Derivatives of boron-containing peptides and pharmaceutical composition having inhibitory activity to trypsinogen serine proteases

 

(57) Abstract:

Usage: in biology and medicine, in particular in the chemistry of boron-containing peptides for compositions that possess inhibitory activity towards trypsin-like serine proteases. The essence of the invention: derivatives of boron-containing peptides total f-ly 1: , where W is hydrogen, N-protecting group: benzyloxycarbonyl, C1-C6-alkanoyl or C1-C6-alkoxycarbonyl; Y - TMSal-Pro (p-TBDPS-O-Me)Phal-Pro, n-OH-Me-Phal-Pro, TMSal-Adgly, or a partial peptide sequence of f-crystals 2: -NH-CH(R3)-C(O)AA, where R3- CH2-Si(CH3)3, -CH2C(CH3)3or methylnaphthalene group, or methylene-substituted; AA is a peptide radical Pro, Gly, Ala, Leu, Val, Ile, Asp, Glu; Q1and Q2together financially or the group-O-CH2CH2-O; R1is hydrogen, R2- AX, where A is (CH2)Z: Z = 2-5; X - NH2, -NH-C(NH)-NH2N3C1-C4-alkoxy, Si(CH3)3or R1and R2- trimethylene group, and a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a boron-containing peptide f-crystals of 1 in the amount of 0.02-15 mg/kg of the body. 2 C and 3 C.p. f-crystals, 2 tab.

The invention relates to new biologically active link is to trypsinogen serine proteases, which can find application in biology and medicine.

Of inhibitors trypsinogen serine proteases important role in the body play thrombin, factor X6, kallickrein, plasmin, and protease similar to prolyl-endopeptidase and the protease IgAI.

Thrombin, the terminal enzyme in the coagulation system, breaks down dissolved fibrinogen into fibrin, which is then cross linked and insoluble gel, which is the matrix for a blood clot. When damage to the vessel specified process is necessary to stop bleeding. Under normal conditions the amount of thrombin in plasma is not very large. Increasing concentrations of thrombin may lead to the formation of blood clots that cause thromboembolic disease, one of the most common and significant health problems of our time.

Thrombin promotes hemostatic regulation through multiple biological reactions. In addition to its primary function, convert fibrinogen to fibrin, thrombin activates factor XIII, which is responsible for binding to fibrin. Thrombin also causes due to a positive feedback mechanism, activation of factors V and VIII, which are necessary (both) DL the Incas initiates their aggregation and thickening, which leads to the cessation of bleeding.

Effects of thrombin are governed by natural inhibitors in plasma. The most important of these are antithrombin III and heparin. These two compounds were isolated and therapeutically and prophylactically used in the absence of equilibrium in the hemostatic mechanism with risk for activation of prothrombin.

Synthetic thrombin inhibitors with oral activity would be useful as an alternative to subcutaneous introduction of these natural inhibitors. As a result of considerable research in this area synthesised good inhibitors of thrombin in vitro, but still no real good product for oral therapeutic applications. Imitation of a number of amino acids of fibrinogen, an important natural substrate of thrombin, were obtained a few good short peptide substrates of thrombin. These peptide substrates can be converted to inhibitors of the enzyme. Thus, the chromogenic substrate D-Phe-Pro-Arg-PNA and D-Phe-Pip-Arg-PNA similar to a number of predecessors splitting blood thrombin. The corresponding reversible and irreversible inhibitors of D-Phe-Pro-Arginal and D-Phe-Pro-Arg-CH2Cl show inhibitory ability in vitro in distribution in therapy, and peptide aldehyde, an example of which is mentioned above, has a sufficiently low value LD50.

Factor Xathe enzyme coagulation responsible for the formation of thrombin by the complete proteolysis of its precursor prothrombin. For the main effect of factor Xaat least 10 times more in vivo thrombogenic than thrombin. This follows from the fact that the factor Xaone stage of the translation referred to thrombin in a large cascade system, as well as taking into account the fact that one molecule of factor Xacan produce many molecules of thrombin from its precursor. Its activity can be explained by a fairly minor removal of factor Xathe organism. Thrombin, not like the X factoraquickly removed from circulating blood and penetrates into the walls of blood vessels. The Central position of the X factorawhen crossing internal and external paths can lead to the appropriate targets to modulate hemostatic mechanism.

Kallickrein is formed from prekallikrein impact factor XII, if you have a negatively charged surface. Kallickrein, in turn, can transform factor XII factor XIIand, which is formed corresponding Akti is ktoi system, likely that the surface serves as a binder in the protective mechanism, and a great level of activation of the system definitely proves the existence of a collision or dispersion when intravascular coagulation. The role of kallikrein in this stage in the removal of kininogen high molecular weight active bradykinin, much less expenditure. Bradykinin also causes increased vascular permeability, pain, and migration of polymorphonuclear leukocytes. Inhibitors of education cenina showed high activity when certain types of inflammation, including arthritis or pancreatitis, and may be suitable also for the treatment of asthma. The substance having a high activity as an inhibitor of kallikrein, Aprotinin (the Drug). Aprotinin polypeptide with molecular weight of 6500, forms a stable complex with proteases, having a stability constant of 10-10-10-13M

Fibrinolysis is the process in which the enzyme dissolution of the clots fibrinogen and fibrin. Plasma contains protein, plasminogen, which when exposed to various activators converted into plasmin, a proteolytic enzyme, whose activity is similar to the activity of trypsin. The plasmin destroys fibrinogen and fibrin the subject is in equilibrium with the coagulation system. Small blood clots forming in the blood stream, can be enzyme dissolved, and the circulation through the blood vessels can be restored by the influence of the fibrinolytic system of the body. If fibrinolytic activity is also high, it can cause or prolong bleeding. The activity can also be inhibited by natural inhibitors in the blood.

