HK1072265B - Selective dipeptide inhibitors of kallikrein - Google Patents

Description

The present invention relates to a series of novel compounds that are selective inhibitors of the enzyme plasma kallikrein, to pharmaceutical compositions comprising these inhibitors, and the use of such compositions in the treatment of human diseases.

BACKGROUND

The enzyme plasma kallikrein, also known by the classification EC.3.4.21.34, is a member of a family of trypsin-like serine protease that also includes tissue kallikrein, thrombin, trypsin and plasmin. It is found in plasma as an inactive zymogen that is activated by Factor XIIa. The enzyme has a broad spectrum of activity. Plasma kallikrein liberates the vasoactive peptide bradykinin from high molecular weight kininogen by cleavage of Lys-Arg and Arg-Ser bonds. The same peptide can also be liberated from low molecular weight kininogen in the presence of neutrophil elastase. It is also capable of activating prourokinase and plasminogen, and is also thought to participate in the conversion of prorenin to renin. Plasma kallikrein is an essential component of the intrinsic blood coagulation cascade although its role does not involve the release of bradykinin or enzymatic cleavage. High molecular weight kininogen, the preferred substrate for plasma kallikrein, is essential for the activation in this cascade (K. D. Bhoola et al., Pharm. Rev., 1992, 44, 1-80).

The physiological effects of plasma kallikrein are likely to result from the proteolytic cleavage of kininogens to liberate kinins or of other substrates, e.g. precursors of growth factors. Kinins such as bradykinin are potent mediators of inflammation. In addition they influence cellular functions such as blood pressure, local blood flow, glucose transport and cell proliferation. These cellular actions which are modified by release of secondary messengers such as platelet activating factor, leukotrienes, prostaglandins, Substance P, acetylcholine and noradrenaline.

Several groups have disclosed synthetic inhibitors of plasma kallikrein. These include arginine ketomethylene derivatives (WO 92/04371 and D. M. Evans et al., Immunopharmacology, 1996, 32, 115-116), noragmatine and agmatine derivatives (WO 95/07291, WO 94/29335), benzamidine derivatives (J. Stürzbecher et al., Brazilian J. Med. Biol. Res. 1994, 27, 1929-1934), boronic acid derivatives (US 5,187,157) and aminomethylcyclohexanoyl derivatives (N. Teno et al., Chem. Pharm. Bull., 1993, 41, 1079-1090). The aminomethylcyclohexanoyl derivatives have been shown to be active in models of collagen-induced arthritis in mice (Y. Fujimora et al., Agents Actions, 1993, 39, 42-48) and endotoxin-induced disseminated intravascular coagulation (DIC) in rats (S. Okamoto et al., Agents Actions (Supplement), 1992, 38(Part 1), 198-205). The boronic acid derivatives are active in models of inflammatory bowel disease (A. Stadnicki et al., Digestive Diseases and Sciences, 1996, 41, 912-920 and FASEB, 1998, 12, 325-333).

Selectivity with respect to the other members of the trypsin-like serine protease family is an important issue. Inhibitors of tissue kallikrein displaying poor plasma kallikrein activity have previously been reported (M. Szelke et al., Brazilian J. Med. Biol. Res. 1994, 27, 1935 and D. M. Evans et al., Immunopharmacology, 1996, 32, 117), but there remains a need for compounds that selectively inhibit plasma kallikrein and not tissue kallikrein.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to a series of acylaminopiperidine-1-carboxamidines that are inhibitors of plasma kallikrein. These compounds demonstrate good selectivity for plasma kallikrein, and are potentially useful in the treatment of inflammatory bowel disease, arthritis, inflammation, septic shock, hypotension, cancer, adult respiratory distress syndrome, disseminated intravascular coagulation, cardiopulmonary bypass surgery and bleeding from post-operative surgery. The invention further relates to pharmaceutical compositions of the inhibitors, to the use of the compositions as therapeutic agents, and to methods of treatment using the compositions.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention comprises a series of novel 4-(dipeptidylamino)-piperidine-1-carboxamidines according to general formula 1.

In general formula 1, R¹ represents a group selected from a hydrogen atom (H), a lower alkyl group, a group according to R⁴-CO, a group according to R⁴-O₂CCH₂, a group according to R⁵-OCO, and a group according to R⁵-SO₂. R² represents a group selected from a lower alkyl group, a cycloalkyl or (C₅-C₁₂)cycloalkylalkyl group, either of which may optionally be substituted with an alkyl or alkoxy group, an aralkyl group which may optionally be substituted with up to three groups chosen from F, Cl, Br, I, OH, lower alkyl, O-(lower alkyl), O-benzyl, NH₂, NO₂, NH-acyl, CN and CF₃, and an aralkyloxymethyl group which may optionally be substituted with up to three groups chosen from F, Cl, Br, OH, lower alkyl and O-(lower alkyl). Alternatively, R¹ and R² together may constitute an ortho-xylylene group (o-C₆H₄(CH₂)₂). The aromatic ring of this xylylene group may optionally be substituted with a group selected from F, Cl, Br, OH, lower alkyl and O-(lower alkyl).

R³ represents a group selected from H, OH and O-(lower alkyl).

R⁴ represents a group selected from H, lower alkyl and phenyl.

R⁵ represents a group selected from lower alkyl, phenyl and benzyl.

In the context of the present disclosure, the terms "alkyl group" and "lower alkyl group" are used interchangeably to denote linear and branched saturated hydrocarbon groups with between 1 and 8 carbon atoms, such as methyl, ethyl, isopropyl, tert-butyl, neopentyl and isooctyl groups.

The term "cycloalkyl group" is used to denote monocyclic or polycyclic saturated hydrocarbon groups with between 3 and 12 carbon atoms, such as cyclopropyl, cyclohexyl, bicyclo[4.4.0]decyl (i.e. decahydronaphthyl) and adamantyl groups.

The term "cycloakylalkyl group" is used to denote alkyl groups that bear a cycloalkyl group as a substituent, such as cyclohexylmethyl and 1-(cyclopentyl)ethyl groups. Where a limit is specified, as in (C_a-C_b)cycloalkylalkyl, this denotes that the cycloalkyl moiety has between a and b carbon atoms.

