CZ20002463A3 - Novel thrombin inhibitors - Google Patents

Abstract

Compounds of formula A-B-D-E-F are described wherein A, B, D, E and F have specific meanings, their preparation. New compounds are useful in the preparation of medicines for the treatment of asthma, arthritis, rhinitis, pancreatitis, tumor growth, thrombosis and thromboembolism.

Description

Technical field

The invention relates to novel five-membered heterocyclic amidines, their preparation and their use as competitive inhibitors of trypsin-like serine proteases, in particular thrombin and kininogenases such as kallikrein. The invention also relates to pharmaceutical compositions comprising these compounds as active ingredients and to the use of these compounds as thrombin inhibitors, anticoagulants and anti-inflammatory agents.

BACKGROUND OF THE INVENTION

Thrombin belongs to the serine protease family and plays a central role in the coagulation cascade as a terminal enzyme. Both the internal and external coagulation cascade leads through a series of enhancing steps to the production of thrombin from prothrombin. Thrombin-catalyzed cleavage of fibrinogen to fibrin then initiates blood clotting and platelet aggregation, which in turn, due to platelet factor 3 binding and clotting factor XIII and a large number of highly active mediators, promote thrombin formation.

The formation and action of thrombin are the main processes involved in the development of white, arterial, and red vein thrombi and are therefore potentially suitable sites for targeting the active substances. Thrombin inhibitors, in contrast to heparin, are capable, independently of cofactors, of simultaneously inhibiting the effects of free thrombin and platelet binding. These inhibitors are able to prevent the acute phase of thromboembolic events following percutaneous transluminal coronary angioplasty (PTCA) and lysis, and to act as anticoagulants in extracorporeal circulation (artificial heart-lung, hemodialysis). They can also be used generally for the prevention of thrombosis, for example after surgery.

Synthetic arginine derivatives are known to affect the enzymatic activity of thrombin by interacting with the active serine residue of protease thrombin. Peptides based on the sequence Phe-Pro-Arg in which the Nterminal amino acid is in the D-configuration have proven to be particularly advantageous. D-Phe-Pro-Arg isopropyl ester is described as a competitive thrombin inhibitor (C. Mattson et al., Folia Haematol, 109 (1983) 43-51).

Substitution at the C-terminus of the arginine to the aldehyde leads to an increased inhibitory effect. For example, a large number of arginals capable of attaching the hydroxyl group of active serine in hemiacetal have been described in EP 185390, 479489, 526877, 542525; and further in WO 93/15756, 93/18060.

The thrombin-inhibitory activity of peptide ketones, fluorinated alkyl ketones and keto esters, boric acid derivatives, phosphoric esters and ketocarboxamides can be similarly explained by this interaction of lilac (EP 118280, 195212, 362002, 364344,

41041 1, 471651, 589741, 293881, 503203, 504064, 530167; WO 92/07869, 94/08941).

The diphenyl esters of the 4-amidinophenylglycine phosphonate peptide described by J. Oleksyszyn et al. in J. Med. Chem. 37 (1994) 226231, are irreversible thrombin inhibitors with insufficient selectivity for other serine proteases.

DE 3 108 810, WO 93/11 152 and EP 601 459 disclose agmatine and the resulting arginine derivatives which are not capable of interacting with active serine in serine proteases.

WO 94/29336, EP 0 601 459 and WO 95/23609 disclose a further development that replaces agmatin with an arylamidine residue.

EP 0 672 658 describes not only thrombin inhibitors having an attached agmatine or benzamidine residue, but also a thrombin inhibitor having amidinothiophene (Example 65).

Kininogenases are serine proteases that release vasoactive peptides, called kinins (bradykinin, calidine and Met-Lys-bradykinin), from kininogens. Kininogens are multifunctional proteins that occur in the coagulation and inflammatory cascade reactions. As inhibitors, they can protect cells from damage by cysteine proteases (Müller Esterl, FEBS Lett. 182, (1985)

310-314). Important kininogenases are plasma kallikrein, tissue kallikrein and mast cell tryptase.

Kinins such as bradykinin and kallidine are vasoactive peptides that affect a large number of biological processes. They play an important role in inflammatory processes. By increasing vascular permeability they cause a decrease in blood pressure and edema. Furthermore, they are very effective as pain-producing substances naturally occurring 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 44 (1) (1992) 1- 80).

Regardless of the underlying mechanism for inflammatory processes, fluid containing all these protein systems, if present in the blood of the circulatory system, exits from the blood vessels. This means that the leakage of plasma fluid from blood vessels is related to diseases such as asthma, runny nose and internal inflammation. Further, mast cell tryptase is released in particular in allergic processes (Salomonsson et al., Am. Rev. Respir. Dis. 146 (1992) 1535-1542).

Argininchlormethylketony H- (D) _{-Pro-Phe-Arg-CH2} Cl and H- (D) -Phe-Phe-Arg-CH2 Cl were described by Kettner and Shaw as plasma kallikrein inhibitors (Biochem. 17 (1978) 47784784 and Meth Enzym 80 (1981) 826-842).

Various other synthetic derivatives of benzamidines and benzylamines have been shown to be plasma kallikrein inhibitors, with benzamidines having a significantly higher inhibitory effect (F. Markward, S. Drawert, P. Walsmann, Biochemical Pharmacology 23 (1974) 2247-2256).

PKSY-527, N- (trans-4-aminomethylcyclohexylcarbonyl) -L-phenylalanine-4-carboxymethylanilide hydrochloride, is also a potent inhibitor of these kininogenases (Wanaka, Ohamoto et al., Thromb. Res., 57 (6) (1990) 889-895).

SUMMARY OF THE INVENTION

The invention relates to compounds of formula I

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

A: r ²

AND

R @ ¹ (CH3). C (CB 3) n

AND

R ^! * · · · ·

5 ····················· · Where m is Ο, 1 or 2, n is 0, 1 or 2,

R ¹ is HOOC-, C 1-6 -alkyl-OOC-, aryl-CO-4-alkyl-OOC or -OH,

R ² is H-, C 1-4 -alkyl- or R ¹ - (CH ₂ ) m and R ³ is H- or C 1-4 -alkyl-,

B: _r6

AND

-N-C-COI

R ⁵ where

R ⁴ is H-, C ^1-4 -alkyl- or R ¹ - (CH ₂ ) m - (wherein R ¹ and m are as defined above), p is 0 or 1,

R ⁵ is H- or C 1-4 -alkyl-,

R ⁶ is H-, C 1-8 -alkyl-, 2-thienyl-, 3-thienyl-, 3-indolyl-, 4-imidazolyl-, 2-pyridyl-, 3-pyridyl-, 4-pyridyl-, phenyl- it may carry up to three identical or different radicals from the group consisting of C 1-4 -alkyl-, CF ₃ -, C 1-4 -alkoxy-, HO-, BnO-, F- or. C1- or C3-8-cycloalkyl which may carry up to four identical or different C1-4-alkyl radicals and / or wherein one or two CC single bonds in the ring may be replaced by a C = C double bond and / or a phenyl ring it may be unfused, C 7 -C ₁₂ -bicycloalkyl- or C 10 -tricycloalkyl- or R ⁴ and R ⁶ together may form an ethylene or propylene group,

R ⁷ is H, C 1-8 -alkyl-, phenyl-, which may carry up to three identical or different radicals from the group consisting of C 1-4 -alkyl-, CF 3 -, C 1-4 -alkoxy-, F- or Cl- or C _3-8 -cycloalkyl-, which can carry up to four identical or different Ci-C4-alkyl radicals,

R ⁸ is H or C 1-4 -alkyl,

where R ²⁰ is H, C 1-4 -alkyl, Bn or BnO (CO) - and wherein the following applies: if D is II, III or XI, then E has the following meaning:

R10

R9 r11 γ —- z where

a) when X = S, O, NH or NR ¹² , then

Y is -CR ¹³ =, -CH = a

Z is -CR ¹⁴ = or

Y is -CR ¹³ = a

From e -CH = or

b) when X = NR ¹² , then

Y is -CH = a

Z is -CH = or

(c) where X = S, O or NH,

Y is -CR ¹⁵ = a

Z is N = or Y is N = and Z is -CR 15 ^S = or

d) if X = -NR ¹² -, then

Y is -N = and Z is -CR ¹³ · =, -N = or Yje -CR ¹⁶ = a Z is N = and ♦ R ⁹ Yippee H- or Ci. _3- alkyl-, R ¹⁰ Yippee H- or C _1-4 -alkyl-, R ¹¹ Yippee H- or Ci. _4- alkyl-, R ¹² Yippee CH ₃ - or C ₂ H ₅ -, R ¹³ Yippee Cl, CF ₃ - or C ₄ -alkyl- R ¹⁴ Yippee CI-, CF ₃ - or Ci. _4- alkyl- R ¹⁵ Yippee CF ₃ - or C1. _4- alkyl-, _{R is} _ on Yippee H-, CF ₃ - or C _1-4 -alkyl-

is as defined above, or, if D is IV, VI, VII, VIII, IX or X, then E is as follows: importance:

.x.

 • · • ··· where

X is O, S or -NR ¹⁷

Y is N = a

Z is -CR ¹⁶ = or -N = or

Y is -CR ¹⁶ = and Z is-N = or

Y is -CR ¹⁸ = and Z is -CR ¹⁹ =

R ⁹ , R ¹⁰ , R ¹¹ , R ¹⁶ and R ²⁰ are as defined above, R ¹⁷ is H, CH _{3 -} or C ₂ H ₅ -,

R ¹⁸ is H-, Cl-, CF ₃ - or C _1-4 -alkyl-,

R ¹⁹ is H-, Cl-, CF ₃ - or C 1-6. _4- alkyl-, or when D is II, III, IV, VI, VII, VIII, IX, X or XI, E has the following meanings:

or

R10

N where

(a) if X = S, then

Y is -CR ¹⁸ = a

Z is -CR ¹⁹ = or

Y is -CR ¹⁶ =

Z is -N = or

b) when X = O or -NR ¹² -, then

Y is-N =, -CR ¹⁶ = a

Z is -N =, -CR ¹⁸ = a

R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹⁶ , R ¹⁸ , R ¹⁹ and R ²⁰ have the meanings given above,

F:

Y is> ^NH, N-OH, or ^NH 2 NH ₂ and their salts with physiologically acceptable acids.

The amino acid derivatives represented by the symbol B preferably have the (D) configuration; the azetidine carboxylic acid, proline and pipecolic acid D preferably have the (L) configuration.

Preferred compounds of formula I are those wherein A to E have the following meanings:

A: ..........

HOOC- (CH ₂₎ t (t = 1, 2 or 3), _{(HOOC-CH2) 2} -CH-,

HOOC-CH ₂ -GH (COOH) -, HOOC-CH (C _1-4 alkyl) -,

HOOC-C (C 1-4 -alkyl) ₂ -, C 1-6 alkyl; _6- alkyl-OOC- (CH ₂ ) _t -.

B is r ⁶

-N-C-CO

AND

R ⁵ p is 0 or 1,

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

«

-10 R ⁶ is Η-, C 1-8 -alkyl-, 2-thienyl-, 3-thienyl-, 3-indolyl-, 4-imidazolyl-, 2-pyridyl-, 3-pyridyl-, 4-pyridyl-, phenyl- which may carry up to three identical or different radicals from the group which includes CH ₃ -, CF ₃ -, CH 3 O-, HO-, BnO-, F or C1, or _C3 .e-cycloalkyl, which may carry up to four methyl radicals, bicyclo [2.2.2] octyl-, bicyclo [2.2.1] heptyl-, adamantyl-, indanyl-, decainyl-, «

R ⁷ is H, C 1-8 -alkyl-, phenyl-, which may carry up to three identical or different radicals from the group consisting of CH ₃ -, CF ₃ -, CH ₃ O-, F- or Cl-, or C 3-8 -cycloalkyl-, which can carry up to four methyl radicals,

R ⁸ is H, C 1-4 -alkyl,

where R ²⁰ is H, CH ₃ , Bn or BnO (CO) - and where:

if D is II, III or XI, then E means:

H

η Η ϊ — z

β • 9 9 9 9 9 9 9 9 9 99 99 where

(a) where X = S, O or NR ¹⁷ , then

Y is -CR ¹³ = or -CH = and Z is -CR ¹⁴ = or

Y is -CR ¹³ = and Z is -CH = or

b) when X = NR ¹² , then

Y is -CH = and Z is -CH =, or

c) when X = S, O or NH, then

Y is -CR ¹⁵ = and Z is N = or

Y is N = and Z is -CR ¹⁵ = or

d) when X = NR ^12,

- Y is N = a

Z is -CR ¹⁶ =, -N = or

Y is -CR ¹⁶ = a.

Z is -N = a

R ¹² is CH ₃ - or C ₂ H ₅ -,

R ¹³ is Cl, CF3 or Ci-4-alkyl,

• 00 00 0 0 0 0 0

R ¹⁴ is Cl, CF3 or alkyl-CI4 · R ¹⁵ is CF 3 or C 1 _ ₄ -a alkyl-,

R ⁶ is H-, CF ₃ - or C _1-4 -alkyl-,

R ¹⁷ is H, CH ₃ - or C ₂ H 5

R ²⁰ is as above, or when D is IV, VI, VII, VIII

E stands for:

, IX or X, then H

where

X is 0, S or -NR ¹⁷ - and wherein Y is -N ^: = a of Yippee or -CR ¹⁶ = or -N = Y Yippee -CR ¹⁶ = a of Yippee or -N = Y Yippee -CR ¹⁸ =, and of Λ Yippee -cr ¹⁹ = 3 R ¹⁶ , R ¹⁷ , R @ ²⁰ have the abovementioned meanings

R ¹⁸ is H-, Cl-, CF 3 - or C 1-4 -alkyl- and R ¹⁹ is H-, Cl-, CF 3 - or Cl. _4- alkyl-, or if D is II, III, IV, VI, VII, VIII, IX, X or XI, then E has the meanings:

where

and)

Y

Z when X = S, -CR ¹⁵ = and -CR ¹⁹ =

• · »·

Y or is -CR ¹⁶ = and of is -N = or

(b) when X = O or -NR ¹² ,

Y is -N = or -CR ¹⁶ = a

Z is -N = or -CR ¹⁵ = and R ¹² , R ¹⁶ , R ¹⁸ , R ¹⁹ and R ²⁰ are as defined above,

F:

NH

NH, or

N-OH

NH2-2 and their salts with physiologically acceptable acids.

The amino acid derivatives represented by the symbol B preferably have the (D) configuration; the azetidine carboxylic acid, proline and pipecolic acid in symbol D preferably have the (L) configuration.

Particularly preferred compounds of formula I are those wherein I

AND; HOOC-CH 2, HOOC-CH ₂ -CH ₂ , HOOC-CH (CH ₃ ), HOOC-CH (C ₂ H ₅ )

B: | f < ^{R 7} -f- ^{R 8) p} - _N - _c - _co - p is 0 or 1,

R 1 is H-, CH ₃

R ⁵ is H-, CH ₃ -,

• · ·················

R ⁶ is C 1-8 -alkyl-, C 3-8 -cycloalkyl-, which may carry up to four methyl radicals, 2-thienyl-, 3-indolyl-, 4-imidazolyl-, 2-pyridyl-, 3-pyridyl-, 4-pyridyl, phenyl, which may carry up to three identical or different radicals from CH ₃ -, CF ₃ -, CH ₃ O-, HO-, BnO-, F- or Cl-, bicyclo [2.2. 2] octyl, bicyclo [2.2.1] heptyl, adamantyl, indanyl, decalinyl, with particular emphasis on cyclopentyl, cyclohexyl and cycloheptyl,

R ⁷ is H, CH ₃ -,

R ⁸ is H, CH ₃ -,

where

R ²⁰ is H, BnO (CO) - and where the following applies:

if D is II, III or XI, then E means:

H

H h Y — z where

X is -S- and where • · · ·

Y is -CH = a

Z is -CR ¹³ = or

Y is -CR ¹³ = a

Z is -CH = or

Y is -CR ¹⁵ = a

Z is -N = or

Y is -N = a

Z is -CR ¹⁵ = a

R ¹³ is CI-, CF ₃ - or CH ₃ ,

R ¹⁵ is CF ₃ - or CH ₃ - and R ²⁰ is as above, or if D is IV, VI, VII, VIII, IX or X, then E represents:

H

where

X is S, and where

Y is N = a

Z is -CR ¹⁶ = or

Y is -CR ¹⁶ = a

Z is -N = or

Y is -CR ¹³ = a

Z is -CH = or

Y is -CH = a

Z is -CR ¹³ = 9 or 9

Y is -CH = a

Z is -CH = a

R ¹³ , R ²⁰ have the above meanings and R ¹⁶ is H-, CF ₃ - or CH _3, or when D is II, III, IV, VI, VII, VIII,

IX, X or XI, then E means:

or

H

AND

N —CII Η H ^X x

a) in the case where X = S, Y is CH and Z is -CR ^{1 S} or Y = is -CR ¹⁶ =, and Z is -N = or

Y is -CR ¹⁸ = a

Z is -CH = or

b) when X = O or NCH ₃ Y is -CH = and Z is -CR ¹⁶ = or Y is -CR ¹⁶ = and Z is -CH = or

c) when X = -NR ¹² Y is -N = a

Z is -CR ¹⁸ = a

R ¹² is CH ₃ - or C ₂ H ₅ - and

NH

R ¹⁸ is H, Cl -, CF ₃ - or CH ₃ -, and

R ¹⁶ , R ²⁰ have the meanings given above

F:

N-OH or 2 nh ₂ and their salts with physiologically acceptable acids.

The amino acid derivatives represented by B preferably have the (D) configuration; the azetidine carboxylic acid, proline and pipecolic acid in symbol D preferably have the (L) configuration.

Highly preferred compounds of formula I are those in which A, B, D, E and F have the following meanings:

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

R ⁴ (R ⁷ -CR ⁸ ) _p

I I

-N-C-COI

R ⁵ p is 0 or 1,

R ⁴ is H-,

R ⁵ is H-,

R 8 is C 1-8 -alkyl-, 2-thienyl-, 3-indolyl-, 4-imidazolyl-, 2-pyridyl-, 3-pyridyl-, 4-pyridyl-, C 3-8 -cycloalkyl, which can carry up to four methyl radicals, phenyl-, which may carry up to three identical or different radicals from the group consisting of CH ₃ -, CF ₃ -, CH ₃ O-, HO-, BnO-, F- or CI-, bicyclo [2.2.2 ] octyl, bicyclo [2.2.1 jheptyl, adamantyl, indanyl, decalinyl, with special emphasis on cyclopentyl, cyclohexyl and cycloheptyl,

D:

knows ··· ♦

IX where the following applies:

if D is II, III or XI, then E means:

H

where

X is S and is -CR ¹³ = is -CH = or

Y is -CH =

Z is -CR ¹³ = or is -CR ¹⁵ = is -N = or

Y is -N = a‘

J u * «·

Z is -CR ¹⁵ = a

R ¹³ is Cl -, CF ₃ - or CH ₃ - a

R ¹⁵ is CF ₃ - or CH ₃ - or if D is IV, VII, VII, VIII, IX or X; then E means:

H

where

X is S and

Y is -N = a

Z is -CR ¹⁶ = or

Y is -CR ¹⁶ = a

Z is -N = or

Y is -CH = a

Z is -CR ¹³ = or

Y is -CR ¹³ = a

Z is -CH = or

Y is -CH = a

Z is -CH = a.

