-ketoamide inhibitors of 20s proteasome

 

(57) Abstract:

Describes new compounds of General formula I

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where X2- Ar or Ar - X3where X3represents-C=O or-CH2CO - and Ar represents a phenyl or indole; R1and R2each independently selected from hydrogen or a side chain of natural amino acids, are represented by linear or branched (C1-C10)alkyl which may be substituted by hydroxy, carboxy, amino, cyclohexyl, phenyl or replacement of the phenyl, biphenyl, naphthyl or pyridium; X1chosen because HE or groups

< / BR>
where X4is a HE; R3selected from hydrogen or a side chain of natural amino acids, are represented by linear or branched (C1-C10)alkyl which may be substituted by hydroxy, phenyl or replacement of the phenyl. It also describes a pharmaceutical composition having the ability to inhibit chymotrypsinogen action 20S proteasome comprising compound I and one or more pharmaceutical excipients, presented in the form of a solution or tablets. 2 C. and 13 C.p. f-crystals, 2 tab.

Background of the invention

Multicatalytic the state for ATP-dependent proteolysis of the majority of cellular proteins (Soy, O., Tanaka, K. and Goldberg, A. 1996 Ann. Rev. Biochem. 65, 801-847). 20S proteasome contains the catalytic core of the complex and was bicrystalline from archaebacteria Thennoplasma acidophilum (Lowe, J., Stock, D., Jap, B., Zwicki, P., Bauminster, W. and Huber, R. 1995 Science 268, 533-539) and Saccharomyces cerevisiae (Groll, M., Ditzel, L., Lowe, J., Stock, D., Bochtler, M., Bartunik HD and Huber, R. 1997 Nature 386, 463-471). Unlike archaebacterial proteasome, which primarily shows chymotrypsinogen proteolytic activity (Dahhnann, C., Corr, F., Kuehn, L., Niedel. C., Pfeifer, G. 1989 FEBS Lett. 251, 125-131; Seemuller, E., Lupas, A., Zuw, F., Zwicki, P and Baumeister, W. FEBS Lett. 359, 173, (1995), eukaryotic proteasome performs at least five identifiable proteolytic activities. Three of these activities are similar in specificity to chymotrypsin, trypsin and peptidylglutamyl. The other two described activity show a preference for cleavage of peptide bonds on the carboxyl end of the branched chain amino acids (UGAR) and in relation to the peptide linkages between neutral amino acids with short-chain (SnAAP) (Orlowski, M. 1990 Biochemistry 29, 10289-10297).

Although the 20S proteasome contains a proteolytic core, it does not destroy the proteins in vivo, if is not in the form of a complex with 19S "cap" on both sides of its structure, which manifests itself Outrushed proteins, subject to destruction, adding a variety of polypeptide molecules with a mass of 8.5 kDa, ubiquitin (presented in Soy, O., Tanaka, K. and Goldberg, A. 1996 Ann. Rev. Biochem. 65, 801-847).

A large number of functional groups of the substrate were used as potential inhibitors of serine and thiol proteases. Several of these motifs have been described as inhibitors of the proteasome. These include peptide aldehydes (Vinitsky, A., Michaud, S. Powers, J. and Orlowski, M. 1992 Biochemistry 31, 9421-9428; Tsubuki, 3., Hiroshi, K., Saito, Y. Miyashita, N., Inomata, M. and Kawashima, S. 1993 Biochem. Biophys. Res. Commun. 196, 1195-1201; Rock, K., I., Gramm, C., Rothstein, L., Clark, K., Stein, R., Dick, L.,Hwang, D. and Goldberg, A. L. (1994) Cell 78, 761-771)), N-acetyl-L-leucinol-L-leucinol-L-norleucinal (ALLN) and N-acetyl-L-leucinol-1-Latinamerican (LLM), the most potent inhibitor of this type is N-carbobenzoxy-1-L-leucinol-L-leucinol-L-Norvaline (MG115). Other reports described a number of dipeptide inhibitors that have values IC50in the range from 10 to 100 nm (Iqbal, M., Chatterjee, S., Kauer, J. C., Das, M., Messina, P. , Freed, B. , Biazzo, W. and Siman, R. 1995 I-Med. Chem. 38, 2276-2277). As strong inhibitors of the proteasome has also been described by a number-Methocarbamol and breefing derivatives of dipeptides (Iqbai, M., Chatterjee, S., Kauer, J. C., Mallamo, J. P., Messina, P. A., Reiboldt, A. and Siman, R. 1996 Bloorg. Med-Chem. Lett 6, 287-290) and apotential, that shows specificity in the inhibition of proteasome activity is lactacystin (Fenteany, G., Standaert, R. F. , Lane, W. S., Choi, S., Corey, E. J. and Schreiber, S. L. 1995 Science 268, 726-731), which is a metabolite of Streptomyces. This molecule was originally discovered by its ability to stimulate axonal growth in cell lines of neuroblastoma (Omura, S., Matsuzaki, K., Fujimoto, T., Kosuge, K., Furuya, T., Fujita, S. and Nakagawa, A. 1991 J. Antibiot. 44, 117-118), and later it was demonstrated that it inhibits proliferation of some cell types (Fentcany, G., Standaert, R. F., Reichard, G. A., Corcy, E. J. and Schrcibcr, S. L. 1994 Proc. Nat'1. Acad. Sci. USA 91, 3358-3362).