Polyangiitis breaks peptide bonds at the carboxylic position of Proline present in the peptide chain. It is a serine protease, which easily destroys neuropeptides, including substance P, narrowsburg, thyrotropin

releasing hormone and luteinizing hormone releasing hormone that enables cells to produce interleukin 2 (IL-2). The enzyme inhibited benzyloxycarbonyl-shed-prolinal with Ki14 nm. Although almost nothing is known about the physiological role of polyangiitis, it can be argued that it plays an important role in the regulation of biological activity of different neuropeptides.

IgA proteases catalyzed destruction of the IgA, the predominant formation of antibodies, which includes initial protection from infection, division Fcand antigestagens Fa what you want to make. All IgA proteases, thus, lead to a Proline residue within the core of human IgA. Peptide prolyl-boranova acid inhibit IgAI proteases gonococci (Neisseria gonorrhola) and Hemophilus influenzae, which suggests that these enzymes belong to the serine proteases, a class of proteolytic enzymes.

Numerous effects caused by thrombin in a variety of physiological processes associated with pathological disorders, such as cancer, inflammation and neural activity, suggest the potential use of inhibitors of thrombin in some cases, not strictly related to the cardiovascular system.

Many tumor cells cause procoagulation activity associated with the formation of thrombin. Consistently occur local fibrin deposition and coagulation, which significantly affect the growth of damage. Further, due to these effects in endothelial cells, thrombin may contribute to bleeding in the damaged cells due to metastasis. Therefore, inhibitors of thrombin can be not only suitable for the treatment of individual cancers, but also to restore sverhkachestvennyh properties, China endothelial cells causes a lot of proinflammatory changes, such as the synthesis and release of interleukin-1, prostaglandins and plate-activating factor. In addition, thrombin causes the exposure of GMP-140 and CD 63, two adhesion molecules responsible for leukocyte adhesion to the endothelial surface. Thrombin also increases vascular permeability proteins, the effect of which binds neutrophils and release interleukin-8 prior to protein, peptide, associated, presumably, with respiratory disorders, rheumatic arthritis and ulcerative colitis.

Participation in all of these Pro-inflammatory processes makes the thrombin potential therapeutic target for the treatment with inhibitors of thrombin with relevance to inflammatory processes pathological disorders.

The activity of protease nexin-1, a modulator of nerve irritation and specific natural antagonist of thrombin, as noted, is particularly reduced in patients with disease of Alzheimer. This, along with observations about the increase trombinoscope activity in the brain disease of Alzheimer, suggests that thrombin inhibitors can be used for irreversible or reversible process neuro-pathological izraz and proteases was studied boric acid. First of boric acid containing amino acid, similar to that of the protease inhibitor was boric acid analogue of N-acetyl-L-phenylalanine, which was used as an inhibitor of chymotrypsin and subtilisin. Peptide-boranova acid were used as inhibitors of chymotrypsin, subtilisin and elastase.

The interaction of boric acid proteases in biological systems is well known, various simple boric acid is practically nontoxic to humans in their application. Peptide boronic acid inhibitors of elastase, have been applied to experimental animals in relation to emphysema. In contrast to peptide-chloromethylketone they were not toxic at biologically effective doses.

In the description of European patent N 293881 provides a method of producing peptides containing the derivative C-terminal boronic acids lysine, ornithine and arginine, and their use as inhibitors trypsinogen serine proteases. Other amino acids in the peptides have either D-or L-forms of any of the 20 amino acids of natural origin.

It was found that substances with high properties as inhibitors trypsinogen serine proteases, palwal.

The purpose of the present invention, the search range of boron-containing peptides of new compounds with high activity inhibition trypsinogen serine proteases.

This goal is achieved by the described derivatives of boron-containing peptides of General formula

< / BR>
where W is hydrogen;

N-protecting group, such as benzyloxycarbonyl: (C1-6)alkanoyl or (C1-6)alkoxycarbonyl;

Y is TMSal-Pro (p-TBDPS-O-Me)Phal-Pro-P-OH-Me-Phal-Pro, TMSal-Adgly or a partial peptide sequence of the formula

< / BR>
where R11is a group of the formula

< / BR>
-CH2-C(CH3)3< / BR>
AA is Pro, Gly, Ala, Leu, Val, Jleu, Asp or Glu;

Q1and Q2are together diol group of the formula

< / BR>
R4represents hydrogen;

R5is a group of the formula-AX and the last is A group -(CH2)z(in this formula, z 2, 3, 4 and 5), and X represents-NH2-, -NH-C(NH)-NH2, -N3C1-4alkoxy, or-Si(CH3)3,

or R4and R5together represent trimethylene group.

The preferred compounds are the compounds listed below:

< / BR>
< / BR>
< / BR>
In addition, preetam, including derived peptide-boric acid and a pharmaceutically acceptable carrier, characterized in that, as specified derivative is used as a compound of General formula I in a quantity of 0.02 to 15 mg/kg body weight.

The compounds of formula I are suitable as inhibitors trypsinogen proteases and can be used for in vitro diagnostic and mechanical radiation emitted by these enzymes. In addition, because of their inhibitory activity they are suitable for use in the prevention or treatment of diseases caused by an excess of enzyme in the regulatory system, for example for monitoring coagulation by fibrinolysis system.

