CA2163011A1 - New peptides derivatives - Google Patents

Abstract

A compound of the general formula (I): A1 - A2 -NH-(CH2)n - NH-C(NH)-NH2, wherein n is an integer 2, 3, 4, 5, or 6; preferably 3 or 4; A1 represents a stuctural fragment of formulae (IIa), (IIb), (IIc), (IId) or (IIe); A2 represents a structural fragment (a), as well as processes for the preparation thereof, the use and the pharmaceutical formulations.

Description

WO 94/2933~ PCT/SE94/00~34 NQW Pepti~e d~riv~tives OESCRIPTION

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 ~owel diseaes, and arthritis.

BACKGROUND

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

Kinins (bradykinin, 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-lOoO). 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 SUB~Ill~TESHEFr wo 94,29335 2 1 6 3 0 1 1 PCT/SEg4/00534 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 A2 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.

Lik~wise, esters and amides were reported by Fareed et al. in Ann. N.Y. Acad. 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 SU8~111~JTESHEET

WO9~/29335 ~i t ~ 2 1 6 3 0 1 1 PCT/SE94/00534 of arginine and isothiouronium analogues thereof have been reported in EP-A2-0,293,881.

In WO 92/04371 a series of kalli~rein 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 stereoisomers, are potent inhibitors of serine proteases and especially kallikreins:

Al _ A2 -NH- (CH2) n~ NH-C (NH) -NH2 Formula I

wherein:

n is an integer 2, 3, 4, 5, or 6; preferably 3 or 4;

A1 represents a structural fragment of Formulae IIa, IIb, IIc, IId or IIe;

S U B ~lll ~ TE S H EEFr WO 94129335 ~ ~ r ~ 2 1 6 3 0 1 1 PCT/SE94/00534 'NJl",t (~q $ ~N

(C (~)p ~ o llC
lla llb C~

I~e nd wherein:

p is an integer 0,1 or 2;

m is an integer 1, 2, 3, or 4, preferably 2;

q is an integer 0,1 or 2, preferably 1;

R1 represents H, an alkyl group having 1 to 4 carbon atoms, a hydroxyalkyl group having 2-3 carbon atoms or RllOOC-alkyl-, where the alkyl group has 1 to 4 carbon atoms and Rll is H or an alkyl group having 1 to 4 carbon atoms, or Rl represents Rl200C-l,4-phenyl-CH2-, wherein R12 is H or an alkyl group having 1 to 4 carbon atoms, or R1 represents R13-NH-CO-alkyl-, wherein the alkyl group has 1 to 4 carbon atoms and is possibly substituted alpha to the carbonyl with an alkyl group having 1 to 4 carbon atoms and wherein R13 is H or an alkyl group having 1 to 4 carbon atoms or -CH2COOR12, wherein R12 is as defined above, or SUB~ I l I ~JTE SHEEr WO94t2933s .~ PCTISE94/00534 Rl represents R14So2-, Ph(4-COOR12)-SO2-, Ph(3-COOR12)-S02-, or Ph(2-COoR12)-so2-, wherein R12 is as defined above and R14 is an alkylgroup having 1-4 carbon atoms, or R1 represents Co-R15, wherein R15 is an alkyl group having 1-4 carbon atoms, or R1 represents CO-ORls, wherein R15 is as defined above, or Rl represent CO-(CH2)p-COOR12, wherein R12 and p are as defined above, or R1 represents -CH2PO(OR16)2, wherein R16 is, individually at each occurrence, H, methyl or ethyl;
R2 represents H or an alkyl group having 1 to 4 carbon atoms or R21OOC-alkyl-, wherein the alkyl group has 1 to 4 carbon atoms and is possibly substituted in the position which is alpha to the carbonyl group, and the alpha substituent is a group R22-(CH2)p-, wherein p is as defined above and R22 is methyl, phenyl, OH, COOR2l, and R21 is H or an alkyl group having 1 to 4 carbon atoms;

R3 represents an alkyl group having 1-4 carbon atoms, or R3 represents a cyclohexyl- or cyclopentyl group, or R3 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 OR21, wherein R21 is as defined above or R3 represents a 1-napthyl, 2-naphtyl, 4-pyridyl, 3-pyrrolidyl, or a 3-indolyl group which may or may not be substituted with OR21 wherin R2l is as defined above and with 3s p ~ 1; or R3 represent a cis- or trans-decalin group with p = 1; or SUE~ TE SHEF~

W094/29335 6 2 1 6 3 0 ~ ~ PCT/SEg4/00534 ~

R3 represents Si(Me)3 or CHtR31)2, wherein R31 is a cyclohexyl- or phenyl group;

A2 represents a structural fragment - NH - CH - C -(CH2) p l3 wherein R3 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 carbon-carbon 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 ksllikrein:
Compounds of Formula I having S-configuration on the A2 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-tR)Cha-Phe-Agm HOOC-CH2-(R)Cha-Phe-Agm H-(R)Cha-Phe-Nag SUB~ 111 ~JTE SHE~

~ W094/29335 -. 2 1 6 3 0 1 1 PCT/SE94/00534 HOOC-CH2-(R)Cha-Phe-Nag CH3-CO-(R)-Cha-Phe-Nag CH3-CH2-(R)-Cha-Phe-Nag HOOC-CO-(R)-Cha-Phe-Nag S HOOC-CH2-(R)Phe-Phe-Agm HOOC-CH2-(R)Phe-Cha-Agm HOOC-CH2-(R)Cha-Cha-Agm HOOC-CH2-(R)Phe-Phe-Nag HOOC-CH2-(R)Phe-Cha-Nag HOOC-CH2-(R)Cha-Cha-Nag HOOC-CH2-(R)Cha-~Nal-Agm HOOC-CH2-(R)Cha-~Nal-Agm H-(R)Phe-Cha-Agm H-(R)Phe-Cha-Nag H-(R)Phe-Phe-Agm H-(R)Phe-Phe-Nag CH3-(R)Phe-Phe-Agm CH3-(R)Cha-Phe-Agm CH3-(R)Phe-Cha-Agm HOOC-CH2-(R)Pro-Phe-Agm HOOC-CH2-(R)Pro-Phe-Nag H-(R)Pro-Phe-Agm H-(R)Pro-Phe-Nag CH3-(R)Pro-Phe-Agm CH3-(R)Pro-Phe-Nag Particularly preferred compounds are:

H-(R)Cha-Phe-Agm HOOC-CH2-(R)Cha-Phe-Agm H-(R)Cha-Phe-Nag HOOC-CH2-(R)Cha-Phe-Nag CH3-CO-(R)-Cha-Phe-Nag CH3-CH2-(R)-Cha-Phe-Nag HOOC-CO-(R)-Cha-Phe-Nag SUB~ 11 l ~TE SHE~

W094/29335 8 1 630 1 ¦ PCT/SEg4/00534 ~

The best mode according to the invention known at present is to use the compound according to Example 4 namely.

