NO300504B1 - Compound and therapeutic composition comprising this - Google Patents

Description

The present invention relates to inhibitors of serine proteases such as thrombin, factor Xa, kallikrein and plasmin as well as other serine proteases such as prolylendopeptidase and lg AI protease. Thrombin, which is the last enzyme in the coagulation system, splits soluble fibrinogen into fibrin, which then cross-links and forms an insoluble gel that forms the matrix for a thrombus. When a blood vessel is destroyed, the above procedure is necessary to stop the bleeding.

Under normal conditions, no measurable amount of thrombin is present in plasma. An increase in thrombin concentration can result in the formation of clots which can lead to thromboembolic disease, one of the most common serious medical problems of our time.

Thrombin contributes to hemostatic control by means of a number of biological reactions. In addition to its primary function, the conversion of fibrinogen to fibrin, thrombin activates factor XIII which is responsible for cross-linking of fibrin. Thrombin also acts by means of a positive feed-back mechanism involving the activation of factors V and VIII, both of which are required for its own formation from prothrombin. Thrombin has another important role: its binding to platelets initiates platelet release and aggregation responsible for primary hemostasis.

The reactions of thrombin are further controlled by natural inhibitors in plasma. The most important of these are anti-thrombin III and heparin. These two compounds have been isolated and are used therapeutically and prophylactically in conditions with an imbalance in the hemostatic mechanism and with a risk of prothrombin activation.

Synthetic thrombin inhibitors with oral activity will be useful as alternatives to the parenteral administration of these natural inhibitors. Research in this area has resulted in the synthesis of good inhibitors of thrombin in vitro, but until now there is no really good candidate for oral therapeutic use. By mimicking amino acid sequences in fibrinogen, the important natural substrate for thrombin, a number of good short peptide substrates for thrombin have been formed. These peptide substrates are also converted into inhibitors of the enzyme. The chromogenic substrates D-Phe-Pro-Arg-pNa and D-Phe-Pip-Arg-PNA thus mimic the sequence before the bond that is cleaved by thrombin. The corresponding reversible and irreversible inhibitors, D-Phe-Pro-Arginal and D-Phe~Pro-Arg-CH2Cl show inhibition in vitro in the range of 10"<8>M.

Chloromethyl ketones are generally far too nonspecifically reactive to be ideal for therapeutic use, and the peptidaldehyde exemplified above has a fairly low LD50 value.

Factor Xa is the coagulation enzyme responsible for the formation of thrombin by limited proteolysis of its zymogen, prothrombin. On a weight-to-weight basis, Xa is at least 10 times more thrombogenic in vivo than thrombin. This is due to the fact that factor Xa is one step above thrombin in the amplifying cascade system, so that one factor Xa molecule can form a number of thrombin molecules from its precursor. Its strength may also be due to the rather slow removal of factor Xa in the body. Unlike factor Xa, thrombin is rapidly removed from the circulating blood to sites of high affinity on the vessel wall. The central position of factor Xa at the junction of the intrinsic and extrinsic tracks should make it a suitable target for modulating the hemostatic mechanism.

Kallikrein is formed from prekallikrein by the action of factor XII when it is located on a negatively charged surface. Kallikrein can in turn cleave factor XII to factor Xlla and thereby form a reciprocal activation system. Factor Xlla is the first enzyme in the intrinsic part of the coagulation system. The importance of the contact system is presumably as a surface-derived defense mechanism, and a broad activation of the system is normally seen in shock or disseminated intravascular coagulation (DIC). Kallikrein's role at this stage is to cleave high molecular weight kininogen and release the vasodilator bradykinin. Bradykinin also causes increased vascular permeability, pain and migration of neutrophilic leukocytes. Inhibitors of kinin formation have been shown to be beneficial in certain types of inflammation, including arthritis and pancreatitis, and may also be used in the treatment of asthma. The only substance that has achieved clinical significance as a kallikrein inhibitor is aprotinin (Trasylol). Aprotinin is a polypeptide with a molecular weight of 6,500 and forms a stable complex with proteases with a binding constant of 10"<10> to 10"<13> M.

Fibrinolysis is the process that results in the enzymatic dissolution of fibrinogen and fibrin clots. Plasma contains a protein, plasminogen, 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 breakdown products.

Under normal conditions, the fibrinolytic system is in balance with the coagulation system. Small thrombi formed in the bloodstream can be dissolved enzymatically and circulation through the vessels can be restored by the activation of the fibrinolytic system in the body. If the fibrinolytic activity is too high, this can cause or prolong bleeding. The activity can be inhibited using natural inhibitors in the blood.

Prolylendopeptidase cleaves peptide bonds on the carboxyl side of proline residues in a peptide chain. It is a serine protease that rapidly degrades a number of neuropeptides including substance P, neurotensin, thyrotropin-releasing hormone and lutenizing hormone-releasing hormone and which is associated with the cell's ability to form interleukin 2 (IL-2). The enzyme is inhibited by benzyloxycarbonyl-prolylprolinal with a Ki of 14 riM. Despite the fact that almost nothing is known about the physiological role of prolyl endopeptidase, it may play a dominant role in the regulation of the biological activities of various neuropeptides.

