AU685465B2

AU685465B2 - New peptides derivatives

Info

Publication number: AU685465B2
Application number: AU69408/94A
Authority: AU
Inventors: Karl Thomas Antonsson; Ruth Bylund
Original assignee: Astra AB
Current assignee: AstraZeneca AB
Priority date: 1993-06-03
Filing date: 1994-06-02
Publication date: 1998-01-22
Anticipated expiration: 2014-06-02
Also published as: AU6940894A; WO1994029335A1; SE9301911D0; EP0701567A1; JPH08511017A

Description

WO 94/29335 PCTISE94/00534 1 New peptide derivatives

DESCRIPTION

In its broad sense this invention relates to protease inhibition and treatment of inflammatory diseaes. More specifically this invention relates to new competitive inhibitors of trypsin-like serine proteases such as kininogenases, their synthesis, pharmaceutical compositions containing the compounds as active ingredients, and the use of the compounds for treatment of inflammatory disorders, e.g. asthma, rhinitis, urticaria, inflammatory bowel diseaes, and arthritis.

BACKGROUND

Kininogenases are serine proteases.that act on kininogens to produce kinins (bradykinin, kallidin, and Met-Lysbradykinin). Plasma kallikrein, tissue kallikrein, and mast cell tryptase represent important kininogenases.

Kinins (bradykinz kallidin) are generally involved in inflammation. For example, the active inflammation process is associated with increased permeability of the blood vessels resulting in extravasation of plasma into the tissue. The ensuing plasma exudate contains all the protein systems of circulating blood. The plasma-derived kininogens inevitably will be interacting with different kallikreins, forming kinins continually as long as the active plasma exudation process is ongoing. Plasma exudation occurs independent of the mechanisms that are involved in the inflammation, whether it is allergy, infection or other factors (Persson et al., Editorial, Thorax, 1992, 47:993-1000). Plasma exudation is thus a feature of many diseases including asthma, rhinitis, common cold, and inflammatory bowel diseases. Particulary in allergy mast cell tryptase will be released (Salomonsson et al., Am. Rev. Respir. Dis., 1992, 146:1535-1542) to SUBSTITUTE

SHEET

WO 94/29335 PCT/SE94/00534 2 contribute to kinin formation and other pathogenic events in asthma, rhinitis, and intestinal diseases.

The kinins are biologically highly active substances with smooth muscle effects, sectretory effects, neurogenic effects, and actions that may perpetuate inflammatory processes including activation of phospholipase A 2 and increasing vascular permeability. The latter action potentially induces a vicious circle with kinins providing for the generation of more kinins etc.

Tissue kallikrein cleaves primarily low molecular weight kininogen to produce kallidin and plasma kallikrein preferably releases bradykinin from high molecular weight kininogen.

PRIOR ART Inhibitors of kallikrein based on the amino acid sequence around the cleavage site (-Ser-Pro-Phe-Arg Ser-Ser-Arg-) have been reported earlier.

The arginine chloromethyl ketones were reported as plasma kallikrein inhibitors by Kettner and Shaw in Biochemistry 1978, 17:4778-4784 and Meth. Enzym. 1981, 80:826-842.

Likewise, esters and amides were reported by Fareed et al. in Ann. N.Y. Arad. Sci. 1981, 370:765-784 to be plasma kallikrein inhibitors.

In EP-A2-0,195,212 protease enzym inhibitors, based on analogues of peptidase substrates, including kallikrein, are described.

Inhibitors of trypsin like serine proteases, such as thrombin and kallikrein, based on C-terminal boronic acid derivatives SUBSTITUTE SHEET WO 94/29335 PCTSE9400534~ 3 of arginine and isothiouronium analogues thereof have been reported in EP-A2-0,293,881.

In WO 92/04371 a series of kallikrein inhibitors with carbonyl-activating or binding groups are described.

DISCLOSURE OF THE INVENTION An objective of the present invention is to provide novel and potent kallikrein inhibitors with competitive inhibitory activity towards the enzyme i.e. causing reversible inhibition. A further objective is to obtain inhibitors which can be given orally, dermally, rectally, or via the inhalation route.

According to the invention it has been found that compounds of the general Formula I, either as such or in the form of physiologically acceptable salts, and including Fpssible stereoisomers, are potent inhibitors of serine proteases and especially kallikreins: 2 Formula I wherein: n is an integer 3, 4, 5, or 6; preferably 3 or 4; Al represents a structural fragment of Formulae IIa, lib, IIc, lid or lie;

M

WO 94129335 PCTISE94O 0534 R2Z flc lib R N, wherein: 9* 9 9 9 p 9 99** 9* 9.

B.

9 a. 9.

*0 *0* eel,.

9* 0 90 9.

*99* 4@ .9 99 9* 9 9* 9

S.

p is an integer 0,1 or 2; m is an integer 1, 2, 3, or 4, preferably 2; q is an integer 011 or 2, preferably 1; RI represents H, an alk~yl group having 1 to 4 carbon atoms, a hydroxyalkyl group having 2-3 carbon atoms or R 1 1 0C-alkyl-, where the alkyl group has 1 to 4 carbon atoms and R 11 is H or an alkyl group having 1 to 4 carbon atoms, or

R

1 represents R 12 00C-.l,4-phenyl-CH 2 wherein R 12 is H or an alkyl. group having 1 to 4 carbon atoms, or

R

1 represents R 13 -M*-Co-alkyl-I wherein the alkyl group has 1 to 4 carbon atoms and is optionally substituted alpha to the carbonyl. with an alkyl group having 1 to 4 carbon atoms and wherein R 1 3 is H or an alkyl group having 1 to 4 carbon atoms Or -CH 2

COOR

1 2 wherein R~ 12 is as defined above, or

'J

WO 94/29335 PCT/SE94/00534

R

1 represents R 1 4 S0 2 Ph(4-COOR 12

)-SO

2 Ph(3-COOR 12

)-SO

2 or Ph(2-COOR 12 )-S0 2 wherein R 1 2 is as defined above and R 14 is an alkylgroup having 1-4 carbon atoms, or

R

1 represents CO-R s 1 wherein R 1 5 is an alkyl group having 1-4 carbon atoms, or

R

1 represents CO-OR 15 wherein R 1 5 is as defined above, or

R

1 represent CO-(CH 2 )p-COOR 1 2 wherein R 12 and p are as defined above, or

R

1 represents -CH 2

PO(OR

1 6) 2 wherein R 1 6 is, individually at each occurrence, H, methyl or ethyl;

R

2 represents H or an alkyl group having 1 to 4 carbon atoms or R 2 1 OOC-alkyl-, wherein the alkyl group has 1 to 4 carbon atoms and isoptionally substituted in the position which is alpha to the carbonyl group, and the alpha substituent is a 20 group R22-(CH 2 wherein p is as defined above and R 2 2 is methyl, phenyl, OH, COOR 21 and R 2 1 is H or an alkyl group having 1 to 4 carbon atoms;

R

3 represents an alkyl group having 1-4 carbon atoms, or

R

3 represents a cyclohexyl- or cyclopentyl group, or 9

R

3 represents a phenyl group which may or may not be substituted with an alkyl group having 1 to 4 carbon atoms, or with a group OR 2 1 wherein R 2 1 is as defined above or

R

3 represents a 1-napthyl, 2-naphtyl, 4-pyridyl, 3pyrrolidyl, or a 3-indolyl group which may or may not be substituted with OR 2 1 wherin R 2 1 is as defined above and with p 1; or

R

3 represent a cis- or trans-decalin group with p 1; or 1 WO 94/29335 PCT/SE94/00534 6

R

3 represents Si(Me) 3 or CH(R31) 2 wherein R 3 1 is a cyclohexyl- or phenyl group;

A

2 represents a structural fragment 0 O

II

NH CH C

I

(CH

2 )p

R

wherein R 3 and p are as defined above; An alkyl group may be straight or branched unless specified otherwise. Alkyl groups having 1 to 4 carbon atoms are methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl and t-butyl. When unsaturation is referred to, a carboncarbon double bond is intended. Abbreviations are listed at the end of this specification.

According to the invention it has been found that compounds of the general Formula I, either as such or in the form of physiologically acceptable salts, and including stereoisomers, are potent inhibitors of trypsin-like serine proteases and especially plasma and/or tissue kallikrein: Compounds of Formula I having S-configuration on the A 2 amino acid are preferred ones, of those compounds also having R-configuration on the Al amino acid are particularly preferred ones.