Now it is clearly established that the proteasome is the main extraditable proteolytic system included in proteolytic cascades required for various cellular functions such as cell division, processing of antigens and degradation of short-lived regulatory proteins, such as products of oncogenes, cycline and transcription factors (Ciechanover, A. (1994) Cell 79, 13-21; Palombell, V. J., Rando, O. J., Goldberg, A. L. and Maniaus. T. 1994, Cell 78, 773-785). For example, the active form of NF-KB is heterodimer containing subunit P65 and P50. The latter are represented in the cytosol as an inactive precursor (R). Proteolytic prose and R65 are contained in the cytosol as an inactive complex, associated with the inhibitory protein I QR. Inflammatory stimuli such as LPS, activates NF-KB by initiating a signaling pathway that leads to the destruction of IQ. These signals also stimulate the processing R in P50. Thus, the two proteolytic process, both managed by the ubiquitin-proteasomal by required for induced activation of NF-KB.

The observation that ubiquitin-mediated proteasomal proteolysis plays a crucial role in the activation of NF-KB could be used in the clinic through the use of inhibitors targeted to the proteasome. Abnormal activation of NF-KB, followed by a stimulation of the synthesis of cytokines was observed in various inflammatory and infectious diseases. Activation of NF-KB is also necessary for angiogenesis and the expression of adhesion molecules (CAM and selection), thus, proteasome inhibitors may also be useful in the treatment of diseases associated with vascular system.

Reliably confirmed that the ubiquitin-proteasomal path is crucial for the controlled destruction of tsiklonov, which causes the exit from mitosis and allows the cells to enter into the next phase of the cell cycle (Glotzer, M. , Murray, A. W. and Kirschn the Directors causes stunting. Therefore, another possible application of proteasome inhibitors is their use in the treatment of diseases caused by abnormal cell division.

Several classes of peptide inhibitors of 20S protease have been described in the modern literature. -Ketoamine group was used in protease inhibitors for many reasons. In particular, the number-Methocarbamol and breefing derivatives of dipeptides (Iqbal, M., Chatterjee, S., Kauer, J. C., Mallamo, J. P. , Messina, P. A., Reiboldt, A. and Siman, R. 1996 Bioorg. Med. Chem. Lett 6, 287-290) have been described as potent inhibitors of 20S proteasomal functions. Derivatives of 3-indoleamines acid were declared as pharmaceutically active compounds for the treatment of disorders of the Central nervous system (De Luca, et al, WO 88/09789) through a mechanism that regulates the content kynurenine acid in the brain.

Although described various compositions, which, to some extent, inhibit cell proliferation, a need remains for more effective compounds that inhibit cell proliferation through the 20S proteasome.

THE INVENTION

The object of this invention is a method of inhibiting cell proliferation in mammals,of these cellular proliferative properties.

It is also an object of this invention is a method of treatment of diseases which can be influenced by inhibition proteasomal functions.

Further, the object of this invention is a method of treating proliferative diseases, which are caused by the inhibition proteasomal functions.

Another object of this invention is the use of a therapeutically effective amount of the compositions described herein, for the suppression of disorders of cell proliferation in humans.

Another object of this invention is the use of a therapeutically effective amount of the compositions described herein, for inhibiting proteasomal functions.

In one embodiment, this invention relates to a compound having the formula

< / BR>
where X2is a AG or AG-X3where X3is C= O or-CH2WITH-and where AG represents phenyl, substituted phenyl, indole, substituted indoles, and any other heteroaryl; R1and R2each independently selected from the side chains known natural amino acids and not natural amino acids, hydrogen, 1-10 carbon linear and branched alkyl, 1-10 carbon l of the aqueous substituted aryl, alkoxyaryl, 3-8 carbon cycloalkyl, heterocycle, substituted heterocycle, heteroaryl and substituted heteroaryl; X1selected from hydroxide, monoalkylamines, dialkylamines, alkoxide, arillashiga and

< / BR>
where X4represents a hydroxide, arylamino, monoalkylamines, dialkylamines, alkoxide or arylalkyl and

R3selected from known natural amino acids, natural amino acids, hydrogen, 1-10 carbon linear and branched alkyl, 1-10 carbon linear and branched substituted alkyl, aryl, substituted aryl, 1-10 carbon linear and branched substituted aryl, alkoxyaryl, 3-8 carbon cycloalkyl, heterocycle, substituted heterocycle, heteroaryl and substituted heteroaryl.