All compounds of the invention are inhibitors of thrombin and factor Xahave anti-thrombogenic properties and can be used if necessary, anti-thrombogenic agents (see tab.1). In General, the compounds can be administered orally or intramuscularly in the body with obtaining anti-thrombogenic effect. In the case of large mammals, including humans, the compounds can be administered alone or in a mixture with pharmaceutical carriers or diluents in a dose of 0.02-15 mg/kg of body weight, mainly 1 to 10 mg/kg, with taking anti-thrombogenic effect and can be given a single dose is reduplicate coagulation. Pharmaceutical diluents are well known and include sugars, starches and water, which can be used for tablets, capsules, injectable solutions, and other Compounds of the invention can be added to the blood with the purpose of preventive blood coagulation in the blood flowing in vessels, pipes or connected equipment in contact with blood.

The advantages of the compounds of the invention are oral activity, the fast action and low toxicity.

In addition, these compounds are particularly advantageous for individual treatment in the event of excessive sensitivity to these substances, such as heparin.

The following examples use symbols that represent the following:

Z benzyloxycarbonyl,

Boc tert-butyloxycarbonyl,

Ac acetyl,

MeOH methyl alcohol,

HONSu N-hydroxy-succinic acid imide,

EtOAc ethyl acetate;

DCC dicyclohexylcarbodiimide,

OPin pinanediol,

THF tetrahydrofuran,

n-Bu n-butyl,

Np n-nitrophenyl,

TLC thin layer chromatography,

Bzl benzyl,

Baa-NH-CH-(CH2CH2CH2Br)B-,

TMSal trimethylsilylethynyl,

Adal adamantylamine,

Naphal 2-nafcillin,

Boro Orn-NH-CH-(Santillian,

Boro Pro-OPin analog of Proline in which the-COOH group is replaced by B-OPin-group,

Boro Lys-NH-CH-(CH2CH2CH2CH2NH2B,

Boro HArg-NH-CH-(CH2CH2CH2CH2NHC (NH)NH2)B-,

Boro Mpg-NH-CH-(CH2CH2CH2-OCH3)B-,

p-OH-Me-Phal n-hydroxymethyl-phenylalanine,

p.TBDPS-O-Me-Phal n-tert.butyl-diphenyl-silyl-oximeter-phenylalanine.

The kinetic parameters Ki, Konand Koffdetermined by the inhibition of the enzyme catalyzed the hydrolysis of peptide-arg-pNA. This hydrolysis results in n-nitroaniline, and at the time of its formation (monitored by optical density measured at 405 nm) velocity is determined inhibited and eingeborenen reactions.

Kinetic studies were conducted in 96-microislet. the Cup in combination with a single-celled kinetic counter. The active area of the test human thrombin is dissolved in a buffer solution of 0.1 m phosphate, pH of 7.4, containing 0.1 M NaCl and 0.1% bovine serum albumin, with the receipt of a solution containing 400 nm of the active enzyme. For chromogenic test this solution is dissolved in the same buffer to 0.4 nm. The substrate, 2AcOH-H-D-cyclohexyl-ala-arg-pNA, is dissolved in a buffer until the concentration is the teaching of 1 mm solution. Further dilution of the lead phosphate buffer solution described above.

Tests faster by adding 100 l of a solution of the enzyme to a mixture containing 100 l of inhibitor and 50 l of substrate solution. The excretion of n-nitroaniline as a result of hydrolysis of substrates peptidyl-n-nitroanilide runs from 30 minutes to 1 hour, with increasing measured optical density (405 nm). The obtained values are used to calculate the kinetic parameters in the presence or in the absence of inhibitor. Though we cannot exclude other mechanisms of action, typical for this class of thrombin inhibitors is that there are two main processes, namely, fast reverse and slow firmly-binding, competitive inhibition. The kinetic constants of all inhibitors acting on reversible-linked mechanism, i.e., fast action (initial velocity v0control > v0with inhibitor) and ItEt(the total concentration of the inhibitor/total concentration of enzyme), were calculated by the method of linear regression when the concentration of inhibitor I/v vS-graphically (I). The value of Kiis calculated from the horizontal section Ki,appusing equation (1):

K who is under the influence of inhibitor) and low (Kiclose to or lower than Et), i.e. stable inhibited state is reached very slowly, the formation of pNA is described by the equation

,

where P is the number of pNA formed at time t,

v0the initial velocity,

vsspeed in the study,

Kobsthe true rate of the overall reaction as a function of Et, It, Ki,appand there is the rate constant of second order for the interaction between inhibitor and enzyme. Measurement data is slow, steady-binding inhibition, calculated according to equation (2) using nonlinear regression allow us to estimate the values of Kobs. The values of Koffand Ki,appthen obtained from the graph KobsvS[1] the Value of Koffgiven a vertical section, and the values and Kicalculated from the inclined and horizontal sections using respectively equation (1).

Example 1. Boc-D-TMSal-Pro-NH-CH[(CH2)3N3]BOPin.