Preferred compounds of the invention include: H-(R)Cha-Phe-Agm

HOOC-CH

2 -(R)Cha-Phe-Agm H-(R)Cha-Phe-Nag SUBSTITUTE SHEET WO 94/29335 7

HOOC-CH

2 Cha-Phe-1Nag

CH

3 -CO- -Cha-Phe-Nag

CH

3

-CH

2 -Cha-Phe-Nag HOOC-CO- -Cha-Phe-Nag

HOOC-CH

2 Phe-Phe-Agin

HOOC-CH

2 Phe-Cha-Agjm

HOOC-CH

2 Cha-Cha-Agm

HOOC-CH

2 Phe-Phe-Nag !!OOC-C11 2 Phe-Cha-Nag

HOOC-CH

2 Cha-Cha-Nag

HOOC-CI

2 Cha-aNal-Ag~m

HOOC-CH

2 Cha-PNal-Agn H- Phe-Cha-Acim H- Phe-Cha-Nag H- Phe-Phe-Agjm Hf- Phe-Phe-Nag

CH

3 Phe-Phe-Agm

CH

3 Cha-Phe-Agm

CH

3 Phe-Cha-Agmn

HOOC-CH

2 Pro-Phe-Agjm

HOOC-CH

2 Pro-Phe-Nag H- Pro-Phe-Agm H- Pro-Phe-Nag

CH

3 Pro-Phe-Agjm

CH

3 Pro-Phe-Nag Particularly preferred compounds are: H- Cha-Phe-Agm

HOOC-C.H

2 Cha-Phe-Agm H- Cha-Phe-Nag

HOOC-CH

2 Cha-Phe-Nag

CH

3 -CO- -Cha-Phe-Nag

CH

3

-CH

2 -cha-Phe-Nag HOOC-CO- -Cha-Phe-Nag PCT/SE94/00534 SUBSTITUTE SHEET WO 94/29335 PCT/SE94/00534 8 The best mode according to the invention known at present is to use the compound according to Example 4 namely.

HOOC-CH

2 -(R)Cha-Phe-Nag Medical and pharmaceutical use The invention also provides compositions and methods for the treatment of physiological disorders and especially inflammatory diseases such as asthma, rhinitis, pancreatitis, uticaria, inflammatory bowel diseaes, and arthritis. An effective amount of Formula I with or without a physiologically acceptable carrier or diluent can be used solely or in combination with other therapeutic agents.

Depending upon the disorder and patient to be treated the compositions may be administered via oral, dermal, nasal, tracheal, bronchial, parenteral, or rectal routes at varying doses.

The compounds inhibit the activity of kallikreins assessed with chromogenic substrates according to known procedures.

The anti-inflammatory actions of the present compounds can for example be studied by their inhibition of allergeninduced exudative inflammatory processes in airway mucosa or gut mucosa.

Determinaton of the inhibition constant KX for plasma kallikrein.

K

i determinations were made with a chromogenic substrate method, and performed on a Cobas Bio centrifugal analyzer manufactured by Roche (Basel, Switzerland). Residual enzyme activity after incubation of human plasma kallikrein with various concentrations of test compound was determined at three different substrate concentrations, and measured as change in optical absorbance at 405 nm and 37 0

C.

SUBSTITUTE SHEET WO 94/29335 P'CT/SE9400534 9 Human plasma kallikrein (E.C.3.4.21.34, Chromogenix AB, M81ndal, Sweden), 250 pl of 0.4 nkat/ml in buffer (0.05 mol/l Tris-HCl, pH 7.4, 1 0.15 adjusted with NaCl) with bovine albumin 5 g/l (cat no 810033, ICI Biochemicals Ltd, High Wycombe, Bucks, GB), was incubated for 300 s with 80 pl of test compound solution in 0.15 mol/l NaCl containing albumin g/l. An additional 10 pl of water was supplied in this step. Then 40 pl of kallikrein substrate (S-2302, Chromogenix AB, 1.25, 2.0 or 4.0 mmol/l in water) was added together with another 20 ip of water, and the absorbance change monitored.

Ki was evaluated from Dixon plots, i.e. diagrams of inhibitor concentration versus 1/ (AA/min), where the data for the different substrate concentrations form straight lines which intercept at x= -K i Pharmaceutical preparations The compounds of the Formula I will normally be administered by the oral, rectal, dermal, nasal or parenteral route in the form of pharmaceutical preparations comprising the active ingredient either as a free base or a pharmaceutical acceptable non-toxic organic or inorganic acid addition salt, e.g. the hydrochloride, hydrobromide, lactate, acetate, citrate and trifluoroacetate and the like in a pharmaceutically acceptable dosage form.

The dosage form may be a solid, semisolid or liquid preparation prepared by per se known techniques. Usually the active substance will constitute between 0.1 and 99 by weight of the preparation, more specifically between 0.1 and 56 by weight for preparations intended for parenteral administration and between 0.2 and 75 by weight for preparations suitable for oral administration.

Suitable daily doses of the compounds of the invention in therapeutical treatment of humans are about 0.001-100 mg/kg SUBSTITUTE SHEET WO 94/29335 PCTSE94/00534 body weight at peroral administration and 0.001-50 mg/kg body weight at paronteral administration.

Preparation A further objective of the invention is the mode of preparation of the compounds. The compounds of Formula I may be prepared by coupling of an N-terminally protected dipeptide (WI-Al-A 2 -OH) or amino acid (W 1

-A

2 when a Nterminally protected amino acid is used a second amino acid is added afterwards using standard methods, to a compound

H

2

N-(CH

2 )n-X wherein Al, A 2 and n are as defined with Formula I and X is an unprotected or protected guanidino group or a protected amino group, or a group transferable into an amino group, where the amino group is subsequently transferred into an unprotected or protected guanidino group, followed by removal of the protecting group(s) or deprotecting of the N-terminal nitrogen followed by alkylation of the N-terminal nitrogen and deprotection by known methods.

The coupling is accordingly done by one of the following methods: Method I Coupling of an N-terminally protected dipeptide, prepared by standard peptide coupling, with either a protected- or unprotected amino guanidine or a straight chain alkylamine carrying a protected or masked amino group at the terminal end of the alkyl chain, using standard peptide coupling,shown in the formula

W

1

A

1

-A

2

-OH

4 H 2

N-(CH

2 )n-X

W

1

A

1

-A

2 -NH6(H 2 a SUBSTITUTE SHEET w I WO 94/29335 PCT/SE94/00534 11 wherein A 1

A

2 and n are as defined in Formula I W 1 is a Nteminal amino protecting group such as tert-butyloxy carbonyl and benzyloxy carbonyl and X is -NH-C(NH)-NH 2

-NH-C(NH)-NH-

W

2

-N(W

2

)-C(NH)-NH-W

2

-NH-C(NW

2

)-NH-W

2 or -NH-W 2 where W 2 is an amine protecting group such as tert-butyloxy carbonyl or benzyloxy carbonyl, or X is a masked amino group such as azide, giving the protected peptide. The final compounds can be made in any of the following ways, depending on the nature of the X- group used: Removal of the protecting group(s) (when X= -NH-C(NH)-NH 2

-C(NH)-NH-W

2

-NH-C(NW

2

)-NH-W

2 or -NH-CWi)-NH-W 2 or a selective deprotection of the W 1 group (e.g when X= -NH-C(NH) -NH-W, -N(W 2 -C(NH)-NH-W -NH-

C(NW

2

)-NH-W

2

W

2 in this case must be orthogonal to W 1 followed by alkylation of the N-terminal nitrogen and deprotection or a selective deprotection/ unmasking of the terminal alkylamino function NH-W 2

W

2 in this case must be orthogonal to W 1 or X= a masked aminogroup, such as azide) followed by a guanidation reaction, using standard methods, of the free amine and deprotection of the Wl-group.

Method II Coupling of an N-terminally protected amino acid, prepared by standard methods, with either a protected- or unprotected amino guanidine or a straight chain alkylamine carrying a protected or masked amino group at the terminal end of the alkyl chain, using standard peptide coupling, shown in the formula

W

1

-A

2

-OH

Sr2N- (CH 2 n-X WI-A (CHf2?-X wherein A 2 n, W and X are as defined above followed by deprotection of the W 1 -group and coupling with the N-terminal amino acid, in a protected form, leading to the protected SUBSTITUTE SHEET

M

WO 94/29335 WO9429335 CT/SE94/00S34 12 peptide described in Method I. The synthesis to the final compounds is then continued according to Method I.

DETAILED DESCRIPTION OF THE INVENTION The following description is illustrative of aspects of the invention.

EXPERIMENTAL PART General Experimental Procedures.

The 1 H NMR and 13 C NMR measurements were performed on BRUKER AC-P 300, BRUKER 200 and BRUKER AM 500 spectrometers, the former operating at a 1H frequency of 500.14 MHz and a 13C freguency of 125.76 MHz and the latter at 1 H and 13 C freguency of 300.13 MHz and 75.46 MHz respectively.

