Cyclopeptide

 

(57) Abstract:

Described cyclopeptide formula I in free form, in salt form or complex ester

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where a denotes the residue of glycolic acid, optionally-substituted stands, ethyl, optionally substituted thiazole; denotes a residue-amino--methylamino octanoic acid; R1denotes methyl; denotes a residue of N-methyltryptophan formula II

< / BR>
where R1denotes alkoxy; R4denotes methyl; R5is methyl; the symbol represents a double bond; X represents-aminosilanes (C2-C14)carboxylic acid, and Y represents the residue of N-methyl--aminosilanes (C2-C10)carboxylic acids, which are inhibitors of the expression of adhesion molecules and inhibitors of the release of TNF and are therefore suitable for the treatment of inflammatory and other diseases caused by elevated levels of expression of adhesion molecules, and/or mediated by TNF. Also described therapeutic composition having cytotoxic activity, activity against inhibition of the expression of I CAM-1, V CAM-1 and E - selectin, release of TNF and proliferation of cells containing a therapeutically effective is to lead and to their therapeutic use as inhibitors of the expression of adhesion molecules.

The cell adhesion molecules such as ICAM-1 (intercellular adhesion molecule), VCAM-1 (molecule vascular adhesion) and E-selectin is expressed on the surface of endothelial cells, and, in the case of ICAM-1 on the surface of keratinocytes in response to mediators, which are precursors of inflammation, such as TNF (factor-alpha tumor necrosis), IFN (interferon), IL1 (interleukin 1) and LPS (lipopolysaccharides). Appropriate inteligency, for example, LFA-1, VLA-4 and SLExis expressed on the surface of cells in the bloodstream.

Transendothelial migration of leukocytes during inflammatory processes, as well as extravascular relationship of cells is regulated as a result of interactions between these adhesion molecules and their inteligently.

Therefore, inhibitors of the expression of adhesion molecules potentially suitable for the treatment of many painful conditions.

Cyclopeptide are cyclic molecules containing amino acid residues linked together by peptide bonds, and at least one hydroxy-group, substituted by a carboxylic acid residue, which is connected via its hydroxyl radical with the adjacent remainder of kislingbury the expression of ICAM-1, VCAM-1 and E-selectin. According to the present invention were created additional new cycloheptatriene from the same General class of compounds, including compounds exhibiting particularly important target properties.

The present invention relates to cycloheptatrien formula I

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where A denotes the residue of glycolic acid, optionally-substituted stands, ethyl or vinyl, optionally substituted with halogen, alkoxygroup, optionally protected hydroxy - or aminosidine group, CSNH2, COOR2, vinyl or-CCH or thiazole,

where R2represents H or (ness.)alkyl, optionally substituted by alkyl, halogen, cycloalkyl, optionally substituted thiazole, COOR2or-CCH, where R2has the above meaning;

B denotes the remainder-amino-methylamino octanoic acid;

R1denotes hydrogen or methyl;

C denotes tryptophan or N-methyltryptophan formula II

< / BR>
where R3denotes hydrogen, alkoxy, alkyl or benzyl, R4denotes hydrogen or halogen, R5denotes hydrogen or methyl and the symbol denotes a single or double bond;

X represents the residue-aminoamides>2-C10carboxylic acid, provided that A does not denote a residue unsubstituted-hydroxy-substituted butyric acid, and provided that when A represents a residue of glycolic acid-substituted ethyl, ethyl residue optionally substituted only amino-, hydroxy-group, chlorine, alkoxygroup, optionally substituted thiazole, optionally substituted vinyl, cyclopropyl, CSNH2or-CCH.

In formula I the orientation of amino acid residues from N-Terminus to the C-end is clockwise, and pettalidae the ester linkage is located between residues A and y When R1denotes methyl, residues R1-Leu and Leu indicate residues N-methyllysine and leucine, respectively.

Preferably A represents the residue of glycolic acid-substituted stands or ethyl, optionally substituted amino, hydroxy-group, chlorine, alkoxygroup, optionally substituted thiazole, optionally substituted vinyl, cyclopropyl, CSNH2or-CCH.

Preferably C denotes the residue of N-methyltryptophan formula II, where R3denotes hydrogen, C1-C4alkoxy (especially methoxy) or alkyl and R4denotes a hydrogen or halogen.

Predpochtitel - or substituted C1-C4the alkyl. Most preferably X represents the residue octane or butyric acid, -amino - or substituted C1-C4by alkyl, preferably by the stands.

Preferably Y represents a residue of N-methyl-aminosilanes C2-C4carboxylic acid, which is not necessarily - or-substituted C1-C4the alkyl. Most preferably Y represents the residue of the N-th methylalanine or N-methylvaline.

The invention includes peptides or pathology with an open circuit, the corresponding compounds of formula I; for example, molecules with an open circuit, the resulting gap ester chemical bond between residues Y and A or rupture of the amide bond between any other adjacent pair of acid residues. Preferably derived from open-chain are compounds of formulas IV or V

H-C-X-Y-A-B-R1Leu-Leu.OR7(IV)

and

HA-B-R1Leu-Leu-C-X-y OR7(V)

where R7denotes hydrogen or alkyl, for example, C1-C4(ness.)alkyl.

In accordance with one embodiments of the invention preferred are the compounds of formula Ip< / BR>
< / BR>
where Apdescribes-methylamino octanoic acid;

R1pdenotes hydrogen or methyl;

C denotes tryptophan or N-methyltryptophan, which is optionally substituted N'-C1-C4alkoxygroup;

Xpdenotes a residue aminosilanes C2-C14carboxylic acid;

Ypdenotes a residue of amino - or N-methyl - N-aminosilanes C2-C10carboxylic acids.

In accordance with another embodiment of the invention preferred are the compounds of formula I'p< / BR>
< / BR>
where Bp, R1pCpXpand Yphave the meanings mentioned above, and A'pdenotes a residue-hydroxy-substituted butyric acid-substituted group of the formula VI

< / BR>
where R2means (NISS. )alkyl group, for example, C1-C4(NISS. )alkyl group.

Most preferably R2denotes methyl or ethyl.

In accordance with another embodiment of the invention preferred are the compounds of formula I"p< / BR>
< / BR>
where Bp, R1pCpXpand Yphave the meanings mentioned above, and A"pdenotes a residue-hydroxy-substituted butyric acid, -smeshannyi group, the second ring, connecting in position 5 with thiazolidine ring.

Compounds of formulas I, IV, V, Ip, I'pand I"pin the context of the present description hereinafter designated as "compound of the invention", and this term also includes all compounds according to the invention are in the form of a salt or a complex ester or free-form.