HOOC-CH2-(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 allergen-induced exudative inflammatory processes in airway mucosa or gut mucosa.

Determinaton of the inhibition constant Ki for ~lasma kallikrein.

Ki 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 37C.

SUB~ JTE 8HEET

~ W094/29335 2 1 6 3 0 1 1 PCTISEg4/00534 Human plasma kallikrein (E.C.3.4.21.34, Chromogenix AB, Molndal, Sweden), 250 ~l 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 ~l of test compound solution in 0.15 mol/l NaCl containing albumin 10 g/l. An additional 10 ~l of water was supplied in this step. Then 40 ~l of kallikrein substrate (S-2302, Chromogenix AB, 1.25, 2.0 or 4.0 mmol/l in water) was added together with another 20 ~l of water, and the absorbance change monitored.

K~ was evaluated from Dixon plots, i.e. diagrams of inhibitor concentration versus 1/ (~A/min), where the data for the different substrate concentrations form straight lines which intercept at x= -K~.

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 50 % 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 SU~5 111 ~ TE SHEEFr W094l29335 ~ ~ 2 1 6 3 0 1 1 PCT/SE94/00534 ~

body weight at peroral administration and 0.001-50 mg/kg body weight at parenteral administration.

P~eparation 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 tWl-A1-A2-OH) or amino acid (Wl-A2-OH), when a N-terminally protected amino acid is used a second amino acidis added afterwards using standard methods, to a compound H2N- (CH2 ) n~X

wherein Al, A2 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 ~ H2N~(CH2)n~X
Wl _ Al_~2--N~ ( C~2 ) n--X

SUB~ ~ JTE SHEEl-~ W094/29335 ~ 2 1 6 3 0 1 1 PCT/SE94/00534 wherein A1, A2 and n are as defined in Formula I , Wl is a N-teminal amino protecting group such as tert-butyloXy carbonyl and benzyloxy carbonyl and X is -NH-C~NH)-NH2, -NH-C(NH)-NH-W2, -N(W2)-C(NH)-NH-W2, -NH-C(NW2)-NH-W2 or -NH-W2, where W2 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)-NH2, -N(W2)-C(NH)-NH-W2, -NH-C(NW2)-NH-W2 or -NH-C(NH)-NH-W2), or a selective deprotection of the Wl-group (e.g when X= -NH-C(NH)-NH-W2,-N(W2)-C(NH)-NH-W2, -NH-C(NW2)-NH-W2, W2 in this case must be orthogonal to Wl) followed by alkylation of the N-terminal nitrogen and deprotection or a selective deprotection/ unmasking of the terminal alkylamino function (X= NH-W2 , W2 in this case must be orthogonal to W1 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 mas~ed amino group at the terminal end of the alkyl chain, using standard peptide coupling, shown in the for~ula ~-A~-O~
~ ~2~ 2)n~X

wherein A2, n, Rl and X are as d~fined above followed by deprotection of the W1-group and coupling with the N-termina3 amino acid, in a protected form, leading to the protected SUB~ 111 ~JTE SHEEl-W094/29335 2 1 6 3 0 1 1 PCT/SE94/00534 ~

peptide described in Method I. The synthesis to the final compounds is then continued according to Method I.

S ~ETAILE~ DESCRIPTION OF THE INVENTION

The following description is illustrative of aspects of the invention.

EXPERIMENTAL PART

GenQral Experime~tal Proce~ures.

The lH NMR and 13C NMR measurements were performed on BRUKER
AC-P 300, BRUKER 200 and BRUKER AM 500 spectrometers, the former operating at a lH frequency of 500.14 MHz and a 13C
freguency of 125.76 MRz and the latter at lH and 13C 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; CDCl3 (isotopic purity > 99.8~, Dr.
Glaser AG Basel), CD30D (isotopic purity > 99.95~, Dr. Glaser AG Basel) or D2O (isotopic purity > 99.98%, Dr. Glaser AG
Basel).

The lH and 13C chemical shift values in CDCl3 and CD30D are relative to tetramethylsilane as an external standard. The 1H
chemical shifts in D2O are relative to the sodium salt of 3-(trimethylsilyl)-d4-propanoic acid and the 13C chemical shifts in D2O are referenced relative to 1,4-dioxane (67.3 ppm), both as external standard. Calibrating with an externa~
standard may in some cases cause minor shift differen~es compared to an internal standard, however, the ~ffferenc~ in lH chemical shift is less than 0.02 pp~ and in ~-~C ~ess than 0.1 ppm.

SUE~:~ 111 UTE SHEET

~ W094l29335 13 2 1630 1 1 PCT/SE94/00534 Thin-Layer Chromatography was carried out on commercial Merck Silicagel 60F254 coated glass or aluminium plates.
- Visualization was by a combination of W-light, followed by spraying with a solution prepared by mixing 372 ml of 5 EtOH(95%), 13.8 ml of concentrated H2S0~, 4.2 ml of concentrated acetic acid and 10.2 ml of p-methoxy benzaldehyde or phosphomolybdic acid reagent (5-10 w.t ~ in EtOH(95~)) and heating.

Flash chromatography was carried out on Merck Silicagel 60 (40-63 mm, 230-400 mesh) under pressure of N2.