Ig A proteinase-catalyzed cleavage of Ig A, the predominant form of antibody comprising the main defense against infection, separates the Fc from the antigen-binding Fab regions of the molecule. Such cleavage would be expected to weaken or abolish its antimicrobial activity. All Ig A proteinases that have been identified thus cleave after a proline residue within the hinge region of human Ig A. Peptide prolyl boronic acids inhibit Ig A 1 proteinases from Neisseria gonorrhea and Hemophilus influenzae, which indicates that these enzymes belong to the serine protease family of proteolytic enzymes.

The complex role that thrombin plays in a number of physiological processes that are associated with pathological disorders such as cancer, inflammation and neuronal activity suggests a potential use of thrombin inhibitors for a number of indications that are not strictly related to the cardiovascular system.

A variety of tumor cells have been shown to elicit procoagulant activity associated with thrombin generation. As a result of this, local fibrin deposition and coagulation occur, which are believed to be important for the tumor's growth. In addition, due to its action on endothelial cells, thrombin may facilitate the extravasation of tumor cells during metastasis. Thus, thrombin inhibitors may prove to be beneficial not only in the treatment of certain types of cancer, but also in connection with the hypercoagulability often observed in patients during therapy with chemotherapeutic agents.

Thrombin activation of endothelial cells induces a number of pro-inflammatory changes such as the synthesis and release of interleukin-1, prostaglandins and platelet activation factor. In addition, thrombin induces the exposure of GMP-140 and CD63, two adhesive molecules responsible for the adhesion of leukocytes to the endothelial surface. Thrombin also increases the vascular permeability of proteins, a mechanism involving neutrophils, and cleaves the interleukin-8 precursor protein, a peptide believed to be involved in respiratory disorders, rheumatoid arthritis and ulcerative colitis.

Its involvement in all these pro-inflammatory processes makes thrombin a potential target for the therapeutic treatment, with thrombin inhibitors, of inflammation-related pathological conditions.

The activity of the protease nexin-1, a modulator of nerve development and a specific natural thrombin antagonist, is marked and particularly reduced in patients with Alzheimer's disease. This, together with the observation that thrombin-like activity was increased in brains with Alzheimer's disease, suggests that thrombin inhibitors may have a potential for limiting or reversing neuronal pathological changes associated with thrombin hyperactivity.

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

The interaction between boric acids and proteases in biological systems is known and various simple boric acids have been shown to be sufficiently non-toxic for use in humans. Peptide boric acid inhibitors of elastase have recently been used in animal experiments in connection with emphysema. Unlike the peptide chloromethyl ketones, no toxicity was reported at biologically effective dose levels.

EP patent application 293 881 describes the preparation of peptides comprising C-terminal boronic acid derivatives of lysine, ornithine and arginine and their use as inhibitors of trypsin-like serine proteases. The other amino acids in the peptides are all either the D or L forms of the 20 naturally occurring amino acids.

It has now been found that compounds with superior properties as inhibitors of trypsin-like serine proteases are obtained when the peptide contains at least one unnatural α-amino acid with a hydrophobic side chain.

The present invention thus relates to a compound which is characterized by having formula I

where W is hydrogen or an N-protecting group,

Y is a sequence of two amino acids of which the N-terminal amino acid is an unnatural amino acid of formula II

where R 11 is a group of formula (c) or (d)

and the other amino acid is L proline,

Qi and Q2 together represent a group of formula (a)

R5 is a group -A-X where

A is -(CH2)Z- where z is 2, 3, 4 or 5, and X is -NH2 or -NH-C(NH)-NH2, and where the asymmetric carbon atom marked with <*> may have D- or L configuration, or represent any mixture of these.

The N-protecting group W has the preferred formula H(CH2CH20)p- where p = 3-30, R6CO-, R70CO- or R8SO2- where R6 is C1-6 alkyl, R7 is C1-6 alkyl, phenyl, benzyl or naphthyl, and R8 is phenyl, naphthyl or <C>1-6 alkylphenyl, where R7OCO is preferred. The most preferred protecting groups are those of the formula R7'OCO- in which R7' is tert-butyl (designated Boe) and in which R7' is benzyl (designated Z).

The most preferred compounds are the compound of formula

III

the compound of formula IV and the compound of formula V

A peptide is said to have an affinity for the active site of trypsin-like proteases if that particular peptide has a value of 10"<3> M or less for the dissociation constant.

The compounds of formula I in which X is -NH-C(NH)-NH2 can be prepared by reacting a compound of formula I in which X is -NH2 with cyanamide in an organic solvent under strongly acidic conditions. The compounds in which X is -NH2 can in turn be prepared by hydrogenating a compound of formula I in which X is -N3. The hydrogenation can be carried out under standard conditions using e.g. a Pd/C catalyst.

The compounds where X is -N3 can be prepared by a compound of formula VI

wherein W, Y, Qx and Q2 are as above and R12 is -A-Br wherein A is as above is reacted with sodium azide in a polar aprotic solvent such as dimethylsulfoxide. The intermediates of formula VI can be obtained by reacting a compound of formula VII in which Q-1 and Q2 are as indicated above and R13 is -A-Br in which A is as indicated above with LiN[Si (CH3)3]2, followed by of hydrolysis with 3 molar equivalents of acid and coupling with a protected peptide of formula VIII

where W and Y are as indicated above.

