EP1049673A1 - Heterocyclic amidines as callicrein protease inhibitors - Google Patents

Abstract

The invention relates to compounds of the formula A-B-D-E-F, in which A, B, D, E and F have the meanings given in the description, and to their production. These new compounds are suitable for producing medicines.

Description

HETEROCYCLIC AMIDINE AS KALLIKREIN PROTEASE INHIBITOR

description

The present invention relates to new five-membered heterocyclic amidines, their preparation and their use as competitive inhibitors of kininogenases such as kallikrein and trypsin-like serine proteases, especially thrombin. The invention also relates to pharmaceutical compositions which contain the compounds as active constituents, and to the use of the compounds as anti-inflammatory agents, thrombin inhibitors and anticoagulants.

Kininogenases are serine proteases that release vasoactive peptides from kininogens, the so-called kinins (bradykinin, kallidin and Met-Lys-bradykinin). Kininogens are multifunctional proteins that occur in cascade coagulation and inflammation reactions. As inhibitors, they protect cells from destruction by cysteine proteases (Müller Esterl, 1985, FEBS Lett. 182, 310-314). Important kininogenases are plasma kallikrein, tissue kallikrein and mast cell tryptase.

Kinins such as bradykinin and kallidin are vasoactive peptides that affect a variety of biological processes. They play an essential role in inflammatory processes. By increasing vascular permeability, they lead to hypotension and edema. Furthermore, they are very potent pain-producing substances in the body and are of great importance as cellular mediators in the pathophysiology of asthma, allergic rhinitis and arthritis (KD Bhoola, CD. Figueroa, K. Worthy, Pharmacological Reviews 1992, 44 (1), 1- 80).

Regardless of the mechanisms underlying inflammatory processes, fluid escapes from the blood vessels that contains all of the protein systems in the circulating blood. This means that the escape of plasma fluid from the vessels plays a role in diseases such as asthma, rhinitis and inflammation-related internal diseases. Mast cell tryptase is released particularly in allergic processes (Salomonsson et al., Am. Rev. Respir. Dis., 1992, 146, 1535-1542). 2

The arginine chloromethyl ketones H- (D) -Pro-Phe-Arg-CH ₂ Cl and H- (D) -Phe-Phe-Arg-CH-Cl were described by Kettner and Shaw as plasma kallikrein inhibitors (Biochem. 1978, 17, 4778-4784 and Meth. Enzym. 1981, 80, 826-842).

Various synthetic derivatives of benzamidines and benzyl aines have been shown to inhibit plasma kallikrein, the benzamidines having a significantly stronger inhibitory activity (F. Markward, S. Drawert, P. Walsmann, Biochemical Pharmacology 1974, 23, 2247-2256).

PKSI-527, the hydrochloride of N- (trans-4-aminomethylcyclohexylcarbonyl) -L-phenylalanine-4-carboxymethyl-anilide, is also an effective inhibitor for this kininogenase (Wanaka, Ohamoto et al., Thromb. Res. 1990 , 57 (6), 889-895).

Thrombin belongs to the group of serine proteases and plays a central role as a terminal enzyme in the blood coagulation cascade. Both the intrinsic and the extrinsic coagulation cascade lead to several reinforcement levels

Formation of thrombin from prothrombin. The thrombin-catalyzed cleavage of fibrinogen to fibrin then initiates blood coagulation and platelet aggregation, which in turn increases the formation of thrombin by binding platelet factor 3 and coagulation factor XIII and a whole series of highly active mediators.

Thrombin formation and action are central events in the development of both white, arterial and red, venous thrombi and therefore potentially effective targets for pharmaceuticals. In contrast to heparin, thrombin inhibitors are able, independently of cofactors, to completely inhibit the effects of free thrombin as well as that bound to platelets. They can prevent thromboembolic events after percutaneous transluminal coronary angioplasty (PTCA) and lysis in the acute phase and serve as anticoagulants in the extracorporeal circulation (cardiopulmonary machine, hemodialysis). They can also be used in general for thrombosis prophylaxis, for example after surgery.

It is known that synthetic arginine derivatives influence the enzyme activity of thrombin by interacting with the active serine residue of the protease thrombin. Peptides based on Phe-Pro-Arg, in which the N-terminal amino acid is in the D form, have proven to be particularly favorable. D-Phe-Pro-Arg-isopropyl ester is more competitive 3

Thrombin inhibitor is described (C. Mattson et al., Folia Haematol, 109, 43 to 51, 1983).

The derivatization of the C-terminus arginine to the aldehyde leads to an increase in the inhibitory effect. A large number of arginals are described which are able to bind the hydroxyl group of the "active" serine semi-acetally (EP 185390, 479489, 526877, 542525; WO 93/15756, 93/18060).

The thrombin-inhibitory activity of peptidic ketones, fluorinated alkyl ketones, and of keto esters, boric acid derivatives, phosphoric acid esters and α-ketocarboxamides can also be explained with this serine interaction (EP 118280, 195212, 362002, 364344, 410411, 471651, 589741, 293881 506420 530167; WO 92/07869, 94/08941).

J. Oleksyszyn et al. in J. Med. Chem. 37, 226 to 231 (1994) described peptidic 4-ZAmidinophenylglycine phosphonate diphenyl esters are irreversible thrombin inhibitors with insufficient selectivity towards other serine proteases.

DE 3 108 810, WO 93/11152 and EP 601 459 describe agmatine and thus arginine derivatives which cannot interact with the active serine of the serine proteases.

WO 94/29336, EP 0 601 459 and WO 95/23609 represent a further development, the agmatine being replaced by an arylamidine residue.

In addition to thrombin inhibitors which carry an agmatine or benzamidine residue, EP 0 672 658 also describes a thrombin inhibitor with an amidinothiophene (Example 65).

The invention relates to the use of the compounds of the formula I.

A — B-D-E — F (I)

for the production of medicaments for diseases whose pathomechanism is based directly or indirectly on the proteolytic action of kininogenases, in particular kallikrein, and

wherein A, B, D, E and F have the following meaning and their use as kallikrein or thrombin inhibitors: A:

R ^

R ¹ (CH ₂ ) _m C (CH ₂ ) _n

R ³ wherein

m 0, 1, 2 or 3, n 0, 1 or 2,

R ¹ HOOC-, Cι- ₆ -alkyl-OOC-, aryl-C ₀ - ₄ -alkyl-OOC or -OH,

R ^{2 is} H, C ₄ alkyl or R ¹ - (CH ₂ ) _m -,

R ³ H or Cι-. ₄ -alkyl-,

represent

B:

R ⁴ R ⁶ II —NC-CO-

wherein

R ⁴ H, C ₄ alkyl or R ¹ - (CH ₂ ) _m - (where R ¹ and m have the meaning given above), R ⁶ -CH ₂ - (CR ⁷ R ⁸ ) -W or - (CR ⁷ R ⁸ ) _q -W with q = 0 or 1 and W = H, C ₈ alkyl, 2-thienyl, 3-thienyl, 3-indolyl, 4-imidazolyl, 2-pyridyl , 3-pyridyl, 4-pyridyl, phenyl, which is up to three identical or different radicals of the group C ₄ alkyl ₄ , CF ₃ , C ₄ alkoxy, HO, BnO, F or Cl- can carry C ₃ - ₈ cycloalkyl, which can carry up to four identical or different C ₄ alkyl residues and / or in which one or two CC single bonds in the ring can be replaced by a C = C double bond and / or to which a phenyl ring can be fused, C 1 -C bicycloalkyl or cio tricycloalkyl, or R ⁴ and R ⁶ together form a butylene group or a - (CH ₂ ) _r - (CR ¹² R ⁸ ) _p -Group with r = 2 or 3 and p = 0 or 1 and R ¹² = cyclohexyl, phenyl, R ⁸ = H or Cι_ ₄ alkyl, R ⁵ H or Cι_ ₄ alkyl, R ⁷ H, Cι - ₈ alkyl, phenyl, which can carry up to three identical or different radicals of the group Cι- ₄ alkyl, CF ₃ -, Cι- ₄ alkoxy-, F- or Cl-, C ₃ _ ₈ -cycloalkyl-, which is up to four identical or different Cι- ₄ alkyl radicals can carry, R ⁸ H or Cι- ₄ alkyl-, 5 mean

D:

E:

R10

, x.

'N

R, -9κR11 Y-Z where

X S, 0 or NH,

Y -N = or -CH =,

Z -N = or -CH = mean

and

R ^{9 is} H or C ₃ alkyl,

R ^{10 is} H or C ₄ alkyl,

R ^{11 is} H or C ₄ alkyl,

F:

_{NH N ^} OH

- or - f

NH _{2 N} μ

and their salts with physiologically acceptable acids.

The amino acid derivatives represented by B are preferably (D) -configured, azetidinecarboxylic acid or proline in D are preferably (L) -configured.

The following compounds are preferred, particularly preferred or very particularly preferred for the abovementioned uses.

Compounds of the formula I in which the groups A, B, D, E and F have the following meanings are preferred: )

A: HOOC- (CH ₂ ) _t - C ₆ alkyl-OOC- (CH ₂ ) _t -, (t = 1, 2 or 3),

(HOOC-CH ₂ ) ₂ -CH-, HOOC-CH ₂ -CH (COOH) -, HOOC-CH (Cι- ₄ -alkyl) -, HOOC-C (Cι_ ₄ -alkyl) ₂ -,

B:

R ⁴ R ⁶ I |

—N-C-CO-

R ⁴ H, C ₄ alkyl, HOOC (CH ₂ ) _m - (m = 1, 2 or 3),

R ⁵ H, methyl

R ⁶ -W, -CH ₂ - (CR ⁷ R ⁸ ) -W with W = Cι_ ₈ alkyl-, 2-thienyl, 3-thienyl,

3-indolyl-, 4-imidazolyl-, 2-pyridyl-, 3-pyridyl-, 4-pyridyl-, phenyl-, which up to three identical or different radicals of the group CH ₃ -, CF ₃ -, CH ₃ O- , HO-, BnO-, F- or Cl- can carry, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, which cycloalkyl radicals can carry up to four methyl radicals, adamantyl, indanyl, or -CH-W with W = Ci-s-alkyl, 2-thienyl, 3-thienyl, 3-indolyl, 4-imidazol-yl, 2-pyridyl, 3-pyridyl, 4-pyridyl, phenyl, which up to can carry three identical or different radicals of the group Cι_ ₄ -alkyl-, CF ₃ -, Cι_ ₄ -alkoxy-, HO-, BnO-, F- or Cl-, cyclopentyl, cycloheptyl, cyclooctyl, which cycloalkyl radicals up to can carry four methyl residues, adamantyl, indanyl,

R ^{7 is} H, C ₈ alkyl, phenyl, which can carry up to three identical or different radicals from the group C ₄ alkyl, CF ₃ , C ₄ alkoxy, F or Cl, C ₃ _ ₈ cycloalkyl, which can carry up to four identical or different C ₄ alkyl radicals,

R ^{8 is} H or C ₄ alkyl,

D:

H

Ϊ -C

HH 7

F:

NH _N -OH or - -

NH ₂ \ JH ₂ or

ß ⁾ A: -C ₆ alkyl-OOC- (CH ₂ ) t-. (t = 1, 2 or 3), HOOC- (CH ₂ ) ₂ -, HOOC- (CH ₂ ) ₃ -, (HOOC-CH ₂ ) ₂ -CH-, HOOC-CH ₂ -CH (COOH) -, HOOC- CH (Cι_ ₄ -alkyl) -, HOOC-C (Cι_ ₄ -alkyl) ₂ -,

B:

R ⁴ R ⁶ II —NC-CO-

R ⁵

R ^{4 is} H, C ₄ alkyl, HOOC (CH ₂ ) _m - (m = 1, 2 or 3),

R ⁵ H, methyl

R ⁶ - (CR ⁷ R ⁸ ) -W with W = cyclohexyl-, which can carry up to four methyl radicals,

R ⁷ H-, Cι- ₈ ^_ alkyl, phenyl, which can carry up to three identical or different radicals of the group Cι_ ₄ -alkyl-, CF ₃ -, Cι_ ₄ -alkoxy-, F- or Cl-, C ₃ _ ₈ -cycloalkyl-, which can carry up to many identical or different Cι- ₄ alkyl radicals,

R ^{8 is} H or C ₄ alkyl,

D:

HI

H 1 H 1 , NH N-OH

"or - NHL ^N N or

γ ⁾

A: HOOC- (CH ₂ ) _t -, Cι_ ₆ -alkyl-OOC- (CH ₂ ) _t -, (t = 1, 2 or 3),

(HOOC-CH ₂ ) -CH-, HOOC-CH ₂ -CH (COOH) -, HOOC-CH (Cι_ ₄ -alkyl) -, HOOC-C (C ₁ _ ₄ -alkyl) ₂ -,

B:

R ⁴ R ⁶ II —NC-CO-

R ⁴ H, C ₄ alkyl or HOOC (CH ₂ ) _m - (m = 1, 2 or 3), R ⁵ H, methyl,

R ⁶ -CH ₂ - (CR ⁷ R ⁸ ) -W or - (CR ⁷ R ⁸ ) _q -W with q = 0 or 1 and W = H-, Ci-g-alkyl-, 2-thienyl, 3- Thienyl, 3-indolyl-, 4-imidazolyl-, 2-pyridyl-, 3-pyridyl-, 4-pyridyl-, phenyl-, which up to three identical or different radicals of the group Cι- ₄ -alkyl-, CF ₃ - , C ₄ alkoxy, HO, BnO, F or Cl can carry, C ₃ _g-cycloalkyl, which can carry up to four identical or different C ₄ alkyl radicals, adamantyl, indanyl -, R ⁷ H-, Cι_ ₈ -alkyl-, phenyl-, which can carry up to three identical or different radicals of the group Cι- ₄ -alkyl-, CF ₃ -, Cι_ ₄ -alkoxy-, F- or Cl- , C ₃ _ ₈ cycloalkyl, which can carry up to four identical or different C ₄ alkyl radicals, R ⁸ H or C ₄ alkyl,

D:

O

NH -OH

- or - <

JN NH ₂ H _^1. or

^δ >

A: HOOC- (CH ₂ ) _t - (t = 1, 2 or 3), (HOOC-CH ₂ ) ₂ -CH-, HOOC-CH ₂ -CH (COOH) -, HOOC-CH (Cι- ₄ - Alkyl) -, HOOC-C (Ci- ₄ -alkyl) ₂ -, Cι_ ₆ -alkyl-OOC- (CH ₂ ) _t -,

B:

R ⁴ R ⁶ II —NC-CO-

RU ⁵

R ⁴ H, C ₄ alkyl or HOOC (CH ₂ ) _m - (m = 1, 2 or 3), R5 H, methyl,

R ⁶ -CH ₂ - (CR ⁷ R ⁸ ) -W or - (CR ⁷ R ⁸ ) _g -W with q = 0 or 1 and W = H-, Ci-g-alkyl-, 2-thienyl, 3- Thienyl, 3-indolyl-, 4-imidazolyl-, 2-pyridyl-, 3-pyridyl-, 4-pyridyl-, phenyl-, which up to three identical or different radicals of the group Cι- ₄ -alkyl-, CF ₃ - , Cι_ ₄ -alkoxy-, HO-, BnO-, F- or Cl- can carry, C ₃ _ ₈ -cycloalkyl-, which can carry up to four identical or different Cι- ₄ alkyl radicals, adamantyl, indanyl -, R ⁷ H-, Ci-s-alkyl, phenyl, which carry up to three identical or different radicals of the group Cι_ ₄ -alkyl-, CF ₃ -, Cι- ₄ -alkoxy-, F- or Cl- can, C ₃ _ ₈ cycloalkyl, which can carry up to four identical or different C ₄ alkyl radicals, R ⁸ H or C ₄ alkyl, 10

D:

O 1 or

_\ HI ^

H H Y-Z where either

a) in the case of X = 0 or NH,

Y -N =, -CH = and

Z -N =, -CH = mean

or

b) in the case of X = S,

Y -N = and

Z -N =, -CH = mean or

Y -CH = and Z -N = mean

, NH N -OH or ^

NH ^N NH.

and their salts with physiologically acceptable acids.

