Low-molecular peptide derivatives as inhibitors in interaction laminine/nidogen

FIELD: peptides, pharmacy.

SUBSTANCE: invention relates to low-molecular derivatives of peptides that are able to act as inhibitors in interaction between laminine and nidogen (interactions laminine/nidogen). Also, invention relates to a method for their preparing, pharmaceutical composition prepared on thereof and their using for preparing pharmaceutical agents, and for identification of inhibitors in interaction laminine/nidogen.

EFFECT: valuable properties of peptides.

5 cl, 12 dwg

 

The object of the present invention are low molecular weight derivatives of peptides that can act as inhibitors of the interaction between laminin and nidorina (laminin/Neogen interaction), the way they are received, prepared on the basis of their pharmaceutical compositions and their use for the manufacture of pharmaceutical products and to identify inhibitors of the interaction of laminin/nidorina.

Association of laminin (glycoprotein 80 kDa) and Neogene (glycoprotein 1-60 kDa) is considered as the most important biomolecular mechanism involved in the synthesis and stabilization of basal membranes (Mayer, U., and Timpl, R., 1994, increasing interest among Matrix Assembly and Structure (P.D. Yurchenco, D. Birk and R.P. Mecham, Ed.), 389-416, Academic Press, Orlando, FL). The ability of Neogene to form ternary complexes with all the basic ingredients of the basal membrane, such as, for example, isoforms, containing γ1-laminin (information item, see: Burgeson, R.E., Chiquet, M., Deutzmann, R., Ekblom, P., Engel, J., Kleinmann, H., Martin, G.R., Meneguzzi, G., Paulsson, M., Sanes, J., Timpl, R., Tryggvasson, K., Yamada, Y., Yurchenco, P.D., 1994, Matrix Biology, 14, 209-211), collagen IV, perlecan and fibulin, and associated structures formed on the basis of each of them, means that it performs the function of the linker that connects the spatial organizes and stabilizes independently existing macromolecules (Fox, J.W., Maer, U., Nischt, R., Aumailley, M., Reinhardt, D., Wiedemann, H., Mann, K., Timpl, R., Krieg, T., Engel, J., and Chu, M.-L., 1991, EMBO J. 10, 3137-3146).

The basal membrane are highly specialized extracellular structures that are related to the implementation of important functions in the control of cell and tissue activity, tissue structure, cell growth, cell transformation, cell migration and tissue-specific gene expression (Adams, J.C. and Watt, F.M., 1993, Development 117, 1183-1198). The experiments carried out using monoclonal antibodies against laminin, have provided clear evidence of the Central role of interaction of laminin/nidorina in the synthesis process of the functioning of the basal membrane. These antibodies are obtained by immunization of rabbits with laminin P1 or received recombinant methods nicogossian domain of laminin (γ1 III 3-5). After concentration of antibodies using affinity chromatography on laminin P1 or laminin γ1 III 3-5 matrix demonstrated in tests on inhibition complete inhibition of the Association type laminin/nitogen. This effect is based on steric blockade by antibodies access Neogene to laminin, the binding regions are located in the immediate vicinity of nitogen-binding sequence of laminin (Mayer, U., Nischt, R., E., Mann, K., Fukuda, K., Gerl, M., Yamada, Y., Timpl, R., 1993, EMBO J. 12, 1879-1885).

In cultures ambr the national authorities indicated antibodies inhibit the development of renal tubules, the formation of pulmonary alveoli, as well as the morphogenesis of the salivary glands in embryos. All three of these models relate to the programs of ontogenesis, which is essential seamless synthesis of basement membrane (Ekblom, P., Ekblom, M., Fecker, L., Klein, G., Zhang, H.-Y., Kadoya, Y., Chu, M.-L., Mayer u, Timpl, R., 1994, Development 120, 2003-2014).

Antibodies acting against γ1 chain sequence of laminin in the region responsible for binding to nidorina also able to inhibit the Association of laminin/nitogen. However, this inhibition is competitive in nature in contrast to the actions of the above antibodies against laminin, because in this case they compete directly with nidorina for the binding site on laminin (WO 98/31709).

Monoclonal antibody of the IgM subclass (against laminin P1 A6/2/4 - DSM ACC2327, see WO 98/31709) inhibits in vitro the interaction of laminin/nidorina value IR50equal to 30 nm. As described above, the preparation of polyclonal antibodies against laminin, it prevents embryonic morphogenesis of the salivary glands in the culture of organs. This fact underlines the specificity of the interaction of laminin/nidorina and the importance of LE-4 module, and also identified the sequence of the region in the domain of laminin γ1 III 4 in the implementation of the specified interaction.

Nitogen-binding domain of laminin b the l is clearly identified and characterized from the point of view of its location, primary sequence and spatial structure (made by x-ray analysis of the crystal lattice and NMR analysis of the primary structure) (Gerl, M., Mann, K., Aumailley, M., Timpl, R., 1991, Eur. J. Biochem., 202, 167-174; Mayer, U., Nischt, R., Pöschl, E., Mann, K., Fukuda, K., Gerl, M., Yamada, Y., Timpl, R., 1993, EMBO J. 12, 1879-1885; Baumgartner, R., Czisch, M., Mayer, U., Pöschl, E., Huber, R., Timpl, R., Holak, T.A., 1996, J. Mol. Biol., 257, 658-668; Stetefeld, J., Mayer, U., Timpl, R., Huber, R., 1996, J. Mol. Biol., 257, 644-657). The specified domain is located in LE module (epidermal rastoptalo factor leninova type) short arm γ1 chain of laminin in the domain γ1 III 4. LE modules represent a structural motif consisting of 50-60 amino acids with a characteristic complex folded structure similar to EGF (epidermal growth factor), with 4 disulfide bridges (Bairoch, A., 1995, Nomenclature of increasing interest among domains. The SWISS-PROT Protein sequence data bank release, 310; Engel, J., 1989, FEBS Letters, 251, 1-7).

High affinity binding of Neogene with complementary leninova domain was found to laminin P1 from EHS tumor mouse for laminin 2 and laminin 4 from human placenta and laminin Drosophila. The reason for this interspecific overlap the binding specificity lies in the extremely high sequence identity γ1 III 4 domain in relation to the studied species. It is 97% for sequences of human and mouse, 61% for mouse and Drosophila, and, as the strange, 51% between mouse and Caenorhabditis elegans, in relation to the entire domain (Pikkarinen, T., Kallunki, T., Tryggvasson, K., 1987, J. Biol. Chem., 263, 6751-6758; Chi, H.-C., Hui, C.-F., 1989, J. Biol. Chem., 264, 1543-1550; Wilson, R. et al., 1994, Nature, 368, 32-38; Pöschl, E., Mayer, U., Stetefeld, J., Baumgartner, R., Holak, T.A., Huber, R., Timpl, R., 1996, EMBO J. 15, 5154-5159).

Research has revealed not only the existence of dependence of the binding process of Neogene from the intact three-dimensional structure, but also led to the identification of well-defined sequences of regions located in the stable with the help of S-S linkages loops a and C domain γ1 III 4. This identified five amino acids, four of which are located within the area of the loop and consisting of 7 amino acids, and one of them is the tyrosine side-chain loop with (Mann, K., Deutzmann, R., Timpl, R., 1988, Eur. J. Biochem., 178, 71-80). Synthetic peptides, which can be obtained on the basis of the relevant regions γ1 III 4 domain and which are able to inhibit the binding of laminin/nidorina in specific tests on the binding, are disclosed in patent Fox and Temple (J.W. Fox and R. Timpl, U.S. patent No. 5493008).

Apparently, to achieve high affinity binding to binding site of Neogene on the molecule laminin requires interaction with tyrosine or histidine in the loop (loop C), neighbouring with a valid binding sequence. This interaction with aromaticheski and amino acids is considered as a precondition of inhibition in the range of values IR 50<500 nm, based on the size γ1 III 3-5 equal to 3 Yes, and based on the known nature of the relationship structure-function, which is described in U.S. patent No. 5493008. However, it still remains unclear the question of whether a loop interacts with directly with nidorina or she is only involved in the stabilization of a suitable spatial patterns NIDPNAV sequence of the corresponding region (Pöschl, E., Fox, J.W., Block, D., Mayer u, Timpl, R., 1994, EMBO J. 13, 3741-3747; Baumgartner. R., Czisch, M., Mayer, U., Pöschl, E., Huber, R., Timpl, R., Holak, T.A., 1996, J. Mol. Biol., 257, 658-668; Stetefeld, J., Mayer, U., Timpl, R., Huber, R., 1996, J. Mol. Biol., 257, 644-657).

Interaction type laminin/Neogen has a strong influence conformational component (Mayer, U., Nischt, R., Pöschl, E., Mann, K., Fukuda, K., Gerl, M., Yamada, Y., Timpl, R., 1993, EMBO J. 12, 1879-1885; Mann, K., Deutzmann, R., Timpl, R., 1988, Eur. J. Biochem., 178, 71-80). Synthetic peptides, which can be obtained on the basis of nitogen-binding site of laminin, not able to form a disulfide bond, such as is present in the LE modules, but they demonstrate activity in tests on inhibition, which is about 400-10000 times weaker than the activity of intact laminin P1 or laminin γ1 III 3-5 (Pöschl, E., Fox, J.W., Block, D., Mayer u, Timpl, R., 1994, EMBO J. 13, 3741-3747; J.W. Fox, and R. Timpl, U.S. patent No. 5493008). This decline in activity is quite unusual, since it is known that in aqueous solution, the peptides can take great is th number of different conformations, and only a small percentage of the peptide is a biologically active conformation. The most active peptide as described to date (with a value of IR50equal to 22 nm) has a molecular weight of about 2700 Yes (i.e. about 50% match LE module). Its structure includes the intact S-S loop, which presumably stabilizes the structure of the sequence of the essential amino acids NIDPNAV in the respective region (Poschl, E., Fox, J.W., Block, D., Mayer u, Timpl, R., 1994, EMBO J. 13, 3741-3747; J.W. Fox, and R. Timpl, U.S. patent No. 5493008).

The chemical formula of the sequence NIDPNAV (Asn-Ile-Asp-Pro-Asn-Ala-Val) has the following form:

Inhibitors of the interaction of laminin/nitogen can be used for the manufacture of pharmaceutical drugs used in diseases associated with increased or undesirable synthesis of basal membranes.

Such diseases include, in particular, all types of diabetes, which are accompanied by thickening of the basal membranes (especially in the kidneys, eyes, cardiovascular system, liver fibrosis, especially fibrosis of the liver caused by alcohol intoxication, which is characterized by the synthesis of a continuous basal membrane in the sinusoids and the corresponding capillariasis, all fibrosis (chronic or iatrogenic), in which there is an increased synthesis of basement membrane and its components (fibrosis of the kidney, lung, skin), atherosclerosis, where and who eat place limiting the regulation of lipid metabolism and which may be caused by, among other impaired filtering lipoproteins through partially capillarian sinusoids of liver pathological changes in the vascular system, observed in atherosclerosis, may also contribute to the modification of the composition and structure of the basal membranes of the blood vessels), diseases in which angiogenesis exacerbates the severity of the clinical picture, such as cancer, when the tumor growth requires neovascularization and diabetic retinopathy, retrolental fibroplasia, a disease with a strong inflammatory component, such as rheumatoid arthritis, osteoarthritis, vasculitis), hemangioma, psoriasis and many others.

However, the use of the peptides described in U.S. patent 5493008, as drugs are limited largely to their conformational flexibility, instability against proteases, poor bioavailability and low pharmacodynamics (Milner-White, E.J., 1989, Trends Pharmacol, Sci., 10, 70-74; Verber, D.F., Freidinger, R., 1985, Trends. Neurosci., 8, 392-396; Hruby, V.J,, 1994, Peptides, Proc. Thirteenth American Symposium (Hodges, R.S., Smith, J.A., Ed.), 3-17, ESCOM, Leiden, Netherlands).

Adaca of the present invention is the detection of low-molecular peptide derivatives, which can specifically interact with nitogen-binding site on laminin and thus competitive inhibition at low concentrations the Association between laminin and nidorina.