The reaction is preferably carried out in a dry aprotic polar solvent such as e.g. tetrahydrofuran and at a temperature between -78°C and room temperature.

The intermediates of formula VII can be obtained by the method of Matteson et al, Organometallics 3 1284-8

(1984).

The protected peptide of formula VIII can be prepared by methods commonly used in peptide chemistry, starting from the desired unnatural amino acid. Such amino acids are either commercially available (e.g. the amino acid in which R1X is the group c) or can be prepared using methods analogous to those described in the literature, e.g. Angew. Chem. 93, 793 (1981) andJ. Am. Chem. Soc. 109, 6881 (1987).

The compounds of formula I can be used as inhibitors of trypsin-like proteases and can be used in vitro for diagnostic and mechanical studies of these enzymes. Due to their inhibitory effect, they are further indicated for use in the prevention or treatment of diseases caused by an excess of an enzyme in a regulatory system, e.g. to regulate the coagulation of the fibrinolytic system.

The invention thus also relates to a therapeutic preparation which is characterized by the fact that it contains a

binding according to the invention in combination with a pharmaceutically acceptable excipient and/or diluent.

The compounds according to the invention which are thrombin or factor Xa inhibitors have anti-thrombogenic properties and can be used when an anti-thrombogenic agent is necessary. These compounds can generally be administered orally or parenterally to a host to achieve an anti-thrombogenic effect. In the case of larger mammals such as humans, the compounds can be administered alone or together with pharmaceutical carriers or diluents at a dose of from 0.02 to 15 mg/kg body weight and preferably from 1 to 10 mg/kg body weight to achieve the anti-thrombogenic effect, and they can be administered in the form of a single dose or in several doses or as a composition with an extended duration of action. When a blood circuit outside the body is to be established in a patient, 0.1-1 mg/kg can be administered intravenously. For use with whole blood, from 1 - 10 mg per liter of blood can prevent coagulation. Pharmaceutical diluents are well known and include sugars, starches and water which can be used for the manufacture of tablets, capsules, injectable solutions and the like. The compounds according to the invention can be added to blood to prevent coagulation of the blood in containers used for the collection or distribution of blood, and in connection with tubes or implantable devices that come into contact with blood.

The advantages of the compounds of the invention include oral activity, rapid onset of activity and low toxicity. In addition, these compounds may have particular utility in the treatment of individuals who are hypersensitive to compounds such as heparin.

The meaning of a number of symbols is as follows:

Z = benzyloxycarbonyl

Boe = t-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-(CH2CH2CH2Br)B-

TMSal = trimethylsilylalanine

Adal = adamantylalanine

Naphal = 2-naphthylalanine

BoroOrn = -NH-CH-(CH2CH2CH2NH2)B-

BoroArg = -NH-CH- [CH2CH2CH2NHC (NH)NH2 ] B-

Adgly = 1-adamantylglycine

BoroPro = analogue of proline in which the -COOH group is OPin

replaced with B-OPin

BoroLys = -NH-CH-(CH2-CH2-CH2-CH2-NH2) B-

BoroHArg = -NH-CH-(CH2CH2CH2CH2NHC (NH)NH2 ) B-

BoroMpg = -NH-CH-(CH2CH2CH2-OCH3)B-

p-OH-Me-Phal = p-hydroxymethyl-phenylalanine p-TBDPS-O-Me-Phal = p-tert.butyl-diphenylsilyloxy-methylphenylalanine

The kinetic parameters<e><K>i( Kon and Koff are determined by inhibiting the enzyme-catalyzed hydrolysis of a peptide-arg-pNa. This hydrolysis gives p-nitroaniline and the time-dependent release, which is quantified by the optical density measured at 405 nm , and determines the rates of inhibited and non-inhibited reactions.

Kinetic measurements are carried out on a plate with 96 micro-wells together with a kinetic meter in the form of a simple cell. Active-site titrated human thrombin is dissolved in 0.1 M phosphate buffer, pH 7.4 containing 0.1 M NaCl and 0.1% bovine serum albumin to a stock solution containing 400 nM active enzyme. For the chromogenic experiment, 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 chromophore/ethanol 1:1 and then diluted with distilled water to a 1 mM stock solution. Further dilution is carried out with the phosphate buffer described above.

Experiments are initiated by adding 100 µl enzyme solution to a mixture containing 100 µl inhibitor and 50 µl of a substrate solution. Release of p-nitroaniline from the hydrolysis of the peptidyl p-nitroanilide substrates is followed for 30 min. to 1 hour by measuring the increase in optical density at 405 nm. The collected data is used to calculate the kinetic parameters in the presence and in the absence of the inhibitor. Although other mechanisms of action are not excluded, the characterization of this group of thrombin inhibitors is limited to two main mechanisms that have been observed, namely fast reversible and slow binding competitive inhibition. Kinetic constants for the inhibitors that show a fast reversible binding mechanism, namely fast binding (initial speed v0 for control > v0 with inhibitor) at It » Et (total inhibitor concentration/total enzyme concentration) are calculated by linear regression from a plot of l/v against inhibitor concentration [IN]. The K± value is calculated from the horizontal intercept Ki app by equation (1)