The samples were 10-50 mg dissolved in 0.6 ml of either of the following solvents; CDCl 3 (isotopic purity 99.8%, Dr.

Glaser AG Basel), CD 3 OD (isotopic purity 99.95%, Dr. Glaser AG Basel) or D 2 0 (isotopic purity 99.98%, Dr. Glaser AG Basel).

The 1H and 13 C chemical shift values in CDCl 3 and CD 3 OD are relative to tetramethylsilane as an external standard. The 1

H

chemical shifts in D 2 0 are relative to the sodium salt of 3- (trimethylsilyl)-d 4 -propanoic acid and the 3 C chemical shifts in D 2 0 are referenced relative to 1,4-dioxane (67.3 ppm), both as external standard. Calibrating with an external standard may in some cases cause minor shift differences compared to an internal standard, however, the difference in 1 H chemical shift is less than 0.02 ppm and in 1 3 C less than 0.1 ppm.

SUBSTITUTE SHEET WO 94/29335 PCTISE94/00534 13 Thin-Layer Chromatography was carried out on commercial Merck Silicagel 60F 254 coated glass or aluminium plates.

Visualization was by a combination of UV-light, followed by spraying with a solution prepared by mixing 372 ml of EtOH(95%), 13.8 ml of concentrated H 2

SO

4 4.2 ml of concentrated acetic acid and 10.2 ml of p-methoxy benzaldehyde or phosphomolybdic acid reagent (5-10 w.t in and heating.

Flash chromatography was carried out on Merck Silicagel (40-63 mm, 230-400 mesh) under pressure of N 2 Freeze-drying was done on a Leybold-Heraeus, model Lyovac GT 2, apparatus.

Protection Procedures Boc-(R)Cha-OH To a solution of H-(R)Cha-OH, 21.55 g (125.8 mmol), in 130 ml 1 M NaOH and 65 ml THF was added 30 g (137.5 mmol) of (Boc) 2 0 and the mixture was stirred for 4.5 h at room temperature.

The THF was evaporated and an additional 150 ml of water was added. The alkaline aqueous phase was washed twice with EtOAc, then acidified with 2 M KHSO 4 and extracted with 3 x 150 ml of EtOAc. The combined organic phase was washed with water, brine and dried (Na 2

SO

4 Evaporation of the solvent afforded 30.9 g (90.5 of the title compound as a white solid.

Preparation of Starting Materials Boc-(R)Cha-OSu Boc-(R)Cha-OH (1 HOSu (1.1 eq) and DCC or CME-CDI (1.1 eq) were dissolved in acetonitrile (about 2.5 ml/mmol acid) and stirred at room temperature over night. The precipitate SUBSTITUTE SHEET WO 94/29335 PCT/SE94/00534 14 formed during ti e raaction was filtered off, the solvent evaporated and the product dried in vacuo. (When CME-CDI was used in the reaction the residue, after evaporation of the

CH

3 CN, was dissolved in EtOAc and the organic phase washed with water and dried. Evaporation of the solvent gave the title compound).

H-NMR (500 MHz, CDC1 3 2 rotamers ca: 1:1 ratio) 6 0.85-1.1 2H), 1.1-1.48 4H), 1.5-1.98 16H; thereof 1.55 (bs, 2.82 (bs, 4H), 4.72 (bs, 1H, major rotamer), 4.85 (bs, 1H, minor).

Boc-(R)Cha-Phe-OH To a stirred mixture of 6.61 g (40 mmol) H-Phe-OH and 1.4 g of NaOH (35 mmol) in 60 ml DMF/H 2 0 at 5 oC was added 3.68 g (10 mmol) Boc-(R)Cha-OSu and the mixture was allowed to reach room temperature. After 3 hours the solvent was evaporated and the residue was dissolved in 150 ml of water.

The basic water phase was washed with 2 x 50 ml EtOAc, acidified with 1 1 KHSO 4 and extracted with 2 x 100 mL EtOAc.

The combined organic phase was washed with 2 x 50 mL water and dried (MgSO 4 Filtration and evaporation of the solvent gave 2.86 g of the title compound.

Boc-(R)Cha-Phe-O8u To a stirred solution of 2.81 g Boc-(R)Cha-Phe-OH (6.71 miol) and 850 mg HOSu (7.38 mmol) in 30 mL of CH 3 CN was added 3.13 g CME-CDI (7.38 mmol) and the reaction was left at room temperature for 15 hours. The precipitate formed during the reaction was filtered off, the solvent evaporated and the reasidue was dissolved in 150 mL EtOAc. The organic phase was washed with 1 x 20 mL water, 1 x 20 mL Na 2

CO

3 2 x 20 mL water, 1 x 20 mL brine and dried (MgSO 4 Filtration followed by evaporation of the solvent gave 2.44 g of the title compound which was used without further purification.

SUBSTITUTE SHEET WO 94/29335 PCT/SE94/00534 Boo-Nag(Z) N-Bensyloxycarbonyl-O-methyl isourea To a stirred solution of concentrated aqueous NaOH (2.8 L, w/w, 19.1 M, 53 mol) and water (32 L) at 180 C was added in two portions o-methylisourea hemisulphate (1.7 kg, 94%, 13.0 mol) and 0-methylisourea hydrogensulphate (1.57 kg, 99%, 9.0 mol). The reaction mixture was cooled to 3-50 C.

Benzyl chloroformiate (3.88 kg, 92%, 20.9 mol) was added over a 20 minutes period under cooling and vigorous stirring. The reaction temperature went from 3 to 80 C during the addition of Z-Cl. The addition funnel was rinsed with 5 litres of water which was added to the reactor. The reaction mixture was stirred at 0-30 C for 18 h, filtered and the crystals was washed with cooled C) water (10 L).

Vacuum drying 250 C, 10-20 mbar) for 48 h gave 3.87 kg (89%) of the title compound as a white crystalline powder.

(ii) Boc-Nag(Z) To a stirred solution Boc-NH-(CH 2 3

-NH

2 x HCl (prepared according to Mattingly Synthesis, 367 (1990)) (3.9 kg, 18.5 mol) in iso-propanol (24 kg) at 60-700 C was added in portions over a 30 minutes period KHCO 3 (4.2 kg, 42 mol). A slow evolution of CO 2 occurs. The mixture was stirred for another 30 minutes followed by addition in portions over a minutes period N-bensyloxycarbonyl-O-methyl isourea (3.74 kg, 18.0 mol). The reaction mixture was stirred at 65-700 C for 16 h, cooled to 200 C and filtered. The precipitate was washed with iso-propanol (10 5 The combined filtrates was concentrated at reduced pressure keeping the heating mantle not warmer than 65-700 C. When approximately 45 litres was distilled off EtOAc (90 L) was added. The reaction mixture was cooled to 20 25 0 C, washed with water (10 and L) and brine (5 and dried with Na 2

SO

4 (2 kg). After stirring the rection mixture was filtered and the filter cake SUBSTITUTE SHEET WO 94/29335 PCT/SE94/00534 16 was washed with EtOAc (11 and 7 The combined filtates were concentrated at reduced pressure keeping the heating mantle not warmer than 40-500 C. When approximately 90 litres of EtOAc was distilled off, toluene (25 L) was added and the evaporation continued. After collection of approximately another 18 litres of destillate, toulene (20 L) was added under vigorous stirring and the resulting mixture was cooled to -1 to 00 C and gently stirred over night (17 The crystal slurry was filtered and the product was washed with cooled toluene (10 and 5 Vacuum drying (10-20 mbar, 400 C) for 24 h gave 4.83 kg (13.8 mol, 76%) of Boc-Nag(Z).

1 H-NMR (300 MHz, CDC1 3 6 1.41 9H), 1.6-1.7 2H), 3.3 4H), 4.8-5.0 (bs, 1H), 5.10 2H), 7.2-7.4 51).

Boc-Agm(Z) Boc-Agm To a slurry of 14.95 g (65.5 mmol, 1 eq.) of agmatine sulphate (Aldrich), 13.7 ml of Et 3 N (98.25 mmol, 1.5 eq.), 165 ml of H 2 0 and 165 ml of THF was added 21.5 g (98.25 mmol, eq.) of (Boc) 2 0 during 5 minutes at room temperature. The mixture was stirred vigorously over night, evaporated to dryness and the residue was washed with 2x100 ml of Et20 to give Boc-Agm as a white powder which was used without further purification in the next step.

