PL167469B1 - Method of obtaining peptidic derivatives of boric acid - Google Patents

Abstract

The method of producing peptide derivatives boric acid of the formula I in which W is 77) hydrogen or nitrogen protecting group, Y is sequence per amino acid such that n + 1 amino acid the Y-Lys or Y-Arg peptide has an affinity for the active parts of the trypsin protease, where n is a number the total of 1-10 and at least one amino acid is unnatural amino acid with a hydrophobic chain side, Qi and Q2 are the same or different and represent -OH, -COR1, -CONR1R2, -NR1R2 or -OR3 groups, or Qi and Q2 together represent the remainder of the diol, Ri, R2 and R3 are such alone or different and represent a C 1-10 alkyl, C 6-10 aryl radical, C 6-10 aralkyl or phenyl substituted with up to three substituents such as C1-4- alkyl, halogen or C1-4 alkoxy, R4 R represents a hydrogen atom or a C1-10 alkyl radical, R5 is the group -A-X, where A is the group - (CH2) z, w which z is 2, 3, 4 or 5, a group -CH (CH3) - (CH2) 2, -CH2-CH (CHa) -CH2-, - (CH2) 2-CH (CH3) -, - (CH2> 2- C (CH3) 2, -CH (CH3H (CH2) 3-, -CH2-CH (CH3) - (CH3) - (CH2) 2 -, - CH2-CH2-CH (CH3) -CH2 -, - (CH2) 3CH (CH3) -, - (CH2) 3-C (CH3) 2; C6-10-aryl, C6-10-aralkyl, X represents the group -NH2 and the asymmetric carbon atoms marked with an asterisk may indicate the D or L configuration or mixtures thereof, characterized in that the compound of formula 7, wherein Qi and Q2 are as defined above a Ri3 is the group -A-Br in which A is as defined above meaning, reacted with LiN [Si (CH3) 312 and then acid hydrolysis and coupling with the protected are carried out a peptide of formula 8, where W and Y are above given meaning, followed by the resulting compound of the formula 6,

Description

The subject of the invention is a process for the production of new peptide boric acid derivatives for pharmaceutical use.

The invention relates to inhibitors of serine proteases such as thrombin, factor Xa, kallikrein and plasmin, as well as other serine proteases such as prolyl endopeptidase and IgAI protease. Thrombin, the last enzyme in the clotting system, cleaves soluble flbrinogen into fibrin, which then cross-links to form an insoluble gel that forms the stroma of the thrombus. When a vessel is damaged, the above process is necessary to stop the bleeding. Under normal circumstances, the amount of thrombin present in plasma is not measurable. The increase in thrombin levels can cause blood clots that can lead to thromboembolism, one of the most widespread serious medical problems of our time.

Thrombin contributes to hemostatic control through a variety of biological responses. In addition to its primary function of converting fibrinogen to fibrin, thrombin activates factor XIII, which is responsible for cross-linking fibrin. Thrombin also works through a positive feedback mechanism to activate factors V and VIII, both of which are essential for it from prothrombin. Thrombin plays another important role: its binding to platelets initiates platelet release and aggregation, which is responsible for primary haemostasis.

In addition, the thrombin reactions are inhibited by natural inhibitors in the plasma. The most important of these are antithrombin III and heparin. These two compounds have been isolated and are used therapeutically and prophylactically in conditions where there is an imbalance in the hemostatic mechanism with the risk of activation of prothrombin.

Orally acting synthetic thrombin inhibitors would be valuable alternatives to parenterally administered natural inhibitors. As a result of numerous studies in this field, beneficial in vitro thrombin inhibitors have been obtained, but so far there is no real good agent for oral therapeutic use. During the reconstitution of the amino acid sequences of fibrinogen, an important natural thrombin substrate, several good short peptide thrombin substrates were obtained. These peptide substrates were also converted into enzyme inhibitors. Thus, the chromogenic substrates D-Phe-Pro-Arg-pNA and D-Phe-Pip-Arg-PNA mimic the sequence preceding thrombin bond cleavage. The corresponding reversible and irreversible inhibitors of D-Phe-Pro-Arginal and D-Phe-Pro-Arg-CHbCl show in vitro inhibition of the order of 1 (T ⁸ M.

Chloromethyl ketones are generally too nonspecifically reactive to be suitable for therapeutic purposes, and the above-mentioned peptide aldehydes have too low an LD50 value.

Factor Xa is the clotting enzyme responsible for the production of thrombin through the limited proteolysis of its zymogen, prothrombin. On a positive basis, factor Xa is at least 10 times more thrombogenic (clot-forming) in vivo than thrombin. This is because factor Xa is one step higher than thrombin in an enhancement cascade such that one factor Xa molecule can produce multiple thrombin molecules from its precursor. Its strength may also be due to the rather slow removal of factor Xa from the body. Thrombin, unlike factor Xa, is rapidly cleared from the bloodstream to sites of high affinity on the vessel walls. Factor Xa's central position at the nodal point of intra- and extrinsic pathways makes it a suitable target for modulating the hemostatic mechanism.

Kallikrein is formed from prekallikrein by factor XII when placed on a negatively charged surface. Kallikrein in turn can convert factor XII to factor XIIa, whereby a reciprocal activation system is formed. Factor XIIa is the first enzyme in the inner part of the clotting system. The importance of the contact system is likely to be in a surface-mediated defense mechanism, and a large-scale activity of this system is typically seen during shock or disseminated intravascular coagulation (DIC). The role of kallikrein at this stage is to cleave the high molecular weight kininogen to release the vasodilating bradykinin. Bradykinin also causes increased vascular permeability, pain and migration of neutrophilic leukocytes. It was found that inhibitors

Kinin formation is beneficial in certain types of inflammation, including pancreatitis and arthritis, and can also be used to treat asthma. The only substance that has gained clinical significance as a kallikrein inhibitor is aprotinin (Trasylol). Aprotinin is a polypeptide with a molecular weight of 6,500, it forms stable complexes with proteases with a binding constant of 10 ¹ ° - 10 ¹³ M.