Freeze-drying was done on a Leybold-Heraeus, model Lyovac GT

2, apparatus.
~rotection Proc~ures 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)2O
and the mixture was stirred for 4.5 h at room t~mp~rature.
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 KHSO4 and extracted with 3 x 150 ml of EtOAc. The combined organic phase was washed with water, brine and dried lNa2SO4). Evaporation of the solvent afforded 30.9 g ~90.5 ~) of the title compound as a white solid.
Pre~arat~on of 8tartinq Haterials - ~oc-(R)Ch~-08u Boc-(R)Cha-OH (1 eq.), 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 SUE~ I I I ~TE 8HEE~

W094/29335 ~ 2 ~ 6 3 0 1 1 PCT/SE94/00534 ~

formed during the reaction 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 CH3CN, 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, CDCl3, 2 rotamers ca: 1:1 ratio) ~ O.85-1.1 (m, 2H), 1.1-1.48 (m, 4H), 1.5-1.98 (m, 16H; thereof 1.55 (bs, 9H)), 2.82 (bs, 4H), 4.72 (bs, lH, major rotamer)~ 4.85 t~s, lH, minor).

Boc-(R)Cha-Phe-O~

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/H2O (1/1) at + 5 C 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 M KHS04 and extracted with 2 x 100 mL EtOAc.
The combined organic phase was washed with 2 x 50 mL water and dried (MgSO4). Filtration and evaporation of the solvent gave 2.86 g (68%) of the title compound.
Boc-(R)Ch~-Phe-08u To a stirred solution of 2.81 g Boc-(R)Cha-Phe-OH (6.71 mmol) and 850 mg HOSu (7.38 mmol) in 30 mL of CH3CN 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 Na2CO3(aq), 2 x 20 mL
water, 1 x 20 mL brine and dried (MgSO4). Filtration followed by evaporation of the solvent gave 2.44 g (70%) of the title compound which was used without further purification.

SUE~ UTE SHEET

~ W094/29335 ~ 2 t 6 3 0 1 1 PCT/SEg4/00534 Boc-N~g(Z) (i) N-Bensyloxycarbonyl-O-methyl isourea To a stirred solution of concentrated aqueous NaOH (2.8 L, 50% w/w, 19.1 M, 53 mol) and water (32 L) at 18 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 3-5 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 8 C during the addition of Z-C1. The addition funnel was rinsed with 5 litres of water which was added to the reactor. The reaction mixture was stirred at 0-3 C for 18 h, filtered and the crystals was washed with cooled (3 C) water (10 L).
Vacuum drying 25 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-(CH2)3-NH2 x HCl (prepared according to Mattingly P.G., Synthesis, 367 (1990)) (3.9 kg, 18.5 mol) in iso-propanol (24 kg) at 60-70 C was added in portions over a 30 minutes period KHC03 (4.2 kg, 42 mol). A
slow evolution of CO2 (g) occurs. The mixture was stirred for another 30 minutes followed by addition in portions over a 30 minutes period N-bensyloxycarbonyl-O-methYl isourea (3.74 kg, 18.0 mol). The reaction mixture was stirred at 65-70 C for 16 h, cooled to 20 C and filtered. The precipitate was washed with iso-propanol (10 + 5 L). The combined filtrates was concentrated at reduced pressure keeping the heating - mantle not warmer than 65-70 C. When approximately 45 litres was distilled off EtOAc (90 L) was added. The reaction mixture was cooled to 20-25 C, washed with water (10 and 5 L) and brine (5 L), and dried with Na2SO~ (2 kg). After stirring the rection mixture was filtered and the filter cake 8 U B ~ll~ ~ TE S H EEFr W094/29335 2 1 6 3 0 1 ~ PCT/SEg4/00534 ~

was washed with EtOAc (11 and 7 L). The combined filtates were concentrated at reduced pressure keeping the heating mantle not warmer than 40-50 C. When approximately 90 litres of EtOAc was distilled off, toluene (25 L) was added and the S 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 0 C and gently stirred over night (17 h). The crystal slurry was filtered and the product was washed with cooled toluene (10 and 5 L). Vacuum drying (10-20 mbar, 40 C) for 24 h gave 4.83 kg (13.8 mol, 76%) of Boc-Nag(Z).

lH-NMR (300 MHz, CDCl3): ~ 1.41 (s, 9H), 1.6-1.7 (m, 2H), 3.0-3.3 (m, 4H), 4.8-5.0 (bs, lH), 5.10 (s, 2H), 7.2-7.4 (m, SH).
Boc-Agm(Z) (i) Boc-Agm To a slurry of 14.95 g (65.5 mmol, 1 eq.) of agmatine sulphate (Aldrich), 13.7 ml of Et3N (98.25 mmol, 1.5 eq.), 165 ml of H20 and 165 ml of THF was added 21.S g (98.25 mmol, l.S e~.) of (Boc)2O during S 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 (+5C) 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 3s temperature for 4 h methanol (150 ml) was added and the stirring was continued for an additional 20 h at room SU8::.111UTESHEEl' W094/29335 2 ~ 6 3 0 1 1 PCT/SE94/00~34 temperature. The organic solvent was evaporated and 200 ml of H2O was added to the residue. The basic water phase was extracted with lx300 ml and 2x200 ml of EtOAc. The combined organic phases was washed with H2O (2xlOOml), brine (lx100 ml) and dried (MgSO4). Evaporation of the solvent and flash chromathography (CH2Cl2/MeOH, a stepwise gradient of 97t3, 95/5 and 9/1 was used) gave 14.63 g (58%) of pure Boc-Agm(Z) as a white powder.

lH-NMR (CDCl3, 500 MHz): ~ 1.35-1.40 (m, 2H), 1.45 (s, 9H), 1.5-1.6 (m, 2H), 3.0-3.2 (m, 4H), 4.65 (bs, lH), 5.1 (s, 2H), 7.2S-7.40 (m, SH).

13C-NMR (CDCl3, 75.5 MHz): ~ 25.44, 27.36, 28.21, 65.83, 79.15, 127.47, 127.66, 128.14, 137.29, 156.47, 161.48, 163.30.

~-(R)Ch~-P~-Nag~Z) (i) 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, l9S 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 5C. After 3 h the solution was evaporated, the residue dissolved in wateF (1000 mL) and extracted with ethylacetate (2 x 300 mL). The aqueous phase was acidified with potassium hydrogensulfate (lM) to pH 3 and extracted with ethylacetate 8 U ~ ~III ~ TE S H EEFr W094/29335 2 1 6 3 0 ~ 1 PCT/SEg4/00534 ~

(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.
S , (ii) Boc-(R)Cha-Phe-NagtZ) Boc-(R)Cha-Phe-OH (20.8 g, 49.7 mmol) was dissolved in acetonitrile (350 mL). The vessel was cooled and 4-dimethylaminopyridine (12.1 g, 99.4 mmol) was added while maintaining a reaction temperature at 2C. 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 (lM, 250 mL), sodium carbonate (lM, 3 x 250 mL), water t200 mL) and finally brine (200 mL). The collected organic layer was evaporated and the title compound, Boc(R)Cha-PXe-Nag(z) (24.9 g, 38 mmol), was isolated in 77%
yield.