In this regard, an object of the present invention is a compound of the formula I:

in which

R1 denotes a group of the formula

ororor

oror

oror

where R4 denotes-A, -NH2, -Other, -NR2And2, -NHR1,

oror

and R5 represents -(CH2)1COOA, - (CH2)1CONH2, -(CH2)1NH2or -(CH2)1-SO3N

or

X denotes a group described by one of the following formulas

ororor

ororor

or

where Y denotes O, S, -N(A)-CO - or -(CH2)r-,

D represents (CH2)r, O, S, NH, NR, (CH2)r-O (CH2)r-S, (CH2)r-NH or (CH2)r-NR and

R2 denotes-A-E-HE-E-COOH or-E-CONH2,

where E appears is t a linear or branched C 1-C10is an alkyl chain, which may not be substituted or replaced by groups-A, -(CH2)2mHE, -(CH2)m-COOH, -(CH2)m-S(O)NA2or5-C10-cycloalkyl group,

or E represents C5-C10-cycloalkyl, which may be substituted or replaced by groups-A, -(CH2)m-OH, -(CH2)m-COOH, - (CH2)m-C(O)NA2or C5-C10-cycloalkyl group,

and R3 represents a group of one of the following formulas:

or

or

where R6 represents-H, -COOH, -CONH2, -CONHR, -CONR2, -CH2OH, or

or

or

and where R7 represents a linear or branched C1-C10is an alkyl group which may be substituted or replaced by groups-A, -(CH2)m-OH, -(CH2)m-COOH, -(CH2)m-S(O)NA2or C5-C10-cycloalkyl group,

or R7 represents C5-C10-cycloalkyl group which may be substituted or replaced by groups-A, -(CH2)m-OH, -(CH2)m-COOH, -(CH2)m-C(O)NA2or5-C10-cycloalkyl what Ruppel,

and R denotes a branched or unbranched C1-C6-alkyl, C2-C6alkenyl,2-C6-quinil,5-C10-cycloalkyl, Het or Ar, which optionally can be substituted by one or more halogen atoms, C1-C6-alkyloxy, branched or non-branched C1-C6-alkyl, C2-C6-alkenylphenol,2-C6-alkenylphenol or5-C10-cycloalkyl groups, or-C1-C6-alkyl-Het, -C1-C6-alkyl-Ar, -O-C1-C6-alkyl-Het, -O-C1-C6-alkyl-Ar, Het or Ar,

where Het denotes a monocyclic or bicyclic, 5-to 10-membered aromatic or nonaromatic ring containing 1 or 2 identical or different heteroatoms as members of the specified ring selected from the group consisting of nitrogen, oxygen and sulfur, which may be unsubstituted or substituted by one or more hydroxy or carboxypropyl, and where

Ar denotes a monocyclic or bicyclic 5-to 10-membered aromatic ring which may be unsubstituted or substituted by one or more hydroxy or carboxypropyl, and

Z represents (CH2)m, O, S, NH, NR, N-C(O)-R or NSO2R,

And denotes N or C1-C4-alkyl and

l, m and r represent integers from 0 to 3,

n and k denote integers from 1 to d is 2,

p denotes an integer from 0 to 1, and

q denotes an integer from 1 to 3

and all their stereoisomeric forms and mixtures thereof in all ratios, including all physiologically tolerant of salt.

Physiologically tolerant of salt include, for example, salts of inorganic and organic acids, for example hydrochloric acid, sulfuric acid, acetic acid, citric acid or p-toluensulfonate acid, or salts of inorganic and organic bases, such as NH4OH, NaOH, KOH, CA(Oh)2, Mg(OH)2, diethanolamine or Ethylenediamine, or salts of amino acids such as arginine, lysine, lysyl-lysine or glutamic acid.

In one preferred embodiment implementing the present invention relates to a compound of formula I in which n is 1.

Another preferred embodiment relates to the compound of formula I, in which R in the group X denotes Het or Ar, which may be optionally substituted C1-C6-alkyl-Het, -C1-C6-alkyl-Ar, -O-C1-C6-alkyl-Het, -O-C1-C6-alkyl-Ar, Het or Ar. More preferably, R in the group X denotes Het. For example, Het can refer to:

or

or

or

or

The preferred implementation of the present invention also includes a compound of formula I, in which R in the group X denotes Ar, which may be optionally substituted C1-C6-alkyl-Ar, -O-C1-C6-alkyl-Ar or Ar. Preferably R in the group X denotes Ar.

For example, Ar can be defined:

or

or

or

The preferred implementation of the present invention also includes a compound of formula I, in which R in the group X represents:

or

In the compound of formula I, X preferably denotes a group of the following formula:

Preferably Y represents -(CH2)rwhere r preferably has the value 0 or 1 and k preferably has a value of 1 or 2.

Another preferred implementation of the present invention relates to the compound of formula 1, in which the group X has the following formula:

and D preferably represents -(CH2)r-where r takes the value 0 or 1.

Also preferred is an implementation option for the present invention, related to the compound of formula I in which R1 denotes a group of the following formula:

where Z denotes preferably (CH2)mand m is 0 or 1. Preferably R5 represents -(CH2)1-COOA where a preferably denotes H, or R5 represents -(CH2)1-COONH2where 1 is set to 0. Preferably R4 represents-NH2or, where a preferably denotes H, or preferably R4 represents-NHR1, where-NHR1 preferably represents:

and where R5 in the group-NHR1 preferably denotes

1 preferably has the value 0, or R5 in the group-NHR1 preferably denotes

1 preferably has the value 0, or R5 in the group-NHR1 preferably denotes (CH2)1-NH2and 1 preferably represents 0.

Another preferred embodiment of the present invention relates to the compound a of the formula I, where R1 denotes a group of the following formula:

where Z represents preferably -(CH2)mand m preferably has a value of 1, R4 preferably denotes-NH2and R5 preferably represents -(CH2)1-COOA where 1 preferably has a C is Uchenie 0, and preferably denotes N.

Another preferred embodiment of the present invention relates to the compound a of the formula I, where R1 denotes a group of the following formula:

where R5 preferably represents -(CH2)1-COOA where 1 preferably has the value 0, And preferably denotes N.

Another preferred embodiment of the present invention relates to the compound of formula I, where R2 denotes A, and And preferably refers to the group-CH3or R2 denotes the group-S-COOH, preferably-CH2-COOH, or R2 denotes the group-S-IT, preferably-CH2HE.

Another preferred embodiment of the present invention relates to the compound of formula I, where R3 denotes a group of the following formula:

where k preferably represents 2.

Another preferred embodiment of the present invention relates to the compound of formula I, where R3 denotes a group of the following formula:

Another preferred embodiment of the present invention relates to the compound of formula I, where R3 denotes a group of the following formula:

where R7 preferably denotes rasvet the military C 1-C10is an alkyl group, preferably-CH(CH3)2- (CH3)3, -CH(CH3)CH2-CH3or-CH2-CH(CH3)2and where R6 preferably denotes-H, -COOH, -CONH2, -CH2OH, -CON(CH3)2or more preferably R6 represents:

where q represents preferably 2.

Another preferred embodiment of the present invention relates to the compound of formula I, where R3 denotes a group of the following formula:

where R7 preferably denotes-CH(CH(CH3)2)2or-CH2C(CH3)3.

Compounds according to the present invention are non-natural (i.e. they do not occur in nature) low-molecular-weight derivatives of peptides, which are able to inhibit the interaction of laminin/nitogen in the nm concentration range. Unexpectedly it was found that the molecular structure was capable of high-affinity binding to the binding sites of Neogene on the molecule laminin, without interaction with tyrosine or histidine from the loop (loop C), neighbouring with a valid binding sequence.

Even more unexpected was the discovery of the fact that considered in the present izopet the Institute of low molecular weight derivatives of peptides with values of molecular weight of from 550 to 800 Yes show ingibirovanie of the same order, as the most active among currently known peptides (IR50approximately 22 nm), which have a molecular weight of about 2700 Yes (i.e. about 50% from the value characteristic of LE module) and include the intact S-S loop, which mainly stabilizes the structure of the region with the sequence NIDPNAV (J.W. Fox, and R. Timpl, U.S. patent 5493008).

The target compound was obtained by specific synthesis, based on knowledge of the nature of the interaction between structure/function and known according to the literature data the three-dimensional structure linking Neogen site, in the form of peptide derivatives on the resin used as the substrate. The building blocks used for peptide synthesis, varied in accordance with predetermined criteria to achieve the desired structural differences and the integration of non-natural blocks. To test whether the obtained peptide derivatives inhibiting activity and then comparing them with each other was used sensitive method of screening analysis, which was applied after removal derived from the resin of the substrate.

Compounds according to the present invention can be used to obtain pharmaceutical drugs used in the treatment of diseases associated with increased or undesirable synthesis of basal membranes./p>

In accordance with the foregoing, the possible areas of therapeutic application of these peptide derivatives and/or their physiologically tolerant of salts are:

1. All types of late complications of diabetes, associated with thickening of the basal membranes (especially in the kidneys, eyes, cardiovascular system).

2. Cirrhosis, especially alcoholic cirrhosis of the liver, characterized by the synthesis of a continuous membrane in the sinusoids and the resulting process by capillarization.

3. All fibrosis (chronic or iatrogenic), which may be increased synthesis of basal membranes and their components (kidney, lung, skin).

4. Atherosclerosis, which is characterized by limited regulation of lipid metabolism, which may be caused including impaired filtration of lipoproteins through partially capillarian sinusoids of the liver. Moreover, pathological changes in the vascular system, which may occur in atherosclerosis, also make a definite contribution to the modification of the composition and structure of the basal membranes of the blood vessels.

5. 3abolevaniya in which angiogenesis contributes to the deterioration of the clinical picture, such as in the case of cancer, when the tumor growth requires neovascularization, in the case of diabetic retinopathy, retrolental fibroplasia, diseases with severe vospalitelnost (for example, rheumatoid arthritis, osteoarthritis, vasculitis), hemangioma, psoriasis and many others.

Thus, the compounds according to the present invention and/or their physiologically tolerant of salt can be used as pharmaceuticals. In this regard, another object of the present invention is a pharmaceutical composition comprising at least one compound according to the present invention and/or physiologically tolerant of salt.

The compounds of formula I and their physiologically tolerant of salt and derivatives can be administered in accordance with the present invention an animal, preferably a mammal, in particular humans, for treatment or prevention. They can be administered per se, as mixtures with one another or in the form of pharmaceutical preparations suitable for enteral or parenteral administration, and, as an active constituent contain an effective dose of at least one of the compounds of formula I and/or physiologically tolerant of salts and derivatives, and may be combined with conventional pharmaceutically innocuous excipients and/or additives.

These pharmaceutical preparations can be administered systemically or topically. For example, they can be used in the form of pills, tablets, coated tablets, tablets coated with sugar film, gra the street, hard and soft gelatin capsules, powders, solutions, syrups, emulsions, suspensions or other pharmaceutical forms. In addition, the introduction can be carried out vnutrivaginalno or rectally, such as suppositories, or parenterally, or by implantation, for example, in the form of solutions for injection or for infusion, microcapsules or sterzhnevykh forms, and in addition, the administration can be local or percutaneous, for example, in the form of ointments, solutions or tinctures, or in any other way, for example in the form of a nasal spray or aerosol mixtures, or in the form of inhaled drugs of dry powders. In the case of parenteral solutions may be selected, for example, intravenous, intramuscular, subcutaneous, intra-articular, intra-articular, or other method, for example by inhalation of wet aerosols or dry powder preparations.

The pharmaceutical preparations according to the present invention are obtained by known in the art ways to use pharmaceutically inert inorganic and/or organic carriers in combination with the compound(s) of formula I and/or their physiologically tolerant salts and derivatives. For more pills, tablets, tablets coated with sugar film, and hard gelatin capsules can be used,for example, lactose, corn starch or its derivatives, talc, stearic acid or its salts and other carriers in the case of soft gelatin capsules and suppositories can, for example, fats, waxes, semisolid and liquid polyols, glycols, natural or hardened oils and other Acceptable fillers in the manufacture of solutions, such as solutions for injection, or of emulsions or syrups are, for example, water, alcohols, glycerol, diols, polyols, sucrose, invert sugar, glucose, vegetable oil and other Acceptable substrates for the production of microcapsules, implants or sterzhnevykh forms include for example, copolymers of glycolic acid and lactic acid. In normal pharmaceutical preparations contain from about 0.5 to 90 wt.% compounds of the formula I and/or their physiologically tolerant of salts and derivatives.