In another embodiment, the invention relates to a method of inhibiting proteasomal proteases factor in humans, including the introduction of a therapeutically effective amount of the composition described above, man.

In another embodiment this invention relates to a pharmaceutical composition containing the compound described above and one or more pharmaceutical excipients.

DESCRIPTION Protectionif diseases and immunological diseases in mammals, in particular, in humans, by using compounds of the following General formula:

< / BR>
where X2is a AG or AG-X3where X3includes-C=O, CH2CO - or (CH2)nwhere n=0-2, and where AG represents phenyl, substituted phenyl, indole, substituted indoles and any other heteroaryl;

R1and R2independently selected from the side chains known natural amino acids and not natural amino acids, hydrogen, 1-10 carbon linear and branched alkyl, 1-10 carbon linear and branched substituted alkyl, aryl and substituted aryl, 1-10 carbon linear and branched substituted aryl, alkoxyaryl, 3-8 carbon cycloalkyl, heterocycle and substituted heterocycle, or heteroaryl and substituted heteroaryl. R2represents preferably biaryl or biphenyl. R1represents preferably isobutyl. X1selected from HE, mono - or dialkylamino, alkoxide, arillashiga and

< / BR>
where X4is a HE, arylamino, mono or dialkylamino, alkoxide or arylalkyl, preferably IT;

R3selected from the side chains known natural amino acids and not natural AMI linear and branched substituted alkyl, aryl and substituted aryl, 1-10 carbon linear, branched substituted aryl, alkoxyaryl, 3-8 carbon cycloalkyl, heterocycle and substituted heterocycle, or heteroaryl and substituted heteroaryl;

R3preferably is the CO2N, CH2CO2H, (CH2)2CO2H, AGD, Lys, Asn, Gln, Asp, Glu, Phe and Nle.

The following enumerations are definitions for some terms used here.

"Halogen" refers to fluorine atoms, bromine, chlorine and iodine.

"Hydroxyl" refers to the group-HE.

"Thiol" or "mercapto" refers to the group-SH.

"Alkyl" refers to a cyclic, branched or straight chain alkyl groups with carbon atoms of from one to ten. This term is further illustrated by such groups as methyl, ethyl, n-propyl, ISO-propyl, n-butyl, tert-butyl, ISO-butyl (or 3-methylpropyl), cyclopropylmethyl, ISO-amyl, n-amyl, n-hexyl and the like.

"Substituted alkyl" refers to lower alcelam, such as have just been described, including one or more groups such as hydroxyl, thiol, alkylthiol, halogen, alkoxy, amino, amido, carboxyl, cycloalkyl, substituted cycloalkyl, hetero, alseny hetaryl, aralkyl, heteroalkyl, alkylaryl, alkylamines, alkylsilanes, alkylcyclopentanes, cyano. These groups can be attached to any carbon atom of the lower alkyl groups.

"Aryloxy" denotes the group-OAS, where AG represents aryl, substituted aryl, heteroaryl or substituted heteroaryl group, as defined below.

"Amino" refers to the group NRR', where R and R' can independently be hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl, hetaryl, or substituted hetaryl as defined below, or aryl.

"Amido" denotes the group-C(O)NRR', where R and R' can independently be hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl, hetaryl, substituted hetaryl as defined below.

"Carboxyl" denotes the group-C(O)OR, where R can independently be hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl, hetaryl, substituted hetaryl, and the like, as defined.

"Aryl" or "AG" refers to an aromatic carbocyclic group having at least one aromatic ring (e.g., phenyl or biphenyl) or multiple to runafter, naphthyl, antrel or financil).

"Substituted aryl" refers to aryl, optionally substituted by one or more functional groups, for example halogen, lower alkyl, lower alkoxy, alkylthio, acetylene, amino, amido, carboxyla, hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano, thiol, sulfamido and the like.

"Heterocycle" refers to a saturated, unsaturated or aromatic carbocyclic group having a single ring (e.g., morpholino, pyridyl or furyl) or multiple condensed rings (e.g., Natterer, Minoxidil, chinoline, indolizinyl or benzo[b]thienyl) and having in the ring at least one heteroatom, such as N, O or S, which may be independently unsubstituted or substituted, e.g. by halogen, lower alkyl, lower alkoxy, alkylthio, acetylene, amino, amido, carboxyla, hydroxyl, aryl, aryloxy, a heterocycle, hetaryl, substituted hetaryl, nitro, cyano, thiol, sulfamido and the like.