The amino acid derivatives represented by B are preferably (D) -configured, azetidinecarboxylic acid or proline in D are preferably (L) -configured.

Compounds of the formula I in which A, B, D, E and F have the following meanings are particularly preferred: 11 α)

A: C ₁ -2-alkyl-OOC-CH, HOOC-CH ₂ , HOOC-CH ₂ -CH ₂ , HOOC-CH (CH ₃ ), HOOC-CH (C ₂ H ₅ ),

B:

R ⁴ R ⁶ II —NC-CO-

R ⁴ H-, CH ₃ -

R ⁵ H-, CH ₃ -,

R ⁶ -W or -CH ₂ - (CR ⁷ R ⁸ ) -W with W = Cι- ₈ alkyl-, 2-thienyl,

3-thienyl, 3-indolyl, 4-imidazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, phenyl, which contains up to three identical or different radicals from the group CH ₃ -, CF ₃ -, CH ₃ can carry 0-, HO-, BnO-, F- or Cl-, cyclopentyl, cyclohexyl, cycloheptyl-, which can carry up to four methyl radicals, or - (CR ⁷ R ⁸ ) -W with W = Cι_ ₈ alkyl, 2-thienyl, 3-thienyl, 3-indolyl, 4-imidazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, phenyl, which is up to three identical or different Radicals of the group CH ₃ -, CF ₃ -, CH ₃ 0-, HO-, BnO-, F- or Cl- can carry, cyclopentyl, cycloheptyl- which can carry up to four methyl radicals,

R ⁷ H-, CH ₃ -,

R ⁸ H-, CH ₃ -,

D:

H

TH 12

F:

NH

- <

NH. or

^{ß) A:} C _1-2 -alkyl-OOC-CH _2, HOOC-CH ₂ -CH _2, HOOC-CH (CH _3), HOOC-CH (C ₂ H ₅

B:

R 1 ⁴ R1 ⁶

—N-C 1-CO-

R ⁴ H-, CH ₃ -

R5 H-, CH ₃ -,

R ⁶ - (CR ⁷ R ⁸ ) -W with W = cyclohexyl, sometimes up to four methyl residues,

R ⁷ H, CH ₃ -, R ⁸ H, CH ₃ -,

D:

O

H

'NC IIHH

NH

- <

NH. or 13 γ ⁾

A: -C ₂ alkyl-OOC-CH ₂ , HOOC-CH, HOOC-CH-CH ₂ , HOOC-CH (CH ₃ ), HOOC-CH (C ₂ H ₅ ),

B:

R ⁴ R ⁶ II —NC-CO-

R ⁴ H-, CH ₃ -

R ⁵ H-, CH ₃ -,

R ⁶ -CH ₂ - (CR ⁷ R ⁸ ) -W or - (CR ⁷ R ⁸ ) _q -W with q = 0 or 1 and W =

Ci-s-alkyl, 2-thienyl, 3-thienyl, 3-indolyl, 4-imidazolyl-2-pyridyl, 3-pyridyl, 4-pyridyl, phenyl, which has up to three identical or different radicals the group CH ₃ -, CF ₃ -, CH ₃ O-, HO-, Bno-, F- or Cl-, C ₅ _ ₇ -cycloalkyl-, which can carry up to four methyl radicals,

R ⁷ H, CH ₃ -,

R ⁸ H, CH ₃ -,

D:

O

E:

H

'NC IHH

y NH

NH.

or 14 δ)

A: -C ₂ alkyl-OOC-CH ₂ , HOOC-CH ₂ , HOOC-CH ₂ -CH ₂ , HOOC-CH (CH ₃ ), HOOC-CH (C ₂ H ₅ ),

B:

R ⁴ R ⁶ II —NC-CO-

R ⁴ H-, CH ₃ -

R ⁵ H-, CH ₃ -,

R ⁶ -CH ₂ - (CR ⁷ R ⁸ ) -W or - (CR ⁷ R ⁸ ) _q -W with q = 0 or 1 and W =

Cι- ₈ alkyl, 2-thienyl, 3-thienyl, 3-indolyl, 4-imidazolyl-2-pyridyl, 3-pyridyl, 4-pyridyl, phenyl, which is up to three identical or different radicals the group CH ₃ -, CF ₃ -, CH ₃ 0-, HO-, BnO-, F- or Cl-, C ₅ _ ₇ -cycloalkyl-, which can carry up to four methyl radicals,

R ⁷ H, CH ₃ -,

R ⁸ H, CH ₃ -,

D:

OO or

H

^• v H-KH-Y wherein VZ

a) in the case of X = 0,

Y -CH = and

Z -CH = mean or b) in the case of X = S

Y -N = and

Z -N =, -CH = mean or 15

Y -CH = and

Z -N =, mean

F:

NH

- NH 2

and their salts with physiologically acceptable acids.

The amino acid derivatives represented by B are preferably (D) -configured, azetidinecarboxylic acid or proline in D are preferably (L) -configured.

In addition to the substances mentioned in the examples, the following substances are particularly preferred:

MeOOC-CH ₂ - (D) -Cha-Pro-NH-CH ₂ -5- (2-.am) -thioph

HOOC-CH ₂ -CH ₂ - (D) -Cha-Aze-NH-CH ₂ -5- (2-am) -thioph

HOOC-CH ₂ - (D) -HCha-Aze-NH-CH ₂ -5- (2-am) -thioph

HOOC-CH ₂ - (D) -HCha-Pro-NH-CH ₂ -5- (2-am) -thioph

HOOC-CH ₂ - (D) -Cheg-Aze-NH-CH ₂ -5- (2-am) -thioph HOOC-CH ₂ - (D) -Chea-Aze-NH-CH ₂ -5- (2- .am) -thioph

CH ₃ OOC-CH ₂ - (D) -Chg-Pro-NH-CH ₂ -5- (2-am) -thioph

HOOC-CH ₂ - (D) -Cog-Pro-MH-CH ₂ -5- (2-am) -thioph

CH ₃ OOC-CH ₂ - (D) -Chg-Aze-MH-CH ₂ -5- (2-am) -thioph

HOOC-CH ₂ - (D) -4-MeCha-Pro-MH-CH ₂ -5- (2-am) -thioph HOOC-CH ₂ - (D) -Cha-Aze-NH-CH ₂ -5- ( 2-ham) -thioph

MeOOC-CH ₂ - (D) -Cha-Aze-NH-CH ₂ -5- (2-ham) -thioph

EtOOC-CH ₂ - (D) -Cha-Aze-NH-CH ₂ -5- (2-ham) -thioph

EtOOC-CH ₂ - (D) -Cha-Pro-NH-CH ₂ -5- (2-am) -thioph

EtOOC-CH ₂ - (D) -Chg-Pro-NH-CH ₂ -5- (2-am) -thioph EtOOC-CH ₂ - (D) -Chg-Aze-NH-CH ₂ -5- (2- am) -thioph

HOOC-CH ₂ - (N-Me) - (D) -Chg-Pro-NH-CH ₂ -5- (2-am) -thioph

HOOC-CH ₂ -CH ₂ - (D) -Cha-Pro-NH-CH ₂ -5- (2-am) -thiaz

HOOC-CH ₂ - (D) -Chg-Pro-NH-CH ₂ -5- (2-am) -thiaz

HOOC-CH ₂ -CH ₂ - (D) -Cha-Pro-NH-CH ₂ -2- (5-am) -thiaz HOOC-CH ₂ - (D) -HCha-Aze-NH-CH ₂ -2- (5-am) thiaz

HOOC-CH ₂ - (D) -HCha-Pro-NH-CH ₂ -2- (5-am) -thiaz

HOOC-CH ₂ - (D) -Cha-Aze-NH-CH ₂ -2- (5-am) -thiaz

HOOC-CH ₂ - (D) -Chg-Aze-NH-CH ₂ -2- (5-am) -thiaz

HOOC-CH ₂ - (D) -Cheg-Aze-NH-CH ₂ -2- (5-am) -thiaz HOOC-CH ₂ - (D) -Chea-Aze-NH-CH ₂ -2- (5- am) -thiaz

HOOC-CH ₂ - (D) -Cpg-Pro-NH-CH ₂ -2- (5-am) -thiaz

HOOC-CH ₂ - (D) -Cheg-Pro-NH-CH ₂ -2- (5-am) -thiaz 16

^• HOOC-CH ₂ - (D) _{-CPA-Pro-NH-CH2} -2- (5-am) -thiaz HOOC-CH ₂ - (D) _{-Chea-Pro-NH-CH2} -2- (5 -am) -thiaz CH ₃ OOC-CH ₂ - (D) -Chg-Pro-NH-CH ₂ -2- (5-am) -thiaz CH ₃ OOC-CH ₂ - (D) -Chg-Aze-NH -CH ₂ -2- (5-am) -thiaz HOOC-CH ₂ - (D) -Cog-Pro-NH-CH ₂ -2- (5-am) -thiaz HOOC-CH ₂ - (D) -4 -MeCha-Pro-NH-CH ₂ -2- (5-.am) -thiaz HOOC-CH ₂ - (N-Me) - (D) -Chg-Pro-NH-CH ₂ -2- (5-am ) -thiaz HOOC-CH ₂ - (D) -Cha-Pro-NH-CH ₂ -5- (2-am) -thiaz HOOC-CH ₂ -CH ₂ - (D) -Chg-Pro-NH-CH ₂ -5- (2-am) -for HOOC-CH ₂ - (D) -Chea-Pro-NH-CH ₂ -5- (2-am) -for HOOC-CH ₂ - (D) -Cpg-Pro NH-CH ₂ -5- (2-am) -for HOOC-CH ₂ - (D) -Cha-Aze-NH-CH ₂ -5- (2-am) -for HOOC-CH ₂ - (D) - Chg-Aze-NH-CH ₂ -5- (2-am) -for HOOC-CH ₂ - (D) -Chea-Pro-NH-CH ₂ -5- (2-am) -for

List of abbreviations:

Adaala: Adamantylalanine

Adagly: Adamantylglycine AIBN: Azobisisobutyronitrile

Ac: acetyl am: amidino

Aze: Azetidine carboxylic acid

Bn: benzyl Boc: tert. Butyloxycarbonyl

Bu: butyl

Cbz: Benzy1oxycarbony1

Cha: cyclohexylalanine

Chea: cycloheptylalanine Cheg: cycloheptylglycine

Chg: cyclohexylglycine

Cog: cyclooctylglycine

Cpa: cyclopentylalanine

Cpg: cyclopentylglycine d: doublet

TLC: thin layer chromatography

DCC: dicyclohexylcarbodiimide

Dch: dicyclohexylalanine

Dcha: dicyclohexylamine DCM: dichloromethane

DMF: dimethylformamide

DIPEA: Diisopropy1eth lamin

Dpa: Diphenylalanine

Et: Ethyl Eq: equivalents

Gly: glycine urine: hydroxyamidino 17

HCha homocyclohexylalanine, 2-amino-4-cyclohexylbutyric acid

HOSucc: hydroxysuccinimide

HPLC: high performance liquid chromatography imi: imidazole iPr: iso-propyl

Leu: Leucine

Solution: Solution m: Multiplet

Me: methyl α-MeCha: α-methylcyclohexylalanine ß, ß-Me ₂ Cha: 2-amino-3-cyclohexyl-3-methyl-butyric acid or ß, ß-dimethylcyclohexylalanine

4-MeCha: (4-methylcyclohex-l-yl) alanine γ-MeCha: (1-methylcyclohex-l-yl) alanine 3,3-Me ₂ Cha: (3, 3-dimethylcyclohex-l-yl) alanine

4-MeChg: (4-methylcyhex-l-yl) glycine

3,3-Me ₂ Chg: (3, 3-dimethylcyclohex-1-yl) glycine

MPLC: medium pressure liquid chromatography

MTBE: methyl tert. -butyl ether NBS: N-bromosuccinimide

Nog: norbornylglycine

Oxaz: oxazole

Ph: phenyl

Phe: phenylalanine PPA: propylphosphonic anhydride

Pro: proline

Py: pyridine

Pyr: 3, 4-dehydroproline pyraz: pyrazole pyrr: pyrrole q ^; quartet

RT: room temperature s: singlet sb: singlet broad t: triplet t: tertiary tBu: tertiary butyl tert: tertiary

TBAB: tetrabutylammonium bromide TEA: trietylamine

TFA: trifluoroacetic acid

TFAA: trifluoroacetic anhydride thiaz: thiazole thioph: thiophene Z: benzyloxycarbonyl 18th

The following definitions apply to the individual substituents in the description and the claims:

The term "cycloalkyl" by itself or as part of another substituent contains saturated or cyclic hydrocarbon groups which contain the stated number of carbon atoms.

C ₃ _ ₈ -cycloalkyl refers to saturated alicyclic rings with 3 to 8 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 4-methyl-cyclohexyl, cycloheptyl or cyclooctyl,

The term "alkyl" by itself or as part of another substituent means a linear or branched alkyl chain radical of the length given in each case. So Cι- ₄ alkyl, for example methyl, ethyl, 1-propyl, 2-propyl, 2-methyl-2-propyl, 2-methyl-1-propyl, 1-butyl, 2-butyl; C; _L _ ₆ alkyl, for example C ₄ alkyl, pentyl, 1-pentyl, 2-pentyl, 3-pentyl, 1-hexyl, 2-hexyl, 3-hexyl, 4-methyl-1-pentyl or 3,3-dimethyl -butyl; C ₈ alkyl, for example C ₆ alkyl, heptyl or octyl.