R ¹³ is as defined above and R ¹⁶ is H, CF ₃ - or CH ₃ - or, if D is II, III, IV, VI, VII, VIII, IX, X or XI, then E represents

X.

N — CG Y ¹ 'V //

Η H Z '' N-C—

I I \ Η H Z

^- 20 ^{- - - - - - - - - - - - - - - - - - - - - - - - - - - -} where

(a) where X = S,

Y is -CH = a

Z is -CR ¹⁸ = or

Y is -CR ¹⁸ = a

Z is -CH = or

Y is -CR ¹⁶ = a

Z is -N = or

(b) where X = O or NCH ₃

Y is -CH = a

Z is -CR ¹⁶ or

Y is -CR ¹⁶ = a

Z is -CH = or

(c) where X = NCH ₃

Y is -N = a

Z is -CR ¹⁶ =

R ¹⁶ is as defined above and R ¹⁸ is H, Cl-CF ₃ - or CH ₃ -,

F: nh

NH ₂ or

NOH

NH,

 ··· <

and salts thereof with physiologically acceptable acids.

The amino acid derivatives represented by B preferably have the (D) configuration; the azetidine carboxylic acid, proline and pipecolic acid in symbol D preferably have the (L) configuration.

The selection of the compounds mentioned in the examples is represented by the following substances which are of great importance:

HOOC-CH ₂ - (D) -Cha-Pro-NH-CH ₂ -5- (2-am-3-CF ₃ ) -thioph

HOOC-CH2- (D) Chg-Pro-NH-CH2-5- (2-am-3-CF ₃₎ -thioph

HOOC-CH 2 - (D) -Cha-Pro-NH-CH ₂ -5- (2-am-4-Me) -thioph

HOOC-CH 2 - (D) -Cha-Pro-NH-CH ₂ -5- (2-am-4-CI) -thioph

HOOC-CH ₂ - (D) -Cha-Pro-NH-CH ₂ -5- (2-am-4-CF ₃ ) -thioph

_HOOC-CH2 - (D) Chg. Pro-NH-CH2-5- (2-am-4-Me) -thioph

_HOOC-CH2 - (D) _{Chg-PRQ-NH-CH2} -5- (2-am-4-Cl) -thioph

HOOC-CH ₂ - (D) -Chg Pro-NH-CH ₂ -5- (2-am-4-CF ₃ ) -thioph

HOOC-CH2- (D) Chg-Pro-NH-GH2-5- (2-amino-3,4-Me ₂₎ -thioph

HOOC-CH 2 - (D) -Cha-Aze-NH-CH ₂ -2- (4-am) -thioph

HOOC-CH 2 - (D) -Chg-Pic-NH-CH 2 -2- (5-am) -thioph

HOOC-CH 2 - (D) -Cha-Pic-NH-CH 2 -2- (4-am) -thioph

_HOOC-CH2 - (D) _{-Chg-Pic-NH-CH2} -2- (4-am) -thioph

HOOC-CH ₂ - (D) -Cha-Pic-NH-CH ₂ -2- (5-am-3-Me) -thioph

HOOC-CH2- (D) -Chg-Pic-NH-CH2-2- (5-am-3-Me) -thioph

HOOC-CH ₂ - (D) -Cha-Pic-NH-CH ₂ -2- (5-am-3-C 1) -thioph

HOOC-CH ₂ - (D) -Chg-Pic-NH-CH ₂ -2- (5-am-3-Cl) -thioph

HOOC-CH ₂ - (D) -Cha-Pic-NH-CH ₂ -2- (5-am-4-Me) -thioph

HOOC-CH ₂ - (D) -Chg-Pic-NH-CH ₂ -2- (5-am-4-Me) -thioph

HOOC-CH ₂ - (D) -Cha-Pic-NH-CH ₂ -2- (5-am-4-Cl) -thioph

HOOC-CH ₂ - (D) -Chg-Pic-NH-CH ₂ -2- (5-am-4-Cl) -thioph

HOOC-CH 2 - (D) -Chea-Pro-N H-CH ₂ -2- (4-am) -thioph

HOOC-CH ₂ - (D) -Cpa -Pro-NH-CH ₂ -2- (4-am) -thioph

- * - * - Λ Λ

HOOC-CH ₂ - (D) -Chg-Pro-NH-CH ₂ -2- (4-am-5-Me) -thioph

HOOC-CH ₂ - (D) -Chg-Pro-NH-CH ₂ -2- (4-am-5-Cl) -thioph

HOOC-CH ₂ - (D) -Chg-Pro-NH-CH ₂ -2- (4-am-5-CF ₃ ) -thioph

HOOC-CH ₂ -CH ₂ - (D) -Chg-Pro-NH-CH ₂ -2- (4-am) -thioph

HOOC-CH ₂ -CH ₂ - (D) -Cha-Pro-NH-CH ₂ -2- (4-am) -thioph

_HOOC-CH2 - (D) -Chea-Pro-NH-CH2-4- (2-am) -thioph

_HOOC-CH2 - (D) -Cpa-NH-CH ₂ -4- (2-am) -thioph

HOOC-CH ₂ - (D) -Chg-Pro-NH-CH ₂ -4- (2-am) -thioph

_HOOC-CH2 - (D) -Cheg-Pro-NH-CH ₂ -4- (2-am) -thioph

_HOOC-CH2 - (D) -Cpg-Pro-NH-CH ₂ -4- (2-am) -thioph

_HOOC-CH2 - (D) _{Cha-Pic-NH-CH2} -2- (4-am) -thiaz

HOOC-CH ₂ - (D) -Chg-Pic-NH-CH ₂ -2- (4-am) -thiaz

HOOC-CH ₂ - (D) -Cha-Aze-NH-CH ₂ -2- (4-am) -thiaz

HOOC-CH ₂ - (D) -Chg-Aze-NH-CH ₂ -2- (4-am) -thiaz

MeOOC-CH ₂ - (D) _{Cha-Pro-NH-CH2} -2- (4-am) -thiaz

Hoog-CH ₂ - (D) _{-Cpg-Pro-NH-CH2} -2- (4-am) -thiaz

HOOC-CH ₂ - (D) -Chea-Pro-NH-CH ₂ -2- (4-am) -thiaz

HOOC-CH ₂ - (D) -Cheg-Pro-NH-CH ₂ -2- (4-am) -thiazate

_HOOC-CH2 - (D) _{Cha-Pic-NH-CH2} -2- (4-am) -thiaz

HOOC-CH ₂ - (D) -Chg-Pic-NH-CH ₂ -2- (4-am) -thiaz

_{HOOC-CH2 -CH2} - (D) _{Cha-Pro-NH-CH2} -2- (4-am) -thiaz

HOOC-CH ₂ -CH ₂ - (D) -Chg-Pro-NH-CH ₂ -2- (4-am) -thiaz

_HOOC-CH2 - (D) _{Cha-Pro-NH-CH2} -2- (4-amino-5-Me) -thiaz

HOOC-CH ₂ - (D) -Chg-Pro-NH-CH ₂ -2- (4-am-5-Me) -thiaz

HOOC-CH ₂ - (D) -Cha-Pro-NH-CH ₂ -2- (4-am-5-CF ₃ ) -thiaz

HOOC-CH ₂ - (D) -Chg-Pro-NH-CH ₂ -2- (4-am-5-CF ₃ ) -thiaz

HOOC-CH ₂ - (D) -Cha-Pro-NH-CH ₂ -2- (5-am-4-Me) -thiaz

HOOC-CH ₂ - (D) -Chg-Pro-NH-CH ₂ -2- (5-am-4-Me) -thiaz

HOOC-CH ₂ - (D) -Cha-Pro-NH-CH ₂ -2- (5-am-4-CF ₃ ) -thiaz

HOOC-CH ₂ - (D) -Chg-Pro-NH-CH ₂ -2- (5-am-4-CF ₃ ) -thia ₂

HOOC-CH ₂ - (D) -Chg-Pro-NH-CH ₂ -5- (3-am) -isox

HOOC-CH ₂ - (D) -Cha-Pro-NH-CH ₂ -2- (4-am) oxaz • 4,444

-23,

4444 44

HOOC-CH 2 - (D) -Chg-Pro-NH-CH 2 -2- (4-am) -oxaz

_{HOOC-CH2 -CH2} - (D) Chg-Pro-NH-CH2-5- (3-am) furo

HOOC-CH ₂ -CH _{2 -} (D) -Cha-Pro-NH-CH ₂ -5- (3-am) -fur

_HOOC-CH2 - (D) -Chea-Pro-NH-CH2-5- (3-am) furo

HOOC-CH ₂ - (D) -Cha-Pic-NH-CH ₂ -5- (3-am) -fur

HOOC-CH ₂ - (D) -Chg-Pic-NH-CH ₂ -5- (3-am) -fur

HOOC-CH ₂ -CH _{2 -} (D) -Cha-Pic-NH-CH ₂ -5- (3-am) -fur

_{HOOC-CH2 -CH2} - (D) -Chg-Pic-NH-CH2-5- (3-am) furo

HOOC-CH ₂ - (D) -Chg-Aze-NH-CH ₂ -5- (3-am) -fur

HOOC-CH 2 - (D) -Cha-Aze-NH-CH ₂ -5- (3-am) -fur

HOOC-CH ₂ - (D) -Cha-Aze-NH-CH ₂ -2- (4-am-1-Me) -pyrr

HOOC-CH 2 - (D) -Chg-Aze-NH-CH ₂ -2- (4-am-1-Me) -pyrr

_HOOC-CH2 - (D) Chg-Pro-NH-CH2-2- (4-am-1-Me) pyrrolo

HOOC-CH ₂ - (D) -Cha-Pic-NH-CH ₂ -2- (4-am-1-Me) -pyrr

HOOC-CH 2 - (D) -Chg-Pic-NH-CH ₂ -2- (4-am-1-Me) -pyrr

HOOC-CH 2 - (D) -Cha-Aze-NH-CH ₂ -2- (5-am-1-Me) -pyrr

_HOOC-CH2 - (D) Chg-Aze-NH-CH2-2- (5-amino-1-Me) pyrrolo

HOOC-CH ₂ - (D) -Chg-Pro-NH-CH ₂ -2- (5-am-1-Me) -pyrr

HOOC-CH 2 - (D) -Cha-Pic-NH-CH ₂ -2- (5-am-1-Me) -pyrr

HOOC-CH 2 - (D) -Chg-Pic-NH-CH ₂ -2- (5-am-1-Me) -pyrr

HOOC-CH ₂ - (D) -Cha-Aze-NH-CH ₂ -4- (2-am-1-Me) -pyrr

HOOC-CH 2 - (D) -Chg-Aze-NH-CH ₂ -4- (2-am-1-Me) -pyrr

HOOC-CH ₂ - (D) -Chg-Pro-NH-CH ₂ -4- (2-am-1-Me) -pyrr

HOOC-CH ₂ - (D) -Cha-Pic-NH-CH ₂ -4- (2-am-1-Me) -pyrr

_HOOC-CH2 - (D) -Chg-Pic-NH-CH ₂ -4- (2-amino-1-Me) pyrrolo

HOOC-CH ₂ - (D) -Cha-Aze-NH-CH ₂ -5- (3-ann-1-Me) -pyraz

HOOC-CH2- (D) -Chg-Aze-NH-CH2-5- (3-am-1-Me) -pyraz

HOOC-CH 2 - (D) -Chg-Pro-NH-CH ₂ -5- (3-am-1-Me) -pyraz

HOOC-CH ₂ - (D) -Cha-Pic-NH-CH ₂ -5- (3-am-1-Me) -pyraz

HOOC-CH 2 - (D) -Chg-Pic-NH-CH ₂ -5- (3-am-1-Me) -pyraz

_HOOC-CH2 - (D) Cha-Aze-NH-CH2-3- (5-amino-1-Me) pyrazolo

HOOC-CH 2 - (D) -Chg-Aze-NH-CH ₂ -3- (5-am-1-Me) -pyraz

···· 4 · ·· ««

HOOC-CH2- (D) -Cha-P.ro-NH-CH2-3- (5-am-1-Me) -pyraz

HOOC-CH 2 - (D) -Chg-Pro-NH-CH ₂ -3- (5-am-1-Me) -pyraz

HOOC-CH ₂ - (D) -Cha-Pic-NH-CH ₂ -3- (5-am-1-Me) -pyraz

HOOC-CH 2 - (D) -Chg-Pic-NH-CH ₂ -3- (5-am-1-Me) -pyraz

HOOC-CH ₂ - (D) -Chg-Pro-NH-CH ₂ -5- (3-am) -oxadiazuBuOOC-CH 2 N-BOC- (D) -Chg-Pro-NH-CH ₂ -2- ( 4-amoxoxaz

HOOC-CH 2 - (D) -Chg-Pro-NH-CH ₂ -5- (3-am-4-CI) -thioph

EtOOC-CH ₂ - (D) _{Chg-Pro-NH-CH2} -5- (3-amino-4-C)) - thioph

HOOC-CH ₂ - (D) -Chg-Pro-NH-CH ₂ -5- (3-am-4-Me) -thioph

EtOOC-CH 2 - (D) -Chg-Pro-NH-CH ₂ -5- (3-am-4-Me) -thiophobuOOC-CH ₂ - (D) -Cha-Pro-NH-CH ₂ -4- (2-ham) -thioph tBuOOC-CH ₂ - (D) -Chg-Pro-NH-CH ₂ -4- (2-ham) -thioph tBuOOC-CH ₂ - (D) -Chg Aze-NH-CH ₂ -4 - (2-ham) -thioph tBuOOC-CH2- (D) -Cha-Aze-NH-CH2-4- (2-ham) -thioph tBuOOCCH2- (D) -Cha-Pro-NH-CH2-4- ( 2-ham-thiaz tBuOOC-CH2- (D) -Chg-Pro-NH-CH2-4- (2-ham) -thiaz tBuOOC-CH2- (D) -Chg-Aze-NH-CH2-4- ( 2-ham-thiaz tBuOOC-CH2- (D) -Cha-Aze-NH-CH2-4- (2-ham) -thiaz

List abbreviations: Adaala: adamantylalanine Adagly: adamantylglycine AIBN: azobisisobutyronitrile Ac: acetyl am: amidino And that: azetidine carboxylic acid Br i: benzyl bs: wide singlet Boc: target. butyl oxy carbonyl Bu: butyl Cbz: benzyl oxy carbonyl 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 Dep: 4,5-dehydropipecolic acid DMF: dimethylformamide DIPEA: diisopropylethylamine Dpa: diphenylalanine EDC: N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide hydrochloride Et: ethyl Eq: equivalents Gly: glycine fur: furan ham: hydroxyamidinyl HOSucc: hydroxysuccinimide HPLC: high performance liquid chromatography imi: imidazole iPr: isopropyl isox: isoxazole Leu: leucine Lsg: solution Me: methyl a-MeCha: α-methylcyclohexylalanine

• «·» ·

P, P-Me2Cha: 2-amino-3-cyclohexyl-3-methylbutyric acid or β, β-dimethylcyclohexylalanine

4-MeCha: (4-methylcyclohex-1-yl) alanine

Y-MeCha: (1-methylcyclohex-1-yl) alanine

3.3-Me2Cha: (3,3-dimethylcyclohex-1-yl) alanine

4-MeChg: (4-methylcyclohex-1-yl) glycine

3.3-Me2Chg: (3,3-dimethylcyclohex-1-yl) glycine

MPLC: liquid chromatography at liquid pressure

MTBE: methyl tert-butyl ether

NBS: N-bromosuccinimide

Nog: norbornylglycine

Ohind: (2) -octahydroindole-2-carboxylic acid

Oxadiazole: 1,2,4-oxadiazole

Oxaz: oxazole

Ph: phenyl

Phe: phenylalanine

Pie: pipecolic acid

PPA: propylphosphonanhydride

For: proline

Py: pyridine pydaz: (3 S) -2,3,4,5-tetrahydropyridazine-3-carboxylic acid

Pyr: 3,4-dehydroproline pyraz: pyrazole pyrr: pyrrole pyzo-3: (3S) pyrazolidine-3-carboxylic acid q: quartet

RT: Room temperature

RP-18: reverse phase C-18 with: singlet str: singlet, wide t: triplet t: tertiary

tBu: tert.butyl tert: tert.

TBAB: tetrabutylammonium bromide

TEA: triethylamine

TFA: trifluoroacetic acid

TFFA; thiazole: thiazole thioph: thiophene trifluoroacetanhydride

Thz-2: thiazoline-2-carboxylic acid

Thz-4: thiazolidine-4-carboxylic acid

5,5-Me2Thz-4: (45) -5,5-dimethylthiazolidine-4-carboxylic acid TOTU: O- (cyanoethoxycarbonylmethylene) amino-N, N, N ', N'-tetramethyluronium tetrafluoroborate triazine: 1,2,4 -triazole

Z: benzyloxycarbonyl

The following definitions of each substituent are used throughout the specification and claims:

The term cycloalkyl alone or as part of another substituent includes saturated or cyclic hydrocarbon groups containing a given number of carbon atoms. C _3-8 cycloalkyl refers to saturated alicyclic rings of 3 to 8 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 4-methylcyclohexyl, cycloheptyl or cyclooctyl.

The term alkyl alone or as part of another substituent means a radical with a linear or branched alkyl chain of the length indicated for each individual case. Thus, C 1-4 alkyl is, for example, methyl, ethyl, 1-propyl,

2-propyl, 2-methyl-2-propyl, 2-methyl-1-propyl, 1-butyl, 2-butyl, C1-6-alkyl is, for example, C1-4-alkyl, pentyl, 1-pentyl, 2-pentyl ,

3-pentyl, 1-hexyl, 2-hexyl, 3-hexyl, 4-methyl-1-pentyl or

3,3-dimethylbutyl. In addition to the radicals listed as possible meanings of C1. ₄ -alkyl, C 1-6 -alkyl means, for example, C 1-6 -alkyl, heptyl or octyl.