If the interaction with the enzyme is slow (V0 is not affected by the inhibitor) and fixed (Kx close to or less than Et) so that the inhibited equilibrium rate is only slowly achieved, the development curve for pNA formation is described by where P is the amount of pNA formed at time 'f, V0 is the initial rate, Vs is the rate at equilibrium and kobs is an apparent global reaction rate as a function of Et, It, Kiapp, and the observed second-order rate constant (k'on) for the interaction between the inhibitor and enzyme. Data from measurements of slow binding inhibition are fitted to equation (2) by a non-linear regression analysis which gives estimates of kobs. Values for k'on, koff and Ki#app are then obtained from a plot of kobs against [I] . The value for koff is given by the vertical intercept, while the values k'on and K.j_ are calculated from the oblique and horizontal intercepts using equation (1).

Reference example 1

Boc-TMSal-Pro-NH-CH [(CH 2 ) 3 N 3 ] BOPin

A. Boc-D-TMSal-OH

D-TMSal ethyl ester (21.5 g, 113.7 mmol) prepared according to the procedure given in Angew, Chem. 93, 793 (1981)

is dissolved in CH 2 Cl 2 and a solution of an excess of Boc 2 O in CH 2 Cl 2 is added. After 15 hours at room temperature, 500 ml of ice-cold 0.25 N hydrochloric acid is added. The organic layer is washed with 5% NaHCO 3 and brine, dried over Na 2 SO 4 and concentrated in vacuo.

The raw material (colorless oil) is used directly in the saponification step. It is dissolved in methanol, cooled to 0°C, mixed with 510 ml of 1 N NaOH and stirred at 0°C for 3 hours. After acidification to pH 1 with 1 N HCl, the mixture is extracted several times with ether. The organic layers are combined, washed with brine, dried over Na 2 SO 4 and concentrated in vacuo. The product (29.7 g of oil) is used in the next step without further purification.

B. Boc-D-TMSal-Pro-ONSu

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

Boc-D-TMSal-ONp (51.6 g, 113.7 mmol) is dissolved in THF and an aqueous solution of equimolar amounts of proline and Et 3 N is added. After 20 hours at room temperature, the THF is removed in vacuo and the aqueous residue is diluted with water and then extracted several times with EtOAc. The pH in the aqueous layer is adjusted to 3 by adding 10% citric acid. The oily product obtained is extracted several times with EtOAc. The combined organic layers are washed with brine, dried over Na 2 SO 4 and concentrated in vacuo. The colorless oil is recrystallized from ether/hexane to give the dipeptide Boc-D-TMSal-Pro-OH as a white crystalline compound, mp: 176°C.

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

After drying over Na 2 SO 4 and concentration in vacuo, Boc-D-TMSal-Pro-ONSu is obtained as a white foam.

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

This procedure is a 3-step procedure which is carried out in one and the same container and which includes the in situ formation of a chiral α-(bistrimethylsilyl)amidoborate, the hydrolysis of the two trimethylsilyl groups and the coupling of the thus formed α-aminoborate with the active the ester (Boc-D-TMSal-Pro-ONSu) prepared in step B. The entire reaction sequence is carried out under an argon atmosphere. The chiral o -chloroborate ((+)-pinanediol-(S)-1-chloro-4-bromo-butane-1-borate) (1.75 g, 5.0 mmol) is dissolved in 2.5 mL of THF and added to a pre-cooled (-78"C) solution of lithium hexamethyldisilazane (5 ml of a 1.0 M solution in hexane, 5.0 mmol). After stirring for 30 min. at -7 8"C, the solution is warmed overnight to room temperature . After cooling again to -78°C, 3 molar equivalents of HC1 in dioxane are added. The mixture is heated to room temperature and stirred for 2 hours at this temperature. After cooling again to -20°C, a solution of the active ester from step B (2.28 g, 5.0 mmol) in 6 mL of CH 2 Cl 2 is added followed by the addition of 1.39 mL (10.0 mmol) triethylamine.

The mixture is stirred for 1 hour at -13°C, heated to room temperature and stirred for 2 hours at this temperature. The mixture is filtered, the filtrate is concentrated in vacuo and the residue is diluted with ether and washed with 2 N HCl, 5% NaHCO 3 and brine. The organic layer is dried over Na 2 SO 4 and concentrated in vacuo. The residue crystallized on standing to give the desired chiral peptide borate as a white crystalline compound.

D) Boc-D-TMSal-Pro-NH-CH[ (CH 2 ) 3 N 3 ]B-OPin

Boc-D-TMSal-Pro-Baa-OPin, the product from step C (804 mg,

1.2 mmol) is dissolved in 13 ml of DMSO and sodium azide (156 mg,

2.4 mmol) is added. The mixture is stirred for 3 hours at room temperature. Ether/ice water is added and white crystals immediately precipitate from the mixture. The white precipitate is filtered off and washed with ether to give 0.6 of the azide as a white crystalline compound.

Example 1

Boc-D-TMSal-Pro-BoroOrn-OPin

The azide from ref. ex. 1 (569 mg, 0.9 mmol) is dissolved in 25 mL of EtOAc and hydrogenated in the presence of 0.5 g of 10% Pd/C. After 2.5 hours, the catalyst is removed and the solution is concentrated in vacuo to give a white foam which is recrystallized from EtOAc/ether to give the desired product as a white crystalline compound, mp: 200 - 202 "C, [0c]D<20 > = -11.6" (c = 0.5 L MeOH).