In addition to the active compounds and media pharmaceuticals may also include AIDS or additives, such as fillers, to contribute to decomposition, binders, oiling agents, moisturizers, stabilizers, emulsifiers, preservatives, sweeteners, colorants, flavorings or aromatizers, thickeners, diluents, sautereau substances, solvents or solubilizing agents, means for Costigan what I depot effect, salt affecting the osmotic pressure, the means used for applying the covering shell, or antioxidants. They can also contain two or more compounds of the formula I and/or their physiologically tolerant of salts and derivatives. In addition, these drugs can include one or more therapeutically or prophylactically active substances in addition, at least one compound of the formula I and/or physiologically tolerant to salts and derivatives. In normal pharmaceutical preparations may contain from 0.2 to 500 mg, preferably from 1 to 100 mg of active compound of the formula I and/or physiologically tolerant of salts and derivatives per dose.

If the compounds of formula I or containing pharmaceuticals are used in the form of aerosols, for example in the form of nasal sprays or wet aerosol or inhalation of the dry powder, the introduction may be carried out by means of a spray, atomizer, pump atomizer, the device for inhalation, the inhaler with measured dose or inhaler for dry powder, respectively. Pharmaceutical forms for administration of the compounds of formula I in the form of an aerosol spray can be prepared using a method known at that level of technology to any specialist in this field. So, for Paul the treatment of solutions or dispersions of the compounds of formula I in water can be used in mixtures of water-alcohol or suitable saline solutions conventional additives, for example benzyl alcohol or other suitable preservatives, enhancers of absorption to improve bioavailability, solubilizing agents, contribute to the dispersion, and others, and in case of appropriate forms - traditional propellants such as chlorofluorocarbons and/or fluorocarbons, while the powder form preparations based on compounds of the formula I and/or their physiologically tolerant of salts can be obtained by freeze drying or preferably during the spray drying of aqueous solutions of compounds of the formula I and/or their physiologically tolerant acceptable salts and water-soluble additives, such as sugar or derivatives of sugars and amino acids.

The dose of the compounds of formula I in the pharmaceutical compositions can vary within wide limits and, as usual, by the attending physician in each case when the specific individual conditions. For example, it depends on the nature and severity of the disease to be treated, the specific compound, in acute or chronic form have to treat disease or to apply the product with the preventive purpose, and whether in combination with the compound of the formula I in the composition other active compounds. In General, in the case of oral introduction is appropriate to achieve effective results in adults is daily dose, component from about 0.01 to 100 mg/kg, preferably from 0.1 to 10 mg/kg, in particular from 0.3 to 2 mg/kg (in each case, the mean dose per kg body weight). In the case of intravenous administration the daily dose is generally from about 0.01 to 50 mg/kg, preferably from 0.01 to 10 mg/kg of body weight. In particular, with the introduction of relatively large amounts of the daily dose can be divided in several doses, for example 2, 3 or 4 separate introduction. In some cases, under appropriate conditions may need to increase or decrease the indicated dosage.

In addition, the compounds of formula I and their salts according to the present invention can be used as intermediate products for the manufacture of other compounds, in particular other pharmaceutically active compounds derived from compounds of the formula I, for example, with appropriate modifications radicals or by introducing them to the right groups, such as the reactions of esterification, reduction, oxidation or other types of transformations of functional groups.

The peptide derivatives according to the present invention can, on the one hand, be used directly as a therapeutic tool, but can also be a basis for the creation of related structures, which can also be used as a Lek is stannage remedy for the treatment of diseases, associated with an increased or undesirable synthesis of basal membranes.

Another object of the present invention is a method of identifying compounds that can inhibit the interaction of Neogene and laminin, namely, that the specified connection according to the present invention is used as a competitive inhibitor. This method may also include the composition of the identified compounds in pharmaceutically acceptable form.

The object of the present invention is also a method of obtaining a pharmaceutical composition intended for the identification of compounds that inhibits the interaction of Neogene and laminin, which consists in the fact that the connection according to the present invention, used as a competitive inhibitor, and/or its physiologically tolerant of salt, mixed with a pharmaceutically acceptable carrier.

The object of the present invention is also a method of obtaining the compounds of formula I according to the present invention.

The compound of the formula I:

according to the present invention receive through fragmentary condensation of the compounds of formula II:

with the compound of the formula III

where the variables R1, X, n, R2 and R3 have specified the s above values, in this regard, the compounds of formulas II and III can be protected on defined above, functional groups using methods known in peptide chemistry (see, for example, Houben-Weyl, Methods der Organischen Chemie, vol. 15/1 and 15/2, Georg Thieme Verlag, Stuttgart, 1974). Suitable condensation methods well known in the art (Houben-Weyl, Methods der Organischen Chemie, vol. 15/1 and 15/2, Georg Thieme Verlag, Stuttgart, 1974). Acceptable condensing or linking reagents are, for example, carbonyldiimidazole, carbodiimides, such as dicyclohexylcarbodiimide or diisopropylcarbodiimide, or O-((cyano(etoxycarbonyl)-methylene)amino)-N,N,N',N'-Tetra-methyl-Urania tetrafluoroborate (TOTU) or anhydride pyrophosphoric acid (PPA). The condensation reaction is carried out in standard conditions. Generally, peptide condensation occurs, the need for protection of amino groups that are not involved in binding assays, with protective groups, which are then easily removed under conditions different from those used for binding assays. The same applies to carboxypropyl that are not involved in binding assays and are preferably protected in the form of C1-C6-alilovic esters, benzyl esters or tert-butyl esters during binding assays. Ashita amino group is not necessary in the case of an amino group present in the form of predestiny the basic amino groups, for example in the form of nitro - or cyano groups. When this amino receive then at the stage of hydration after the condensation reaction. Upon completion of the stage of condensation of the protective group is removed using a known and acceptable methods, for example benzyloxycarbonyl and benzyl groups can be removed by hydration in benzyl esters, and protective tert-butylene group mainly broken down in acidic conditions, whereas 9-fluorenylmethoxycarbonyl disposed in the secondary amines have had.

Obtaining the compounds of formula I according to the present invention can also be carried out with stepwise addition of the respective components, for example natural and unnatural amino acids and their derivatives in the solid phase, these components can be added in different order.

Order to obtain the compounds of formula I may also be useful to conduct no direct coupling components of the formulae I and II in fragmentary condensation, and carry out the specified reaction with their respective predecessors, receiving the desired intermediate, which then can be converted to a compound of formula I, for example, by appropriate reactions.

The above-described method of introducing a molecule functional groups not directly, but through sootvetstvuetopredelennyj order to obtain the intermediate compounds of which can then be easily obtained final product under transformation groups predecessor to the respective functional groups and subsequent condensation reaction, may be used in application to different parts of the molecule of the compounds of formula I, i.e. with respect to the side chain of the compounds of formula I: R1 or R1-X, respectively.

Examples

The following abbreviations have the following meanings:

Agents and solvents:

Asónacetic acid
aqwater
BSA (BSA)bovine serum albumin
DCC (DCC)N,N'-dicyclohexylcarbodiimide
DHM (DCM)dichloromethane
DIPEA (DIPEA)N,N-diisopropylethylamine
DMAP (DMAP)4-dimethylaminopyridine
DMF (DMF)N,N-dimethylformamide
DMSO (DMSO)the sulfoxide
Et2Odiethyl ether
EtOAcetretinate (ethyl ester of acetic acid)
EtOHethanol
Fmoc-OSuccFmoc-O-succinimide
HOBT1-hydroxybenzotriazole
KHMDS (KHMDS)hexamethyldisilazide potassium
n-Bulin-butyl lithium
MeOHmethanol
MTBE (MTBE)methyl tert-butyl ether
The tea (TEA)the triethylamine
TFU (TFA)triperoxonane acid
THF (THF)tetrahydrofuran
TMEDA (TMEDA)tetramethylethylenediamine
TMSCltrimethylsilane
TOTUO-((cyano(etoxycarbonyl)methylene)amino)-
N,N,N',N'-tetramethylurea tetrafluoroborate
The N3(TrisN3) trisilicate

Chemical group:

MemethylCH3-
EtethylCH3-CH2-
nPrn-propylCH3CH2CH2-
iPrisopropyl(CH3)2CH-
NBun-butylCH3CH2CH2CH2-
iBuisobutyl(CH3)2SNSN2-
tButert-butyl(CH3)3With-
PhphenylWith6H5-
Fmoc9-fluorenylmethoxycarbonyl
ZbenzyloxycarbonylWith6H5-CH2-O-CO-
SIDE (EAST)tert-butyloxycarbonyl(CH3)3C-O-CO-

1. Screening of libraries of inhibitors of the interaction of laminin/nidorina

The library was created to search for smaller, more powerful and metabolically more stable peptides relative to the known level of heptapeptide NIDPNAV (Pöschl, E., Fox, J.W., Block, D., Mayer u, Timpl, R. (1994) EMBO J. 13, 3741-3747, Pöschl, E., Mayer, U., Stetefeld, J., Baumgartner, R., Holak, T.A., Huber, R., Timpl, R. (1996) EMBO J. 15, 5154-5159; Baumgartner, R., Czisch, M., Mayer, U., Pöschl, E., Huber, R., Timpl, R., Holak, T.A. (1996) J. Mol. Biol. 257, 658-668). Synthesis and screening of libraries was performed in three podubniak: pentameron, hexamers and heptamers. Below is a description of the screening strategies pentamers of medbibliotekoy. The specified method is representative of the methods used in relation to the other two podubniak, except that screening hexamers in the first stage, use approximately 50 beads per cell, and screening of heptameron use about 100 Sha is the ISR for the cell.

1.1. Screening pentamers library

Pentamers library contains 2160 different connections.

1) Approximately 8800 separate balls suspended in 0.1% HCl and distributed to seven filters at the bottom of the 96-alopecia microtiter tablet approximately 14 beads per cell.

2) the Beads are washed two times with 200 μl of deionized water and then add 50 ál of 500 mm HEPES, pH 7.0. Used in the library the linker releases one aliquot connection with the increase of pH to 7.0, and the specified phase splitting is carried out throughout the night.

3) Tablets fold vertices of the filter plate with the bottom U-shaped and centrifuged. The mixture of compounds released from the beads collect at the bottom of the tablet, whereas the corresponding balls remain on the source filter of the tablet.

4) Add the balls 25 ál DMSO to flush the remaining free connection of the balls, and the tablets again centrifuged to separate the compounds in the solution from the beads. The resulting preparation for storage is a predominantly 27 μm concentration of the compound in 333 mm HEPES, 33% DMSO.

5) Conduct pre-incubation solution for storing, containing connection with nidorina (10 ál connection of the storage solution with 90 ál of a solution of Neogene) and analyzed as described in the attached Protocol research is Finance, with the final concentration at screening, 2.7 μm on the connection.

6) In 25 cells for analysis, where achieved reproducible inhibition ≥62%, corresponding balls of the original filter plates suspended in a mixture of 0.05% HCl and 0.1% tween-20 and pipette transfer on five new filter plates in the number 1 bead per cell. To each plate as controls add two control ball with the parent connection on the same linker.

7) Beads are washed two times with 200 μl of deionized water and then to each cell add 25 ál of 50 mm NaOH. The linker used in the library releases the second equimolar an aliquot connection with the increase of pH values from 7.0 to 10.0 or more. Phase splitting should be performed within 3 hours.

8) Tablets put at the top of the plate with the bottom U-shaped and centrifuged. Compounds released from the beads collect at the bottom of the tablet, whereas the corresponding balls remain on the source filter of the tablet.

9) Balls washed with 20 µl of 50 mm HEPES (initial pH 7.0) with the addition of 50 mm HCl, and the solution was centrifuged in the bottom plate and combine with the first released break.

10) Balls washed a third time 25 μl of DMSO to balance with balls for 10 minutes before centrifugation.

11) Received the haunted released samples analyzed in 1/10 volume as indicated in stage 5.

12) Solutions that inhibit as well or better than the control balls (about 50%inhibition)are considered as the best. Restore 23 ball from among the best, while the other two potentially running the ball is defined as additional weak inhibitors in separate cells.

13) Most active solutions are subjected to mass spectrophotometric analysis to determine their molecular weight.

14) Corresponding to a separate active beads are subjected to degradation adman to determine peptide sequences.

15) Analyze the combined MS data and the results of the analysis Adminu to identify the structures of the active compounds.

Below you can find the patterns and frequency of their recovery. G-Nora denotes hydroxypropylamino glycine, linker element.

Legend:

Na12=L-3-(2-naphthyl)alanyl:

G-Hopa=glycine-3-hydroxypropionic:

D=Asp (aspartic), P=Pro (shed), N=Asn (asparagine), A=Ala (alanyl), V=Val (valinol), S=Ser (ceril), I=Ile (isoleucyl).