(iii) H2N-(R)Cha-Phe-~ag(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 4C and HCl (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 temperatur. After 1.5 h all starting material was consumed. The solvent was decanted from the HCl-salt of the deprotected peptide and the crude product was dissolved in water (50 mL) 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).

SU~ UTE SHEEr' ~ W094/29335 ,t ~ 2 ~ 6 3 0 ~ I PCT/SEg4/00534 The collected organic phase was evaporated and H2N-(R)Cha-Phe-~ag(Z) (3 g, 5.5 mmol), was isolated after purification by chromatography on silicaqel (230-400 mesh) eluting with CH2C12:MeOH:NH4OH (100:4:1 to 100:15:1) in 36% yield.
lH NMR (200 MHz, CDC13) ~ (ppm) 7.82 (d, lH), 7.4-7.1 (m, lOH), 5.09 (s, 2H), 4.42 (q, lH), 3.4-2.9 (m, 7H), 1.8-0.7 (m, 15H~.

Workin~ Ex~mplQs ExamPle ~-(R)Ch~-Phe-Agm x 2 TFA

(~) Boc-(R)Cha-Phe-Agm(Z) A solution of 729 mg (2 mmol) Boc-Agm(Z) in 15 mL TFA/CH2C12 (1:4) was stirred at room temperature for about 2 h. The solvent was evaporated and the product was dissolved together with 1.03 g (2 mmol) of Boc-(R)Cha-Phe-OSu in 10 mL DMF , the pH was adjusted with NMM to about 9 and the mixture was stirred at room temperature for 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 M ~HSO4, 1 M NaOH, water and dried (MgSO4). Evaporation of the solvent followed by flash chromatography (70 g SiO2) using a stepwise gradient of 100 mL CH2C12/MeOH (95/5) followed by 250 mL CH2C12/MeOH (9/1) gave 1.09 g (96 %) of the title compound.
1H-NMR (500 MHz, CDCl3, mixture of two rotamers): major rotamer: ~ 0.7-0.9 (m, 2H), 1.0-1.8 (m, 25H; thereof 1.39 (s, 9H)), 2.9-3.25 (m, 6 H), 4.02 (m, lH), 4.71 (q, 1 H), 5.05 (s, 2 H), 7.1-7.4 (m, 10H).

S U B ~lII ~ T E S H EE~r W094/2933~ ? 2 1 6 30 ll PCT/SE94/00534 _ 13C-NMR (125 MHz, D2O): car~onyl and guanidine carbons:
161.7, 163.6, 172.0, 172.7 and 174.9.

(ii) H-(R)Cha-Phe-Agm x 2 TFA
A solution of lOO mg (0.15 mmol) Boc-(R)Cha-Phe-Agm(Z) in 10 mL CH2C12/TFA ~4/1) was stirred at room temperature for 2 h 45 min after which the solvent was evaporated. The residue was dissolved in 9 ml EtOH/H2O (8/1) and hydrogenated over 40 mg 5 % Pd/C at athmospheric pressure for 3 h. The catalyst wa~
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.

lH-NMR (500 MHz, D2O, mixture of two rotamers): major rotamer:
0.65-1.7S (m, 17H), 2.86-3.23 (m, 6H), 3.96 (t, lH), 4.59 ~dd, lH), 7.1~-7.4 (m, 5~).

13C-NMR (125 MH2~ D2O): guanidine ~ 157.3; carbonyl carbons: t 171.0 and 173.1.

ExamPle ~OOC-C~2-~R)Cha-Phe-Agm ~ 2 TFA
(i) H-(R)Cha-Phe-Agm~Z) A solution of 0.99 g (1.49 mmol) Boc-(R)Cha-Phe-Agm(Z) in 30 mL CH2C12/TFA (4/1) was stirred at room temperature for 3 h after which the solvent was evaporated and the residue dissolved in 100 mL CH2C12. The organic phase was washed with 1 x 30 mL 5 M NaOH, 2 x 30 mL water and dried (MgS04).
Filtration and evaporation of the solvent gave 825 mg (98 %) of the title compound as a white powder.

SUE~ UTE SHEET

~ WO 94/2933~ 2 1 6 3 0 1 1 PCT/SEg4/00534 H-NMR (300 ~z, CDC13): ~ 0.75-1.0 (Dl, 2H), 1.05-1.75 (m, 15H), 2.93-3.34 (m, 7H), 4.56 (q, lH), 7.13-7.39 (m, 10H).

13C-N~ (75 ~z, CDCL3): carbonyl and guanidine carbons: ô
161.8, 163.8, 171.8 and 176.5.

(ii) BnOOC-CH2-(R)Cha-Phe-Agm(Z) A mixture of 282 mg (0.5 mmol) H-(R)Cha-Phe-Agm(Z), 173 mg (1.25 mmol) K2C03 and 137.5 mg (0.6 mmol) BnOOC-CH2-Br in 16 mL C~I3CN/DMF (15/1) was heated to 50 C 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 (MgSO4). Evaporation of the solvent followed by flash chromatography (37 g SiO2) using CH2C12/MeOH(NH3-saturated) (95/5) as eluent afforded 230 mg (64 %) of the desired compound.

lH-N~ (300 ~z, CDCl3): ~ 0.7-O.9S (m, 2H), 1.05-1.75 (m, 15H), 2.84-3.25 tm, 8H), 4.56-4.68 (m, lH), 4.95 (s, 2H), 5.12 (s, 2H), 7.1-7.45 (m, l5H).

13C-N~ (75 MHZ, CDCL3): car~onyl and guanidine carbons:
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 mmol) BnOOC-CH2-(R)Cha-Phe-Agm(Z) in 18 mL EtOH/H20 (5/1) was added a small amount (15 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.