Compounds according to the invention contain asymmetric atoms and can therefore exist in different epimeric forms. All possible epimere and their diastereoisomers mixture fall under the scope of the invention. Preferred are epimer, which have the ability to inhibit the expression of adhesion molecules. In General, for example, for pharmaceutical use in accordance with the invention, the preferred will be epimer, which have the ability to inhibit the expression of adhesion molecules in pure or almost pure form (i.e. free or almost free from epimeres that do not have the ability to inhibit the expression of adhesion molecules), for example, containing at least 90%, e.g. at least 95%, of active epimer (i.e., containing less than 10%, e.g. less than 5%, inactive epimer). Most of prezza, which is typical of most preferred compounds of formula VII, below.

Particularly preferred compounds according to the invention are compounds of formulas VIII, IX and X

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The compounds of formula VIII were isolated from cultures of a strain of fungi F/94-499709, examples of which in accordance with the Budapest agreement was deposited in the German collection of microorganisms and cell cultures (DSM) September 18, 1995, and received a registration number DSM 10275.

Characteristics of the strain of fungi F/94-499709 below in example 1 of the present description. The compound of formula VIII is deserving of special attention by the connection according to the invention.

Samples of strain F/94-499709 can also be obtained at the company Sandox Ltd., CH-4002, Basel, Switzerland.

It should be noted that access to samples DSM 10275 limited in accordance with the terms of rule 28(4) and (5) of the European patent Convention.

The invention includes a strain F/94-499709 (DSM 10275) in a highlighted form and its mutants and derivatives, as well as new cyclopeptide, which are produced by this strain.

The compounds of formula VIII and related compounds can be obtained by cultivation of the strain F/94-texts of her connections, for example, according to example 2.

Characteristics of the compounds of formula VIII shown in example 3.

Compounds according to the invention can be obtained by derivation of the compounds of formula XI or XII (as presented below in the present description) or VIII of the way, including

a) to obtain compounds of the formula I, where A is substituted by a group COOR2the interaction of compounds of formula I, where A is substituted by CN, with nucleophiles, preferably with alcohol, in the presence of a suitable basic or acidic catalyst, preferably hydrochloric acid, in an organic solvent, preferably in a simple ether, or

b) for compounds of formula I, where A is replaced by alkoxylation, the interaction of the compounds of formula I, where A is substituted by a group CH2-OH, alkylating compounds, such as alkylhalogenide or diazocompounds, in the presence of a catalyst or without it, or

to obtain compounds of the formula I, where A is substituted by a group COOR2the formation of esters of the corresponding compounds of formula I, where A is substituted by a COOH group, by standard methods, preferably by conversion into the acid chloride with, for example, thionyl chloride, and processing corresponding IPN is n CH2OH, the recovery of the corresponding compounds of formula I, where A is substituted by a group COOR2, metal hydrides or borohydrides, preferably a complex of borane-dimethyl sulphide, organic solvents, or

d) for compounds of formula I, where A is substituted by an optionally substituted vinyl, the interaction of the compounds of formula I, where A is substituted by a group CHO, with a Wittig reagent, or

e) for compounds of formula I, where A is A substituted CH2NH2the recovery of the corresponding compounds of formula I, where A is substituted by a group CH2N3or

g) to obtain the compounds of formula I, where A is A substituted group, the dehydrogenation of the corresponding compounds of formula I, where A is substituted by the group CH= CBr2or

C) for compounds of formula I, where A is replaced by cyclopropyl, the interaction of the corresponding compounds of formula I, where A is A substituted vinyl, with diazomethane, or

and) to obtain compounds of the formula I, where A is A substituted group CSNH2the interaction of the corresponding compounds of formula I, where A is A substituted group CN, sulfur derivatives, preferably with diphenylphosphinite acid, for example, by boiling under reflux solution serosoderjaschei with the rd formula I"p< / BR>
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where the substituents have the meanings mentioned above, the interaction of the compounds of formula I"pwhere A"pdenotes a residue-hydroxy-substituted butyric acid, is substituted by a group-CS-NH2with-haloalkaliphilic compound of formula XIII

Hal-CH2-CO-R6, (XIII)

where R6matter mentioned above, a Hal denotes halogen, or acetal compounds of the formula XIII

(the reaction can be carried out in accordance with known methods, for example, the interaction of a solution of the compounds of formula II in a solvent, inert under the reaction conditions, for example, dimethylformamide or pyridine, at an elevated temperature, preferably at 60-100oC; the final product may be isolated and purified by using common methods), or

l) to obtain preferred compounds of formula I'p< / BR>
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where the substituents have the meanings mentioned above, the interaction of the compounds of formula I'pwhere A'pdenotes a residue-hydroxy-substituted butyric acid, is substituted by the group-CHO, alkoxycarbonylmethyl and isolation of compounds of formula I'por

m) to obtain the compounds of formula I, where R3denotes hydrogen, UDA is ineni formula I, in which the symbol refers to a simple link, the recovery of compounds of the formula I, in which the symbol represents a double bond, or

o) to obtain the compounds of formula I, where R3denotes alkyl or benzyl, the introduction of these groups in the compounds of formula I, where R3denotes hydrogen, or

p) to obtain the compounds of formula I, where R4denotes halogen, halogenoalkane compounds of formula I, where R4denotes hydrogen, or

p) to obtain the compounds of formula I, where R3denotes alkoxy and the symbol represents a double bond, the interaction of the compounds of formula I,

where R3denotes hydrogen and the symbol indicates a simple relationship with alkaline tungstate and hydrogen peroxide and alkylation of N-hydroxyindoles intermediate product and, if necessary, the allocation of the compounds of formula I.

In a preferred above embodiments, the implementation of a) and the compounds of formula I are those compounds of formula I in which A represents the residue-hydroxybutiric acid-substituted by a group COOR2CN, alkoxymethyl, CH2-OH, COOH, optionally optionally substituted vinyl, CHO, CH2NH2CH2N3, CCH, CH=CBr2, ticleni as follows:

(I) to obtain an intermediate product in which A substituted-CHO, oxidation of the corresponding compounds of formula I in which A is substituted by-CH2OH, and

(II) to obtain an intermediate product in which A is substituted by-COOH by hydrolysis of the corresponding compounds of the formula I, in which A substituted COO-alkyl, with inorganic acids, for example HCl in aqueous alcohol solution or with Foundation.

Intermediates for producing compounds of formula I in which A is substituted by-CN, include natural compounds. For example, the compounds of formulas XI and XII

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can be obtained as isolates from cultures of a strain of the fungus F92-4471/08 deposited according to the Budapest Treaty in the culture Collection of the Department of agriculture USA (NRRL) on 2 July 1993 and received a registration number NRRL 21123. Characteristics of the strain of the fungus F92-4471/08 and isolation of compounds XI and XII are described in detail in international application WO 96/03430.

Compounds according to the invention can also be obtained by chemical synthesis, for example, using conventional methods of peptide synthesis. Usually the final stage in the formation of compounds is phase cyclization in which the linear peptide is atom, cyclist using reactions leading to the formation of amide or ester bonds.

