3) C 67,92; H 6,6; N 6,6? found: C 67,94; H 6,79; N 6,59.

Mass spectrometry: (ESI+): calculated 424,4 found 425,2.

Example 8

Synthesis of methyl ester of N-[N'-(4-methoxyphenylacetyl)-piperidin-4-carbonyl] -indolin-2-(R, S)-carboxylic acid (this compound can be used as the source for obtaining the amides of General formula (I)

1.2 g (3.0 mmol) of methyl ester of N-[1-Boc-piperidin-4-carbonyl]-indolin-2-(R, S)-carboxylic acid are dissolved in 30 ml of CH2Cl2at room temperature, mixed with of 1.14 g (10 mmol) TFU and stirred for 24 h, the Reaction mixture is evaporated in vacuo on a rotary evaporator, treated with 100 ml of IT and washed 3 times with saturated aqueous NaHCO3and 1 time with saturated aqueous NaCl solution. The organic phase is dried over MgSO4and the solvent is removed in vacuo on a rotary evaporator. The residue is dissolved in 30 ml of CH2Cl2mixed with 1.01 g (10 mmol) of triethylamine and 366 mg (3.0 mmol) of 4-dimethylaminopyridine, cooled to 0oC and mixed with a solution of 606 mg (3.3 mmol) of 4-methoxyphenylacetylene in 10 ml of CH2Cl2. After stirring for 24 h the reaction mixture was evaporated on a rotary evaporator, treated with 100 ml ether, acetic acid and s ' solution of NaCl. Then the solvent is distilled off in vacuo on a rotary evaporator and the residue chromatographic 80 g Flashgel (kieselgel Flash) using as eluent n-hexane/AcOEt. The appropriate fractions are collected and evaporated on a rotary evaporator, followed by drying in high vacuum (oil pump). Obtain 1.1 g of product as a white foam.

TLC: AcOEt, Rf= 0,22

1H-NMR (DMSO-d6270 MHz): 1,35-of 1.85 (m, 13, Boc, 4 Pip); 2,7-2,8 (m, 4, H-C(3), H-C(5)); of 3.25 (m, 1, H-C(3)-Ind); the 3.65 (m, 1, H-C(3')-Ind); and 3.8 (s, 3, SOON3); 3,95 (m, 1, H-C(4)-Pip); the 5.45 (d, 1, H-C(2)-Ind); 7,05 (m, 1, Ar); 7,1-7,3 (m, 2, Ar); and 8.1 (d, 1, Ar).

Elemental analysis: calculated for C25H28N2O5(436,51): C 68,70; H 6,47; N, 6.42 per found: C 69,97; H 6,98; N 5,27.

Example 9

Synthesis of methyl ester of N-(4-methoxyphenylacetyl)-indolin-2-(R,S)-carboxylic acid (this compound can be used as the source for obtaining the amides of General formula (I)

B odnogolosy flask 100 ml septum was placed 1 g of the hydrochloride of the methyl ester of (R,S)-indoline-2-carboxylic acid and 1.14 g (9,36 mmol) DMAP in 25 ml of dry CH2Cl2. Then with a syringe prikatyvajut 1.04 g (856 ál) 4-methoxyphenylacetylene at 0oC for 30 minutes the Mixture is stirred will versaut purification by chromatography on 150 g of kieselgel Flash (CH2Cl2/MeOH, 9:1). After removal of solvents in vacuo on a rotary evaporator obtain 830 mg (59%) powder light gray in color.

TLC: CH2Cl2; Rf= 0,31

1H-NMR (DMSO-d6, 270 MHz): 3,15-3,3 (m, 1, H-C(3)-Ind); of 3.5-3.7 (m, 3, CH2+ H-C(3')-Ind); 5,0 (m, 1, H-C(2)-Ind); 6,85 (m, 2, Ar-Ind); 7,0 (m, 1, Ar-Ind); 7,1-7,3 (m, 4, Phenyl); of 8.25 (d, 1, Ar-Ind).

Elemental analysis: calculated for C19H19N1O4(325,3); C 70,15; H 5,85; or 4.31 N, found: C 70,34; H 5,78; N 4,22.

Mass spectrometry: (ESI+) calculated 325,3 found to 326.1.