The term "alkoxy" by itself or as part of another substituent means a linear or branched alkyl chain radical which has the length indicated in each case and is bonded to the parent compound in question via an oxygen atom. So Cι_ ₄ -alkoxy means, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy, 2-methyl-2-propoxy, 2-methyl-l-propoxy, 1-butoxy, 2-butoxy.

The invention further relates to compounds that form the structural element

NH

D '/

Contain NH ₂ , in which D and E have the meaning given above and on the nitrogen atom of building block D there is a hydrogen atom, a protective group, an optionally substituted natural or unnatural amino acid, an optionally substituted carboxylic acid or an optionally substituted alkyl radical. The structural fragment is valuable as a component of serine protease inhibitors and in particular thrombin and kallikrein inhibitors. 19

The invention further relates to the intermediates of the formulas Ha and Ilb

A B D E CN Ha,

ABDE CSNH ₂ Ilb,

wherein A, B, D and E have the meaning given above.

The new intermediates are used to prepare compounds I and are valuable building blocks for the synthesis of serine protease inhibitors.

The compounds of the formula I can be present as such or in the form of their salts with physiologically tolerated acids. Examples of such acids are: hydrochloric acid, citric acid, tartaric acid, lactic acid, phosphoric acid, methanesulfonic acid, acetic acid, formic acid, maleic acid, fumaric acid, succinic acid, succinic acid, hydroxy acid, sulfuric acid, glutaric acid, aspartic acid, pyruvic acid, benzoic acid, glucuronic acid, oxalic acid, ascorbic acid and acetylglycine.

If in the compounds of the formula IR ¹ is C ₆ alkylOOC, aryl Co ₄ alkylOOC and / or F is hydroxyamidine, these compounds can act in vivo as prodrugs from which the corresponding carboxylic acid R ¹ = HOOC is produced enzymatically - or the corresponding amidines (F = C (= NH) NH ₂ ) are formed.

Furthermore, those compounds which are converted in vivo into pharmacologically active compounds of the general formula I are regarded as prodrugs of the compounds of the general formula I. In vivo metabolism can e.g. take place in the liver during the "first pass" metabolism.

The invention also relates to compounds and medicaments of the general formula I, but I not (N- (carboxy (CH ₂ ) _n ) -D-cyclohexylalanyl-N [[5-aminoiminomethyl) thiophen-2-yl] methyl] -L-prolinamide, with n = 1, 2 or 3.

The compounds of formula I are competitive inhibitors of trypsin-like serine proteases, especially thrombin and kininogenases such as kallikrein. They can be used for the following indications: 20th

Diseases whose pathomechanisr.rαs is based directly or indirectly on the proteolytic effects of thrombin

Diseases whose pathomechanism is based on thrombin-dependent activation of receptors and signal transduction

Diseases associated with stimulation [e.g. PAI-1, PDGF (platelet derived growth factor), P-selectin, ICAM-1, tissue factor] or inhibition (e.g. NO synthesis in smooth muscle cells) are associated with gene expression in body cells,

Diseases based on the mitogenic effects of thrombin

Diseases that are based on a throbin-dependent change in contractility and permeability of epithelial cells (e.g. vascular endothelial cells),

- Thrombin-dependent, thromboembolic events such as deep

Venous thrombosis, pulmonary embolism, myocardial or cerebral infarction, atrial fibrillation, bypass occlusion,

disseminated intravascular coagulation (DIC),

Reocclusion and to shorten the reperfusion time when comedicated with thrombolytics such as streptokinase, urokinase, prourokinase, t-PA, APSAC, plasminogen activators from the salivary glands of animals and the recombinant and mutant forms of all these substances,

the occurrence of early reocclusion and later restenosis after PTCA,

the thrombin-dependent proliferation of smooth muscle cells,

the accumulation of active thrombin in the CNS (e.g. in M. Alzheimer's),

- Tumor growth and against the adhesion and metastasis of tumor cells.

In particular, the new compounds for the therapy and prophylaxis of thrombin-dependent thromboembolic events such as deep venous thrombosis, pulmonary embolism, myocardial or cerebral infarction and unstable angina can continue to be used for the therapy of disseminated intravascular coagulation (DIC). Further They are suitable for combination therapy with thrombolytics such as streptokinase, urokinase, prourokinase, t-PA, APSAC and other plasminogen activators to shorten the reperfusion time and extend the reocclusion time.

Further preferred fields of application are the prevention of thrombin-dependent early reocclusion and late restenosis after percutaneous transluminal coronary angioplasia, the prevention of thrombin-induced proliferation of smooth muscle cells, the prevention of active thrombin accumulation in the CNS (eg in M. Alzheimer's disease), the fight against tumors and the prevention of mechanisms that lead to adhesion and metastasis of tumor cells.

The new compounds can also be used to coat artificial surfaces such as hemodialysis membranes and the hose systems and lines required for them, as well as oxygenators for extravascular circulation, stents and heart valves.

The new compounds can also be used in diseases whose pathomechanism is based directly or indirectly on the proteolytic action of kininogenases, in particular kallikrein, e.g. for inflammatory diseases such as asthma, pancreatitis, rhinitis, arthritis, urticaria and other internal inflammatory diseases.

The compounds according to the invention can be administered in the usual way orally or parenterally (subcutaneously, intravenously, intramuscularly, intraperitoneally, rectally). It can also be applied with vapors or sprays through the nasopharynx.

The dosage depends on the age, condition and weight of the patient and on the type of application. As a rule, the daily dose of active ingredient per person is between about 10 and 2000 mg with oral administration and between about 1 and 200 mg with parenteral administration. This dose can be given in 2 to 4 single doses or once a day as a depot form.

The new compounds can be used in the customary pharmaceutical application forms in solid or liquid form, for example as tablets, film tablets, capsules, powders, granules, dragées, suppositories, solutions, ointments, creams or sprays. These are manufactured in the usual way. The active ingredients can be combined with the usual pharmaceutical auxiliaries such as tablet binders, fillers, preservatives, tablet disintegrants, flow regulators, plasticizers, wetting agents, dispersants, 22

Emulsifiers, solvents, retardants, antioxidants and / or propellants are processed (see H. Sucker et al.: Pharmaceutical Technology, Thieme-Verlag, Stuttgart, 1978). The application forms thus obtained normally contain the active ingredient in an amount of 0.1 to 99% by weight.

Experimental part

The compounds of the formula I can be prepared in accordance with schemes I-III.

The building blocks A, B, D and E are preferably constructed separately beforehand and used in a suitably protected form (see scheme I-III).

Scheme I

A B D

OH H-CN

^• CN

-OHH ^• CN

^• CN

(P) (U) H ^• CN

(P) • CN

(P) _ *? H ₂ .S-alkyl

(P)

NH

NH (P) <NH ₂ ^ NH

H

NH ₂

(P = protecting group, (P) = protecting group or H)

Scheme I describes the linear structure of molecule I by coupling the amine HE-CN with the N-protected amino acid PD-OH to PDE-CN, splitting off the N-terminal protective group to HDE-CN, coupling with the N-protected amino acid PB- OH to PBDE-CN, splitting off of protective group P to HBDE-CN, 23 closing alkylation with the optionally protected (P) -AU building block (U = leaving group) or reductive alkylation with (P) -A'-U (U = aldehyde, ketone) or Michael addition with a suitable (P) -A "-C = C derivative to (P) -ABDE-CN. The nitrile function is converted into the amidine group either via the classic Pinner synthesis (R. Boder, DG Neilson, Chem. Rev. 1962, 6_1, 179) or via a modified Pinner synthesis which proceeds as an intermediate via iminothioester salts (H. Vieweg et al., Pharmazie 1984, 3.9, 226) or directly according to the method of A. Eschenmoser Helv. Chimica Acta £ 9 (1986) 1224. Subsequently protecting groups still present in the molecule are preferably cleaved off by acid hydrolysis.

If building block E is not installed as HE-CN, but as HE-CONH ₂ in the synthesis, one of the protected ones is used

Intermediate stages are the dehydration of the amide to nitrile function or the conversion into the thioamide function using Lawesson's reagent. Alternatively, building block E can be used as HE-CSNH ₂ in the synthesis.

Scheme II

B

(P) (U) H

(P)

(P) OH H '

(P) ^• OH H- ^• CN (P) • CN

(P). ^

NH ₂ -S-alkyl

(P)

^ NH

^ NH

(P) ~ ^ NH ₂ _ ^ NH

H

NH ₂ 24

Scheme II describes the linear structure of the molecule I by alkylation, reductive amination or Michael addition of HBP to suitable, optionally protected A building blocks to (P) -ABP, cleavage of the C-terminal protective group to (P) -AB-OH, Coupling with HDP to (P) -ABDP, cleavage of the C-terminal protective group to (P) -ABD-OH, coupling with HE-CN to (P) -ABDE-CN and conversion of this intermediate to the end product analogous to Scheme I.

In the case of compounds (P) -A-B-P with still free NH function on B, this must be provided with a suitable protective group before the C-terminal protective group is split off. The protective groups used in each case must be orthogonal to one another.

As an alternative to the HE-CN building block, HE-CONH ₂ , HE-CSNH _{2 /} HEC (NH) NH ₂ , HEC (NP) NH ₂ , HEC (NP) NHP can also be used, in which case the coupled intermediate (P ) -ABDE-CONH ₂ dehydrated to (P) -ABDE-CN or converted to (P) -ABDE-CSNH ₂ using Lawesson's reagent.

Scheme III

A B D

OH H ^• CN

(P) • CN

(P) y

NH ₂ • S-alkyl (P)

NH

^ NH (P) ^" \ NH ₂

_ ^ NH H NH ₂

Scheme III describes a very efficient way of preparing the compounds I by a convergent synthesis. The appropriately protected building blocks (P) -AB-OH and HDE-CN are coupled together and the resulting intermediate (P) -ABDE-CN is converted into the end product in accordance with Scheme I. 25th

As an alternative to HDE-CN, HD-Ξ-CONH ₂ or HDE-CSNH ₂ can also be used, in which case the coupled intermediate (P) -ABDE-CONH _{2 is} dehydrated to (P) -ABDE-CN or in (P) -ABDE-CSNH _{2 is} transferred.

Boc, Cbz or Fmoc, preferably Boc, are used as N-terminal protective groups; C-terminal protective groups are methyl, tert-butyl and benzyl. If there are several protective groups in the molecule, they must be orthogonal to one another if they are not to be split off simultaneously.

The required coupling reactions and the usual reactions of the introduction and deprotection of protective groups are carried out according to standard conditions of peptide chemistry (see M. Bodanszky, A. Bodanszky "The Practice of Peptide Synthesis", 2nd edition, Springer Verlag Heidelberg, 1994).

Boc protective groups are removed by means of dioxane / HCl or TFA / DCM, Cbz protective groups by hydrogenolysis or using HF. The saponification of ester functions takes place with LiOH or NaOH in an alcoholic solvent or in dioxane / water. t-Butyl esters are cleaved with TFA or HC1.

The reactions were monitored by TLC, the following eluents usually being used:

A. DCM / MeOH 95: 5

B. DCM / MeOH 9: 1

C. DCM / MeOH 8: 2 D. DCM / MeOH / 50% HOAc 40: 10: 5 E. DCM / MeOH / 50% HOAc 35: 15: 5

If column chromatographic separations are mentioned, these were separations over silica gel, for which the above-mentioned solvents were used.

Reversed phase HPLC separations were carried out with acetonitrile / water and HOAc buffer.

All reactions were routinely carried out under a nitrogen atmosphere. 26

The starting compounds can be prepared using the following methods:

As building blocks A for the alkylation e.g. -Bromacetic acid- tert-butyl ester, ß-bromopropionic acid-tert. -butyl ester, α-bromo-propionic acid tert. -butyl ester, γ-bromobutyric acid tert-butyl ester, -bromobutyric acid tert. -butyl ester, THP-protected bromoethanol, THP-protected γ-bromopropanol, for reductive amination e.g. Dihydroxyacetone, acetone dicarboxylic acid di-tert. butyl ester and for Michael addition e.g. Acrylic acid tert-butyl ester, methacrylic acid tert-butyl ester, fumaric acid di-tert-butyl ester. Unless they are commercially available, the tert-butyl esters mentioned are prepared analogously to G. Uray, W. Lindner, Tetrahedron 1988, AA, 4357-4362.

B building blocks:

There are many possibilities available in the literature for the general and special synthesis of amino acids. An overview of this includes Volume El6d / Part 1 - Houben-Weyl, p. 406 ff.

Commonly used starting materials were ethyl benzophenone imine acetate, diethyl acetamidomalonate and ethyl isonitrile acetate.

Various racemic glycine and alanine derivatives were prepared e.g. starting from ethyl isonitrile acetic acid and a corresponding ketone or aldehyde (see H.-J. Prätorius, J. Flossdorf, M.-R. Kula Chem. Ber. 1975, 10_8, 3079).

The syntheses of cyclooctylglycine, cycloheptylglycine, 2-norbonylglycine, adamantylalanine, γ-methylcyclohexylalanine, 4-isopropylcyclohex-1-yl-alanine, 4-methylcyclohex-l-yl-alanine, 4-methylcyclohex-1-ylglycine , Cyclo-eptylalanine and cyclopentylalanine were synthesized via the corresponding ethyl 2-formylamino-acrylic acid (U. Schöllkopf and R. Meyer, Liebigs Ann. Chem. 1977, 1174 and H.-J. Prätorius, J. Flossdorf, M. Kula, Chem. Ber. 1985, 108, 3079) starting from ethyl isocyanate with the respective carbonyl compounds cyclooctanone, cycloheptanone, 2-norbornanone, 1-formyladamantane, 1-formyl-1-methyl-cyclohexane, l-formyl-4-isopropyl-cyclohexane, l-formyl -4-methyl-cyclohexane, 4-methylcyclohexanone, formylcycloheptane and formylcyclopentane according to the following general rules: 27

General procedure for the synthesis of the 2-formylamino-acrylic acid ethyl ester:

The solution of 100 mmol of ethyl isocyanate in 50 mL of THF was added dropwise to 100 mmol of potassium tert-butoxide in 150 mL of THF at 0 to -10 ° C. After 15 min, 100 mmol of the corresponding carbonyl compound in 50 mL THF were added at the same temperature, the reaction mixture was allowed to slowly rise to RT and the solvent was removed on a rotary evaporator. The residue was mixed with 50 mL water, 100 mL acetic acid and 100 mL DCM and the

Product extracted with DCM. The DCM phase was dried over a ₂ SO ₄ and the solvent was removed on a rotary evaporator. If necessary, the almost pure products could be further purified by column chromatography over silica gel (eluent: mixtures of ether / petroleum ether).