1.2. Procedure: obtain a peptide library

Peptide library synthesized using the technique of splitting/mixing (Lam, for K.S., Salmon, S.E., Hersh, E.M., Hruby, V.J., Kazmierski, W.M. and Knapp, R.J. (1991) Nature 34, 82; Furka, A., Sebestyen, F., Asgedom, M., and Dibo, G. (1991) Int. J. Pept. Protein Res. 37, 487) using the methods of tverdofaznoi chemistry of peptide synthesis using Fmoc (Stewart, J.M., and Young, J.D. (1984) Solid Phase Peptide Synthesis. Pierce Chemical Co., Rockford, IL., Atherton, E. and Sheppard, R.C. (1989) Solid Phase Peptide Synthesis. IRL Press, Oxford). In each cycle of binding of each resin bead is exposed to only one of the activated amino acids. In this regard, at the completion of the synthesis library each resin bead is only one peptide form. Since it was not possible to separately test all connections, the authors on each Smolyan ball built in two copies of the same patterns with differentially degradable linker, figure 1 (Kocis, P., Krchnak., V., and Lebl, M. (1993) Tetr. Lett. 34, 7251; Lebl, M., Krchnak, V., Salmon, S.E., and Lam, for K.S. (1994) A Companion to Methods in Enzymology 6, 381). 3 Quiroga can be achieved by the release of the peptide from the resin ball during a number of successive stages using different splitting mechanisms. The release of the first portion of the peptide in the form of hydroxypropylamino carried out in buffer at pH 7-9. The release of the second portion of the peptide is activated when using higher values of pH (Scheme 1).

Peptide libraries using polystyrene beads with grafted polyethylene glycol or TentaGel®S NH2. In fact acceptable to use any resin beads, compatible with the are conditions of peptide synthesis and screening in aqueous media.

Penta-, hexa - and heptaminol libraries receive from one fixed position (L-asparagine). Hydroxypropylamino glycine at the C-end is the part of the linker;

H-X4X3-Asn-X2X1-Gly-NH(CH2)3OH (2160 peptides)

H-X5X4X3-Asn-X2X1-Gly-NH(CH2)3OH (25920 peptides)

H-X6X5X4X3-Asn-X2X1-Gly-NH(CH2)3OH (311040 peptides)

X1: N-Fmoc-L-amino acids (9)used in the first randomization: valine, isoleucine, threonine, phenylalanine, β(2-naphthyl)alanine, 2-azetidinone acid, Proline, cyclohexylglycine, phenylglycine.

X2: N-Fmoc-L-amino acids (4)used in the second randomization: alanine, glycine, serine, aspartic acid.

X3=X5=X6: N-Fmoc-L-amino acids (12)used in the third, fifth and sixth randomization: pipecolinate acid, P(2-naphthyl)alanine, glutamic acid, lysine, 2-azetidinone acid, threonine, Proline, asparagine, isoleucine, 3,5-diyodtirosin, citrulline, arginine.

X4: N-Fmoc-L-amino acids (5)used in the fourth randomization: aspartic acid, glutamic acid, 2-aminoadenosine acid, O-sulfate of tyrosine, γ-carboxy-glutamic acid.

Resin (PEG-PS·HCl, Millipore®, 20 g load of 0.58 mmol/g, the average particle size of 220 μm) swells in N,N-dimethylformamide for 2 hours, after which it is neutralized with 10% N,N-diisopropylethylamine in dichloromethane. Next, the resin was washed with dichloromethane and N,N-dimethylformamide. SV is to provide a linker (figure 1, 3 EQ.) using 1,3-diisopropylcarbodiimide and 1-hydroxybenzotriazole (each 3 EQ.) in N,N-dimethylformamide at room temperature for 12 hours. The progress of the reaction is followed by test with bromophenol blue (Krchnak, V., Vagner, J., Safar, P., and Lebl, M. (1988) Collec. Czech. Cem. Commun. 53, 2542). The end of the reaction link is determined by the ninhydrin test (Kaiser, E., Colescott, R.L., Bossinger, C.D., and Cook, P.I. (1969) Anal. Biochem. 34, 595). After washing N,N-dimethylformamide remove Fmoc-protective group in the processing of 50% piperidine in N,N-dimethylformamide for 15 minutes. 3 Quiroga resin washed with N,N-dimethylformamide and determine the number of released fulvin-piperidinol adduct on UV spectrophotometer (302 nm). Stable load level of the resin (mmol/g)determined by this method during the entire process of synthesis of the library, is one of the controlled parameters.

The resin is divided into 9 equal portions. 3 Quiroga in each aliquot of resin added separately nine Fmoc-protected amino acids (X1) and within 2 hours conduct binding by the above procedure. Next, the resin is collected in a cylindrical glass vessel, Frit attached to the bottom. Tar bubbled with dry nitrogen to mix. 3 Quiroga remove Fmoc-protective group as described above.

After that, the resin is divided into 4 equal portions. To each aliquot of resin add CTD is the super four Fmoc-protected amino acids (x2) and spend the linking by the same procedure, as previously. Remove the Fmoc-protective group, and determine the level of loading of the resin. In the next cycle associated L-asparagine by the above mentioned procedure. 3 Quiroga resin divided into aliquots for use in another cycle of binding. After completion of all stages of randomization removes the Fmoc protective group, the protective groups of the side chain break down for 2.5 hours the mixture triperoxonane acid (82,5%), anisole (5%), water (5%), thioanisole (5%) and ethicial (2.5 percent). Next, the resin is washed triperoxonane acid, dichloromethane, N,N-dimethylformamide and methanol. The resulting library is stored at a temperature of 4°C.

To confirm the quality of the library conduct sample analysis several balls in the framework of the degradation of Edman and mass spectrophotometric analysis.

1.3. The results (see also figure 2-12)

2. Synthesis on a large scale

2.1. Synthesis of N-Fmoc-TRANS-3-(2'-naphthyl)-L-Proline (A8)

Brief description: N-Fmoc-TRANS-3-(2'-naphthyl)-L-Proline (A8) get in the way, VK is causea 10 stages:

2.1.1. Ethyl TRANS-3-(2'-naphthyl)-propenoate (A1)

To a stirred solution of 2-naphthaldehyde (7.8 g, 50 mmol) in 50 ml ethanol add (carletonville)triphenyl-fastran (18,3 g of 52.5 mmol). There is a slight exothermic reaction. While stirring the mixture overnight, a precipitate may form. Next, the reaction mixture was diluted with Et2O (500 ml) and washed with 1M H3RHO4(2×100 ml), then with saturated solution of NaHCO3(1×100 ml), water (100 ml) and brine (100 ml). The organic fraction is dried (MgSO4) and concentrate under reduced pressure. The residue is passed through the gasket with SiO2and elute with hexane:EtOAc (9:1). After concentration under vacuum get almost quantitative yield in the form of a mixture in the ratio of 85:15 of geometric isomers (most likely in the TRANS-form according to NMR). This material is re-crystallized from a mixture of hexane/EtOAc (enrichment hexane) to obtain 4.5 g of the desired product as a mixture of isomers in a ratio of 97:3 (according to NMR). The mother liquid is concentrated and re-crystallized as described previously, obtaining another 2.9 g (a total of 7.4 g, 33 mmol, 65% yield). NMR (CDCl3) δ to 7.93 (s, 1H), 7,88-7,83 (s, 4H), to 7.67 (DD, 1H, J=1,6, 8.6 Hz), 7,53-7,50 (s, 2H), 6,55 (d, 1H, J=16.0 Hz), 4,30 (q, 2H, J=7,1 Hz)of 1.42 (t, 3H, J=7,1 Hz).

2.1.2. TRANS-3-(2'-naphthyl) propane the traveler acid (A2)

To a solution of ester A1 (4,24 g of 18.8 mmol) in THF (75 ml) is added LiOH·N2On (2,36 g, 56,3 mmol) in water (19 ml). Initially heterogeneous mixture was vigorously stirred overnight, after which it becomes homogeneous. Next, the reaction mixture is acidified with concentrated HCl (pH≈2), and therefore a precipitate. Specified heterogeneous mixture is transferred into a separating funnel and extracted with EtOAc (3×150 ml). The combined extracts dried (MgSO4) and concentrate under vacuum to obtain carboxylic acid as a white solid (3,66 g, 98% yield). NMR (CDCl3) δ of 7.97 (d, 1H, J=15.7 Hz), of 7.90 (d, 1H, J=15.3 Hz), of 7.90-7,83 (s, 3H), of 7.70 (DD, 1H, J=1,6; 8.6 Hz), EUR 7.57-7,50 (s, 2H), return of 6.58 (d, 1H, J=16.0 Hz).

2.1.3. TRANS-(4S)-3-(3'-(2"-naphthyl)-propenyl)-4-phenyl-2-oxazolidinone (A3)

A solution of carboxylic acid A2 (3,66 g, 18.5 mmol) and triethylamine (1,87 g of 2.56 ml, 18.5 mmol) in anhydrous THF (74 ml) cooled to -78°C. for 2 minutes add pivaloate (2.35 g, 2,40 ml of 19.4 mmol), which is accompanied by the formation of a white precipitate. After 10 minutes, the flask is placed in a bath with a temperature of 0°10 minutes, after which the flask was again cooled to -78°for 1.5 hours. In a separate flask oxazolidinone derived from L-phenylglycinol (of 3.31 g, 20.3 mmol)in anhydrous THF (74 ml) cooled to -78°C. then add a solution of n-BuLi (1.6 M in hexane, to 11.6 ml, 18.5 mmol) and stirring p. who borrow for about 1 hour, accompanied by deposition metallizovannogo oxazolidinone from solution THF/hexane. The mixed anhydride is added by cannula to metallizovannogo the oxazolidinone and the reaction mixture is placed in a bath with a temperature of 0°C. After 1 hour, the bath removed and the mixture warmed to room temperature over night. Then the reaction quenched by adding 50 ml of saturated NH4Cl. THF is removed under reduced pressure and after transfer to a separating funnel, the mixture is extracted with CH2Cl2(3×75 ml). The combined organic fractions washed with 1M NaOH (2×50 ml), dried (MgSO4) and concentrate. The residue is re-crystallized from a mixture of EtOAc/hexane to obtain white solids (a 3.87 g, and 11.2 mmol, 61% yield). NMR (CDCl3) δ with 8.05 (d, 1H, J=15.7 Hz), 7,94 (d, 1H, J=15,4 Hz), 7,87-7,81 (s, 3H), 7,76 (DD, 1H, J=1.5 and 8.6 Hz), 7,53-7,47 (s, 2H), 7,41-7,34 (s, 5H), to 5.58 (DD, 1H, J=8,7; 3,9 Hz), was 4.76 (t, 1H, J=8.7 Hz), to 4.33 (DD, 1H, J=8,8; 3,9 Hz).

2.1.4. (3'R4S)-3-(3'-(2"-naphthyl)-4'-pentenyl)-4-phenyl-2-oxazolidinone (A4)

To a solution of CuI (3,96 g of 20.9 mmol) and TMEDA (2.66 g, of 3.46 ml, with 22.9 mmol) in anhydrous THF (92 ml) at -78°With add vinylboronic magnesium (1.0 M in THF, to 20.9 ml, of 20.9 mmol). The mixture is stirred for 15 minutes. In a separate flask, to a solution of the unsaturated imide A3 (a 3.87 g, 11.3 mmol) in anhydrous THF (42 ml) add trimethylsilane (5,69 g, 6,64 ml, at 52.2 mmol). Due to the insolubility of imide membrane ale is HT bulb, containing cuprate reagent is removed and added as one portion of the slurry of imide followed by rapid washing a large amount of THF. The bath temperature was raised to -30°and stirring is continued for 1 hour. Next, the reaction mixture is poured into 250 ml of a mixture of saturated NH4Cl/concentrated NH4OH in the ratio 3:2 respectively. Separate the layers, and the aqueous fraction extracted with EtOAc (3×200 ml). The combined organic fractions washed sequentially with saturated NH4Cl (1×100 ml) and water (1×100 ml). The organic fraction is dried (MgSO4) and concentrate under reduced pressure. The residue is purified by passing through the gasket with SiO2and elute with hexane:EtOAc 4:1. The eluate concentrated under vacuum to obtain white solids (of 3.64 g, 9,81 mmol, 87% yield). NMR (CDCl3) δ 7,87-of 7.82 (s, 3H), 7,72 (s, 1H), 7,54-7,27 (s, 8H), 6,11 (DDD, 1H, J=6,7; 10,4, of 17.0 Hz), of 5.34 (DD, 1H, J=8,6, 3.5 Hz), 5,10 (d, 1H, J=8,2 Hz), to 5.08 (d, 1H, J=and 17.2 Hz), 4,56 (t, 1H, J=8,8 Hz), 4.26 deaths (DD, 1H, J=8,8, 3.5 Hz), of 4.16 (DDD, 1H, J=8,1; 7,0; 6.9 Hz), 3,68 (DD, 1H, J=8,4; and 16.5 Hz), 3,50 (DD, 1H, J=6,5% to 16.5 Hz).