The term alkoxy, alone or as part of another substituent, a radical with a linear or branched alkyl chain of the length indicated in each case and which is bound to the corresponding basic compound via an oxygen atom. Thus, C 1-4 -alkoxy means, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy, 2-methyl-2-propoxy, 2-methyl-1-propoxy, 1-butoxy, 2-butoxy.

The invention further relates to compounds comprising the following structural element:

nh ₂ wherein D and E are as defined in any one of claims 1 to 4, and wherein at least one of the radicals selected from the group consisting of hydrogen, a protecting group, an unsubstituted or substituted natural or unnatural amino acid, an unsubstituted or substituted carboxylic acid the acid, unsubstituted or substituted alkyl radical is attached to the nitrogen atom of structural block D.

The invention further relates to compounds which comprise the following structural element:

-29 -E

NH wherein E is as defined in any one of claims 1 to 4 and wherein at least one of the radicals selected from the group consisting of hydrogen, protecting group, unsubstituted or substituted natural or unnatural amino acid, unsubstituted or substituted carboxylic acid, unsubstituted or substituted the alkyl radical is attached to the nitrogen atom of the NR ⁹ group

Finally, the invention also relates to compounds having one of the following structural elements:

which contains

wherein Q is CH ₃ or Cl, T is NCH ₃ , O or S and W is NCH ₃ or S.

The invention further relates to intermediates of formulas Va and Vb1; 7 · «,« ϊ

-30 A - Β - D - EN - CN Va,

A - B - D - E - CSNH ₂ Vb, wherein A, B, D and E are as defined above.

These novel intermediates are used to prepare compounds I and are valuable structural blocks for the synthesis of serine protease inhibitors.

These compounds of formula I may exist as such or in the form of salts with physiologically acceptable 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, hydroxysuccinic acid, sulfuric acid, glutaric acid, aspartic acid , pyruvic acid, benzoic acid, glucurohic acid, oxalic acid, ascorbic acid and acetylglycine.

When in the compounds of formula I, R 1 is C 1-6 -alkyl-OOC, aryl-Co-4-alkyl-OOC and / or F is hydroxyamidine, these compounds may act as prodrugs in vivo to produce enzymatically corresponding carboxyl derivatives R ¹ = HOOC- or corresponding amidines F = -C (= NH) -NH ₂ .

Prodrugs of compounds of formula I are those compounds that are metabolized in vivo to provide pharmacologically active compounds of formula I. This conversion may be due, for example, to the first metabolism in the liver.

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

-cavobes whose pathogenetic mechanism is directly or indirectly derived from the thrombin's proolytic effect,

- diseases whose pathogenetic mechanism is derived from thrombin-dependent receptor activation and signaling,

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

- diseases derived from the mitogenic effect of thrombin,

- diseases derived from thrombin-dependent changes in the contractility and permeability of epithelial cells,

- thrombin-dependent thromboembolic events,

- Multiple intravascular coagulation (DIC),

- reocclusion and to reduce reperfusion time for concomitant administration of thrombolytics such as streptocinase, urokinase, prourokinase, t-PA, APSAC, salivary gland plasminogen activators and the recombinant and mutated forms of all these substances,

- signs of early reocclusion and subsequent restenosis after PTCA,

- thrombin-dependent proliferation of smooth muscle cells,

- accumulation of active thrombin in the CNS (eg in Alzheimer's disease),

- tumor growth and prevention of tumor cell adhesion and metastasis.

The novel compounds can be used for the treatment and prevention of thrombin-dependent thromboembolic events such as thrombosis of the major vessels of pulmonary embolism, myocardial or brain infarction and unstable angina, as well as for the treatment of multiple intravascular

-32 coagulation (DIC). Further, they can be used for combination therapy with thrombolytics such as streptokinase, urokinase, prourokinase, t-PA, APSAC and other plasminogen activators, to reduce the time required for reperfusion and to prolong the reocclusion time.

Other preferred fields of application are prevention of thrombin-dependent early reocclusion and subsequent restenosis following percutaneous transluminal coronary angioplasty, prevention of thrombin-induced smooth muscle cell proliferation, prevention of active thrombin accumulation in the CNS (e.g., Alzheimer's disease), tumor suppression and preventive leading to tumor cell adhesion and metastasis.

The novel compounds can also be used for coating artificial surfaces such as hemodialysis membrane surfaces and the surfaces of tubing systems and fasteners required for blood conduction, as well as for coating oxygenator apparatus surfaces in extracorporeal circulation, implants and heart valves.

Furthermore, the novel compounds can be used for the treatment of diseases whose pathogenetic mechanism is directly or indirectly derived from the proteolytic effect of kininogenases, in particular kallikrein, for example inflammatory diseases such as asthma, pancreatitis, rhinitis, arthritis, urticaria and internal diseases in which applies kallikrein.

The compounds of the invention may be administered orally or parenterally (subcutaneously, intravenously, intramuscularly, interperitoneally, rectally) by conventional means. They can also be administered in gaseous or spray form through the nasopharynx space.

-33 · · · · 33 33 * * * * * · 33 33 33 •• · ·· ·· ·· - ·· ··

The dosage depends on the age, condition and weight of the patient and the type of administration. As a rule, the daily dose of active ingredient per person is 10-2000 mg for oral administration and about 1-200 mg for parenteral administration. This dose can be divided into 2 to 4 individual doses or can be administered once daily in a storage form.

The novel compounds can be used in the usual solid or liquid pharmaceutical form, e.g., as uncoated or (film) coated tablets, capsules, powders, granules, sugar coated tablets, supositories, solutions, ointments, creams or sprays. These molds are produced by known methods. For this purpose, the active substances can be mixed with conventional pharmaceutical additives such as tablet binders, blowing agents, preservatives, tablet disintegrators, flow regulators, emollients, wetting agents, dispersants, emulsifiers, solutions, release retardants, antioxidants and and / or propellants (cf. H. Sucker et al .; Pharmazeutische Technologie, Thieme-Verlag, Stuttgart, 1978). The forms for administration obtained in this way normally contain from 0 _# 1 to 99% by weight of active ingredient.

Experimental part

Compounds of formula I may be prepared, for example, as depicted in Schemes III-III.

Structural blocks A, B, O and E are preferably assembled separately beforehand and used in a suitably protected form (see Schemes III-III).

• · · · · · φ φ

-34 • · · ··

Scheme I

A Β D Ε

(Ρ '= protecting group, (Ρ) = protecting group or Η)

Scheme I describes the linear assembly of molecule I by coupling the amine HE-CN to the N-protected amino acid PD-OH to obtain PDE-CN, removing the N-terminal protecting group to form HDE-CN, coupling the N-protected amino acid PB-OH, thereby is obtained by PBDE-CN, deprotection to obtain HBDE-CN, followed by alkylation with unprotected or protected (P) -AU structural block (U = cleavable group) or reductive alkylation of (P) -A'-U (U = aldehyde, ketone) or Michael addition with a suitable (P) -AC = C- derivative to give (P) -ABDE-CN. Conversion of a nitrile functionality to an amidine group is accomplished by either classical Pinner synthesis (R. Boder, D. G. Neilson, Chem. Rev. 61 (1962) 179).

4 4 4 «• · 4 4

444 4 4 9 9

4,444 4,444

4444 44 44 44 44 44

-35 or a modified Pioneer synthesis which proceeds via intermediates which are salts of iminothioesters (H. Vieweg et al., Pharmazie 57 (1984) 226) or directly by the A. Eschenmoser method (Heiv. Chimica Acta 69 (1986) 1224). Subsequently, the protecting group still present in the molecule is cleaved, preferably by acid hydrolysis.

When HE-CONH _{2 is} used as the E structural block in the synthesis, the dehydration of the amide group to the nitrile group or conversion to the thioamide functionality is carried out on protected intermediates. As an alternative, structural block E may be used in the form of HE-CSNH2 in the synthesis.

Scheme II

-36 Scheme II depicts linear assembly of a compound of formula I by alkylation, reductive amination, or Michael addition of HBP to the appropriate suitable unprotected or protected structural blocks A, providing (P) -ABP, a C-terminal protecting group tear to yield (P) - AB-OH, binding to GDP, providing (P) -ABDP, tearing off the C-terminal protecting group, providing (P) -ABD-OH, linking HE-CN, providing structure of formula (P) -ABDE-CN and reaction of these intermediates to give the final product as shown in Scheme I.

If the (P) -A-B-P compounds still have a free NH functionality on B, the NH functionality must first be protected before the C-terminal protecting group is peeled off. The protecting groups used in this case must be perpendicular to each other.

As an alternative to this HE-CN block can also be used HE-CONH2., HE-CSNH2, HEC (NH) NH _2, HEC (NP) _NH2, HEC (NP) NHP, with the following binding arises intermediate type (P) -ABD-E'CONH2, which in one embodiment is dehydrated to (P) -ABDE-CN or in the other embodiment directly converted to (P) -ABDE-CSNH ₂ , for example by reaction with Lawesson's reagent.

 • ·

-37 Scheme III

A B D E

Scheme III illustrates a very efficient route for the preparation of compounds of Formula I by convergent synthesis. The suitably protected (P) -A-B-OH and H-D-E-CN structural blocks are combined and the resulting intermediate of formula (P) -A-B-D-E-CN is reacted to give the final product as shown in Scheme I.

Alternatively, HDE-CONH2 or HDE-CSNH2 can also be used instead of HDE-CN, whereby the intermediate (P) -ABDE-CONH2 is formed upon coupling, which in the first embodiment dehydrates to (P) -ABDE-CN or is converted to (P) -ABDE-CSNH ₂ .

The N-terminal protecting groups used are Boc, Cbz or Fmoc, preferably Boc and the C-terminal protecting groups are methyl, tert-butyl and benzyl. If multiple protecting groups are present in the molecule, they must be perpendicular to each other if they are not to be cleaved simultaneously.

-38 * * »» »» »» 38 38

The desired coupling reactions and other reactions for the deprotection and deprotection reactions are carried out under conventional peptide chemistry conditions (see M. Bodanszky, A. Bodanszky, The Practice of Peptide Synthesis, 2nd edition, Springer Verlag Heidelberg, 1994).

The Boc protecting groups are cleaved by treatment with dioxane / HCl or TFA / DCM, and the Cbz protecting groups are cleaved by hydrogenolysis or by treatment with HF. Hydrolysis of the ester functions is accomplished by treatment with LiOH in an alcoholic solvent or dioxane / water mixture. TFA or HCl is used to cleave tert-butyl esters.

with Reaction progress was monitored by TLC, usually using the following mobile phases: AND. 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 separation is mentioned, these separations were carried out on silica gel using the mobile phases mentioned above.

Separation by reverse phase HPLC was carried out using acetonitrile / water and using HOAc buffer.

All reactions were routinely carried out under a nitrogen atmosphere.

The starting compounds can be prepared by the following methods:

Examples of structural blocks A used for alkylation are tert-butyl α-bromoacetate, tert-butyl β-bromopropionate, tert-butyl α-bromopropionate, tert-butyl γ-bromobutyrate, tert-butyl α-bromobutyrate, THP-protected bromethanol THP-protected γ-bromopropanol, α-bromo-γ-butyrolactone, dihydroxyacetone, di-tert-butyl acetone dicarboxylate and tert-butyl acrylate, tert-butyl methacrylate and di-tert-butyl fumarate are used for reductive amination. Those of these tert-butyl esters which cannot be purchased can be prepared by methods similar to those described in G. Uray, W. Lindner, Tetrahedron, 44, 1988 4357-4362.

Structural blocks B:

A wide variety of options for general and specific amino acid synthesis are described in the literature. They are reviewed, among others, in Houben-Weyl, Volume E16d / Part 1, pages 406 and others.

The precursors that have been used are benzophenone imine ethyl acetate, diethyl acetamidomalonate and ethyl isonitrilacetate.

Various racemic glycine and alanine derivatives have been prepared, for example starting from ethyl isonitrile acetate and a suitable ketone or aldehyde (see H.-J. Pratorius, J. Flossdorf, M.-R. Kula Chem. Ber. 108 (1975) 3079).

«T · τ * ·

-40 in e ·

The syntheses of cyclooctylglycine, cycloheptylglycine, 2-norbonylglycine, adamantylalanine, γ-methylcyclohexylalanine, 4-isopropyl-1-cyclohexyl alanine, 4-methyl-1-cyclohexyl alanine, 4-methyl-1-cyclohexylglycine and cycloheptylalanine 2-formylaminoacrylates (U. Schollkopf and R. Meyer, Liebigs Ann. Chem. 1977, 1174 and H.-J. Pratorius, J. Flossdorf, M.-R. Kula Chem. Ber. 108 (1985) 3079), wherein starting from ethyl isocyanoacetate and the relevant carbonyl compounds of cyclooctanone, cyclohexanone, 2-norbornanone, 1-formyladamantane, 1-formyl-1-methylcyclohexane, 1-formyl-4-isopropylcyclohexane, 1-formyl-4-methylcyclohexane and 4-methyl -cyclohexanone, formylcyclohexane and formylcyclopentane using the following general methods:

General method for the synthesis of ethyl 2-formylaminoacrylates:

A solution of 100m ethyl isocyanoacetate in 50mL THF was added dropwise to 100m potassium butoxide potassium in 150mL THF at 0-10 ° C. After 15 minutes at the same temperature, 100 mmol of the appropriate carbonyl compound in 50 ml of THF was added, the reaction mixture was allowed to warm slowly to room temperature and the solvent was removed by rotary evaporation. The residue was mixed with 50 mL of water, 100 mL of acetic acid and 100 mL of DCM, and the product was extracted with DCM. The DCM phase was dried over Na 2 SO 4 and the solvent was stripped off on a rotary evaporator. The resulting products were almost pure, but after cooling were further purified, if necessary, by column chromatography on silica gel (mobile phase: ether / petroleum ether mixtures).

General method for obtaining amino acid hydrochlorides starting from ethyl 2-formylaminoacrylates • 4 »4 ·

• 4 • 44 »44

100m ethyl 2-formylaminoacrylates were hydrogenated with Pd / C (10%) and hydrogen in 200mL glacial acetic acid until reaction was complete. The catalyst was then filtered off, the acetic acid was stripped off as much as possible on a rotary evaporator and the residue was refluxed in 200 ml of 50% concentrated hydrochloric acid for 5 hours. The hydrochloric acid was stripped off on a rotary evaporator and the product was dried at 50 ° C under reduced pressure and then washed several times with ether. The hydrochlorides were obtained as brownish 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. From 16.5 g (150 mmol) of 2-norbornanone, 26.6 g of 2-norbonylglycine hydrochloride was obtained. From 19.7 g (120 mmol) of 1-formyladamantane, 26.0 g of adamantylalanine hydrochloride were obtained. From 2.6 g (100 mmol) of 1-formyl-1-methylcyclohexane, 16.6 g of γ-methylcyclohexytalanine hydrochloride was obtained. From 16.8 g (150 mmol) of 4-methylcyclohexanone, 25.9 g of 4-methylcyclohexylglycine hydrochloride was obtained. 18 g of trans-4-methyl-1-cyclohexylalanine hydrochloride were obtained from 15 g of trans-1-formyl-4-methylcyclohexane. Starting from 9 g of 3,3-dimethyl-1-formylcyclohexane, 10 g of 3,3-dimethyl-1-cyclohexylalanine hydrochloride was obtained.

1-Formyl-3,3-dimethylcyclohexane aldehyde, which is required for synthesis, was prepared by a method based on the publication of Moskal and Lensen (Rec. Trav. Chim. Pays-Bas 106 (1987) 137-141).

A solution of n-butyllithium in n-hexane (72 mL, 115 mmol) was added dropwise over 10 minutes to the stirred solution.

-42 diethyl isocyanomethylphosphonate (17 mL, 105 mmol) in 280 mL of anhydrous diethyl ether at -60 ° C. The resulting suspension was then stirred at -60 ° C for 15 min and a solution of 3,3-dimethylcyclohexanone (13 g, 105 mmol) in 100 mL of anhydrous diethyl ether was added over 10 min while keeping the temperature below -45 ° C. . The reaction mixture was allowed to warm to 0 ° C and after stirring at this temperature for 90 min, 150-200 mL of a 38% aqueous hydrochloric acid solution was added. The resulting mixture was vigorously stirred at room temperature for 15 h to complete the hydrolysis. The organic phase was then separated and washed with 200 ml each of water, saturated sodium bicarbonate solution and saturated sodium chloride solution. It was then dried over magnesium sulfate, filtered, and concentrated on a rotary evaporator to remove the solvent. The resulting residue was used without further purification as the starting material for amino acid synthesis.

Cyclopentylglycine was prepared by hydrolysis using N-acetyl- (D, L) -cyclopentylglycine with 6N hydrochloric acid, wherein said N-acetyl- (D, L) -cyclopentylglycine was prepared as described in J.T. Hill and F.W. Dunnem, J. Org. Chem., 30 (1965), 1321.

Boc- (D) -α-methylcyclohexylalanine

3.4 g (12.2 mmol) of Boc- (D) -α-methyl-Phe-OH was hydrogenated in 100 mL of MeOH in the presence of 250 mg of 5% Rh on Al 2 O 3 at 10 bar with hydrogen at 50 ° C for Filtration and evaporation of the solvent gave 2.8 g of Boc- (D) -amethyl-Cha-OH. ¹ 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))

-43 • 4 ·· 4'4 ·

4 · · 4 · 4 '· ♦ ·· .; 4.

• 4 * 4 4 4 4 · »4 4 V • 4 · 4 · 4 · 4 · 4 · 4 · 4 · 4 · 4 · 4 · 4 · 4 · 4 · 4 · > * 4.4 * · <· 4

Boc- (3-Ph) -Pro-OH was synthesized by a method similar to that described in J.Y.L. Chung et al. (J.Y. L. Chung et al., J. Org. Chem. 55 (1990) 270).

Preparation of Boc-1-tetralinylglycine

Boc-1-tetralinylglycine was prepared from 1,2-dihydronaphthalene. 1,2-Dihydronaphthalene was first converted to 1-tetralyl bromide by treatment with HBr (similar to the reaction in J. Med. Chem. 57 (1994) 1586). The bromide was then reacted with diethyl acetamidomalonate and, after hydrolytic cleavage, the resulting amino acid was converted to the Boc-protected form of conventional conditions. Another possible preparation is described by E. Reimann and D. Voss (E. Reimann, D. Voss, Arch. Pharm. 310 (1977) 102).

Preparation of Boc- (D, L) Dch-OH ‘

Boc- (D, L) -Dpa-OH (1 mmol) was added. hydrogenated in 12 ml MeOH together with 5% Rh / Al 203 catalyst additive at 5 bar. Filtration and removal of the solvent under reduced pressure resulted in an almost quantitative yield.