Fibrinolysis is the process that causes the enzymatic dissolution of fibrinogen and fibrin clots. Plasmin contains the plasminogen protein, which under the influence of various activators is converted into plasmin, a proteolytic enzyme whose activity is similar to that of trypsin. Plasmin breaks down fibrinogen and fibrin into fibrin / fibrinogen degradation products.

Under normal conditions, the fibrinolytic system is in equilibrium with the coagulation system. The small clots formed in the bloodstream can be dissolved enzymatically and the circulation in the vessels can be restored to normal by activating the fibrinolytic system in the body. If the fibrinolytic activity is too high, it may cause or prolong bleeding. This activity can be inhibited by natural inhibitors in the blood.

Prolyl endopeptidase cleaves peptide bonds at the carboxy portion of proline residues in the peptide chain. It is a serine protease that readily degrades a number of neuropeptides containing substance P, neurotenin, thyrotropin releasing hormone and luteinizing hormone releasing hormone, and which is related to the cell's ability to produce interleukin 2 (IL-2). The enzyme is inhibited by benzyloxycarbonyl-prolyl-prolinal with an inhibitor constant Ki of 14 nM. Despite the fact that almost nothing is known about the physiological role of prolylendopeptidase, it may play an important role in regulating the biological activity of various neuropeptides.

IgA-catalyzed cleavage of IgA, the predominant form of antibodies as the first line of defense against infection, separates the Fc from the antibody-binding Fab regions of the molecule. Such cleavage would be expected to reduce or abolish antimicrobial activity. All identified IgA proteinases are cleaved after a proline residue in the human IgA axis. Peptide prolylboronic acids inhibit IgA and proteinases from Neisseria gonorrhoea and Hemophilus influenzae, indicating that these enzymes belong to the serine protease family of proteolytic enzymes.

The multiple roles played by thrombin in various physiological processes related to pathological conditions, such as cancer, inflammation and neuronal activity, suggest that thrombin inhibitors may be used for numerous non-cardiovascular indications.

Many neoplastic cells have been found to exhibit procoagulant activity related to the production of thrombin. As a result, local fibrin deposition and clotting occurs, which is believed to be important for tumor growth. In addition to its action on endothelial cells, thrombin may facilitate extravasation of tumor cells during metastasis. Thus, thrombin inhibitors may be beneficial not only in treating certain cancers, but may also reduce the hypercoagulability frequently observed in patients during therapy with chemotherapeutic agents.

Activation of endothelial cells with thrombin induces a number of pro-inflammatory changes such as the synthesis and release of interleukin-1, prostaglandins and the platelet activating factor. In addition, thrombin exposes GMP-140 and CD63, two adhesion molecules responsible for the adhesion of leukocytes to the endothelial surface. Thrombin also enhances vascular permeability of proteins, an action that involves neutrophils, and cleaves the interleukin-8 precursor protein, a peptide that is possibly involved in respiratory disorders, rheumatoid arthritis, and ulcerative colitis.

The involvement of thrombin in all these pro-inflammatory processes makes it a potential target for therapeutic treatment with thrombin inhibitors in inflammatory pathological disorders.

The activity of the nexin-1 protease, which is a nerve growth modulator and a specific natural thrombin antagonist, is significantly and specifically reduced in Alzheimer's patients. This along with the observation that the activity of the thrombin type increases in

167,469 in Alzheimer's disease brains suggests that thrombin inhibitors may limit or reverse the meuronic lesions associated with hyperthrombin hyperfunction.

Boric acids have been studied as inhibitors of various serine esterases and proteases. The first boric acid-containing amino acid analog to be used as a protease inhibitor was the boric acid analog of N-acetyl-L-phenylalanine, which was used as chymotrypsin and subtilisin inhibitor. Peptide boric acid derivatives have been used as chymotrypsin, subtilisin and elastase inhibitors.

The interaction of boric acids with proteases in biological systems is known and various simple boric acids have been found to be sufficiently non-toxic to humans. Peptide boric acid inhibitors of elastase have recently been used in animal experiments on emphysema. Unlike the peptide chloromethyl ketones, there is no mention of toxicity at the biologically effective dosage level.

European Patent No. 293881 describes a process for the preparation of peptides containing C-terminal boric acid derivatives of lysine, ornithine and arginine and their use as inhibitors of serine proteases of the trypsin type. The other amino acids in these peptides are the D or L form of the 20 naturally occurring amino acids.

It has been found that compounds with excellent trypsin type serine protease inhibitor properties can be obtained if the peptide comprises at least one unnatural α-amino acid having a hydrophobic side chain.

According to the invention, peptide derivatives of boric acid of the formula I are obtained, in which W is a hydrogen atom or a nitrogen protecting group, Y is a sequence of n amino acids chosen such that the n + 1 amino acid peptide Y-Lys or Y-Arg has an affinity for the active side of the protease type trypsin, wherein n is an integer of 1-10, preferably 1-4 and wherein at least one amino acid is an unnatural amino acid having a hydrophobic side chain, Q1 and Q2 are the same or different and represent the groups -OH, -CORi, -CONR1R2, - NR1R2 and OR3, or Q1 and Q2 together form a diol group, Ri, R2 and R3 are the same or different and represent C1-10-alkyl, C6-10-aralkyl, or phenyl radicals substituted with up to three substituents such as C1-10 -4-alkyl, halogen, and C 1-16, and C 1-6 coxy; R4 represents a hydrogen atom or a C1-10 aalkyl radical, R5 represents a group -AX wherein A is a group - (CH2) z-, where z is 2, 3, 4 or 5; -CH (CH ₃ ) - (CH2) 2-, -CH2-CH (CH ₃ ) -CH2-, - (CH ₂ ) 2-CH (CH ₃ ) -, - (CH2) 2-C (CH3) 2 -, -CH (CH3H (CH2) 3, -CH2-CH (CH3) - (CH2) 2, -CH2-CH2-CH (CH3) -CH2, (CH2) 3cH (CH3) - (CH2) 3-C (CH3) 3, C6-10 aryl, Ca10-aralkyl, X is -NH2 and the asymmetric carbon atom marked with a asterisk may have the D or L configuration, or a mixture thereof.