Example 10

Synthesis of N-Boc-indoline-2-(R,S)-carboxylic acid-4-pyridyl-amide

B odnogolosy flask of 100 ml was placed 2,63 g (10.0 mmol) of 1-Boc-(R, S)-indoline-2-carboxylic acid, 1.13 g (12,0 mmol) of 4-aminopyridine, 1.47 g (12,0 mmol) of 4-dimethylaminopyridine in 30 ml of CH2Cl2at 0oC and mixed with a solution of 2.48 in (12,0 mmol) dicyclohexylcarbodiimide in 5 ml of CH2Cl2. After 48 h, the reaction mixture was filtered through celite, the solvent is removed in vacuo on a rotary evaporator, the residue is treated with 100 ml of ether acetic acid. Washed 2 times with 10% aqueous HCl solution, 2 times with saturated aqueous NaHCO3and 1 time with saturated aqueous NaCl solution. After distillation of the solvent in vacuum HB as eluent n-hexane/AcOEt. After removal of solvents in vacuo on a rotary evaporator the residue is crystallized from AcOEt/ether and obtain 2.4 g of product.

TLC: CH2Cl2/MeOH, 95:5; Rf= 0,19

1H-NMR (CDCl3, 270 MHz): was 1.58 (s, 9H, Boc); 3,43-of 3.54 (m, 2H, H-C(3)-Ind); 5,0 (m, 1, H-C2-Ind); 7,02 (m, 1H, H-C(7)-Ind); 7,17-7,26 (m, 3H, H-C(6), H-C(5), H-C(4)-Ind); 7,45 (q, 2H, H-C(3), H-C(5)-Py); EUR 7.57 (NH); of 8.47 (q, 2H, H-C(2), H-C(6)-Py).

Elemental analysis: calculated for C19H21N3O3(339,40): C 67,84; H 6,29; N 12,49, found: C 67,75; H 6,33; N 12,53.

Example 11

Synthesis of N-Boc-indoline-2-(R, S)-carboxylic acid-[piperazinone acid-morpholide]-amide

B odnogolosy flask of 100 ml was placed 2,63 g (10.0 mmol) of 1-Boc-(R, S)-indoline-2-carboxylic acid, of 2.56 g (12,0 mmol) morpholine piperazineethanol acid, 1.47 g (12,0 mmol) of 4-dimethylaminopyridine in 30 ml of CH2Cl2at 0oC and mixed with a solution of 2.48 in (12,0 mmol) dicyclohexylcarbodiimide in 5 ml of CH2Cl2. After 48 h, the reaction mixture was filtered through celite, the solvent is removed in vacuo on a rotary evaporator, the residue is treated with 100 ml of HER. Washed 2 times with 10% aqueous HCl solution, 2 times with saturated aqueous NaHCO3and 1 time with saturated aqueous NaCl solution. After distillation of the solvent under vacuum on a rotary Ispat n-hexane/AcOEt. After removal of solvents in vacuo on a rotary

evaporator the residue is crystallized from AcOEt/ether and obtain 2.4 g of product.

TLC: CH2Cl2/MeOH, 95:5; Rf= 0,19

1H-NMR (CDCl3, 270 MHz): 1,48-1,58 (d, 9H, Boc); is 3.21 (s, 2H, H-C(2")); 3,42-3,69 (m, 16H); 5,1 (br, 2H, H-C(3)-Ind); 6.48 in (q, 1H,); 6,90 (q, 1H); 7,14 (m, 1H); by 8.22 (q, 1H).

Elemental analysis: calculated for C24H34N4O5(458,56): C 62,86; H 7,47; N 12,21 found: C 63,21; H of 7.48; N 13,61.