A. Boc-D-TMSal-OH.

Ethyl ester of D-TMSal (21,5 g 113,7 mmol), obtained according to the method given in Angew, Chem 93,793 (1981), dissolved in CH2Cl2and add to the solution an excess of Boc2O in CH2Cl2. After soaking for 15 hours when the room is 3
and brine, and then dried over Na2SO4and concentrated in vacuo. The crude product (colorless oil) directly used at the stage of saponification. It is dissolved in methanol, cooled to 0oC, mixed with 510 ml of 1 n NaOH and stirred at 0oC for 3 hours After acidification to pH 1 1 n HCl, the mixture is repeatedly extracted with ether. The organic layers are combined, washed with brine, dried over Na2SO4and concentrated in vacuo.

B. Boc-D-TMSal-Pro-ONSu.

Boc-D-TMSal-OH (29,7 g 113,7 mmol) and n-NITROPHENOL (19 g, 136,3 mmol) dissolved in EtOAc. After cooling to 0oC was added DCC (23,4 g, 113,6 mmol) and the mixture is stirred for 1 hour at 0oC, and then 15 hours at room temperature. The precipitate is filtered off, washed his EtOAc and the filtrate concentrated in vacuo. The resulting oil purified chromatography (9:1 hexane: EtOAc) to give the desired Boc-D-TMSal-ONp in the form of white crystals.

Boc-D-TMSal-ONp (51,6 g 113,7 mmol) is dissolved in THF and added dropwise an aqueous solution of equimolar amounts of Proline and Et3N. After exposure for 20 h at room temperature THF is distilled off in vacuum and the remaining aqueous layer was diluted with water, and then extracted several times with EtOAc. Using lemon sour EtOAc. The combined organic layers washed with brine, dried over Na2SO4and concentrated in vacuo. A colourless oil is recrystallized from ether/hexane to obtain the dipeptide Boc-D-TMSal-Pro-OH in the form of white crystals, so pl. 176oC.

The resulting dipeptide (26 g, 72,5 mmol) dissolved in EtOAc. After cooling to 0oC add HONSu (9,8 g of 85.5 mmol) and DCC (14.9 g, 72,3 mmol). The mixture is stirred for 3 hours at 0oC and a further 15 h at room temperature. Then the mixture is again cooled to 0oC, the filtered dicyclohexylphosphino and washed several times with EtOAc. The filtrate was washed with aqueous 0.1 M Na2CO3and then water 2% KHSO4.

After drying over Na2SO4and concentration in vacuo receive in the form of a white compound (foaming) of Boc-D-TMSal-Pro-ONSu.

C. Boc-D-TMSal-Pro-Baa-OPin.

This operation is the first stage of 3-stage process, which involves the formation of in situ chiral -(bestemmelser) aminomalonate, hydrolysis of two trimethylsilyl groups and the combination thus obtained a-aminoboronic with the active ester (Boc-D-TMsal-Pro-ONSu) obtained in stage B. the consecutive reaction is carried out in an argon atmosphere. Chiral a-harborone ((+)-pinardi solution hexamethyldisilazane lithium (5 ml of 1 M solution in hexane, 5 mmol). After stirring for 30 minutes at -78oC solution per night is heated to room temperature. Then again cooled mixture to -78oC, was added 3 mol equivalent of HCl in dioxane. The mixture is heated to room temperature and at this temperature, stirred for 2 hours, After cooling to -20oC add a solution of the active ester from step B (2.28 g, 5 mmol) in 6 ml of CH2Cl2and then add to 1.39 ml (10 mmol) of triethylamine.

The mixture is stirred for 1 h at -13oC, warmed to room temperature and stirred at this temperature for another 2 hours the Mixture is filtered, the filtrate was concentrated in vacuo, the residue diluted with ether and washed with 2 n HCl, 5% NaHCO3and brine. The organic layer is dried over Na2SO4and concentrated in vacuo. The residue is crystallized, getting the desired chiral pamidronate in the form of a white crystalline substance.

D. Boc-D-TMSal-Pro-NH-CH[(CH2)3N3]BOPin.

Boc-D-TMSal-Pro-Baa-OPin product stage (804 mg) was dissolved in 13 ml of DMSO and added sodium azide (156 mg, 2.4 mmol). The mixture is stirred for 3 h at room temperature. Add ether/ice water, right visadelta from a mixture of white crystals. White LASS="ptx2">

Example 2. Boc-D-TMSal-Pro-Boro Orn-OPin.

Azide of example 1 (569 mg, 0.9 mmol) dissolved in 25 ml EtOAc and hydronaut in the presence of 0.5 g of 10% Pd/C. After 2.5 h, the catalyst was removed and the solution was concentrated in vacuo to obtain a white foamy substance, which is recrystallized from EtOAc/ether to obtain the desired product as a white crystalline substance, so pl. 200-202oC []2D0= -11,6(0.5 in MeOH).

Example 3. Boc-D-TMSal-Pro-Boro Arg-OPin (bansilalpet).

Boc-D-TMSal-Pro-Boro Orn-OPin example 2 (250 mg, 0,412 mmol) dissolved in 2 ml ethanol. Add benzosulfimide (65,2 mg, 0,412 mmol). After stirring for 15 minutes at room temperature add cyanamide (86,6 mg of 2.06 mmol) and the mixture heated under reflux. The course of the reaction is controlled by using RP-TLC until the disappearance of the spot of ninhydrin for Amin (educt) and the appearance of bands Sakaguchi product. After 7 days, when Amin is found in small quantities, the solution was concentrated in vacuo. The residue is dissolved in MeOH and purified chromatography on columns h cm Sephadex LH-20 with MeOH.