Thus, the invention relates to a method for producing a cyclic peptoid formula I, comprising the cyclization of the linear peptide or peptoid containing residues of acids A, B, R1Leu, Leu, C, X, or Y, are related to each other accordingly.

Compounds according to the invention possess pharmacological activity and, therefore, suitable for use as pharmaceuticals. In particular, the compounds according to the invention are inhibitors stimulated the expression of cell adhesion molecules, especially effective inhibitors of the expression of VCAM-1 compared with E-selectin and ICAM-1. In particular, the compounds according to the invention are also inhibitors of the release of TNF, for example, inhibitors of the release of TNF.

Studies, which can be used to detect the inhibition of the expression of ICAM-1, VCAM-1 and E-selectin and to identify inhibiting the release of TNF compounds according to the invention is described below in the examples.

Thus, from the point of view of their activity as inhibitors of expression of cell adhesion molecules of the compound suitable for alasannya condition include a number of acquired and congenital diseases/disorders, when the activation of leukocytes and their transport plays a major role in the pathogenic process, including the majority of known acute and chronic inflammation (e.g., allergies, asthma, dermatitis, psoriasis, damage during reperfusion and septic shock), autoimmune conditions (such as diabetes, multiple sclerosis and rheumatoid arthritis) and mediated by the immune system neurodegeneration (for example, acquired disorders associated with immunodeficiency). Other indications for the compounds according to the invention include the metastasis of tumor (eg, melanoma, osteocarcinoma), atherosclerosis and rejection of the allograft/xenograft, since it is known that inhibition of molecules vascular adhesion can significantly improve the prognosis for these processes.

Compounds according to the invention also have therapeutic potential against hyperproliferative skin diseases (e.g. psoriasis), as well as various malignant diseases, taking into account their inhibitory activity in submicromolar concentrations, which identified during testing within 72 h in experiments to study the proliferation of keratinocytes and other cells.

Thus, taking into account their activity as inhibitors of the release of TNF, the compounds according to the invention are suitable for the prevention and treatment of diseases or pathological conditions mediated by TNF, especially TNF , for example, inflammatory conditions, autoimmune diseases, severe infections and rejections of transplant organ or tissue, including the rejection of the allograft, and xenograft, for example, for the treatment of recipients with heart transplant, lung, combination heart-lung, liver, kidney, pancreatic, skin or corneal, and to prevent graft versus host such how occur after bone marrow transplantation.

Compounds according to the invention is particularly suitable for the treatment, prevention or reduction of the intensity of the disease and of inflammatory conditions, in particular inflammatory conditions, etiology including an autoimmune component such as arthritis (nalewanyj. Specific autoimmune diseases for which can be used compounds of the invention include autoimmune haematological disorders (including e.g. hemolytic anaemia, aplastic anaemia, pure anemia red cells and idiopathic thrombocytopenia), lupus erythematosus, polyhedric, sclerodoma, Wegener's granulomatosis, dermatomyositis, chronic active hepatitis, severe pseudoparalysis the gravis, psoriasis, and syndrome of Stevens-Johnson, idiopathic sprue, autoimmune inflammatory disease of the digestive tract (including e.g. ulcerative colitis and Crohn's disease), endocrine opthalmopathy, graves disease, sarcoidosis, multiple sclerosis, primary biliary cirrhosis, juvenile diabetes (diabetes mellitus type I), uveitis (anterior and posterior choroid eyeball), dry keratoconjunctivitis and vernal keratoconjunctivitis, interstitial lung fibrosis, psoriatic arthritis and glomerulonephritis (with and without nephrotic syndrome or nephrotic syndrome, e.g. including idiopathic nephrotic syndrome or minimal change nephropathy).

Compounds according to the invention is also suitable for the treatment, prevention or Ulu the diseases of the respiratory tract.

Compounds according to the invention is suitable for treatment of undesirable acute and overactive inflammatory responses that are mediated by TNF, especially TNF , such as acute infections, including septic shock (such as endotoxic shock and respiratory distress syndrome of adults), meningitis, pneumonia; and severe burns; and for the treatment of cachexia or wasting syndrome associated with pathological release of TNF, as a result of infection, cancer or organ dysfunction, especially AIDS-related cachexia, for example, associated with HIV infection or caused by it.

Thus, the invention also relates to therapeutic compositions containing the compounds according to the invention and their therapeutic application.

In particular, the invention relates to a method of treatment or prevention of diseases in which the expression of adhesion molecules, including introduction to the patient a therapeutically effective or efficient from the point of view of prevention of a number of compounds according to the invention.

The invention also provides therapeutic compositions containing a therapeutically effective amount of the compounds according to the invention.

In particular, the invention also relates to several additional objects, which are the following.

A. Method of inhibiting production of soluble TNF, primarily FBL , or reducing inflammation in a patient (i.e., in a mammal, especially humans) in need of such treatment, comprising the introduction of this patient an effective amount of the compounds according to the invention, or a method of treatment of any of the above conditions, in particular, a method of treating inflammatory or autoimmune disease or condition, for example, multiple sclerosis or rheumatoid arthritis, or alleviate one or more symptoms of any of the above conditions.

B. the Connection according to the invention for use as a pharmaceutical, e.g. for use for the prevention or treatment of diseases or pathological conditions mediated by TNF, for example, as immunodepressant or anti-inflammatory agent, or for use to prevent, reduce or treat any ASS="ptx2">

Century Pharmaceutical composition comprising the compound according to the invention together with a pharmaceutically acceptable diluent or carrier, for example, intended for use for the prevention or treatment of diseases or pathological conditions mediated by TNF, for example, as immunodepressant or anti-inflammatory agent, or for use to prevent, reduce or treat any disease or condition as described above, for example, autoimmune or inflammatory disease or condition.

, The Use of compounds according to the invention in the manufacture of a medicinal product intended for use for the prevention or treatment of diseases or pathological conditions mediated by TNF, for example, as immunodepressant or anti-inflammatory agent, or for use to prevent, reduce or treat any disease or condition as described above, for example, autoimmune or inflammatory disease or condition.

The composition can be intended for parenteral, oral administration, for use in aerosol form for inhalation representatives or excipients and may contain additives such as stabilizers, etc.

The applied dose of the compounds may vary depending on the condition or disease to be treated, depending on whether they apply to treatment or prevention, including, depending on the mechanism and route of administration. In General, although satisfactory results are obtained with the introduction of oral doses of from about 0.05 to about 10 mg/kg/day, it is preferable to use from about 0.1 to about 7.5 mg/kg/day, more preferably from about 0.1 to about 2 mg/kg/day, using single or divided doses (2-4 times a day). An alternative to this for parenteral administration, for example, using an intravenous drip or infusion, may be applied dose from about 0.01 to about 5 mg/kg/day, preferably from about 0.05 to about 1 mg/kg/day and more preferably from approximately 0.1 to approximately 1.0 mg/kg/day.