General instructions for the amino acid hydrochlorides based on the 2-formylamino-acrylic acid ethyl esters:

100 mmol of the 2-formylamino-acrylic acid ethyl ester were hydrogenated with Pd / C (10%) hydrogen in 200 mL glacial acetic acid until the reaction was complete. The catalyst was then filtered off, the acetic acid was drawn off as far as possible on a rotary evaporator and the residue was heated to reflux in 200 ml of semi-concentrated hydrochloric acid for 5 h. The hydrochloric acid was removed on a rotary evaporator, the product was dried at 50 ° C. in vacuo and washed several times with ether. The hydrochlorides were obtained as weakly colored crystals.

Starting from 18.9 g (150 mmol) of cyclooctanone, 25.0 g of cyclooctylglycine hydrochloride were obtained. Starting from 22.4 g (200 mmol of cycloheptanone, 36.2 g of cycloheptylglycine hydrochloride were obtained. Starting from 16.5 g (150 mmol) of 2-norbornanone, 26.6 g of 2-norbonylglycine hydrochloride were obtained. Starting from 19.7 26.0 g of adamantylalanine hydrochloride were obtained in g (120 mmol) of 1-formyladamantane, and 16.6 g of γ-methylcyclohexylalanine were obtained starting from 12.6 g (100 mmol) of 1-formyl-1-methylcyclohexane Starting from 16.8 g (150 mmol) of 4-methylcyclohexanone, 25.9 g of 4-methylcyclohexylglycine hydrochloride were obtained, and starting with 15 g of trans-l-formyl-4-methylcyclohexane, 18 g of trans-4-methylcyclohexane were obtained -1-yl-alanine hydrochloride Starting from 9 g of 3,3-dimethyl-1-formylcyclohexane, 10 g of 3,3-dimethylcyclohex-1-yl-alanine hydrochloride were obtained. 28

The required for the synthesis of aldehyde, l-formyl-3, 3-dimethyl cyclohexane, following Moskal and Lensen (Rec. Trav Chim Pays-Bas, 1987, 106, _137-141...) Was made:

5 A solution of n-butyllithium in n-hexane (72 mL, 115 mmol) was added dropwise within 10 min at -60 ° C to a stirred solution of diethyl isocyanomethylphosphonate (17 mL, 105 mmol) in 280 mL anhydrous diethyl ether. The resulting suspension was stirred for 15 min at -60 ° C and within 10 min with

10 a solution of 3, 3-dimethylcyclohexanone (13 g, 105 mmol) in 100 ml of anhydrous diethyl ether were added, the temperature being kept below -45 ° C. The reaction mixture was allowed to come to 0 ° C., was stirred at this temperature for 90 min and 150-200 ml of 38% strength aqueous hydrochloric acid were carefully added. For complete

Hydrolysis was stirred vigorously at RT for 15 h. The organic phase was separated off and washed with 200 ml of water, saturated sodium bicarbonate solution and saturated sodium chloride solution. It was dried over magnesium sulfate, filtered off and concentrated on a rotary evaporator to remove the solvents.

20 distant. The residue obtained was used without further purification as the starting material for the synthesis of the amino acid.

Cyclopentylglycine was prepared by hydrolysis with 6N hydrochloric acid from N-acetyl- (D, L) -cyclopentylglycine, which 25 according to the literature specification of J.T. Hill and F.W. Thin,

J. Org. Chem. (1965), 30, 1321.

Boc- (D) -oc-methyl-cyclohexylalanine

3.4 g (12.2 mmol) of Boc- (D) - α-methyl-Phe-OH were added in 100 mL MeOH at 50 ° C in the presence of 250 mg of 5% Rh on Al ₂ 0 _{3 for} 24 h 30 10 bar hydrogenated with hydrogen. After filtration and

Stripping off the solvent 2.8 g of Boc- (D) - α-methyl-Cha-OH. ^i- H NMR (DMSO-d ⁶ , δ in ppm): 12 (very broad signal, COOH);

1.7-0.8 (25H); 1.35 (s, Boc); 1.30 (s, Me).

35 Boc- (3-Ph) -Pro-OH was analogous to a procedure by J.Y.L. Chung et al. (J.Y.L. Chung et al. J.Org.Chem. 1990, 5j5, 270).

Preparation of Boc-1-tetralinylglycine:

40

Boc-1-tetralinylglycine was prepared starting from 1,2-dihydronaphthalene. 1,2-Dihydronaphthalene was first converted into 1-tetralyl bromide with HBr (analogous to J. Med. Chem 1994, 37, 1586). The bromide was then reacted with diethyl acetamidomalonate 45, hydrolytically cleaved and the a-amino acid obtained was converted into the Boc-protected form under standard conditions. Another display option is from E. Reimann and 29

D. Voss (E. Reimann; D. Voss Arch. Pharm 1977, 310, 102).

Representation of Boc- (D, L) Dch-OH:

Boc- (D, L) -Dpa-0H (1 mmol) was hydrogenated in 12 mL MeOH together with catalytic amounts of 5% RI1 / Al ₂ O ₃ at 5 bar. After filtration and removal of the solvent in vacuo, the product was obtained in quantitative yield.

Representation of H- (D, L) -Chea-OH:

4.0 g of cycloheptylmethyl methanesulfonate (19.39 mmol), prepared from cycloheptylmethanol and methanesulfonic acid chloride, were together with 4.9 g of benzophenoneimmethyl methyl ester (18.47 mmol), 8.9 g of dry, finely powdered potassium carbonate (64.65 mmol) and 1 g Tetrabutylammonium bromide (3 mmol) in 50 mL dry acetonitrile heated to reflux for 10 h in an inert gas atmosphere. The potassium carbonate was then filtered off, the filtrate was evaporated to dryness, and the crude product was hydrolyzed directly with 20 ml of 2N hydrochloric acid in 40 ml of ethanol with stirring at RT for 1.5 h. After the reaction solution had been diluted, benzophenone was extracted with ethyl acetate, then H-D, L-chea-OEt was extracted in the alkaline range (pH = 9) with DCM, and the solution was dried over magnesium sulfate and evaporated. Yield 3.7 g ≤ 95% of theory.

Boc- (D, L) - (3, 4, 5- (MeO) ₃ ) Phe-OH was prepared by alkylation of methyl benzophenone with trimethoxybenzyl chloride, subsequent introduction of Boc protective groups and ester saponification.

H- (D, L) - ß, ß-Me ₂ Cha-OH was prepared according to U. Schöllkopf, R. Meyer, L. Ann. Chem. 1977. 1174-82.

The amino acids mentioned were converted into the Boc-protected form in each case using di-tert-butyl dicarbonate in water / dioxane and then recrystallized from ethyl acetate / hexane mixtures or column chromatographically on silica gel (mobile phase: ethyl acetate / petroleum ether - Mixtures) cleaned.

The Boc-protected amino acids were used as B building blocks in accordance with Scheme I. 30th

Some of the amino acids mentioned were converted as B building blocks into the corresponding benzyl esters and linked to the appropriately protected A building blocks. In the case of compounds with still free NH function, this was then protected with a Boc group, the benzyl ester group was hydrogenated, and the AB-OH building block was purified by crystallization, salt precipitation or column chromatography. This route is exemplified for tBuOOC-CH ₂ - (Boc) (D) Cha-OH described below.

Synthesis of (D) -cyclohexylalanine benzyl ester:

A suspension of 100 g (481 mmol) (D) -cyclohexylalanine hydrochloride, 104 g (962 mmol) benzyl alcohol and 109.7 g (577 mmol) p-toluenesulfonic acid monohydrate in 2200 mL toluene was slowly refluxed on a water separator heated. In a temperature range of 80-90 ° C, evolution of hydrogen chloride and the dissolution of the suspension to a clear solution were observed. When no more water separated (approx. 4 h), 500 ml of toluene were distilled off, the reaction mixture was allowed to cool overnight, the residue formed was filtered off and washed twice with 1000 ml of hexane each time. The residue obtained (195 g) was then slurried in 2000 ml of dichloromethane, mixed with 1000 ml of water and adjusted to pH 9-9.5 with stirring by successive addition of 50% sodium hydroxide solution. The organic phase was separated off, washed twice with 500 ml of water each time, dried over sodium sulfate, filtered off from the drying agent and concentrated the filtrate, giving 115 g (94%) of the title product as a light oil.

N- (tert-butyloxycarbonylmethylene) - (D) -cyclohexylalanine benzyl ester:

115 g (440 mmol) of (D) -cyclohexylalanine benzyl ester were added in

2000 ml of acetonitrile were dissolved, 607.5 g (4.40 mol) of potassium carbonate and 94.3 g (484 mmol) of tert-butyl bromoacetate were added at RT and the mixture was stirred at this temperature for 3 days. The carbonate was filtered off, washed with acetonitrile, the mother liquor was concentrated (30 ° C., 20 mbar), the residue was taken up in 1000 ml of methyl tert. -butyl ether and extracted the organic phase with 5% citric acid and saturated sodium bicarbonate solution. The organic phase was dried over sodium sulfate, the drying agent was filtered off, the mixture was concentrated and the oil obtained (168 g) was used directly in the following reaction. 31

N-Boc-N- (tert-butyloxycarbonylmethylene) - (D) -cyclohexylalanine benzyl ester:

The oil obtained in the previous synthesis (168 g, 447 mmol) was dissolved in 1400 mL acetonitrile, 618 g (4.47 mol) potassium carbonate powder and 107.3 g (492 mmol) di-tert-butyl dicarbonate were added, and 6 Stirred at RT for days. The potassium carbonate was suctioned off, washed with about 1000 ml of acetonitrile and the filtrate was concentrated. 230 g of the desired product were obtained.

N-Boc-N- (tert-butyloxycarbonylmethylene) - (D) -cyclohexylalanine-cyclohexylai-monium salt:

115 g of N-Boc-N- (tert-butyloxycarbonylmethylene) - (D) -cyclohexylalanbenzyl ester were dissolved in 1000 ml of pure ethanol and at 25-30 ° C. in the presence of 9 g of 10% Pd on activated carbon for 2 h Normal pressure hydrogenated with hydrogen. After filtration and removal of the solvent on a rotary evaporator, 100 g (260 mmol) of a yellow oil were obtained, which was taken up in 1600 ml of acetone and heated to reflux. The heating bath was removed and a solution of 27 g (273 mmol) of cyclohexylamine in acetone was added rapidly via a dropping funnel. When the reaction mixture was cooled to RT, the desired salt crystallized out. The solid was filtered off, washed with 200 ml of acetone and recrystallized again from acetone for final purification. After drying the residue in a vacuum drying cabinet at 30 ° C., 70.2 g of the desired salt were obtained as a white powder.

N-Boc-N- (tert-butyloxycarbonylmethylene) - (D) -cyclohexylglycine-cyclohexylammonium salt was prepared in an analogous manner from cyclohexylglycine as a starting material.

The N-Boc-N- (tert-butyloxycarbonylmethylene) - (D) -cylcoheptyl-glycine or N-Boc-N- (tert-butoxycarbonylmethylene) - (D) -cyclopentylglycine derivative were derived from the corresponding Cycloheptyl and cyclopentylglycine compounds prepared.

N-Boc-N- (tert-butyloxycarbonylethylene) - (D) -cyclohexylalanine-cyclohexylammonium salt:

a) 3-Bromo-propionic acid tert-butyl ester

16.64 g (109 mmol) bromopropionic acid, 150 mL condensed 2-methylpropene and 2mL concentrated sulfuric acid were placed at -30 ° C in a nitrogen countercurrent in a glass vessel suitable for an autoclave, sealed tightly and stirred for 72 h at RT. For working up, the reaction vessel was opened again 32

Cooled to -30 ° C. and the reaction solution was carefully poured into 200 ml of an ice-cold, saturated sodium bicarbonate solution. Excess 2-methylpropene was allowed to evaporate with stirring, the residue was extracted three times with 50 ml of dichloromethane each time, the combined organic phases were dried over sodium sulfate, the desiccant was filtered off and the mixture was concentrated under a water pump vacuum. The oily residue was purified by column chromatography (mobile phase n-hexane, later n-hexane / diethyl ether 9: 1). 18.8 g of the title compound were obtained.

b) N- (tert-Butyloxycarbonylethylene) - (D) -cyclohexylalanine benzyl ester

49.4 g (189 mmol) (D) -cyclohexylalanine benzyl ester were in

250 mL acetonitrile dissolved, at room temperature with 31.6 g (151 mmol) bromopropionic acid tert. -butyl ester and boiled under reflux for 5 days. The precipitate formed was filtered off, washed several times with acetonitrile, the filtrate was concentrated under water pump vacuum, the residue was taken up in 350 ml of dichloromethane and the organic phase was extracted with 5% citric acid and saturated sodium hydrogen carbonate solution. The organic phase was dried over sodium sulfate, the drying agent was filtered off and the mixture was concentrated. The oily residue was purified by column chromatography (mobile phase dichloromethane, later dichloromethane / methanol 95: 5). A slightly contaminated oil was obtained which was used directly in the following reaction.

c) N-Boc-N- (tert-butyloxycarbonylethylene) - (D) -cyclohexylalanine benzyl ester

The oil obtained in the previous synthesis (30 g, max. 70 mmol) was dissolved in 150 mL acetonitrile, with 28 mL (160 mmol) di-isopropylethylamine and 19.2 g (88 mmol) di-tert. butyl dicarbonate were added and the mixture was stirred at RT for 3 days. The reaction mixture was concentrated on a rotary evaporator in a water-jet vacuum, the residue was taken up in n-hexane and washed five times with 3 ml of a 5% citric acid solution, the combined organic phases were dried over sodium sulfate, the anticaking agent was filtered off, and the mixture was concentrated and subjected the residue of a column chromatography separation (eluent hexane / ethyl acetate 95: 5). 32.66 g (64 mmol) of the desired product were obtained. 33 d) N-Boc-N- (tert-butyloxycarbonylethylene) - (D) -cyclohexylalanine cyclohexylammonium salt

32.66 g (64 mmol) of N-Boc-N- (tert-butyloxycarbonylethylene) - (D) -cyclohexylalanine benzyl ester were dissolved in 325 ml of pure ethanol and at 25-30 ° C. in the presence of 3 g of 10% strength Pd on activated carbon hydrogenated with hydrogen for 14 h at normal pressure. After filtration of the solution through Celite ^®, washing with ethanol and removal of the solvent on a rotary evaporator to give 26.7 g of a yellow oil which was taken up in acetone and heated to reflux. The heating bath was removed and a solution of 7 g (70 mmol) of cyclohexylamine in acetone was added rapidly via a dropping funnel. When the reaction mixture was cooled to RT, the desired salt crystallized out. The solid was filtered off, washed with 25 ml of acetone and recrystallized again from acetone for final purification. After drying the residue in a vacuum drying cabinet at 30 ° C., 26.6 g (54 mmol) of the desired salt were obtained as a white powder.