The desired product is obtained in the form of white matter []2D0= -45,3(1 in CH2Clthe-pentane-1-boronate-4-bromo-1-butene 3 (20,8 ml, 203,3 mmol) is reacted with cateriniana (24.4 g, 203,3 mmol) at 100oC for 16 hours the Crude product is distilled under vacuum to obtain 4-bromo-butane-1-boronate in the form of a white crystalline substance.

(+)-Pinanediol (27.7 g, 163 mmol) was dissolved in THF and added to the synthesized above 4-bromo-butane-1-boronate (41,6 g, 163 mmol). After soaking for 1 h at room temperature THF is distilled off in vacuum and the residue purified chromatography (90:10 hexane/ EtOAc) to give a colourless oil (+)-pinanediol-4-bromo-butane-1-boronate.

The desired (+)-pinanediol-(S)-1-chloro-5-bromo-pentane-1-boronate receive in accordance with the method available in Organometallic 3, 1284 (1984). This is cooled to -100oC methylene chloride (9.8 ml) in THF and within 20 minutes was added n-utility (71,6 ml of a 1.6 M solution of 114.5 mmol). After 15 minutes at -100oC was added a cold (-78oC) a solution of (+)-pinanediol-5-bromo-pentane-1-boronate (32,8 g, 104,1 mmol) in THF. After holding the reaction mixture at -100oC (for 1 h) is added anhydrous ZnCl2(7,1 g, 52 mmol) in THF. After additional exposure for 15 minutes at -100oC, the reaction mixture was warmed to room temperature and then stirred for 2 hours at this temperature. The solvent is distilled in UB>2
SO4and removal of solvent in vacuo receive (+)-pinanediol-(S)-1-chloro-5-bromo-pentane-1-boronate in the form of a yellow oil, which without further purification sent directly to the next stage.

B. Boc-D-TMSal-Pro-NH-CH((CH2)4Br)B-OPin.

A solution of LiN(SiMe3)2(65,2 ml of 1 M solution of 65.2 mmol) in THF cooled to -78oC. Add-harbron with stage A (23.7 g, to 65.2 mmol). After stirring for 1 h at -78oC the mixture is stirred at room temperature for 15 hours Then the reaction mixture was again cooled to -78oC, was added HCl (29,8 ml 6,56 n solution, 196 mmol) in dioxane, the solution is stirred for 45 minutes at -78oC and then 2 h at room temperature. The mixture is cooled to -15oC, was added Boc-TMSal-Pro-ONSu (and 29.7 g of 65.2 mmol) of example 1 in CH2Cl2and then added triethylamine (18,1 ml, 30.4 mmol) to the reaction had begun condensation. After stirring at -15oC for 1 h the mixture is stirred for 2 h at room temperature. The mixture is filtered through Hyflo and concentrated in vacuo. The residue is diluted with ether/water and extracted several times with ether. After drying over Na2SO4and concentration in vacuum with carrying out the crystallization from the floor.sq. 74oC.

C. Boc-D-TMSal-Pro-NH-CH((CH2)4N3B-OPin.

In DMSO dissolve the product of stage B (33,3 g, to 48.6 mmol) and added sodium azide (6,3, 97,2 mmol). The mixture is stirred for 6 h at room temperature. Add ether/ice water and the mixture extracted several times with ether. After drying over Na2SO4and concentration get oil, which crystallized to obtain Boc-D-TMSal-Pro-NH-CH((CH2)4N3B-OPin in the form of a white crystalline substance, so pl. 69-70oC;D= -74,4(c 1 in MeOH).

Example 5. Boc-D-TMSal-Pro-Boro Lys-OPin.

In EtOAc dissolve azide of example 4 (22 g, 34 mmol) and hydronaut in the presence of 4 g of 10% Pd/C. After 9 h, the catalyst was removed and the solution concentrated in vacuo. The resulting mass was dissolved in EtOAc and crystallized to obtain the desired Boc-D-TMSal-Pro-Boro Lys-OPin in the form of white crystals, so pl. 128-129oC;D= -59,6(c 1 in MeOH).

Example 6. Boc-D-TMSal-Pro-Boro HArg-OPin (bansilalpet).

In ethanol dissolve benzoylphenyl Boc-D-TMSal-Pro-Boro Lys-OPin example 5 (800 mg, 1,03 mmol). Added cyanamide (210 mg, 5 mmol) and the mixture heated under reflux. The progress of the reaction is controlled by using RP-TLC, and see the emergence of bands Sakaguchi n is I slightly the solution was concentrated in vacuo. The residue is dissolved in MeOH and chromatographically purified on a column of h cm Sephadex LH-20 with MeOH.

The desired product is obtained as a white foamy substance D= -40,8(c 0.5 in CH2Cl2).

Examples 7-29.

Methods, such as the one outlined in examples 1-6 can be obtained the compounds of formula

< / BR>
in which R4, , R11, Q1+ Q2and AA have the values listed in table 2.

< / BR>
< / BR>
-CH2-C(CH3)3f)

Example 30. Boc-D-TMSal-Pro-Boro Mpg-OPin.

A. (+)-Pinanediol-(S)-1-chloro-methoxy-butane-1-boronate-3-methoxy-1-propene (6 g, and 83.3 mmol) is reacted with catecholborane (10 g, and 83.3 mmol) at 100oC for 24 h the Crude product is distilled in vacuo to obtain 3-mitochon-propane-1-boronate in the form of a colorless oil.