By unnatural amino acid is meant an amino acid other than D- or L-Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val.

Preferably, the nitrogen protecting group W is a group of the formula H (CH2CH2O) p, where p is 3-30, the group RaCO-, R7OCO- or R8SO2-, in which R _e is a C1- a-alkyl radical, R7 is a C1 radical - α -alkyl, phenyl, benzyl or naphthyl and Re is phenyl, naphthyl or C1-4alkylphenyl radical, with R7OCO- being preferred. The most preferred protecting groups are those of the formula R7 ^{, (} COC ~, where R7 'is tertiary butyl (denoted Boc) and where R7' is benzyl (denoted Z).

Preferably R5 is R5 'where R5' is the group - (CH2) _Z '-X' where X 'is the group -NH2 and az' is 2, 4 or 4.

Compounds of formula la are preferred, where W, Y, R4 and R5 are as defined above and R9 and R10 represent the remainder of the dihydroxy compound.

Examples include 2,3-butanediol, catechol, 2,3-dimethylbutanediol-2,3, cyclohexanediol, ethylene glycol, 1,2-hexanediol, 2,3-hexanediol, diethanolamine or aliphatic or aromatic compounds containing hydroxyl groups which are substituted on adjacent carbon atoms or on carbon atoms substituted with other carbon atoms.

Particularly preferred are compounds in which Qi and Q2 together represent an OPin group of formula 9 or a group of formula 10.

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Y-forming amino acids are α-amino acids, such as naturally occurring L-amino acids in proteins, corresponding enantiomeric D-amino acids or chemically modified alpha-amino acids such as glutamic acid gamma-piperidide of formula 11 or pipecolic acid (Pip), with the proviso that at least one amino acid is an unnatural amino acid having a hydrophobic side chain.

The preferred unnatural amino acids are compounds of Formula 2 wherein R11 is hydrophobic. A preferred hydrophobic group is a methylene group bound to an optionally substituted polar aromatic group or an alicyclic group having at least two rings and no polar substituents, or to a tertiary group. butyl or trimethylsilyl group. Preferably Rn is R11 ', where R11' is a group of formula 12, a group of formula -CH2-Si (CH3) 3, a group of formula 13, a group of formula -CH2-C (CH3) 3, a group of formula 14, 15 or 16.

The unnatural amino acids of formula II may exist in the D or L form or as mixtures thereof, but preferably in the D form.

Preferred compounds are the thrombin inhibitors of formula I wherein Y is a sequence of two amino acids where the N-terminal amino acid is an unnatural amino acid and the other amino acid is L-proline (pro) of formula 17.

More preferred compounds are represented by the formula 1 'wherein N, R4, R5, R11, Q1 and Q2 are as defined above.

Particularly preferred are compounds of formula I wherein W, R4, R5 'and R11 are as defined above.

The most preferred compounds are the compound of formula 3, the compound of formula 4 and the compound of formula 5.

A peptide should have an affinity for the active part of the trypsin-type protease if the dissociation constant of the peptide in question is 10-3 M or lower.

The process according to the invention for the preparation of compounds of formula 1 in which the substituents are as defined above consists in that the compound of formula 7 in which Qi and Q2 are as defined above, and R13 is the group -A-Br in which A is as above given meaning, reacted with LiN [Si (CH3) 3] 2, followed by acid hydrolysis and coupling with a protected peptide of formula 8, in which W and Y are as defined above, and then the obtained compound of formula 6, where R12 is -A-Br and A, W, Y, Q1 and Qa are as defined above, is reacted with sodium azide and the resulting compound of formula I wherein R5 is AX in which A is is hydrogenated as defined above and X is N3 and the remaining substituents have the same meaning as defined above. The hydrogenation can be carried out in a known manner using, for example, a Pd / C catalyst.

The reaction of the compound of formula 6 with sodium azide is carried out in a polar aprotic solvent such as dimethyl sulfoxide.

The reaction of the compound of formula 7 with the compound LiN [Si (CH3) 3] 2 and subsequent hydrolysis with 3 molar equivalents of the acid and coupling with the protected peptide of formula 8 is preferably carried out in a dry aprotic polar solvent such as tetrahydrofuran at a temperature of - 78 ° C to room temperature.

The starting compounds of formula 7 are obtained by the method of Matteson et al., Organometallica 3, 1284-8 (1984).

The protected peptide of formula 8 can be produced by methods known in peptide chemistry, starting from the desired unnatural amino acid. Such amino acids are commercially available, for example the amino acid of formula II in which R1 is the group of formula 12, or can be prepared by methods analogous to those described in the literature, e.g. Angew, Chem. 93, 793 (1981) and J. Am. Chem. Soc. 109, 6881 (1987).

The compounds of formula I may find use as inhibitors of trypsin-type proteases and may be used in vitro for diagnostic and mechanistic studies of these enzymes. Moreover, due to their inhibitory activity, they are indicated for use in the prevention or treatment of disorders caused by an excess of an enzyme in the regulatory system, for example for the control of coagulation in the fibrinolytic system.

The compounds according to the invention that are inhibitors of thrombin or factor Xa exhibit anti-thrombogenic properties and can be used when it is essential.