2.1.5. (2'S3'R4S)-3-(2'-azido-3'-(2"-naphthyl)-4'-pentenyl)-4-phenyl-2-oxazolidinone (A5)

To anhydrous THF (34 ml) at -78°add in one portion hexamethyldisilazide potassium (0.5 M in toluene, of 25.5 ml, 12.8 mmol). Make a slurry of imida A4 (of 3.64 g, 9,81 mmol) in THF (34 ml) and injected through the cannula, rinsing the I THF (2× 11 ml) to achieve a more complete transfer. After 30 minutes trisilane (and 4.40 g of 14.2 mmol) dissolved in THF (34 ml), cooled to -78°and added via cannula. Thirty minutes add Asón (1,41 g of 1.34 ml of 23.4 mmol) to extinguish the reaction. After that, the mixture was stirred at room temperature overnight. The mixture is partitioned between CH2Cl2(300 ml) and dilute brine (150 ml). The layers separated, and the aqueous phase extracted with CH2Cl2(3×150 ml). The combined organic fractions are dried (MgSO4) and concentrate under reduced pressure. The residue is purified with flash chromatography to obtain the product (3,41 g of 8.28 mmol, 84% yield). NMR (CDCl3) δ a 7.85-of 7.82 (s, 3H), 7,72 (s, 1H), 7,53-7,47 (s, 2H), 7,42 (DD, 1H, J=1,7; 8,5 Hz), 7,37-7,31 (s, 3H), 7.18 in-7,15 (s, 2H), 6,28 (DDD, 1H, J=8,2; 10,2; and 17.1 Hz), 5,63 (d, 1H, J=10,2 Hz), lower than the 5.37 (d, 1H, J=17,0 Hz), of 5.34 (d, 1H, J=10,2 Hz), a 4.83 (DD, 1H, J=3,0, 8,3 Hz), 4,14 (t, 1H, J=7,2 HZ), 4,07 (DD, 1H, J=9,3; and 17.9 Hz), of 3.94 (DD, 1H, J=3,0; 5.8 Hz), 3,68 (t, 1H, J=8.6 Hz).

2.1.6. Methyl-(2S3R)-2-azido-3-(2'-naphthyl)-4-pentenoate (A6)

To a solution of imide A5 (3,41 g of 8.28 mmol) in THF (62 ml) is added water (21 ml), 35% N2O2(2.7 ml) and LiOH·H2O (695 mg, of 16.6 mmol). After 2 hours add Na2SO3(4,17 g, up 33.1 mmol) in aqueous solution (41 ml). This mixture is stirred for 15 minutes, and the THF removed under reduced pressure. The aqueous solution is acidified with HCl and extracted with EtOAc (2 on; 150 ml). The combined extracts dried (MgSO4) and concentrate under reduced pressure. The resulting residue is passed through a column spacer SiO2, elwira a mixture of hexane:EtOAc (1:1), obtaining after concentration of white solids, which is predominantly a mixture of carboxylic acids and chiral auxiliary reagent. Re-crystallization from a mixture of hexane:EtOAc gives the chiral auxiliary substance in the form of needle crystals. The mother liquid is concentrated and transferred to the stage of esterification. The residue containing the crude carboxylic acid was dissolved in anhydrous Meon (46 ml) and cooled to 0°S. 3 Quiroga added thionyl chloride (1.18 g, 725 μl, 9,94 mmol) and after 10 minutes the mixture is heated at the boiling point under reflux for 2 hours. To this mixture is added water (1.0 ml), stirred all within 10 minutes, and the contents of the flask are concentrated under reduced pressure. The residue is partitioned between EtOAc (150 ml) and brine (100 ml). Separate the layers, and the organic fraction dried (MgSO4) and concentrate under reduced pressure. The residue is purified with flash chromatography (hexane:EtOAc, 19:1) to give the methyl ester (1.54 g, of 5.48 mmol, 66% yield). NMR (CDCl3) δ 7,84-7,80 (s, 3H), 7,71 (s, 1H), 7,50-7,46 (s, 2H), 7,39 (DD, 1H, J=1,8; 8,5 Hz), 6,23 (DDD, 1H, J=8,3; 10,9; and 17.6 Hz), 5.0mm (d, 1H, J=9.9 Hz), 5,28 (d, 1H, J=17.7 and Hz), 4,22 (d, 1H, J=7.5 Hz), 4,06 (t, 1H, J=7.9 Hz).

2.1.7. Methyl ester of TRANS-3-(2'-naphthyl)-L-Proline (A7)

Borane-methylsulfinyl complex (2.0 M in THF, to 6.57 ml, of 13.1 mmol) was diluted with anhydrous THF (26 ml) and cooled to 0°C. Gently syringe add cyclohexen (2.16 g, 2.66 ml of 26.3 mmol). After 30 minutes, a white precipitate is formed. Stirring is continued for another three hours. Next, the contents of the flask are concentrated under vacuum. The reagent was diluted in CH2Cl2to a state of pulp (36 ml) and cooled to 0°C. Dissolved in CH2Cl2(9 ml) vinylated A6 (1,23 g of 4.38 mmol) and add it to the cannula. The reaction mixture becomes pale yellow and starts noticeable gas evolution. The mixture is warmed to room temperature over night. Add Meon (26 ml) and stirred for 15 more minutes. 3 Quiroga mixture is concentrated under reduced pressure. The obtained residue selected from the Et2O (25 ml) and extracted with 0.1m HCl (5×25 ml). Water extracts alkalinized with saturated NaHCO3and extracted with CH2Cl2(3×100 ml). The organic extracts are dried (MgSO4) and concentrate under vacuum to obtain the cyclic product with some impurities in the form of derivatives of dicyclohexylurea (974 mg, 3,82 mmol, 87% yield of crude material), NMR (CDCl3) δ 7,84 for 7.78 (s, 3H, 7,71 (s, 1H), 7,49-7,41 (s, 3H), 3,91 (d, 1H, J=6.9 Hz), of 3.69 (s, 3H), 3,63 (m, 1H), 3,48 (DD, 1H, J=8,2; to 15.4 Hz), with 3.27 (d, 1H, J=7,8 Hz)at 3.25 (d, 1H, J=7.8 Hz), 2,33 (m, 1H), 2,09 (m, 1H).

2.1.8. N-Fmoc-TRANS-3-(2'-naphthyl)-L-Proline (A8)

510 mg (2 mmol) of the methyl ester (A7) in 12 ml of 6 N. HCl is heated at 100°C for 10 hours. 3 Quiroga reaction solution was concentrated under reduced pressure, and the obtained solid residue is suspended in 15 ml of acetone. Later in the suspension was adjusted pH to 9-10 using a 2 n solution of Na2CO3. Then slowly add 742 mg (2.2 mmol) of Fmoc-O-succinimide. Nacina pH is then adjusted to 9-10, and the mixture is stirred at room temperature for 4 hours, then incubated at room temperature overnight. 3 Quiroga pH value was adjusted to 2 using concentrated HCl, and the resulting mixture is stirred with ethyl acetate. Is filtered off with suction 560 mg of precipitated product. The aqueous phase is extracted three times with ethyl acetate and then mixed with methylene chloride. This procedure results in another 185 mg of the product as a precipitate. The output is 745 mg (80,4%). NMR (d6-DMSO) δ 7,95-7,80 (C, 6N), to 7.68 (d, 1H, J=7,3 Hz), 7,60 (d, 1H, J=7.4 Hz), 7,50-7,34 (C, 6N), 7,25 (m, 1H), 4,39-to 4.15 (s, 4H), 3,70-of 3.48 (s, 3H), to 2.29 (m, 1H), and 2.14 (m, 1H).

2.2. N-succinyl-TRANS-3-(2'-naphthyl)-L-prolyl-L-asparaginyl-L-alanyl-L-valine-amide (34)

2.2.1. N-Fmoc-the Rance-3-(2'-naphthyl)-L-Proline-L-asparagine-L-alanyl-L-valine-amide (B1)

463,5 mg (1 mmol) of N-Fmoc-TRANS-3-(2'-naphthyl)-L-Proline (A8), 338 mg of H-Asn-Ala-Val-NH2hydrochloride (obtained using conventional methods of peptide chemistry) and 135 mg NOT dissolved in 20 ml of DMF. When 0°type of 0.13 ml of N-ethylmorpholine and 220 mg DCC. The mixture was stirred at 0°C for 1 hour and then at room temperature for 3 hours, then incubated at room temperature overnight. The precipitate is filtered off with suction, and the resulting solution was concentrated under high vacuum. The resulting residue is distributed between pentanol and solution of NaHCO3. Pentelow phase is washed with a solution of KHSO4and a solution of H2O/NaCl. The precipitate is filtered off with suction, and the resulting material was triturated with diethyl ether. This procedure results in 473 mg of product. Pentelow phase is dried with Na2SO4and concentrate. The residue is triturated twice with diethyl ether. This procedure gives another 257 mg of the product.

Output: 730 mg (97.7 per cent)

2.2.2. TRANS-3-(2'-naphthyl)-L-Proline-L-asparagine-L-alanyl-L-valine-amide (B2)

248 mg (of 0.332 mmol) of N-Fmoc-TRANS-3-(2'-naphthyl)-L-Proline-L-asparagine-L-alanyl-L-valine-amide B1 is taken in 5 ml of DMF. Add 0.35 ml (3,32 mmol) of diethylamine, and the mixture is stirred at room temperature for 15 minutes. Next, the mixture is filtered with suction Cherepovetsky layer and concentrated under high vacuum. The solid residue triturated with diethyl ether and filtered off with suction.

Yield: 141 mg (81%)

2.2.3. Methyl tert-butylacrylate (B3).

In an argon atmosphere suspended 13,2 g (100 mmol) of monomethylamine in 500 ml of methylene chloride. Within 30 minutes is added dropwise to 12.9 ml (150 mmol) of oxalicacid, and then the mixture is stirred at room temperature for 6 hours. After about 3.5 hours formed a clear solution. Then added dropwise to 300 ml of tert-butanol. This mixture stand at room temperature for 21 hours and concentrate transparent solution. The resulting residue is dissolved in ethyl acetate and washed with N2O, the solution of NaHCO3and again N2O. 3 Quiroga above solution is dried using Na2SO4and concentrate.

Output: 21,6 g (crude oily product)

2.2.4. Mono-tert-butylacrylate (B4)

9.4 g (50 mmol) of methyl tert-butylacrylate (B3) is dissolved in 115 ml of 1,4-dioxane. After that add 110 ml of 0.5 N. NaOH. This mixture is maintained at room temperature, and the product precipitates. Next, the mixture was kept at room temperature for one week and then concentrated. The aqueous solution is extracted with diethyl ether. The aqueous phase is cooled to 0°and acidified to pH 4 with 2 N. H2SO4. 3 Quiroga mixture is extracted five is ü once diethyl ether. The combined organic phases, washed with N2O, dried with Na2SO4and concentrate. Output: 5,62 g butter (64,5%).

2.2.5. N-tert-butyl-succinyl-TRANS-3-(2'-naphthyl)-L-Proline-L-asparagine-L-alanyl-L-valine-amide (B5)

262 mg (0.5 mmol) of TRANS-3-(2'-naphthyl)-L-Proline-L-asparagine-L-alanyl-L-valine-amide (B2), to 87.1 mg (0.5 mmol) of mono-tert-butylacrylate (B4) and 67.5 mg of HOBt are dissolved in 5 ml of DMF. When 0°add 110 mg DCC, and the mixture was stirred at 0°C for 1 hour and then at room temperature for 2 hours, then incubated at room temperature overnight. The precipitate is filtered off with suction, and the filtrate concentrated under high vacuum. The residue is triturated with a solution of NaHCO3, is filtered off with suction, washed with water and dried in a desiccator.