N-Boc-N- (tert.butyloxycarbonylmethylene) - (D) -cyclohexylalanyl-3, 4-dehydroproline:

a) N-Boc-Pyr-OH (5 g, 23.45 mmol) was dissolved in MeOH (50 mL) and HC1 in dioxane (4N, 30 mL) was added. The mixture was then heated under reflux for 12 h. The solvent was spun off and H-Pyr-OMe hydrochloride was obtained as the product. Yield: 3.84 g (100%).

b) N- (t-Bu0 ₂ C-CH ₂ ) -N-Boc- (D) -Cha-OH (8 g, 20.75 mmol) was dissolved in dichloromethane (75 mL) and at -10 ° C with Ethyldiisopropylamine (15.5 mL, 89.24 mmol) was added. After stirring for 5 min at this temperature, a solution of H-Pyr-OMe hydrochloride (3.4 g, 20.75 mmol) in dichloromethane (25 mL) was added dropwise. A solution of propanephosphonic anhydride in ethyl acetate (50%, 20 ml, 26.96 mmol) was then added dropwise and the mixture was stirred at -10 to 0 ° C. for 2 h. The mixture was diluted with dichloromethane and washed with saturated sodium hydrogen carbonate solution (2 x 80 mL), 5% citric acid solution (2 x 15 mL) and saturated sodium chloride solution (1 x 20 mL). The organic phase was dried over sodium sulfate and the solvent was spun off. The crude product was purified by means of flash chromatography (silica gel, dichloromethane / methanol = 95/5). Yield: 6.2 g (60%). 34 c) N- (t-Bu0 ₂ C-CH ₂ ) -N-Boc- (D) -Cha-Pyr-OMe (5.5 g, 11, 12 mmol) was dissolved in dioxane (40 mL) with Sodium hydroxide solution (IN, 22.2 mL, 22.24 mmol) was added and the mixture was stirred at RT for 2 h. The dioxane was spun off, the aqueous phase was washed with ethyl acetate and acidified to pH 1-2 with potassium hydrogen sulfate solution (20%). The aqueous phase was extracted with dichloromethane and the combined organic phases were dried over sodium sulfate. Yield: 5 g (94%), colorless foam. Recrystallization from water-saturated n-hexane gave the corresponding carboxylic acid as colorless crystals (mp = 158-160 ° C).

N-Boc-N- (tert-butyloxycarbonylmethylene) - (D) -cyclohexylglycyl-3, 4-dehydroproline and N-Boc-N- (tert.-butyloxycarbonylmethylene) - (D) -cyclohexylglycyl-proline:

These compounds were prepared in an analogous manner from N-Boc-N- (tert-butyloxycarbonylmethylene) - (D) -cyclohexylglycm and 3, 4-dehydro-prolin methyl ester or proline methyl ester.

N-Boc-N- (tert.butyloxycarbonylmethylene) - (D) -cyclohexylalanyl-proline:

a) N- (t-Bu0 ₂ C-CH ₂ ) -N-Boc- (D) -Cha-OH (20 g, 51.88 mmol) was dissolved in dry methylene chloride (100 mL). After cooling down

-5 ° C, N-ethyldiisopropylamine (90 mL, 518.88 mmol) was added dropwise and the mixture was stirred for 5 min. H-Pro-OBn x HC1 (12.54 g, 51.88 mmol) was then added and, after stirring for 5 min, 50% propanephosphonic anhydride solution in ethyl acetate (45.1 mL, 62.26 mmol) was diluted with Methylene chloride (45 mL) was added dropwise over 30 min. After stirring for 1 h at 0-5 ° C, the mixture was slowly warmed to RT and stirred for 12 h at RT. The mixture was diluted with methylene chloride and successively with sat. Sodium bicarbonate solution, 5% citric acid solution and sat. Washed sodium chloride solution. After drying over sodium sulfate, the solvent was distilled off in vacuo. Yield: 28.9 g (yellowish oil, 97%).

b) The product obtained according to a) (28.5 g, 49.76 mmol) was dissolved in methianol (650 mL), 10% Pd on carbon (1.8 g) was added and hydrogenation was carried out at RT and 1 atmosphere. The catalyst was then filtered off through Celite® and the filtrate was concentrated in vacuo. Yield: 22.2 g (colorless foam, 92%). 35

D building blocks:

The compounds (L) -proline and (L) -azetide carboxylic acid used as D building blocks can be purchased either as free amino acids or as Boc-protected compounds or as corresponding methyl esters. If the (L) -3, 4-dehydroproline or a correspondingly protected derivative was used as the D building block, the compounds shown were generally hydrogenated on the final stage to give the corresponding proline derivatives.

E modules:

The synthesis of the E building blocks was carried out as follows:

5-aminomethyl-2-cyanothiophene:

This building block was synthesized in accordance with the regulation in WO 95/23609

5-aminomethyl-2-cyanofuran:

a) 5-Cyanofuran-2-carbaldehyde

165 mL (264 mmol) of a 1.6 molar solution of n-butyllithium in n-hexane were added to a solution of 26.7 g (264 mmol) of diisopropylamine in 600 mL of tetrahydrofuran cooled to -78 ° C. within 20 min. The solution was allowed to come to -20 ° C, cooled again to -75 ° C and slowly added a solution of 22.3 g (240 mmol) of 2-cyano-furan in 100 mL tetrahydrofuran at this temperature. The mixture was allowed to stir for 30 min, 93 mL of dimethylformamide were slowly added dropwise and the mixture was stirred again for 30 min. For working up, a solution of 40 g of citric acid in 200 ml of water was added at -70 ° C. The mixture was concentrated on a rotary evaporator, mixed with 600 ml of saturated sodium chloride solution and extracted three times with 200 ml of diethyl ether each. The combined organic extracts were dried over magnesium sulfate. After the desiccant had been filtered off, the solvent was distilled off in a water jet vacuum and the residue was purified by column chromatography (mobile phase dichloromethane). The eluate was concentrated and the residue was subjected to steam distillation (boiling range of the azeotrope with water: 60-65 ° C. at p = 0.1 mm Hg). After extraction of the distillate with diethyl ether, drying of the organic phase and concentration of the solution, 10.6 g (88 mmol, 36%) were obtained. 36 of the title link. ⁱ H-NMR (270 MHz, d ₆ -DMSO): δ = 7.7 (d, 1H), 7.8 (d, 1H), 9.75 (s, 1H).

b) 5-hydroxymethyl-2-cyanofuran

2.34 g (62 mmol) of sodium borohydride were added in portions at -30 ° C. to a solution of 30 g (0.25 mol) of 5-cyanofuran-2-carbaldehyde in 500 ml of absolute ethanol. The mixture was stirred at -30 ° C. for 2 h and the cooled reaction solution was brought to pH 7 with the aid of a 5% citric acid solution in water. The reaction mixture was concentrated in a water jet vacuum, the residue was mixed with saturated sodium chloride solution, extracted several times with 150 ml of diethyl ether, dried the combined organic phases over magnesium sulfate, filtered off the drying agent and distilled off the solvent in a water jet vacuum at RT. 27 g (22 mmol, 88%) of the title compound were obtained as a dark red oil, which was used in the following reactions without further purification. ⁱ H-NMR (250 MHz, d ₆ -DMSO): δ = 4.4 (m, 2H), 5.6 (bs, 1H), 6.6 (d, 1H), 7.5 (d, 1H) ).

c) 5-Bromomethyl-2-cyanofuran

38 g (145 mmol) of triphenylphosphine were added to a solution of 15 g (121 mol) of 5-hydroxymethyl-2-cyanofuran in 250 mL of tetrahydrofuran. The mixture was cooled to -10 ° C. and a solution of 48 g (145 mmol) of tetrabromomethane in 100 ml of tetrahydrofuran was added. The mixture was allowed to warm to RT and was stirred at this temperature for 3 h. The reaction mixture was concentrated on a rotary evaporator in a water jet vacuum and the residue was purified by column chromatography (mobile phase petroleum ether: dichloromethane 1: 1, R _f = 0.5). 11.5 g of the title compound were obtained. ⁱ H-NMR (250 MHz, d ₆ -DMSO): δ = 4.8 (m, 2H), 6.7 (d, 1H), 7.7 (d, 1H).

d) 5-N, N-bis (tert-butoxycarbonyl) aminomethyl-2-cyanofuran: A solution of 22.9 g (123 mmol) of 5-bromo-methyl-2-cyanofuran cooled to 0 ° C in 400 mL tetrahydrofuran 4.0 g (135 mmol) of sodium hydride (80% suspension in mineral oil) were added in portions. Then a solution of

29.4 g (135 mmol) of di-tert-butyl-iminodicarboxylate in 200 ml of tetrahydrofuran were added dropwise, the temperature not exceeding 5 ° C. The mixture was allowed to warm to RT and stir overnight. Since the conversion was incomplete (TLC control), a total of 1.2 g of sodium hydride were added in three portions over a period of 9 h. To complete the conversion, the mixture was heated at 35 ° C. for a further 3 h and then left at RT 37 cool and slowly added 600 mL of a saturated ammonium chloride solution. The solvent was distilled off in a water jet vacuum, the residue was extracted several times with ethyl acetate, the combined organic phases were washed with saturated sodium chloride solution, dried over magnesium sulfate and concentrated on a rotary evaporator. This gave 37.3 g of an oily residue which was still di-tert. -butyl iminodicarboxylate contained and was used as a crude product in the following reaction. ^X H-NR (250 MHz, d ₆ -DMSO): δ = 1.40, 1.45 (s, 18H), 4.75 (s, 2H), 6.55 (d, 1H), 7.55 (d, 1H).

e) 5-aminomethyl-2-cyanofuran hydrochloride

37.3 g of 5-N, N-bis (tert-butoxycarbonyl) aminomethyl-2-cyano-furan (crude product from d), maximum 123 mmol) were dissolved in 600 ml of ethyl acetate and cooled to 0 ° C. It was saturated with hydrogen chloride gas, a white precipitate separating out after 30 min. The mixture was allowed to come to RT, stirred overnight, and the resulting suspension was then concentrated

Rotary evaporator, stirred the residue with diethyl ether, filtered off the solvent and dried the solid residue at RT in vacuo. 15.1 g (77% yield over two steps) of the title compound were obtained as a slightly ocher powder. ⁱ H-NMR (250 MHz, d ₆ -DMSO): δ = 4.15 (bs, 2H), 6.85 (d, 1H), 7.65 (d, 1H), 8.8-9.0 (bs, 3H).

5-aminomethyl-2-amidothiocarbonyl-l, 3, 4-thiadiazole hydrochloride:

a) N-chloroacetyl-N'-carbamoylcarbonyl hydrazine

55.1 g (0.66 mol, 1.3 equivalents) of sodium hydrogen carbonate were added in portions to a solution of 52 g (0.5 mol) of oxamic acid hydrazide in 700 ml of dirnethylformamide. With gentle cooling (10 ° C.), 52.2 ml (0.66 mol) of chloroacetyl chloride were added dropwise over the course of 45 minutes. For working up, the cloudy solution was concentrated. The residue still containing dirnethylformamide was mixed with 700 ml of diethyl ether and stirred vigorously. The precipitate was filtered off and recrystallized from water. Since the solid obtained could not be dried, it was taken up again in acetone, stirred well, suction filtered and washed with acetone and diethyl ether. After drying the residue at 50 ° C. in a high vacuum, 68.6 g (76%) of the desired product were obtained. 38 b) 5-chloromethyl-2-amidothiocarbonyl-1,3,4-thiadiazole

A suspension of 23.0 g (128 mmol) of N-chloroacetyl-N'-carbamoylcarbonyl-hydrazine in 240 mL of dioxane was treated in succession with 14.1 g (141 mmol) of potassium hydrogen carbonate and 28.5 g (64 mmol) of phosphorus pentasulfide . The mixture was stirred at 50 ° C. for 2 h and at RT overnight. The mixture was neutralized with saturated sodium hydrogen carbonate solution. It was extracted several times with ethyl acetate, the combined organic phases were dried over sodium sulfate and concentrated on a rotary evaporator. The residue was taken up in diethyl ether, stirred vigorously and filtered off. After drying, 21 g (85%) of the desired product were obtained, which was reacted further without further purification.

5-azidomethyl-2-amidothiocarbonyl-1,3,4-thiadiazole

The product obtained according to b) (21 g, 108 mmol) was dissolved in 300 mL dry acetone, 17.6 g (271 mmol) sodium azide was added and the mixture was heated to 50-60 ° C. The mixture was stirred at this temperature for 11 h, during which the initially yellow solution turned deep orange. The mixture was stirred at RT overnight, filtered off from the resulting solid, washed with acetone, concentrated, taken up in 400 ml of ethyl acetate and stirred with 50 ml of water. Insoluble portions of the two-phase mixture were suctioned off, then the phases were separated. The ethyl acetate phase was dried and concentrated, and the residue was purified by column chromatography (silica gel, dichloromethane / methanol). 11.6 g of the desired product were obtained.

d) 5-aminomethyl-2-amidothiocarbonyl-1,3,4-thiadiazole

5.3 g (26.5 mmol) of 5-azidomethyl-2-amidothiocarbonyl-1,3,4-thiadiazole were dissolved in 26 ml of methanol and initially at

RT with 11 mL (79 mmol) triethylamine, then at 0 ° C slowly added 8.0 mL (79 mmol) propanedithiol. The reaction was highly exothermic, the reaction solution turned black. The mixture was stirred at RT for a further two hours, the precipitate formed was filtered off with suction, washed with methanol and diethyl ether, taken up in 50 ml of ethanol, stirred at RT overnight, filtered off with suction and washed with a little ethanol. The residue was stirred in diethyl ether / petroleum ether, suction filtered and washed with petroleum ether. After drying in vacuo, 2.4 g of the desired product were obtained, which despite 39 existing impurities were directly implemented further (Example 10).