Thus, an acceptable daily dose for sick people range from approximately 2.5 to approximately 500 mg orally, preferably from about 5 to about 250 mg orally, more than before 250 mg intravenously, preferably from about 2.5 to about 125 mg intravenously and more preferably from about 2.5 to about 50 mg intravenously.

Connections can be entered using any method adopted, including interline, parenterally or topically or by using an inhaler. Acceptable forms for enteral introduction are solutions for drinking, tablets or capsules. Acceptable forms for parenteral administration are injectable solutions or suspensions. Acceptable forms for local application include creams, lotions, etc., in the concentration range of compounds from 0.01 to 10%, preferably from 0.1 to 1 wt.% in terms of the weight of such compositions. Suitable standard dosage forms for oral administration may contain from 1 to 50 mg of the compound, usually from 1 to 10 mg.

Below the invention is explained in more detail based on examples with reference to the accompanying drawings on which is shown:

in Fig. 1 - UV spectrum of the compound of formula VIII,

in Fig. 2 is the IR spectrum of the compound of formula VIII,

in Fig. 3 - FD-mass spectrum of the compound of formula VIII,

in Fig. 4 - FD-mass spectrum (Appendix LiI) the compounds of formula VIII and

in Fig. the 51H-NMR spectrum of the compound Faure what I characteristics of strain F/94-499709 use the following Wednesday, where the concentration of the components of the environment are listed in % in relation to the mass-to-volume (wt. /about. in deionized water, and heat sterilization is carried out by exposure for 20 min at 121oC:

MEA: 2% malt extract, and 0.4% yeast extract, 2% agar.

At the point of inoculation on Wednesday MEA in Petri dishes and incubating in the dark for strain F/94-499709 received the following characteristics:

Optimal growth temperature is 24-30oC.

After 14 days of incubation the colonies reached a diameter 25-32 mm at 24oC, 30-37 mm 27oC and 7-15 mm at 33oC. At temperatures above 37oC and below 13oC did not reveal any growth of strain F/94-499709.

Colonies growing in the dark at 27oC usually have color from cream to pale buff skin, retain their shape from relatively flat to slightly expanded with the development in the centre of a small and limited aerial mycelium color from whitish to light gray. Radial grooves can become noticeable, and when the bottom darker concentric zones can be dominant. In Mature cultures of aerial and substrate mycelium in the Central parts of the colonies may become dark gray, scopacasa study has not found any spirulina structures, and, therefore, strain F/94-499709 can be called mycelium sterilum (strain with sterile mycelium).

Example 2: Fermentation of strain F/94-499709

To obtain the compounds of formula VIII by fermentation of strain F/94-499709 suitable for use are the following environment and processes. Unless otherwise noted, all concentrations of the components of the environment are listed in % in relation to the mass-to-volume (wt./about.) in deionized water, and heat sterilization is carried out by exposure for 20 min at 121oC:

PCM: (precultural and intermediate cultural environment): 2% malt extract, and 0.4% yeast extract, 0.1% agar.

PRM (producing environment): 2% solution of starch, 0.5% yeast extract, 2% glucose, 2% extract-soaked maize, 0.5% peptone, and 0.2% calcium carbonate.

Preculture obtained by thawing 2 ml of inoculated suspensions of strain F/94-499709, frozen with liquid nitrogen, depositing seed into the Erlenmeyer flask 500 ml, containing 200 ml of medium PCM, and incubation at 24oC for 7 days on a rotary shaker at 200 rpm

To obtain primary intermediate culture in each of the fourteen Erlenmeyer flasks 500 ml, containing the s is obtained by making as inoculum 1.4 l primary intermediate crops in each of the two 50-liter fermentors, containing PCM. The fermentation is carried out for 6 days under the following conditions: 24oC, 1 l of air/min/1 l medium, a blade stirrer rotating at a speed of 150 rpm, and a pressure of 0.5 bar. To obtain the compounds of formula VIII and related compounds 13 l secondary intermediate culture make in the quality of seeds in each of three 500-liter fermentors containing the PRM. The fermentation is carried out in the following conditions: 21oC, 1 l of air/min/1 l medium, blade mixer, a first rotating with a speed of 100 rpm with a gradual increase speed to 150 rpm, and a pressure of 0.5 bar. After 96 h, collected 1500 l of fermentation and unite for the selection of the target compounds of formula VIII and related compounds.

Example 3: Selection peptoid formula VIII from strain F/94-499709

The liquid medium of 1500 l of fermentation together with 1700 l of ethyl acetate homogenized reactor type Dispax and intensively stirred for 3 hours the Organic phase is separated using the separator of the type Westfalia. This stage extraction is repeated and the organic phase together evaporated under reduced pressure, getting 2745 g of extract. Extract degrease by a three-stage extraction with 40 l of methanol/Vya 960 g of skim milk extract. This extract chromatographic on a column containing 15 kg of Sephadex LH20 in a solution of methanol, receiving a fraction of the total mass of 135 g containing peptoid formula VIII. 300 g of silica gel impregnorium the whole fraction by weight of 135 g and the impregnated silica gel was then added to the upper part of the column containing 1.5 kg of silica gel firm Merk with a grain size of 0.04-0,063 mm and chromatographic using 1 l of a mixture of methyl tert-butyl ether/cyclohexane in the ratio 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 3 l MTBE and 3 l of a mixture of MTBE/methanol (95: 5). Fraction 1 liter gather and analyze using GHUR (liquid chromatography high resolution) and TLC (thin layer chromatography). Fractions 8 and 9 are combined and evaporated to dryness. After crystallization from a simple ester gain of 21.9 g of pure peptoid formula VIII. Further purification of the mother liquor and fractions 10-13 using chromatography on silica gel H Merck (750 g), which is carried out similarly to the above method, allows you to receive an additional portion of the crystalline cyclopeptide formula VIII.

It is established that during cleaning of the simple ester peptoid has a melting temperature (tPL) 143-146oC and optical rotation []2D0=-233,9o(C = 0,9 VCAM-1 using the cellular enzyme-linked immunosorbent assay (ELISA).

Molecular formula: C51H83N7O9(938,3)

UV-spectrum in methanol: = 290 (5,4), 279 (5,08), 220 (38,1), 197 (55,7) (see Fig. 1).

IR (KBr) spectrum shown in Fig. 2.

MS-BT spectrum shown in Fig. 3.

MS-BT-range (LiI) is shown in Fig. 4.

1H-NMR spectrum in CDCl3shown in Fig. 5.

MTBE denotes methyl tert-butyl ether.

VCAM refers to a molecule vascular adhesion.