Example 12

Synthesis of N-[1-Boc-piperidin-4-carbonyl] -indolin-2-(R, S)-carboxylic acid-[piperazinone acid-morpholide]-amide

458,56 mg (1.0 mmol) of 1-Boc-indolin-2-(R, S)-carboxylic acid-[piperazinone acid morpholide]-amide are dissolved at room temperature in 20 ml of CH2Cl2mixed with some of 1.14 g (10 mmol) TFU and stirred for 24 h, the Reaction mixture was evaporated on a rotary evaporator, treated with 10 ml of IT and washed two times with saturated aqueous NaHCO3and once saturated aqueous NaCl. The organic phase is dried over MgSO4and the solvent is removed in vacuo on a rotary evaporator. The residue is dissolved in 10 ml of CH2Cl2mixed with 505 mg (5 mmol) of triethylamine, 320,7 mg (1.4 mmol) 4-Boc-piperidinecarboxylic acid and 357,7 mg is, then evaporated in vacuum on a rotary evaporator and treated with 100 ml of ether acetic acid. Washed twice with water, once with 10% aqueous HCl solution, twice with saturated aqueous solution of NaHCO3and once with saturated aqueous NaCl, dried over MgSO4. After distillation of the solvent under vacuum on a rotary evaporator the residue is crystallized from a mixture of ether acetic acid/isopropanol.

Elemental analysis: calculated for C25H28N2O5(557,70): C 62,46; H to 7.77; N 12,56, found: C 61,56; H a 7.62; N 11,96.

Example 13

Synthesis of N-[N'-(4-methoxyphenylacetyl)-piperidin-4-carbonyl]-indolin-2-(R, S)-carboxylic acid-[S-(N--Z)-lysimetry ether]-amide

Stage 1: N-[1-Boc-piperidin-4-carbonyl] -indolin-2-(R, S)-carboxylic acid-[S-(N--Z)-lysimetry ether]-amide

3,74 g (10 mmol) N-[1-Boc-piperidin-4-carbonyl]-indolin-2- (R,S)-carboxylic acid and of 3.31 g (10 mmol) of the hydrochloride of the methyl ester N---Z-lysine was dissolved in 20 ml of CH2Cl2and added dropwise at room temperature within 30 min to a suspension containing 5,11 g (20 mmol) of 2-chloro-1-methylpyridinium iodide and 4.04 g (40 mmol) of triethylamine in 30 ml of CH2Cl2. The reaction mixture was refluxed for 8 h, then the solvent UDA the DOI, twice polysystem aqueous solution of KHSO4twice with an aqueous solution of 2N NaOH, once with saturated aqueous NaCl. After drying over MgSO4the solvent is removed in vacuo on a rotary evaporator and the residue was subjected to purification by chromatography on 400 g of kieselgel using as eluent CH2Cl2/MeOH, 95:5. The appropriate fractions are collected, evaporated under vacuum on a rotary evaporator. After drying in high vacuum using an oil pump gain of 4.2 g of powder light brown color.

TLC: CH2Cl2/MeOH, 95:5 Rf= 0,41

Elemental analysis: calculated for C32H48N4O8(650,78): C 64,60; H 7,13; N 8,61 found: C 64,73; H 7,01; N 8,64.

Stage 2: N-[N'-(4-methoxyphenylacetyl)-piperidin-4-carbonyl]- indolin-2-(R,S)-carboxylic acid-[S-(N--Z)-lysimetry ether]-amide

3.25 g (5.0 mmol) of N-[1-Boc-piperidin-4-carbonyl]-indolin-2- (R,S)-carboxylic acid-[S-(N--Z)-lysimetry ether]-amide are dissolved at room temperature in 50 ml of CH2Cl2mixed with 2.28 g (20 mmol) TFU and stirred for 4 h, the Reaction mixture is evaporated in vacuo on a rotary evaporator and treated with 100 ml and then washed three times with saturated aqueous Storytell removed in vacuum on a rotary evaporator. The residue is dissolved in 30 ml of CH2Cl2mixed with 1.01 g (10 mmol) of triethylamine and 366 mg (3.0 mmol) of 4-dimethylaminopyridine, then the mixture is cooled to 0oC and add a solution of 1.01 g (5.5 mmol) of 4-methoxyphenylacetylene in 10 ml of CH2Cl2. After 24 h stirring, the reaction mixture is evaporated in vacuo on a rotary evaporator, treated with 100 ml of IT and washed twice with an aqueous solution of 1N HCl, twice with saturated aqueous solution of NaHCO3and once saturated aqueous NaCl. After removal of the solvent under vacuum on a rotary evaporator the residue is purified by chromatography on 80 g of kieselgel Flash using as eluent n-hexane/AcOEt. The appropriate fractions are collected, evaporated on a rotary evaporator, dried in high vacuum using an oil pump and get the product as white foam.