2-aminomethyl-5-cyanothiazole hydrochloride

a) 2- (Boc-aminomethyl) thiazole-5-carboxylic acid methyl ester

10.2 g (74.7 mmol) of methyl 2-chloro-3-oxopropionate and 14.2 g (74.7 mmol) of N-Boc-glycine-thioamide were suspended in 100 ml of dichloroethane, and the mixture was stirred at 70 ° C. for 6 h (TLC

Control), concentrated in vacuo, the residue taken up in water, extracted several times with dichloromethane at PH 5, the organic phases dried over magnesium sulfate and again concentrated in vacuo. The crude product (15.3 g) was dissolved in diethyl ether, then n-hexane was added, the solution was decanted and the residue was subjected to this process again. After repeating twice, the collected solutions were combined and evaporated in vacuo. 8.1 g of the desired compound were obtained.

b) 2- (Boc-aminomethyl) thiazole-5-carboxylic acid

6.5 g (23.8 mmol) of 2- (boc-aminomethyl) thiazole-5-carboxylic acid methyl ester were dissolved in 50 ml of THF, 11.9 ml of 2N aqueous lithium hydroxide solution (23.8 mmol) were added, the mixture was stirred at room temperature for 3 h (TLC control) and then with 50 ml of water are added, the mixture is extracted three times with dichloromethane to remove non-polar by-products, the aqueous phase is acidified to PH3 and the product of value is isolated by triple extraction with dichloromethane. After drying over

Magnesium sulfate, the organic phase was spun in and stirred in a little diethyl ether, giving 4.8 g of the desired compound.

c) 2- (Boc-aminomethyl) thiazole-5-carboxamide

4.95 g (19.16 mmol) of 2- (bocaminomethyl) thiazole-5-carboxylic acid were suspended in 300 ml of dichloromethane, saturated with ammonia gas, and dropwise with 44.2 ml of propanephosphonic anhydride (50% solution in ethyl acetate, 57.4 mmol)) and then stirred for 3 hours at room temperature. After adding 50 ml of water and stirring for 30 minutes, the precipitate was filtered off with suction (3.2 g), the organic phase was separated off, the aqueous phase was extracted four times with dichloromethane, the collected organic phases were dried over magnesium sulfate and concentrated in vacuo. 40 narrowed (1.7 g). The two crude product parts were extracted from dichloromethane and thus obtained in pure form.

d) 2- (Boc-aminomethyl) - 5-cyanothiazole

To a mixture of 50 ml dichloromethane, 5.5 g (21.4 mmol) 2- (Boc-aminomethyl) thiazole-5-carboxamide and 13.8 g (106.9 mmol, 18.3 ml) diisopropylethylamine was added dropwise at 0 ° C 1.2 g (53.4 mmol, 7.5 ml) trifluoroacetic anhydride and the solution was then stirred for 24 hours at room temperature. The

Reaction solution was diluted with dichloromethane, washed twice with water, three times with aqueous hydrochloric acid solution (PH ~ 2), once with water, dried over magnesium sulfate and concentrated in vacuo. The crude product (8.0 g) was used in the subsequent reaction without further purification.

e) 2-aminomethyl-5-cyanothiazole hydrochloride

2- (Boc-aminomethyl) - 5-cyanothiazole (8.0 g crude product) were dissolved in 150 ml isopropanol, mixed with 67 ml 5M isopropanolic hydrochloric acid solution and stirred overnight in a sealed vessel. The solution was concentrated in vacuo, codistilled several times with dichloromethane and extracted from ether. 4.0 g of the desired compound were obtained in pure form.

Example 1: N- (Hydroxycarbonylmethylene) - (D) -cyclohexylglycyl-prolyl- [5- (2-amidino) -thienylmethyl] amide hydroacetate:

a) 3,4-Dehydroprolyl- [5- (2-cyano) thienylmethyljamide

Boc-3, 4-dehydroproline (5 g, 23.4 mmol) and 5-aminomethyl-2-cyanothiophene hydrochloride (4.5 g, 25.8 mmol) were dissolved in dichloromethane (25 mL) and at 0 ° C with ethyldiisopropylamine (28 mL, 163.8 mmol) with a 50% solution of propanephosphonic anhydride in ethyl acetate (24.8 mL, 117 mmol). After stirring at 0 ° C. for 1 h, the mixture was warmed to RT and stirred at RT for 12 h. The reaction mixture was diluted with dichloromethane, washed with sodium hydrogen sulfate solution (4x), sodium hydrogen carbonate solution (3x) and saturated sodium chloride solution (lx). After drying over sodium sulfate and filtering off the drying agent, the solvent was distilled off in a water jet vacuum. To split off the Boc group, the residue was added to dichloromethane (95 mL), stirred at RT, evaporated to dryness, codistilled twice with dichloromethane, concentrated again and purified by column chromatography. 6.6 g of the 41 wanted a product that was still slightly solvent-based.

b) N- (tert-butoxycarbonylmethylene) - (N-Boc) - (D) -cyclohexylglycyl-3, 4-dehydroprolyl- [5- (2-cyano) -thienyl] methylamide

t-Bu0 ₂ C-CH ₂ -Boc- (D) -Chg-OH (6.5 g, 17.5 mmol) and H-pyr-NH-CH ₂ -5- (2-CN) -thioph-hydrochloride (4.72 g, 17.5 mmol) were suspended in dichloromethane (90 mL) and ethyldiisopropylamine (11.3 g, 87.5 mmol) was added to give a clear, slightly reddish solution. The reaction mixture was cooled to about 5 ° C. and a 50% solution of propanephosphonic anhydride in ethyl acetate (18 ml) was added dropwise. After stirring overnight at RT, the mixture was diluted with dichloromethane (100 ml) and washed with dilute sodium hydrogen sulfate solution (3x), saturated sodium hydrogencarbonate solution (2x) and water (lx). After drying over sodium sulfate, the solvent was distilled off in a water jet vacuum after the desiccant had been separated off. 11.15 g of a slightly reddish brown oil were obtained.

c) N- (tert.butoxycarbonylmethylene) - (N-Boc) - (D) -cyclohexyl-glycyl-3,4-dehydroprolyl- [5- (2-amidothiocarbonyl) thienylmethyljamide

The product obtained according to b) was dissolved in pyridine (68 ml) and triethylamine (11.5 ml). The reaction mixture was cooled to 0 ° C and saturated with hydrogen sulfide (the solution turned green). The reaction solution stood at RT overnight. The excess hydrogen sulfide was with

Nitrogen is displaced and the solvent is distilled off in a water jet vacuum. The residue was dissolved in diethyl ether (500 mL), washed with dilute sodium hydrogen sulfate solution (3x), saturated sodium hydrogen carbonate solution (2x) and water (lx). After drying over sodium sulfate, the solvent was distilled off in a water jet vacuum. The yellow tough crude product (10.92 g) was used in the next step without further purification.

d) N- (tert-Butoxycarbonylmethylene) - (N-Boc) - (D) -cyclohexyl-glycyl-3,4-dehydroprolyl- [5- (2-S-methyliminothiocarbonyl) thienylmethyl] amide hydroiodide

The crude product obtained according to c) was dissolved in dichloromethane (115 ml) and methyl iodide (14.99 g, 105.6 mmol) was added. After stirring over the weekend at RT that was 42

Distilled off solvent in a water jet vacuum. 12.6 g of a yellowish solid foam were obtained.

e) N- (tert-butoxycarbonylmethylene) - (N-Boc) - (D) -cyclohexyl-glycyl-3, 4-dehydroprolyl- [5- (2-amidino) thienylmethyl] amide hydroacetate

The crude product obtained according to d) was mixed with 25.5 ml of a 10% solution of ammonium acetate in methanol (2.55 g ammonium acetate, 38.12 mmol). Because after stirring over

At RT at night, starting material was still present, a further 3.0 ml of a 10% strength solution of ammonium acetate in methanol were added and the mixture was stirred at RT overnight. The solvent was then distilled off in a water jet vacuum, the residue was taken up in dichloromethane, the salts were filtered off with suction and the filtrate was concentrated, 13.3 g of crude product being obtained in the form of a yellow solid foam.

f) N- (Hydroxycarbonylmethylene) - (D) -cyclohexylglycyl-3, 4-dehydroprolyl- [5- (2-amidino) thienylmethyl] amide hydroacetate

The product obtained according to e) was dissolved in 180 ml dichloromethane and dropwise mixed with ethereal hydrochloric acid solution (46.5 ml, approx. 5 mol / 1). After stirring overnight at RT, the solvent was distilled off in a water jet vacuum, dichloromethane was added to the residue and the solvent was distilled off in a water jet vacuum (2x). The crude product (8.96 g) was dissolved in methanol and converted into the acetate salt via an ion exchanger (Fluka, order no. 00402).

g) N- (Hydroxycarbonylmethylene) - (D) -cyclohexylglycyl-prolyl- [5- (2-amidino) thienylmethyl] amide hydroacetate

1.5 g (2.96 mmol) of the crude product obtained according to f) were dissolved in 60 ml of dry methanol and 15 ml of glacial acetic acid, 0.75 g of 10% palladium on activated carbon was added and the mixture was hydrogenated at RT under a slight hydrogen overpressure for 24 h. The catalyst was then replaced twice and hydrogenated for a further two days. After filtering off the catalyst and concentrating the solution, the crude product was purified by means of MPLC (RP-18, acetonitrile / water) and the fractions were freeze-dried. 0.5 g of the target product was obtained as an amorphous white solid, FAB-MS (M + H +): 450 43

As an alternative to the form of presentation described here, Boc-prolin can be used instead of Boc- (L) -3, 4-dehydroproline, in which case the hydrogenation on the final stage is omitted.

The following was prepared as in Example 1:

Example 2:

N- (Hydroxycarbonylethylene) - (D) -cyclohexylalanyl-prolyl- [5- (2-amidino) thienylmethyl] amide hydroacetate, FAB-MS (M + H ⁺ ): 478

The preparation was carried out starting from N- (tert-butoxycarbonylethylene) - (N-Boc) - (D) -cyclohexylalanine and 3,4-dehydroprolyl- [5- (2-cyano) -thienylmethyl] amide including over several stages borrowed hydrogenation analogously to Example 1.

The following compounds can be prepared analogously to Example 1:

Example 3: N- (Hydroxycarbonylmethylene) - (D) -cycloheptylglycyl-prolyl-

[5- (2-amidino) thienylmethyl] mid hydroacetate or N- (hydroxycarbonylmethylene) - (L) -cycloheptylglycyl-prolyl- [5- (2-amidino) - thienylmethyl] amide hydroacetate. The two diastereomeric compounds are separated on the final stage by means of reversed phase HPLC (acetonitrile / water and HOAc buffer).

Example 4:

N- (hydroxycarbonylmethylene) - (D) -cyclopentylglycyl-prolyl- [5- (2-amidino) -thienylmethyl] amide hydroacetate or N- (hydroxycarbonylmethylene) - (L) -cyclopentylglycyl-prolyl- [5- (2-amidino) thienylmethyl] amide hydroacetate. The two diastereomeric compounds are separated on the final stage by means of reversed phase HPLC (acetonitrile / water and HOAc buffer).

Example 5:

N- (Hydroxycarbonylmethylene) - (D) -cycloheptylalanyl-prolyl- [5- (2-amidino) -thienylmethyl] amide hydroacetate or N- (hydroxycarbonylmethylene) - (L) -cycloheptylalanyl-prolyl- [5 - (2-amidino) thienylmethyl] amide hydroacetate. The two diastereomeric compounds are separated on the final stage by means of reversed phase HPLC (acetonitrile / water and HOAc buffer).

Example 6:

N- (Hydroxycarbonylmethylene) - (D) -cyclopentylalanyl-prolyl- [5- (2-amidino) -thienylmethyl] amide hydroacetate or N- (hydroxycarbonylmethylene) - (L) -cyclopentylalanyl-prolyl- [5 - (2-amidino) thienylmethyl] amide hydroacetate. The separation of the two 44 diastereomeric compounds takes place on the final stage by means of

Reversed phase HPLC (acetonitrile / water and HOAc buffer).

Example 7: N- (Hydroxycarbonylmethylene) - (D) -adamantylalanyl-prolyl- [5- (2-amidino) -thienylmethyl] amide hydroacetate or N- (hydroxycarbonylmethylene) - (L) -adamantylalanyl-prolyl - [5- (2-amidino) thienylmethyl] amide hydroacetate. The two diastereomeric compounds are separated on the final stage by means of reversed phase HPLC (acetonitrile / water and HOAc buffer).

Example 8:

N- (Hydroxycarbonylmethylene) - (D) -cyclohexylglycyl-prolyl- [5- (2-.amidino) furylmethyl] amide hydroacetate:

a) Prolyl- [5- (2-cyano) furylmethyl] amide hydrochloride

At RT, 1.0 g (4.6 mmol) of Boc-ProOH and 5.57 mL (432.5.) Were added to a suspension of 737 mg (4.6 mmol) of 5-aminomethyl-2-cyanofuran hydrochloride in 20 mL of dichloromethane mmol) of ethyl diisopropylamine. With gentle cooling, 4.92 mL (23.2 mmol) of a 50% solution of propanephosphonic anhydride in ethyl acetate were added dropwise at 5 ° C. After stirring overnight at RT, the mixture was evaporated to dryness, the residue was taken up in ethyl acetate and the

Destruction of the excess PPA stirred in water for 30 min. After the phases had been separated, the organic phase was freed from ethyldiisopropylamine using 20% sodium hydrogen sulfate solution. After drying over sodium sulfate and filtering off the drying agent, the solvent was distilled off in a water jet vacuum and the residue was dried in a high vacuum. 1.43 g (97%) of a yellowish oil were obtained, which was directly reacted further.

The oil obtained according to a) (1.43 g, 4.48 mmol) became

Cleavage of the Boc group dissolved in 45 mL dichloromethane and mixed with 9 mL of an approx. 5 m solution of hydrogen chloride in diethyl ether at RT. The mixture was allowed to stir overnight and the reaction mixture was concentrated to dryness on a rotary evaporator. 1.14 g (83%) of the title compound were obtained.

b) N- (tert-butoxycarbonylmethylene) - (N-Boc) - (D) -cyclohexyl-glycyl-prolyl- [5- (2-cyano) -furylmethyl] amide

1.66 g (4.48 mmol) t-Bu0 ₂ C-CH ₂ - (Boc) - (D) -Chg-OH and 1.38 g

(4.48 mmol) prolyl- [5- (2-cyano) furylmethyl] amide hydrochloride were suspended in 20 mL dichloromethane and with 5.37 mL 45

(31.3 mmol) of ethyldiisopropylamine were added. The reaction mixture was cooled to about 5 ° C. and 4.75 ml of a 50% solution of propanephosphonic anhydride in ethyl acetate were added dropwise, the solution clarifying. After stirring overnight at RT, the mixture was evaporated to dryness, the residue was taken up in ethyl acetate and the mixture was stirred in water for 30 min to destroy the excess PPA. The organic phase was separated off and washed with 20% sodium hydrogen sulfate solution in order to remove residues of ethyldiisopropyl-a in. After drying over sodium sulfate, the solvent was distilled off in a water jet vacuum after the desiccant had been separated off. 2.67 g (96%) of the desired product were obtained as an oil.

c) N- (tert-butoxycarbonylmethylene) - (N-Boc) - (D) -cyclohexylglycyl-prolyl- [5- (2-amidothiocarbonyl) furylmethyl] amide

The product obtained according to b) was dissolved in pyridine (16 ml) and triethylamine (8 ml). The reaction mixture was saturated with hydrogen sulfide at RT and stirred at RT overnight. The excess hydrogen sulfide was displaced with nitrogen and the reaction mixture was poured onto 200 ml of ice-cooled 5% sodium hydrogen sulfate solution. The mixture was extracted three times with 50 ml of ethyl acetate and the combined ethyl acetate phases were washed again with 5% sodium hydrogen sulfate solution in order to remove residues of pyridine. It was then washed again with saturated sodium bicarbonate solution, dried over sodium sulfate and the solvent was distilled off in a water jet vacuum. The crude product obtained (2.55 g) was used in the next step without further purification.