Output: 169 mg (49,6%).

2.2.6. N-succinyl-TRANS-3-(2'-naphthyl)-L-Proline-L-asparagine-L-alanyl-L-valine-amide (34)

316 mg of N-tert-butylbenzoyl-TRANS-3-(2'-naphthyl)-L-Proline-L-asparagine-L-alanyl-L-valine-amide (B5) is dissolved in 2 ml of 90%-triperoxonane acid and incubated at room temperature for 1 hour. After that, the mixture is filtered through a clarifying layer and concentrate. The residue is triturated with diethyl ether and filtered off with suction. This procedure gives 159 mg of the crude product. For purification, the substance chromatographic on the Sefa is EXE (Sephadex® LH20) using a mixture of butanol/glacial acetic acid/water.

Output: 27,5 mg (9,5%),

m/z 625,298949 (M+N)+(the mass spectrum of the high-resolution)

Data of NMR for compound 34:

Chemical shifts for compound 34 in DMSO at 300 K:

Cm. the formula above1H13C
TRANSCISTRANSCIS
But-1--173,88173,92
But-22,54/2,462,61/2,2028,6428,37
But-32,70/2,542,56/2,3928,8028,80
But-4--170,67170,22
Pro-α4,39to 4.6866,1865,46
Pro-C'--171,03170,94
Pro-β3,553,6847,4249,45
Pro-γ2,40/2,162,33/1,9432,0230,41
Pro-δ3,81/3,72 3,59/3,5346,1245,40
Nap-1--138,79139,55
Nap-27,767,78125,14124,79
Nap-2a--132,97132,97
Nap-37,877,87127,66127,66
Nap-47,497,49126,03126,03
Nap-5of 7.48of 7.48125,63125,63
Nap-67,887,88127,33127,33
Nap-6a--131,98131,98
Nap-77,877,89127,97127,97
Nap-87,457,46125,96125,96
Asn-NH8,318,50--
Asn-αof 4.44with 4.6450,13of 49.79
Asn-C'--170,58170,29
Asn-β2,64/2,462.57 m/2,4536,9636,25
Asn-γ-C'--171,73171,44
Asn-δ-NH27,41/6,937,33/6,93--
Ala-NH7,748,02--
Ala-α4,194,2748,7148,46
Ala-C'--171,84171,78
Ala-β1,221,1917,4918,04
Val-NHof 7.487,70--
Val-α4,044,0857,6957,57
Val-C'--172,77172,73
Val-β1,971,9730,0830,29
Val-γ0,820,8619,2419,27
Val-γ'0,820,8417,8917,97
Val-NH27,15/6,997,27/7,00--

2.3. N-succinyl-L-(2-naphthyl) alanyl-L-asparaginyl-L-serinol-L-valinol-glycine-3-hydroxypropylamino (24)/p>

2.3.1. Benzyloxycarbonyl-glycine-(3-propanol)amide (C1)

627 g (30 mmol) Gly-OH, of 2.45 ml of 3-amino-1-propanol and of 4.05 g of HOBt are dissolved in 60 ml of DMF. Further, when 0°With added 6.6 g DCC. The mixture is stirred for 1 hour at 0°and for 3 hours at room temperature, and then incubated at room temperature overnight. The formed precipitate is filtered off with suction, and the filtrate concentrated under high vacuum. The residue is distributed between ethyl acetate and a solution of NaHCO3. 3 Quiroga organic phase is washed with a solution of NaHCO3and a mixture of H2O/NaCl, dried using Na2SO4and concentrate. The residue is triturated with diethyl ether.

Output: 7,05 g (88,2%).

2.3.2. Benzyloxycarbonyl-glycine-(3-propanol tert-butyl ester)amide (C2)

7 g (26,28 mmol) benzyloxycarbonyl-glycine-(3-propanol)amide (C1) is dissolved in 60 ml of dioxane. At low temperature (liquid CO2slowly add 6 ml of N2SO4. Then add 60 ml of condensed isobutylene. This mixture is shaken in an autoclave at room temperature under a nitrogen pressure of about 20 bar within 3 days. 3 Quiroga mixture is stirred with diethyl ether and extracted three times 2 N. a solution of Na2CO3. The aqueous solution was washed with diethyl ether. Next, organic the ski phases unite, washed with water, dried using Na2SO4and concentrate.

Output: 7.98 g (94,2%)

2.3.3. Glycine-(3-propanol tert-butyl ester) amide hydrochloride (C3)

7.98 g (24,75 mmol) benzyloxycarbonyl-glycine-(3-propanol tert-butyl ester) amide (C2) is dissolved in 80 ml Meon, mixed with Pd on coal and hydrogenizing in autotitrator using a methanolic solution of HCl and H2. 3 Quiroga catalyst is filtered off with suction and the filtrate is concentrated. The resulting residue is dried under high vacuum.

Yield: 4.7 g (84,5%).

2.3.4 Benzyloxycarbonyl-L-valine-glycine-(3-propanol tert-butyl ester)amide (C4)

5,13 g (20,43 mmol) benzyloxycarbonyl-Val-OH, 4.59 g (20,43 mmol) glycine-(3-propanol tert-butyl ester) amide hydrochloride (C3) and 2.75 g of HOBt are dissolved in 60 ml of DMF. Next add at 0°From 2.65 g of N-ethylmorpholine and 4.5 g DCC. The resulting mixture was stirred at 0°C for 1 hour and then at room temperature for 2 hours. Next, the mixture was kept at room temperature overnight, then concentrated under high vacuum. The residue is partitioned between glacial acetic acid and a solution of NaHCO3. 3 Quiroga phase of glacial acetic acid is washed with a solution of NaHCO3, solution of KHSO4and a mixture of H2O/NaCl, dried using Na2SO4and concentrate. Solid estato is triturated with diethyl ether and filtered off with suction.

Output: to 7.32 g (85%).

2.3.5. L-valine-glycine-(3-propanol tert-butyl ester) amide hydrochloride (C5)

7,29 g (17.3 mmol) benzyloxycarbonyl-L-valine-glycine-(3-propanol tert-butyl ester) amide (C4) is dissolved in 90 ml Meon, mixed with Pd on coal and hydrogenizing in autotitrator using a methanolic solution of HCl. 3 Quiroga catalyst is filtered off with suction, and the filtrate concentrated. The residue (amorphous) is dried under high vacuum, triturated with diethyl ether and filtered with suction.

Output: 5,22 g (93,2%).

2.3.6. Benzyloxycarbonyl-L-serine(tert-butyl ether)-L-valine-glycine-(3-propanol tert-butyl ester)amide (C6)

5,46 g (18.5 mmol) of Z-Ser(But)HE, 6 g (18.5 mmol) of L-valine-glycine-(3-propanol tert-butyl ester) amide hydrochloride (C5) and 2.5 g of HOBt are dissolved in 60 ml of DMF. 3 Quiroga at 0°add 2.4 ml of N-ethylmorpholine and 4,07 g DCC. The resulting mixture was stirred at 0°C for 1 hour and then at room temperature for another 3 hours. Next, this mixture stand at room temperature overnight, then concentrated under high vacuum. The obtained solid residue partitioned between glacial acetic acid and a solution of NaHCO3. 3 Quiroga phase of glacial acetic acid is washed with a solution of NaHCO3, solution of KHSO4and a mixture of H2O/NaCl, and then dried using Na2SO and concentrated, the Residue triturated with diethyl ether and filtered off with suction.

Output: 9,74 (93,2%)

2.3.7. L-serine(tert-butyl ether)-L-valine-glycine-(3-propanol tert-butyl ester) amide hydrochloride (C7)

9,74 g (17,25 mmol) benzyloxycarbonyl-L-serine(tert-butyl ether)-L-valine-glycine-(3-propanol tert-butyl ester) amide (C6) is dissolved in about 100 ml of the Meon, mixed with Pd on coal and hydrogenizing in autotitrator using a methanolic solution of HCl. Then the catalyst is filtered off with suction, and the filtrate concentrated. The residue (amorphous) is dried under high vacuum, triturated with diethyl ether and filtered off with suction.

Output: 8,02 g (99,6%).

2.3.8. Benzyloxycarbonyl-L-asparagine-L-serine(tert-butyl ether)-L-valine-glycine-(3-propanol tert-butyl ester)amide (C8)

a 4.53 g (17 mmol) of Z-Asn-OH, 7,94 g L-serine(tert-butyl ether)-L-valine-glycine-(3-propanol tert-butyl ester) amide hydrochloride (C7) and 2.3 g of HOBt are dissolved in 60 ml of DMF. When 0°type of 2.21 ml of N-ethylmorpholine and 3.74 g DCC. The resulting mixture was stirred at 0°C for 1 hour and then at room temperature for 3 hours, then concentrated under high vacuum. The resulting residue is distributed between pentanol and solution of NaHCO3. Pentelow phase is washed with a solution of NaHCO3, solution of KHSO 4and a mixture of H2O/NaCl, and then dried with Na2SO4and filtered with suction, and the filtrate concentrated under high vacuum. The residue is triturated with diethyl ether, cooled and filtered with suction. The product is dried in a desiccator over P2O5.

Yield: 10.8 g (93,6%).

2.3.9. L-asparagine-L-serine(tert-butyl ether)-L-valine-glycine-(3-propanol tert-butyl ester) amide hydrochloride (C9)

10.8 g (15.9 mmol) benzyloxycarbonyl-L-asparagine-L-serine(tert-butyl ether)-L-valine-glycine-(3-propanol tert-butyl ester) amide (C8) is dissolved in approximately 160 ml of warm Meon, mixed with Pd on coal and hydrogenizing in autotitrator using a methanolic solution of HCl. 3 Quiroga catalyst is filtered off with suction, and the filtrate concentrated. The amorphous residue is dried under high vacuum, triturated with diethyl ether, cooled and filtered with suction.

Output: 8,96 g (97%).

2.3.10. Benzyloxycarbonyl-L-2-nafcillin-L-asparagine-L-serine(tert-butyl ether)-L-valine-glycine-(3-propanol tert-butyl ester)amide (C10)

of 5.24 g (15 mmol) benzyloxycarbonyl-2-Nal-OH, 8,72 g (15 mmol) of L-asparagine-L-serine(tert-butyl ether)-L-valine-glycine-(3-propanol tert-butyl ester) amide hydrochloride (C9) and 2,04 g of HOBt are dissolved in 60 ml of DMF. 3 Quiroga at 0°add 1,95 ml of N-ethylmorpholine is 3.3 grams DCC. The resulting mixture was stirred at 0°C for 1 hour and then at room temperature for another 3 hours. Next, this mixture stand at room temperature overnight, diluted with DMF and a little heat. The formed precipitate is filtered off with suction, and the filtrate concentrated under high vacuum. The resulting residue is triturated with a solution of NaHCO3and filtered with suction, and then triturated with a solution of KHSO4, filtered with suction, triturated with N2O, filtered with suction, washed with N2O and dried in a desiccator over P2O5.

Output: 13,25 g (>99%).

2.3.11. L-2-nafcillin-L-asparagine-L-serine(tert-butyl ether)-L-valine-glycine-(3-propyl alcohol-tert-butyl ester)amide hydrochloride (C11)

cent to 8.85 g (10.1 mmol) benzyloxycarbonyl-L-2-nafcillin-L-asparagine-L-serine(tert-butyl ether)-L-valine-glycine-(3-propanol tert-butyl ester) amide (C10) partially dissolved in 270 ml Meon, mixed with Pd on coal and hydrogenizing in autotitrator using a methanolic solution of HCl. The resulting suspension was diluted with DMF. After about 6 hours the mixture is concentrated to half the original volume. All material is dissolved. The mixture was incubated at room temperature overnight. Then the catalyst is filtered off with suction, and Phil is spending diluted with the same amount Meon, mix with the new catalyst (Pd on coal) and again hydrogenizing on autotitrator. After 7 hours the mixture is placed at night in conditions of room temperature. This mixture hydrogenizing for 4 hours, the catalyst is filtered off with suction, and the filtrate concentrated. The residue (amorphous) sumat under high vacuum, triturated with diethyl ether and filtered off with suction.

Output: 7,56 g (96,2%).