SUE~ ~TE SHEE~

W094/29335 2 ~ 6 3 0 1 ~ PCTtSEg4/00534 ~

H-NMR (300 MHz, CD30D): ~ 0.65-1.85 (m, 17H), 2.8-3.0 (m, lH), 3.0-3.3 (m, SH), 3.78 (bs, 2H), 4.0 (bs, lH), 4.62 (m, lH), 7.1-7.4 (m, 5H).

13C-NMR (75 MHz, CD30D): guanidine: ~ 158.6; carbonyl carbons:
~ 168.9, 169.6 and 173.4.

ExamPle 3 ~-~R)C~a-Phe-Nag x 2 ~FA

(i) Boc-(R)Cha-Phe-Nag(Z) Prepared in the same way as described for Boc-(R)Cha-Phe-Agm(Z) in Example 1 (i) from Boc-(R)Cha-Phe-OSu (2 mmol) and Boc-Nag(Z) (2 mmol).Yield S 1.02 g (78 %).

(ii) H-(R)Cha-Phe-Nag x 2 TFA

A solution of 100 mg (0.15 mmol) Boc-(R)Cha-Phe-Nag(Z) in 10 mL CH2C12/TFA (4/1) was stirred at room temperature for 3 h 35 min after which the solvent was evaporated. The residue was dissolved in 9 ml EtOH/H20 (8/1) 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 97 mg (98 %) of the title compound as a white powder.

lH-NMR (500 MHz, D20, mixture of two rotamers): major rotamer:
~ 0.75-1.85 (m, 15H), 2.9-3.45 (m, 6H), 4.05 (t, lH), 4.6-4.8 (m, lH; partially hidden by the H-O-D signal), 7.3-7.6 (m, 5H).

13C-MMR (75 MHz, D20): guanidine ~ 157.6; carbonyl carbons:171.3 and 173.5.

SUE~ I I UTE SHEEI' ~ W094/29335 2 1 6 3 0 ~ 1 PCT/SEg4/00534 Example 4 HOOC-CH2-~R)Ch~-Phe-N~g x 2 TFA

(i) H-(R)Cha-Phe-Nag(Z) Prepared in the same way as described for H-(R)Cha-Phe-Agm(Z) in Example 2 (i) from Boc-(R)Cha-Phe-Nag(Z). Yield 90 %.

13C-NMR ( 75 MHz, CDC13): ~ 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, 137.5, 161.8, 163.7, 171.5 and 176.6.

lS (ii) BnOOC-CH2-(R)Cha-Phe-Nag(Z) A mixture of 275 mg (0.5 mmol) H-(R)Cha-Phe-Nag(Z), 173 mg (1.25 mmol) X2CO3 and 137.5 mg (0.6 mmol) BnOOC-CH2-Br in 15 mL CH3CN was heated to 50 C for 3 h and 50 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 (MgSO4). Evaporation of the solvent followed by flash chromatography (37 g Sio2) using CH2Cl2/MeOH(NH3-saturated) (95/S) as eluent afforded 209 mg (60 %) of the desired compound.

1H-NMR (300 MHz, CDCl3): ~ 0.72-0.93 (m, 2H), 1.0-1.72 tm, 13H), 2.83-3.25 (m, 9H), 4.54 (q, lH), 5.09 (s, 2H), 5.11 (s, 2H), 7.05-7.4 (m, lSH), 7.59 (d, lh; NH).

3C-NMR (75 MHz, CDC13): carbonyl and guanidine carbons:
161.8, 163.6, 171.3, 171.6 and 175.2.

(iii) HOOC-CH2-(R)Cha-Phe-Nag x 2 TFA

8UB~ 1 1 1 UTE SHEE~

W094/2933S ~ 2 ~ 6 3 0 1 1 PCT/SE94/00534 To a solution of 209 mg (0.3 mmol) BnOOC-CH2-(R)Cha-Phe-Nag(Z) in 18 mL EtOH/H20 (S/l) was added a small amount (15 drops) of TFA and the mixture was hydrogenated over 70 mg S %
Pd/C at athmospheric pressure for 4 h. The catalyst was S filtered off, the solvent was evaporated and the residue was dissolved in water and freeze dried to afford 190 mg (90%) off the title compound as a white powder.

lH-NMR (300 MHz, CD30D): ~ O.6-1.38 (m, 6H), 1.4-1.9 (m, 9H), 2.9-3.4 (m, 6H), 3.9 (bs, 2H), 4.1 ~bs, lH), 4.7 (m, lH;
partially hidden by the H-O-D signal), 7.1-7.45 (m, 5H).

3C-NMR (75 MHz, CD30D): guanidine: ~ 157.5; carbonyl carbons:
~ 169.3, 169.5 and 173.2.
ExamDle 5 ~-~R)Cha-Phe-N~g H2N-(R)Cha-Phe-Nag(Z) (300 mg, 0.55 mmol) was dissolved in ethanol (50 mL) and trifluoroaceticacid (56 ~L, 0.73 mmol) was added. The mixture was sonicated and palladium on charcoal (5%, 50 mg) 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 ~mol) was isolated in 56% yield.
H NMR (200 MHz, d-HCl+d2-H20) ~ (ppm) 7.40-7.00 (m, 5H), 4.47 (t, lH), 3.86 (t, lH), 3.25-2.65 (m, 6H), 1.7S-O.S0 (m, lSH).
TsP-MS ~ound (m/z)=417 (calc. for MH+(C22H37N602~417).

Example 6 CH3-C0-~R)Cha-Phe-N~g (i) CH3-C0-(R)Cha-Phe-Nag(Z) SUB~ 111 ~JTE SHEET

W094/2933~ 2 ~ 6 3 0 ~1 PCT/SE94/00534 H2N-(R)Cha-Phe-Nag(Z) (500 mg, 0.91 mmol) was dissolved in acetonitrile (7.5 mL). Acetylchloride (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 HCl-salt. Diethylether (5 mL) was added 20 minutes later. The precipitate was filtered off and dried under vacuum at 35C overnight and the dry product, CH3-C0-(R)Cha-Phe-Nag(Z)xHCl (407 mg, 0.69 mmol) was isolated in 76%
yield.
lH NMR (200 MHz, CDC13) ~ (ppm) 7.4-7.1 (m, lOH), 5.1 (q, 2H), 4.7 (m, lH), 4.0 (m, lH), 3.5-2.9 (m, 6H), 2.0-0.6 (m, 18H). TSP-MS found (m/z)=593 (calc. for MH~(C32H45N605)593).