d) N- (tert-Butoxycarbonylmethylene) - (N-Boc) - (D) -cyclohexylglycyl-prolyl- [5- (2-S-methyliminothiocarbonyl) -furylmethyl] amide hydroiodide

The crude product obtained according to c) was dissolved in 40 ml acetone and mixed with 2.89 ml (46.2 mmol) methyl iodide. After stirring overnight at RT, the solvent was distilled off in a water jet vacuum. After drying at RT in vacuo, 2.57 g of a solid foam were obtained. 46 e) N- (tert.butoxycarbonylmethylene) - (N-Boc) - (D) -cyclohexylglycyl-prolyl- [5- (2-amidino) furylmethyl] amide hydroacetate

The crude product obtained according to d) (2.57 g, 3.43 mmol) was dissolved in 25 mL acetonitrile, mixed with 0.53 g (6.87 mmol) ammonium acetate and stirred for eight hours at 40 ° C. and overnight at RT. The solvent was then distilled off in a water jet vacuum, the residue was taken up in dichloromethane, the salts were filtered off with suction and the filtrate was concentrated. The crude product was purified by reversed phase HPLC (acetonitrile / water and acetic acid buffer) to give 67 mg of a yellow solid foam.

f) N- (Hydroxycarbonylmethylene) - (D) -cyclohexylglycyl-prolyl- [5- (2-amidino) furylmethyl] amide hydroacetate

The product obtained according to e) (67 mg, 0.1 mmol) was mixed with 5 ml of a 1 N aqueous hydrochloric acid solution and 2 ml of dioxane. After stirring overnight at RT, the mixture was diluted with water and the mixture obtained was freeze-dried. The crude product thus obtained was purified by MPLC (RP-18, acetonitrile / water) to give 12 mg of the desired product. FAB-MS (M + H ⁺ ): 434

Example 9: N- (Hydroxycarbonylmethylene) - (D) -cycloexylalanyl-prolyl- [5- (2-amidino) furylmethyl] amide hydroacetate

FAB-MS (M + H ⁺ ): 448

The representation was carried out analogously to Example 8, with b)

N- (tert.butoxycarbonylmethylene) - (N-Boc) - (D) -cyclohexylalanine was used instead of N- (tert.butoxycarbonylmethylene) - (N-Boc) - (D) - cyclohexylglycm.

Example 10: N- (Hydroxycarbonylmethylene) - (D) -cyclohexylalanyl-prolyl- [5- (2-amidino) -l, 3, 4-thiadiazolmethyl] amide hydroacetate, FAB-MS (M + H ⁺ ): 466:

The illustration was carried out analogously to Example 9, with prolyl- [5- (2-amidothiocarbonyl) -1,4,4 thiadiazolmethyl] amide hydrochloride being used instead of prolyl- [5- (2-cyano) furylmethyl] amide hydrochloride in b), so that c) was eliminated. In e) methanol was used as the solvent instead of acetonitrile. In f) the mixture was stirred at 60 ° C. for 3 h instead of at RT overnight. 47

Example 11: 1- [N- (Hydroxycarbonylmethylene) - (D) -cyclohexyl-glycyl] -azetidine-2-cabonic acid- [5- (2-amidino) thienylmethyl] amide

a) 1- [N-t-Butoxycarbonyl) - (D) -cyclohexylglycyl] -azetidine-2-carboxylic acid- [5- (2-cyano) thienylmethyl] amide:

To a solution of 3.9 g (11.5 mmol) of 1- [N- (t-butoxycarbonyl) - (D) -cyclohexylglycyl] -azetidine-2-carboxylic acid

(WO 9429336) and 2 g (11.5 mmol) of 5-aminomethyl-2-cyanothiophene hydrochloride in 40 ml of methylene chloride were added dropwise

-5 ° C 5.9 g (45.8 mmol) diisopropylethylamine and then 11.5 mL (14.9 mmol) 50% propanephosphonic anhydride solution in ethyl acetate. The mixture was stirred for 2 h, during which the temperature rose to 10 ° C. The organic phase was washed with water, 5% sodium bicarbonate and 5% citric acid solution, dried over sodium sulfate and evaporated to dryness. Purification by column chromatography (silica gel, eluent: ethyl acetate) of the residue gave 4.5 g (85% of theory) of white, amorphous powder, FAB-MS: 461 (M + H ⁺ ).

b) 1- [N- (t-Butoxycarbonylmethylene) - (D) -cyclohexylglycyl] - azetidine-2-carboxylic acid- [5- (2-cyano) thienylmethyl] amide

4.5 g (3.8 mmol) of the above compound were dissolved in 70 ml of isopropanol, 12.3 ml of 4N hydrochloric acid in dioxane were added and the mixture was left to stand at RT overnight. After the solvent had been distilled off, the residue was dissolved in methylene chloride and extracted 3 times with water. The combined aqueous extracts were made alkaline with 1N sodium hydroxide solution, the separated oily base was extracted 3 times with methylene chloride and the solvent was then distilled off. 2.9 g (8 mmol) of oil remained. This was dissolved in 50 ml of methylene chloride and 10 ml of acetonitrile and, after addition of 2.1 g (16 mmol) of diisopropylethylamine and 1.5 g (7.6 mmol) of t-butyl bromoacetate, left to stand at RT for 24 h.

The organic phase was washed twice with 5% citric acid solution, 5% sodium hydrogen carbonate solution and water, dried over sodium sulfate and the solvent was distilled off. 3.3 g (92% of theory) of slightly yellowish oil remained. FAB-MS: 475 (M + H ⁺ ). 48 c) 1- [N-Hydroxycarbonylmethylene] - (D) -cyclohexylglycyl] -azetidine-2-carboxylic acid- [5- (2-amidino) thienylmethyl] amide

The conversion of the above product into the amidine was carried out analogously to Example 1. The crude amide obtained contained considerable amounts of a by-product and had to be purified by column chromatography (silica gel, eluent: methylene chloride: methanol: acetic acid = 24: 6: 1.5). 0.9 g was isolated as a white, amorphous powder. The t-butyl ester group was split off by standing in 3N hydrochloric acid for 12 hours. After the hydrochloric acid had been distilled off at the end with the addition of toluene, the hydrochloric acid residue was converted into the betaine by chromatography on a silica gel column with a methanol / 25% ammonia eluent (50 / 2.5). 0.45 g of white, amorphous powder, FAB-MS: 463 (M + H ⁺ ) was obtained.

Example 12:

1- [N- (Hydroxycarbonylmethylene) - (D) -cyclohexylalanyl] -azetidine-2-carboxylic acid- [5- (2-amidino) -thienylmethyl] amide can be prepared analogously to Example 11.

Example 13:

Analogously to Example 8, N- (hydroxycarbonylethylene) - (D) - cyclohexylalanyl-prolyl- [5- (2-amidino) furylmethyl] amide hydroacetate can be prepared.

Example 14:

N- (Hydroxycarbonylmethylene) - (D) -cyclohexylalanyl-prolyl- [2- (5-amidino) -thiazolylmethyl] amide can be prepared according to the following reaction sequence: Coupling of (t-Bu0C-CH ₂ -) - (Boc) - (D) -Cha-Pro-OH with H ₂ N-CH ₂ - (5-CN) -2-thiaz to (t-Bu0 ₂ C-CH ₂ -) - (Boc) - (D) -Cha- Pro-NH-CH- (5-CN) -2-thiaz, amidine formation and subsequent deprotection analogous to Example 8c-f.

Example 15:

N- (Hydroxycarbonylmethylene) - (D) -cyclohexylglycyl-prolyl- [2- (5-amidino) -thiazolylmethyl] amide can be prepared according to the following reaction sequence: Coupling of (t-Bu0 ₂ C-CH-) - (Boc ) - (D) -Chg-Pro-OH with H ₂ N-CH ₂ - (5-CN) -2-thiaz to (t-Bu0 ₂ C-CH ₂ -) - (Boc) - (D) -Chg -Pro-NH-CH ₂ - (5-CN) -2-thiaz, amidine formation and subsequent deprotection analogous to Example 8c-f. 49

Example 16:

N- (Hydroxycarbonylmethylene) - (D) -p-metoxyphenylalanyl-prolyl- [2- (5-amidino) -thienylmethyl] amide can be prepared analogously to WO98 / 06741 (Example 14) from the corresponding ABD and EF building blocks .

Example 17:

N- (Hydroxycarbonylmethylene) - (D) -p-trifluoromethylphenylalanyl-prolyl- [2- (5-amidino) -thienylmethyl] amide can be prepared analogously to WO98 / 06741 (Example 14) from the corresponding ABD and EF building blocks .

Example 18:

N- (Hydroxycarbonylmethylene) - (D) -cyclohexylalanyl-prolyl- [5- (2-hydroxyamidino) thienylmethyl] amide can be passed through

Reaction of (t-Bu0C-CH ₂ -) - (Boc) - (D) -Cha-Pro-NH-CH ₂ - (2-CN) -5-thioph with hydroxylamm hydrochloride (methanol, diisopropylethylamine, room temperature) and subsequent deprotection (HCl in dichloromethane at room temperature).

Example 19:

N- (methoxycarbonylmethylene) - (D) -cyclohexylalanyl-prolyl- [5- (2-hydroxyamidino) thienylmethyl] amide can be obtained by reacting (t-Bu0 ₂ C-CH ₂ -) - (Boc) - ( D) -Cha-Pro-NH-CH- (2-CN) -5- thioph with hydroxylamm hydrochloride (methanol, diisopropylethylamine, room temperature) and subsequent deprotection and transesterification (HCl in methanol at room temperature).

Example 20: N- (Ethoxycarbonylmethylene) - (D) -cyclohexylalanyl-prolyl- [5- (2-hydroxyamidino) thienylmethyl] amide can be obtained by reacting (t-Bu0 ₂ C-CH ₂ -) - (Boc ) - (D) -Cha-Pro-NH-CH ₂ - (2-CN) -5- thioph with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) and subsequent deprotection and transesterification (HCl in ethanol at room temperature).

Example 21:

N- (Hydroxycarbonylmethylene) - (D) -cyclohexylglycyl-prolyl- [5- (2-hydroxyamidino) thienylmethyl] amide can be obtained by reacting (t-Bu0 ₂ C-CH ₂ -) - (Boc) - ( D) -Chg-Pro-NH-CH ₂ - (2-CN) -5- thioph with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) and subsequent deprotection (HCl in dichloromethane at room temperature). 50

Example 22:

N- (methoxycarbonylmethylene) - (D) -cyclohexylglycyl-prolyl- [5- (2-hydroxyamidino) thienylmethyl] amide can be obtained by reacting (t-Bu0 ₂ C-CH ₂ -) - (Boc) - ( D) -Chg-Pro-NH-CH ₂ - (2-CN) -5- thioph with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) and subsequent deprotection and transesterification (HCl in methanol at room temperature).

Example 23: N- (Ethoxycarbonylmethylene) - (D) -cyclohexylglycyl-prolyl- [5- (2-hydroxyamidino) thienylmethyl] amide can be obtained by reacting (t-Bu0 ₂ C-CH ₂ -) - (Boc ) - (D) -Chg-Pro-NH-CH ₂ - (2-CN) -5- thioph with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) and subsequent deprotection and transesterification (HCl in ethanol at room temperature). Example 24:

N- (Hydroxycarbonylmethylene) - (D) -cyclohexylglycyl-azetidine-2-carboxylic acid- [5- (2-hydroxyamidino) -thienylmethyl] amide can be obtained by reacting (t-Bu0 ₂ C-CH ₂ -) - ( Boc) - (D) -Chg-Aze-NH-CH ₂ - (2-CN) -5-thioph with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) and subsequent deprotection (HCl in dichloromethane at room temperature).

Example 25: N- (Methoxycarbonylmethylene) - (D) -cyclohexylglycyl-azetidine-2-carboxylic acid- [5- (2-hydroxyamidino) -thienylmethyl] amide can be obtained by reacting (t-Bu0C-CH ₂ -) - (Boc) - (D) -Chg-Aze-NH-CH ₂ - (2-CN) -5-thioph with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) and subsequent deprotection and transesterification (HCl in methanol at room temperature) .

Example 26:

N- (Ethoxycarbonylmethylene) - (D) -cyclohexylglycyl-azetidine-2-carboxylic acid - [5- (2-hydroxyamidino) thienylmethyl] amide can be obtained by reacting (t-Bu0 ₂ C-CH ₂ -) - ( Boc) - (D) -Chg-Aze-NH-CH ₂ - (2-CN) -5-thioph with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) and subsequent deprotection and transesterification (HCl in ethanol at room temperature). 51

Pharmacological examples

Example A

Chromogenic test for kallikrein inhibitors

Reagents: Kallikrein from human plasma (No. K 3126, Sigma, Deisenhofen, Germany)

Substrate: Chromozym GK (No. 709875, Boehringer, Mannheim, Germany)

Buffer: 20mM Tris (HCl pH = 8.50

Experimental procedure:

The chromogenic test for determining kallikrein activity is carried out in microplates. 2 μl of the substance solution in DMSO are added to 93 μl buffer, mixed with a final concentration of 0.01 units / ml kallikrein. It is incubated at 20 to 25 ° C for 10 minutes. The test is started by adding 100 μl substrate (500 μmol / 1 final

Concentration) can be added. After a further 30 minutes of incubation, the absorption is measured at 405 nm in the photometer.

Example B thrombin time

Reagents: Thrombin Reagent (Cat. No. 126 594, Boehringer, Mannheim, Germany)

Preparation of citrate plasma:

9 parts of venous human blood of the V. cephalica are mixed with one part of sodium citrate solution (0.11 mol / 1). Then centrifuged. The plasma can be stored at -20 ° C.

Experimental procedure:

50 μl test substance solution and 50 μl citrate plasma are incubated for 2 minutes at 37 ° C. (CL8, ball type, Bender & Hobein, Munich, FRG). Then 100 μl thrombin reagent (37 ° C.) is added. The time until the fibrin clump is formed is determined. 52

Example C

Chromogenic test for thrombin inhibitors

Reagents: Human Plasma Thrombin (No. T-8885, Sigma, Deisenhofen, Germany)

Substrate: H-D-Phe-Pip-Arg-pNA2HCl (S-2238,

Chromogenix, Mölndahl, Sweden)

Buffer: Tris 50 mmol / 1, NaCl 154 mmol / 1, pH 8.0

Experimental implementation:

The chromogenic test can be carried out in microtiter plates. 10 μl of substance solution in DMSO are added to 250 μl of buffer with thrombin (final concentration 0.1 NIH units / ml) and incubated for 5 minutes at 20 to 28 ° C. The test is carried out by adding 50 μl

Substrate solution in buffer (final concentration 100 μmol / 1) started, incubated at 28 ° C and stopped after 5 minutes by adding 50 μl citric acid (35%). The absorption is measured at 405/630 nm.