The term "alkoxy group" is used to denote O-(alkyl) groups.

The term "acyl group" is used to denote formyl (H-CO) and alkyl-CO groups.

The term "aralkyl group" is used to denote alkyl groups that bear an aryl group as a substituent, such as benzyl and 1-naphthylmethyl groups. The term "aryl group" includes phenyl, naphthyl, furyl, thienyl, pyrrolyl and pyridyl groups.

The term "aralkyloxymethyl group" is used to denote aralkyl-OCH₂ groups.

The compounds of the present invention all have a guanidine functional group and so can form addition salts with acids. To the extent that such acids are pharmaceutically acceptable then these salts fall within the scope of the invention. Examples of suitable acids include acetic acid, trifluoroacetic acid, fumaric acid, malic acid, citric acid, benzoic acid, benzenesulphonic acid, hydrochloric acid, sulphuric acid and phosphoric acid. Certain compounds within the invention have an acidic functional group and so can form salts with alkaline and alkaline earth metals. Again, insofar as these are pharmaceutically acceptable they are included in the scope of the invention. Examples of such salts include the sodium, potassium and calcium salts.

The compounds of the present invention all have at least two stereogenic centres (asymmetric carbon atoms) and so can exist as optical isomers, such as enantiomers, diastereomers and epimers. All such isomers are included in the scope of the present invention. Mixtures of such isomers, including (but not limited to) racemic mixtures are also included in the scope of the invention.

In a preferred embodiment, the present invention comprises compounds according to general formula 1 in which R¹ is selected from H, lower alkyl and R⁴-O₂CCH₂.

In another preferred embodiment, the present invention comprises compounds according to general formula 1 in which R² is selected from (C₆-C₁₀)cycloalkylalkyl, benzyl optionally substituted with up to three groups chosen from F, Cl, Br, OH, lower alkyl and O-(lower alkyl), phenethyl optionally substituted with up to three groups chosen from F, Cl, Br, OH, lower alkyl and O-(lower alkyl), and benzyloxymethyl optionally substituted with up to three groups chosen from F, Cl, Br, OH, lower alkyl and O-(lower alkyl). More preferably R² is selected from cyclohexylmethyl, decahydronaphth-2-ylmethyl, benzyl, 4-fluorobenzyl, 4-chlorobenzyl, 4-hydroxybenzyl, 4-(lower alkyl)oxybenzyl, α-hydroxybenzyl, α-methoxybenzyl, phenethyl and benzyloxymethyl.

In another preferred embodiment, the present invention comprises compounds according to general formula 1 in which the absolute stereochemistry is as depicted in general formula 1A. More preferably, the absolute stereochemistry is as depicted in general formula 1B.

In another preferred embodiment, the present invention comprises a compound selected from:

• (2'S,2"R)-4-(2'-(2"-amino-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine;
• (2'S,2"R)-4-(2'-(2"-carboxymethylamino-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine;
• (2'S,2"R)-4-(2'-(3"-(4"'-ethoxyphenyl)-2"-(methyloxycarbonylmethylamino)-propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine;
• (2'S,2"R)-4-(2'-(2"-amino-3"-cyclohexylpropanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine;
• (2'S,2"R)-4-(2'-(2"-carboxymethylamino-3"-cyclohexylpropanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine;
• (2'S,2"R)-4-(2'-(3"-cyclohexyl-2"-(methyloxycarbonylmethylamino)propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine;
• (2'S,2"R)-4-(2'-(2"-amino-3"-phenylpropanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine;
• (2'S,2"R)-4-(2'-(2"-carboxymethylamino-3"-phenylpropanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine;
• (2'S,2"R)-4-(2'-(2"-(methyloxycarbonylmethylamino)-3"-phenylpropanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine;
• (2'S,2"R)-4-(2'-(2"-amino-3"-decahydronaphth-2"'-ylpropanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine;
• (2'S,2"R)-4-(2'-(2"-carboxymethylamino-3"-decahydronaphth-2"'-ylpropanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine;
• (2'S,2"R)-4-(2'-(3"-decahydronaphth-2"'-yl-2"-(methyloxycarbonylmethylamino)propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine;
• (2'S,2"R,3'R)-4-(2'-(2"-amino-3"-cyclohexylpropanoylamino)-3'-hydroxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine;
• (2'S,2"R,3'R)-4-(2'-(2"-carboxymethylamino-3"-cyclohexylpropanoylamino)-3'-hydroxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine;
• (2'S,2"R,3'R)-4-(2'-(3"-cyclohexyl-2"-(methyloxycarbonylmethylamino)propanoylamino)-3'-hydroxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine;
• (2'S,2"R,3'R)-4-(2'-(2"-amino-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-methoxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine;
• (2'S,2"R,3'R)-4-(2'-(2"-carboxymethylamino-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-methoxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine; and
• (2'S,2"R,3'R)-4-(2'-(3"-(4"'-ethoxyphenyl)-2"-(methyloxycarbonylmethylamino)-propanoylamino)-3'-methoxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine.

The compounds of the present invention may be prepared by the methods generally known in the art, and particularly those methods used in the field of peptide chemistry. A useful starting material is 4-amino-1-benzylpiperidine (2). Protection of the primary amine with a tert-butyloxycarbonyl (Boc) group to give 3 and hydrogenolysis provides piperidine derivative 4. This can be treated with isothiourea derivative 5 to give the carboxamidinopiperidine derivative 6 in which the amine and guanidine functional groups are differentially protected.

The carboxamidinopiperidine derivative 6 can then be selectively deprotected and coupled to an N-protected amino acid 7 to give intermediate 8.

The elaboration of intermediate 8 to give the final product depends to some extent on the nature of R¹. When R¹ is R⁵-OCO then the synthesis may conveniently proceed via intermediate 9.

When R¹ is H, R⁴-CO, R⁴-O₂CCH₂ or R⁵-SO₂ then the synthesis may conveniently proceed via intermediates 10 and 11.