(ii) CH3-C0-(R)Cha-Phe-Nag CH3-C0-(R)Cha-Phe-Nag(Z) (400 mg, 0.68 mmol) was dissolved in ethanol (60 mL) and palladium on charcoal (5%, 80 mg) 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 mg) was collected. The crude product (150 mg) was purified by reveresed phase chromatography (C8-gel) eluting with MeCN:NH40Ac (O.lM) (40:60) and the product (100 mg, 0.22 mmol) in 64% yield.
lH NMR (200 MHz, d4-CH30H) ~ (ppm) 7.25-6.85 (m, 5H), 4.44 (dd, lH), 4-02 (t, lH), 3.30-2.90 (m, 5H), 2.72 (dd, lH), 2.0-0.5 (m, 18H). TSP-MS found (m/z)459 (calc. for MH (C24H39N6O3)459)-Example 7 C~3cH2-~R)ch~-ph~-N~g (i) CH3CH2-(R)Cha-Phe-Nag(Z) H2N-(R)Cha-Phe-Nag(Z) (500 mg, 0.91 mmol), p-toluenesulphonic acid (173 mg, o.9l mmol) and methanol (7.5 mL) were added to 8UE~ JTE SHE~

W094/2933~ ~ 2 1 6 3 01 ~ PCT/SE94/00534 a reaction vessel which was cooled with ice. Acetaldehyde (51 ~L, 0.91 mmol) was added and finally after another 30 min., sodium cyanoborohydride (86 mg, 1.36 mmol) 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 Me~12: MeOH: NH40H (90:10:1) yielding CH3CH2-(R)Cha-Phe-Nag(Z) (lS0 mg, 0.26 mmol) in 29% yield.
lH NMR (200 MHz, d4-MeOH) ~ (ppm) 7.39-7.28 (m, 10H), 5.11 (s, 2H), 4.63 (t, lH, 3.2-2.9 (m), 2.42 (m, 2H), 1.8-0.8 (m, 18H).

(ii) CH3CH2-(R)Cha-Phe-Nag(Z) 150 mg, 0.26 mmol) was dissolved in EtOH 840 mL) and acetic acid (1 mL). Palladium on charcoal (5%, 51 mg) 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 washed with MeOH/AcOH (2:1, 40 mL). The title compound (32 mg, 0.072 mmol) was isolated in 28% yield by chromatography on silicagen (230-400 mesh) eluting with heptane: EtOAc: TEA
(30:70:1)-H NMR (200 MHz, d4-MeOH) ~ (ppm) 7.50-7.10 (m, 5H), 4.62 (q, lH), 3.76-3.67 (m, lH), 3.65-3.56 (m, lH), 3.51 (t, lH), 3.35-2.95 (m, 6H), 2.70 (q, 2H), 2.0-0.5 (m, 18H).
~xam~7e 8 HOOC-CO-~R)Cha-Phe-Nag (i) HOOC-CO-(R)Cha-Phe-Nag(Z) H2N-(R)Cha-Phe-Nag(Z) (500 mg, 0.91 mmol) was dispersed in acetonitrile (5 mL). Methyloxalylchloride (104 ~L, 1.14 mmol) was added to the slurry. After 60 minutes the starting material was consumed, confirmed by HPLC, and the clear solution was evaporated.

SUB~ ~ TE SHE~

~ W094/29335 ~s 2 ~ 6 3 0 1 1 PCT/SEg4/00534 The crude methylester was hydrolyzed by dissolving the residue in tetrahydrofuran (4 mL) and adding LioH (115 mg, 2.73 mmol) d$ssolved in water (2 mL). After 90 min. more LioH
(70 mg, 1.7 mmol) was added and 30 minutes later water (10 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.
lH NMR (200 MHz, d6-DMSO) ~ (ppm) 8.7 (d, lH), 8.2-7.6 (m), 7.31-7.22 (m, 10H), 4.94 (s, 2H), 4.32 (m, lH), 4.00 (m, lH), 3.3-2.6 (m, 6H), 1.7-0.6 (m, 15H). TSP-MS found (m/z) 623 (calc. for MH (C32H43N607)623)-(ii) HOOC-CO-(R)Cha-Phe-Nag HOOC-CO-(R)Cha-Phe-Nag(Z) (210 mg, 0.34 mmol) was dispersed in tetrahydrofuran (25 mL) and acitic acid (20 mL) was added.
Palladium on charcoal (5%, 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 85%
yield.
1~ NMR (200 MHz, D~-MeOH) ~ (ppm) 7.19 (m, SH), 4.54 (dd, lH), 4.00 (t, lH), 4.00 (t, lH), 3.50-2.90 (m, 5H), 2.70 (t, lH), 1.90-0.60 (m, 15H). TSP-NS found (m/z) 489 (calc. for MH~(C24H36N6O5)489)-Ph~m~c~utical preparat~ons 3s A. The compounds according to the invention can be formulated in solid dosage forms for oral administration or for topical administration to the intestines.

8 U 8 ~lII ~ T E S H EEFr -ExamPle A1 Plain t~blet ~ininogenase inhibitor10 mg/tablet 5 Lactose anhydrous250 mg/tablet Microcrystalline cellulose 60 mg/tablet Magnesium stearate 6 mg/tablet The active constituent is mixed with lactose and micro-crystalline cellulose and magnesium stearate is admixed and tablets are compressed from the mixture.

Example A2 Coated tablet Kininogenase inhibitor100 mg/tablet Lactose 300 mg/tablet Polyvinylpyrrolidone40 mg/tablet ~agnesium stearate8 mg/tablet Hydroxypropylmethylcellulose 8 mg/tablet Polyethyleneglycol 1 mg/tablet Talc 1 mg/tablet Titandioxid 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.

Example A3 Gastro-resist~nt tAblet 3s Kininogenase inhibitor 10 mg/tablet Lactose 200 mg/tablet Polyvinylpyrrolidone 40 mg/tablet SUE~.JTE SHEEr W094/2933~ ~ i 2 1 6 3 0 ~ 1 PCT/SE94/00534 Microcrystalline cellulose 50 mg/tablet Magnesiumistearate 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.