Preparation of H-D, L-Chea-OH

4.0 g of cycloheptylmethyl methanesulfonate (19.39 mmol), prepared from cycloheptylmethanol and methanesulfonyl chloride, was refluxed for 10 hours under an inert gas atmosphere together with 4.9 g of benzophenone iminglycine ethyl ester (18.47 mmol), 8.9 g of dry fine powder. potassium carbonate (64.65 mmol) and 1 g tetrabutylammonium bromide (3 mmol) in 50 mL dry. The potassium carbonate was then filtered off, and the filtrate was evaporated

-44 «» · ¹ I I I I I I. 9. The crude product was directly hydrolyzed by treatment with 20 ml of 2N hydrochloric acid in 40 ml of ethanol, using stirring at room temperature for 1.5 hours. The reaction solution was then diluted and benzophenone was extracted into ethyl acetate in an acidic reaction, then extracted with HD, L-Chea-OEt into DCM under alkaline reaction (pH = 9) and the solution was dried over magnesium sulfate and concentrated in a rotary evaporator. Yield 3.7 g ξ 95% of theory.

Boc- (D, L) - (3,4,5- (MeO) 3) Phe-OH was prepared by alkylating benzophenone iminglycine ethyl ester with trimethoxybenzyl chloride, followed by addition of a Boc protecting group and ester hydrolysis.

H- (D, L) -p, p-Me ₂ Cha-OH was prepared according to the procedure described by U. Schollkopf, R. Meyer, L. Ann. Chem. (1977) 1174-82.

Said amino acids were converted to Boc-protected form using di-tert-butyl dicarbonate by conventional procedures and then recrystallized from ethyl acetate / hexane or purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether).

Boc-protected amino acids were used as B structural blocks as shown in Scheme I.

Said amino acids such as B structural blocks have also in some cases been converted to the corresponding benzyl esters and bound to correspondingly protected A structural blocks. In the case of N-H compounds which were still free, they were subsequently protected with a Boc group,

-45 · 9 · 9 · 9 · * 9 * * 9 9 9 * 9 9 9 9 9 9 9 9 9 fc '*' · The benzyl ester group was removed by hydrogenation and the AB-OH structural block was purified by crystallization, salt precipitation or column chromatography. This procedure is described in the example below for tBuOOC-CH2- (Boc) (D) Cha-OH.

Synthesis of (D) -cyclohexylalanine benzyl ester

A suspension of 100 g (481 mmol) of the (D) -cyclohexylalanine product, 104 g (962 mmol) of benzyl alcohol and 109.7 g (577 mmol) of p-toluenesulfonic acid monohydrate in 2200 ml of toluene was slowly heated at reflux with a water separator. In the temperature range of 80-90 ° C, formation of hydrogen chloride and dissolution of the suspension were observed to form a clear solution. When water was no longer separated (about 4 h), 500 ml of toluene was distilled off, the reaction mixture was allowed to cool overnight and the resulting residue was filtered off and washed twice with 1000 ml of hexane each time. The resulting residue (195 g) was then suspended in 2000 ml of dichloromethane and after addition of 1000 ml of water, the pH was adjusted to 9-9.5 by the stepwise addition of 50% sodium hydroxide solution with stirring. The organic phase was removed, washed twice, each time with 500 ml of water, dried over sodium sulphate and filtered to remove the desiccant, by concentrating the filtrate to dryness to give 115 g (94%) of the desired product as a light oil.

N- (tert-butyloxycarbonylmethylene) - (D) -hexylalanine benzyl ester

115 g (440 mmol) of (D) -cyclohexyialanine benzyl ester were dissolved in 2000 ml of acetonitrile, and 607.5 g (4.40 mol) of potassium carbonate and 94.3 g (484 mmol) of tert. butyl bromoacetate and the mixture was stirred at this temperature

• · ♦ t ·

-46 ° C 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 the organic phase was extracted with 5% citric acid and saturated sodium bicarbonate solution. The organic phase was dried over sodium sulfate, filtered to remove the desiccant, concentrated, and the resulting oil (168 g) was used directly in the next reaction.

N-Boc-N- (t-butyloxycarbonylmethylene) - (D) -cyclohexylalanine benzyl ester

The oil (168 g, 447 mmol) obtained in the previous synthesis was dissolved in 1400 mL of acetonitrile and after addition of 618 g (4.47 mmol) of potassium carbonate powder and 107.3 g (492 mmol) of di-tert. The butyl dicarbonate was stirred at room temperature for 6 days. The potassium carbonate was filtered off with suction, washed with approximately 1000 ml of acetonitrile and the filtrate was concentrated. 230 g of the expected product were obtained.

N-Boc-N- (t-butyloxycarbonylmethyl-T) - (D) -cyclohexylalanine cyclohexylammonium salt

115 g of N-Boc-N- (tert-butyloxycarbonylmethylene) - (D) -cyclohexylalanine benzyl ester was dissolved in 1000 ml of pure ethanol and hydrogenated in the presence of 9 g of 10% Pd on activated carbon with hydrogen at atmospheric pressure at 25-30 ° C Filtration and removal of the solvent in a rotary evaporator gave 100 g (260 mmol) of a yellow oil which was taken up in 1600 ml of acetone and heated at reflux. The heating bath was removed and a solution of 27 g (273 mmol) of cyclohexylamine in acetone was quickly added via a dropping funnel. The desired salt crystallized upon cooling the reaction mixture to room temperature.

-47 © · ·· · '♦ · *. ♦ *. ··.

«· 4 * 4 · *? · * * · · 4 4 4 4 4 4 4 4 • * · 4 · · 4 4 _S · 4 9

The solid was filtered off, washed with 200 ml of acetone and the final purification was carried out again by recrystallization from acetone. Drying the residue in a vacuum evaporator at about 30 ° C afforded 70.2 g of the desired salt as a white powder.

The cyclohexylammonium salt of N-Boc-N- (tert-butyloxycarbonylmethylene) - (D) -cyclohexylglycine was prepared analogously from cyclohexylglycine as a precursor. N-Boc-N- (tert-butyloxycarbonylmethylene) - (D) -cycloheptylglycine and N-Boc-N (tert-butyloxycarbonylmethylene) - (D) -cyclopentylglycine derivatives were prepared from the corresponding cycloheptyl- and cyclopentylglycine compounds.

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

a) tert-butyl 3-bromopropionate

16.64 g (109 mmol) of brompropionic acid, 150 ml of condensed 2-methylpropene and 2 ml of concentrated sulfuric acid were added to a glass autoclave under a countercurrent of nitrogen at -30 ° C, the autoclave was sealed and the mixture was stirred for 72 hours. at room temperature. For further work-up, the reaction vessel was recooled to -30 ° C and the reaction solution was poured cautiously into 200 mL of ice saturated sodium bicarbonate solution. Excess 2-methylpropene was evaporated with stirring, the residue was extracted three times, each time with 50 ml of dichloromethane and the combined organic phases were dried over sodium sulfate, filtered to remove the desiccant and concentrated with a vacuum pump. The oily residue was purified by column chromatography

··············

-48 (mobile phase n-hexane, later n-hexane / diethyl ether 9: 1). Thus 18.9 g of the title compound are obtained.

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

49.4 g (189 mmol) of (D) -cyclohexylalanine benzyl ester was dissolved in 250 ml of acetonitrile, treated with 31.6 g (151 mmol) of tert-butyl bromopropionate at room temperature, and the mixture was refluxed for 5 days. The mixture was filtered to remove the precipitate which was washed repeatedly with acetonitrile, the filtrate was concentrated under a vacuum water pump, the residue was taken up in 350 ml of dichloromethane and the organic phase was extracted with 5% citric acid and saturated sodium bicarbonate solution. The organic phase was dried over sodium sulfate, filtered to remove the desiccant, and concentrated. The oily residue was purified by column chromatography (eluent dichloromethane, additional dichloromethane / methanol 95: 5). A slightly contaminated oil was obtained and used directly in the next reaction.

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

The oil obtained in the above synthesis (30 g, max. 70 mmol) was dissolved in 150 mL of acetonitrile and treated with 28 mL (160 mmol) of diisopropylethylamine and 19.2 g (88 mmol) of di-tert-butyl dicarbonate and the mixture. was stirred for 3 days at room temperature. The reaction mixture was concentrated with a vacuum pump on a rotary evaporator,

4 »»

-49 »·» · κ ·· residue was taken up in n-hexane, the mixture was washed 5 times using each 3 ml of 5% citric acid solution, the combined organic phases were dried over sodium sulfate, _and filtered to remove desiccant and concentrated, and the residue was subjected to column chromatography (hexane / ethyl acetate 95: 5). This resulted in 32.66 g (64 mmol) of the desired product.

d) N-Boc-N- (t-butyloxycarbonylethylene) - (D) -cyclohexylalanine cyclohexylammonium salt ·

32.66 g (64 mmol) of N-Boc-N benzyl ester (tert-butyloxycarbonyl ethylene) (D) -cyclohexylaluminum was dissolved in 325 ml of pure ethanol and hydrogen hydrogen for 14 hours at atmospheric pressure at 25 ° C. -30 ° C in the presence of 3 g of 10% pure Pd on activated carbon. Filtration of the solution through Celite®, washing with ethanol and removal of the solvent in a rotary evaporator gave 26.7 g of a yellow oil which was taken up in acetone and heated at reflux. The heating bath was removed and a solution of 7g (70 mmol) of cyclohexylamine in acetone was quickly added via a dropping funnel. The desired salt crystallized by cooling the reaction mixture to room temperature. The solid was filtered off, washed with 25 ml of acetone and, for final purification, recrystallized once more from acetone. Drying the residue in a vacuum evaporator at 30 ° C gave 26.6 g (54 mmol) of the desired salt as a white powder.

4 «

N-Boc-N- (tert-butyloxycarbonylmethylene) - (D) -cyclohexyl alanyl 3,4-dehydroproline:

a) N-Boc-Pyr-OH (5 g, 23.45 mmol) was dissolved in MeOH (50 mL), and HCl in dioxane (4N, 30 mL) was added. The mixture was then heated under reflux for 12 hours. Removal of the solvent in a rotary evaporator gave the H-Pyr-OMe product as the final product. Yield: 3.84 g (100%).

b) N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Cha-OH (8 g, 20.75 mmol) was dissolved in dichloromethane (75 mL) and at -10 ° C was ethyldiisopropylamine (15.5 mL, 89.24 mmol) was added. Then the mixture was stirred for 5 minutes at this temperature and a solution of the H-Pyr-OMe product (3.4 g, 20.75 mmol) in dichloromethane (25 mL) was added dropwise. Subsequently, a solution of propanephosphonic anhydride in ethyl acetate (50%, 20 mL, 26.96 mmol) was added dropwise and the mixture was stirred for 2 h at temperatures from -10 to 0 ° C. The batch was diluted with dichloromethane and washed with saturated sodium bicarbonate 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 removed in a rotary evaporator. The crude product was purified by flash chromatography (silica gel, dichloromethane / methanol = 95/5). Yield: 6.2 g (60%).

c) N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Cha-Pyr-OMe (5.5 g, 11.12 mmol) was dissolved in dioxane (40 mL) then aqueous sodium hydroxide solution (1N, 22.2 mL, 22.24) was added

0'0 • '· 0 0 .0 0 * • * ··· · .0 · ·'!

0 0 0 0 0 0 '0 ·

The mixture was stirred for 2 hours at room temperature. The dioxane was removed on a rotary evaporator and the aqueous phase was washed with ethyl acetate and acidified to pH 1-2 with potassium hydrogen sulphate 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 (m.p. - 158-160 ° C).

N-Boc-N- (tert-butyl oxycarbonylmethylene) - (D) -cyclohexylglycyl-3,4-dehydroproline:

This compound was synthesized analogously from NBoc-N- (tert-butyl oxycarbonylmethyl) - (D) -cyclohexylglycine and 3,4-dehydroproline methyl ester.

N-Boc-N- (tert-butyloxycarbonylmethylene) - (D) cyclohexylalanylproline:

a) N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Cha-OH (20 g, 51.88 mmol) was dissolved in dry methylene chloride (100 mL). After cooling to -5 ° C, Nethyldiisopropylamine (90 mL, 518.88 mmol) was added dropwise and stirring was continued for 5 minutes. H-Pro-OBn x HCl (12.54 g, 51.88 mmol) was then added at -5 ° C and the mixture was stirred for 5 minutes. A 50% solution of propanephosphonic anhydride in ethyl acetate (45.1 mL, 62.26 mmol) diluted with methylene chloride (45 mL) was added dropwise over 30 minutes. Then the mixture was stirred after

52 hours at 0-5 ° C, then slowly transferred to room temperature and stirred for 12 hours at room temperature. The batch was diluted with methylene chloride and washed sequentially with saturated sodium bicarbonate solution, 5% citric acid solution and saturated sodium chloride solution. After drying over sodium sulfate, the solvent was distilled off in vacuo. Yield: 28.9 g (light yellow oil, 97%).

b) The product obtained as described in a) (28.5 g, 49.76 mmol) was dissolved in methanol (650 mL), 10% pure Pd on carbon (1.8 g) was added, and the mixture was hydrogenated at room temperature and under a hydrogen atmosphere at 100 kPa. The catalyst was then removed by filtration through a Celite® filter and the filtrate was concentrated in vacuo. Yield: 22.2 g (colorless foam, 92%).

N-Boc-N- (tert-butyloxycarbones Imethylene) - (D) cyclohexylglycylproline:

This compound was prepared analogously from N-Boc-N (tert-butyloxycarbonylmethylene) - (D) -cyclohexylglycine and proline methyl ester.

D structural blocks:

Compounds utilized as D structural blocks, (L) -proline, (L) -pipecolinic acid, and (L) -azetidine carboxylic acid are commercially available either as free amino acids such as Boc

The compounds to be protected or the corresponding methyl esters. When (L) -3,4-dehydroproline or (D, L) -4,5-dehydropipecolinic acid or the corresponding protected derivatives were used as D structural blocks, the prepared compounds were hydrogenated in the last step to produce the corresponding proline derivatives. (L) -3,4-dehydroproline (H-Pyr-OH) is commercially available; (D, L) -4,5-dehydropipecolinic acid (H- (D, L) -Dep-OH) can be prepared according to the procedures described in A. Burgstahler, CE Aiman J. Org. Chem. 25 (1960), 489 or by C. Herdeis, W. Engel Arch.

Pharm 326 (1993), 297.

E structural blocks were synthesized as follows:

5-Aminomethyl-2-cyanothiophene:

This structural block was synthesized as described in WO 95/23609.

4-Aminomethyl-2-cyanothiophene:

a) 2-bromo-4-formylthiophene g (320 mmol) of 3-formylthiophene was dissolved in 600 ml of methylene chloride, the solution was cooled to 5 ° C, then 100 g (750 mmol) of aluminum chloride was added in small portions and the reaction mixture was subsequently refluxed. A solution of 59 g (19 mL, 360 mmol) of bromine in 40 mL of methylene chloride was added dropwise over 45 minutes and the mixture was allowed to react under reflux for 4 hours. After cooling, the reaction solution was quenched with 600 g of ice water and extracted with methylene chloride, the organic phase was washed with a saturated solution.

- 54 • · 0 0 0 0 0 · 0 · * '0 0 · 0 000 000 · «0 0 ^{! L.}

0 · 0 · 0 · 0 · 0 · 0 · 0 · 0 · 0 · 0 · 0 · 0 · 0 · 0 · 0 · 0 · 00 · 00 · 00 · 00 · * ·· sodium bicarbonate, dried over magnesium sulphate and concentrated in a rotary evaporator under vacuum . 64.5 g of crude product were obtained, which was purified by column chromatography (silica gel, methylene chloride / petroleum ether). Overall, 56.5 g of somewhat contaminated product was obtained.

b) 2-cyano-4-formylthiophene

Copper cyanide (7.6 g, 85 mmol) was added to a solution of 2-bromo-4-formylthiophene (13.53 g, 70.82 mmol) in DMF (25 mL) and the reaction mixture was refluxed for 3.5 hours, during which time the initially light green suspension turned into a dark solution. After addition of water, the reaction mixture was repeatedly extracted with ethyl acetate, the organic phases were combined, washed with saturated sodium chloride solution, dried over sodium sulfate and concentrated under somewhat reduced pressure. Purification of the residue with ether (7 g) gave 1.6 g of pure product. The mother liquor together with the crude products from the other batches was purified by chromatography. (silica gel, methylene chloride / petroleum ether 1: 1). A total of 56.5 g of 2-bromo-4-formylthiophene was reacted to give 2-cyano-4-formylthiophene to give 12.6 g of pure product (31% yield).

c) 2-cyano-4-hydroxymethylthiophene

3.47 g (91.8 mmol) of sodium borohydride was added portionwise to a suspension of 12.6 g (91.8 mmol) of 2-cyano-4-formylthiophene in 200 ml of ethanol and stirred at room temperature for 2 hours during which time the reaction mixture slowly turned into a clear solution. After concentration

- 55 · ♦ 55 55 55 55 55 55 55 55 55 55 55

in vacuo, the residue was taken up in ethyl acetate, washed successively with saturated sodium chloride solution, 5% citric acid and saturated sodium chloride solution, and the organic phase was dried using sodium sulfate and concentrated in vacuo. It was obtained

11.7 g of predominantly pure product (yield 91.5%).

d) 4-bromomethyl-2-cyanothiophene

11.7 g (84.07 mmol) of 2-cyano-4-hydroxymethylthiophene together with 24.1 g (91.87 mmol) of triphenylphosphine were dissolved in 100 ml of THF at room temperature and successively cooled with cooling (ice bath). 30.47 g (91.87 mmol) of tetrabromomethane are added. After stirring for 3 hours at room temperature, the mixture was concentrated in vacuo and purified by silica gel chromatography (methylene chloride / petroleum ether system). 18.8 g of a crystalline pale yellow product which still contained petroleum ether were obtained.

e) 4-N, N-bis (tert-butoxycarbonyl) aminomethyl-2-cyanothiophene

18.81 g of 4-bromomethyl-2-cyanothiophene (crude product, maximum 84.07 mmol) was dissolved in 160 mL of THF, the solution was cooled to 5 ° C and 3.07 g (102.4 g) was added portionwise. mmol) 80% sodium hydride suspension. 22.25 g (102.4 mmol) of di-tert-butyl iminodicarboxylate dissolved in 160 ml of THF were then added dropwise and the mixture was then stirred at room temperature overnight at 5 ° C. Since TLC revealed that the reaction was not complete, the batch was heated at 30-35 ° C for 4.5 hours. After cooling to 0-5 ° C, it was

33 ml of saturated ammonium chloride solution was slowly added dropwise, THF was distilled off in vacuo, the residue was extracted repeatedly with ethyl acetate and the ethyl acetate phases were washed with saturated sodium chloride solution, dried over sodium sulfate and evaporated in a rotary evaporator. The red viscous residue (34.61 g) was used as a crude product in a subsequent reaction.

f) 4-aminomethyl-2-cyanothiophene hydrochloride

34.61 g of 4-N, N-bis (tert-butoxycarbonyl) aminomethyl-2-cyano-thiophene (crude product, maximum 84.07 mmol) was dissolved in 600 ml of ethyl acetate and the solution was cooled to 0-5 ° C, saturated with gaseous HCl and was warmed to room temperature. After 3 hours, the resulting suspension was evaporated on a rotary evaporator, the product was distilled together with methylene chloride, the residue was extracted by stirring with ether and dried in vacuo. 13.85 g of product were obtained in the form of a light powder. Two-step yield: 94.3%.