167 469 anti-thrombogenic factor. In general, these compounds can be administered orally or parenterally to achieve an anti-thrombogenic effect. For larger mammals such as humans, the compounds can be administered alone or in conjunction with pharmaceutical carriers or diluents at dosages of 0.02-15 mg / Kg of body weight, preferably 1-10 mg / Kg in order to obtain an anti-thrombogenic effect and can be they should be administered in the form of a single dose or in divided doses or in the form of a prolonged release. When extracorporeal blood circulation is used, 0.1-1 mg / Kg is administered intravenously. When used at a dose of 1-10 mg per liter for whole blood, clotting is prevented. Pharmaceutical diluents are sugars, starches and water, which can be used in the preparation of tablets, capsules, injectable solutions, etc. The compounds of the present invention can be added to blood to prevent blood from clotting in blood collection or separation containers, tubing or implanted appliances that come in contact with blood.

The advantages of the new compounds are oral activity, quick onset of action and low toxicity. Additionally, these compounds may find special use in the treatment of subjects that are hypersensitive to compounds such as heparin.

In the following examples, the symbols mean the following:

Z = benzyloxycarbonyl

Boc = Tertiary butyloxycarbonyl

Ac = acetyl

MeOH = methyl alcohol

EtOAc = ethyl acetate

DCC = dicyclohexylcarbodiimide

HONSu = N-hydroxysuccinimide

OPin = pinanediol

THF = tetrahydrofuran

N-Bu = n-butyl

Np = p-nitrophenyl

TLC = thin layer chromatography

Bzl = benzyl

Baa = -NH-CH- (CH ₂ CH ₂ CH ₂ Br) BTMSal = trimethylsilylalanine

Adal = adamantylalanine

Naphal = 2-naphthylalanine

BoroOrn = -NH-CH- (CH ₂ CH ₂ CH ₂ NH ₂ ) BBoroArg = -NH-CH- [CH ₂ CH ₂ CH ₂ NHC (NH) NH ₂ ] BAdgly = 1-adamantylglycine

Boro-Pro-OPin = proline analog in which the -COOH group is replaced with B-OPin

BoroLys = -NH-CH- (CH ₂ -CH2-CH ₂ -CH2-NH ₂ ) BBoroHArg = -NH-CH- (CH ₂ CH ₂ CH ₂ CH ₂ NHC) NH (NH ₂ ) BBoroMpg = -NH-CH - (CH ₂ CH ₂ CH ₂ -OCH3) Bp-OH-Me-Phal = p-hydroxymethyl-phenylalanine p-TBDPS-O-Me-Phal = p-tert-butyl-diphenyl-silyl-oxymethyl-phenylalanine.

The kinetic parameters of Ki, Kon and koff are determined by inhibition of enzyme catalyzed peptide-arg-pNA hydrolysis. This hydrolysis yields p-nitroaniline and its time-dependent release, quantified by the optical density measured at 405 nm, determines the degree of inhibited and uninhibited reaction.

Kinetic measurements are performed on a 96 microwell plate in combination with a single-cell kinetic reader. Human thrombin, titrated on the active part, is dissolved in 0.1 M phosphate buffer, pH 7.4 containing 0.1 M NaCl and 0.1% bovine serum albumin in a stock solution containing 400 nM active enzyme.

167 469

For the chromogenic assay, this solution is dissolved in the same buffer to 0.4 nM. The substrate, 2AcOH-H-D-cyclohexyl-ala-arg-pNA, is dissolved in the same buffer to a concentration of 0.5 mM. Inhibitors are first dissolved in a 1: 1 creamophore / ethanol system and then diluted with distilled water to a 1 mM stock solution. Further dilutions are carried out with the phosphate buffer described above.

Assays are started by adding 100 µl of enzyme solution to a mixture containing 100 µl of inhibitor and 50 µl of substrate solution. Release of p-nitroaniline from the hydrolysis of the peptidyl-p-nitroanilide substrate occurs within 30 minutes to 1 hour and the increase in optical density at 405 nm is measured. The collected data is used to calculate kinetic parameters in the presence and absence of the inhibitor. While other mechanisms of action are not excluded, the characterization of this class of thrombin inhibitors is limited to the two main mechanisms observed, namely fast, reversible, and slow tightly binding competitive inhibition. The kinetic constants of these inhibitors showing a fast, reversible binding mechanism, namely fast binding (initial amount Vo of inhibition> Vo with inhibitor (with It >> Et (total inhibitor concentration / total enzyme concentration) are calculated by linear regression derived from the function plot 1 / vvs Inhibitor concentration (I) The Ki value is calculated from the horizontal determinant of Ki, app by equation (1)

Ki, app = Ki (1 + S / Km) (1)

If the enzyme interaction index is slow (Vo with no inhibitor effect) and compact (Ki close to or lower than Et) such that the steady state inhibition rate is slowly achieved, the rate of pNA formation is described by equation (2) (V ₀ -Vs)

P = Vst + --- [1-exp (-kobst)] kob3 (2) where: P is the amount of pNA formed at time t, Vo is the initial amount, Vs is the equilibrium amount and kobs is the global amount as a function of Et , It, Ki, app and the observed secondary constant (k'on) for the interaction between the inhibitor and enzyme. The data from slow, firmly binding braking measurements are applied to equation (2) by nonlinear regression analysis from which approximate kobs values are obtained. The values for k'on, koff and Ki, app are then obtained from the plot of the function kobs vs (I). The koff value is obtained from the vertical determinant, while the k'on and Ki values are calculated from the slope and horizontal determinants, respectively, using equation (1).

The following examples explain the invention in more detail.

Example! . Boc-TMSai-Pro-NH-CHKCHz ^ jBOPin.