2.3.12. N-tert-butyl-succinyl-L-2-nafcillin-L-asparagine-L-serine-L-valine-glycine-(3-propanol)amide (C12)

523 mg (3 mmol) of L-2-nafcillin-L-asparagine-L-serine(tert-butyl ether)-L-valine-glycine-(3-propanol tert-butyl ester) amide hydrochloride (C11), 2,33 g mono-tert-butylacrylate (B4) and 405 mg of HOBt are dissolved in 20 ml of DMF. When 0°add to 0.39 ml of N-ethylmorpholine and 660 mg DCC. The resulting mixture was stirred at 0°C for 1 hour and at room temperature for 2 hours, then incubated at room temperature overnight. This mixture concentrated under high vacuum, the solid residue triturated with a solution of NaHCO3and filtered with suction. 3 Quiroga received the product triturated with a solution of KHSO4, is filtered off with suction, washed with N2O and dried in a desiccator over P2O5.

Output: 3.04 from g (crude product).

2.3.13. N-succinyl-L-2-nafcillin-L-and Paragon-L-serine-L-valine-glycine-(3-propanol)amide (C24)

3 g (crude product) of N-tert-butyl-succinyl-L-2-nafcillin-L-asparagine-L-serine-L-valine-glycine-(3-propanol) amide (C12) is dissolved in 30 ml of 90% triperoxonane acid and all stand at room temperature for 1 hour. 3 Quiroga this mixture concentrated and the resulting residue triturated with diethyl ether and filtered off with suction. This procedure leads to obtain 2.6 g of the crude product. For purification of 250 mg of the specified product is dissolved in warm glacial acetic acid and chromatographic on Sephadex (Sephadex® LH20) using a mixture of butanol/glacial acetic acid/water.

Yield: 103 mg

m/z 730,341246 (M+N)+(the mass spectrum of the high-resolution)

Data of NMR for compound 24:

Chemical shifts for compound 24 in DMSO at 300 K:

td align="center"> 171,20
Cm. the formula above1H13C
But-1-173,71
But-2to 2.2928,99
But-32,32/of 2.2629,91
But-4-171,15
Nap-NH8,19-
Nap-α4,6053,92
Nap-C'-
Nap-β3,19/2,9137,56
Nap-1-135,62
Nap-27,42127,85
Nap-37,80127,30
Nap-3a-131,75
Nap-4the 7.85127,38
Nap-57,44125,31
Nap-67,47125,83
Nap-77,82127,38
Nap-7a-132,91
Nap-87,73127,38
Asn-NH8,35-
Asn-α4,6049,73
Asn-C'-170,92
Asn-β2,60/2,4937,03
Asn-γ-C'-171,73
Asn-δ-NH27,44/6,99-
Ser-NH7,89-
Ser-α4,3355,15
Ser-C'-170,13
Ser-β3,66/3,5661,52
Ser-OHis 4.93-
Val-NH 7,82-
Val-α4,1058,37
Val-C'-171,02
Val-β2,0429,91
Val-γ0,8619,17
Val-γ'0,8618,11
Gly-NHof 8.06-
Gly-αthe 3.6541,97
Gly-C'-168,45
Xxx-NH7,63-
Xxx-2'3,1035,78
Xxxx-3'1,5432,24
Xxx-4'3,40with 58.33
Xxx-4'-HE4,40-

3. Inhibition of the interaction of laminin/nitogen and biological activity

Unless specifically stated otherwise used chemicals are purchased from Merk (Merk (Darmstadt), Sigma (Sigma (Munich)or Riedel de Hine (Riedel de Haen (Seelze)).

Selection laminina P1 from human placenta, human Neogene from transfected SOME 293 cells and mouse laminin γ1 III 3-5 of SOME 293 cells described in document WO 98/31709.

Example 3.1

Test for inhibition of the detection of inhibition of binding of l is Minin/nidorina peptide derivatives

3.1.1. HTS-screening (high-sensitivity version of the test):

The method of fluorescence analysis of the process of dissolution over time

The coating on the inspected tubes

Microtiter plates (for example, FluoroNunc®) cover 75 μl of 0.1 μg/ml solution of laminin P1 (strength of 0.159 g of Na2CO3, 0,293 g NaHCO3, 0.02 g NaN3/l, pH of 9.2) at room temperature for 1 hour. 3 Quiroga solution dig, and free binding sites blocked by incubation with 0.5% BSA (in a medium containing 7.9 g NaCl, 1.2 g of Na2HPO4, 0.31 g of KCl, 0,23 g NaH2PO4, 0.04% tween-20/l, pH of 7.2) at room temperature for 0.5 hours. Upon completion of the reaction block hold the decantation of the solution and washed tablets once with 250 μl of wash buffer (FBI/0.04% twin).

Sequential inhibition

In parallel with the application of the sample onto the tablet perform preliminary incubation 85-100 μl of 0.25 nm solution of Neogene (obtained by recombinant method of human Neogene) inhibitor or standard in a separate reaction vessel at room temperature for 1 hour in medium containing 7.9 g NaCl, 1.2 g of Na2HPO4, 0.31 g of KCl, 0,23 g NaH2PO4, 0.04% tween-20/l, 0.5% BSA, pH 7,2).

75 μl Preincubation environment (Neogen +inhibitor or standard) is transferred to the filled cells microtiter plansheta incubated at room temperature for 1 hour. After this is performed twice with a wash using FBI/0.04% twin.

Detection of bound Neogene performed during incubation (at room temperature) for 1 hour with 75 µl of specific drug antibodies derived from chicken egg yolks are immunized with human nidorina. The IgY fraction is used in the dilution 1:500 in a mixture FBI/0.04% twin. The complex of Neogene and specifically related antibodies after washing twice FBI/0.04% reveal twin adding Antiquing IgY-Biotin (75 ál dilution 1:2500; Promega, Madison, WI 53711, 608-274-4330). With this purpose, after incubation for 1 hour and washing twice with a mixture of FBI/0.04% twin carry out incubation with streptavidin-europium (Wallac, 1 hour at room temperature) and washed twice with a mixture of FBI/0.04% twin. In the end, it becomes possible after adding 100 ál of the amplifying solution (Wallac) and stirring for 5 minutes to measure the fluorescent signal on the meter Victor for counting multiple labels using the discovery Protocol europium. Determine the relationship between the number of associated Neogene in solutions with inhibitor and Neogene without added inhibitor.

3.1.2. A three-day analysis to balance

Selected inhibitors examined for their inhibitory activity in the specified case analysis. The analysis is described in U.S. patent 5493008.

Given lower the table gives comparative values IR 50selected substances together with the results of the HTS-screening. They show that the 3-day analysis gives a somewhat lower value and, as expected, it is more sensitive than the screening test. However, what is also clear from the above comparison, inhibiting patterns can be reliably identified when conducting the screening analysis, developed by the authors.

Characteristics of specific inhibitors of the Association of laminin/nitogen; values IR50(μm) in different studies
StructureHTS test3-day analysis to balance
NIDPNAVa 3.91,2
DPNAV7,75,0
Connection 240,360,09
The connection 310,190,085

Example 3.2 (hypothetical)

Testing the biological activity of peptide derivatives

Several models that have been described in the literature, can be used to determine biological activity of peptide derivatives.

Below are some representative works:

Formation of tubuli in cultures of embryonic kidneys (the Formation of tubules in cultures of embryonic p. the check).

Grobstein, C. (1956) Exp. Cell Res. 10: 424-440.

Ekblom, P. et al. (1994) Development 120: 2003-2014.

Branching morphology in embryonic lungs. (Ramified morphology embryonic lung).

Grobstein, C. (1953) J. Exp. Zool. 124: 383-413.

Kadoya, Y. et al., (1997) Development 124: 683-691.

Basement membrane assembly in a organotypic skin culture. (Build basal membrane in organotypic culture of the skin).

Smola, H., Stark, H.-J, Thiekötter, G., Mirancea, N., Krieg, T., Fusenig, N.E. (1998) Exp. Cell. Res. 239: 399-410.

Reconstitution of hydra from disintegrated cells. (Recovery Hydra from disintegrated cells).

Yang, Y.G., Mayura, K., Spainhour, C.B., Edwards, Jr., J.F., Phillips, T.D. (1993) Toxicology 85: 179-198.

Thickening of basement membranes in hydra after culturing at increased glucose concentration.(Thickening bazalnich membranes the Hydra after cultivation with high concentration of glucose).

Zhang, X.; Huff, J.K.; Hudson, B.G.; Sarras Jr.; M.P. (1990) Diabetologia 33: 704-707.

All kinds of quantitative analysis of angiogenesis summarized in a review article Jain, R.K. et al., Nature Medicine (1997) Vol.3, No.11, for example:

Induction of Haemangiomes in mice by implantation of cells from spontaneous hemangioendotheliomes,

O'reilly, M.S., Brem -, M.S., Folkman, J. (1995) J. Pediatr. Surg. 30:2, 325-329.

Growth of micro-vessels in a serum-free culture of rat aorta,

Nicosia, R.F., Ottinetti, A. (1990) Lab. Invest Vol.63, No.1, 115-122.

Formation of capillaries of endothelic cells on micro-carriers after imbedding into a fibrin gel

Nehls, V.; Drenckhahn, D. (1995) Microvascular Research 50: 311-322.

1. The compound of the formula I

in which R1 denotes a group one of the following formulas

oror

where Z represents -(CH2)m;

R4denotes-A, -NH2,

and R5represents -(CH2)1Sooa, (CH2)1CONH2, -(CH2)1NH2or

or

X denotes a group described by one of the following formulas:

where D denotes (CH2r and

R2denotes-A-S-HE, where E denotes a linear or branched C1-C10is an alkyl chain,

and R3represents a group of one of the following formulas

or

or

where R6 denotes

and where R7 represents a linear or branched C1-C10is an alkyl group which may be unsubstituted or substituted groups, -(CH2)mHE

and R denotes Het or Ar, which optionally can be substituted by-O-C1-C6-alkyl-Ar or Ar, where Het denotes a monocyclic or bicyclic the conglomerate, 5-10-membered aromatic or nonaromatic ring containing 1 or 2 identical or different heteroatoms as members of the specified ring selected from the group consisting of nitrogen and sulfur, and where

Ar denotes a monocyclic or bicyclic, 5-to 10-membered aromatic ring,

Z represents (CH2)m;

And denotes N or C1-C4-alkyl;

l, m and r represent integers from 0 to 3;

n and k denote integers from 1 to 2;

p denotes an integer from 0 to 1;

q denotes an integer from 1 to 3,

including their physiologically tolerant of salt.

2. The compound of formula I according to claim 1, wherein n denotes 1.

3. The compound of formula I according to claim 1 or 2, characterized in that R in the group X denotes Het.

4. Pharmaceutical composition having the ability to inhibit the interaction of laminin/nidorina, characterized in that it contains at least one compound of the preceding claims 1 to 3 and/or its physiologically tolerant of salt.

5. A method of obtaining a pharmaceutical composition, comprising mixing the compound according to claim 1 and/or physiologically tolerant of salt with a pharmaceutically acceptable carrier.



 

Same patents:

FIELD: organic chemistry, peptides, medicine, pharmacy.

SUBSTANCE: invention relates to peptide derivatives named as memnopeptides that are used as an active component for manufacturing a medicinal preparation used in treatment of bacterial infection. Invention proposes compound of the formula (I): wherein radicals R1, R2, R3, R4, R5, R6, R7, R8 and (A)n have corresponding values, or its salt. Compounds of the formula (I) are prepared by culturing microorganism Memnoniella echinata FH 2272, DSM 13195 under suitable conditions in the nutrient medium containing at least one source of carbon atoms and at least one source of nitrogen atoms and the process is carrying out until the accumulation of at least one compound of the formula (I) in the nutrient medium followed by isolation of indicated compound. The attained technical result involves the development of a pharmaceutical composition eliciting an antibacterial activity. The development of the preparation provides expanding assortment of agents used in treatment of diseases said above.

EFFECT: improved preparing method, valuable medicinal properties of compounds.

10 cl, 2 tbl, 7 ex

Oligopeptides // 2260597

FIELD: organic chemistry, peptides, biochemistry.