Deprotection of the guanidine functional group gives compounds according to general formula 1 in which R¹ is H. Derivatisation of the primary amine prior to deprotection of the guanidine gives access to other embodiments of R¹.

Intermediates 12, 13 and 14 may then be deprotected to give the corresponding compounds according to general formula 1.

When R¹ is alkyl, or when R¹ and R² together form a xylylene group, the synthesis may conveniently proceed via intermediate 15.

Two deprotection steps then give the corresponding compounds according to general formula 1.

The compounds of the present invention are potent and selective inhibitors of plasma kallikrein. They are therefore useful in the preparation of medicaments for treatment of disease conditions for which over activity of plasma kallikrein is a causative factor. Generally, for use in such treatment the compounds will be formulated for administration to the patient. The pharmaceutical formulation may be a solid or liquid, such as a tablet, capsule, solution or suspension. Methods of preparing such formulations are well known in the pharmaceutical art.

The compositions will be administered to the patient under the supervision of the attending physician.

EXAMPLES

The following abbreviations have been used:

AcOH

acetic acid

Boc-DCha-OH

N-(tert-butyloxycarbonyl)-3-cyclohexyl-D-alanine

Boc-DTyr(Et)-OH

N-(tert-butyloxycarbonyl)-O-ethyl-D-tyrosine

Boc-Phe-ONSu

N-(tert-butyloxycarbonyl)-phenylalanine succinimidyl ester

DMF

dimethylformamide

H-thPse-OH

threo-3-phenylserine

mplc

medium pressure liquid chromatography

TFA

trifluoroacetic acid

• "Celite" is a registered trademark of Celite Corp.
• "Vydac" is a registered trademark of W.R. Grace & Co.

EXAMPLE 1 (2'S,2"R)-4-(2'-(2"-Amino-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine trifluoroacetate

1A. 1-Benzyl-4-(tert-butyloxycarbonylamino)piperidine

4-Amino-1-benzylpiperidine (3.2g, 16.8mmol) was dissolved in CH₂Cl₂ (100ml). Di-tert-butyl dicarbonate (3.7g, 17.0mmol) and N,N-diisopropylethylamine (1.9g, 19mmol) were added. The mixture was stirred for 18h at room temperature then the solvent was removed in vacuo and the residue was taken up in ethyl acetate (150ml). This solution was washed with 0.3M KHSO₄ (2 x 30ml), sat. NaHCO₃ (2 x 30ml), water (2 x 30ml) and brine (1 x 30ml), dried (Na₂SO₄) and evaporated in vacuo to give a yellow oil which was purified by flash chromatography on silica gel (eluant: 70% chloroform, 30% cyclohexane) to give a yellow solid identified as 1-benzyl-4-(tert-butyloxycarbonylamino)piperidine (4.9g, 18.9 mmol, 100%).

1B. 4-(tert-Butyloxycarbonylamino)piperidine

1-Benzyl-4-(tert-butyloxycarbonylamino)piperidine (4.9g, 18.9mmol) was dissolved in ethanol (100ml). This solution was hydrogenated over 10% palladium on charcoal at 60 psi. After 18h at room temperature the mixture was filtered through Celite and the residue washed with ethanol (100ml). The combined filtrates were evaporated in vacuo to give a white solid identified as 4-(tert-butyloxycarbonylamino)piperidine (2.3g, 7.1mmol, 51 %).

1C. N,N'-Di(benzyloxycarbonyl)-4-(tert-butyloxycarbonylamino)piperidine-1-carboxamidine

4-(tert-Butyloxycarbonylamino)piperidine (1.5g, 7.5mmol) was dissolved in ethanol (100ml). N,N'-Bis(benzyloxycarbonyl)-S-methylisothiourea (3.1g, 8.7mmol) and mercuric oxide (1.9g, 8.8mmol) were added. The mixture was stirred at 40°C for 4h then the solid was filtered off and washed with ethanol (50 ml). The combined filtrates were evaporated in vacuo to give a colourless oil which was purified by flash chromatography on silica gel (eluant: 90% pet ether 60-80, 10% ethyl acetate) to give a colourless oil identified as N,N'-di(benzyloxycarbonyl)-4-(tert-butyloxycarbonylamino)piperidine-1-carboxamidine (3.3g, 6.6mmol, 87%).

1D. (2'S)-N,N'-Di(benzyloxycarbonyl)-4-(2'-(tert-butyloxycarbonylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine

N,N'-Di(benzyloxycarbonyl)-4-(tert-butyloxycarbonylamino)piperidine-1-carboxamidine (3.1g, 6.1mmol) was dissolved in 4M HCl/dioxan (70ml). After 30min at room temperature the solvent was evaporated in vacuo and the residue was dissolved in CH₂Cl₂ (60ml). This solution was cooled to 0°C, Boc-Phe-ONSu (2.2g, 6.1 mmol) was added and the pH adjusted to 9 with N-methylmorpholine. The mixture was stirred at room temperature for 4 h, the solvent was evaporated in vacuo and the residue dissolved in ethyl acetate (200ml). This solution was washed with 0.3M KHSO₄ (2 x 30ml), sat. NaHCO₃ (2 x 30ml), water(2 x 30ml) and brine (1 x 30ml), dried (Na₂SO₄) and evaporated in vacuo to give a white solid which was purified by flash chromatography on silica gel (eluant: 70% chloroform, 30% cyclohexane) to give a white solid identified as (2'S)-N,N'-di(benzyloxycarbonyl)-4-(2'-(tert-butyloxycarbonylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine (3.56g, 5.4 mmol, 89%).