(+)-Pinanediol (10.6 g, 62.5 mmol) was dissolved in THF and added to the synthesized above 3-methoxy-propane-1-boronate (12 g, 62.5 mmol). After stirring at contact temperature for 1 h in vacuum distilled THF and the residue purified khromotograficheskie (80:20 hexane/EtOAc) to give (+)-pinanediol-3-methoxypropan-1 boronate in the form of a colorless oil.

The desired (+)-pinanediol-(S)-1-chloro-4-m (2.2 ml) in THF cooled to -100oC for 20 minutes was added n-utility (13,8 ml of 1.6 M solution, 22 mmol). The mixture is maintained at -100oC and add a solution of (+)-pinanediol-3-methoxy-propane-1-boronate (5,04 g, 20 mmol) in THF, and then anhydrous ZnCl2(1.42 g, 10 mmol). Stand the mixture for 15 minutes at -100oC, it is heated to room temperature and stirred at this temperature for 2 hours the Solvent is distilled off in vacuo, the residue diluted with ether and washed with water. The organic layer is dried over Na2SO4and concentrate to obtain the oil, which is purified khromotograficheskie (9:1 hexane/EtOAc), allocating the result in the form of a colorless oil required (+)-pinanediol-(S)-1-chloro-4-methoxy-butane-1-boronate.

B. Boc-D-TMSal-Pro-Boro Mpg-OPin.

A solution of LiN (SiMe3)2(5 ml, 1 M solution, 5 mmol) in THF cooled to -78oC. Add-chloro-boronat with stage A (1,53 g, 5 mmol) in THF. After stirring for 1 h at -78oC the mixture is stirred for another 15 h at room temperature. Then the reaction mixture was again cooled to -78oC, was added HCl (2.7 ml 5,65 n solution, 15 mmol) in dioxane, the solution is stirred 30 minutes at -78oC and then 2 h at room temperature. The mixture is cooled to -15oC, was added Boc-TMSal-Pro-ONSu (2.28 g, 5 mmol) use the 15oC for 1 h the mixture is stirred for further 2 h at room temperature. The mixture is filtered through Hyflo and concentrated in vacuo. The residue was diluted with EtOAc and washed with 0.2 n HCl 5% NaHCO3and finally with brine. After removal of the solvent receive oil, which is purified khromotograficheskie (EtOAc), highlighting in the form of a white foamy substance Boc-D-TMSal-Pro-Boro Mpg-OPin D= -48,8(c 0.25 CH2Cl2).

Example 31. Boc-D-(n-TBDPS-O)methyl)Phal-Pro-Boro Orm-OPin.

A. Boc-D-(n-((1,1-dimethylethyl)diphenyl-silyl)oxy)methyl-phenylalanine.

For selective recovery of the azide group in the substrate add thiophenol (7,27 g, 66 mmol) to a suspension of SnCl2(3.12 g, 16.5 mmol) in CH2Cl2. Add triethylamine (6.8 ml, a 49.5 mmol) to give a yellow solution. Boc-anhydride (4.8 g, 22 mmol) is added to the mixture, and then in the form of a solution in CH2Cl2add prepared according to the method described in J Am. Chem. Soc. 1096881 (1987), 3(2S), 4S-3-(2-azido-3-(n-((1,1-dimethyl)diphenyl) hydroxy-methyl)phenyl-1-oxo-propyl)-4-(phenylmethyl)-2-oxazolidinone (%de>95; 6.8 g, 11 mmol). The mixture is stirred at room temperature, diluted with EtOAc/2HNaOH and filtered through Hyflo. The organic layer was washed with 2% aqueous solution of NaHSO4a 5% aqueous NaHCO3oC. After holding at 0oC for 1.75 h add Na2CO4(1.25 g, 9.9 mmol) in water. In vacuum distilled THF, bringing pH balance to 1-2, and the mixture is extracted three times with EtOAc. The combined organic layers washed with water, dried over Na2SO4and concentrated in vacuo. After crystallization from hexane/ether get oxazolidinone in the form of white crystals. The filtrate was concentrated in vacuo to obtain the desired compound as a white foamy substance.

B. Boc-D-(n(((1,1-dimethylethyl)diphenyl-silyl) oxy)-methyl)-phenylalanine-Pro-ONSu.

DCC (0,59 g is 2.88 mmol) was added to a mixture of the title compound of stage A (1.6 g, is 2.88 mmol) and n-NITROPHENOL (0,43 g of 3.12 mmol) in EtOAc, and the temperature is 0oC. the Reaction mixture was stirred 16 h at room temperature. After cooling to 0oC the precipitate is filtered off and washed it with cold EtOAc. The filtrate conc stage.

Boc-D-(n-TBDPS-O-Me)-Phal-ONP (2.2 g, is 2.88 mmol) was dissolved in THF and added an aqueous solution of L-Proline (365 mg, 3,17 mmol) and Et3N (0,88 ml, 6,33 mmol). After soaking for 15 h at room temperature in vacuum distilled THF. Bring the pH to 3 with 10% citric acid. The obtained oily product is extracted several times with EtOAc. The combined organic layers washed with brine, dried over Na2SO4and concentrated in vacuo. Colorless oil cleanse chromatography (9:1 CH2Cl2/EtOH) to give a white foaming substances Boc-D-(n-TBDPS-O-Me)-Phal-Pro-OH.