Example 2

Boc-D-TMSal-Pro-BoroArg-OPin (benzenesulfonate) Boc-D-TMSal-Pro-boroOrn-OPin from ex. 1 (250 mg, 0.412 mmol) is dissolved in 2 ml of ethanol. Benzenesulfonic acid (65.2 mg, 0.412 mmol) is added. After stirring for 15 min. at room temperature, cyanamide (86.6 mg, 2.06 mmol) is added and the mixture is heated at reflux. The progress of the reaction is measured by RP-TLC in which the disappearance of the ninhydrin spot for the amine starting material and the appearance of the Sakaguchi band for the product are observed. After 7 days, amine can no longer be detected and the solution is concentrated in vacuo. The residue is dissolved in MeOH and chromatographed on a 5 x 55 cm Sephadex LH-20 column with MeOH.

The desired product is obtained as a white foam,

[Cc]D<20> = -45.3" (c = 1 in CH 2 Cl 2 ).

Reference example 2

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

A) (+)-Pinanediol-(S)-1-chloro-5-bromopentane-1-borate-4-bromo-1-butene (20.8 ml, 203.3 mmol) is reacted with catecholborane (24.4 g , 203.3 mmol) at 100°C for 16 hours. The crude product is distilled in vacuo to give 4-bromobutane-1-borate as a white crystalline compound. (+)-Pinanediol (27.7 g, 163 mmol) is dissolved in THF and the above synthesized 3-bromobutane-1-borate (41.6 g, 163 mmol) is added. After 1 hour at room temperature, the THF is removed in vacuo and the residue is purified by flash chromatography (90:10 hexane/EtOAc) to give (+)-pinanediol-4-bromobutane-1-borate as a colorless oil.

The desired (+)-pinanediol-(S)-1-chloro-5-bromopentane-1-borate is prepared according to the procedure given in Organometallics 3, 1284 (1984). Therefore, methylene chloride (9.8 ml) in THF is cooled to -100°C and n-butyllithium (71.6 ml, 1.6 M solution, 114.5 mmol) is added over 20 min. After 15 min. at -100°C, a cold (-78°C) solution of (+)-pinanediol-5-bromopentane-1-borate (32.8 g, 104.1 mmol) in THF is added.After a further 1 hour at -100 C, anhydrous ZnCl2 (7.1 g, 52.0 mmol) in THF is added. After a further 15 min. at -100 C, the reaction mixture is warmed to room temperature and stirred for 2 hours at this temperature. The solvent is removed in vacuo, 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-bromopentane-1-borate is obtained as a yellow oil which is used directly in the next step without further purification.

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

A solution of LiN(SiMe3)2 (65.2 mL of 1.0 M solution,

65.2 mmol) in THF is cooled to -78°C. The cc-chloroborate from step A) (23.7 g, 65.2 mmol) in THF is added. After stirring for 1 hour at -78°C, the mixture is stirred for 15 hours at room temperature.

After this period, the reaction mixture is cooled to -78°C, HCl (29.8 ml of 6.56 N solution, 196 mmol) in dioxane is added, the solution is stirred for 45 min. at -78°C and then for 2 hours at room temperature. The mixture is cooled to -15°C, Boc-TMSal-Pro-ONSu (29.7 g, 65.2 mmol) from ex. 1 in CH 2 Cl 2 is added before triethylamine (18.1 mL, 130.4 mmol) is added to initiate the coupling reaction. After stirring at -15°C for 1 hour, the mixture is stirred for 2 hours at room temperature. The mixture is filtered over Hyflo and concentrated in vacuo. The residue is diluted with ether/water and extracted several times with ether. After drying over Na2SO4 and concentration in vacuo, the desired Boc-D-TMSal-Pro-NH-CH ( (CH2)4Br)B-OPin is obtained after crystallization from ether/hexane as a white crystalline compound, mp: 74°C.

C) Boc-D-TMSal-Pro-NH-CH((CH2)4N3)B-OPin

The product from step B) (33.3 g, 48.6 mmol) is dissolved in DMSO and sodium azide (6.3 g, 97.2 mmol) is added. The mixture is stirred for 6 hours at room temperature. Ether/ice water is added and the mixture is extracted several times with ether. After drying over Na2SO4 and concentration in vacuo, the obtained oil is recrystallized to give Boc-D-TMSal-Pro-NH-CH ( (CH2) 4N3 ) B-Pin as a white crystalline compound,

mp: 69-70°C, αD = -74.4° (c=1.0 in MeOH).

Example 3

Boe- D- TMSal- Pro- BoroLys- OPin

The azide from ref. ex. 2 (22.0 g, 34.0 mmol) is dissolved in EtOAc and hydrogenated in the presence of 4.0 g of 10% Pd/C. After 9 hours, the catalyst is removed and the solution is concentrated in vacuo. The resulting foam is dissolved in EtOAc and crystallized to give the desired Boc-D-TMSal-Pro-BoroLys-OPin as white crystals, mp: 128-129°C, (XD = -59.6* (c = 1.0 in MeOH).