~ample A4 G~stro-resistant exten~ed release granulas for the small ~ntestine Kininogenase inhibitor 100 mg/g Lactose 448 mg/g Microcrystalline cellulose 200 mg/g Hydroxypropyl celluloseSO mg/g Ethylcellulose 20 mg/g Acetyltributylcitrate 2 mg/g Eudragit L30D 50 mg/g 25 Triethylcitrate 5 mg/g Talc 25 mg/g The active constituent is mixed with lactose and micro-crystalline 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 L3OD dispersion with triethylcitrate and talc. The granules are filled in gelatin capsules each containing 10 mg of active constituent.

SUE~ I UTE 8HEI~

W094/29335 2 t 6 3 0 1 I PCT/SE94/00534 ~

F~ample A5 G~stro-r~3~st~nt extended release granule~ for th~ colon Kininogenase inhibitor 200 mg/g 5 Lactose 400 mg/g J
Microcrystalline cellulose 200 mg/g Hydroxypropyl cellulose 50 mg/g Eudragit NE30D 50 mg/g Eudragit S100 50 mg/g 10 Talc 50 mg/g The active constituent is mixed with lactose and micro-crystalline cellulose and granulated with hydroxypropyl cellulose in water. The granulation is extruded, spheronized 15 and dried. The granules are coated with a dispersion of Eudragit NE30D, Eudragit S100 and talc in water. The granules sre filled in gelatin capsules each containing 100 mg of active constituent.

20 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 < 4~m).
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 30 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 35 11) and dichlorodifluoromethane (propellant 12) and dichlorotetrafluoroethane (propellant 114). The most frequent SUB~ 111 ~JTE SHEE'r 21630~ 1 W094/29335 PCTISE94/00~34 used HFAs are tetrafluoromethane (propellant 134a) and heptafluoropropane tpropellant 227).

Low concentrations of surfactant such as sorbitan trioleate, ` 5 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.

10 Example B1 per cent (w/w) Kininogenase inhibitor 0.5 Trichloromonofluoromethane 15 (propellant 11) Dichlorodifluoromethane 84 15 (propellant 12) Sorbitan trioleate 0.5 Example B2 per cent (w/w) Kininogenase inhibitor 0.5 20 Trichloromonofluoromethane 25 (propellant 11) Dichlorodifluoromethane 74.48 (propellant 12) Oleic acid 0.02 Example B3 per cent (w/w) Kininogenase inhibitor 0.2 Trichloromonofluoromethane 15 (propellant 11) 30 Dichlorodifluorometane 64.78 - Ethanol 20 Oleic acid 0.02 Example B4 per cent (w/w) 35 Kininogenase inhibitor 0.4 Tetrafluoroethane 59.58 (propellant 134a) 8 U B ~ll~ ~ T E SH EE--Heptafluoropropane 20 (propellant 227) Ethanol 20 Oleic acid 0.02 ExamPle B5 per cent (w/w) Kininogenase inhibitor l.O
Heptafluoropropane 93.5 (propellant 227) lO Ethanol 5 Sorbitan trioleate 0.5 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 TurbuhalerG. 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 < 4~m).
lO0 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 1 mg.

Exam~le B7 The kininogenase inhibitor is micronized in a jet mill to a particle size suitable for inhalation (mass diameter < 4~m).
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.

SUE~ 1 a 1 ~.)TE SHE~

~ W094/29335 ~ ` 2 ~ 630 1 1 PCT/S~94/00~34 ABBREVIATIONS
Ac = Acetyl Agm = Agmatine Aqm(Z) = ~-N-benzyloxycarbonyl agmatine Boc = tertiary butoxy carbonyl Brine = saturated water/NaCl solution Bn = benzyl Cha = (S)-~-cyclohexyl alanine CME-CDI z 1-Cyclohexyl-3-(2-morpholino-ethyl)carbodiimide metho-p-toluenesulfonate DCC = dicyclohexyl carbodiimide DMF - dimethyl formamide Et = ethyl lS EtOAc = ethyl acetate HOSu ~ N-hydroxysuccinimide HPLC = High Performance Liquid Chromatography LioH = Lithium hydroxide Me - methyl Nag 5 noragmatine Nag(Z) = ~-N-benzyloxycarbonyl-noragmatine Nal (S)-naphthylalanine NMM - N-methyl morpholine Ph = phenyl 2S Phe = (S)-phenylalanine Pro = (S)-proline Ser = (S)-serine TFA = trifluoracetic acid THF = tetrahydrofuran Z = benzyloxycarbonyl Prefixes n, s, i and t have their usual reAnings: normal, iso, sec and tertiary. The stereochemistry for the amino acids is by default (S) if not otherwise stated.

S U B~ ~ T E SH EEFr

Claims (27)

1. A compound of the general formula A1-A2-NH-(CH2)n-NH-C(NH)-NH2 Formula I

wherein:

n is an integer 2, 3, 4, 5, or 6; preferably 3 or 4;

A1 represents a structural fragment of Formulae IIa, IIb, IIc, IId or IIe;

IIa IIb IIc nd Ib wherein:

p is an integer 0,1 or 2;

m is an integer 1, 2, 3, or 4, preferably 2;

q is an integer 0-2, preferably 1;

R1 represents H, an alkyl group having 1 to 4 carbon atoms, a hydroxyalkyl group having 2-3 carbon atoms or R11OOC-alkyl-, where the alkyl group has 1 to 4 carbon atoms and R11 is H or an alkyl group having 1 to 4 carbon atoms, or R1 represents R12OOC-1,4-phenyl-CH2-, wherein R12 is H or an alkyl group having 1 to 4 carbon atoms, or R1 represents R13-NH-CO-alkyl-, wherein the alkyl group has 1 to 4 carbon atoms and is possibly substituted alpha to the carbonyl with an alkyl group having 1 to 4 carbon atoms and where R13 is H or an alkyl group having 1 to 4 carbon atoms or -CH2COOR12, wherein R12 is as defined above, or R1 represents R14SO2-, Ph(4-COOR12)-SO2-, Ph(3-COOR12)-SO2-, or Ph(2-COOR12)-SO2-, wherein R12 is as defined above and R14 is an alkyl group having 1-4 carbon atoms, or R1 represents CO-R15, wherein R15 is an alkyl group having 1-4 carbon atoms, or R1 represents CO-OR15, wherein R15 is as defined above, or R1 represent CO-(CH2)p-COOR12, wherein R12 and p are as defined above, or R1 represents -CH2PO(OR16)2, wherein R16 is, individually at each occurrence, H, methyl or ethyl;