A. Boc-D-TMSal-OH.

21.5 g (113.7 mmol) of D-TMSal ethyl ester prepared according to Angew. Chem. 93, 793 (1981) was dissolved in CH ₂ Cl ₂ and a solution of an excess of BoczO CH2G2. The mixture is kept for 15 hours at room temperature, then 500 ml of ice-cooled 0.25 N hydrochloric acid are added. The organic layer was washed with 5% NaHCO3 and brine, dried over NazSO ₄ and concentrated in vacuo. The crude product in the form of a colorless oil is used directly for the saponification step. The product was dissolved in methanol, cooled to 0 ° C, mixed with 510 ml of 1N NaOH and stirred at 0 ° C for 3 hours. After acidification to pH 1 with 1N HCl, the mixture was extracted several times with ether. The organic layers are combined, washed with brine, dried over Na ₂ SO _4, and concentrated in vacuo. The 29.7 g oil product is used in the next step without further purification.

B. Boc-D-TMSal-Pro-ONSU 29.7 g of Boc-D-TMSal-OH (113.7 mmol) and 19.0 g (136.3 mmol) of p-nitrophenol are dissolved in EtOAc. After cooling to 0 ° C, 23.4 g (113.6 mmol) of DCC are added and the mixture is stirred for 1 hour at 0 ° C and then for 15 hours at room temperature. The precipitate was filtered off and washed with EtOAc, and the filtrate was concentrated under reduced pressure. The resulting oil was purified by flash chromatography (9: 1 hexane / EtOAc) to afford the desired Boc-D-TMSal-ONp as white crystals.

51.6 g (113.7 mmol) of Boc-D-TMSal-ONp are dissolved in THF and an aqueous solution of equimolar amounts of proline and EteN is added. After the mixture was kept for 20 hours at room temperature, the THF was removed under reduced pressure and the aqueous residue was diluted with

167,469 with water then extracted several times with EtOAc. The pH of the aqueous layer is adjusted to 3 by adding 10% citric acid. The resulting oily product was extracted several times with EtOAc. The combined organic layers are washed with brine, dried over Na ₂ SO 4, and concentrated under reduced pressure. The colorless oil was recrystallized from ether / hexane to give the Boc-D-TMSal-Pro-OH dipeptide as a white crystalline compound, m.p. 176 ° C.

26.0 g (72.5 mmol) of the obtained dipeptide are dissolved in EtOAc. After cooling to 0 ° C, 9.8 g (85.5 mmol) of HONSu and 14.9 g (72.3 mmol) of DCC are added. The mixture is stirred for 3 hours at 0 ° C and then for a further 15 hours at room temperature. The mixture was cooled again to 0 ° C, the dicrochexy and urea was filtered off and washed several times with EtOAc. The filtrate was washed with an aqueous solution of 0.1 M Na ₂ CO ₃ and then with an aqueous 2% solution of KHSO 4.

After drying over Na ₂ SO 4 and concentration under reduced pressure, Boc-DTMSal-Pro-ONSu is obtained as a white foam.

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

This process is a one-pot reaction involving the formation in situ of chiral α- (bistrimethylsilyl} -amido-borane, hydrolysis of two trimetrylsilyl groups and coupling of the thus obtained α-amino borane with an active ester (Boc-D-TMSal-Pro-ONSu) prepared in step B. The initial reaction sequence is carried out under an argon atmosphere. 1.75 g (5.0 mmol) of a chalchloroborate (1-borate (+ - pinanediol- (SZ-1-chloro-4-bromo) -butane was dissolved in 2.5 ml of THF and added to a pre-cooled to -78 ° C solution of lithium hexamethyldisilazane (5 ml of a 1.0 M solution in hexane, 5.0 mmol). The mixture was stirred for 30 minutes at at -78 ° C, the solution is then brought to room temperature overnight. After cooling again to -78 ° C, 3 molar equivalents of HCl in dioxane are added. The mixture is warmed to room temperature and stirred for 2 hours at this temperature. After cooling back to -20 ° C, a solution of 2.28 g (5. 0 mmol) of the active ester from step B in 6 ml of CH ₂ Cl ₂ followed by 1.39 ml (10.0 mmol) of methylamine.

The mixture is stirred for 1 hour at -13 ° C, warmed to room temperature and stirred for 2 hours at this temperature. The mixture was filtered, the filtrate was concentrated under reduced pressure, the residue was diluted with ether and washed with 2N HCl, 5% NaHCO3 and brine. The organic layer was dried over Na ₂ SO 4 and concentrated under reduced pressure. The residue crystallized on standing to afford the desired chiral peptide borate as a white crystalline material.

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

804 mg (1.2 mmol) of Boc-D-TMSal-Pro-Baa-OPin product from step C are dissolved in 13 ml of DMSO and 156 mg (2.4 mmol) of sodium azide are added. The mixture was stirred for 3 hours at room temperature. A mixture of ether and ice water is added, whereupon white crystals precipitate. The precipitate was filtered off and washed with ether to give 0.6 g of the azide as a white crystalline material.

Example II. Boc-D-TMSal-Pro-BoroOrn-OPin.

569 mg (0.9 mmol) of the azide from Example 1 are dissolved in 25 ml of EtOAc and hydrogenated in the presence of 0.5 g of 10% Pd on carbon. After 2.5 hours, the catalyst was removed and the solution concentrated in vacuo to give a white foam which was recrystallized with EtOAc / ether to give the desired product as a white crystalline compound, mp 200-202 ° C (αD> = 11) . 6 ° (c == 0.5 in MeOH).

Example III. Boc-D-TMSal-Pro-NH-CH ((CH2) 3N3) B-OPin.

A) 1-borate (4 --- pmanodiol- (S) ~ 1-chloro-5-bromo-pentane.

20.8 ml (203.3 mmol) of 4-bromo-1-butene are reacted with 24.4 g (203.3 mmol) of catecholborane at 100 ° C for 16 hours. The crude product was distilled under reduced pressure to give 4-bromo-butane-1-borate as a white crystalline material. 27.7 g (163 mmol) of (+) -pinanediol were dissolved in THF and 41.6 g (163 mmol) of Abromobutane 1-borate obtained above was added. The mixture is kept for 1 hour at 10 ° C

After removal of the THF under reduced pressure, the residue was purified by flash chromatography (90:10 hexane / EtOAc) to afford (+) -pinanediol-4-bromobutane 1-borate as a colorless oil.