Elemental analysis: calculated for C39H46N4O8H2O (716,84): C 65,35; H of 6.75; N 7,82 found: C 65,47; H 6,89; N 7,81.

The authors found that the compounds synthesized according to examples 1-7 and 10-13 are highly effective modulators, binding immunophilins, these compounds being immobilized on koceski liquids.

To search for ligands of General formula I, having the greatest affinity for Cyp B and, respectively, FKBP receptors immobilized ligands incubated with cell homogenate and carry out electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate (LTOs) (EF in PAG-ordinator) (see drawing).

Immobilized on the carrier ligands have a particularly high affinity for immunophilins, the binding specificity significantly exceed CsA and, therefore, FK 506. High affinity immobilized on the carrier ligands of the formula I is demonstrated by the method of EF in PAG-ordinator (see drawing)

Explanations to the data of electrophoresis in SDS page-ordinator

(a) Homogenate of cells

b) the Eluate homogenate of cells after incubation with immobilized on the carrier ligands of General formula I

(C) Removing cyclophilin B with the matrix described in paragraph (b), with LTOs at 25oC

(d) SDS-control

e) Removing cyclophilin B with the matrix described in paragraph (b), with LTOs at 95oC

f) Protein markers of known molecular weight (Sigma: 12 kDa, 18 kDa, 25 kDa, 45 kDa, 66 kDa)

g) the Eluate homogenate of cells after incubation with immobilized CsA

h) Removing cyclophilin B CsA-matrix described in paragraph (g), with LTOs at ntrol

Compounds according to the invention of General formula I exhibit an extremely high affinity for immunophilins and inhibit peptidyl-prolyl-CIS-TRANS-isomerase (PPI-activity). For the primary screening of these compounds (1 µmol/l) determine the inhibition of cyclophilin B person using the method definition PPI activity. To determine the PPI activity using the conventional methods described in the literature: G. Fischer, H. Bang, C. Mech, Biomed. Biochim. Acta, 43, 1101-1111; G. Fischer, H. Bang, A. Schellenberger, Biochim. Biophys. Acta, 791, 87-97, 1984; D. H. Rich et al., J. Med.Chem. 38, 4164-4170, 1995.

Compounds according to the invention of General formula I pre-incubated with 10 nmol Cyp B for 15 min at 4oC. the Enzymatic reaction is performed by adding chymotrypsin, HEPES buffer and peptide substrate Suc-Ala-Ala-Pro-Phe-Nan. Then evaluate the change in absorption at 390 nm. The change of absorption is the result of two partial reactions: a) rapid hydrolysis by chymotrypsin TRANS-peptide; (b) non-enzymatic CIS-TRANS isomerization catalyzed by cyclophilin. The magnitude of PPI activity of the compounds according to the invention of General formula 1 are given in table. 1.

Known immunodepressant action CsA, apparently, can be explained by the formation of nadmo the compounds according to the invention of General formula I in the supramolecular complex CsA-CypB or CsA-Anything-calcineurin compounds of General formula I is incubated with the homogenate of T-cells in the presence of 3H-CsA (100 nmol). After gel filtration on Superose 12 in the obtained fractions measured radioactivity and compared with untreated control. The corresponding value replacement3H-CsA compounds according to the invention of General formula I in the supramolecular complex CypB-CsA or CypB-CsA-calcineurin are given in table. 2.

The method of determining the proliferation of IL-2 is based on embedding3H-thymidine in stimulated OKT-3 (anti-CD-3-human antibodies) T cells. The method consists in the following: in cell microplate seeded 100000 T cells in 150 μl of culture medium, stimulate them by adding OCT-3 (1 μg/ml) and incubated with the corresponding one compound of General formula I proposed in the invention, within 45 am followed in each cell pipette, add 10 ál of solution 3H-thymidine (with 0.5 µci). The plate is incubated in an atmosphere of 5% CO2for 6 h at 37oC. After collecting the cells measure the radioactivity using a counter. Corresponding values of inhibition CD3induced proliferatio for compounds according to the invention of General formula I are given in table. 3.

In the experiment on animals show that the compounds according to the invention of General formula I, aerovane inflammatory processes.