(ii) Boc-Agm(Z) To a cold (+5 0 C) slurry of the crude Boc-Agm from the previous step (ca: 65.5 mmol) in 180 ml of 4N NaOH and 165 ml of THF was added 24 ml (169 mmol, 2.5 eq) of benzyl chloroformate during 10 minutes. After stirring at room temperature for 4 h methanol (150 ml) was added and the stirring was continued for an additional 20 h at room SUBSTITUTE SHEET WO 94/29335 PCT/SE94/00534 17 temperature. The organic solvent was evaporated and 200 ml of was added to the residue. The basic water phase was extracted with 1x300 ml and 2x200 ml of EtOAc. The combined organic phases was washed with H20 (2xl00ml), brine (1x100 ml) and dried (MgSO 4 Evaporation of the solvent and flash chromathography (CH 2 Cl 2 /MeOH, a stepwise gradient of 97/3, 95/5 and 9/1 was used) gave 14.63 g of pure Boc-Agm(Z) as a white powder.

1 H-NMR (CDC13 500 MHz): 6 1.35-1.40 2H), 1.45 9H), 1.5-1.6 2H), 3.0-3.2 4H), 4.65 (bs, 1H), 5.1 2H), 7.25-7.40 1 3 C-NMR (CDC1 3 75.5 MHz): 6 25.44, 27.36, 28.21, 65.83, 79.15, 127.47, 127.66, 128.14, 137.29, 156.47, 161.48, 163.30.

H-(R)Cha-Phe-Nag(Z) Boc-(R)Cha-Phe-OH Boc-(R)Cha-.OH was dissolved in acetonitrile (200 mL), N-hydroxisuccinimide (9.9 g, 81 mmol) was added.

Dicyclohexylcarbodiimide (17.8 g, 81 mmol) was then added slowly and the reaction mixture was stirred overnight at room temperature. The precipitate was filtered off and the Boc- (R)Cha-OSu containing solution was evaporated. Phe-OH (48.7 g, 195 mmol), sodiumhydroxide (10.3 g, 258 mmol), water (270 mL) and finally dimethylformamide (70 mL) were added to a reaction vessel while stirring. Boc-(R)Cha-OSu was dissolved in dimethylformamide (200 mL) and added slowly to the reaction vessel while maintaining the reaction temperature below 5°C. After 3 h the solution was evaporated, the residue dissolved in water (1000 mL) and extracted with ethylacetate (2 x 300 mL). The aqueous phase was acidified with potassium hydrogensulfate (iM) to pH 3 and extracted with ethylacetate SUBSTITUTE SHEET WO 94/29335 PCT/SE9/00534 18 (2 x 700 mL). The pooled organic layer was washed with water (2 x 300 mL) and dried over magnesiumsulfate. After filtration and evaporation the title product Boc-(R)Cha-Phe- OH (21 g, 50 mmol) was isolated in 67% yield.

(ii) Boc-(R)Cha-Phe-Nag(Z) Boc-(R)Cha-Phe-OH (20.8 g, 49.7 mmol) was dissolved in acetonitrile (350 mL). The vessel was cooled and 4dimethylaminopyridine (12.1 g, 99.4 mmol) was added while maintaining a reaction temperature at 2 0 C. Nag(Z) (12.4 g, 49.7 mmol) was added resulting in a white slurry. Finally 1- (3-dimethylaminopropyl)-3-ethylcarbodiimide (8.6 g, 52 mmol) was added slowly over a 10 minute period. The solution was allowed to reach room temperature and stirred overnight. The solution was evaporated and the residue was dissolved in ethylacetate (400 mL) and water (150 mL). The organic layer is washed with potassium hydrogensulfate (1M, 250 mL), sodium carbonate (IM, 3 x 250 mL), water (200 mL) and finally brine (200 mL). The collected organic layer was evaporated and the title compound, Boc(R)Cha-PHe-Nag(Z) (24.9 g, 38 mmol), was isolated in 77% yield.

(iii) H2N-(R)Cha-Phe-Nag(Z) Boc-(R)Cha-Phe-Nag(Z) (10 g, 15.4 mmol) was dissolved in ethylacetate (50 mL). The reaction vessel was cooled with ice-water bath to 4OC and HC1 (46 mL, 152 mmol, 3,3 M in ethylacetate) was then added. The ice container 'was removed and the solution was allowed to reach room teprpratur. After h all starting material was consumed. Tha solvent was decanted from the HCl-salt of the deproteced peptide and the crude product was dissolved in water (50 gaL) and extracted with ethylacetate(50 mL). The collected aqueous phase was neutralized by addition of potassium carbonate (4.3 g, 30.8 mmol) and then it extracted with dichloromethane (150 mL).

SUBSTITUTE SHEET WO 94/29335 PCTISE94/00534 19 The collected organic phase was evaporated and H 2 N-(R)Cha- Phe-Nag(Z) (3 g, 5.5 inmol), was isolated after purification by chromatography on silicagel (230-400 mesh) eluting with

CH

2 C1 2 :MeOH:NH 4 OH (100:4:1 to 100:15:1) in 36% yield.

1 H &MR (200 MHz, ODC1 3 6 (ppm) 7.82 1H), 7.4-7.1 (m, 5.09 2H), 4.42 1H), 3.4-2.9 (mn, 7H), 1.8-0.7 (in, iSH).

Working Exan-1e Examp~le I H-(R)Cha-Phe-Agjm x 2 TVA Boc-(R)Cha-Phe-Agn(Z) A solution of 729 ing (2 minol) Boc-Agm(Z) in mL TFA/CH 2 Cl 2 was stirred at room temperature for about 2 h. The solvent was evaporated and the product was dissolved together with 1.03 g (2 mmiol) of Boc-(R)Cha-Phe-OSu in 10 mL DMF I the PH was adjusted with NIM to about 9 and the mixture was stirred at room teinperaturr; fo 5 days. The solvent was evaporated in vacuo and the residue was dissolved in 200 mL EtOAc. The organic phase was washed with 2 x 10 mL of water, 1 14 KHS0 4 I M NaOH, water and dried (MgS0 4 Evaporation of the solvent followed by flash chromatography (70 g Si0 2 using a stepwise gradient of 100 mL CH 2 Cl 2 /MeOH (95/5) followed by 250 mL CH 2 Cl 2 /MeOH gave 1.09 g (96 of the title compound.

1 H-NMM (500 M4Hz, CDC1 3 mixture of two rotainers): major rotamer: 6 0.7-0#9 214), 1.0-1.8 (in, 25H4; thereof 1.39 (sl 2.9-3.25 (in, 6 H)l 4.02 (mn, 1H), 4.71 1 5.05 2 14), 7.1-7.4 (mn, 10H4).

SUBSTITUTE SHEET WO 94/29335 WO 42933 PCT/SE94/00534 1 3 C-NMR (125 MHz, D 2 carbonyl and guanidine carbons: 6 161.7, 163.6, 172.0, 172.7 and 174.9.

(ii) H-(R)Cha-Phe-Agm x 2 TFA A solution of 100 mg (0.15 mmol) Boc-(R)Cha-Phe-Agm(Z) in mL CH 2 C1 2 /TFA was stirred at room temperature for 2 h min after which the solvent was evaporated. The residue was dissolved in 9 ml EtOH/H 2 0 and hydrogenated over 40 mg 5 Pd/C at athmospheric pressure for 3 h. The catalyst was filtered off the solvent evaporated and the residue was dissolved in water and freeze dried to give 93 mg (94 of the title compound as a white powder.

1 H-NMR (500 MHz, D 2 0, mixture of two rotamers): major retainer: 6 0.65-1.75 17H), 2.86-3.23 6H), 3.96 1H), 4.59 (dd, 1H), 7.15-7.4 13 C-NMR (125 MHz, D 2 guanidine 6 157.3; carbonyl carbons: 6 171.0 and 173.1.

Example 2

HOOC-CH

2 -(R)Cha-Phe-Agm x 2 TFA H-(R)Cha-Phe-Agm(Z) A solution of 0.99 g (1.49 mmol) Boc-(R)Cha-Phe-Agm(Z) in mL CH 2 C1 2 /TFA was stirred at room temperature for 3 h after which the solvent was evaporated and the residue dissolved in 100 mL CH 2 Cl 2 The organic phase was washed with 1 x 30 mL 5 M NaOH, 2 x 30 mL water and dried (MgS0 4 Filtration and evaporation of the solvent gave 825 mg (98 of the title compound as a white powder.

SUBSTITUTE SHEET WO 94/29335 PCT/SE94/00534 21 1 H-NMR (300 MHz, CDC1 3 6 0.75-1.0 2H), 1.05-1.75 (m, 2.93-3.34 7H), 4.56 1H), 7.13-7.39 1 3 C-NMR (75 MHz, CDCL 3 carbonyl and guanidine carbons: 6 161.8, 163.8, 171.8 and 176.5.