1E. (2'S,2"R)-N,N'-Di(benzyloxycarbonyl)-4-(2'-(2"-(tert-butyloxycarbonylamino)-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine

(2'S)-N,N'-Di(benzyloxycarbonyl)-4-(2'-(tert-butyloxycarbonylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine (2.5g, 3.85mmol) was dissolved in 4M HCl/dioxan (70ml). After 30min at room temperature the solvent was evaporated in vacuo and the residue dissolved in CH₂Cl₂/DMF (9:1, 50ml). This solution was cooled to 0°C and Boc-DTyr(Et)-OH (1.2g, 3.84mmol) was added followed by 1-hydroxybenzotriazole hydrate (680mg, 5.0mmol) and water-soluble carbodiimide (1.0g, 5.0mmol). After 15min the pH adjusted to 8 with N-methylmorpholine The mixture was stirred at room temperature for 18 h, after which time the solvent was evaporated in vacuo and the residue dissolved in chloroform (200ml). This solution was washed with 0.3M KHSO₄ (2 x 30ml), sat. NaHCO₃ (2 x 30ml), water (2 x 30ml) and brine (1 x 30ml), dried (Na₂SO₄) and evaporated in vacuo to give a white solid which was purified by flash chromatography on silica gel (eluant: 85% chloroform, 15% hexane) to give a white solid identified as (2'S,2"R)-N,N'-di(benzyloxycarbonyl)-4-(2'-(2"-(tert-butyloxycarbonylamino)-3"-(4"'-ethoxyphenyl)-propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine (2.47g, 3.2mmol, 65%).

1F. (2'S,2"R)-4-(2'-(2"-Amino-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine trifluoroacetate

(2'S,2"R)-N,N'-Di(benzyloxycarbonyl)-4-(2'-(2"-(tert-butyloxycarbonylamino)-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine (2.1g, 2.7mmol) was dissolved in 4M HCl/dioxan (50ml). After 30min at room temperature the solvent was evaporated in vacuo and the residue dissolved in AcOH/water (95:5, 50ml). This solution was hydrogenated over 10% palladium on charcoal. After 2h at room temperature the mixture was filtered through Celite and the residue washed with AcOH/water (9:1, 30ml). The combined filtrates were evaporated in vacuo and the residue purified by mplc on Vydac C₁₈ (15-25µ) using MeCN/H₂0/TFA to give a white solid identified as H-DTyr(Et)-Phe-4-amino-1-amidinopiperidine trifluoroacetate (1.12g). [M+H]⁺ = 480.6

EXAMPLE 2 (2'S,2"R)-4-(2'-(3"-Cyclohexyl-2"-(methyloxycarbonylmethylamino)propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine trifluoroacetate

2A. (2'S,2"R)-N,N'-Di(benzyloxycarbonyl)-4-(2'-(2"-(tert-butyloxycarbonylamino)-3"-cyclohexylpropanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine

(2'S)-N,N'-Di(benzyloxycarbonyl)-4-(2'-(tert-butyloxycarbonylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine (from Example 1D, 1.9g, 2.99mmol) was dissolved in 4M HCl/dioxan (70ml). After 30min at room temperature the solvent was evaporated in vacuo and the residue dissolved in CH₂Cl₂/DMF (9:1, 50ml). This solution was cooled to 0°C and Boc-DCha-OH (900mg, 3.3mmol) was added followed by 1-hydroxybenzotriazole hydrate (820mg, 6.1 mmol) and water-soluble carbodiimide (730mg, 3.6mmol). After 15min the pH was adjusted to 8 with N-methylmorpholine. The mixture was stirred at room temperature for 18 h, after which time the solvent was evaporated in vacuo and the residue dissolved in chloroform (200ml). This solution was washed with 0.3M KHSO₄ (2 x 30ml), sat. NaHCO₃ (2 x 30ml), water (2 x 30ml) and brine (1 x 30ml), dried (Na₂SO₄) and evaporated in vacuo to give a white solid which was purified by flash chromatography on silica gel (eluant: 90% chloroform, 10% hexane) to give a white solid identified as (2'S,2"R)-N,N'-di(benzyloxycarbonyl)-4-(2'-(2"-(tert,butyloxycarbonylamino)-3"-cyclohexylpropanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine (1.73g, 2.14 mmol, 72%).

28. (2'S,2"R)-N,N'-Di(benzyloxycarbonyl)-4-(2'-(3"-cyctohexyl-2"-(methyloxycarbonylmethylamino)propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine

(2'S,2"R)-N,N'-Di(benzyloxycarbonyl)-4-(2'-(2"-(tert-butyloxycarbonylamino)-3"-cyclohexylpropanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine (1.73g, 2.14mmol) was dissolved in 4M HCl/dioxan (50ml). After 30min at room temperature the solvent was evaporated in vacuo and the residue dissolved in acetonitrile (100ml). Methyl bromoacetate (400mg, 2.6mmol) and N,N-diisopropylethylamine (440mg, 4.4mmol) were added. The reaction mixture was stirred at 60°C for 5h after which time the solvent was evaporated in vacuo and the residue dissolved in ethyl acetate (200ml). This solution was washed with 0.3M KHSO₄ (2 x 30ml), sat. NaHCO₃ (2 x 30ml), water (2 x 30ml) and brine (1 x 30ml), dried (Na₂SO₄) and evaporated in vacuo to give a yellow oil which was purified by flash chromatography on silica gel (eluant: 90% chloroform, 10% hexane) to give a white solid identified as (2'S,2"R)-N,N'-di(benzyloxycarbonyl)-4-(2'-(3"-cyclohexyl-2"-(methyloxycarbonylmethylamino)propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine (1.65g, 2.11 mmol, 98%).

2C. (2'S,2"R)-4-(2'-(3"-Cyclohexyl-2"-(methytoxycarbonylmethylamino)propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine trifluoroacetate

(2'S,2"R)-N,N'-Di(benzyloxycarbonyl)4-(2'-(3"-cyclohexyl-2"-(methyloxycarbonylmethylamino)propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine (1.62g, 2.11mmol) was dissolved in AcOH/water (95:5, 50ml). This solution was hydrogenated over 10% palladium on charcoal. After 2h at room temperature the mixture was filtered through Celite and the residue washed with AcOH/water (9:1, 30ml). The combined filtrates were evaporated in vacuo and the residue purified by mplc on VydacC₁₈ (15-25µ) using MeCN/H₂O/TFA to give a white solid identified as (2'S,2"R)-4-(2'-(3"-cyclohexyl-2"-(methyloxycarbonylmethylamino)propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine trifluoroacetate (570mg). [M+H]⁺ = 515