Example 4

Boc-D-TMSal-Pro-BoroHArq-OPin (benzenesulfonate) The benzenesulfonate of Boc-D-TMSal-Pro-BoroLys-OPin from ex. 3 (800 mg, 1.03 mmol) is dissolved in ethanol. Cyanamid (210 mg, 5.0 mmol) is added and the mixture is heated to reflux. The progress of the reaction is measured by means of RP-TLC where the disappearance of the ninhydrin spot for the amine starting material and the presence of the Sakaguchi line for the product are observed. After 7 days, amine can no longer be detected and the solution is concentrated in vacuo. The residue is dissolved in MeOH and chromatographed on a 5 x 55 cm Sephadex LH-20 column with MeOH.

The desired product is obtained as a white foam, aD = -40.8'

(c=0.5 in CH 2 Cl 2 ).

Examples 5-10

By methods analogous to those described in examples 1-6, compounds of formula were obtained:

wherein <R>4, <R>5', Rn, Qx<+> Q2 and AA have the meanings shown in the following Table I.

Reference example 3

Boc-D-TMSal-Pro-BoroMpq-OPin

A) (+)-Pinanediol-(S)-1-chloro-4-methoxybutane-1-borate-3-methoxy-1-propene (6.0 g, 83.3 mmol) is reacted with catecholborane (10.0 g , 83.3 mmol) at 100°C for 24 h. Crude product is distilled in vacuo to give 3-methoxypropane-1-borate as a colorless oil. (+)-Pinanediol (10.6 g, 62.5 mmol ) is dissolved in THF and the above synthesized 3-methoxypropane-1-borate (12.0 g, 62.5 mmol) is added. After 1 h at room temperature, the THF is removed in vacuo and the residue is purified by flash chromatography (80:20 hexane/EtOAc) to give (+)-pinanediol-3-methoxypropane-1-borate as a colorless oil.

The desired (+)-pinanediol-(S)-1-chloro-4-methoxybutane-1-borate is prepared according to the procedure given in Organometallics 3, 1284 (1984). Methylene chloride (2.2 ml) in THF is cooled to -100°C and n-butyllithium (13.8 ml 1.6 M solution, 22.0 mmol) is added over 20 min. After 15 min. at -100"C, a solution of (+)-pinanediol-3-methoxypropane-1-borate (5.04 g, 20 mmol) in THF is added followed by anhydrous ZnCl2 (1.42 g, 10.0 mmol). After another 15 min

-100'C, the reaction mixture is heated to room temperature and

stirred for 2 hours at this temperature. The solvent is removed in vacuo, the residue is diluted with ether and washed with water. The organic layer is dried over Na 2 SO 4 and concentrated to give an oil which is purified by flash chromatography (9:1 hexane/EtOAc) to give the desired (+)-pinanediol-(S)-1-chloro-4-methoxybutane-1- borate as a colorless oil.

B) Boc-D-TMSal-Pro-BoroMpg-OPin

A solution of LiN(SiMe3)2 (5 ml of 1.0 M solution,

5.0 mmol) in THF is cooled to -78 °C. (X-chloroborate from step A) (1.53 g, 5.0 mmol) in THF is added. After stirring for 1 hour at -78°C, the mixture is stirred for 15 hours at room temperature. After this period, the reaction mixture is cooled to

-78°C, HCl (2.7 ml 5.65 N solution, 15.0 mmol) in dioxane is added, the solution is stirred for 30 min. at -78°C and then for 2 hours at room temperature. The mixture is cooled to -15°C, Boc-TMSal-Pro-ONSu (2.28 g 5.0 mmol) from ref. 1 in CH 2 Cl 2 is added before triethylamine (1.39 mL, 10.9 mmol) is added to initiate the coupling reaction. After stirring at -15 "C for 1 hour, the mixture is stirred for 2 hours at room temperature. The mixture is filtered over Hyflo and concentrated in vacuo. The residue is diluted with EtOAc and washed with 0.2 N HCl, 5% NaHCO 3 and finally with brine. After that the solvent is removed, an oil is obtained which is purified by flash chromatography (EtOAc) to give Boc-D-TMSal-Pro-BoroMpg-OPin as a white foam, aD = -48.8" (c=0.25 in CH2Cl2 ).

Reference example 4

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

A) Boc-D-(p-((1,1-dimethylethyl)diphenylsilyl)oxy)methylphenyl-alanine

To selectively reduce the azido group in the substrate, thiophenol (7.27 g, 66.0 mmol) is added to a suspension of SnCl2 (3.12 g, 16.5 mmol) in CH2Cl2. Triethylamine (6.8 mL, 49.5 mmol) is added and a yellow solution is obtained. Boc anhydride (4.8 g, 22.0 mmol) is added before (3 (2S), 4S-3-(2-azido-3-(p-((1,1-dimethyl)diphenylsilyl)oxymethyl)phenyl- 1-oxopropyl)-4-(phenyl-methyl)-2-oxazolidinone (%de>95, 6.8 g, 11.0 mmol), prepared according to the procedure given in J. Am. Chem. Soc. 109 6881