Example 4: Synthesis of N1'-dismatching derivative compounds of formula VIII

The solution containing 4.9 g of the compound of formula VIII is dissolved in 3 ml of methanol and add 8 g of palladium on coal (10%). The mixture is stirred in hydrogen atmosphere for 2 h, washed by a stream of argon, filtered and evaporated, getting mentioned in the title compound as a colourless foam. The compound analyzed by thin-layer chromatography and NMR spectroscopy, obtaining the following results:

TLC: silica gel, toluene/methanol 9/1, Rf=0.28 in;

1H-NMR (3 conformer 56:37:7, indicated by the symbols*,o, ', shows characteristic signals): 8,72*(d, J=10 Hz, NH); 8,08 (s, br, indole NH); 6,97*, 6,90o(2d, J=2 Hz, indole H-2); 6,34o(d, J=9.5 Hz, NH); 6,00*(d, J=6.5 G is-H); of 3.42 (q, J=7 Hz, MeAla-H); 3,43o, 3,19o, 3,12*, 2,93*, 2,53*, 2,35o(6s, NMe); 1,54*, 1,50o(2d, J=7 Hz, MeAla-H); 1,38o, 1,24*(2d, J=7 Hz, lactic acid,- N); 0,57*that -0,01*(2d, J=6.5 Hz, Me); -0,19*(ddd, Leu-H).

Example 5: Synthesis of N1'-methyl derivative compounds of formula VIII

A solution containing 5 mg of the product obtained in example 4 in 0.5 ml of anhydrous DMF is mixed with 100 ml of iodomethane and add a solution containing 3 mg of bis(trimethylsilyl)amide sodium in 0.3 ml of DMF. After stirring the reaction mixture for 1.5 h at room temperature the mixture is poured into 0.1 M aqueous HCl solution, extracted with ethyl acetate and distributed between ethyl acetate and saturated aqueous sodium bicarbonate. The organic phase is washed with brine, dried over sodium sulfate and evaporated in vacuum. The crude product was then purified using chromatography on silica gel (gradient: toluene/methanol in a ratio of from 100/0,25 to 100/2,5), receiving specified in the title compound as a colourless foam. The compound analyzed by thin-layer chromatography and NMR spectroscopy to obtain the following results:

TLC: silica gel, toluene/methanol 9/1, Rf=0,40;

1H-NMR (2 to the C, NH); 6,79*, 6,74o(2s, indole H-2); 6,35o(d, J=9.5 Hz, NH); 5,98*(d, J=6,5 Hz, NH); 5,32o(ddd, PrLeu-H); 5,12othat 5,08*(2q, J=7 Hz, lactic acid, Me); 4,50*(dd, MeLeu-H); 4,06*(ddd, Leu-H); to 3.73 (s, indole, NMe); 3,44o, 3,19o, 3,15*2,93*, 2,53*, 2,35o(6s, NMe); 1,55*and 1.51o(2d, J=7 Hz, MeAla-H); 1,39o, 1,24*(2d, J= 7 Hz, lactic acid-H); 0,57*, -0,09*(2d, J=6.5 Hz, Me); -0,32*(ddd, Leu-H).

Example 6: Methyl ester 5-[8,11-Diisobutyl-14-(1-methoxy - 1H-indol-3-ylmethyl)-7,13,9,20-tetramethyl-5,17-bis(2-etylhexyl)- 3,6,9,12,15,18,21-gataka-1-oxa-4,7,10,13,16,19 - hexasaccharides-2-yl]-Penta-2-ene acid

The solution containing 185 mg of the compounds of formula XV (see below) and 1.26 g of methoxycarbonylmethylene in toluene, stirred at room temperature for 1 h Then the solvent is evaporated in vacuo, the residue chromatographic on a column for gel filtration type LH-20 in methanol and the fractions containing the product, evaporated in vacuum and optionally purified using chromatography on silica gel (eluent: gradient of a mixture of toluene/methanol from 99.5/0.5 to 96/4), getting listed in title product as a colorless solid foam. This product is subjected to freeze-drying from benzene.

Working analogously to example 6, to obtain ethyl ester.

The initial product of the formula XV

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is known (WO 96/03430). In this formula, the substituents have the following meanings:

B' means

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X means

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C' means

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Y' means

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In the following examples 7 to 11 apply the same reduction, except that

A" means

THE
IS A", R8means thiazol-2-yl, B means In', R1means CH3C means C, R3means OCH3the symbol indicates a double bond, X is X', Y means Y').

A solution containing 400 mg of the compounds of formula I in which A denotes A", R8means-CSNH2, B means In', R1means CH3C means C, R3means OCH3the symbol indicates a double bond, X is X', Y means Y', and 0.5 ml of hydrate chloroacetaldehyde in 8 ml of anhydrous dimethylformamide is stirred with 0.5 g of molecular sieves with pore size of 4 and heated to 60oC for 5 hours the mixture is Then diluted with ethyl acetate, filtered and extracted with 0.1 G. of HCl. The organic phase is washed with brine, dried and evaporated in vacuum. The crude product is purified by chromatography on silica gel (eluent: gradient of a mixture of toluene/methanol from 99.5/0.5 to 98/2), getting mentioned in the title compound as a solid foam.

Working analogously to example 7, the following compounds of formula I (A denotes the A, b means In', R1means CH3C means C, R3means OCH3the symbol indicates a double bond, X is X', Y means Y') (PL. 1).

The original product, i.e. compound of formula I, in SUB> means OCH3the symbol indicates a double bond, X is X', Y means Y', obtained as follows.

A solution containing 1.1 g of the compound of formula XI (A denotes the residue-hydroxy-substituted butyric acid, is substituted by-CN, means In', R1means CH3C means C', R4means OCH3the symbol indicates a double bond, X is X', Y means Y') and 1.8 g diphenylphosphino acid in 25 ml of isopropanol is heated under reflux for 7 hours, the Reaction mixture was evaporated in vacuo, dissolved in ethyl acetate and extracted with sodium bicarbonate solution and saline. After evaporation of the organic layer, the crude product is purified by chromatography on silica gel (eluent: gradient of a mixture of toluene/methanol from 99.5/0.5 to 98/2) to give the original connection in the form of a colorless foam.

Compounds of examples 4-11 have activity similar to that of the compounds of formula VIII when determining VCAM-1 using cell ELISA.