Then dipeptide (1.3 g, of 2.06 mmol) was dissolved in EtOAc. After cooling to 0oC add HONS4(220 mg, 2,47 mmol) and DCC (330 mg, of 2.06 mmol). The mixture is again cooled to 0oC, the filtered dicyclohexylphosphino and washed several times with cold EtOAc. The filtrate was washed with aqueous 0.1 M Na2CO38% NaHSO4and then brine. After drying over Na2SO4and concentration in vacuo obtain the target compound Boc-D-(n-TBDPS-O-Me)-Phal-Pro-ONSu in the form of a white foaming product.

C. Boc-D-(n-TBDPS-O-Me)-Phal-Pro-Baa-OPin.

A named connection get similar to the first stage 3-stage receiving, described in the synthesis of Boc-D-TMSal-Pro-Baa-the and (+)-pinanediol-(S)-1-chloro-4-bromo-butane-1-boronate) (659 mg, 2 mmol) with LiN(SiMe3)2(2 mmol) and hydrolysis with NCl; this intermediate is reacted with an active ester of stage B (1.45 g, 2 mmol) in the presence of Et3N (4 mmol) with receipt of a named connection that cleanse chromatography (1:1 hexane/EtOAc).

D. Boc-D-(n-TBDPS-O-Me)-Phal-Pro-Boro Orn-OPin.

The product of stage C (680 mg, to 0.72 mmol) was dissolved in DMSO and added sodium azide (94 mg, 1.44 mmol). The mixture is stirred for 4 h at room temperature. Add ether/ice water and this mixture stand out white crystals. Their is filtered off and washed with water to obtain a white crystalline substance Boc-D-(n-TBDPS-O-Me)-Phal-Pro-NH-CH((CH2)3N3B-OPin. This azide (272 mg, 0.3 mmol) was dissolved in EtOAc and hydronaut in the presence of Lindlar catalyst. After 8 h, the catalyst was removed and the solution concentrated in vacuo. The crude product is purified chromatography (EtOAc, then EtOH) to give the desired compound as a white foamy substanceD= -32,4(c 0.25 MeOH).

Example 32. Boc-D-(n-OH-Me)-Phal-Pro-Boro Orn-OPin.

Bor-ornithine example 31 (132 mg, 0.75 mmol) is dissolved in THF and conduct the reaction with n-Bu4NF (0.3 ml of a 1.1 M solution, 0.3 mmol). After 45 minutes at room temperature, add ice water and polycentric in vacuum. The resulting oil is purified chromatographically on short columns (EtOAc, and then EtOH) to give the desired title compound as a white foamy productD= -34,0C (c 0.1 in MeOH).

Example 33. Boc-D-TMSal-Adgly-Boro Pro-OPin.

A. L-1-adamantylamine.

(3(2S)-4S)-3-(2-azido-2-adamant-1-yl-1-oxoethyl)-4-phenylmethyl) -2-oxazolidone (% de>95; 9,86 g, 25 mmol), obtained according to the method described in J. Arn. Chem. Soc. 109, 6881 (1987), dissolved in 320 ml of a mixture of THF/H2O (3:1), cooled to 0oC, mixed with 4 equiv. of hydrogen peroxide and 2 equiv. LiOH. The resulting mixture was stirred at 0oC until complete consumption of the substrate (30 minutes) and peroxyl (percarboxylic) decompose at 0oC with a 10% excess of 1.5 n aq. Na2SO3. After mixing with the aqueous NaHCO3(pH 9-10), the mixture is extracted several times with EtOAc to remove oxazolidinone (chiral) formed side. The product carboxylic acid, isolated by extraction with EtOAc from acidified (pH 1-2) of the aqueous phase, dried over Na2SO4and concentrated in vacuo. The desired (S)-azido-adamant-1-luksusowe acid is isolated in the form of white crystals (5.29 g) and used at a later stage without additional purification. 2 (S)-azido-adamant-1-iloks 2.5 h, the catalyst was removed and the solution was concentrated in vacuo to obtain the desired amino acid in the form of cleaners containing hydrochloride salt. The obtained hydrochloride are suspended in 40 ml of H2O and treated with 1.9 grams of solid NaHCO3. The resulting amino acid is filtered and washed several times with water. After drying in vacuo get white crystals of L-1-adamantylamine, []2D0= +3.0V (C 1 in MeOH).

B. Boc-D-TMSal-Adgly-ONSu.

B THF dissolve Boc-D-TMSal-ONP (7,71 g, a 20.2 mmol) of example 1 and added an aqueous solution of equimolar amounts of 1-adamantylamine and Et3N. After 20 h of exposure at room temperature THF is distilled off in vacuum and the aqueous residue diluted with 150 ml of 0.1 n HCl, and then extracted several times with EtOAc. The combined organic layers washed with brine, dried over Na2SO4and concentrated in vacuo. The oily product is purified chromatographically on silica gel (CH2Cl2) to produce the dipeptide Boc-D-TMSal-Adgly-OH in the form of oil. This dipeptide (6,9 g of 15.2 mmol) is dissolved in 80 ml of EtOAc. After cooling to 0oC add HONS4(2.1 g, 18 mmol) and DCC (3.1 g, of 15.2 mmol). The mixture was stirred at 0oC 3 h and then at room temperature for another 15 hours the Mixture is again cooled to 0oC, the filtered dicyclohexylphosphino and washed with EtOAc. The filtrate was washed with aqueous 0.1 M NaHCO3and then water 2% KHSOthe VA Boc-D-TMSal-Adgly-ONSu (7.2 g).