(ii) BnOOC-CH 2 -(R)Cha-Phe-Agm(Z) A mixture of 282 mg (0.5 mmol) H-(R)Cha-Phe-Agm(Z), 173 mg (1.25 mmol) K 2 C0 3 and 137.5 mg (0.6 mmol) BnOOC-CH2-Br in 16 mL CH 3 CN/DMF (15/1) was heated to 50 oC for 4 h and 15 minutes after which the solvent evaporated and the residue dissolved in 70 mL EtOAc. The organic phase was washed with 4 x 10 mL water, 10 mL Brine and dried (MgSO 4 Evaporation of the solvent followed by flash chromatography (37 g SiO 2 using

CH

2 C12/MeOH(NH 3 -saturated) (95/5) as eluent afforded 230 mg (64 of the desired compound.

1 H-NR (300 MHZ, CDCI 3 6 0.7-0.95 2H), 1.05-1.75 (m, 15H), 2.84-3.25 8H), 4.56-4.68 1H), 4.95 2H), 5.12 2H), 7.1-7.45 13 C-NMR (75 MHz, CDCL 3 carbonyl and guanidine carbons: 6 161.7, 163,6, 171.56, 171.61 and 175.1.

(iii) HOOC-CH2-(R)Cha-Phe-Agm x 2 TFA To a solution of 230 mg (0.323 nmmol) BnOOC-CH2-(R)Cha-Phe- Agm(Z) in 18 mL EtOH/H 2 0 was added a small amount drops) of TFA and the mixture was hydrogenated over 70 mg 5 Pd/C at athmospheric pressure for 6 h. The catalyst was filtered off, the solvent was evaporated and the residue was dissolved in water and freeze dried to afford 223 mg (96%) off the title compound as a white powder.

SUBSTITUTE SHEET WO 94/29335 PCT/SE94/00534 22 1 H-NNR (300 Miz CD 3 OD): 0. 65-1. 85 (in, 17H) 2 0 (in, 1H) 3. 0-3. 3 (mn, 5H-) 3. 78. 2H) 4. 0 (bs, 1H-) 4 .62 (mn, 7.1-7.4 (mn, 1 3 C-NMR (75 Ml~z, CD 3 0D) guanidine: 6 158.6; carbonyl carbons: 6 168.9, 169.6 and 173.4.

Exampl1e 3 H-(R)Cha-Phe-Nag X 2 TPA Boc- Cha-Phe-Nag (Z) Prepared in the same way as described for Boc- CR) Cha-Phe- Agin(Z) in Example 1 from Boc-(R)Cha-Phe-OSu (2 minol) and Boc-Nag(Z) (2 mmol).Yield =1.02 g (78%) (ii) H-(R)Cha-Phe-Nag x 2 TFA A solution of 100 mng (0.15 mmuol) Boc-(R)Cha-Phe-Nag(Z) in mL CH 2

CI

2 /TFA was stirred at room temperature for 3 h min after which the solvent was evaporated. The residue was dissolved in 9 ml EtOH/H 2 0 and hydrogenated over 40 mng Pd/C at athinospheric pressure for 3 h. The catalyot was filtered off the solvent evaporated and the residue was dissolved in water and freeze dried to give 97 mng (98 of the title compound as a white powder.

IH-NM4 (500 MHz, D 2 0, mixture of two rotamers) major rotamer: 6 0.75-1.85 15H), 2.9-3.45 (in, 6H), 4.05 111)i 4.6-4.8 (in, 1H; partially hidden by the H-0-D signal) 7.3-7.6 (mi, 511).

13 C-N1MR (75 M4Hz, D)20): guanidine 6 157.6; carbonyl carbons:171.3 and, 173.5.

SUBSTITUTE SHEET WO 94/29335 PCT/SE94/00534 23 Example 4 H00C-CH 2 -(R)Cha-Phe-Nag X 2 TPA H-(R)Cha-Phe-Nag(Z) Prepared in the same way as described for H-(R)Cha-Phe-Aqjm(Z) in Example 2 from Boc-(R)Cha-Phe-Nag(Z). Yield 90 13 C-N4I 75 MHz, CDCl 3 6 26.0, 26.2, 26.4, 29.5, 32.2, 34.0, 34.2, 36.7, 37.7, 38.3, 42.6, 52.7, 55.1, 66.3, 126.9, 127.7, 127.9, 128.3, 128.6, 129.1, 136.7, 137.5t 161.8, 163.7, 171.5 and 176.6.

(11) BnOOC-CH 2 Cha-Phe-Nag (Z) A mixture of 275 mg (0.5 mmol) H-(R)Cha-Phe-Nag(Z), 173 mg (1.25 mmuol) K 2 C0 3 and 137.5 mg (0.6 nunol) BnOOC-CH 2 -Br in mL CH 3 CN was heated to 50 0 C for 3 h and 50 minutes after which the solvent evaporated and the residue dissolved in inL EtOAc. The organic phase was washed with 4 x 10 mL water, mL Brine and dried (MgSO 4 Evaporation of the solvent followed by flash chromatography (37 q SiO 2 using

CH

2 Cl 2 /MeOH(NH 3 -saturated) (95/5) as eluent affordo-d 209 mg (60 of the desired compound.

1 H-NI4R (300 MHz, CDC1 3 6 0.72-0.93 (in, 2H), 1.0-1.72 (in, 13H)o 2.83-3.25 (in, 9H), 4.54 (qt 1H), 5.09 2H), 5.11 (so 2H1), 7.05-7.4 (mn, 15H1), 7.59 1h; NH).

13 C-MM (75 Mz, CDC1 3 carbonyl and guanidine carbons: 6 161.8, 163.6, 171.31 171.6 and 175.2.

(iii) H00C-CH 2 -(R)Cha-Phe-Nag x 2 TFA SUBSTITUTE SHEET WO 94/29335 PCTISE94/00534 24 To a solution of 209 mg (0.3 mxnol) BnOOC-CH 2 -(R)Cha-Phe- Nag(Z) in 18 mL EtOH/H 2 0 was added a small amount drops) of TFA and the mixture was hydrogenated over 70 mg Pd/C at athmospheric pressure for 4 h. The catalyst was filtered off, the solvent was evaporated and the residue was dissolved in water and freeze dried to afford 190 mg off the title compound ais a white powder.

1 H-NMR (300 MHz, CD 3 OD): 6 0.6-1.38 (in, 6H1), 1.4-1.9 (in, 9H1), 2.9-3.4 (mn, 6H), 3.9 (bs, 211), 4.1 (bs, 1H1), 4.7 (mn, 1M; partially hidden by the H-0-D signal), 7.1-7.45 (mn, 13 C-N14R (75 Mlqz, CD 3 OD): guanidine: 6 157.5; carbonyl carbons: 6 169.3, 169.5 and 173.2.

is E9xample H-CR) Cba-Pho-Nag

H

2 N-(R)Cha-Phe-Nag(Z) (300 mg, 0.55 imol) was dissolved in ethanol (50 niL) and trifluoroaceticacid (56 pL, 0.73 mmuol) was added. The mixture was sonicated and palladiumi on charcoal 50 mng) was charged before it was hydrogenated at 45 psi hydrogen pressure in a Parr shaking apparatus for 19 h. The suspension was filtered through celite and after the solvent was evaporated the title compound (0.13 g, 0.31 mmol) was isolated in 56% yield.

'H1 NNTR (200 MHz, d-HC1+d 2

-H

2 0) 8 (ppm) 7.40-7.00 (in, 511), 4.47 111), 3.86 111), 3.25-2.65 (in, 611), 1.75-0.50 (mn, 1511).

TSP-MS found (i/z)=417 (calc. for MH+(C 2 2

H

3 7

N

6 0 2 )417).

Example 6

CR

3 -CO- CR) Cha-Plie-Nag C11 3 -CO- Cha-Phe-Nag (Z) SUBSTITUTE SHEET WO 94/29335 PCT/SE94/00534

H

2 N-(R)Cha-Phe-Nag(Z) (500 mg, 0.91 minol) was dissolved in acetonitrile (7.5 mL). Ac etyichioride (107 mg, 1.36 mmol) dissolved in acetonitrile (1 mL) was then transferred to the reaction vessel. After 30 min. the acylated peptide precipitated as an Md1-salt. Diethylether (5 mL) was added minutes later. The precipitate was filtered off and dried under vacuum at 35 0 C overnight and the dry product, CH 3

-CO-

(R)Cha-Phe-Nag(Z)xHCl (407 mg, 0.69 mniol) was isolated in 76* yield.