EXAMPLE 3 (2'S,2"R)-4-(2'-(2"-(Carboxymethylamino)-3"-cyclohexylpropanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine trifluoroacetate

3A. (2'S,2"R)-N,N'-Di(benzyloxycarbonyl)-4-(2'-(2"-(carboxymethylamino)-3"-cyclohexylpropanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine

(2'S,2"R)-N,N'-Di(benzyloxycarbonyl)-4-(2'-(3"-cyclohexyl-2"-(methyloxycarbonylmethylamino)propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine (from Example 2B, 1.6g, 2.1 mmol) was dissolved in tetrahydrofuran (50ml). 1 M Lithium hydroxide (3ml, 3mmol) was added. After 18h the solvent was evaporated in vacuo and the residue dissolved in ethyl acetate (150ml). This solution was washed with 1 M citric acid (1 x 30ml), water (2 x 30ml) and brine (1 x 30ml), dried (Na₂SO₄) and evaporated in vacuo to give a white solid identified as (2'S,2"R)-N,N'-di(benzyloxycarbonyl)-4-(2'-(2"-(carboxymethylamino)-3"-cyclohexylpropanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine (1.62g, 2.11mmol, 100%).

3B. (2'S,2"R)-4-(2'-(2"-(Carboxymethylamino)-3"-cyclohexylpropanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine trifluoroacetate

(2'S,2"R)-N,N-Di(benzyloxycarbonyl)-4-(2'-(2"-(carboxymethylamino)-3"-cyclohexylpropanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine (1.62g, 2.11mmol) was dissolved in AcOH/water (95:5, 50ml). This solution was hydrogenated over 10% palladium on charcoal. After 2h at room temperature the mixture was filtered through Celite and the residue washed with AcOH/water (9:1, 30ml). The combined filtrates were evaporated in vacuo and the residue purified by mplc on Vydac C₁₈ (15-25µ) using MeCN/H₂0/TFA to give a white solid identified as (2'S,2"R)-4-(2'-(2"-(carboxymethylamino)-3"-cyclohexylpropanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine trifluoroacetate (570mg). [M+H]⁺ = 501

EXAMPLE 4 (2'S,2"R)-4-(2'-(2"-Benzoylamino-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-phenyl-propanoylamino)piperidine-1-carboxamidine trifluoroacetate

4A. (2'S,2"R)-4-(2'-(2"-Benzoylamino-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-phenylpropanoylamino)-N,N'-di(benzyloxycarbonyl)piperidine-1-carboxamidine

(2'S,2"R)-N,N'-Di(benzyloxycarbonyl)-4-(2'-(2"-(tert-butyloxycarbonylamino)-3"-(4'''-ethoxyphenyl)propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine(from Example 1F, 100mg, 0.12mmol) was dissolved in 4M HCl/dioxan (20ml). After 30min at room temperature the solvent was evaporated in vacuo and the residue dissolved in CH₂ Cl₂ (20ml). This solution was cooled to 0°C and benzoyl chloride (19.7mg, 0.141mmol) and triethylamine (36mg, 0.36mmol) were added. The mixture was stirred at room temperature for 18 h, the solvent was evaporated in vacuo and the residue dissolved in ethyl acetate (70ml). This solution was washed with 0.3M KHSO₄ (2 x 20ml), sat. NaHCO₃ (2 x 20ml), water (2 x 20ml) and brine (1 x 20ml), dried (Na₂SO₄) and evaporated in vacuo to give a white solid which was purified by flash chromatography on silica gel (eluant: 85% chloroform, 15% cyclohexane) to give a white solid identified as (2'S,2"R)-4-(2'-(2"-benzoylamino-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-phenylpropanoylamino)-N,N'-di(benzyloxycarbonyl)piperidine-1-carboxamidine (65mg, 0.072 mmol, 60%).

4B. (2'S,2"R)-4-(2'-(2"-Benzoylamino-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine trifluoroacetate

(2'S,2"R)-4-(2'-(2"-Benzoylamino-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-phenylpropanoylamino)-N,N'-di(benzyloxycarbonyl)piperidine-1-carboxamidine (65mg, 0.072mmol) was dissolved in AcOH/water (95:5, 25ml). This solution was hydrogenated over 10% palladium on charcoal. After 1 hour at room temperature the mixture was filtered through Celite and the residue washed with AcOH/water (9:1, 20ml). The combined filtrates were evaporated in vacuo and the residue purified by mplc on VydacC₁₈ (15-25µ) using MeCN/H₂0/TFA to give a white solid identified as (2'S,2"R)-4-(2'-(2"-benzoylamino-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine trifluoroacetate (44mg). [M+H]⁺ = 585

EXAMPLE 5 (2'S,2"R,3'R)-4-(2'-(2"-Amino-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-hydroxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine trifluoroacetate

5A. (2S,3R)-2-(tert-Butyloxycarbonylamino)-3-hydroxy-3-phenylpropanoic acid

H-thPse-OH (J. Biol. Chem., 1953, 204, 323) (1.4g, 7.73mmol) was dissolved in dioxan (75ml). Sodium hydroxide 820mg, 20.5mmol) in water (75ml) was added followed by di-tert-butyl dicarbonate (2.1g, 9.6mmol). The mixture was stirred for 18h at room temperature then the dioxan was removed in vacuo and the residue was washed with diethyl ether (1 x 100ml), acidified to pH 4 with 1M HCl and extracted with CHCl₃ (3 x 100ml). The combined extracts were washed with water (1 x 50ml) and brine (1 x 50ml), dried (Na₂SO₄) and evaporated in vacuo to give a white solid identified as (2S,3R)-2-(tert-butyloxycarbonylamino)-3-hydroxy-3-phenylpropanoic acid (1.6g, 5.7 mmol, 74%).