1H-NMR spectrum (CDCL3)

Examples

7. 3 conformer 46:51:3, denoted by the symbols*,o, ': 8,70*(d, J=10 Hz, LeuPr NH); 7,89*(d, J=10 Hz, NH); 7,83o(d, J=9 Hz, NH); 7,69, 7,66 (2d, J=3.3 Hz, thiazole H); 7,58 (d, J=10 Hz, NH); 7,53*, 7,00o(2dd, Methrone H-5'); 6,98o(s, Methrone H-2'); 6,18o(d, J=10 Hz, Leu NH); 6,03*(d, J=7 Hz, Leu NH); 5,80' (d, J=10 Hz, Leu NH); 5,30o(ddd, LeuPr-H); 5,11*(dd, hydroxipropionic acid-H); of 5.05-to 4.98 (m, -N); 4,94 (dd, Methrone-H); 4,86o(ddd, LeuPr-H); to 4.73 (m-H); 4,49*(dd, MeLeu-H); 4,23*(ddd, Leu-H); was 4.02, 4,00 (2s, N-OMe); 3,84o(m-H); 3,59 is 3.40 (m); to 3.38 (q, J=7 Hz, MeAla-H); to 3.33 (s, N-Me); a 3.2-2,85 (m); is 3.21 (s, N-Me); of 3.07 (s, N-Me); 2,93*(s, Methrone N-Me); 2,53*(s, MeLeu N-Me); 2,49 (s, N-Me); 2,43 - 2,28 (m); 2,18 (m); 1,98 (m); 1,81 (m); 1,7-1,1 (m); 1,48, 1,46 (2d, J=7 Hz, MeAla-Me); 1,06*(d, J= 6,5 Hz, MeLeu Me); 1,00 - 0,84 (m); 0,61*(d, J=6.6 Hz, Leu Me); 0,54' (d, J=6.6 Hz, Leu Me); 0,35' (d, J=6.6 Hz, Leu Me); 0,10*(d, J=6.6 Hz, Leu Me); -0,12*(ddd, Leu-CH).

8. 3 conformer 47:48:5, denoted by the symbols*,o, ': 8,64*(d, J=10 Hz, LeuPr NH); of 7.82 (2d, J=10 Hz, NH); 7,63o(d, J=10 Hz, NH); 7,23, 7,17, 7,08, 6,97 (4t, arene. H); 7,32, 7,28 (2s, thiazole H); 7,07 (s, Methrone H-2'); for 6.81 (s, Methrone H-2'); 6,22o(d, J=10 Hz, Leu NH); 6,07*(d, J=7 Hz, Leu NH); 5,80' (d, J=10 Hz, Leu NH); from 5.29o(ddd, LeuPr-H); 5,20*(dd, Methrone-H); 5,12o(dd, hydroxipropionic acid-H); 5,03o(ddd, LeuPr-H); equal to 4.97*(ddd, LeuPr-H); 4,85 (m, hydroxipropionic acid + LeuPr-H); 4,71o(ddd, Leu-H); to 4.52*(dd, MeLeu-H); 4,21*(ddd, Leu-H); was 4.02, 3,90 (2s, N-OMe); 3,34 (s, N-Me); 3,22 (s, N-Me); to 3.02 (s, N-Me); of 2.93 (s, N-Me); 2,53 (s, N-Me); 2,48 (s, N-Me); 1,43, 1,42 (2d, J=7 G2">

9. 3 conformer 42:52:6, indicated by the symbols*, oC, ': 8,67 (d, J=10 Hz, LeuPr NH); 7,83, 7,80 (2d, J=10 Hz, NH); 7,63o(d, J=10 Hz, NH); 7,55, 7,42, 7,39, 7,36 (4d, Methrone arene.); 7,22, 7,18, 7,05, 6,97 (4dd, Methrone H-5', H-6'); 7,06o(s, Methrone H-2'); 6,88*(s, Methrone H-2'); 6,22o(d, J= 10 Hz, Leu NH); 6,02*(d, J=7 Hz, Leu NH); 5,77' (d, J=10 Hz, Leu NH); 5,30o(ddd, LeuPr-H); to 5.21*(dd, Methrone-H); 5,0 (m-H); 4,85 (m-H); the 4.65 (ddd, Leu-H); 4,49*(dd, MeLeu-H); 4,13*(ddd, Leu-H); 4,03, 3,98 (2s, N-OMe); 3,70 (m); 3,55 (m); 3,45

(q, J=7 Hz, MeAla-H); 3,37, 3,20, 3,11, 2,93, 2,52, 2,43 (6s, N-Me); 2,73 (m, tetrahydrobenzoic); 1,50, 1,48 (2d, J=7 Hz, MeAla-Me); 1,04*(d, J= 6,5 Hz, MeLeu Me); 0,58*(d, J=6.6 Hz, Leu Me); 0,50' (d, J=6.6 Hz, Leu Me); 0,30' (d, J=6.6 Hz, Leu Me); 0,03*(d, J=6.6 Hz, Leu Me); -0,22*(ddd, Leu-CH).

10. 3 conformer 44:51:5, denoted by the symbols*, o, ': 8,66*(d, J=10 Hz, LeuPr NH); 7,83, 7,81 (2d, J=10 Hz, NH); 7,63o(d, J=10 Hz, NH); EUR 7.57*, 7,43o, 7,38*that was 7.36o(4d, J=8 Hz, indole-H); 7,21*, 7,18o, 7,05, 6,97 (4t, indole-H); 7,06*(s, Methrone H - 2'); 6,88o(s, Methrone H-2'); 6,70o, 6,69*(2q, J= 1 Hz, thiazole-H); 6,23o(d, J=10 Hz, Leu NH); 6,05o(d, J=7 Hz, Leu NH); 5,80' (d, J=10 Hz, Leu NH); from 5.29o(ddd, LeuPr-H); 5,11o(dd, hydroxipropionic acid-N); 5,01 (m); equal to 4.97 (dd, -H); is 4.85 (m, 2 H); 4,69o(ddd, Leu NH); 4,57' (dd, -H); 4,48*(dd, MeLeu-H); 4,16*(ddd, Leu-H); 4,03, 3,98 (2s, N-OMe); of 3.43 (q, J=7 Hz, /SUP> (d, J= 6.6 Hz, Leu d-Me); 0,52', 0,33' (2d, J=6.5 Hz, Leu d-Me); 0,05*(d, J=6.6 Hz, Leu d-Me); -0,14*(ddd, Leu-CH).

11. 3 conformer 47:50:3, denoted by the symbols*,o, ': 8,69 (d, J=10 Hz, LeuPr NH); 7,79, 7,78 (2d, J=10 Hz, NH); 7,65o(d, J=10 Hz, NH); 7,51*, 7,41o, 7,39*, 7,38o(4d, J=8 Hz, indole-H); 7.23 percent*, 7,20o, 7,07, 7,00 (4t, indole-H);? 7.04 baby mortality*(s, Methrone H-2'); 6,85o(s, Methrone H-2'); OF 6.71*(s, thiazole-H); 6,25o(d, J=10 Hz, Leu NH); 6,04*(d, J=7 Hz, Leu NH); 5,80' (d, J= 10 Hz, Leu NH); 5,30o(ddd, LeuPr-H); 5,10o(dd, hydroxipropionic acid-N); 5,02 (m); equal to 4.97 (dd, -H); 4,85 (m, 2x-H); 4.72 ino(ddd, Leu-H); 4,60' (dd, -H); 4,50*(dd, MeLeu-H); 4,17*(ddd, Leu-H); 4,04, 4,00 (2s, N-OMe); of 3.48 (q, J=7 Hz, MeAla-H); 3,41, 3,21, 3,17, 2,92, 2,53, 2,47 (6s, N-Me); 0,97, of 0.96 (2s, tert-Bu-thiazole); 1,50, 1,49 (2d, J=7 Hz, MeAla-Me); 1,06*(d, J=6,5 Hz, MeLeu-d-Me); 0,62*(d, J=6.6 Hz, Leu d-Me); 0,53', 0,35' (2d, J=6.5 Hz, Leu d-Me); 0,07*(d, J=6.6 Hz, Leu d-Me); -0,08*(ddd, Leu-CH).