1H NI4R (200 M4Hz, CDCl3) 6 (ppm) 7.4-7.1 (i 10H), 5.1 (q, 2H), 4.7 (in, 1H), 4.0 (mn, 1ff), 3.5-2.9 (in, 6Hf), 2.0-0.6 (mn, 18Hf). TSP-MS f ound =593 (calc. f or NM+(C 32

H

45

N

6

O

5 593) (ii) CH 3 -Co- Cha-Phe-Nag

CH

3 -CO-(R)Cha-Phe-Nag(Z) (400 mng, 0.68 rnmol) was dissolved in ethanol (60 mnL) and palladium on charcoal 80 ing) was added. The mixture was hydrogenated at 45 psi hydrogen pressure in a Parr shaking apparatus for 20 h. the suspension was filtered through celite and after the solvent was evaporated a crude mixture (300 mng) was collected. The crude product (150 mng) was purified by reveresed phase chromatography (C8-gel) eluting with MeCN:NH4OAc (0.1M) (40:60) and the product (100 mng, 0.22 iniol) in 64% yield.

1 Hf NMlR (200 MHz, d 4

-CH

3 OH) 6 (ppm) 7.25-6.85 (mn, 5H), 4.44 (dd, 1Hf), 4-02 (tt 1ff), 3.30-2.90 (mn, 5ff), 2.72 (dd, 1Hff) 2.0-0.5 (mn, 18Hf). TSP-MS found (infm)459 (calc. for MH+ (C 24 ff3 9 6

O

3 4 59) Example 7

CR

3

CR

2 CR) Cha-Pha-Nag Mi CH 3

CH

2 Cha-Phe-Nag (Z)

H

2 N-(R)Cha-Phe-Nag(Z) (500 mng, 0.91 mmxol), p-toluenesulphonic acid (173 ing, 0.91 nunol) and methanol (7.5 mL) were added to SUBSTITUTE SHEET WO 94/29335 PCT/SE94/00534 26 a reaction vessel which was cooled with ice. Acetaldehyde (51 AL, 0.91 mmol) was added and finally after another 30 min., sodium cyanoborohydride (86 mg, 1.36 minol) was added. The mixture was stirred at room temperature for four days and then evaporated. The crude product was purified by chromatography on silicagel (230-400 mesh) eluting with MeC1 2 MeOH: NH 4 0H (90:10:1) yielding CH 3

CH

2 -(R)Cha-Phe-Nag(Z) (150 mg, 0.26 uiuol) in 29% yield.

1 H NI4R (200 Mliz, d4-MeOH) 6 (ppm) 7.39-7.28 (in, 10H), 5.11 (s, 211), 4.63 1H, 3.2-2.9 2.42 (in, 2H), 1.8-0.8 (in, 18H).

(ii) CH 3

CH

2 -(R)Cha-Phe-Nag(Z) 150 mng, 0.26 minol) was dissolved in EtOX 840 mL) and acetic acid (1 mL). Palladium on charcoal 51 mng) was charged before it was hydrogenated at 45 psi hydrogen pressure in a Parr shaking apparatur for 2 days. The mixture was filtered and the filter cake was wasiied with MeOH/AcOH 40 inL). The title compound (32 mng, 0.072 mmiol) was isolated in 28% yield by chromatography on silicagen (230-400 mesh) eluting with heptane: EtOAc: TEA (30:70:1).

'H NI4R (200 Mifz, d 4 -MeOH) 6 (ppm) 7.50-7.10 (in, 5H), 4.62(q 1H), 3.76-3.67 (mn, 1H), 3.65-3.56 (mn, 1H), 3.51 1H), 3.35-2.95 (in, 6H), 2.70 2H), 2.0-0.5 (mn, 181H).

Examl~le 8 HOOC-CO- Cha-P130-Nag HOO0C-CO-(R)Cha-Phe-Nag(Z)

H

2 N-(R)Cha-Phe-Nag(Z) (500 mng, 0.91 mmiol) was dispersed in acetonitrile (5 inL). Methyloxalyichioride (104 pL, 1.14 imol) was added to the slurry. After 60 minutes the starting material was consumed, confirmed by HPLC, and the clear solution was evaporated.

SUBSTITUTE SHEET WO 94/29335 PCT/SE94/00534 27 The crude methylester was hydrolyzed by dissolving the residue in tetrahydrofuran (4 mL) and adding LiOH (115 mg, 2.73 mmol) dissolved in water (2 mL). After 90 min. more LiOH mg, 1.7 mmol) was added and 30 minutes later water mL) was added and the insoluble material were dissolved.

After evaporation the dry uncolored powder was slurried in water (10 mL) containing ammonium chloride (150 mg). The mixture was stirred for 30 min. and then the precipitate was filtered and washed with two portions of water.

1 H NMR (200 MHz, d 6 -DMSO) 6 (ppm) 8.7 1H), 8.2-7.6 7.31-7.22 10H), 4.94 2H), 4.32 1H), 4.00 1H), 3.3-2.6 6H), 1.7-0.6 15H). TSP-MS found 623 (calc. for MH+(C 3 2

H

4 3

N

6 0 7 )623).

(ii) HOOC-CO-(R)Cha-Phe-Nag HOOC-CO-(R)Cha-Phe-Nag(Z) (210 mg, 0.34 mmnol) was dispersed in tetrahydrofuran (25 mL) and acitic acid (20 mL) was added.

Palladium on charcoal 30 mg) was charged before it was hydrogenated at 45 psi hydrogen pressure in a Parr shaking apparatus for 25 h. The suspension was filtered through celite and the filter cake was washed with tetrahydrofuran and after the solvent was evaporated the crude product (257 mg) was collected. After azeotropic evaporation with three portions of toluene (tot; 50 mL) and overnight drying under vacuum the product (140 mg, 0.29 mmol) was isolated in yield.

1 H NMR (200 MHz, D 4 -MeOH) 6 (ppm) 7.19 5H), 4.54 (dd, 1H), 4.00 1H), 4.00 1H), 3.50-2.90 5H), 2.70 1H), 1.90-0.60 15H). TSP-MS found 489 (calc. for

MH+(C

24

H

36 N60 5 )489).

Pharmaceutical preparations A. The compounds according to the invention can be formulated in solid dosage forms for oral administration or for topical administration to the intestines.

SUBSTITUTE SHEET WO 94/29335 PCT/SE94/00534 Example Al Plain tablet Kininogenase inhibitor Lactose anhydrous Microcrystalline cellulose Magnesium stearate 10 mg/tablet 250 mg/tablet 60 mg/tablet 6 mg/tablet The active constituent is mixed with lactose and microcrystalline cellulose and magnesium stearate is admixed and tablets are compressed from the mixture.

Example A2 Coated tablet Kininogenase inhibitor Lactose Polyvinylpyrrolidone Magnesium stearate Hydroxypropylmethylcellulose Polyethyleneglycol Talc Titandioxid

C

4 4 0 mg/tablet 0 mg/tablet 0 mg/tablet 8 mg/tablet 8 mg/tablet 1 mg/tablet 1 mg/tablet 1 mg/tablet The active constituent is mixed with lactose and granulated with polyvinylpyrrolidone in water. After drying and milling magnesium stearate is admixed and tablets are compressed. The tablets are coated with a solution of hydroxypropylmethylcellulose, polyethyleneglycol, talc and titandioxide in water.

Examrlee A3 Gastro-resistant tablet Kininogenase inhibitor Lactose Polyvinylpyrrolidone 10 mg/tablet 200 mg/tablet 40 mg/tablet SUBSTITUTE SHEET WO 94/29335 PCT/SE94/00534 29 Microcrystaliine cellulose 50 mg/tablet Magnesium stearate 8 mg/tablet Eudragit L 10 mg/tablet Dibutylphtalate 1 mg/tablet Talc 2 mg/tablet The active constituent is mixed with lactose and granulated with polyvinylpyrrolidone in water. After drying and milling microcrystalline cellulose and magnesium stearate is admixed and tablets are compressed. The tablets are coated with a solution of Eudragit L, dibutylphtalate and talc in isopropanol/aceton.

Example A4 Gastro-resistant extended release granules for the small intestine Kininogenase inhibitor 100 mg/g Lactose 448 mg/g Microcrystalline cellulose 200 mg/g Hydroxypropyl cellulose 50 mg/g Ethylcellulose 20 mg/g Acetyltributylcitrate 2 mg/g Eudragit L30D 50 mg/g Triethylcitrate 5 mg/g Talc 25 mg/g The active constituent is mixed with lactose and microcrystalline cellulose and granulated with hydroxypropyl cellulose in water. The granulation is extruded, spheronized and dried. The granules are first coated with ethylcellulose dispersion with acetyltributylcitrate and then with Eudragit dispersion with triethylcitrate and talc. The granules are filled in gelatin capsules each containing 10 mg of active constituent.