5B (2'S,3'R)-N,N'-Di(benzyloxycarbonyl)-4-(2'-(tert-butyloxycarbonylamino)-3'-hydroxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine

N,N'-Di(benzyloxycarbonyl)-4-(tert-butyloxycarbonylamino)piperidine-1-carboxamidine (from Example 1C, 2.3g, 4.5mmol) was dissolved in 4M HCl/dioxan (70ml). After 30min at room temperature the solvent was evaporated in vacuo and the residue dissolved in CH₂Cl₂/DMF (9:1, 50ml). This solution was cooled to 0°C and (2S,3R)-2-(tert-butyloxycarbonylamino)-3-hydroxy-3-phenylpropanoic acid (1.6g, 5.6mmol) was added followed by 1-hydroxybenzotriazole hydrate (1.1g, 8.1mmol) and water-soluble carbodiimide (1.4g, 75.0mmol). After 15 min the pH adjusted to 8 with N-methylmorpholine. The mixture was stirred at room temperature for 18 h, after which time the solvent was evaporated in vacuo and the residue dissolved in ethyl acetate (200ml). This solution was washed with 0.3M KHSO₄ (2 x 30ml), sat. NaHCO₃ (2 x 30ml), water (2 x 30ml) and brine (1 x 30ml), dried (Na₂SO₄) and evaporated in vacuo to give a white solid which was purified by flash chromatography on silica gel (eluant: 50% ethyl acetate, 50% pet. ether) to give a white solid identified as (2'S,3'R)-N,N'-di(benzyloxycarbonyl)-4-(2'-(tert-butyloxycarbonylamino)-3'-hydroxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine (1.1g, 1.6 mmol, 36%).

5C. (2'S,2"R,3'R)-N,N'-Di(benzyloxycarbonyl)-4-(2'-(2"-(tert-butyloxycarbonylamino)-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-hydroxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine

(2'S,3'R)-N,N'-Di(benzyloxycarbonyl)-4-(2'-(tert-butyloxycarbonylamino)-3'-hydroxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine (1.1g, 1.6mmol) was dissolved in 4M HCl/dioxan (70ml). After 30min at room temperature the solvent was evaporated in vacuo and the residue dissolved in CH₂Cl₂/DMF (9:1, 50ml). This solution was cooled to 0°C and Boc-DTyr(Et)-OH (620mg, 2.0mmol) was added followed by 1-hydroxybenzotriazole hydrate (270mg, 2.0mmol) and water-soluble carbodiimide (420mg, 2.1mmol). After 15min the pH was adjusted to 8 with N-methylmorpholine The mixture was stirred at room temperature for 18 h, after which time the solvent was evaporated in vacuo and the residue dissolved in ethyl acetate (200ml). This solution was washed with 0.3M KHSO₄ (2 x 30ml), sat. NaHCO₃ (2 x 30ml), water (2 x 30ml) and brine (1 x 30ml), dried (Na₂SO₄) and evaporated in vacuo to give a white solid which was purified by flash chromatography on silica gel (eluant: 60% ethyl acetate, 40% pet. ether) to give a white solid identified as (2'S,2"R,3'R)-N,N'-di(benzyloxycarbonyl)-4-(2'-(2"-(tert-butyloxycarbonylamino)-3"-(4'''-ethoxyphenyl)propanoylamino)-3'-hydroxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine (1.2g, 1.3 mmol, 80%).

5D. (2'S,2"R,3'R)-4-(2'-(2"-Amino-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-hydroxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine trifluoroacetate

(2'S,2"R,3'R)-N,N'-Di(benryloxycarbonyl)-4-(2'-(2"-(tert-butyloxycarbonylamino)-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-hydroxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine (1.2g, 1.3mmol) was dissolved in 4M HCl/dioxan (50ml). After 30min at room temperature the solvent was evaporated in vacuo and the residue dissolved in AcOH/water (95:5, 50ml). This solution was hydrogenated over 10% palladium on charcoal. After 2h at room temperature the mixture was filtered through Celite and the residue washed with AcOH/water (9:1, 30ml), The combined filtrates were evaporated in vacuo and the residue purified by mplc on Vydac C₁₈ (15-25µ) using MeCN/H₂0/TFA to give a white solid identified as (2'S,2"R,3'R)-4-(2'-(2"-amino-3"-(4"'-ethoxyphenyl)-propanoylamino)-3'-hydroxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine trifluoroacetate (540mg). [M+H]⁺ = 497.0

EXAMPLE 6 (2'S,2"R,3'R)-4-(2'-(2"-Amino-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-methoxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine trifluoroacetate

6A. (2'SR,3'RS)-N,N'-Di(benzyloxycarbonyl)-4-(2'-(tert-butyloxycarbonylamino)-3'-hydroxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine

(2'SR,3'RS)-N,N'-Di(benzyloxycarbonyl)-4-(2'-(tert-butyloxycarbonylamino)-3'-hydroxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine was prepared by the same method as described in Example 5 but starting with racemic H-thPse-OH.

6B. (2'SR,3'RS)-N,N'-Di(benzyloxycarbonyl)-4-(2'-(tert-butyloxycarbonylamino)-3'-methoxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine

(2'SR,3'RS)-N,N'-Di(benzyloxycarbonyl)-4-(2'-(tert-butyloxycarbonylamino)-3'-hydroxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine (180mg, 0.27mmol), was dissolved in CH₂Cl₂ (30ml). lodomethane (190mg, 1.3mmol) and silver oxide (132mg, 0.8mmol) were added. After 18h at 60°C the mixture was filtered and the filtrate was evaporated in vacuo to give a brown oil which was purified by flash chromatography on silica gel (eluant: 50% ethyl acetate, 50% pet. ether) to give a white solid identified as (2'SR,3'RS)-N,N'-di(benzyloxycarbonyl)-4-(2'-(tert-butyloxycarbonylamino)-3'-methoxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine (112mg, 0.16mmol, 61%).