"Heteroaryl" or "Garni" refers to a heterocycle in which at least one heterocyclic nucleus is aromatic. Preferred heteroside to the heterocycle, optionally mono - or polishmaster one or more functional groups, for example halogen, lower alkyl, lower alkoxy, alkylthio, acetylene, amino, amido, carboxyla, hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano, thiol, sulfamido and the like.

"Cycloalkyl" refers to divalent cyclic or polycyclic alkyl group containing 3 to 15 carbon atoms.

"Substituted cycloalkyl" refers to cycloalkyl group that includes one or more substituents, for example halogen, lower alkyl, substituted lower alkyl, alkoxy, alkylthio, acetylene, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano, thiol, sulfamido and the like.

Examples of compounds which can be used in therapeutic methods according to this invention, in particular as inhibitors proteasomal functions presented in table 1.

The compounds described above can be used to treat diseases and disorders mediated 20S proteasome, such as proliferative diseases, cancer, inflammation. Preferably the compositions of this izobreteniya according to this invention are used to treat inflammatory diseases.

Compounds of the present invention can be used for the treatment of diseases mediated 20S proteasome in mammals.

Compounds according to this invention can be introduced mammals for and prevention and treatment, any method of administration, which can be delivered to the 20S proteasome, at least one compound according to this invention. Non-limiting examples of methods of introduction include oral, parenteral, dermal, intradermal, rectal, nasal, or any other, suitable for the introduction of a pharmaceutical composition method, which is known to specialists in this field.

The compositions of this invention can be administered in the form of suitable pharmaceutical dosage forms. Pharmaceutical dosage form largely depends on the method of introduction. The term pharmaceutical dosage form refers to objects, such as tablets, capsules, liquids and powders, including inhibitors of the 20S proteasome of the present invention, alone or in the presence of one or more pharmaceutical excipients. A variety of additives, such as excipients and adjuvants, in znachitelnaya variety of drugs and carriers for administration of compositions according to this invention.

The route of administration selected for the compounds according to this invention determines the final shape and composition of the pharmaceutical dosage forms containing inhibitors of 20S proteasome in this invention. For example, the internal introduction of compounds according to this invention is administered orally in the form of powders, tablets, capsules, pastes, drinks, granules or solutions, suspensions and emulsions, which can be administered orally or in the form of a bolus, containing a medicinal substance, food or drinking water. Internal administration can also be performed using the drug with delayed release containing additives, such as surfactants or coated with starch capsules, or using the compositions quick release, such as freeze dried fast dissolve tablets. Skin injection is carried out, for example, in the form of transdermal patches, spray or "irrigation" or "spraying". Parenteral administration is carried out, for example, in the form of injections (intramuscularly, subcutaneously, intravenously, intraperitoneally) or by implantation.

Suitable pharmaceutical dosage forms containing inhibitors of the 20S FR the social solutions concentrates for oral administration after dilution, solutions for use on the skin or in body cavities, preparations for "irrigation" or "spraying", the gels; emulsions and suspensions for oral or dermal administration and for injection; semi-solid preparations; preparations in which the active compound is included in a base cream or in the basis of emulsion oil-in-water or water-in-oil; solid preparations such as powders, pre-prepared mixtures or concentrates, granules, pills, tablets, boluses, capsules; aerosols and inhalations, and the product which has a definite form containing the active substance.

Pharmaceutical dosage forms, which represent solutions can be introduced by intravenous, intramuscular and subcutaneous injection. Solutions for injection is obtained by dissolving the active compound in a suitable solvent and, if appropriate, by adding adjuvants such as solubilization, acids, bases, buffer salts, antioxidants and preservatives. The solution is sterile filtered and degassed.

Alternative solutions, including the composition of this invention can be administered orally. Concentrates composename concentration. Oral solutions and concentrates are given as described above for the case of solutions for injection. Solutions for use on the skin is applied dropwise, RUB, RUB, sprinkle or spray. These solutions prepared as described above for the case of solutions for injection.

Gels applied to the skin or injected into the body cavity. The gels obtained by processing solutions, which were obtained as described for the case of solutions for injection, with the same amount of thickener that is formed of a transparent substance smetanopodobnogo consistency, or by any other methods known to experts in this field.

Solutions for "irrigation" and "spraying" pour or sprinkle on a limited area of skin, the active compound penetrates the skin and acting systemically. Solutions for "irrigation" and "spraying" is produced by dissolution, suspension or emulsion of the active compound in suitable solvents or mixtures of solvents, which are tolerant to the skin. If applicable, add other additives such as colorants, resorption accelerators, antioxidants, stabilizers against the action of light and substances that increase the adhesiveness.