SUBSTITUTE SHEET WO 94/29335 PCT/SE9400534 Example Gastro-resistant extended release granules for the colon Kininogenase inhibitor 200 mg/g Lactose 400 mg/g Microcrystalline cellulose 200 mg/g Hydroxypropyl cellulose 50 mg/g Eudragit NE30D 50 mg/g Eudragit S100 50 mg/g Talc 50 mg/g The active constituent is mixed with lactose and microcrystalline cellulose and granulated with hydroxypropyl cellulose in water. The granulation is extruded, spheronized and dried. The granules are coated with a dispersion of Eudragit NE30D, Eudragit S100 and talc in water. The granules are filled in gelatin capsules each containing 100 mg of active constituent.

B. The compounds according to the invention can be formulated in pressurized aerosols or in dry powder inhalers for oral or nasal inhalation. The kininogenase inhibitor is micronized to a particle size suitable for inhalation therapy (mass median diameter 4pm).

For pressurized aerosols the micronized substance is suspended in a liquid propellant mixture and filled into a container which is sealed with a metering valve.

Alternatively, the kininogenase inhibitor can be dissolved in the liquid propellant mixture with the aid of ethanol.

The propellants used may by chlorofluorocarbons (CFCs) or hydrofluoroalkanes (HFAs) of different formulae. The most frequent used CFCs are trichloromonofluoromethane (propellant 11) and dichlorodifluoromethane (propellant 12) and dichlorotetrafluoroethane (propellant 114). The most frequent SUBSTITUTE SHEET WO 94/29335 PCT/SE94/00534 31 used HFAs are tetrafluoromethane (propellant 134a) and heptafluoropropane (prope~llant 227).

Low concentrations of surfactant such as sorbitan trioleate, lecithin, oleic acid or other suitable substance may be used to improve the physical stability. Etanol may be used as surfactant or as a medium to increase the solubility of active substance in the propellant mixture.

Example--B1 Kininogenase inhibitor Trichloromonofluoromethale (propellant 11) Dichlorodifluoromethane (propellant 12) Sorbitan trioleate Examp~le B Kininogenase inhibitor Trichloromonofluoromethale (propellant 11) Dich lorodif luoromethane (propellant 12) Oleic acid Examiple B3j Ki~ninogenase inhibitor Trichloromonofluoromethale (propellant 11) Dichlorodifluorometale Ethanol Oleic acid Kininogenase inhibitor Tetraf luoroethane (propellant 134a) per cent (w/w) 84 per cent (w/w) 74.48 0.02 per cent (w/w) 0.2 64.78 0.02 per cent (w/w) 0.4 59.58 SUBSTITUTE SHEET WO 94/29335 PCTSE94/005344 32 Heptafluoropropane (propellant 227) Ethanol Oleic acid 0.02 Example B5 per cent (w/w) Kininogenase inhibitor Heptafluoropropane 93.5 (propellant 227) Ethanol Sorbitan trioleate In a dry powder inhaler the micronized kininogenase inhibitor may be used alone or mixed with a carrier substance such as lactose, mannitol or glucose. Another possibility is to process the micronized powder into spheres which break up during the dosing procedure. This powder or spheronized powder is filled into the drug reservoir in a singledose or multidose inhaler, e.g. the latter being Turbuhaler®. A dosing unit meters the desired dose which is inhaled by the patient.

Example B6 The kininogenase inhibitor is micronized in a jet mill to a particle size suitable for inhalation (mass diameter 4pm).

100 mg of the micronized powder is filled into a powder aultidose inhaler (Turbuhaler®). The inhaler is equipped with a dosing unit which delivers a dose of 1 mg.

Example B7 The kininogenase inhibitor is micronized in a jet mill to a particle size suitable for inhalation (mass diameter 4pm).

150 mg of the micronized powder is filled into a powder multidose inhaler (Turbuhaler®). The inhaler is equipped with a dosing unit which delivers a dose of 0.5 mg.

SUBSTITUTE SHEET WO 94/29335 PCT/SE94/00534 33

ABBREVIATIONS

Ac Acetyl Agm Agmatine Agrn(Z) w -N-benzyloxycarbolyl agmatine Boc tertiary butoxy carbonyl Brine saturated water/Naci solution Bn benzyl Cha (S)-p-cyclohexy1 alanine CME-CDI I-Cyclohexyl-3- (2-morpholinoethyl) carbodiimide inetho-ptoluenesul fonate DCC dicyclohexyl carbodiimide DMF dimethyl formamide Et ethyl EtOAc ethyl acetate HOSU N-hydroxysuccinimide HPLC High Performance Liquid chromatography LiOH Lithium hydroxide Me methyl Nag noragmatine Nag(Z) =w-N-benzyloxycarbonyl-noragnatine Hal -naphthylalanine NM N-methyl morpholine Ph phenyl Phe (S)-phenylalanine Pro (S)-proline Ser (s)-serine TFA trifluoracetic acid THF tetrahydrofuran Z benzyloxycarbonyl Prefixes no so i and t have their usual meanings: normalo iso, sec and tertiary. The stereochemistry for the amino acids is by default if not otherwise stated.