To determine the inhibition of cell division in the processing of compounds according to the invention of General formula I 500000 human tumor cells, cultured in the presence of compounds of General formula I according to the invention within 48 h, the cells are then stained 10 μl of a solution of the yellow salt of tetrazole (MTT) and continue incubation at 37oC for 4 h in an atmosphere of CO2. The resulting violet color is measured photometrically at 570 nm. The amount of staining after incubation was measured photometrically after adding 100 μl of SDS solution. General cytotoxicity of the compounds according to the invention of General formula I was unable to determine.

1. The immunophilin-specific ligands of General formula I,

< / BR>
where R1- the remainder of the methyl ester of the amino acids selected from-NH2-lysine, -Z-NH-lysine, phenylalanine, R2is hydrogen, R3-butyloxycarbonyl, carboxybenzoyl, the remainder of the 4-piperidinecarboxylic acid, 2-indolinecarboxylic acid4is hydrogen, A is an aromatic group; B-D=-CH-C; X = O, Y = C.

2. The immunophilin-specific ligand under item 1, characterized in that it is a N-[1-Vos-piperidyl-4-carbonyl]-indolin-2(R,S)-carboxylic acid - [S-(N-BOC]-lysine-IU the t of a N-[piperidyl-4-carbonyl] -indolin-2-(R, S)-carboxylic acid-[S-(-NH2)-lysine methyl ester]-amide.

4. The immunophilin-specific ligand under item 1, characterized in that it is a N-[-1-BOC-indolin-2-(R, S)-carbonyl] -indolin-2-(R,S)-carboxylic acid-[S-(N-BOC)-lysine-methyl ester]-amide.

5. The immunophilin-specific ligand under item 1, characterized in that it is a N-[indolin-2-(R,S)-carbonyl]-indolin-2-(R,S)-carboxylic acid -[S-(-NH2)-lysine-methyl ester]-amide.

6. The immunophilin-specific ligand under item 1, characterized in that it is a N-[1-Vos-indolin-2-(R,S)-carbonyl]-indolin-2-(R,S)-carboxylic acid - (S-(N--Z)-lysine-methyl ester)-amide.

7. The immunophilin-specific ligand under item 1, characterized in that it is a 1-Vos-indolin-2-(R,S)-carboxylic acid - (S-(N--Z)-lysine-methyl ester)-amide.

8. The immunophilin-specific ligand under item 1, characterized in that it is a 1-Vos-indolin-2-(R,S)-carboxylic acid -(S-phenylalanine methyl ester)-amide.

9. The immunophilin-specific ligand under item 1, characterized in that it is an N-Vos-indolin-2-(R,S)-carboxylic acid-4-pyridyl-amide.

10. The immunophilin-specific ligand under item 1, otlichuy the]-amide.

11. The immunophilin-specific ligand under item 1, characterized in that it is a N-[1-Vos-piperidyl-4-carbonyl]-indolin-2-(R,S)-carboxylic acid - [piperazinone acid-morpholide]-amide.

12. The immunophilin-specific ligand under item 1, characterized in that it is a N-[N'-(4-methoxyphenylacetyl)-piperidin-4-carbonyl]-indolin-2-(R,S)-carboxylic acid -[S-(N--Z)-lysine-methyl ester]-amide.

13. The immunophilin-specific ligand according to any one of paragraphs.1 - 12, characterized in that it is designed for the manufacture of finished pharmaceuticals.

14. The immunophilin-specific ligand on p. 13, characterized in that the manufactured drugs have anti-asthma and immunodepressant effect or used in combination with a therapeutically known anti-asthma and immunodepressant drugs.

15. Pharmaceutically prepared form containing immobilized on the carrier connection PP.1 - 12, designed to bind pathogenic of immunophilins in liquids, particularly in physiological fluids.

16. Drug with anti-asthma and immunodepressants action, including, p is x2">

17. Drug under item 16, characterized in that it is made in the form of tablets or coated tablets, capsules, solutions, and/or capsules, suppositories, patches, or powder preparations for inhalation.

18. The method of obtaining drugs having anti-asthma and immunodepressants the action used for the preparation of pharmaceutically prepared forms or therapeutically applicable forms, characterized in that the connection on one of the PP.1 - 12 are processed together with commonly used pharmaceutical carriers and diluents and actually other auxiliary substances.