The desired (+) -pinanediol- (S) -1-chloro-5-bromo-pentane-1-borate was prepared according to Organometallics 3.1284 (1984). 9.8 ml of methylene chloride in THF is cooled to -100 ° C and 71.6 M n-butyllithium solution (114.5 mmol) is added over 20 minutes. The mixture is kept for 15 minutes at -100 ° C, then a cold (-78 ° C) solution of 32.8 g (104.1 mmol) of (+) -pinanediol-5-bromo-pentane 1-borate is added in THF. After an additional 1 hour hold of the mixture at -100 ° C, 7.1 g (52.0 mmol) of anhydrous ZnCl 2 are added. The mixture is kept for an additional 15 minutes at -100 ° C, then warmed to room temperature and stirred for 2 hours at this temperature. The solvent is removed under reduced pressure, the residue is diluted with hexane / water and extracted several times with hexane. After drying over Na ₂ SO 4 and removal of the solvent in vacuo, (+) -pinanediol- (S) -1-chloro-5-bromo-pentane-1-borate was obtained as a yellow oil which was used directly in the next step without further purification.

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

Cool 65.2 ml of a 1.0 M solution of LiN (SiMe3) 2 (65.2 mmol) in THF to -78 ° C. 23.7 g (65.2 mmol) of σ-chloroborane from step A in THF are added. The mixture is stirred for 1 hour at -78 ° C and then for 15 hours at room temperature. The mixture is then cooled back to -78 ° C, 29.8 ml of 6.56 N HCl solution (196 mmol) are added, the solution is stirred for 45 minutes at -78 ° C and then 2 hours at room temperature. The mixture is cooled to -15 ° C, 29.7 g (65.2 mmol) of Boc-TMSal-Pro-ONSu from Example 1 in CH2Cl2 are added, followed by 18.1 ml (130.4 mmol) of triethylamine to initiate reaction. The mixture was stirred at -15 ° C for 1 hour and then for 2 hours at room temperature. The mixture was filtered over Hyflo and concentrated under reduced pressure. The residue is diluted with ether / water and extracted several times with ether. The mixture was dried over NazSCL and concentrated in vacuo to afford the desired Boc-D-TMSal-ProNH-CH ((CH2) 4-br) B-OPin which, upon crystallization from ether / hexane, gave a white crystalline material, m.p. 74 ° C .

C) Boc-D-TMSal-Pro-NH-CH ((CH ₂₎ 4 N ₃₎ B-Opin.

33.3 g (48.6 mmol) of the product from step B are dissolved in DMSO and 6.3 g (97.2 mmol) of sodium azide are added. The mixture was stirred for 6 hours at room temperature. Ether / ice water is added and the mixture is extracted several times with ether. After drying over Na ₂ SO 4 and concentration under reduced pressure the resulting oil is crystallized to give Boc-D-TMSałPro-NH-CH ((CH2) 4N3) B-Pin as a white crystalline substance melting at 69 - 70 ° C, ff _D = -74.4 ° (c = 1.0 in MeOH).

Example IV. Boc-D-TMSal-Pro-BoroLys-OPin.

22.0 g (34.0 mmol) of the azide from Example 4 was dissolved in EtOAc and hydrogenated in the presence of 4.0 g of 10% Pd on carbon. After 9 hours, the catalyst was removed and the solution was concentrated under reduced pressure. The resulting foam is dissolved in EtOAc and crystallized to give the desired Boc-D-TMSal-Pro-BoroLys-OPin as white crystals, m.p. 128-129 ° C, σο = -59.6 ° (c = 1.0 in MeOH) .

Examples V-XV. By methods analogous to those described in przykładachI-IV can be prepared the compounds of formula Ib, wherein R 4, R ', R11, Qi + _Q2 and AA have the meanings given in the following table.

167 469

Table

No

example in R11 R4 Re' Qi + Q2 AA (Ooh " C. Solvent 1 2 3 4 5 6 7 8 9 10 V Boc pattern 12 H. - (CH2) 3-NH2 pattern 9 Pro -27.8 ° 0.5 EtOH VI Boc pattern 13 H. ^ CH2) 4-No pattern 9 Pro -64.7 ° 0.51 CC2CI2 VII Boc pattern 13 H. - (CHj), - NH ₂ pattern 9 Pro -52.4 ° 0.54 CH2Cl2 VIII Boc pattern 13 H. - {CH2) 3-NH2 pattern 9 Pro -10.6 ° 0.5 CH2Cl2 IX Ac pattern 12 H. - <CH ₂ ) 3-NH ₂ pattern 9 Pro -78.6 ° 0.75 CH2Cl2 X Boc pattern 13 H. - (CH2) 3-NH2 pattern 10 Gly -4.4 ° 0.5 CH2Cl2 XI Boc -CH2-C (CH3) 3 H. - {CH2) -N3 pattern 9 Pro -76.0 ° 0.5 CH2Cl2 XII Boc -CHi-C (CH3) 3 H. CCH ₂ ) nHH2 pattern 9 Pro -25.4 ° 0.5 CH2Cl2 XIII Boc CHz-SKCHa)! H. - (CK2) 4-NH2 pattern 9 Gly -30.6 ° 0.5 EtOH XIV Boc CH2-Si (CH3) 3 H. - <CH2) 4NH2 pattern 9 Asp -18.4 ° 0.5 MeOH XV H. CH2-Si (CH3) 3 H. - <CH2) 3NH2 pattern 9 Pro -53.6 ° 0.32 MeOH

Example XVI. Boc-D- (p-) TBDPS-O (-methyl) Phal-Pro-BoroOrn-OPin.