C. Boc-D-TMSal-Adgly-Boro Pro-OPin.

A named connection receive, using a technique similar to that described for the first stage of 3-stage get in the synthesis of Boc-D-TMSal-Pro-Baa-OPin in example 1/C. given the low reaction activity spatially constrained active ester phase B (2.7 g, 5 mmol) of intermediate-aminoboronic, which is formed due to the interaction of chiral a-chloro-boronate (+)-pinanediol-(S-1-chloro-4-bromo-butane-1-boronate) (1.7 g, 5 mmol) with hexamethyldisilazane lithium (5 mmol) and hydrolysis with HCl, reacts with the active ester of stage B with obtaining as the main product Boc-D-TMSal-Adgly-Boro Pro-OPin. The crude product is purified chromatographically (2:1 hexane/EtOAc) to give the title product (0,48 g) as a white foamy substance, which is further purified by recrystallization from ether/hexane to obtain the target compound Boc-D-TMSal-Adgly-Boro Pro-OPin in the form of a white crystalline substance. So pl. 187-188oC []2D0= +2,8(1 in CH2Cl2).

1. Derivatives of boron-containing peptides of General formula I

< / BR>
where W is hydrogen or

N-protecting group, such as benzyloxycarbonyl,1- C6-alkanoyl or1WITH6-alkoxycarbonyl; Y TM Sal-Pro - (P-TBDPS-O-Me)Ph>/BR>< / BR>
< / BR>
-CH2-Si(SH3)3< / BR>
or

CH2-C(CH3)3;

AA Pro, Gly, Ala, Leu, Val, Jle, Asp or Glu;

Q1and Q2together diol group of the formula

< / BR>
R4hydrogen;

R5a group of the formula OH and the last And the group -(CH2)7where Z 2,3,4 and 5 and X NH2, -NH-C(=NH)-NH2, -N3WITH1C4- alkoxy or-Si(CH3)3< / BR>
or

R4and R5together trimethylene group.

2. Derivatives under item 1, characterized in that it corresponds to the formula II

< / BR>
3. Derivatives under item 1, characterized in that it corresponds to the formula III

< / BR>
4. Derivatives under item 1, characterized in that it corresponds to the formula IV

< / BR>
5. Pharmaceutical composition having inhibitory activity to trypsinogen serine proteases, including derived peptide boronic acids and pharmaceutically acceptable carrier, characterized in that as a derived peptide boronic acids it contains a derived boron-containing peptide of General formula I

< / BR>
where W is hydrogen or

N-protecting group, such as benzyloxycarbonyl,1- C6-alkanoyl or1WITH6-alkoxycarbonyl;

Y TM Sal-Pro, (P-1
-CH2C(CH3)3, -CH2Si(CH3)3< / BR>
< / BR>
or

< / BR>
AA Pro, Gly, Ala, Leu, Val, Jle, Asp, Gly;

Q1and Q2together diol group of the formula

< / BR>
or

< / BR>
R4hydrogen;

R5a group of the formula OH and the last is a group

-(CH2)z,

where Z is 2, 3, 4, 5;

X-NH2, -NHC(= NH)NH2N3C1- C4-alkoxy or-Si(CH3)3or R4and R5together trimethylene group,

in the amount of 0.02 to 15 mg/kg of body weight.

 

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FIELD: organic chemistry, medicine.

SUBSTANCE: invention relates to applying compounds of the formula (I) for preparing an antibacterial composition and veterinary composition eliciting with the enhanced activity.

EFFECT: valuable properties of agents.

4 cl, 3 tbl, 78 ex

FIELD: organic chemistry, biochemistry, medicine, pharmacy.

SUBSTANCE: invention relates to macrocyclic peptides of the general formula (I): wherein W means nitrogen atom (N); R21 means hydrogen atom (H), (C1-C6)-alkoxy-, hydroxy-group or N-(C1-C6-alkyl)2; R22 means hydrogen atom (H), (C1-C6)-alkyl, CF3, (C1-C6)-alkoxy-group, (C2-C7)-alkoxyalkyl, C6-aryl or Het wherein het means five- or six-membered saturated or unsaturated heterocycle comprising two heteroatoms taken among nitrogen, oxygen or sulfur atom and wherein indicated Het is substituted with radical R24 wherein R23 means hydrogen atom (H), -NH-C(O)-R26, OR26, -NHC(O)-NH-R26, -NHC(O)-OR26 wherein R26 means hydrogen atom, (C1-C6)-alkyl; R3 means hydroxy-group or group of the formula -NH-R31 wherein R31 means -C(O)-R32, -C(O)-NHR32 or -C(O)-OR32 wherein R32 means (C1-C6)-alkyl or (C3-C6)-cycloalkyl; D means a saturated or unsaturated alkylene chain comprising of 5-10 carbon atoms and comprising optionally one-three heteroatoms taken independently of one another among oxygen (O), sulfur (S) atom, or N-R41 wherein R41 means hydrogen atom (H), -C(O)-R42 wherein R42 means (C1-C6)-alkyl, C6-aryl; R4 means hydrogen atom (H) or one-three substitutes at any carbon atom in chain D wherein substitutes are taken independently of one another from group comprising (C1-C6)-alkyl, hydroxyl; A means carboxylic acid or its alkyl esters or their derivatives. Invention relates to pharmaceutical compositions containing indicated compounds and eliciting activity with respect to hepatitis C virus and these peptides inhibit activity of NS3-protease specifically but don't elicit significant inhibitory activity with respect to other serine proteases.

EFFECT: valuable biochemical and medicinal properties of peptides.

106 cl, 9 tbl, 61 ex

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