R2 represents H or an alkyl group having 1 to 4 carbon atoms or R21OOC-alkyl-, wherein the alkyl group has 1 to 4 carbon atoms and is possibly substituted in the position which is alpha to the carbonyl group, and the alpha substituent is a group R22-(CH2)p-, wherein p is as defined above and R22 is methyl, phenyl, OH, COOR21, and R21 is H or an alkyl group having 1 to 4 carbon atoms;

R3 represents an alkyl group having 1-4 carbon atoms, or R3 represents a cyclohexyl- or cyclopentyl group, or R3 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 OR21, or R3 represents a 1-napthyl, 2-naphtyl, 4-pyridyl, 3-pyrrolidyl, or a 3-indolyl group which may or may not be substituted with OR21 and with p = 1; or R3 represent a cis- or trans-decalin group with p = 1; or R3 represents Si(Me)3 or CH(R31)2, wherein R31 is a cyclohexyl- or phenyl group;

A2 represents a structural fragment wherein R3 and p are as defined above.

2. A compound according to claim 1 wherein A1 represents IIa or IIb.

3. A compound according to claim 1 wherein A1 represents IIa.

4. A compound according to claim 1 wherein R3 is cyclohexyl, cyclopentyl, phenyl, substituted phenyl or other aryl systems and p i 1.

5. A compound according to claim 2 wherein R3 is cyclohexyl, cyclopentyl, phenyl, substituted phenyl or other aryl systems and p is 1.

6. A compound according to claim 3 wherein R3 is cyclohexyl, cyclopentyl, phenyl, substituted phenyl or other aryl systems and p is 1.

7. A compound according to one or more of the preceding claims wherein R1 represents R11OOC-alkyl-, wherein the alkyl group has 1 to 4 carbon atoms and R11 is H.

8. A compound according to one or more of the preceding claims wherein R3 is cyclohexyl or substituted phenyl.

9. A compound according to one or more of the preceding claims wherein n is 3 or 4.

10. A compound according to one or more of the preceding claims wherein n is 4.

11. A compound according to one or more of the preceding claims wherein p is 1.

12. A compound according to one or more of the preceding claims wherein q is 1.

13. A compound according to one or more of the preceding claims having R-configuration on the amino acid fragment in the A1 position.

14. A compound according to one or more of the preceding claims having S-konfiguration on the amino acid fragment in the A2 position.

15. A compound selected from H-(R)Cha-Phe-Agm HOOC-CH2-(R)Cha-Phe-Agm H-(R)Cha-Phe-Nag HOOC-CH2-(R)Cha-Phe-Nag CH3-CO-(R)Cha-Phe-Nag CH3-CH2-(R)Cha-Phe-Nag HOOC-CO-(R)Cha-Phe-Nag either as such or in the form of a physiologically acceptable salt and including stereoisomers.

16. The compound HOOC-CH2-(R)Cha-Phe-Nag, either as such or in the form of a physiologically acceptable salt and including stereoisomers.

17. A process for preparing a compound according to claim 1, which process comprises coupling of a N-terminally protected dipeptide (W1-A1-A2-OH) or amino acid (W1-A1-OH) when a N-terminally protected amino acid is used a second amino acid is added afterwards using standard methods, to a compound H2N- (CH2) n-X
wherein A1, A2 and n are as defined in Formula I, W1 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 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.

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.

18. A process according to claim 17 which process comprises a) 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 wherein A1, A2 and n are as defined in Formula I , W1 is a N-teminal amino protecting group such as tert-butyloxy carbonyl and benzyloxy carbonyl and X is -NH-C(NH)-NH2, -NH-C(NH)-NH-W2, -N(W2)-C(NH)-NH-W2, -NH-C(NW2)-NH-W2 or -NH-W2, where W2 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, and further depending on the nature of the X- group used: Removal of the protecting group(s) (when X= -NH-C(NH)-NH2, -N(W2)-C(NH)-NH-W2, -NH-C(NW2)-NH-W2 or -NH-C(NH)-NH-W2), or a selective deprotection of the W1- group (e.g when X= -NH-C(NH)-NH-W2,-N(W2)-C(NH)-NH-W2, -NH-C(NW2)-NH-W2, W2 in this case must be orthogonal to W1) followed by alkylation of the N-terminal nitrogen and deprotection or a selective deprotection/ unmasking of the terminal alkylamino function (X= NH-W2, W2 in this case must be orthogonal to W1 or X= a masked aminogroup, such as azide) followed by a guanidation reaction, using standard methods, of the free amine and deprotection of the W1-group, or 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 wherein A2, n, W1 and X are as defined above followed by deprotection of the Wl-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.

19. Use of a compound of the formula I

according to claim 1, either as such or in the form of a salt, and as such or having the guanidino group either mono protected at the .delta.-nitrogen or diprotected at the .delta.-nitrogens or the .gamma., .delta.-nitrogens, as a starting material in synthesis of a serine protease inhibitor, and in particular in synthesis of a kininogenase inhibitor.

20. Use according to claim 19, wherein the serine protease inhibitor is a peptidic compound.

21. A compound according to claim 1 for use in therapy.

22. A compound according to claim 21 for use as an antiinflammatory agent.

23. A pharmaceutical preparation comprising an effective amount of a compound according to claims 1 in conjunction with one or more pharmaceutical carriers.

24. A pharmaceutical preparation according to claim 23 for use as an antiinflammatory agent.

25. Use of compound according to claim 1 as an active ingredient for manufacture of a pharmaceutical preparation for inhibition of serine proteases and in particular kininogenases in a human or animal organism.

26. A method for obtaining inhibition of serine proteases and in particular kininogenases in a human or animal organism in need of such inhibition, comprising administering to said organism an inhibitory effective amount of a compound according to claim 1.

27. A compound, a process, a pharmaceutical preparation, a use and a method as claimed in any of claims 1-26 and substantially as described.