Suspension can be administered orally, percutaneously or by injection. They are obtained by suspension of the active compound in the liquid, if appropriate with addition of additional adjuvants, such as moisturizing agents, colorants, resorption accelerators, preservatives, antioxidants and stabilizers against the action of light.

The pharmaceutical compositions according to this invention can include one or more additives in the form of pharmaceutically acceptable additives. Used additives include solvents, soljubilizatory, preservatives, substances that increase the viscosity, moisturizing agents, colorants, resorption accelerators, antioxidants, stabilizers against the action of light, substances that enhance adhesiveness, viscosity, fillers, flavoring agents, lubricating agents and any other additives to pathelem, such as water, alcohols, such as ethanol, butanol, benzyl alcohol, glycerol, propylene glycol, polyethylene glycol, N-organic, alkanols, glycerol, aromatic alcohols such as benzyl alcohol, phenylethanol, Phenoxyethanol, esters such as ethyl acetate, butyl acetate, benzyl benzoate, ethers, such as alkalemia esters of alkalophilus, such as onomatology broadcast dipropyleneglycol, monobutyl ether of diethylene glycol, ketones, such as acetone, methyl ethyl ketone, aromatic and/or aliphatic hydrocarbons, vegetable or synthetic oils, DMF, dimethylacetamide, N-organic, 2,2-dimethyl-4-oxymethyl-1,3-dioxolane.

The following supplements can be used as solubilization compositions of this invention include solvents that improve the solubility of the active compound in the main solvent or which prevent its deposition. Examples include polyvinylpyrrolidone, polyoxyethylene castor oil, polyoxyethylene sorbitane ethers.

Used preservatives are, for example, benzyl alcohol, trichloroethanol, p-oxybenzone esters and n-butanol.

Used thickeners include nesticle, such as cellulose derivatives, polyvinyl alcohols and their copolymers, acrylates and methacrylates.

Other liquids that can be used in pharmaceutical dosage forms according to this invention, are, for example, homogeneous solvents, mixtures of solvents and wetting agents, which are usually surface-active substances.

Used colorants are all colorants which are non-toxic and which can be dissolved or suspended.

Used resorption accelerators are DMSO, liquid oils, such as isopropylmyristate, pelargonate dipropyleneglycol, silicone oils, fatty acid esters, triglycerides, fatty alcohols.

Used antioxidants are sulfites or metabisulfite, such as potassium metabisulfite, ascorbic acid, equivalent, butylhydroxyanisole, tocopherol.

Applied by the stabilizer against the action of light is eventially acid.

The substances applied, increase stickiness, include cellulose derivatives, starch derivatives, polyacrylates, natural polymers, such as ale as polyoxyethylene castor oil, polyoxyethylene sorbitan monooleate, sorbitan the monostearate, glycerol monostearate, polyoxyethylene, alkylphenol esters of polyglycol; impolitically surfactants, such as Di-Na N-lauryl-beta-iminodipropylamine or lecithin; anionic surfactants, such as Na lauryl sulphate, sulfate esters of fatty alcohols, monoethanolamine Sol simple ether mono/dialkylphosphites complex phosphoric esters; cationic surfactants, such as cetyltrimethylammonium.

Used substances that increase the viscosity, and substances which stabilize therapeutic emulsion include carboxymethylcellulose, methylcellulose and other cellulose, starch derivatives, polyacrylates, alginates, gelatin, Arabic gum, polyvinylpyrrolidone, polyvinyl alcohol, copolymers metilfenidato ether and maleic anhydride, polyethylene glycols, waxes, colloidal silica or mixtures of the mentioned substances.

To obtain a solid pharmaceutical dosage forms, the active compound is mixed with suitable additives, if appropriate, with addition of adjuvants and the mixture is desirable. Examples of fisiy, the bicarbonates, oxides of aluminum, silica gels, clays, precipitated or colloidal silicon dioxide and phosphates. Examples of solid organic additives include sugar, pulp, food products such as milk powder, animal flour, grain flour and coarse grain flour and starch. Other suitable additives include lubricating agents and give the property slip agents, such as magnesium stearate, stearic acid, talc, bentonites; disintegrating agents such as starch or poperechnyy polyvinylpyrrolidone; binding agents such as starch, gelatin or linear polyvinylpyrrolidone; dry binders such as microcrystalline cellulose.