A. 3 conformer 44: 30: 26, indicated by the symbols*,o, ': cent to 8.85 (d, CSNH2); 8,60*(d, LeuPr NH); 8,17 (d, CSNH2); 8,03, 8,00 (2d, NH); 7,88 (m, CSNH2); 7,6-7,1 (arene.); 6,28*(d, 10 Hz, Leu NH); 6,07o(d, 7 Hz, Leu NH); by 5.87 (d, 9 Hz, Leu NH); 5,26*(ddd, LeuPr-H); 5,22 (dd, hydroxipropionic acid-N); 5,15-4,95, 5,08*(dd, hydroxipropionic acid-H); a 4.83o(ddd, LeuPr-N); 4,50*(ddd, Leu-H); 4,37*(dd, Methrone-H); 4,25o, 0,55', 0,23', 0,17o(4d, 7 Hz, Leu d-Me); -0,15o, -0,17' (ddd, Leu-CH). PKF 285-916 (thiazole)

Biological activity

The active compounds according to the invention are tested in terms of cytotoxicity and inhibition of expression of ICAM-1, VCAM-1 and E-selectin, cell proliferation, and for inhibiting the release of TNF and related cytotoxicity.

The analyses carried out as follows.

Cell line Nasat, randomly transformed, non-carcinogenic cell line of human keratinocytes, which largely preserved the characteristics of phenotypic differentiation of normal keratinocytes (Boukamp and others, 1988, J. Cell Biol. 106, 761-771), used for analysis of cell proliferation and analysis of ICAM-1 using cell ELISA.

A. Analysis of ICAM-1 using cell ELISA

I. Analysis of ICAM-1 keratinocytes by the method of cellular ELISA

Analysis of ICAM-1 using cell ELISA is used to determine the inhibition of the expression of ICAM-1 is basically similar to the method described in Winiski and Foster (1992, J. Invest. Dermatol., 99, 48-52). Cell line Nasat plated on 96-well microtiter plates (2x104cells/well in the following culture medium: modify, 100 mg/ml streptomycin, 2 mm glutamine, 1 mm sodium pyruvate), grown to confluence and then incubated for approximately 24 h in fresh medium for analysis (same environment, and culture, but containing 0.5% FCS instead of 5%) with the addition of environment, stimulated IFN - a /TNF - a (a framework for analysis + 1000 units /ml IFN- /3 ng/ml TNF - a ), or without it, in the presence of test compounds or without him. Then Wednesday, washed and the cell monolayers fixed with 1% paraformaldehyde. Monolayers incubated with saturating amounts of primary antibody (mouse anti-ICAM-1 monoclonal) and secondary antibodies (goat antimurine associated with peroxidase). Then hold peroxidase reaction using as the substrate 3 - amino-9-ethylcarbazole (AEC) and receive an insoluble, colored product that can be easily measured in a standard apparatus for reading microtiter plates.

II. Evaluation of cytoxicity

Upon completion of the reaction using AEC for detection of ICAM-1 monolayers Nasal washed SFR (phosphate buffered saline) solution (200 ml), SFR shed from the plates, which are then dried by the end of the paper towels to remove excess liquid. The bottom surface of microtitration the sorption at 492 nm. Before the layers can dry each well add 0.1 ml of 0.1% solution of crystal violet in ZFA (previously passed through a filter with pore size 0.2 mm). Then the tablets incubated at room temperature for 10 min, thoroughly washed five times SFR, the excess liquid is removed, as described above, and again determine the absorbance at 492 nm before the layers can dry. The difference between the optical densities before and after dyeing gives the values obtained by crystal violet staining, and therefore, they are related to the size of the cell monolayer present in the wells. These values are used to adjust the values obtained for the AEC.

B. Analysis of VCAM-1, ICAM-1 and E-selectin in endothelial cell by the method of cellular ELISA

The analysis is based on the method of cell ELISA on 96-well tablet using cell line microvascular endothelial person (HMEC-1) and cells of the umbilical vein endothelium human (HUVEC). Cells pretreated for 4 h test connection, stimulate over the next 6 to 16 h of TNF , and then fixed with paraformaldehyde for subsequent evaluation of the expression of VCAM-1, ICAM-1 and E-selectia the relative number of cells (staining of the nucleus on Giemsa) after exposure of the test compounds, compared with the control wells (containing only the solvent and the environment). Compounds are considered as giving a positive reaction, if they inhibit VCAM-1, ICAM-1 or E-selectin by 50%, while the loss of cells is <25%.
I. cell Line: For the analysis of VCAM-1 and ICAM-1 using immortalizing (large T-angilena virus SV-40) cell line microvascular endothelial person (HMEC-1; Ades and others, J. Invest. Dermatol. 99: 683-690, 1992). Cell line HMEC-1 constantly Express low levels of ICAM-1, activated by inflammatory mediators. However, they Express only VCAM-1 after stimulation with cytokines. To determine the optimal conditions for the induction of the expression of VCAM-1 and ICAM-1 was performed experiments to determine the dependence of dose-response and time.

II. Growing conditions: Cell line HMEC-1 grown in flasks of the type T-75 (Nunc) under standard conditions (37oC, 5% CO2at the rate of 1,5x106cells per ml of culture medium (COP means a principal medium for endothelial cells [computers; firm Clonetics], supplemented with 10% FCS, 10 ng/ml human epidermal growth factor (EGF) (firm Boehringer), 1 mg/ml hydrocortisone (firm Sigma N 0888), 2.2 g/l NaHCO3, 15 mm Hepes, 0.11 g/l sodium pyruvate, 4 mm glutamine, 100 units/m is e 8 min) and resuspendable cells subcultured every 2-3 days with a dilution factor of 1:3.

III. Method of cell ELISA for VCAM-1 and ICAM-1

96-well flat-bottomed microtiter tablets pre-cover bovine fibronectin (FN, firm Sigma, N F1141), and then seeded based 2x104cells/well in 200 ml of medium for the cultivation of computers and incubated over night. The next day the culture medium (COP) first substitute at the rate of 200 ml/well of medium for analysis computers (CS, supplemented with 5% FCS instead of 10%) and then replace 180 ml of medium containing either (1) certain concentrations of the tested compounds, or (2) the appropriate concentration of the solvent/environment, extracted with methanol, or (3) one environment to analyze computers, and incubated for 4 h at 37oC. Each assay 96-well tablet exercise of duplicate wells. The cells are then stimulated by adding 20 ml of concentrated solution of cytokine (2000 units/ml TNF ), and incubated for 16 h at 37oC.