A) Boc-D- (p -) (1,1-dimethylethyl) diiphenylsilyl (-oxy) methylphenylalanine.

For the selective reduction of the azide group in the substrate, 7.27 g (66.0 mmol) of thiophenol are added to a suspension of 3.12 g (16.5 mmol) of SnCl2 in CH2 Cl2. 6.8 ml (49.5 mmol) of triethylamine are added and a yellow solution is obtained. Then 4.8 g (22.0 mmol) of Boc anhydride are added, followed by 6.8 g (11.0 mmol,% de> 95) of (3- (2S), 4S-3- (2-azido-3 - (p-) (1,1-dimethyl) diphenylsilyl (-oxy-methyl) phenyl-1-oxo-propyl - (- 4-) phenylmethyl) -2-oxazolidinone, prepared according to J. Am. Chem. Soc. 109, 6881 (1987) as a solution in CH2 Cl2 The mixture was stirred for 2.5 hours at room temperature, then diluted with EtOAc / 2N NaOH and filtered through Hyflo. The organic layer was washed with 2% aqueous NaHSO4, 5% aq. NaHCOa solution and finally brine. The yellow oil obtained after drying over NasSO4 and concentration in vacuo was purified by flash chromatography to afford (3 (2S), 4S (-3 ° C) 2 ° C) ((Tert.butyloxy ) -carbonyl) -ammo (-3- (pX (1,1-dimethylethyl) -diphenylol-silyl (-oxy} -methyl) -phenyl-1-oxopropyl) -4- (phenylmethyl) -2-oxazolidinone as white foam. 2.0 g (2.88 mmol) of this compound are dissolved in THF / water and hydrolyzed with 5.76 mmol of in situ generated LiOOH in at 0 ° C. After 1.75 hours at 0 ° C, 1.25 g (9.9 mmol) of Na2SO3 in water are added. The THF was removed under reduced pressure, the pH of the residue was adjusted to 1-2 and the mixture was extracted three times with EtOAc. The combined organic layers are washed with water, dried over Na2SO4, and concentrated under reduced pressure. After crystallization from hexane / ether, the oxazolidinone is obtained in the form of white crystals. The filtrate was concentrated under reduced pressure to afford the desired title compound as a white foam.

B) Boc-D- (p -) (1,1-dimethylethyl) diphenylsilyl- (oxy) methyl-phenylalamno-Pro-ONSu.

0.59 g (2.88 mmol) of DCC is added to a mixture of 1.6 g (2.88 mmol) of the title compound from step A) and 0.43 g (3.12 mmol) of p-nitrophenol in EtOAc at 0 ° C. The mixture was stirred for 16 hours at room temperature. After cooling to 0 ° C, the precipitate was filtered off and washed with cold EtOAc. The filtrate was concentrated under reduced pressure. The resulting Boc-D- (p-TBDPS-O-Me) -Phal-ONp oil is used in the next step without further purification.

2.2 g (2.88 mmol) of Boc-D- (p-TBDPS-O-Me) -Phal-ONp are dissolved in THF and an aqueous solution of 365 mg (3.17 mmol) of L-proline and 0.88 ml ( 6.33 mmol) Et3N. The mixture was stirred for 15 hours at room temperature, then the THF was removed under reduced pressure. The pH is adjusted to 3 by the addition of 10% citric acid. The resulting oily product was extracted several times with EtOAc. The combined organic layers are washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The colorless oil is purified by flash chromatography (9: 1 CH2Cl2) EtOH to elute p-nitrophenol followed by 80:20 CH2Cb (EtOH) to give Boc-D- (p-TBDPS-O-Me) -Phal-Pro- Oh in the form of white foam.

1.3 g (2.06 mmol) of this dipeptide are dissolved in EtOAc. After cooling to 0 ° C, 220 mg (2.47 mmol) of HONSu and 330 mg (2.06 mmol) of DDC are added. The mixture is cooled back to 0 ° C, the dicyclohexylurea is filtered off and washed several times with cold EtOAc. The filtrate was washed with an aqueous 0.1 M NaCO 3 solution, 8% NaHSO 4 solution, and then with brine. After drying over Na2SO4 and concentration in vacuo, the title compound Boc-D- (p-TBDPS-O-Me) -Phal-Pro-ONSu is obtained as a white foam.

167 469

C) Boc-D- (p-TBDPS-0-Me) -Phal-Pro-Baa-OPin.

The title compound is prepared using an analogous 3-step one-pot process as described for the synthesis of Boc-D-TMSal-Pro-Baa-OPin in Example IC. So the intermediate α-aminoborane, which is obtained by reacting 659 mg (2.0 mmol) of chiral α-chloro-borane (1borane (+ --pinanediolO ((S - 1-chloro44-bromo-butane)) with 2.0 with HCl, reacted with 1.45 g (2.0 mmol) of Lin (SiMe3) 2 and ^^ <rn ^^ in the presence of 4.0 mmol of EteN active ester, giving the compound title which is purified by flash chromatograph (1: 1 hexane / EtOAc).

D) Boc-D- (plTBDPS-O-Me) -Phal-PrOlBoroOrn-OPm.

680 mg (0.72 mmol) of the product from step C) are dissolved in DMSO and 94 mg (1.44 mmol) of sodium azide are added. The mixture was stirred for 4 hours at room temperature. Then ether / ice water is added and white crystals immediately precipitate. The white precipitate is filtered off and washed with water to give Boc-D- (p-TBDPS-O-Me) -Phal-Pro-NH-CH ((CH2) 3N3) B-OPm as a white crystalline material.

272 mg (0.3 mmol) of the azide is dissolved in EtOAc and hydrogenated in the presence of Lindlar's catalyst. After 8 hours, the catalyst was removed and the solution was concentrated under reduced pressure. The crude product was purified by flash chromatography (EtOAc then EtOH) to afford the desired title compound as a white foam, Ud = -32.4 ° (c = 0.25 in MeOH).