19. The method of obtaining the immunophilin-specific ligands of General formula I according to p. 1, in which R1, R2, R3, R4X, Y, A, B, and D have the meanings specified in paragraph 1, namely, that indole derivatives of the formula II

< / BR>
in which R4, A, B, D, X and Y have the above values, etherification alcohol III with the length of the hydrocarbon chain C1-C12< / BR>
AlOH III

with the formation of ether IV, in which R4, A, B, D, X and Y have the above values

< / BR>
indole ester IV is introduced into the reaction with a substance V, in which R3X and Y have X and Y are the specified values

< / BR>
finally, the intermediate product VI omelet with the formation of compound VII in which R3, R4, A, B, D, X and Y are the specified values

< / BR>
after which the compound VII is introduced into the reaction with a substance VIII, in which R1and R2have the specified values

< / BR>
and get the desired product of formula I.

 

Same patents:

The invention relates to dipeptide compounds, which are stimulators of growth hormone and can be used for the treatment and prevention of osteoporosis

form with antitumor activity and method thereof" target="_blank">

The invention relates to crystalline trihydrochloride doxicillinform:

< / BR>
having the following parameters of x-ray diraction processes in the powdered state:

15,77; 10,91; 4,88; 4,67;

I/II0,32; 0,42; 0,38; 0,53;

4,14; 3,66; 3,31; 3,21;

I/II0,70; 1,00; 0,52; 0,52,

with antitumor activity and method thereof, which consists in the fact that the powder or syrup-like aqueous suspension of trihydrochloride doxicillin crystallized from aqueous ethanol at a concentration of water of not more than 15 vol.%, 2 S. p.

The invention relates to biotinyl compounds of General formula

< / BR>
where Q is absent or represents-NH-(CH2)5-CO-,

R1denotes X-Arg-Gly-Asp-y,

X - Tripeptide: Gly-Gly-Gly-,

y - dipeptide: -Ser-PrO

or R1denotes A-Cys(R2)-B-U,

R2denotes H, Trt;

And represents Asp or peptide fragment selected from the group consisting of:

Ala-Asp, Thr-Ala-Asp, Lys-Thr-Ala-Asp, Lys-Ala-Ala-Asp,

Arg-Thr-Ala-Asp, Ser-Ala-Asp, Gln-Ser-Ala-Asp,

Gly-Lys-Thr-Ala-Asp, Ile-Ser-Ala-Gly, Arg-Ser-Ala-Gly

Gly-Lys-Thr-Cys (Trt)-Asp

In tstststs or represents Pro or N-methylated derivative of Ala,

moreover, if R1denotes A-Cys(R2)-B-U, only one of the residues a or b may not appear,

U indicates HE or NH2,

or R1means cyclo (Arg-Gly-Asp-Z), Z in the side chain is linked to Q, or if Q is absent, with Biotin,

Z denotes a di - or tripeptides the rest,

moreover, amino acids, independently of one another, are selected from the group consisting of Ala, Val, Tight, Trp, Phe, Cys, Lys, M, these amino acids can be derivatization, and amino acid residues are linked to each other peptidome through N-amino - and = R6- R4< / BR>
where R4= HE

R6= alkylether with 7-14 C atoms,

R8= H, alkyl with 1-6 C-atoms,

moreover, if we are talking about the balance of optically active amino acids and amino acid derivatives, are included as D-, L-forms;

and their salts

-(3-n-methylpyridin s)-alanine as-(l)- rarn-Сhg-Рal iu(3)-nh2" target="_blank">

The invention relates to a new method of obtaining N-acetyl-(L)-4-cyanopyrrolidine separation of the racemate ethyl ester of N-acetyl-(D,L)-4-cyanopyrrolidine and to a new method of obtaining stereoisomer Ac-(L)-pAph-Chg-Me Pal(3)-NH2using as an intermediate compound N-acetyl-(L)-4-cyanopyrrolidine

The invention relates to products derived from histamine and, in particular, the condensation products of histamine or methylsiloxanes histamine and amino acids, the method of their preparation and use as active principle in areas such as therapy and cosmetology, as well as the factor (agent), improving the stability of compositions used in therapy, cosmetology, agriculture and food industry (region)