6C. (2'SR,2"R,3'RS)-N,N'-Di(benzyloxycarbonyl)-4-(2'-(2"-(tert-butyloxycarbonylamino)-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-methoxy-3'-phenylpropanoylamino)piperidine-1 -carboxamidine

(2'SR,3'RS)-N,N'-Di(benzyloxycarbonyl)-4-(2'-(tert-butyloxycarbonylamino)-3'-methoxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine (112mg, 0.16mmol) was dissolved in 4M HCl/dioxan (20ml). After 30min at room temperature the solvent was evaporated in vacuo and the residue dissolved in CH₂Cl₂ (30ml). This solution was cooled to 0°C and Boc-DTyr(Et)-OH (50mg, 0.16mmol) was added followed by PyBrop (76mg, 0.16mmol). The pH adjusted to 9 with N,N-diisopropylethylamine. The mixture was stirred at room temperature for 18 h, after which time the solvent was evaporated in vacuo and the residue dissolved in ethyl acetate (70ml). This solution was washed with 0.3M KHSO₄ (1 x 20ml), sat. NaHCO₃ (1 x 20ml), water (1 x 20ml) and brine (1 x 20ml), dried (Na₂SO₄) and evaporated in vacuo to give a white solid which was purified by flash chromatography on silica gel (eluant: 65% chloroform, 15% hexane) to give a white solid identified as (2'SR,2"R,3'RS)-N,N'-di(benzyloxycarbonyl)-4-(2'-(2"-(tert-butyloxycarbonylamino)-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-methoxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine (108mg, 0.12 mmol, 75%).

6D. (2'S,2"R,3'R)-4-(2'-(2"-Amino-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-methoxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine trifluoroacetate

(2'SR,2"R, 3'RS)-N,N'-Di(benzyloxycarbonyl)-4-(2'-(2"-(tert-butyloxycarbonylamino)-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-methoxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine (108mg, 0.12mmol) was dissolved in 4M HCl/dioxan (20ml). After 30min at room temperature the solvent was evaporated in vacuo and the residue dissolved in AcOH/water (95:5, 20ml). This solution was hydrogenated over 10% palladium on charcoal. After 2h at room temperature the mixture was filtered through Celite and the residue washed with AcOH/water (9:1, 30ml). The combined filtrates were evaporated in vacuo and the residue purified by mplc on Vydac C₁₈ (15-25µ) using MeCN/H₂0/TFA to give a white solid identified as (2'S,2"R,3'R)-4-(2'-(2"-amino-3"-(4"'-ethoxyphenyl)-propanoylamino)-3'-methoxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine trifluoroacetate (18mg). [M+H]⁺ = 511.3

The following compounds were prepared using analogous methods.

Examples 7 - 17

	H		403.3
	H		429.3
	H		443.4
	H		457.4
	H		487.4
	H		487.4
	H		497.4
			555
			569
	H		451.2
	H		467.2

Examples 18 - 52

	H	H	H	H	H	437.3
	H	H	H		H	479
	H	H	H		H	482.3
	H	H	H		H	452.3
	H	H	H	I	H	563.1
	H	H	H	F	H	455.2
	H	H	H	CN	H	462.3
	H	H	H	Cl	H	471.6
	H	H	H		H	505.3
	H	H	H		H	494.3
	H	H	F	H	H	455
	H	H	Cl	Cl	H	505.1
	H	Cl	H	Cl	H	505.1
	H	H	H	OH	H	453.3
	H	H	H		H	543.5
	H	H	H		H	509.3
	H	H	H		H	495.4
	H	H	H		H	467.3
	H	H	H		H	495.2
	H	H	H		H	537.3
	H	H	I		I	733.1
		H	H		H	493.3
		H	H		H	559.3
		H	H		H	573.3
		H	H		H	621
		H	H		H	523.3
		H	H		H	551
		H	H		H	537
		H	H		H	615
		H	H		H	553
		H	H		H	539
	H	-CH=CH-CH=CH-		H	H	487.4
	H	H	-CH=CH-CH=CH-		H	487.3
			H	H	H	449.3
			H		H	493.3

Examples 53 - 54

	OH	497.4
	OMe	511.3

Examples 55 - 58

	H	OH	453.3
	H	OMe	467.3
	OH	H	453.3
	OMe	H	467.3

Example 59 Determination of the inhibition constant K _i for plasma kallikrein

Inhibition of plasma kallikrein activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Stürzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human plasma kallikrein (Callbiochem) was incubated at 37°C with three different concentrations of the chromogenic substrate S-2302 (Chromogenix AB) and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 405nm and the inhibitory constant K_i for the test compound as determined from a Dixon plot (Dixon, Biochem. J., 1953, 55, 170). Typical results are presented in the Table.

	4.5			7.0
	66.0			15.0
	3.0			9.0
	1.4			4.5
	6.6			7.3
	25.0			4.1
	2.6			3.0
	19.0			18.0
	5.5

Example 60 Determination of enzyme selectivity

Selected compounds were further screened for inhibitory activity against other trypsin-like proteases following the method of Example 59 and using the appropriate enzyme and chromogenic substrate (Chromogenix AB). Representative results are presented in the Table. The selectivity is given by: $\mathrm{Selectivity}=\left(K_i\hspace{0.17em}\mathrm{for\; test\; enzyme}\right)/\left(K_i\hspace{0.17em}\mathrm{for\; plasma\; kallikrein}\right)$

Human Tissue	S-2266		45,000	10,000
Kallikrein			6,000	2,000
			18,500	13,000
			>70,000	>10,000
			14,000	930
			1,950	210
			6,800	1,500
			69,000	9,400
			49,000	16,000
Thrombin	S-2238		310	100
			16,500	1,800
Plasmin	S-2390		3,200	1,000
			1,440	160
Trypsin	S-2222		825	270
			3,500	380

Claims (12)

1. A compound according to general formula 1, or a pharmaceutically acceptable salt thereof, wherein

   R¹ is selected from H, lower alkyl, R⁴-CO, R⁴-O₂CCH₂, R⁵-OCO and R⁵-SO₂;

   R² is selected from lower alkyl, cycloalkyl optionally substituted with an alkyl or alkyloxy group, (C₅-C₁₂)cycloalkylalkyl optionally substituted with an alkyl or alkyloxy group, aralkyl optionally substituted with up to three groups chosen from F, Cl, Br, I, OH, lower alkyl, O-(lower alkyl), O-benzyl, NH₂, NO₂, NH-acyl, CN and CF₃, and aralkyloxymethyl optionally substituted with up to three groups chosen from F, Cl, Br, OH, lower alkyl and O-(lower alkyl); or