After that, the cell monolayer was washed with 1% paraformaldehyde in an environment of computers, fixed in 2% paraformaldehyde for 15 min at room temperature (RT) and washed several times with SFR. SFR removed from the cells and monolayer incubated for 30 min in SFR containing 10% normal goat serum> throughout the night. The solution Mat (monoclonal antithical) then removed and cells washed several times with SFR, and then incubated with SFR containing 10% NGS for 30-60 min at RT. The solution NGS remove and add 100 ml associated with horseradish peroxidase goat F(Ab')2antimisting IgG antibodies (firm Tago; dilution 1: 500 in SFR containing 5% NGS) and tablets incubated for 1 h at RT. Then the secondary antibody is removed and the cells washed in SFR, which then replaces the calculation of 150 ml/well of freshly prepared and filtered solution AEC (3-amino-9-ethylcarbazole; firm Sigma) and tablets incubated for 45-60 min at RT. The substrate for peroxidase were removed and the cells washed in SFR. The absorbance value of the AEC determined using an apparatus for reading microtiter tablets at 550 nm and correct with regard to "pure" or reference values obtained at 690 nm.

IV. Analysis of E-selectin

Analysis of E-selectin performed using freshly isolated cell line HUVEC basically the same way as is described for the analysis of VCAM-1 and ICAM-1, except for short-time stimulation of TNF (6-8 hours).

V. Evaluation of cytotoxicity (loss of cells based on staining of nuclei)

Entitelments control using microscopy. The cells are then washed with distilled water (Aquadest) and monolayer at CT cover for 5 min 33% Giemsa solution in Aquadest. Then the wells are washed with Aquadest and air-dried for at least 15 minutes To verify that a colored core and almost no staining of the cytoplasm, using microscopic examination. The absorbance value of the solution Giemsa determined using an apparatus for reading microtiter tablets at 550 nm and correct with regard to "pure" values (rows without cells) obtained at 690 nm.

VI. Data analysis

The AEC values for the constitutive expression of VCAM-1 or E-selectin (control wells without stimulation) almost coincide with those for izotopicheskii similar control pad and represent background staining. In each 96-well tablet average constitutive value is subtracted from the average value of AEC for each group, stimulated cytokine (control group with computers and solvent, as well as the group with the test compound), receiving a number that represents the expression of cell adhesion molecules (CAM) activated in the case of ICAM-1 and induced in the case of VCAM-1 or E-selectin (labeled AEC-ITSELF). Each mn is yet to assess the relative levels of expression of HIMSELF when the density of cells, based on the number of cores (denoted as the ratio of the AEC:Giemsa)

AEC (stimulation) - AEC (without stimulation) = AEC-CAM

AEC-HIMSELF/Giemsa = ratio AEC:Giemsa.

Thus, the "actual value" IC50the indicator ITSELF is determined by comparing the values of AEC:Giemsa for test compounds with a value for stimulated control (computers, solvent). These values are then analyze the relative values of the IC50for option only with Giemsa solution. The exact criterion allows to reveal a good correlation between inhibition of HIMSELF and the profile of cytotoxicity (using Giemsa solution), the study of which may be continued.

C. Analysis of cell proliferation lines Nasat

Cell line Nasat cultured in DMEM, Gibco N 074-02100) supplemented with 2.2 g/l NaHCO3, 0.11 g/l sodium pyruvate, 15 mm Hepes, 5% fetal calf serum (FCS), penicillin (100 units/ml), streptomycin (100 mg/ml) and glutamine (to increase the final concentration to 4 mm). For analysis of cell proliferation separated by treatment with trypsin, suspended in fresh medium and plated on 96-well microtiter tablets with a finite density of 4000 cells/0.2 ml/well what about the connection. After three days incubation at 37oC/5% CO2the degree of cell proliferation compared with that in the control group, which used only the solvent, assessed by the colorimetric method, which allows us to measure relative cell mass using dye sulforhodamine In (Skehan and others, 1990, J. Natl. Cancer Inst. 82, 1107-1112). "The initial number of cells is determined by measuring the relative cell mass at day 0. Results expressed as follows: % inhibition = 100% absorption in control (where controls with solvent = 100%) and expressed as the average standard deviation of three measurements. Curve based dose-response build in semi-logarithmic scale and using linear interpolation to determine the concentration required for inhibition, which is the half of the maximum (IC50).

The maximum inhibition in the absence of the net loss of cells, which represented the initial number of cells, is typically 90-98%.

, Inhibition of release of TNF

I. Odnoletnie cells obtained from peripheral blood of healthy volunteers based on the separation density in picola-hipace in accordance Soi 10% FCS. Cells incubated with serial dilutions of test compounds for 30 min at 37oC before adding INF (100 units/ml) and LPS (5 mg/ml) and then further incubated for 3 h Incubation stop by centrifugation at 1400 rpm for 10 minutes, the Content of TNF in the supernatant was measured using a commercial kit for ELISA (Innotest hTNF supplied by the company Innogenetics N.V., Zwijnaarde, Belgium). Compounds are tested at concentrations from 0 to 10 mm. The examples of the compounds of formula I, especially preferred compounds of formulae I, I'pI"p, VII, IX and X, in this experiment inhibit the release of TNF, with values IC50range from about 5 to about 1 nm.

II. Cytotoxicity

The cytotoxicity determined in cell line THP1 (5104cells/well), which are incubated in the presence of INF (100 units/ml) and LPS (5 mg/ml) and in the presence of test compounds or without for 24 h at 37oC. the Percent of live and dead cells was determined by colorimetric marker (MTT) [bromide 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] , which allows us to measure the mitochondrial dehydrogenase in living cells, as described in Mosman, J. Imm. Methods (1983) 65:55. Preferred compounds of PNA IC50is from about 100 to 1000 nm.

1. Cycloheptadecane formula I in free form, in salt form or complex ester

< / BR>
where a denotes the residue of glycolic acid, optionally-substituted stands, ethyl, optionally substituted thiazole;

In denotes the residue Aime--methylamino octanoic acid;

R1denotes methyl;

C indicates the residue of N-methyltryptophan formula II

< / BR>
where R3denotes alkoxy;

R4denotes hydrogen;

R5is methyl;

the symbol represents a double bond;

X represents the residue-aminosilanes (C2- C14)carboxylic acid;

Y denotes a residue of N-methyl--aminosilanes (C2- C10)carboxylic acid.

2. Cyclopeptide under item 1 of formulas VIII, IX or X

< / BR>
< / BR>
< / BR>
3. A therapeutic composition having cytotoxic activity against inhibition of the expression of ICAM-1, VCAM-1 and E-selectin, TMF release and proliferation of cells containing a therapeutically effective amount of the compounds under item 1 or 2.

Priority points:

21.11.1995 - p. 2 (formula VIII);

01.03.1996 - p. 2 (formula IX);

04.07.1996 - p. 2 (formula X).

 

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< / BR>
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< / BR>
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< / BR>
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