SUBSTITUTE SHEET

Claims

3. A compound according to claim a structural fragment of Formula 1 wherein A 1 represents IIa 0 R2 1",al Fe'-' )P~a
4. A compound according to claim cyclohexyl, cyclopentyl, phenyl, other aryl systems and p is 1. 1 wherein R 3 is substituted phenyl or 2 wherein R 3 is substituted phenyl or A compound according to claim cyclohexyl, cyclopentyl, phenyl, other aryl systems and p is 1. .o a a S* a a* a S *u a a a.a *a a a. sa
6. A compound according to claim 3 wherein R 3 is cyclohexyl, cyclopentyl, phenyl, substituted phenyl or other aryl systems and p is 1.
7. A compound according to any one of the preceding claims wherein R 1 represents PR 1 00C-alkyl-, wherein the alkyl group has 1 to 4 carbon atoms and R 11 is H.
8. A compound according to any one of the preceding claims wherein R 3 is cyclohexyl or substituted phenyi.
9. A compound according to any one of the preceding claims wherein n is 3 or 4.
10. A compound according to claim 9 wherein n is 4.
11. A compound according to any one of the preceding claims wherein p is 1. V '3 WO 94/29335 PCT/SE94/00534 38
12. A compound according to any one of the preceding claims wherein q is 1.
13. A compound according to any one wherein m is 2.
14. A compound according to any one having R-configiiration on the amino position. A compound according to any one having S-configuration on the amino position. of the preceding claims of the preceding acid fragment in of the preceding acid fragment in claUE; the *s X L claims the A 2
16. A serine protease-inhiibitor compound selected from H-(R)Cha-Phe-Agm HOOC-CH 2 -(R)Cha-Phe-Agm H- Cha-Phe-Naq HOOC-CH 2 Cha-Phe-Nag cH 3 -CO-(R)Cha-Phe-Nag CH 3 -CH 2 Cha-Phe-Nag HOOC-CO- cha-Phe-Nag a Ph-either as such, or in--the form of a physiologically cpal s ae-alt and including stereoisomers. 2517hihe erices copries coulingt~ o aun-teOOC-CH 2 Irtcted dipeptide or a'mino acid (WI-Al-OH) when a N- terminally protected amino acid is used a second amino acid is added afterwards using standard methods, to a compound H 2 N- 2 WO 94/29335 PCT/SE94/00534 39 wherein A 1 A 2 and n are as defined in Formula I, W I is an amino protecting group and X is an unprotected or protected guanidino group or a protected amino group, or a group transferable into an amino group, where the amino gronp is subsequently transferred into an unprotected or protected guanidino group, followed by removal of the protecting group(s) or deprotecting of the N-terminal nitrogen followed by alkylation of the N-terminal nitrogen and deprotection by known methods. and if desired forming a physiologically acceptable salt, and in those cases where the reaction results in a mixture of stereoisomers, these are optionally separated by standard chromatographic or re-crystallisation techniques, and if desired a single stereoisomer is isolated. 15 19. A process according to claim 18 which process comprises: 4 «*t a) Method I Coupling of an N-terminally protected dipeptide, prepared by standard peptide coupling, with either a protected- or unprotected aminc guanidine or a straight chain alkylamine 20 carrying a protected or masked amino group at the terminal end of the alky? chain, using standard peptide coupling,shown in the formula 4 W 1 A 1 -A 2 -OH I H 2 N-(CH 2 )n-X WI- A-A 2 -NH(CH 2 n -X wherein A 1 A 2 and n are as defined in Formula I W1 is a N- teminal amino protecting group such as tert-butyloxy carbonyl iH,^b and benzyloxy carbonyl and X is -NH-C(NH)-NH 2 -NH-C(NH)-NH- i f -N(W 2 )-C(NH)-NH-W 2 -NH-C(N 2 )-H-W 2 or -NH-W 2 where W 2 is WO 94/29335 PCT/SE94/00534 an amine protecting group such as tart-butyloxy carbonyl or benzyloxy carbonyl, or X is a masked amino group such as azide, giving the protected peptide, and further depending on the nature of the X- group used: Removal of the protecting group(s) (when X= -NH-C(NH)-NH 2 -N(W 2 )-C(NH)-NH-W 2 -NH- C(NW 2 )-NH-W 2 or -NH-C(NH)-NH-W 2 or a selective deprotection of the Wj- group (e.g when X= -NH-C(NH)-NH-W 2 ,-N(W 2 )-C(NH)-NH- W 2 -NH-C(NW 2 )-NH-W 2 W 2 in this case must be orthogonal to W 1 followed by alkylation of the N-terminal nitrogen and deprotection or a selective deprotection/ unmasking of the terminal alkylamino function NH-W 2 W 2 in this case must be orthogonal to W 1 or X= a masked aminogroup, such as azide) followed by a guanidation reaction, using standard methods, of the free amine and deprotection of the W 1 -group, or 15 b) Method II Coupling of a N-terminally protected amino acid, prepared by standard methods, with either a protected- or unprotected amino guanidine or a straight chain alkylamine carrying a protected or masked amino group at the terminal end of the alkyl chain, using standard peptide coupling, shown in the formula W -A -OH 4 H 2 N-(CH 2 )n-X W 1 -A 2 -NH (CH 2 -X wherein A 2 n, W 1 and X are as defined above followed by deprotection of the W 1 -group and coupling with the N-terminal amino acid, in a protected form, leading to the protected peptide described in Method I, whereafter the synthesis to the final compounds is continued according to Method I.
20. A compound of Formula I according to claim 1, when obtained by the process according to claim 18 or 19. 4 WO 94/29335 41
21. Use of a compound of the formula I PCT/SE94/00534 A 1 -A 2 -NH- (CH 2 )n-NH-C (NH) -NH 2 ft ft 20 according to claim 1 or 20, as a starting material in the synthesis of a serine protease inhibitor, particularly in the synthesis of a kininogenase inhibitor, employing the steps of an appropriate known route of synthesis for the purpose, said compound being employed either as such or in the form of a salt, and as such or having the guanidino group either mono protected at the 6-nitrogen or diprotected at the 6-nitrogens or the Y, 6 -nitrogens.
22. Use according to claim 21, wherein the serine protease inhibitor is a peptidic compound.
23. A pharmaceutical preparation comprising an effective amount of a compound according to any one of claims 1 to 17 or 20, in association with one or more pharmaceutical carriers.
24. Use of a compound according to any one of claims 1 to 17 or 20, in the manufacture of a pharmaceutical preparation, said pharmaceutical preparation suitable for inhibition of serine proteases, particularly kininogenases, in a human or animal organism. A method for obtaining inhibition of serine proteases, particularly kininogenases, in a human or animal organism in need of such inhibition, comprising administering to said human or animal organism, an inhibitory effective amount of a compound according to any one of claims 1 to 17 or 20, or of a pharmaceutical preparation according to claim 23. A WO 94/29335 PCT/SE94/0 0534
26. A compound according to any one of claims 1-18 or or a process according to claim 18 or 19; or a use according to claim 21 or 22; or a pharmaceutical preparation according to claim 23; or a method according to claim 25; in each instance substantially as described' herein. DATED this 3rd day of October 1997 ASTRA AKTIEBOLAG, By its Patent Attorneys, E. F. WELLINGTON CO., ~By: S. 4 0 4 40 0* 0 0 0 0**0 0*0 *000 .4 0* 0 *090 *40004 0 00 00 0 0 0 (Bruce wellingto)- C/BA! 6087 f r INTERNATIONAL SEARCH,1 REPORtT interntionail applicatiott No, PCT/SE 94/00534 A. CLASSIFICATION OF SUBJECT MATTER IPC5: C07K 5/06 C07K( 5/02, A61K 37/64 According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) A61K, C07K Docuimentation searched other than minimum documentation to the extent that such documents are included In the fields searched SE,DK,FI,NO classes as above Electronic data base consulted during the International search (name of data base and, where practicable, search terms used) BIOSIS, MEDLINE, EMBASE, CA, WPI, CLAIMS C. DOCUMENTS CONSIDERED TO BE RELEVANT Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. X BRAZILIAN J MED BIOL RES, Volume 20, 1987, 1,2,4,5, L.A.F. FERREIRA et al, "Kallikrein isolated from 8-10,12-14, commersial crystalline pepsin preparations" 19,20,25 page 511 page 520 A GB, A, 2085444 (RICHTER GEDEON VEGYESZETI GYAR RT), 1-25 28 April 1982 (28.04.82) A WO, Al, 9204371 (FERRING PEPTIDE RESEARCH 1-25 PARTNERSHIP KB), 19 March 1992 (19.03.92) Further documents are listed in the continuation of Box C. 2 See patent familyP annex. Specil categories of cited documents: 1"l later document published after the International flung date or priority WA document defining the general state of the art which Is not considered date and not In conflict with the application but cited to undersand to be of particular relevance the principle or theory underlying the Invention ertier document but published on or after the International filing date IX document of particular relevance: the claimed invention cannot be document which may throw doubts on priority claims) or wich Is considered novel or cannot be considered to involve an inventive cited to establish the publication date of another citation or other step when the document is taken alone special reason (as specified!) 'YI document of particular relevance: the claimed Invention cannot be O0' document refcrring to an oral disclosure, use, exhibition or other considered to Involve an inventive stet when the document is means r-zoblned with one or more other such documtents, such combination 1P. document publied prior to the International filing date but later than being obvious to a person skilled 1st the art the priority data claimed document member of the same patent failly Date of the actual completion of the international search Date of mailing of die international search report 08 -09- 1994 Name and mailing address or the ISA/ Authorized officer Swedish Patent Office Box 5055, S-102 42 STOCKHOLM Eli sabeth Cav'lborg IFacsimile No. 46 8 666 02 86 Telephone No. 46 8 782 25 00 Form PcT/ISA/210 (second sheet) (July 1992) INTERNATIONAL SEARCH REPORT International application No, PCT/SE 94/00534 Box I Observations where certain claims were found unsearchable (Continuation of Item 1 of first sheet) This international search report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons: 1. [71 Claims Nos.: 26 because they relate to subject maner not required to be searched by this Authority, namely: See PCT Rule 39.1(iv): Methods for treatment of the human or animal body by surgery or therapy, as well as diagnostic methods. 2. Claims Nos.: 27 because they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be carried out, specifically: Claim 27, which concerns different kinds of categories in the same claim, is not searchable. See PCT Article 6. 3. Claims Nos.: Sbecause they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a). Box II Observations where unity of Invention is lacking (Continuation of Item 2 of first sheet) This International Searching Authority found multiple inventions in this international application, as follows: 1. As all required additional search fees were timely paid by the applicant, this international search report covers all searchable claims. 2. As all searchable claimscouldbesearchedwithout effort justifying an additionalfee,this Authority didnotinvitepayment of any additional fee. 3. F] As only some of the required additional search fees were timely paid by the applicant, this international search report covers only those claims for which fees were paid, specifically claims Nos.: 4. "l No required additional search fees were timely paid by the applicant. Consequently, this international search report is S restricted to the invention first mentioned in the claims; it is covered by claims Nos.: Remark on Protest The additional search fees were accompanied by the applicant's protest. SNo protest accompanied the p l.yment of additional search fees. I Form PCT/ISA10 (continuation of first sheet (July 1992) 1.NTr1,RNArTIONAL SEARCH REP~ORT Inrormation on patent, family members 02/07/941 International applicallon No. PCT/SE 94/00534 Patent document 1 Publication Patent family Publication cited In search report date member(i) date GB-A- 2085444 28/04/82 AT-B- AU-B- AU-A- BE-A- CA-A- DE-A- FR-A ,B- JP-A- NL-A- SE-B,C- SE-A- SU-A- 384228 535688 6672581 887224 1158641 3108810 2491463 57064653 8100391 452326 8100302 1178322 12/10/87 29/03/84 22/04/82 27/07/81 13/12/83 19/05/82 09/04/82 19/04/82 03/05/82 23/11/87 08/04/82 07/09/85 WO-Al- 9204371 19/03/92 AU-A- 8438791 30/03/92 JP-T- 6501461 17/02/94 Form PCTJISA/210 (patent family ArmCX) (July 1992)