   R¹ and R² together are an o-xylylene group optionally substituted on the aromatic ring with a group selected from F, Cl, Br, OH, lower alkyl and O-(lower alkyl);

   R³ is selected from H, OH and O-lower alkyl;

   R⁴ is selected from H, lower alkyl and phenyl; and

   R⁵ is selected from lower alkyl, phenyl and benzyl.
2. A compound according to Claim 1 wherein R¹ is selected from H, lower alkyl, and R⁴-O₂CCH₂.
3. A compound according to either of Claims 1 and 2 wherein R² is selected from (C₆-C₁₀)cycloalkylmethyl, benzyl optionally substituted with up to three groups chosen from F, Cl, Br, OH, lower alkyl and O-(lower alkyl), phenethyl optionally substituted with up to three groups chosen from F, Cl, Br, OH, lower alkyl and O-(lower alkyl)and benzyloxymethyl optionally substituted with up to three groups chosen from F, Cl, Br, OH, lower alkyl and O-(lower alkyl).
4. A compound according to any of Claims 1 to 3 wherein R² is selected from cyclohexylmethyl, decahydronaphth-2-ylmethyl, benzyl, 4-fluorobenzyl, 4-chlorobenzyl, 4-hydroxybenzyl, 4-(lower alkyl)oxybenzyl, α-hydroxybenzyl, α-methoxybenzyl, phenethyl and benzyloxymethyl.
5. A compound according to any of Claims 1 to 4 wherein the absolute stereochemistry is as depicted in general formula 1A.
6. A compound according to any of Claims 1 to 5 wherein the absolute stereochemistry is as depicted in general formula 1B.
7. A compound according to any of Claims 1 to 6 selected from (2'S,2"R)-4-(2'-(2"-amino-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine; (2'S,2"R)-4-(2'-(2"-carboxymethylamino-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine; (2'S,2"R)-4-(2'-(3"-(4"'-ethoxyphenyl)-2"-(methyloxycarbonylmethylamino)-propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine; (2'S,2"R)-4-(2'-(2"-amino-3"-cyclohexylpropanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine; (2'S,2"R)-4-(2'-(2"-carboxymethylamino-3"-cyclohexylpropanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine; (2'S,2"R)-4-(2'-(3"-cyclohexyl-2"-(methyloxycarbonylmethylamino)propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine; (2'S,2"R)-4-(2'-(2"-amino-3"-phenylpropanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine; (2'S,2"R)-4-(2'-(2"-carboxymethylamino-3"-phenylpropanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine; (2'S,2"R)-4-(2'-(2"-(methyloxycarbonylmethylamino)-3"-phenylpropanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine; (2'S,2"R)-4-(2'-(2"-amino-3"-decahydronaphth-2"'-ylpropanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine; (2'S,2"R)-4-(2'-(2"-carboxymethylamino-3"-decahydronaphth-2"'-yl-propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine; (2'S,2"R)-4-(2'-(3"-decahydronaphth-2"'-yl-2"-(methyloxycarbonylmethylamino)propanoylamino)-3'-phenylpropanoylamino)piperidine-1-carboxamidine; (2'S,2"R, 3'R)-4-(2'-(2"-amino-3"-cyclohexylpropanoylamino)-3'-hydroxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine; (2'S,2"R,3'R)-4-(2'-(2"-carboxymethylamino-3"-cyclohexylpropanoylamino)-3'-hydroxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine; (2'S,2"R,3'R)-4-(2'-(3"-cyclohexyl-2"-(methyloxycarbonylmethylamino)-propanoylamino)-3'-hydroxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine; (2'S,2"R,3'R)-4-(2'-(2"-amino-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-methoxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine; (2'S,2"R,3'R)-4-(2'-(2"-carboxymethylamino-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-methoxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine; and (2'S,2"R,3'R)-4-(2'-(3"-(4"'-ethoxyphenyl)-2"-(methyloxycarbonylmethylamino)-propanoylamino)-3'-methoxy-3'-phenylpropanoylamino)piperidine-1-carboxamidine or a pharmaceutically acceptable salt thereof.
8. A pharmaceutical composition comprising a compound according to any of Claims 1 to 7 or a pharmaceutically acceptable salt thereof.
9. Use of a compound of general formula 1, or a pharmaceutically acceptable salt thereof, wherein

   R¹ is selected from H, lower alkyl, R⁴-CO, R⁴-O₂CCH₂, R⁵-OCO and R⁵-SO₂;

   R² is selected from lower alkyl, cycloalkyl optionally substituted with an alkyl or alkyloxy group, (C₅-C₁₂)cycloalkylalkyl optionally substituted with an alkyl or alkyloxy group, aralkyl optionally substituted with up to three groups chosen from F, Cl, Br, I, OH, lower alkyl, O-(lower alkyl), O-benzyl, NH₂, NO₂ NH-acyl, CN and CF₃, and aralkyloxymethyl optionally substituted with up to three groups chosen from F, Cl, Br, OH, lower alkyl and O-(lower alkyl); or

   R¹ and R² together are an O-xylylene group optionally substituted on the aromatic ring with a group selected from F, Cl, Br, OH, lower alkyl and O-(lower alkyl);

   R³ is selected from H, OH and O-lower alkyl;

   R⁴ is selected from H, lower alkyl and phenyl; and

   R⁵ is selected from lower alkyl, phenyl and benzyl; for the manufacture of a pharmaceutical for the treatment of a disease condition for which over activity of plasma kallikrein is a causative factor.
10. A use according to claim 9 for the treatment of a disease selected among inflammatory bowel disease, arthritis, inflammation, septic shock, hypotension, cancer, adult respiratory distress syndrome, disseminated intravascular coagulation, cardiopulmonary bypass surgery and bleeding from post-operative surgery.
11. The use, in the manufacture of a medicament for treatment of disease in a human or animal, of a compound according to any of claims 1 to 7.
12. A pharmaceutical composition according to claim 8 for the treatment of a disease condition for which over activity of plasma kallikrein is a causative factor.