(1987), is added as a solution in CH2C12. After stirring for 2.5 hours at room temperature, the mixture is diluted with EtOAc/2 N NaOH and filtered over Hyflo. The organic layer is washed with 2% aqueous NaHSO 4 , 5% aqueous NaHCO 3 and finally with brine. After drying over Na2SO4 and concentration in vacuo, the yellow oil obtained is purified by flash chromatography to give (3(2S),4S)-3-(2-(((tert-butyloxy)carbonyl)amino)-3-(p -(((1,1-dimethylethyl)diphenylsilyl)oxy)methyl)phenyl-1-oxopropyl)-4-(phenylmethyl)-2-oxazolidinone as a white foam. This compound (2.0 g, 2.88 mmol) is dissolved in THF/water and hydrolyzed with in situ formed LiOOH (5.76 mmol) at 0'C. After 1.75 hours at 0°C, Na 2 SO 3 (1.25 g, 9.9 mmol) in water. The THF is removed in vacuo, the pH of the residue is adjusted to 1-2 and the mixture is extracted 3 times with EtOAc. The combined organic layers are washed with water, dried over Na 2 SO 4 and concentrated in vacuo. After crystallization from hexane/ether, the oxazolidinone is obtained as white crystals. The filtrate is concentrated in vacuo to give the desired title compound as a white foam.

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

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

(Boc-D-(p-TBDPS-O-Me)-Phal-ONp) (2.2 g, 2.88 mmol) is dissolved in THF and an aqueous solution of L-proline (365 mg, 3.17 mmol) and Et 3 N (0.88 mL, 6.33 mmol) is added. After 15 hours at room temperature, the THF is removed in vacuo. The pH is adjusted to 3 by adding 10% citric acid. The oily product obtained is extracted several times with EtOAc. The combined organic layers are washed with brine, dried over Na 2 SO 4 and concentrated in vacuo. The colorless oil is purified by flashk-

chromatography (9:1 CH2Cl2/EtOH to elute p-nitrophenol, then 80:20 CH2Cl2/EtOH) to give Boc-D-(p-TBDPS-O-Me)-Phal-Pro-OH as a white foam. This dipeptide (1.3 g, 2.06 mmol) is dissolved in EtOAc. After cooling to 0°C, HONSu (220 mg, 2.47 mmol) and DCC (330 mg, 2.06 mmol) are added. The mixture is cooled again to 0"C, dicyclohexylurea is filtered off and washed several times with cold EtOAc. The filtrate is washed with aqueous 0.1 M Na2CO3, 8% NaHSO4 and then with brine. After drying over Na2SO4 and concentration in vacuo, the title compound Boc-D is obtained -(p-TBDPS-O-Me)-Phal-Pro-ONSu as a white foam.

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

The title compound is obtained by means of the 3-step one-pot procedure analogous to that described for the synthesis of Boc-D-TMSal-Pro-Baa-OPin in ref. ex. L/C. The intermediate α-aminoborate, which results from the reaction of the chiral α-chloroborate (( + )-pinanediol-(S)-l-chloro-4-bromobutane-l-borate) (659 mg, 2.0 mmol) with LiN-(SiMe3)2 (2.0 mmol) and hydrolysis with HCl, is reacted with the active ester from step B) (1.45 g, 2.0 mmol) in the presence of Et3N (4.0 mmol) to give the title compound as purified by flash chromatography (1:1 hexane/EtOAc).

D) Boc-D-(p-TBDPS-O-Me)-Phal-Pro-BoroOrn-OPin

The product from step C) (680 mg, 0.72 mmol) is dissolved in DMSO and sodium azide (94 mg, 1.44 mmol) is added. The mixture is stirred for 4 hours at room temperature. Ether/ice water is added and white crystals precipitate immediately from the mixture. 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-OPin as a white crystalline compound. This azide (272 mg, 0.3 mmol) is dissolved in EtOAc and hydrogenated in the presence of a Lindlar catalyst. After 8 hours, the catalyst is removed and the solution is concentrated in vacuo. The crude product is purified by flash chromatography in (EtOAc, then EtOH) to give the desired title compound as a white foam, (XD = -32.4° (c = 0.25 in MeOH).

Reference example 5

Boc-D-(p-OH-Me)-Phal-Pro-BoroOrn-OPin

Borornithine from ref. ex. 4 (132 mg, 0.15 mmol) is dissolved in THF and reacted with n-Bu4NF (0.3 mL of 1.1 M solution, 0.3 mmol). After 45 min. at room temperature ice water is added and the resulting mixture is extracted several times with EtOAc. The combined organic layers are dried over Na 2 SO 4 and concentrated in vacuo. The oil obtained is purified by chromatography (EtOAc then EtOH) to give the desired title compound as a white foam, αD = -34.0' (c=0.1 in MeOH).