Example XVII. Bo - D- (p-OH-Me) -Phal-Pro-BoroOra-OPin.

132 mg (0.15 mmol) of the boro-ornithine from Example 16 are dissolved in THF and reacted with 0.3 ml of a 1.1 M n-Bu4NF solution (0.3 mmol). The mixture was kept for 45 minutes at room temperature, then ice water was added and the resulting mixture was extracted several times with EtOAc. The combined organic layers were dried over Na _a SO 4 and concentrated under reduced pressure. The resulting oil was purified by short chromatography (EtOAc then EtOH) to afford the desired title compound as a white foam, Ud = -34.0 ° (c = 01 in MeOH).

, Q1

WYN-CH - Β II r ₄ r ₅ “Q ₂

PATTERN 1 of ^0R 9

W-Y-N-CH - B

Ra R5 'OR

MODEL 1 a

W-NH-CH-CO-AA-N-CH-B ^R 11

R / R5 *

MODEL 1 b

W-NH-CHtR ^ J-CO-Pro-N-CHR ₅ - Β * 1

Q ₂

PATTERN 1 '

W - NH - CH (R2) -CO- Pro -N- CHR ₅ Ra °

PATTERN 1 "

R11

AND

-NH-CH-CII O

PATTERN 2

MODEL 3

l-) ^x O

MODEL 4

16 * 7469

MODEL 5

WY-NH - CH - Β I ^r 12

MODEL 6

Ol ^Q 2

Cl - CH - BI ^r 13

Ol at ₂

ABOUT

W-Y-O-N

ABOUT

MODEL 8

MODEL 7

MODEL 9

MODEL 10

Glu

MODEL 12

PATTERN

MODEL 4

MODEL 15

FORMULA 16 x ^OH H < ₀

MODEL 17

Publishing Department of the UP RP. Circulation of 90 copies. Price PLN 1.50

Claims (7)

Patent claims A method for the preparation of new peptide boric acid derivatives of formula I, wherein W is a hydrogen atom or a nitrogen protecting group, Y is an n amino acid sequence such that n +1 amino acid peptide Y-Lys or Y-Arg has an affinity for the active part of the protease of the trypsin type, where n is an integer of 1-10 and at least one amino acid is an unnatural amino acid with a hydrophobic side chain, Qi and Q2 are the same or different and represent -OH, -COR1, -CONR1R2, -NR1R 2 or - OR3 or Q1 and Q2 together represent the rest of the diol, R1, R2 and R3 are the same or different and represent a C1-10 alkyl, C6-10 aryl, C6-aralkyl or phenyl radical substituted with up to three substituents, such as a C1-4alkyl group, halogen or a C1-4alkoxy group, R4 is a hydrogen atom or a C1-10alkyl radical, R5 is a group -AX, where A is a group - (CH2) z in which z is 2,3,4 or 5, a group -CH (CH3) - (CH2) 2, -CH2-CH (CH3) -CH2-, - (CH2) ₂ -CH (CH3) -, - (CH2) 2-C (CH3) 2, CH (C H3) - (CH2) 3-, -CH2-CH (CH3) - (CH3) - (CH2) 2-, -CH2-CH2-CH (CH3) -CH2-, - (CH2) 3CH (CH3) -, - (CH2) 3-C (CH3) 2; Ce-10-aryl, C-10-aralkyl, X is -NH2, and the asymmetric carbon atom marked with an asterisk may represent the D or L configuration or mixtures thereof, characterized in that the compound of formula 7 wherein Q1 and Q2 are as defined above and R13 is the group -A-Br, in which A is as defined above, is reacted with LiN [Si (CH3) 3] 2, followed by acidic hydrolysis and coupling with a protected peptide of formula 8, in wherein W and Y are as defined above, and then the resulting compound of formula 6, wherein R12 is -A-Br and A, W, Y, Q1 and Q2 are as defined above, is reacted with sodium azide, and then the obtained compound of formula I, in which R5 is the group -AX, in which A is as defined above, and X is N3, and the remaining substituents are as defined above, is subjected to hydrogenation. 2. The method according to p. A method according to claim 1, characterized in that the starting compounds of formula 8 are used, in which W is the group H (CH2CH2O) -, ReCO-, R7OCO- or R8SO2-, in which p is the number 3 - 30, Re is the group Ge- alkyl, R7 is C1-6alkyl, phenyl, benzyl or naphthyl and R8 is phenyl, naphthyl or C1-4alkylphenyl and Y is as defined in Claim 1. 3. The method according to p. For the preparation of a compound of formula la, wherein W, Y, R4 and R5 are as defined in claim 1 1 or 2, and R9 and R10 represent the rest of the dihydroxy compound, the starting compound of formula 7 is used in which Q1 and Q2 are groups of the formulas -OR9 and -OR10, where R9 and R10 are as defined above, and R13 in the formula 7 is as defined in claim 7 1. 4. The method according to p. According to claim 1, characterized in that the starting compounds of formula 7 are used in which Q1 and Q2 together represent a group of formula 9 or 10, and R13 is as defined in claim 1 1. 5. The method according to p. A process according to claim 1, characterized in that the starting compounds of formula 8 are used, wherein W and Y are as defined in claim 1, 1, wherein the unnatural amino acid in Y is a compound of Formula 2 in which R11 is hydrophobic. 6. The method according to p. The process according to claim 5, characterized in that R11 is a group of the formula 12, a group of the formula -CH2Si (CH3) 3, a group of the formula 13, a group of the formula -CH2-C (CH3) 3, a group of the formula 14, 15 or 16. 7. The method according to p. The method according to claim 1, wherein the starting compounds of formula 8 are used, wherein Y is a sequence of two amino acids of which the N-terminal amino acid is an unnatural amino acid and the other amino acid is L-proline, and W is as defined in claim 1. 1 or 2. 167 469 3