In pharmaceutical dosage forms described herein, the active compounds can be present in the form of a mixture of at least one other inhibitor of the 20S proteasome. Alternative or in addition, the pharmaceutical dosage forms according to the invention, in addition to at least one inhibitor of the 20S proteasome, can include any pharmaceutical compound that is able to cure any known disease or disorder, in those cases, when put together creates no unacceptable adverse effectivnogo the number of selected compounds or combinations, preferably focused in pharmaceutical dosage form. Ready-to-use pharmaceutical dosage forms according to this invention contain the active compound in concentrations of from 10 meters on up to 20 percent by weight and preferably from 0.1 to 10 percent by weight. The pharmaceutical dosage forms according to this invention, which is diluted before administration, preferably contain the active compound in concentrations of from 0.5 to 90 percent by weight and preferably from 5 to 50 percent by weight. Usually, it is proved that to achieve effective results favorably to enter amount from about 0.01 mg to about 100 mg of active substance per 1 kg of body weight per day.

The number and frequency of introduction to pharmaceutical dosage forms comprising the inhibitors of the 20S proteasome in this invention, can be easily determined by any person skilled in the art depending upon, among other factors, routes of administration, the age and condition of the patient. These dosage units may be administered once or ten times daily in acute or chronic disease. No unacceptable Toxicological effects are expected when the connection Isuzu, containing inhibitors of 20S proteasome in this invention is obtained in accordance with conventional techniques of pharmacy involving milling, mixing, granulating and compressing, when necessary, in the case of tabloids or milling, mixing and filling in the case of a hard gelatin capsule forms. When using a liquid additive, the drug will be in the form of a syrup, elixir, emulsion, or aqueous or non-aqueous suspensions. Such liquid drug may be injected directly R. O. or enclosed in a soft gelatin capsule.

Although the compositions described herein can be entered as described above, it is preferable that the method according to this invention was carried out by introducing the compounds described herein are oral. When you select oral route of administration, it is necessary to introduce a greater number of reactive agents for the same effect, which is achieved by a smaller number entered, for example, parenteral. In accordance with a wide clinical practice, preferably the introduction of a connection on the given method in the concentration, which would give an effective therapeutic results without showing rubyfortune. The compositions of this invention can be used as analytical standards for the study of inhibitor of 20S proteasome.

Example 1.

The compounds used in therapeutic method according to this invention, receive conventional methods of organic chemistry. Links to which you can turn for help when describing the synthesis of these compounds in this area include Bodansky''s "The Practice of Peptide Synthesis," Springer-Verlag, First Edition, 1984; "Protective Groups in Organic Synthesis", Second Edition, John Wiley and Sons, New York, 1991. All reactions attaching peptides are performed at room temperature in low and constant stirring. The reaction of a combination of peptides and remove protection control using the Kaiser test for amines. XAA refers to any of the commercially available amino acids, which can be used as a pre-attached to MVNA the polymer. Yaa and Zaa are any of the commercially available amino acids.

Compounds according to this invention can be obtained by solid-phase peptide synthesis (PPPS) in the usual way, which is as follows: weigh XAA-MBNA-polymer and placed in a syringe equipped frittoli filter. The polymer give pre labuhn the creative with the removed protecting group is removed. The polymer with the remote protection is washed with DMF five times, five times Meon and then five times with DMF. Amino acid Yaa can then be attached to the polymer with the remote protection using Yaa in DMF containing Yaa, carbodiimide linking reagent and NOT (hydroxybenzotriazole), 3 equivalents of each. Successful linking solutions Yaa may be necessary to achieve the efficiency of the combination that passes the test Kaiser. Removal of N-terminal protective group and Yaa linking can be repeated to attach the third amino acid Zaa. At the last stage of the combination use of the ketoacid, carbodiimide and NOT in DMF and repeat this stage until such time as the reaction of joining the test will not Kaiser. Complete peptide sequence in the polymer is dried in vacuum for at least six hours and then otscheplaut processing for 2.5 hours or 95/5 triperoxonane acid/water, or freshly prepared solution of 90% triperoxonane acid, 3% identicial, 5% thioanisole and 2% anisole. The products of cleavage allocate any lyophilizate of water, or by rubbing with diethyl ether. The purity of the products is assessed by thin layer chromatography. The selected peptide samples analyzed photoshopdownloads acid)-N-biphenylene-D-Leu-Asp-OH get according to the method of example 1.