2-Aminomethyl-4-cyanothiophene:

a) 4-cyanothiophene-2-carbaldehyde

49.3 g (258.05 mmol) of 4'-bromothiophene-2-carbaldehyde a

Copper cyanide (27.8 g, 310.41 mmol) was suspended in absolute DMF (130 ml) and the suspension was refluxed for 8 hours. The solvent was evaporated in vacuo on a rotary evaporator at 40 ° C, the residue was suspended in ethyl acetate and the suspension was transferred to a Soxleth apparatus. The residue was extracted overnight, the yellow solution was dried over sodium sulfate and evaporated in vacuo. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - and the resulting yellow solid was recrystallized from ether. Yield: 25.3 g (80% of theory).

b) 4-cyanothiophene-2-carbaldehyde oxime

11.6 g (84.6 mmol) of 4-cyanothiophene-2-carbaldehyde was dissolved in 140 ml of methanol and 12.3 g (116.1 mmol) of sodium carbonate was added. Then, 6.5 (93.5 mmol) hydroxylamine hydrochloride was added portionwise with cooling at 15 ° C and the mixture was stirred for 2 hours at 10 ° C. After 80 ml of water was added, the reaction mixture was extracted five times using 50 ml of diethyl ether each time, the organic phase was dried over sodium sulfate and the solvent was removed in vacuo. 12.5 g of the desired product were obtained as a yellow crystalline powder (96% of theory).

c) 2-aminomethyl-4-cyanothiophene hydrochloride

11.22 g (171.64 mmol) of fine zinc powder was carefully added in several small portions to a solution of 4.65 g (30.60 mmol) of 4-cyanothiophene-2-carbaldehyde oxime in 50 mL of trifluoroacetic acid cooled to 0-5 ° C so that the temperature does not exceed 15 ° C. After stirring for 3 hours at room temperature, the excess zinc was decanted, most of the trifluoroacetic acid was removed in vacuo (oil pump), the remaining oil was cooled to 0 ° C, and a mixture of 150 mL of 3N aqueous sodium hydroxide solution and 2 I methylene chloride, previously cooled to 0 ° C. After • ·

The insoluble matter was filtered off, the organic phase was discarded, the aqueous phase was extracted eight times using 20 ml of methylene chloride, the combined organic phases were dried over sodium sulfate and then 20 ml of 6M hydrochloric acid in methanol was added under ice-cooling. During this process, the product precipitated as hydrogen chloride as a white solid, and the suspension was cooled to 4 ° C overnight to complete the crystallization. 2.2 g of product were obtained as colorless needles (50% of theory).

5-Aminomethyl-3,4-dimethylthiophene-2-carboxamide hydrochloride:

g (105.42 mmol) of 5-cyano-3,4-dimethylthiophene-2-carboxamide was suspended in 760 ml of methanol and 110 ml of 2N hydrochloric acid solution, then 9.5 g of Pd on carbon (10%) was added and the mixture was hydrogenated at room temperature. After 4.7 L of hydrogen (4 h) had passed, the methanol was distilled off in vacuo and the aqueous phase was extracted three times with ethyl acetate and then freeze-dried. Yield: 16.3 g (70.4% of theory).

5-Aminomethylisoxazole-3-carboxamide:

a) Ethyl 5-chloromethylisoxazole-3-carboxylate

21.2 g (210 mmol) of triethylamine was added dropwise with stirring to a mixture of 30 g (198 mmol) of ethyl 2-chloro-2-hydroxyiminoacetate and 150 ml of propargyl chloride, cooled to 10-15 ° C. Stirring was continued for 1 hour at room temperature, then

- 59 • water, mixture was added - 59 It was extracted with ether and the organic phase was dried over magnesium sulfate and concentrated in vacuo on a rotary evaporator. The residue was distilled under vacuum at 0.5 torr, the product distilled at 116-122 ° C.

b) 5-chloromethylisoxazole-3-carboxylic acid g (250 mmol) of potassium hydroxide was added to 47.3 g (250 mmol) of ethyl 5-chloromethylisoxazole-3-carboxylate in 150 ml of ethanol and the reaction mixture was stirred for 6 hours at 60-60 ° C. 70 ° C. After cooling, the mixture was concentrated in vacuo, the residue was taken up in water and extracted with ether, the aqueous phase was acidified with hydrochloric acid and then extracted repeatedly with ether, the ether phase was dried over sodium sulfate and concentrated in vacuo (oil pump, 50 ° C). Yield: 31 g (77% of theory);

c) 5-Chloromethylisoxazole-3-carboxylic acid chloride

120 g (743 mmol) of 5-chloromethylisoxazole-3-carboxylic acid together with 500 ml of thionyl chloride and 2 drops of pyridine were refluxed for 10 hours, concentrated in vacuo and then distilled at 20 torr. The product distilled at 125-133 ° C. Yield: 78 g (58% of theory).

d) 5-chloromethylisoxazole-3-carboxamide

- 60 444 · · 4444 44 «4 · 4 4 β

443 4 4 4 4

445 44 4

4 4 4 4 4 5

4 «43 * ·» 4

Ammonia was passed for 1 hour at 10-15 ° C with a solution of 10 g (55.56 mmol) of 5-chloromethylisoxazole-3-carboxylic acid chloride in 100 ml of methylene chloride, and then stirring was continued for 1 hour at room temperature. After the solution was cooled to 0 ° C, the precipitate was filtered off with suction and washed with some cold methylene chloride, and the residue was extracted twice with water while stirring while removing the ammonium salts. Vacuum drying 6.58 g of pure product was obtained as a pale powder (74% of theory).

d) 5-aminomethylisoxazole-3-carboxamide hydrochloride

2.44 g (15.2 mmol) of 5-chloromethylisoxazole-3-carboxamide was added to a mixture of 100 ml of concentrated ammonia solution and 72 ml of methanol, and the reaction solution was heated to 40 ° C and constantly saturated with ammonia gas throughout the reaction. After 6 hours the precursor reacted. The methanol was removed in vacuo and the aqueous phase was extracted twice using methylene chloride and then evaporated to dryness in a rotary evaporator under vacuum under mild conditions. The residue, which was a white solid, was used as a crude product in the coupling reactions.

2-Aminomethyloxazole-4-thiocarboxamide and 2 aminomethylthiazole-4-thiocarboxamide were prepared according to the procedure of G. Videnov, D. Kaier, C. Rempter and G. Jung Angew. Chemie (1996) 108, 1604, the N-Boc protected compounds described therein were deprotected using ethereal hydrochloric acid in methylene chloride.

4-Aminomethylthiazole-2-thiocarboxamide:

(a) Monothio-oxalic acid diamide

The monothiophthalic acid diamide was prepared using ethyl thiooxamidate as feedstock according to the procedure of W. Walter, K.-D. Bode Liebigs Ann. Chem. 660 (1962) 74-84.

b) 2-carbamoyl-4-chloromethylthiazole g (96 mmol) of ethyl thiooxamidate was transferred to 170 ml of n-butanol, 26 g (204 mmol) of 1,3-dichloroacetone were added and the mixture was heated at 112 ° C under nitrogen for 90 minutes atmosphere. The reaction mixture was then concentrated in vacuo and the residue was extracted by stirring with n-hexane (120 mL). 10 g of pure product were obtained.

c) 4-Boc-aminomethyl-2-carbamoylthiazole.

g (56.6 mmol) of 2-carbamoyl-4-chloromethylthiazole was added to an ammonia saturated solution of 350 ml of methanol and 80 ml of a 25% aqueous ammonia solution. The reaction mixture was heated for 6 hours at 4042 ° C while saturated with ammonia, then concentrated in vacuo and distilled together with methanol, and the residue was then extracted by stirring with ether and then acetone. 7.6 g of crude product which still contained a small amount of ammonium chloride was isolated. To remove this

The secondary product was allowed to react, the crude product with (Boc) ₂ O in aqueous dioxane and the protected compound was purified by column chromatography. 4.95 g of pure product were obtained.

d) 4-Boc-aminomethyl-2-cyanothiazole

4.95 g (19.24 mmol) of 4-Boc-aminomethyl-2-carbamoylthiazole was added to 90 ml of methylene chloride and 16.7 ml (97.44 mmol) of diisopropylethylamine, the mixture was cooled to 0 ° C, dropwise at A solution of 6.35 ml of trifluoroacetic anhydride in 10 ml of methylene chloride was added to 5 ° C and the mixture was then warmed to room temperature (TLC control). Then 25 ml of water was added, the mixture was stirred for 30 minutes at room temperature and the pH was adjusted to 2.5 with 10% citric acid solution and the organic phase was washed repeatedly, dried using magnesium sulfate and concentrated in vacuo. 5.4 g of a viscous, light brown crude product were obtained, which was used in the next step without further purification.

E). 4-Boc-Aminomethyl-2-thiocarbamoyl thiazole

The crude product obtained from (d) (max 19.24 mmol) was dissolved in 65 mL of pyridine and 5 mL of triethylamine, saturated with hydrogen sulfide and then allowed to stand at room temperature over the weekend. The reaction mixture was then evaporated in vacuo on a rotary evaporator, the residue was taken up in a mixture of ether and ethyl acetate, and the mixture was washed with 10% citric acid solution and water, dried over sulfate.

CO 4 4 · ♦ · ···. ·

Ο W% · Ο 4 4 4 0 0 0 0 · 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 6.0 g was obtained as a light yellow solid foam.

e) 4-Aminomethyl-2-thiocarbamoylthiazole hydrochloride

The product obtained from the previous experiment was transferred to 100 mL of methylene chloride, 30 mL of an approximately 5 molar solution of ethereal hydrochloric acid was added, and the mixture was stirred overnight at room temperature. The reaction mixture was then evaporated to dryness under vacuum in a rotary evaporator, co-distilled repeatedly with ether and then extracted by stirring with methylene chloride. 4.15 g of the desired product was obtained as a pale yellow amorphous substance.

4-amidino-2- (N-Boc-aminomethyl) -5-methylthiazole x HOAc

(a) α-acetylglycine methyl ester hydrochloride

Potassium tert-butylate (17.8 g, 157.9 mmol) was added to THF (120 mL) and then a solution of N-diphenylmethyldlidengiycin methyl ester (40 g, 157.9 mmol) in THF (60 mL) was added at -70 ° C. . After the yellowish solution was stirred for 30 minutes at this temperature, a solution of acetyl chloride (12.4 g, 157.9 mmol) in THF (70 mL) was added dropwise at -70 ° C. After the mixture was stirred at this temperature for 1.75 hours, 3N HCl (160 mL) was added and the yellowish suspension was stirred for an additional 10 minutes at room temperature. THF was removed at room temperature on a rotary evaporator and the remaining aqueous phase was washed 3 times with diethyl ether. The aqueous phase was freeze-dried and the residue was extracted by stirring with

methanol. The methanolic solution of the product was concentrated on a rotary evaporator at 35 ° C. Yield: 26.4 g (157.9 mmol, quantitative, yellowish solid).

b) BOC-Gly- (α-acetyl-Gly) -OMe

HOC-Gly-OH (24.05 g, 137.27 mmol) was added to THF (400 mL) and triethylamine (13.87 g, 137.19 mmol) was added. The colorless solution was cooled to -20 ° C and a solution of isobutyl chloroformate (18.75 g, 137.28 mmol) in THF (20 mL) was added dropwise at this temperature. The colorless suspension was stirred for an additional 30 minutes at -20 ° C and then acetylglycine methyl ester hydrochloride (23.0 g, 137.3 mmol) was added portionwise. The mixture was stirred for 30 min at -20 ° C and then a solution of triethylamine (13.87 g, 137.19 mmol) in THF (20 mL) was added dropwise over 45 min. The mixture was then stirred for 4 hours at -20 ° C, stirring was continued for a further 12 hours at room temperature. The residue was filtered off with suction, washed with THF and the combined THF phases were concentrated on a rotary evaporator.

Yield: 44.1 g (light brown oil).

¹ H NMR (270 MHz, CDCl ₃ ) δ 1.45 (s, 9H), 2.40 (s, 3H), 3.85 (s, 3H), 3.90 (d, J = 6.5 Hz) 2 H, 5.25 (d, J = 6.5 Hz, 1 H), 7.30 (sbr, 1 H).

c) Methyl 2- (N-Boc-aminomethyl) -5-methylthiazole-4-carboxylate

BOC-Gly- (α-acetyl-Gly) -OMe (39.8 g, 138.2 mmol) was added to THF (400 mL) and Lawesson's reagent (96.6 g, 238 mL) was added in portions at room temperature. 8 mmol).

and

0 '0 ·' * β c · · · ·

999 »999 9 · · · (

• »

000

The yellowish solution was then refluxed for 1.5 hours. THF was removed on a rotary evaporator. The residue (red-brown oil) was extracted by stirring with diethyl ether (600 mL). The ether phase was decanted from undissolved brownish oil and washed successively with 5% citric acid (2x), saturated NaHCO ₃ solution (9x) and water (2x). After drying (MgSO4) the solvent was removed on a rotary evaporator. Yield: 22.0 g (77 mmol, 56%, brownish solid).

¹ H NMR (270 MHz, CDCl ₃ ) δ = 1.50 (s, 9H), 2.75 (s, 3H), 3.95 (s, 3H), 4.55 (d, J = 6.5) Hz, 2H), 5.45 (t, J = 6.5)

Hz, 1H). (Main rotamer on Boc group).

d) 2- (N-Boc-aminomethyl) -5-methylthiazole-4-carboxylic acid

Methyl 2- (N-Boc-aminomethyl) -5-methylthiazole-4-carboxylate (22.0 g, 77 mmol) was dissolved in ethanol (100 mL) and a solution of LiOH (2.2 g, 92 mmol) in water ( 50 ml). The mixture was stirred for 30 minutes at room temperature and then the ethanol was removed on a rotary evaporator and the remaining solution was diluted with water (70 mL). The aqueous phase was washed with ethyl acetate (3X) and the pH was adjusted to 2 with 20% NaHSO ₄ solution, during which a light brown oil separated. The aqueous phase was extracted with dichloromethane and the combined organic extracts were dried (MgSO ₄ ) and concentrated in vacuo. The light brown residue was extracted by stirring in diisopropyl ether. The remaining colorless precipitate was filtered off with suction and washed with diisopropyl ether. Yield: 6.9 g (25.4 mmol, 33%, colorless solid).

¹ H NMR (270 MHz, DMSO-d 6) δ = 1.40 (s, 9H), 2.65 (s, 3H), 4.30 (d, J = 6.5 Hz, 2H), 7.80 (t, J = 6.5 Hz, 1 H).

e) 2- (N-Boc-aminomethyl) -5-methylthiazole-4-carboxamide

2- (N-Boc-aminomethyl) -5-methylthiazole-4-carboxylic acid (6.8 g, 25 mmol) was dissolved in THF (100 mL) and triethylamine (2.53 g, 25 mmol) was added. The mixture was cooled to -20 ° C, and a solution of isobutyl chloroformate (3.41 g, 25 mmol) in THF (10 mL) was added dropwise. The mixture was stirred for 30 minutes at -20 ° C and then ammonia gas was introduced into the light brown suspension for 45 minutes. The mixture was then warmed to room temperature. The residue was filtered off with suction and extracted with THF, the filtrates were concentrated. Yield: 6.9 g (25 mmol, quantitative).

@ ¹ H NMR (270 MHz, DMSO-d6) .delta. = 1.40 (s, 9H), 2.65 (s, 3H), 4.30 (m, 2H), 7.40 (sbr, 1H); 7.50 (sbr, 1H); 7.80 (t, J = 6.5 Hz, 1H).

f) 4-cyano-2- (N-Boc-aminomethyl) -5-methylthiazole

2- (N-Boc-Aminomethyl) -5-methylthiazole-4-carboxamide (6.8 g, 25 mmol) was added to dichloromethane (120 mL). After cooling the mixture to 0 ° C, diisopropylethylamine (15.84 g, 122.8 mmol) was added dropwise. A solution of trifluoroacetic anhydride (8.25 g, 39.3 mmol) in dichloromethane (20 mL) was added dropwise over 30 minutes at -5 ° C. The mixture was stirred for 30 minutes at 0 ° C while warming to room temperature, then stirring was continued for a further 12 hours. The mixture was diluted with dichloromethane (100 mL) and washed with 20% • · ^w · 99 7 · _ · ί - ····

Citric acid, saturated NaHCO ₃ solution and saturated NaCl solution. The organic phase was dried (MgSO ₄ ) and concentrated in vacuo. Yield: 6.3 g (25 mmol, quantitative).

(g) 4-amidino-2- (N-Boc-aminomethyl) -5-methylthiazole x CH ₃ COOH

4-Cyano-2- (N-Boc-aminomethyl) -5-methylthiazole (5.5 g, 21.74 mmol) was dissolved in methanol (15 mL) and N-acetylcysteine (4.1 g, 25 mL) was added. , 12 mmol). The mixture was then heated to 60 ° C with ammonia bubbling for 22 hours. The batch was diluted with methanol and passed through an acetate ion exchanger. The methanol was removed on a rotary evaporator and the residue extracted with acetone. The colorless residue was filtered off with suction and dried in vacuo. Yield: 4.75 g (14.4 mmol, 66%, colorless solid).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ = 1.40 (s, 9H), 1.80 (s, 3H), 2.60 (s, 3H), 4.35 (d, J = 6) 1.5 Hz, 2H), 7.90 (t, J = 6.5 Hz, 1H).