Example D

Platelet aggregation in platelet-rich plasma

Reagents: Human Plasma Thrombin (No. T-8885, Sigma, Deisenhofen, Germany)

Production of the citrate-rich platelet-rich plasma:

Venous blood from the cephalic vein of healthy drug-free test subjects is collected. The

Blood is mixed 9 to 1 with 0.13 molar trisodium citrate.

Platelet-rich plasma (PRP) is produced by centrifugation at 250 x g (10 minutes at room temperature). Platelet-poor plasma (PPP) is through

Centrifugation for 20 minutes at 3600 x g. PRP and PPP can be stored for 3 hours at room temperature in closed PE containers. The platelet concentration is measured with a cell counter and should be between 2.5 to

2.8-10 ° 7ml.

Experimental procedure:

The platelet aggregation is measured turbitrimetrically at 37 ° C. (PAP 4, Biodata Corporation, Horsham,

PA, USA). Before thrombin is added, 215.6 ul PRP for 3 minutes with 2.2 ul test substance 53 incubated and then stirred at 1000 rpm for 2 minutes. At a final concentration of 0.15 NIH-Units / ml, 2.2 μl thrombin solution lead to the maximum agregation effect at 37 ° C / 1000 rpm. The inhibited effect of the test substances is determined by the aggregation rate (slope) of thrombin without substance with the Rate of thrombin is compared with test substance at different concentrations.

Claims

54

claims

1. Use of compounds of formula I.

A — B-D — E — F I where A, B, D, E, and F have the following meanings:

A:

R ^

R ¹ (CH ₂ ). (CH ₂

R ^J in

m 0, 1, 2 or 3, n 0, 1 or 2,

R ¹ HOOC-, Cι- ₆ -alkyl-OOC-, aryl-C ₀ - ₄ -alkyl-OOC or -OH, R ² H-, Cι- ₄ -alkyl- or R ¹ - (CH ₂ ) _m -,

R ^{3 is} H or C ₄ alkyl,

represent

B:

R ⁴ R ⁶ II -NC-CO-

R5 in which

R ^{4 is} H, C ₄ alkyl or R ¹ - (CH ₂ ) _m - (where R ¹ and m have the meaning given above), 55

R ⁵ -CH ₂ - (CR ⁷ R ⁸ ) -W or - (CR ⁷ R ⁸ ) _g -W with q = 0 or 1 and W = H-, Ci-g-alkyl-, 2-thienyl, 3- Thienyl, 3-indolyl-, 4-imidazolyl-, 2-pyridyl-, 3-pyridyl-, 4-pyridyl-, phenyl-, which up to three identical or different radicals of the group Cι_ ₄ -alkyl-, CF ₃ -, Cι- ₄ alkoxy, HO, BnO, F or

Cl- can carry C ₃ - ₈ cycloalkyl, which can carry up to four identical or different C ₄ alkyl residues and / or in which one or two CC single bonds in the ring can be replaced by a C = C double bond and / or to which a phenyl ring can be fused, C ₂ -C bicycloalkyl or CiQ tricycloalkyl, or

R ⁴ and R ⁶ together represent a butylene group or one

- (CH ₂ ) _r - (CR ¹² R ⁸ ) _p group with r = 2 or 3 and p = 0 or 1 and R ¹² = cyclohexyl, phenyl, R ⁸ = H or Cι- ₄ alkyl, R ⁵ H or C ₁ _ ₄ alkyl,

R ⁷ H, Cι_ ₈ -alkyl-, phenyl-, which carry up to three identical or different radicals of the group Cι- ₄ -alkyl-, CF ₃ -, C ₁ - ₄ -AI- koxy-, F- or Cl- can, C ₃ _ ₈ -cycloalkyl-, which can carry up to four identical or different Cι_ ₄ -alkyl radicals,

R ^{8 is} H or C ₄ alkyl,

D:

O or

Rio

I ¹ /.X ^X , ^* N \ ^

R ⁹ R ¹¹ Y-Z

X S, 0 or NH,

Y -N = or -CH =, z mean -N = or -CH =,

and

R ^{9 is} H or C ₃ alkyl, R ¹⁰ H or C ₄ alkyl, R ¹¹ H or C ₄ alkyl, 56

, NH N '-OH

-y or ~~ - f

NH, NH. as well as their produgs and their salts with physiologically compatible acids, for the manufacture of medicaments for diseases whose pathomechanism is based directly or indirectly on the proteolytic action of kininogenases, in particular kallikrein.

2. Use of compounds of formula I according to claim 1 for the manufacture of medicaments for the treatment of inflammatory

"diseases such as asthma, pancreatitis, rhinitis, arthritis, urticaria and other internal diseases in which kallikrein plays a role.

3. Use according to one of claims 1 or 2, wherein in the 20 compounds of formula I the groups A, B, D, E and F have the following meaning:

A: HOOC- (CH ₂ ) _t -, -C-6-alkyl-OOC- (CH ₂ ) t ", (t = 1, 2 or 3),

(HOOC-CH ₂ ) ₂ -CH-, HOOC-CH ₂ -CH (COOH) -, HOOC-CH (Cι- ₄ alkyl) -, ²⁵ HOOC-C (Cι_4-alkyl) ₂ -,

B:

30 R ⁴ R ⁶ II —NC-CO-

R ⁴ H-, Ci- ₄ -alkyl-, HOOC- (CH ₂ ) _m - (m = 1, 2 or 3),

₃₅ R ⁵ H, methyl

R ⁶ -W, -CH ₂ - (CR ⁷ R ⁸ ) -W with W = Cι_ ₈ alkyl-, 2-thienyl, 3-thienyl, 3-indolyl-, 4-imidazolyl-, 2-pyridyl-, 3rd -Pyridyl-, 4-pyridyl-, phenyl-, which up to three identical or different radicals of the group CH ₃ -, CF ₃ -,

₄₀ CH ₃ O-, HO-, BnO-, F- or Cl- can carry, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, which cycloalkyl radicals can carry up to four methyl radicals, adamantyl, indanyl, or -CH-W with W = Cι_ ₈ -alkyl-, 2-thienyl, 3-thienyl, 3-indolyl-, 4-imidazolyl-,

45 2-pyridyl-, 3-pyridyl-, 4-pyridyl-, phenyl-, which up to three identical or different radicals of the group Cι- ₄ -alkyl-, CF ₃ -, Cι- ₄ -alkoxy-, HO-, BnO-, F- or Cl- 57 can carry cyclopentyl, cycloheptyl, cyclooctyl, which cycloalkyl radicals can carry up to four methyl radicals, adamantyl, indanyl,

R ^{7 is} H-, Ci-s-alkyl-, phenyl-, which can carry up to three identical or different radicals from the group Cι_ ₄ -alkyl-, CF ₃ -, Cι- ₄ -alkoxy-, F- or Cl-, C ₃ _ ₈ cycloalkyl, which can carry up to four identical or different C ₄ alkyl radicals,

R ^{8 is} H or C ₄ alkyl,

D:

H

H 1 H 1

NH K _^ OH

- or - NH NH,

4. Use according to one of claims 1 or 2, wherein in the compounds of the formula I the groups A, B, D, E and F have the following meaning:

A: -C 6 alkyl-OOC- (CH ₂ ) t-, (t = 1, 2 or 3), HOOC- (CH ₂ ) ₂ -, HOOC- (CH ₂ ) ₃ -, (HOOC-CH ₂ ) ₂ -CH-, HOOC-CH ₂ -CH (COOH) -, HOOC-CH (Cι- ₄ -Alky1) -, HOOC-C (Cι_ ₄ -alkyl) ₂ -,

B:

R ⁴ R ⁶ II —NC-CO-

R ⁴ H, C ₄ alkyl, HOOC (CH ₂ ) _m - (m = 1, 2 or 3), R ⁵ H, methyl

R ⁶ - (CR ⁷ R ⁸ ) -W with W = cyclohexyl-, which can carry up to four methyl radicals, 58

R ^{7 is} H, C ₈ alkyl, phenyl, which can carry up to three identical or different radicals of the group C ₄ alkyl, CF ₃ , C ₄ alkoxy, F or Cl, C ₃ _ ₈ cycloalkyl, which can carry up to four identical or different C ₄ alkyl radicals,

R ^{8 is} H or C ₄ alkyl,

D:

H

I.

Η- -C

H H

NH N --- OH

\ or -

NH. NH.

5. Use according to one of claims 1 or 2, wherein in the compounds of the formula I the groups A, B, D, E and F have the following meaning:

A: HOOC- (CH ₂ ) _t -, -C ₆ -alkyl-00C- (CH ₂ ) _t ", (t = 1, 2 or 3),

(HOOC-CH ₂ ) ₂ -CH-, H00C-CH ₂ -CH (C00H) -, HOOC-CH (Cι- ₄ alkyl) -, HOOC-C (Cι- ₄ alkyl) ₂ -,

B:

R ⁴ R ⁶ II —NC-CO- |

R ⁵

R ^{4 is} H, C ₄ alkyl or HOOC (CH ₂ ) _m - (m. = 1, 2 or 3),

R ⁵ H, methyl,

R ⁶ -CH ₂ - (CR ⁷ R ⁸ ) -W or - (CR ⁷ R ⁸ ) _g -W with q = 0 or 1 and W = H-, Cχ- ₈ -alkyl-, 2-thienyl, 3- Thienyl, 3-indolyl, 4-imidazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, phenyl, which is up to three identical or different 59

Residues of the group Cι- ₄ alkyl-, CF ₃ -, Cι- ₄ alkoxy-, HO-, BnO-, F- or Cl- can carry, C ₃ _ ₈ cycloalkyl-, which up to four identical or different Cι- ₄ alkyl radicals can carry, adamantyl, indanyl, R ⁷ H, Cι- ₈ alkyl, phenyl, which up to three identical or different radicals of the group Cι_ ₄ alkyl, CF ₃ -, Cι - ₄ -alkoxy-, F- or Cl- can carry, C ₃ _ ₈ -cycloalkyl-, which can carry up to four identical or different Cι- ₄ -alkyl radicals, R ⁸ H- or Cι- ₄ -alkyl- ,

D:

O

E:

H

^' NC

H H

NH | \ r ^0H

- or - ^

NH _{2 N} μ

6. Use according to one of claims 1 or 2, wherein in the compounds of the formula I the groups A, B, D, E and F have the following meaning:

A: HOOC- (CH ₂ ) _t - (t = 1, 2 or 3), (HOOC-CH ₂ ) ₂ -CH-, HOOC-CH ₂ -CH (COOH) -, HOOC-CH (Cι- ₄ - Alky1) -, HOOC-C (Cι- ₄ alkyl) ₂ -, C ^ g-Alky1-OOC- (CH ₂ ) _t -,

B:

R ⁴ R ⁶ II —NC-CO- 60

R ^{4 is} H-, Cι_4-alkyl- or HOOC- (CH_,) _m - (m = 1, 2 or 3),

R ⁵ H, methyl,

R ⁶ -CH ₂ - (CR ⁷ R ⁸ ) -W or - (CR ⁷ R ⁸ ) _g -W with q = 0 or 1 and W = H-, Ci-s-alkyl-, 2-thienyl, 3- Thienyl, 3-indolyl, 4-imidazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,

Phenyl-, which can carry up to three identical or different radicals from the group Cι_-alkyl-, CF ₃ -, Cι- ₄ -alkoxy-, HO-, BnO-, F- or Cl-, C ₃ _ ₈ -cycloalkyl- which can carry up to four identical or different C ₄ alkyl radicals, adamantyl, indanyl,

R ^{7 is} H, C ₈ alkyl, phenyl, which can carry up to three identical or different radicals from the group C ₄ alkyl, CF ₃ , C ₄ alkoxy, F or Cl, C ₃ _ ₈ cycloalkyl, which can carry up to four identical or different C ₄ alkyl radicals,

R ^{8 is} H or C ₄ alkyl,

D:

or

E:

rH Y-V z where either

a) in the case of X = 0 or NH,

Y -N =, -CH = and

Z -N =, -CH = mean

or

b) in the case of X = S,

Y -N = and z -N =, -CH = mean or

Y -CH = and z -N = mean 61

F:

, NH N _^ OH

- ^ or <*

NH. NH.

Compounds of the formula I according to one of claims 1, 3, 4, 5 or 6, but I not being (N- (carboxy (CH ₂ ) _n ) -D-cyclohexylalanyl-N [[5-aminoiminomethyl) thiophene-2 -yl] methyl] -L-

10 prolinamide, with n = 1, 2 or 3.

Medicaments containing compounds according to claim 7 in addition to conventional carriers and auxiliaries.

15 9. Use of the compounds of formula I according to claim 7 for the manufacture of medicaments against:

• Diseases whose pathomechanism is based directly or indirectly on the proteolytic effects of

20 thrombin based,

Diseases whose pathomechanism is based on thrombin-dependent activation of receptors and signal transduction,

Diseases associated with stimulation or inhibition ² of gene expression in body cells,

• diseases based on the mitogenic effects of thrombin,

Diseases which are based on a thrombin-dependent change in contractility and permeability 0 of epithelial cells,

Thrombin-dependent, thromboembolic events,

Disseminated intravascular coagulation,

• reocclusion and to shorten the reperfusion time in the case of comedication with thrombolytics,

"• the appearance of early reocclusion and late restenosis after PTCA,

Thrombin-dependent proliferation of smooth muscle cells,

The accumulation of active thrombin in the CNS,

* 0 • tumor growth as well as against the adhesion and metastasis of tumor cells.

10. Compounds of formula I according to one of claims 1, 3, 4, 5 or 6 for coating surfaces.

45 62

11. Intermediates of the formula Ha and Ilb

A B D E CN Ha,

A B D E CSNH2 Ilb,

wherein A, B, D and E have the meaning given in one of claims 1, 3, 4, 5 or 6.

10

12. Use of connections that make up the structural element

NH

D E (I

NH2

15 contain, wherein D and E have the meaning given in one of claims 1, 3, 4, 5 or 6 and on the nitrogen atom of building block D is a hydrogen atom, a protective group, an optionally substituted natural or 20 unnatural amino acid, an optionally substituted carboxylic acid or sulfonic acid or an optionally substituted alkyl radical for the production of medicaments which contain kallikrein inhibitors.

25 13. Use of connections that make up the structural element

, NH D E ('

NH2

30 contain, in which D and E the in one of claims 1, 3,

4, 5 or 6 have the meaning given and there is a hydrogen atom, a protective group, an optionally substituted natural or unnatural amino acid, an optionally substituted atom on the nitrogen atom of component D.

35 carboxylic acid or sulfonic acid or an optionally substituted alkyl radical for the production of medicaments which contain thrombin inhibitors, the structural element D not being proline if E is 5- (aminoiminomethyl) thiophene-2yl-methylamine.

40

45