Fmoc-N-Asp(Ot-Bu)-MBNA-polymer (20 mg) is weighed and placed in a syringe equipped frittoli filter. The polymer give pre swell in 1 ml DMF for 30 minutes. Remove the Fmoc (fertilityscore)protective group by treatment with 20% piperidine in DMF for 30 minutes. The solution with remote protective group is removed. The polymer with the remote protection is washed with DMF five times, five times Meon and then five times with DMF. Fmoc-D-Leu-OH, is attached to the polymer with the remote protection (1 EQ), using a solution of Fmoc-D-Leu-OH (3 EQ) in 1 ml DMF containing carbodiimide (3 EQ) and NOT (hydroxybenzotriazole) (3 EQ). Second or third binding solutions of Fmoc-D-Leu-OH may be necessary to achieve the efficiency of the combination that passes the test Kaiser. The removal of the protective group with Fmoc and stage combination with amino acid repeat to attach Fmoc-N-(4,4-biphenyl)alanine. At the last stage of accession use indoleamines acid (5 EQ), diisopropylcarbodiimide (5 EQ) and NOT (5 EQ) in DMF and repeat this stage until such time as the reaction mix does not pass the test Kaiser. Complete peptide sequence in the polymer is dried in vacuum for at least six hours and then otscheplaut processing for 2.5 hours or 95/5 of triparanol and 2% anisole. Derived products emit any lyophilizate of water, or by treatment with diethyl ether. The purity of the products is assessed by thin layer chromatography.

1H NMR (400 MHz, d6-DMSO): about 6.5 to 7.7 (m, 14N), and 4.5 (m, 1H), 4,1 (m, 2H), 3,4 (m, 2H), 3 (m, N), and 2.7 (m, 1H), 1,1-1,5 (m, 3H), 0.5 and 0.9 (m, 6N).

Example 3.

In this example, the get (3'-indoleamines acid)-N-biphenylene-D-Leu-Asp-OH, using Chiron Mimotopes Pin Technology.

The first amino acid residue XAA added to 4-(hydroxymethyl)phenoxyacetamide-marker)polymer needles (5.7 mmol/needle) by associating each needle in 800 ál of binding solution (100 mm amino acids, 100 mm DIC, 10 mm DMAP, 1/4 DMF/CH2C12within two hours. The needle is then rinsed for 5 minutes DMF washing twice for 5 min Meon leaching and 15 minutes and air-dried. The Fmoc group is removed from the protection process within 30 minutes 800 ál of 20% piperidine in DMF. Repeat the washing needle (1 DMF washing, 2 MEON wash, 15 minutes air drying). The second amino acid residue Yaa associate (100 mm Yaa, 100 mm DIC, 100 mm NOWT and indicator bromophenol blue in DMF) until then, until the blue color no longer remains on the surface of the needle. Linking if necessary, repeat. The rinse cycle and udler washing as in the case of Yaa, repeating the reaction attach if necessary. The last remnant attached with indoleamines acid 15 EQ 100 mm, 15 EQ DIC, 15 EQ NEWT and the indicator Bromphenol blue in DMF. The reaction attach if necessary, repeat. After the last washing orange needles removed from their substrates and otscheplaut in separate 2 ml plastic centrifuge tube with 1.5 ml of freshly prepared solution of 90% triperoxonane acid, 5% thioanisole, 3% ethicial and 2% anisole for 2.5 hours. The needle is removed from the tubes and the mixture is blown almost to dryness by a stream of nitrogen. Triturated with Et2O and centrifuged each tube. This stage is repeated three times for each tube. The precipitated peptides sobirat, lyophilizers, weighed and used. Product purity is assessed by thin-layer chromatography. Educt gather and analyze relatively reliable samples obtained in example 1.

Example 4.

Compounds according to this invention obtained in accordance with the method of example 1, was analyzed as follows. 20S catalytic subunit of the proteasome (also known as multicatalytic proteinase complex) purified to homogeneity from bovine seraut by the increase in fluorescence upon cleavage of the substrate peptide succinyl-leucine-leucine-valine-tyrosine-7-amino-4-methylcoumarin. Standard in vitro studies consists of 2 µg 20S proteasome, 0.1 to 100 μg/ml of the inhibitor of the proteasome in 200 μl of 50 mm HEPES, containing sodium dodecyl sulphate of 0.1%, pH 7.5. The proteolytic reaction was initiated by adding 50 μm fluorogenic peptide substrate and left to develop for 15 minutes at 37oC. the Reaction is completed by addition of 100 ál of 100 mm acetate buffer, pH 4.0. The rate of proteolysis is directly proportional to the number of free aminoethylamino, which is measured by fluorescent spectroscopy (Ex 370 nm, EM 430 nm).

The results of the study of inhibitor of 20S proteasome are presented in table 2.

Compounds according to this invention obtained in accordance with the method of example 1, were also investigated as follows. 20S catalytic subunit of the proteasome (also known as multicatalytic proteinase complex) was purified to homogeneity from bovine brain according to published methods (S. Wilk and Orlowski, M. 1983, 40 842 J. Neurochem). Trypticase activity of the complex is measured by the increase in fluorescence upon cleavage of the substrate peptide CBZ-D-Leu-Arg-(7-amino-4-methylcoumarin). Standard in vitro studies consists of 2 µg 203 proteasome, 0.1 to 100 μg/ml of inhibitory by adding 50 μm fluorogenic peptide substrate and left to develop for 15 minutes at 37oC. the Reaction was completed by addition of 100 ál of 100 mm acetate buffer, pH 4.0. The rate of proteolysis is directly proportional to the number of free aminoethylamino, which was measured using fluorescent spectroscopy (EX 370 nm, EM 430 nm). Connection 1-207 were investigated for inhibition of tripticase activity and activity as inhibitors in > 10 μg/ml.