2-Aminomethyl-5-amidino-4-methylthiazole x 2 HCl

a) N-BOC-glycine thioamide

N-BOC-glycinonitrile (12.0 g, 76.8 mmol) and diethylamine (0.16 mL, 2.1 mmol) were dissolved in toluene (100 mL). The solution was cooled to -10 ° C, saturated with hydrogen sulfide and then stirred overnight at room temperature. The resulting precipitate was filtered off with suction and washed with toluene. The product was dried under vacuum at 45 ° C.

·· · ·· ♦ · 9 ti ti. · · Ti •

ti

Yield: 13.2 g (69.4 mmol, 90.3%, yellowish solid).

¹ H NMR (270 MHz, DMSO-d ₆ ) δ = 1.40 (s, 9H), 3.80 (d, J = 7 Hz, 2H), 7.05 (t, J = 7 Hz, 1H) 9.0 (sbr, 1H); 9.65 (sbr, 1H).

b) Methyl 2- (N-BOC-aminomethyl) -4-methylthiazole-5-carboxylate

N-BOC-glycine thioamide (10.0 g, 52.6 mmol) was added to methanol (70 mL) and methyl 2-chloroacetoacetate (7.9 g, 52.6 mmol) was added. The mixture was heated for 2 hours at 60 ° C and then stirred for 48 hours at room temperature. The methanol was removed on a rotary evaporator and the residue was extracted by stirring with acetone / diethyl ether. The residual precipitate was filtered off with suction and the filtrate was concentrated. The solid obtained from the filtrate was the desired product (purified by TLC and HPLC). Yield: 8.7 g (30.4 mmol, 57.8%).

ESI-MS: 287 (M + H &lt; + &gt;).

c) 2- (N-BOC-aminomethyl) -4-methylthiazole-5-carboxylic acid

Methyl 2- (N-BOC-aminomethyl) -4-methylthiazole-5-carboxylate (2.8 g, 9.74 mmol) was dissolved in 1,4-dioxane (30 mL) and 1N sodium hydroxide solution (19 mL) was added. ). The mixture was stirred for 4 hours at room temperature and 1,4-dioxane was removed on a rotary evaporator. The mixture was diluted with water and washed with ethyl acetate. The aqueous phase was acidified with a 20% potassium bisulfate solution and the precipitate obtained during this process was filtered off with suction and washed with water. Thus

The product obtained was dried in a vacuum evaporator at 40 ° C. Yield: 2.5 g.

d) 2- (N-BOC-aminomethyl) -4-methylthiazole-5-carboxamide

2- (N-BOC-Aminomethyl) -4-methylthiazole-5-carboxylic acid (12.6 g, 46.27 mmol) was dissolved in dichloromethane (460 mL) and dimethylformamide (0.4 mL). The mixture was cooled to 0 ° C and a solution of oxalyl chloride (6.46 g, 50.90 mmol) in dichloromethane (40 mL) was added dropwise over 30 minutes. The mixture was stirred for 2 hours at 0 ° C, then cooled to -20 ° C and ammonia was bubbled through at this temperature until the reaction was complete. The mixture was then warmed to room temperature and washed with water. The precipitate formed during this process was filtered off with suction. The organic phase was washed with 5% citric acid solution, dried (MgSO4) and concentrated on a rotary evaporator. The resulting solid was combined with the precipitate that was previously obtained by filtration, dried at 50 ° C in a vacuum oven. Yield: 9.8 g (36.12 mmol, 78%).

e) 2- (N-BOC-aminomethyl) -5-cyano-4-methylthiazole

2- (N-BOC-aminomethyl) -4-methylthiazole-5-carboxamide (11.13 g, 41.02 mmol) was suspended in dichloromethane (75 mL) and cooled to 0 ° C. At this temperature, ethyldiisopropylamine (17.86 mL, 102.55 mmol) was added first and then slowly a solution of trifluoroacetic anhydride (6.56 mL, 47.17 mmol) in dichloromethane (20 mL). After stirring for 1 hour, the mixture was diluted

Dichloromethane and washed with 5% citric acid solution. After drying (MgSO 4), the solvent was removed by rotary evaporation and the crude product was purified by flash chromatography.

Yield: 6.5 g (25.66 mmol, 63%).

f) 2- (N-BOC-aminomethyl) -4-methylthiazole-5-thioamide

2- (N-BOC-aminomethyl) -5-cyano-4-methylthiazole (7.5 g, 29.61 mmol) was dissolved in pyridine (30 mL) and triethylamine (27 mL) was added. The solution was saturated with hydrogen sulfide at 0 ° C and then allowed to stand for 48 hours at room temperature. The solvent was then removed on a rotary evaporator and the residue was taken up in ethyl acetate, washed with 20% potassium hydrogen sulfate solution and dried over magnesium sulfate. The solvent was removed on a rotary evaporator and the crude product was dissolved in dichloromethane and precipitated with petroleum ether. The product that precipitated was filtered off with suction, dried in a vacuum oven at 40 ° C. Yield: 7.1 g (24.7 mmol, 83%).

g) 5-amidino-2- (N-BOC-aminomethyl) -4-methylthiazole x HOAc

2- (N-BOC-aminomethyl) -4-methylthiazole-5-thioamide (7.1 g, 24.70 mmol) was dissolved in dichloromethane (40 mL) and iodomethane (17.5 g, 123.52 mmol) was added. ). The mixture was stirred for 56 hours at room temperature, the solvent was removed on a rotary evaporator. The residue was dissolved in a 10% methanolic ammonium acetate solution (29 mL) and stirred at 40 ° C until the reaction was complete. Solvent was removed • Φ

Rotary evaporator, the residue was extracted by stirring with dichloromethane and the resulting solid was filtered off with suction and washed with dichloromethane.

. The residue was dissolved in methanol and converted into the corresponding acetate by means of an ion exchanger saturated with acetate. The solvent was removed on a rotary evaporator and the resulting red-brown oil was extracted by stirring with dichloromethane. The product was obtained as a colorless solid which was dried under vacuum at 40 ° C. Yield: 5.3 g (16.04 mmol, 65%).

h) 5-amidino-2-aminomethyl-4-methylthiazole x 2 HCl

5-amidino-2- (N-BOC-aminomethyl) -4-methylthiazole x HOAc (1.6 g, 4.84 mmol) was suspended in dichloromethane (20 mL), and 4M hydrochloric acid in 1M was added at room temperature. Of 4-dioxane (4.84 mL, 19.37 mmol) and the mixture was stirred for 3 hours at this temperature. The product was filtered off, washed with dichloromethane and dried under vacuum at 40 ° C. Yield: 0.73 g (3.00 mmol, 62%).

2-Aminomethyl-5-amidino-4-trifluoromethylthiazole x 2 HCl

a) Ethyl 2- (N-BOC-aminomethyl) -4-trifluoromethylthiazole-5-carboxylate

N-BOC-glycine thioamide (5.0 g, 26.28 mmol) was dissolved in acetonitrile (60 mL) and a solution of ethyl 2-chloro-4,4,4-trifluoroacetoacetate (6, 38 g, 26.28 mmol). The mixture was then stirred for additional minutes at 5 ° C and for 12 hours at temperature

• 9 rooms. The batch was then cooled to 0 ° C and triethylamine (12 mL, 86.77 mmol) was added dropwise. The mixture was stirred for 20 minutes at 0 ° C, during which time the yellow suspension turned into a clear red-brown solution. Thionyl chloride (2.1 mL, 28.89 mmol) was then added dropwise at 0 ° C. The mixture was stirred for 20 minutes at 0 ° C, then allowed to warm to room temperature over the next hours. The solvent was removed on a rotary evaporator and the residue was taken up in water (100 mL) and extracted repeatedly with ethyl acetate. The combined organic phases were dried (Na ₂ Al ₄ ) and concentrated. The crude product was purified by chromatography (silica gel MeOH / DCM = 2:98). Yield: 2.2 g (6.4 mmol, 24.5%).

¹ H NMR (270 MHz, DMSO-d 6) δ = 1.30 (t, J = 6.5 Hz, 3H), 1.45 (s, 9H), 4.35 (q, J = 6.5 Hz) 2H, 4.45 (d, J = 6.5)

Hz, 2H), 7.95 (t, J = 6.5 Hz, 1H).

b) 2- (N-BOC-aminomethyl) -4-trifluoromethylthiazole-5-carboxamide

Ethyl 2- (N-BOC-aminomethyl) -4-trifluoromethylthiazole-5-carboxylate (15 g, 42.33 mol) was dissolved in methanol. Ammonia was passed through the solution at room temperature until all ester was converted to carboxamide. The solvent was removed on a rotary evaporator and the crude product was purified by flash chromatography. Yield: 4.6 g (14.14 mmol, 33%).

c) 2- (N-BOC-aminomethyl) -5-cyano-4-trifluoromethylthiazole

4 «* ·

2- (N-BOC-Aminomethyl) -4-trifluoromethylthiazole-5-carboxamide (4.6 g, 14.14 mmol) was dissolved in dichloromethane (30 mL) and cooled to -5 ° C. At this temperature, ethyl diisopropylamine (4.6 g, 35.35 mmol) and a solution of trifluoroacetic anhydride (3.4 g, 16.26 mmol) in dichloromethane (10 mL) were added. Then the mixture was stirred for an additional 2 hours at 0 ° C. It was washed successively with saturated sodium bicarbonate solution and 5% citric acid solution. After drying (MgSO ₄ ) the solvent was removed on a rotary evaporator. The crude product was extracted by stirring with diethyl ether / petroleum ether. The supernatant was separated from the oil and concentrated on a rotary evaporator. Yield: 1.9 g (6.18 mmol, 44%).

d) 2- (N-BOC-aminomethyl) -4-trifluoromethylthiazole-5-thioamide

2- (N-BOC-aminomethyl) -5-cyano-4-trifluoromethylthiazole (4.6 g, 14.97 mmol) was dissolved in pyridine (20 mL), triethylamine (24 mL) was added, and the solution was saturated with hydrogen sulfide . After two days at room temperature, the solvent was removed on a rotary evaporator. The crude product was taken up in ethyl acetate and washed successively with 20% sodium hydrogen sulfate solution and water. After drying (MgSO ₄ ) the solvent was removed on a rotary evaporator. The crude product was purified by flash chromatography. Yield: 2.5 g (7.32 mmol, 49%).

e) 5-amidino-2- (N-BOC-aminomethyl) -4-trifluoromethylthiazole

2- (N-HOC-aminomethyl) -4-trifluoromethylthiazole-5-thioamide (2.5 g, 7.32 mmol) was dissolved in dichloromethane.

methane (10 mL) and iodomethane (10.4 g, 73.24 mmol) was added. The mixture was then stirred at room temperature for 48 hours. The solvent was removed on a rotary evaporator and the residue was taken up in methanol (5 mL) and a 10% solution of ammonium acetate in methanol (8.5 mL, 10.98 mmol) was added. The mixture was stirred for 4 days at room temperature, then the crude product solution was passed through an acetate ion exchanger and the solvent was removed on a rotary evaporator. The crude product was purified by flash chromatography. Yield: 0.8 g (2.08 mmol, 28%).

f) 5-amidino-2-aminomethyl-4-trifluoromethylthiazole x 2 HCl

5-Amino-2- (N-BOC-aminomethyl) -4-trifluoromethyl thiazole (0.8 g, 2.08 mmol) was dissolved in dichloromethane and a 4M solution of hydrochloric acid in 1,4-dioxane (2M) was added. , 1 mL, 4.2 mmol). The mixture was stirred for 1 hour at room temperature, then the solvent was removed on a rotary evaporator. The crude product thus obtained was used in further reactions without further purification. Yield: 0.6 g (2.0 mmol, 97%).

ESI-MS: 225 (M + H &lt; + &gt;)

5-Aminomethyl-3-methylthiophene-2-carbonitrile

(a) 5-formyl-3-methylthiophene-2-carbonitrile;

112 mL (179 mmol) of a 1.6 molar solution of n-butyllithium in n-hexane was added to the solution over 20 minutes

25.1 mL (179 mmol) of diisopropylamine in 400 mL of tetrahydrofuran cooled to -78 ° C. The solution was left

The mixture was cooled to -78 ° C and a solution of 20.0 g (162 mmol) of 2-cyano-3-methylthiophene in 80 ml of tetrahydrofuran was slowly added dropwise at this temperature. During this process, the color of the solution turned dark red. Stirring was continued for 45 minutes, while 63 ml (811 mmol) of dimethylformamide was slowly added dropwise and the mixture was stirred for an additional 30 minutes. For working-up, a solution of 27 g of citric acid in 160 ml of water was added at -70 ° C. The mixture was concentrated in a rotary evaporator, 540 ml of saturated sodium chloride solution was added, and the batch was extracted three times using 250 ml of diethyl ether each time. The combined organic extracts were dried over magnesium sulfate. After the desiccant was filtered off, the solvent was distilled off with a vacuum pump and the residue was purified by column chromatography (eluent was hexane / ethyl acetate 4/1). 23 g (94%) of the produced compound were obtained.

¹ H NMR (270 MHz, DMSO-d 6): δ = 2.4 (s, 3H), 8.0 (s, 1H), 9.8 (s, 1H).

(f) 5-hydroxymethyl-3-methylthiophene-2-carbonitrile:

5.75 g (152 mmol) of sodium borohydride was added portionwise at room temperature to a solution of 23 g (152 mmol) of 5-formyl-3-methylthiophene-2-carbonitrile in 300 ml of absolute ethanol. The reaction mixture was stirred for 5 minutes, concentrated with a vacuum pump, taken up in ethyl acetate and extracted with 5% citric acid solution and saturated sodium chloride solution. The organic phase was dried over magnesium sulfate, the desiccant was filtered off and the solvent was removed

distilled off with vacuum pump at room temperature. This gave 24 g of the title compound as a dark red oil, which still contained the solvent and was used in subsequent reactions without further purification.

¹ H NMR (270 MHz, DMSO-d ₆ ): δ = 2.4 (s, 3H), 4.7 (m, 2H), 5.9 (m, 1H), 7.0 (s, 1H) .

(c) 5-bromomethyl-3-methylthiophene-2-carbonitrile:

g (167 mmol) of triphenylphosphine was added to a solution of 24 g (152 mmol) of 5-hydroxymethyl-3-methylthiophene-2-carbonitrile in 180 ml of tetrahydrofuran. A solution of 55 g (167 mmol) of tetrabromomethane in 100 ml of tetrahydrofuran was then added. The mixture was stirred for 90 minutes at room temperature. The reaction mixture was concentrated on a rotary evaporator with a vacuum pump and the residue was purified by column chromatography (eluent hexane: ethyl acetate 8: 2). 34 g of the desired compound were obtained which still contained a small amount of solvent.

¹ H NMR (270 MHz, DMSO-d 6): δ = 2.4 (s, 3H), 5.0 (s, 2H), 7.3 (s, 1H).

(d) 5-N, N-bis (tert-butoxycarbonyl) aminomethyl-3-methylthiophene-2-carbonitrile:

5.0 g (167 mmol) of sodium hydride (80% suspension in mineral oil) was added portionwise to a solution of 33.8 g (152 mmol) of 5-bromomethyl-3-methythiophene-2-carbonitrile in 255 ml of tetrahydrofuran cooled to 0 °. C. Then a solution of 36.4 g (167 mmol) of di-tert-butyl iminodicarboxylate in 255 ml of tetrahydrofuran was added dropwise so that the temperature during this process

-77 • 9 ·· 9 · 9 · 9 9 · 9 · did not rise above 5 ° C. The mixture was allowed to warm to room temperature and was stirred overnight. To complete the reaction, the mixture was heated for an additional 3 hours at 35 ° C and then allowed to cool to room temperature and 510 mL of saturated ammonium chloride solution was slowly added. The solvent was distilled off with a vacuum pump, the residue was extracted repeatedly with ethyl acetate, and the combined organic phases were washed with saturated sodium chloride solution, dried over magnesium sulfate and concentrated on a rotary evaporator. 57.6 g of an oily residue were obtained which still contained di-tert-butyl iminodicarboxylate and was used as a crude product in the next reaction.

¹ H NMR (270 MHz, DMSO-d ₆ ): δ = 1.45 (s, 18H), 2.35 (s, 3H), 4.85 (s, 2H), 7.05 (s, 1H) .

(e) 5-aminomethyl-3-methylthiophene-2-carbonitrile hydrochloride:

52.6 g of 5-N, N-bis (tert-butoxycarbonyl) aminomethyl-3-methylthiophene-2-carbonitrile (crude product from d), no more than 139 mmol) were dissolved in 950 ml of ethyl acetate and the solution was cooled to 0 ° C. The solution was saturated with hydrogen chloride gas. During this process, a white precipitate was separated over 10 minutes. The mixture was. After stirring for two hours at room temperature and for one hour at 30 ° C, the resulting suspension was then concentrated on a rotary evaporator, the residue was extracted by stirring with diethyl ether, the solvent was filtered off and the solid residue was dried at room temperature in vacuo. 24.7 g (94%) of the title compound was obtained as a white powder.

• 0 • 000 ·

¹ H NMR (270 MHz, DMSO-d ₆ ): δ = 2.4 (s, 3H), 4.25 (s, 2H), 7.3 (s, 1H), 8.8-9.0 (bs, 3 H).

¹³ CNMR (DMSO-d 6): 15.0 (CH ₃ ), 36.4 (CH ₂ ), 104.8 (C-2), 113.8 (CN), 131.5 (C-4), 142 8 (C-5), 149.6 (C-3).

5-Aminomethyl-3-chlorothiophene-2-carbonitrile hydrochloride

This compound was synthesized in the same manner as described for 5-aminomethyl-3-methylthiophene-2-carbonitrile, using 3-chloro-2-cyanothiophene prepared by dehydrating 3-chlorothiophene-2-carboxamide with trifluoroacetic anhydride.

5-Aminomethyl-4-methylthiophene-3-thiocarboxamide

a) Ethyl 2-amino-3-cyano-4-methylthiophene-5-carboxylate

Ethyl 2-amino-3-cyano-4-methylthiophene-5-carboxylate was synthesized as described in Organic Chemistry, 19th Edition, Dt. Verlag der Wissenschaften, Leipzig, Heidelberg, Berlin, 1993, Chapter 6, page 374375, starting with 130 g (1.0 mol) of ethylacetoacetate, 66 g (1.0 mol) of malononitrile, 32 g (1.0 mol) sulfur and 80 g (0.92 mol) of morpholine.