Reference example 6

Boc-D-TMSal-Adqly-boroPro-OPin

A. L-l-Adamantyl glycine

(3-(2S),4S)-3-(2-azido-2-adamant-1-yl-1-oxoethyl)-4-(phenyl-methyl)-2-oxazolidone (% de > 95, 9.86 g, 25.0 mmol), prepared according to the procedure described in J. Am. Chem. Soc. 109, 6881 (1987), dissolved in 320 ml of a mixture of THF/H 2 O (3:1), cooled to 0'C, mixed with 4 equivalents of hydrogen oxide and 2.0 equivalents of LiOH. The obtained mixture is stirred at 0°C until the substrate is used up (30 min), and the peroxide (percarboxylate) is suppressed at 0°C with a 10% excess of 1.5 N aqueous Na 2 SO 3 . After buffering with aqueous NaHCO 3 (pH 9-10), the mixture is extracted several times with EtOAc to remove the oxazolidinone chiral auxiliary. The product carboxylic acid is isolated by EtOAc extraction of the acidified (pH 1-2) aqueous phase, dried over Na2SO4 and concentrated in vacuo. The desired (S)-azidoadamant-1-yl-acetic acid is isolated as white crystals (5.29 g) and used in the next step without further purification. 2(S)-azidoadamant-1-yl-acetic acid (5.29 g, 22.5 mmol) is dissolved in 110 mL of EtOH and 11.3 mL of 2 N HCl and hydrogenated in the presence of 0.7 g of 10% Pd/C . After 2.5 hours, the catalyst is removed and the solution is concentrated in vacuo to give the desired amino acid as the hydrochloride salt. The hydrochloride obtained is suspended in 40 ml of H 2 O and treated with 1.9 g of solid NaHCO 3 . The amino acid obtained is filtered off and washed several times with water. After drying in vacuo, L-1-adamantylglycine is obtained as a white crystalline compound, [oc]D<20> = + 3.0<*> (c = 1.0 in MeOH).

B. Boc-D-TMSal-Adgly-ONSu

Boc-D-TMSal-ONp (7.71 g, 20.2 mmol) from ref. ex. 1 is dissolved in THF and an aqueous solution of equimolar amounts of 1-adamantylglycine and Et3N is added. After 20 hours at room temperature, the THF is removed in vacuo and the aqueous residue is diluted with 150 ml of 0.1 N HCl and then extracted several times with EtOAc. The combined organic layers are washed with brine, dried over Na 2 SO 4 and concentrated in vacuo. The oily product is chromatographed on silica gel (CH 2 Cl 2 ) to give the dipeptide Boc-D-TMSal-Adgly-OH as an oil. Boc-D-TMSal-Adgly-OH (6.9 g, 15.2 mmol) is dissolved in 80 mL of EtOAc. After cooling to 0°C, HONSu (2.1 g, 18.0 mmol) and DCC (3.1 g, 15.2 mmol) are added. The mixture is stirred for 3 hours at 0°C and then for a further 15 hours at room temperature . The mixture is cooled again to 0°C, the dicyclohexylurea is filtered off and washed with EtOAc. The filtrate is washed with aqueous 0.1 M NaHCO 3 and then with aqueous 2% KHSO 4 . After drying over Na 2 SO 4 and concentration in vacuo, Boc-D- TMSal-Adgly-ONSu (7.2 g) as a white foam.

C. Boc-D-TMSal-Adgly-boroPro-OPin

The title compound is obtained using the analogous three-step one-pot procedure as described for the synthesis of Boc-D-TMSal-Pro-Baa-OPin in ref. ex. L/C. Due to the low activity of the sterically hindered active ester from step B (2.7 g, 5.0 mmol), the intermediate α-aminoborate, which results from the reaction of the chiral α-chloroborate ((+)-pinanediol- (S)-1-chloro-4-bromobutane-1-borate)

(1.7 g, 5.0 mmol) with lithium hexamethyldisilazane (5.0 mmol) and hydrolysis with HCl, cyclizes to the boroproline derivative which then reacts with the active ester from step B to give the unexpected Boc-D-TMSal-Adgly- boroPro-OPin as the main product. Flash chromatography (2:1 hexane/EtOAc) of the crude product gives the title compound (0.48 g) as a white foam which is further purified by recrystallization from ether/hexane to give the desired product Boc-D-TMSal-Adgly-boroPro-OPin as a white crystalline compound.

mp: 187-188'C [Ct]D<20> = +2.8° (c= 1.0 in CH2<C>12).

Claims (6)

1. Connection, character, characterized by having formula I where W is hydrogen or an N-protecting group,• Y is a sequence of two amino acids of which the N-terminal amino acid is an unnatural amino acid of formula II where R 11 is a group of formula (c) or (d) and the other amino acid is L proline, Qi and Q2 together represent a group of formula (a) R5 is a group -A-X where A is -(CH2)Z- where z is 2, 3, 4 or 5, and X is -NH2 or -NH-C(NH)-NH2, and wherein the asymmetric carbon atom marked with <*> may have D or L configuration, or represent any mixture thereof. 2. Connection as stated in claim 1, characterized in that W is H (CH2CH20)-, R6CO-, R7OCO- or R8SO2-, where: p = 3-30, R6 = C1-6 alkyl, R7 = C1-6 alkyl, phenyl, benzyl or naphthyl, and R8 = phenyl , naphthyl or C1-5 alkylphenyl. 3. Compound as stated in claim 1, characterized in that it has formula III: 4. Compound as stated in claim 1, characterized in that it has formula IV 5. Compound as stated in claim 1, characterized in that it has formula V 6. Therapeutic preparation, characterized in that it contains a compound as stated in claim 1 in combination with a pharmaceutically acceptable excipient and/or diluent.