Example 5.

Compounds according to this invention obtained in accordance with the method of example 1, were also investigated as follows. 20S catalytic subunit of the proteasome (also known as multicatalytic proteinase complex) purified to homogeneity from bovine brain according to published methods (S. Wilk and Orlowski, M. 1983, 40 842 J. Neurochem). Trypticase activity of the complex was measured by the increase in fluorescence upon cleavage of the substrate peptide CBZ-D-Ala-Leu-Arg-(7-amino-4-methylcoumarin). Standard in vitro studies consists of 2 µg 20S proteasome, 0.1 to 100 μg/ml of the inhibitor of the proteasome in 200 μl of 50 mm HEPES, containing sodium dodecyl sulphate of 0.1%, pH 7.5. The proteolytic reaction was initiated by adding 50 μm fluorogenic peptide substrate and left to develop for 15 minutes at 37oC. the Reaction of zavershivshesya of aminoethylamino, which was measured using fluorescent spectroscopy (EX 370 nm, EM 430 nm). Connection 1-207 were investigated for inhibition of tripticase activity and activity as inhibitors in > 10 μg/ml.

Example 6.

Compounds according to this invention obtained in accordance with the method of example 1, were also investigated as follows. 20S catalytic subunit of the proteasome (also known as multicatalytic proteinase complex) purified to homogeneity from bovine brain according to published methods (S. Wilk and Orlowski, M. 1983, 40 842 J. Neurochem). Trypticase activity of the complex is measured by the increase in fluorescence upon cleavage of the substrate peptide CBZ-D-Ala-Leu-Glu-(7-amino-4-methylcoumarin). Standard in vitro consists of 2 µg 20S proteasome, 0.1 to 100 μg/ml of the inhibitor of the proteasome in 200 μl of 50 mm HEPES, containing sodium dodecyl sulphate of 0.1%, pH 7.5. The proteolytic reaction was initiated by adding 50 μm fluorogenic peptide substrate and left to develop for 15 minutes at 37oC. the Reaction is completed by addition of 100 ál of 100 mm acetate buffer, pH 4.0. The rate of proteolysis is directly proportional to the number of free aminoethylamino, which was measured using fluorescent spectrosc > 10 μg/ml of Compound 190 was actively at 5 µg/ml

1. The connection formulas

< / BR>
where X2is a AG or AG-X3where X3represents the C= O or-CH2CO - and ar represents a phenyl or indole;

R1and R2each independently selected from hydrogen or a side chain of natural amino acids, are represented by linear or branched (C1-C10) alkyl which may be substituted by hydroxy, carboxy, amino, cyclohexyl, phenyl or replacement of the phenyl, biphenyl, naphthyl or pyridium;

X1selected from HE, or groups

< / BR>
where X4is a HE;

R3selected from hydrogen or a side chain of natural amino acids, are represented by linear or branched (C1-C10the alkyl which may be substituted by hydroxy, phenyl or replacement of the phenyl.

2. Connection on p. 1, where X1represents a

< / BR>
where X4and R3specified in paragraph 1.

3. Connection on p. 2, where X4is a HE.

4. Connection on p. 3, where R1selected from linear or branched (C1-C10) alkyl.

5. Connection on p. 1, is n (C1-C10) alkyl.

6. Connection on p. 5, where AG is an indole.

7. Connection on p. 5, where AG represents phenyl.

8. Connection on p. 7, where X1represents a

< / BR>
where X4is a HE and R3represents N.

9. Connection on p. 8, where AG is a 3-indole, R1represents isobutyl and X4is a HE.

10. Connection on p. 1, where AG is an indole, R3represents a biphenyl, R1represents isobutyl, R2represents CH2CO2N and X4is a HE.

11. Connection on p. 1, where AG represents phenyl, R2represents a biphenyl, R1represents isobutyl, R3represents CH2CO2N and X4is a HE.

12. Connection on p. 1, where AG is an indole, X3represents CH2CO, R2represents a 4,4'-biphenyl, R1represents isobutyl, R3represents CH2CO2N and X4is a HE.

13. The pharmaceutical composition possessing the n or more pharmaceutical excipients.

14. The pharmaceutical composition according to p. 13, where the pharmaceutical composition is presented in the form of a solution.

15. The pharmaceutical composition according to p. 13, where the pharmaceutical composition is presented in the form of tablets.

 

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< / BR>
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