¹ H NMR (270 MHz, DMSO-d 6): δ = 1.25 (t, 3Hj, 2.3 (s,

3H), 4.2 (q, 2H), 7.9 (bs, 2H).

b) Ethyl 4-cyano-3-methylthiophene-2-carboxylate

A solution of 20.5 g (97.5 mmol) of ethyl 2-amino-3-cyano-4-methylthiophene-5-carboxylate in 600 mL of a 1: 1 mixture of acetonitrile and dimethylformamide was cooled to 5 ° C and

15.7 g (146 mmol) of tert-butyl nitrite was added dropwise during this process, increasing the temperature of the reaction mixture and vigorous gas evolution. The mixture was stirred for seven hours at room temperature and concentrated on a rotary evaporator under high vacuum, the residue was purified by column chromatography (eluent dichloromethane). 9.1 g (48%) of the title compound was obtained as a yellow oil.

@ ¹ H NMR (270 MHz, DMSO-d6): _.delta . = 1.3 (t, 3H), 2.55 (s,

3H), 4.3 (q, 2H), 8.8 (s, 1H).

(c) 5-hydroxymethyl-4-methylthiophene-3-carbonitrile:

2.44 g (64 mmol) of lithium aluminum hydride was added portionwise at 0 ° C to a solution of 25.1 g (129 mmol) of ethyl 3-cyano-4-methylthiophene-5-carboxylate in 400 ml of tetrahydrofuran. The mixture was stirred for 5 hours at room temperature, excess excess reagent was removed by addition of 0.5 N hydrochloric acid and the reaction mixture was concentrated with a vacuum pump, diluted with water and extracted three times with ethyl acetate. The combined organic phases were then washed with 0.5 N hydrochloric acid solution and saturated sodium chloride solution. The organic phase was dried over sulfate, filtered off and the solvent was vacuumed at room temperature.

The residue was purified by column chromatography (eluent dichloromethane / methanol 95: 5). 16.1 g (83%) of the title compound was obtained as a slightly yellow oil.

¹ H NMR (270 MHz, DMSO-d ₆ ): δ = 2.2 (s, 3H), 4.6 (d, 2H), 5.7 (m, 1H), 8.35 (s, 1H) .

Magnesium, desiccant was distilled off with

-80. ·· 0

000

0 0 0 00 00 0 »

0 0 0 0 · 1 ·

(d) 5-bromomethyl-4-methylthiophene-3-carbonitrile:

g (115 mmol) of triphenylphosphine was added at 5 ° C to a solution of 16 g (104 mmol) of 5-hydroxymethyl-4-methylthiophene-3-carbonitrile in 300 ml of tetrahydrofuran. A solution of 38 g (115 mmol) of tetrabromomethane in 100 ml of tetrahydrofuran was then added. The mixture was stirred overnight at room temperature. The reaction mixture was concentrated on a rotary evaporator with a vacuum pump and the residue was purified by column chromatography (eluent petroleum ether: dichloromethane 1: 1). 17 g (76%) of the title compound was obtained as a yellow oil.

@ ¹ H NMR (270 MHz, DMSO-d6): _.delta . = 2.25 (s, 3H), 5.0 (s, 2H), 8.5 (s, 1H).

(d) 5-N, N-bis (tert-butoxycarbonyl) aminomethyl-4-methylthiophene-3-carbonitrile:

3.5 g (103 mmol) of sodium hydride (without oil) was added in portions to a solution of 17.2 g (79.5 mmol) of 5-bromomethyl-4-methylthiophene-3-carbonitrile in 250 ml of tetrahydrofuran cooled to 0 ° C. Then a solution of 22.5 g (103 mmol) of di-tert-butyl iminodicarboxylate in 100 ml of tetrahydrofuran was added dropwise, during which the temperature did not rise above 5 ° C. The mixture was allowed to warm to room temperature and stirred for 2 hours. 400 mL of saturated ammonium chloride solution was added slowly. The solvent was distilled off with a vacuum pump and the residue was diluted with some water and extracted three times with ethyl acetate. The combined organic phases were washed with saturated ammonium chloride solution and saturated solution

Ρ ·

4 «·· • 8 ·

4 4 44 • «• s>

. 4 * 4 4 · • · 4

4444

4 4 4

4 «4 4

Sodium chloride, dried over magnesium sulfate and concentrated on a rotary evaporator. 28 g of an oil which still contained di-tert-butyl iminodicarboxylate were obtained and used as a crude product in the next reaction.

¹ H NMR (270 MHz, DMSO-d ₆ ): δ = 1.4 (s, 9H), 1.45 (s, 9H), 2.3 (s, 3H), 4.8 (s, 2H), 8.4 (s, 1 H).

f) 5-N, N-bis (tert-butoxycarbonyl) amino-methyl-4-methylthiophene-3-thiocarboxamide

The crude product obtained in (e) (no more than 79 mmol) was dissolved in 280 ml of pyridine and 140 ml of triethylamine and the solution was saturated with hydrogen sulfide at room temperature. The originally yellow color of the solution gradually changed to green. The mixture was stirred overnight at room temperature. To complete the reaction, hydrogen sulfide was passed through the mixture for an additional 15 minutes and stirring was continued for two hours at room temperature. Excess hydrogen sulfide was removed using a stream of nitrogen in the scrubber. Then the reaction mixture was concentrated on a rotary evaporator and the concentrate was concentrated. taken up in ethyl acetate, washed repeatedly with 20% sodium bisulfate solution, dried with magnesium sulfate and concentrated on a rotary evaporator. 27 g of a light yellow solid foam were obtained, which was used in subsequent reactions without further purification.

@ ¹ H NMR (270 MHz, DMSO-d6): _.delta . = 1.4 (s, 18H), 2.15 (3,

3H), 4.8 (s, 2H), 7.5 (s, 1H), 9.3 (bs, 1H), 9.75 (bs, 1H).

g) 5-aminomethyl-4-methylthiophene-3-thiocarboxamide hydrochloride

-82 27 g of 5-N, N-bis (tert-butoxycarbonyl) -aminomethyl-4-methylthiophene-3-thiocarboxamides; (crude product from f), no more than 70 mmol) was dissolved in 400 mL ethyl acetate and the solution was cooled to 0 ° C. The solution was saturated with hydrogen chloride gas and during this process a white precipitate separated over 10 minutes. The mixture was stirred for two hours at room temperature, the precipitate was filtered off and washed with ethyl acetate, and the solid residue was dried under vacuum at room temperature. 13.6 g (87%) of the title compound was obtained as a white powder. EI-MS: M &lt; + &gt; = 186.

5-Aminomethyl-4-chlorothiophene-3-thiocarboxamide

(a) 5-formyl-4-chlorothiophene-3-carbonitrile:

g (325 mmol) of tert-butyl nitrite was added dropwise at room temperature to a solution of 53.0 g (250 mmol) of 2-amino-4-chloro-5-formylthiophene-3-carbononitrile (the synthesis of this compound is described in the patent DB 3738910) in 600 ml of a 1: 1 mixture of acetonitrile and dimethylformamide. During this process, the temperature of the reaction mixture rose from 20 ° C to 37 ° C and a strong gas evolution occurred. The mixture was cooled to 25 ° C and stirred for 7 hours at room temperature, the black solution was concentrated on a rotary evaporator under high vacuum, the residue was purified by column chromatography (eluent dichloromethane). 29 g (68%) of the title compound was obtained as a yellow oil.

¹ H NMR (270 MHz, DMSO-d 6): δ = 9.1 (s, 1H), 10.0 (s,

1H).

(b) 5-Hydroxymethyl-4-chlorothiophene-3-carbonitrile:

Sodium borohydride (6.3 g, 166 mmol) was added portionwise at 5 ° C to a solution of 5-methyl-4-chlorothiophene-3-carbonitrile (28.5 g, 166 mmol) in absolute methanol (400 mL). The temperature of the reaction mixture rose slowly and the color changed to dark red. Strong gas evolution was observed. After 10 minutes, the reaction mixture was concentrated with a vacuum pump, taken up in 200 mL of ethyl acetate, extracted with 200 mL of 1 M hydrochloric acid, and washed twice with 250 mL of water each time and saturated sodium chloride solution. The organic phase was dried over magnesium sulfate, the desiccant was filtered off, and the solvent was distilled off with a vacuum pump at room temperature. 22 g (76%) of the title compound was obtained as a dark red oil which was used in subsequent reactions without further purification.

¹ H NMR (270 MHz, DMSO-d ₆ ): δ = 4.65 (bs, 1H), 5.95 (t, 2H), 8.6 (s, 1H).

(c) 5-bromomethyl-4-chlorothiophene-3-carbonitrile:

36.1 g (137 mmol) of triphenylphosphine was added at 5 ° C to a solution of 21.7 g (125 mmol) of 5-hydroxymethyl-4-chlorothiophene-3-carbonitrile in 250 ml of tetrahydrofuran. A solution of 45.6 g (137 mmol) of tetrabromomethane in 100 ml of tetrahydrofuran was then added. The mixture was stirred overnight at room temperature. The precipitate was filtered off, the filtrate was concentrated on a rotary evaporator with a vacuum pump, and the residue was purified by column chromatography (eluent petroleum ether: dichloromethane 1: 1). 26.0 g (88%) of the title compound was obtained as an oil.

 ý

-84 • 9 • · »9 <

¹ H NMR (270 MHz, DMSO-d ₆ ): δ = 4.95 (s, 2H), 8.8 (s, 1H).

(d) 5-N, N-bis (tert-butoxycarbonyl aminomethyl-4-chlorothiophene-3-carbonitrile):

6.9 g (159 mmol) of sodium hydride (oil-free) was added portionwise to a solution of 25.0 g (106 mmol) of 5-bromomethyl-4-quiorthiophene-3-carbonitrile in 300 ml of tetrahydrofuran cooled to 0 ° C. A solution of 34.4 g (159 mmol) of di-tert-butyliminodicarboxylate in 100 ml of tetrahydrofuran was then added dropwise, during which the temperature did not rise above 5 ° C. The mixture was allowed to warm to room temperature and stirred for two hours. 300 ml of saturated ammonium chloride solution was then slowly added. The solvent was distilled off with a vacuum pump and the residue was diluted with some water and extracted three times with ethyl acetate. The combined organic phases were washed with saturated ammonium chloride solution and saturated sodium chloride solution, dried over magnesium sulfate and concentrated on a rotary evaporator. 51.3 g of an oil which still contained di-tert-butyl iminodicarboxylate and solvent residues were obtained and used as a crude product in the next reaction.

¹ H NMR (270 MHz, DMSO-d ₆ ): δ = 1.4 (s, 9H), 1.45 (s, 9H), 4.8 (s, 2H), 8.65 (s, 1H) ).

e) 5-N, N-bis (tert-butoxycarbonyl) amino methyl-4-methylthiophene-3-thiocarboxamide

-85 · «... 9 9 9 9 9 85 85 85 85 85 85

A little of the crude product obtained in d) (39.4 g, no more than 106 mmol) was dissolved in 400 ml of pyridine and 40 ml of triethylamine and the solution was saturated with hydrogen sulfide at room temperature. The original yellow color of the solution turned green. The mixture was stirred overnight at room temperature. Excess hydrogen sulfide was removed using a nitrogen stream in the scrubber. The reaction mixture was then poured into ice-cold 20% sodium bisulfate solution and extracted three times with ethyl acetate. The organic phase was then washed repeatedly with a 20% sodium bisulfate solution, dried over magnesium sulfate and concentrated on a rotary evaporator. 49.0 g of a solvent-containing residue was obtained, which was used in the next reaction without further purification.

¹ H NMR (270 MHz, DMSO-d 6): δ = 1.4, 1.45 (s, 18H), 4.8 (s, 2H), 7.75 (s, TH), 9.4 (bs) 1 H, 10.0 (bs, 1 H).

f) 5-aminomethyl-4-chlorothiophene-3-thiocarboxamide hydrochloride

38.0 g of the crude product from e), no more than 93 mmol, was dissolved in 400 mL of ethyl acetate and cooled to 0 ° C. The solution was saturated with hydrogen chloride gas, during which 10 minutes a white precipitate separated. Since the reaction was still not complete, 200 mL of ethyl acetate was added and the mixture was saturated again with hydrogen chloride gas and stirred overnight at room temperature. The precipitate was filtered off, washed with petroleum ether and dried in vacuo at room temperature. 21.1 g of the title compound was obtained as a white powder which was contaminated with ammonium chloride. EI-MS: M &lt; + &gt; = 206.

Ά.

-86 ··

5-Aminomethyl-T-methyl-1H- [1,2,4] triazole-3-carboxamide

(a) Ethyl aminothioxoacetate

A solution of 29.1 g (294 mmol) of ethyl cyanoformate and 0.4 g (0.57 mL, 5.1 mmol) of diethylamine in 20 mL of benzene was passed at 0 ° C until saturation, during which the solution changed color. on orange. The mixture was stirred at room temperature over the weekend, the reaction mixture was cooled to 0 ° C, and the resulting precipitate (29.1 g) was filtered off and washed with cold benzene. The mother liquor was concentrated and recooled to 0 ° C. The mixture was filtered, the residue was washed with petroleum ether to give 5.7 g of the title compound as a pale yellowish solid (Rf = 0.7, dichloromethane / methanol 9: 1). Total yield: 89%.

¹ H NMR (270 MHz, DMSO-d 6): δ = 1.25 (t, J = 7 Hz, 3H), 4.2 (q, J = 7 Hz, 2H) 9.9 (bs, 1 Η, NH), 10.4 (bs, 1H, NH).

b) Ethyl methyloxamidrazone carboxylate

A solution of 11.93 g (13.6 mL, 259 mmol) of methylhydrazine in 100 mL of ethanol was added dropwise at room temperature to a solution of 34.5 g (259 mmol) of ethyl aminothioxoacetate in 400 mL of ethanol, during which the reaction mixture temperature somewhat increased. The mixture was stirred for three hours at room temperature and concentrated and the residue was used in reaction c) without further purification.

c) Ethyl amino [(2-tert-butoxycarbonylaminoacetyl) methyl] hydrazonoacetate

-87 • ···. »Β β β β β β β β β β

Activation of Boc-Gly-OH and reaction with b):

37.7 g (51.7 mL, 373 mmol) of triethylamine was added at room temperature to a solution of 54.46 g (311 mmol) of Bocglycine in 400 mL of tetrahydrofuran. The mixture was cooled to -5 ° C and a solution of 40.47 g (35.5 mL, 311 mmol) of ethyl chloroformate in 100 mL of tetrahydrofuran was slowly added dropwise over 40 minutes. The mixture was stirred for 30 minutes at -5 ° C; the resulting precipitate was filtered off and washed with a small amount of tetrahydrofuran and the filtrate was treated directly with a solution of b) (259 mmol) in 300 mL of tetrahydrofuran, which was added slowly dropwise at room temperature. The mixture was stirred overnight and concentrated to dryness on a rotary evaporator under reduced pressure, and the residue was purified by column chromatography (silica gel, dichloromethane / methanol 95: 5, R f = 0.26). 15.7 g of an oil was obtained, which was taken up in diethyl ether and the precipitate was filtered off (8.5 g, 11%).

¹ H NMR (270 MHz, DMSO-d ₆ ): δ = 1.25 (t, J = 7 Hz, 3H), 1.35 (s, 9H), 2.9 (s, 3H), 3.6 (d, J = 5 Hz, 2H), 4.3 (q, J = 7H _2, 2H), 6.6 (t, J = 5 Hz 1H), 7.3 (bs, 2H).

d) Ethyl 5-aminomethyl-1-methyl-1H- [1,2,4] triazole-3-carboxylate

7.0 g (23.2 mmol) of ethyl amino [(2-tert-butoxycarbonylaminoacetyl) methyl] hydrazonoacetate was suspended in 30 ml of xylene and the suspension was immersed for 10 minutes in a silicone oil bath which had previously been heated to 180 ° C. Then the solvent was distilled directly from the reaction mixture and

The residue was stirred for an additional 10 minutes at 180 ° C. Residual solvent was removed at 50 ° C under high vacuum. 6.8 g (> 95%) of a dark oil was obtained, which was used in the next reaction without further purification. The sample was filtered through silica gel and analyzed by NMR spectroscopy.

¹ H NMR (270 MHz, DMSO-d ₆ ): δ = 1.25 (t, J = 7 Hz, 3H), 1.35 (s, 9H), 3.9 (s, 3H), 4.2 -4.4 (m, 4H), 7.5 (t, J = 5Hz, 1H).

e) 5-Aminomethyl-1-methyl-1H- [1,2,4] triazole-3-carboxamide

Ammonia gas was passed through a solution of 6.8 g (no more than 23.2 mmol) of ethyl 5-aminomethyl-1-methyl-1H- [1,2.4] triazole-3-carboxylate for 20 minutes at -10 ° C. in 200 ml of ethanol. Stirring was continued for one hour at 0 ° C and overnight at room temperature. Since the reaction was still not complete, gas bubbling was repeated two more times (as described above) and the mixture was stirred overnight at 0 ° C. The mixture was concentrated on a rotary evaporator and the residue was purified by column chromatography (dichloromethane + 5-10% methanol, Rf = 0.3 in dichloromethane / methanol 9: 1). 4.71 g were obtained as a colorless oil.

¹ H NMR (270 MHz, DMSO-d ₆ ): δ = 1.4 (s, 9H), 3.85 (s, 3H), 4.3 (d, J = 5 Hz, 3H), 7.4 (bs, 1H), 7.6 (bs, 1H), 7.65 (bs, 5 H, J = _2, 1H).

f) 5-Aminomethyl-1-methyl-1H- [1,2.4] -triazole-3-carboxamide hydrochloride

► · »

-89 ·· · '·

Hydrogen chloride was passed at 5 ° C until saturated with a solution of 4.7 g (no more than 18.4 mmol) of 5-aminomethyl-1-methyl-1H- [1,2,4] triazole-3-carboxamide in 600 mL ethyl acetate, a white precipitate formed during this process. The mixture was stirred overnight at room temperature and concentrated on a rotary evaporator, treated with diethyl ether, the mixture was concentrated again ^converted: into diethyl ether and the precipitate was filtered off and dried. 3.7 g of a white solid were obtained which still contained ammonium chloride.

¹ H NMR (270 MHz, DMSO-d 6): δ = 3.95 (s, 3H), 4.3 (bs, 2H), 7.6 (bs, 1H), 7.75 (bs, 1H) 8.7-8.9 (m, 2H).