SUBSTANCE: invention describes oligopeptide or its salt taken among the group consisting of oligopeptide (1) and (2): Lys-Ser-Ile-Glu-Gln-Ser-Cys-Asp-Gln-Asp-Glu (I), Ser-Ile-Glu-Gln-Ser-Cys-Asp-Gln-Asp-Glu (II); Ser-Ile-Glu-Gln-Ser-Cys-Asp-Gln-Asp (III); Ser-Ile-Glu-Gln-Ser-Cys-Asp-Gln (IV); Ser-Ile-Glu-Gln-Ser-Cys-Asp (V); Ser-Ile-Glu-Gln-Ser-Cys (VI); Ile-Glu-Gln-Ser-Cys-Asp-Gln-Asp-Glu (VII); Glu-Gln-Ser-Cys-Asp-Gln-Asp-Glu (VIII); Gln-Ser-Cys-Asp-Gln-Asp-Glu (IX); 2) oligopeptide with amino acid sequence obtained by deleting by C- or N-end of one or some amino acids in any amino acid sequence (I)-(IX), and the modified oligopeptide representing oligopeptide biotinylated or dimerized by sulfhydryl group of cysteine residue based on oligopeptide determined in (1) or (2). Oligopeptides elicit activity with respect to hair growth stimulation.

EFFECT: valuable properties of oligopeptides.

11 cl, 6 dwg, 4 ex

FIELD: bioorganic chemistry.

SUBSTANCE: invention provides somatostatin agonists of general formula: A1-cyclo{Cys-A2-D-Trp-A3-A4-Cys}-A5-Y1 (I), wherein A1 represents aromatic D- or L-α-amino acid selected from Phe, D-Phe, Tyr, D-Tyr, β-Nal, D-β-Nal, Cha, or D-Cha; A2 aromatic α-amino acid selected from Phe, Tyr, β-Nal, and Cha; A3 Lys or Orn; A4 β-hydroxyvaline, Ser, hSer, or Thr; A5 β-hydroxyvaline, Ser, hSer, or Thr; and Y1 represents NH2; amide nitrogen atoms of peptide groups and amine group of A1 in compound I are optionally substituted by methyl group, provided that at least one methyl group is available and that compound I cannot have following formula: D-Phe-cyclo{Cys-Phe-D-Trp-Lys-(N-Me-Thr)-Cys}-Thr-NH-2. pharmaceutically acceptable salts of compound I are also claimed.

EFFECT: expanded synthetic possibilities in peptide synthesis.

24 cl, 2 tbl, 18 ex

The invention relates to the field of biotechnology, specifically to peptides active in the attachment, respectiveiy and detaching cells, and can be used to study activity in the attachment of cells, mediated by different proteins of the extracellular matrix, to develop peptides, adhesion, and also for medicinal purposes

The invention relates to peptide compositions with delayed release, representing a compound (I) containing the compound (A) formula

and the polymer containing lactide links, glycolide links and links tartaric acids - which are found in the polymer at the next sootnoshenii: lactide units constitute from about 71% to about 73%, glycolide links from about 26% to about 28%; and the parts of tartaric acid from about 1% to 3%, and the amino group of the compound (a) relate ionic bond with the carboxyl groups of the acid units of the polymer; the particles of compound I, an average size of from about 10 microns to about 100 microns; pharmaceutical composition with delayed release and two methods of treatment of various diseases, including the introduction to the patient an effective amount of compound A, or microparticles

The invention relates to medicine and relates to methods of treatment of tumors and metastases using a combination of antiangiogenic therapy and immunotherapy

The invention relates to new peptides having the amino acid sequence of the 9-55 amino acid residues, comprising the amino acid sequence FTLASAETT (SEQ ID NO:l), pharmaceutical compositions for treatment of autoimmune diseases, containing one or more of these peptides and a pharmaceutically acceptable carrier

FIELD: organic chemistry, medicine.

SUBSTANCE: invention represents ligands MC-4 and/or MC-3 of the formula (I): , wherein X means hydrogen atom, -OR1, -NR1R1' and -CHR1R1' wherein R1 and R1' are taken among the group: hydrogen atom, (C1-C6)-alkyl and acyl; (1) each R2 is taken independently among the group: hydrogen atom, (C1-C6)-alkyl; or (2) (a) R2 bound with carbon atom that is bound with X and Z1 and substitute R5 can be optionally bound to form carbocyclic or heterocyclic ring that is condensed with phenyl ring J; or (b) R2 bound with carbon atom that is bound with ring Ar can be bound with R7 to form ring condensed with ring Ar; each among Z1, Z2 and Z3 is taken independently from the following groups: -N(R3e)C(R3)(R3a)-, -C(R3)(R3a)N(R3e)-, -C(O)N(R3d)-, -N(R3d)C(O)-, -C(R3)(R3a)C(R3b)(R3c)-, -SO2N(R3d)- and -N(R3d)SO2- wherein each among R3, R3a, R3b and R3c, R3d, R3e when presents is taken independently among hydrogen atom and (C1-C6)-alkyl; p is a whole number from 0 to 5 wherein when p above 0 then R4 and R4' are taken among hydrogen atom, (C1-C6)-alkyl and aryl; R5 represents 5 substitutes in phenyl ring J wherein each R5 is taken among hydrogen atom, hydroxy-, halogen atom, thiol, -OR12, -N(R12)(R12'), (C1-C6)-alkyl, nitro-, aryl wherein R12 and R12' are taken among hydrogen atom and (C1-C6)-alkyl; or two substitutes R5 can be bound optionally to form carbocyclic or heterocyclic ring that is condensed with phenyl ring J; q = 0, 1, 2, 3, 4 or 5 wherein when q above 0 then R6 and R6' are taken among hydrogen atom and (C1-C6)-alkyl; Ar is taken among the group consisting of phenyl, thiophene, furan, oxazole, thiazole, pyrrole and pyridine; R7 are substitutes at ring Ar wherein each R7 is taken among hydrogen, halogen atom, -NR13R13', (C1-C6)-alkyl and nitro- wherein R13 and R13' are taken among hydrogen atom and (C1-C6)-alkyl; r is a whole number from 0 to 7 wherein when r is above 0 then R8 and R8' are taken among hydrogen atom and (C1-C6)-alkyl; B is taken among -N(R14)C(=NR15)NR16R17, -NR20R21, heteroaryl ring and heterocycloalkyl ring wherein R14-R17, R20 and R21 are taken independently among hydrogen atom and (C1-C6)-alkyl; s = 0, 1, 2, 3, 4 or 5 wherein when s is above 0 then R and R9' are taken among hydrogen atom and (C1-C6)-alkyl; R10 is taken among the group consisting of optionally substituted bicyclic aryl ring and optionally substituted bicyclic heteroaryl ring; D is taken among hydrogen atom, amino- and -C(O)R11 wherein R11 is taken among the following group: hydroxy-, alkoxy-, amino-, alkylamino-, -N(R19)CH2C(O)NH2 wherein R19 represents (C1-C6)-alkyl, -NHCH2CH2OH and -N(CH3)CH2CH2OH, or its isomers, salts, hydrates or biohydrolysable ester, amide or imide.

EFFECT: valuable medicinal properties of compounds.

18 cl, 107 ex

FIELD: medicine, immunology, peptides.

SUBSTANCE: invention relates to a new composition of biologically active substances. Invention proposes the composition comprising of peptides of the formula: Arg-Gly-Asp and H-Tyr-X-Y-Glu-OH wherein X means Gln and/or Glu; Y means Cys(acm) and/or Cys that elicits ability to inhibit the proliferative response for phytohemagglutinin, to induce the suppressive activity of mononuclear cells and ability of peptides to induce secretion of immunosuppressive cytokines of grouth-transforming factor-β1 and interleukin-10 (IL-10). The composition can be prepared by a simple procedure.

EFFECT: valuable biological properties of composition.

3 cl, 16 tbl, 9 ex

The invention relates to compounds of the prodrugs of inhibitors dipeptidylpeptidase IV (DP IV) the General formula a-b-C, and And denotes the amino acid refers to a chemical bond between a and C or the amino acid and stable inhibitor of DP IV with the missing C-terminal phosphonate residue, which represents AMINOETHYLPIPERAZINE, aminoacetanilide or N-dipeptidyl, O-arylhydroxylamine

The invention relates to compounds of unstable inhibitors dipeptidylpeptidase IV (DP IV), which have the General formula a-b-C, where a denotes an amino acid, A denotes a chemical bond between a and C or the amino acid and C indicates an unstable inhibitor of DP IV, namely the derived dipeptidylpeptidase, except derived peralkylated as derived dipeptidylpeptidase, the rest of peptidylglycine, dipeptidase or dipeptidylpeptidase

The invention relates to a synthetic amide of opioid peptide or its pharmaceutically acceptable salt having affinity against-opioid receptor, which is at least 1000 times greater than its affinity in relation to-opioid receptor, and which reveals long-term effect when introduced in vivo, and this peptide has the following formula:

H-Xaa1-Xaa2-Xaa3-Xaa4-Q

where Xaa1shown are (A)D-Phe,D-Tyr, D-Tic, or D-Ala (cyclopentyl or thienyl), and A - N, NO2, F, Cl or CH3; Xaa2(A')D-Phe, D-1Nal, D-2Nal, D-Tyr or D-Trp, where A' - or 3,4 Cl2; XAA3- D-Nle, (B)D-Leu, D-Hle, D-Met, D-Val, D-Phe or D-Ala (cyclopentyl), and In - N or CMe; XAA4represents the D-Arg, D-Har, D-nArg, D-Lys, D-i.l.y bit, D-Arg (Et2), D-Har(Et2), D-Amf, D-Gmf, D-Dbu, D-Orn or D-Ior; a Q - NR1R2morpholinyl, thiomorpholine, (C)piperidinyl, piperazinil, 4-one or 4,4-disubstituted piperazinil or-lysyl, where R1is lower alkyl, substituted lower alkyl, benzyl, substituted benzyl, and the laminitis)-polymethene or 4-polyoxyethylene group, a R2is H or lower alkyl; C - H, 4-hydroxy or 4-oxo
The invention relates to a group of new protected linear peptides containing the amino acid sequence, which can be used as starting compounds to obtain RGD-containing cyclopeptides, the General formula

R3-Arg-Gly-Asp(OR1)-OR2,

where R1is benzyl or tert-butyl; R2not equal to R1and is selected from the group of tert-butyl; benzyl; 4-methoxybenzyl; 4-nitrobenzyl; diphenylmethyl; 2,2,2-trichloroethyl; 2,2,2-trichloro-1,1-dimethylethyl; allyl; 9-fluorenylmethyl; carboxamidine; substituted 2-sulfonylated type AND-SO2-CH2-CH2- where a is substituted or unsubstituted phenyl or benzyl; R3is a hydrogen atom or a urethane protective group of the B1O-CO-, where1not equal to R1and can take values tert-butyl, benzyl, 4-methoxybenzyl, 9-fluorenylmethyl, 2-(4-nitrophenyloctyl)ethyl; or is a peptidyl containing from one to three amino acid residues; and the peptides, where R3- peptidyl structure E-Z-Y-X-, in which E is a hydrogen atom or a urethane protective group IN2O-CO-, where2not equal to R1and can take values tert-butyl; benzyl; 4-methoxybenzyl; 9-fluorenylmethyl; 2-(4-nitrophen is IN3O-CO-, in which3= R1attached to the omega-amino group; Z can take values Phe or D-Phe

The invention relates to Bioorganic chemistry and pharmacology, namely the tools, potentiating the analgesic effect of morphine hydrochloride

The invention relates to a new compound - tritium-labeled tartino and method for determining taphrina in biological samples, including the introduction of pre in the sample vysokobarnogo tritium captina, extraction, transformation into the prepared extract captina and its labeled analog in them benzoic or ortho-derivative, which is then analyzed by HPLC with fluorescence detection

The invention relates to peptides-immunoregulators and their therapeutic use

The invention relates to compounds of the formula Y-(CR2)n-X-NHJ, where X represents C=O or CR2; n is an integer having a value of from 1 to 6; Y is an L(A)mor R1R2CR-, where L - metallocomplexes agent, And represents-CR2-, -NRCO-, -CONR - or polyalkyleneglycol; m is an integer having a value of from 0 to 10, where one of R1and R2is-NH(B)pZ1and the other is-CO(B)qZ2where p and q are integers having a value of from 0 to 20, and each independently selected from Q or amino acid residue, where Q is a cyclic peptide; Z1and Z2- protective groups which are biocompatible group which inhibits or suppresses the metabolism of the peptide in vivo; J and each R group is independently selected from H, C1-4the alkyl or C1-4alkoxyalkyl; provided that (i) the total number of amino acid residues in R1and R2the group does not exceed 20; (ii) if X is CR2then Y is-CRR1R2and Z2is metallocomplexes agent; (ii) if Y is-CRR1R2then at least one of R1and R2carries at least one frequent detektiruya
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