The invention relates to biotechnology and can be used for Introduzione nucleic acids into cells

The invention relates to new derivatives of chloropyridinyl formula I where Het is a group of formula a, b, C, d or e, R1is hydrogen, unsubstituted or substituted C1- C6alkyl, and the substituents selected from the group comprising halogen, phenyl, cyano, C1- C4alkoxy, C1- C4alkylthio,1- C4alkylsulphonyl; C2- C4alkenyl, unsubstituted or substituted C1- C4alkoxygroup; phenyl or unsubstituted or substituted 1 or 21- C4alkoxygroup, n = 1 or 2, and their acid additive salts

The invention relates to new tetrahydropyridine - or 4-hydroxypiperidine-alkylation formula I, where R1, R2, R3and R6denote hydrogen, halogen, C1-C6-alkyl, C1-C6-perfluoroalkyl, C1-C6-alkoxyl or two adjacent radicals can form precondensation benzene ring, And denotes the carbon atom, and the dotted line denotes an optional bond, or a denotes a carbon atom that is associated with a hydroxyl group (C-OH), and the dotted line indicates the absence of coupling, n = 2 to 6, Z1, Z2and Z3represent a nitrogen atom or a substituted carbon atom, or a physiologically favourable salts, which possess antipsychotic or anxiolytic activity

The invention relates to novel triazole compounds of the General formula (1), where a denotes a linear or branched C1-C18-alkylenes group which may comprise at least one group which is selected from O, S, CONH, COO,3-C6-cycloalkene or double or triple bond; In denotes the radical of formula (a), (b) or (C); R1denotes H, NH2WITH3-C6-cycloalkyl or1-C8-alkyl, which is not substituted or substituted OS1-C8-alkyl; R2denotes H, HE, C1-C8-alkyl, C3-6-cycloalkyl, CF3, CN, NR3R4, SR3or CO2R3where R3denotes N or C1-C8-alkyl, a R4denotes H, C1-C8-alkyl, or COR3where R3stands WITH1-C8-alkyl; Ar represents naphthyl, phenyl with 1-2 substituent selected from C1-C8-alkyl, CF3, CHF2, NO2, SR3, SO2R3where R3means1-C8-alkyl; and pyridyl, pyrimidyl or triazinyl, which have from 1 to 3 substituents selected from C1-C8-alkyl, C2-C6-alkenyl, C2-C6-quinil, halogen, CN, CF3, OR4where R43-C6-lalouche possibly condensed, phenylalkylamine or 5-membered aromatic heterocycle with 1 to 2 nitrogen atoms, which may be condensed with a benzene ring

The invention relates to new N-substituted azaheterocyclic carboxylic acids f-crystals (I) or their salts, in which R1and R2independently represent a hydrogen atom, halogen atom, trifluoromethyl, C1-C6-alkyl or C1-C6-alkoxy: Y is the grouporin which only the underlined atom participates in the cyclic system; X is a group-O-, -S-, -CR7R8, -CH2-CH2-, -CH=CH-CH2-, -CH2-CH=CH-, -CH2CH2CH2-, -CH=CH-, -NR9-(C= O)-, -O-CH2-, -(C= O)- or -(S=O)-, where R7, R8and R9independently represent a hydrogen atom or a C1-C6-alkyl; z = 1, 2, or 3; m = 1 or 2, n = 1 when m = 1 and n = 0 when m = 2; R4and R5each represents a hydrogen atom or, when m = 2, can both work together to develop a bond; R6is hydroxyl or C1-C8-alkoxygroup, or its pharmaceutically acceptable salt, provided that is not included compound 10-(3-(3-carbomethoxy-1-piperidyl) propyl) phenothiazines and 10-(3-(3-carborexics-1-piperidyl) propyl) phenothiazines

The invention relates to Hinayana and hinokitiol, compositions containing them, and methods of producing these compounds

The invention relates to the derivatives of diphenylbutylpiperidine formula I where n is 1 or 2, or pharmaceutically acceptable salts
The invention relates to medicine, in particular to pharmacology concerns intersolubility composition, including fluoxetine in the form intersolubility of granules, which intersolubility layer includes acetate-succinate the hypromellose
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