5-Aminomethyl-3-cyanofuran hydrochloride:

a) 5-N, N-bis (tert-butoxy carbonyl Jamino methyl-3-cyanofuran)

A solution of 20.5 g (0.11 mol) of 5-bromomethyl-3-cyanofuran (ZM Pevzner, VM Ignatiev, I.Lonin, Russ, J. of Gen. Chem. 1994, 64, 2, 125-128) in 50 mL of tetrahydrofuran, cooled to 0 ° C, was added with stirring over 30 minutes at 0 ° C to a suspension of 4.8 g (0.12 mol) of sodium hydride (60% dispersion in mineral oil) in 30 mL of tetrahydrofuran. Subsequently, a solution of 26.2 g (121 mmol) of di-tert-butyliminodicarboxylate in 50 ml of tetrahydrofuran was added dropwise, during which the temperature did not exceed 5 ° C. The mixture was stirred for 3 hours at 5-10 ° C, then allowed to warm to room temperature and stirred overnight. Then, 150 ml of saturated ammonium chloride solution was added slowly. The solvent was distilled off with a vacuum pump, the rest was

- tJU -. ··· ti. Extracted three times using 60 ml of ethyl acetate each time and the combined organic phases were washed twice with 50 ml of ethyl acetate. using a saturated sodium chloride solution, dried over magnesium sulfate and concentrated in a rotary evaporator. After drying for three hours at room temperature in vacuo (1 mm Hg), 33.2 g of a dark syrup still containing di-tert-butyliminodicarboxylate was obtained and used as a crude product in the next reaction.

¹ H NMR (250 MHz, d ₆ -DMSO): δ = 1.40, 1.45 (s, 18H), 4.70 (s, 2H), 6.70 (s, 1H), 8.6 ( s, 1H).

b) 5-aminomethyl-3-cyanofuran hydrochloride

12.89 g of 5-N, N-bis (tert-butoxycarbonyl) aminomethyl-3-cyanofuran (crude product from a) was dissolved in 80 ml of ethyl acetate and cooled to -10 ° C. The mixture was saturated with hydrogen chloride gas and after 15 minutes a white precipitate was separated. The mixture was allowed to warm to room temperature and stirred for two hours, the resulting suspension was then concentrated on a rotary evaporator, the residue (7 g) was extracted by stirring with diethyl ether. The solvent was removed by filtration and the solid residue was dried under vacuum at room temperature. 5 g (79%) of the title compound was obtained as a light ocher powder.

¹ H NMR (250 MHz, d ₆ -DMSO): δ = 4.15 (bs, 2H), 7.0 (s, 1H), 8.6-8.9 (m, 4H).

5-Aminomethyl-1-methyl-pyrrole-2-carbonitrile:

a) 5-cyano-1-methylpyrrole-2-carbaldehyde

1-Methylpyrrole was converted to 2-cyano-1-methylpyrrole by reaction of chlorosulfonyl isocyanate and dimethylformamide in acetonitrile (see, e.g., C.E. Loader et al. Can. J. Chem. (1981), 59,2673-6).

To THF (100 mL) was added diisopropylamine (17.5 mL, 124.38 mmol) under nitrogen. A solution of N-butyllithium in hexane (15%, 75.9 mL, 124.38 mmol) was added dropwise at -78 ° C. The mixture was then stirred at -20 ° C for 45 minutes and then recooled to -78 ° C. A solution of 1-methylpyrrole-2-carbonitrile (12 g, 113.07 mmol) in THF (50 mL) was added dropwise at this temperature. After stirring for 45 min at -78 ° C, DMF (43.9 mL, 546.46 mmol) was added dropwise and the mixture was stirred at this temperature for an additional 2 hours. After citric acid monohydrate (20.56 g) was added, the mixture was warmed to room temperature and water (112 mL) was added. THF was removed by rotary evaporation and the aqueous phase was saturated with sodium chloride and extracted with diethyl ether (3 x 200 mL). The combined organic phases were washed with saturated sodium chloride solution and dried removed and purified with sodium sulfate. The solvent was rotary evaporated and the crude product was flash chromatography (silica gel, dichloromethane). Yield: 8.25 g (54%).

¹ H NMR (CDCl ₃ ) δ = 4.1 (s, 3H), 6.8 (d, 1H), 6.9 (d, 1H), 9.7 (s, 1H).

b) 5-Hydroxymethyl-1-methylpyrrole-2-carbonitrile

-92 removed cleaned

The product obtained under a) (8.2 g, 61.1 mmol) was dissolved in ethanol (200 mL) and sodium borohydride (2.31 g, 61.13 mmol) was added at -10 ° C. After stirring for 1.5 hours at 0-5 ° C, the solvent was removed on a rotary evaporator and ice water and 20% sodium bisulfate solution were added to the residue. The aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with saturated sodium bicarbonate and water until neutral and then dried over sodium sulfate. The solvent was rotary evaporated and the crude product was flash chromatography (silica gel, dichloromethane / methanol = 97.5 / 2.5). Yield: 7.6 g (91%).

1 H NMR (CDCl ₃ ) δ = 1.9 (t, 1H), 3.75 (s, 3H), 4.6 (d, 2H), 6.1 (d, 1H), 6.7 (d, 1H).

c) 5-azidomethyl-1-methylpyrrole-2-carbonitrile

The product obtained under b) (7.5 g, 55.08 mmol) was dissolved in DMF (220 mL) and triphenylphosphine (43.34 g, 165.25 mmol) was added at 0 ° C. After stirring for 5 minutes at the indicated temperature, it should be removed. tetrabromomethane (54.8 g, 165.25 mmol) was added. The mixture was then stirred for 30 minutes at 0 ° C and 1.5 hours at room temperature. After cooling to 0 ° C, sodium azide (4.37 g, 67.21 mmol) was added. The mixture was then stirred for 4.5 hours at room temperature. Saturated sodium chloride solution was added dropwise at 0 ° C and the batch was diluted with ethyl acetate. The organic phase was removed and the aqueous phase was extracted with diethyl ether. The combined organic phases were washed with water and dried over sodium sulfate. The solvent was

removed on a rotary evaporator and the crude product was purified by flash chromatography (silica gel, ethyl acetate / hexane = 1/20). Yield: 5.6 g (63%).

@ 1 H NMR (CDCl3) .delta. = 3.75 (s, 3H), 4.35 (s, 2H), 6.2 (d, 1H), 6.7 (d, 1H).

d) 5-aminomethyl-1-methylpyrrole-2-carbonitrile

The product obtained under c) (4.71 g, 29.25 mmol) was dissolved in methanol (100 mL) and palladium on carbon (10%, 1 g) was added. The mixture was then hydrogenated with hydrogen at 100 kPa for 4 hours. The catalyst was removed by filtration through Celite® and the filtrate was evaporated in a rotary evaporator. The residue was extracted by stirring with dichloromethane / diethyl ether = 1/1. The product was suction filtered, dried at 35 ° C in a vacuum oven. Yield: 2.7 g (68%).

@ 1 H NMR (CDCl3) .delta. = 3.75 (s, 3H), 3.85 (s, 2H), 6.05 (d, 1H), 6.7 (d, 1H).

4-Aminomethyl-1-methylpyrrole-2-carbonitrile:

(a) 5-cyano-1-methylpyrrole-3-carbaldehyde

Aluminum chloride (24.24 g, 180.86 mmol) was dissolved in nitromethane / dichloromethane (1/1, 320 mL), the solution was cooled to -20 ° C and 1-methylpyrrole-2-carbonitrile (8 g, 75, 36 mmol). Then, α, α-dichlorodimethyl ether (10.4 g, 90.43 mmol) dissolved in dichloromethane (42 mL) was added dropwise. After stirring for 4 h at 0 ° C, the mixture was poured on ice (200 g). The aqueous phase was extracted with diethyl ether. Organic Combined * *

The -94 phases were washed with saturated sodium bicarbonate solution, water and saturated sodium chloride solution until neutral. After drying over sodium sulfate, the solvent was removed on a rotary evaporator. The crude product was used in the next reaction without further purification. Yield: 9.2 g (91%).

@ 1 H NMR (CDCl3) .delta. = 3.8 (s, 3H); 7.2 (s, 1 H); 7.4 (s, 1 H); 9.85 (s, 1 H).

b) Analogous to the synthesis of 5-aminomethyl-1-methylpyrrole-2-carbonitrile, 4-aminomethyl-1-methylpyrrole-2-carbonitrile was prepared using 5-cyano-1-methylpyrrole-3-carbaldehyde as starting material. However, 4-azidomethyl-1-methylpyrrole-2-carbonitrile was preferably reduced in the Staudinger reaction (see S. Nagarajan et al. J. Org. Chem. 1987, 52, 5044-6).

1 H NMR (DMSO-d 6) δ = 3.77 (s, 3H), 3.84 (sbr, 2H), 7.00 (sbr, 1H), 7.26 (s, 1H), 8.05 (sbr, 2H).

5-Aminomethyl-1-methyl-pyrrole-3-carbonitrile:

a) 4-cyano-1-methylpyrrole-2-carbaldehyde

1-Methylpyrrole-2-carbaldehyde (10 g, 91.6 mmol) was dissolved in acetonitrile (100 mL) and cooled to -45 ° C. Chlorosulfonyl isocyanate (38.9 g, 274.9 mmol) in acetonitrile (40 mL) was added dropwise over 40 minutes. The mixture was then stirred for 12 hours at room temperature. After addition of dimethylformamide (35 mL) dropwise, the mixture was heated to 50 ° C for 1 hour. After cooling to room temperature, it was reacted

The mixture was poured onto ice (200 mL) and 2N sodium hydroxide solution (286 mL). The resulting precipitate was filtered off with suction. The filtrate was extracted with diethyl ether. The combined ether phases were washed until neutral with dilute sodium bicarbonate solution and water and dried over sodium sulfate. The solvent was distilled off in a vacuum pump and the residue was combined with the previously obtained precipitate. Recrystallization of petroleum ether gave 4-cyano-1-methylpyrrole-2-carbaldehyde (4.3 g) (see, for example, C.E. Loader et al. Can. J. Chem. (1981), 59, 2673-6)

@ 1 H NMR (CDCl3) .delta. = 4.0 (s, 3H); 7.2 (s, 1H); 7.3 (s, 1 H); 9.6 (s, 1 H).

13-C NMR (CDCl 3) δ = 37.4; 94.1; 114.7; 125.8; 132.2; 135.8; 179.7.

b) Analogous to the synthesis of 5-aminomethyl-1-methylpyrrole-2-carbonitrile, 5-aminomethyl-1-methylpyrrole-3-carbonitrile was prepared using 4-cyano-1-methylpyrrole-2-carbaldehyde as starting material.

@ 1 H NMR (DMSO-d6) .delta. = 3.6 (s, 3H), 3.8 (s, 2H), 4.2 (sbr, 2H), 6.4 (s, 1H), 7.6 ( s, 1H).

5-Aminomethyl-3-cyano-1,2,4-oxadiazole hydrochloride:

a) N-Boc-5-aminomethyl-3-cyano-1,2,4-oxadiazole

Ethyl N-Boc-5-aminomethyl-1,2,4-oxadiazole-2-carboxylate (S. Borg et al. J. Org. Chem. 1995, 60, 3112-20) was dissolved in methanol (50 mL). Ammonia was passed through the solution at a temperature of from -10 ° C to room temperature until the reaction was complete. The solvent was

-96 • φ · · · · φ φ φ φ odstran odstran odstran odstran odstran φ φ φ φ odstran φ φ φ odstran φ φ odstran φ odstran odstran odstran φ φ φ φ odstran odstran odstran φ φ φ φ odstran odstran odstran odstran. The resulting crude product was dissolved in dichloromethane (70 mL) and diisopropylethylamine (2.9 mL, 16.55 mmol) was added at -5 ° C. Trifluoroacetic anhydride (1.06 mL, 7.61 mmol), dissolved in dichloromethane (10 mL) was then added dropwise. After stirring for 1.5 hours at 0 ° C, the batch was diluted with dichloromethane, washed 2x with saturated sodium bicarbonate solution, 2x 5% citric acid solution and 1x saturated sodium chloride solution and then dried with sodium sulfate. The solvent was removed by rotary evaporation and the crude product was purified by chromatography (silica gel, dichloromethane: methanol = 97.5: 2.5). Yield: 1.2 g (80%).

b) 5-Aminomethyl-3-cyano-1,2,4-oxadiazole hydrochloride

The product obtained under a) (0.9 g, 4.0 mmol) was dissolved in dichloromethane (45 mL) and 4M hydrochloric acid in dioxane (3.9 mL, 15.61 mmol) was added at room temperature. After stirring for 16 hours at room temperature, the solvent was removed on a rotary evaporator. Yield: 645 mg (100%).

1 H NMR (DMSO-d 6) δ = 4.6 (s, 2H), 9.2 (s, 3H).

-methyl-5-aminomethylpyrazole-3-carboxamide:

a) Methyl 1-methyl-5-amidopyrazole-3-carboxylate

1-Methyl-3-methoxycarbonylpyrazole-5-carboxylic acid chloride (prepared from 3.7 g, 20.09 mmol, 1-methyl-3-methoxycarbonyl-3-carboxylic acid, J. Org. Chem.

φ ·

-97

1989, 54, 428) was dissolved in toluene and the solution was cooled to -10 ° C. Ammonia was then passed through at temperatures from -10 ° C to 0 ° C until the reaction was complete. The solvent was removed in a rotary evaporator. The residue was taken up in ethanol. After stirring for 15 minutes, ethanol was removed on a rotary evaporator and the residue was dissolved in upper water and precipitated by cooling the solution to 0 ° C. The precipitate was filtered off with suction, washed with acetone and dried under vacuum at 45 ° C. Yield: 1.5 g (41%).

b) Methyl 1-methyl-5-cyanopyrazole-3-carboxylate

The product obtained under a) (1.5 g, 8.19 mmol) was taken up in dichloromethane (20 mL). Diisopropylethylamine (3.85 mL, 22.11 mmol) was added at -10 ° C. A solution of trifluoroacetic anhydride (1.3 mL, 9.44 mmol) in dichloromethane (5 mL) was added dropwise at this temperature over 45 minutes. Stirring was then continued for 1 hour at 0 ° C. The batch was diluted with dichloromethane and washed 2x with saturated sodium bicarbonate solution, 2x 5% citric acid solution and 1x saturated sodium chloride solution. After drying over sodium sulfate, the solvent was removed on a rotary evaporator. Yield: 1.35 g (100%).

c) 1-methyl-5-cyanopyrazole-3-carboxamide

The product obtained under b) (1.35 g, 8.19 mmol) was added to methanol (50 mL) and cooled to -10 ° C. Ammonia was then passed over 8 hours. After stirring for 12 hours at room temperature reaction

-98 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·· ·· precursor ended. The precipitated product was filtered off with suction, washed with cold methanol and dried in vacuo. Yield: 1.22 g (100%).

1 H NMR (DMSO-d 6) δ = 4.0 (s, 3H), 7.4 (s, 1H), 7.5 (s,

H, 7.8 (s, 1H).

d) 1-methyl-5-aminomethylpyrazole-3-carboxamide

The product obtained under e) (0.4 g, 2.66 mmol) was dissolved in acetic acid (30 mL) and 10% palladium on carbon (78 mg) was added. The mixture was then hydrogenated at room temperature at atmospheric pressure until completion of the reaction. The catalyst was removed by filtration through Celite® and the solvent was removed in a rotary evaporator. Yield: 0.4 g (100%), FAB-MS (M + H &lt; + &gt;): 155.

-methyl-3-aminomethyl-pyrazole-5-carboxamide

a) Methyl 1-methyl-3-amidopyrazole-5-carboxylate

1-Methyl-5-methoxycarbonylpyrazole-3-carbonyl chloride (synthesized from 4.17 g, 22.6 mmol, 1-methyl-5-methoxycarbonyl-3-carboxylic acid, J. Org. Chem. 1989, 54, 428) was dissolved in toluene and the solution was cooled to -10 ° C. At temperatures from -10 ° C to 0 ° C, ammonia was then passed through until the reaction was complete. The solvent was removed in a rotary evaporator. The residue was taken up in ethanol. The mixture was stirred for 15 minutes, the ethanol was removed by rotary evaporation, and the residue was dissolved in hot water and precipitated by cooling to 0 ° C. The precipitate was filtered off with suction, washed

-99 • · · <

acetone and dried under vacuum at 45 ° C. Yield: 3.36 g (18.4 mmol, 81%).

¹ H NMR (270 MHz, DMSO-d 6) δ = 3.85 (s, 3H), 4.15 (s, 3H), 7.20 (s, 1H), 7.4 (sbr, 1H) 7.7 (sbr, 1H).

b) Methyl 1-methyl-3-cyanopyrazole-5-carboxylate

The product obtained in a) (3.36 g, 18.4 mmol) was reacted by a procedure similar to the synthesis of methyl 1-methylcyanopyrazole-3-carboxylate previously described. Yield: 2.59 g (15.7 mmol, 85%).

@ ¹ H NMR (250 MHz, DMSO-d6) .delta. = 3.90 (s, 3H), 4.15 (s, 3H), 7.60 (s, 1H).

c) 1-methyl-3-cyanopyrazole-5-carboxamide

The product obtained in b) (2.56 g, 15.5 mmol) was reacted in a manner similar to that previously described for the synthesis of 1-methyl-5-cyanopyrazole-3-carboxamide. Yield: 2.3 g (15.3 mmol, 99%).

@ ¹ H NMR (250 MHz, DMSO-d6) .delta. = 4.15 (s, 3H), 7.45 (s, 1H), 7.70 (sbr, 1H), 8.15 (sbr, 1H).

d) 1-methyl-3-aminomethylpyrazole-5-carboxamide x HCl

The product obtained in c) (1.0 g, 6.7 mmol) was reacted in a manner similar to that previously described for the synthesis of 1-methyl-5-aminomethylpyrazole-3-carboxamide. Yield: 1.5 g (5.6 mmol, 83%).

¹ H NMR (270 MHz, DMSO-d 6) δ = 4.00 (q, J = 6.5 H ₂ , 2H), 4.10 (s, 3H), 6.90 (s, 1H), 7, 60 (sbr, 1H), 8.05 (sbr, 1H), 8.25 (sbr, 3H).

The product can be converted to the corresponding hydrochloride by repeated treatment with HCl in 1,4-dioxane followed by concentration of the mixture.

Example 1: N- (hydroxycarbonylmethylene) - (D) -cyclohexylalanylpoly-1- [2- (4-amidino) thiazolyl methyl] amide hydrochloride

a) N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Cha-Pro-NH-CH ₂ -2- (4-CS.NH ₂ ) thiaz

N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Cha-Pro-OH (2.0 g, 4.14 mmol), 2-H ₂ N-CH ₂ -thiaz-4 -CSNH ₂ (1.0 g, 4.56 mmol) and diisopropylethylamine (5.5 mL, 32.53 mmol) were dissolved in 25 mL methylene chloride, the solution was cooled to 0 ° C and 4.8 mL was added dropwise (6.21 mmol) of a 50% solution of propanephosphonic anhydride in ethyl acetate. The reaction mixture was stirred for 1 hour at 0 ° C and for 1 hour at room temperature and then concentrated in vacuo. The residue was taken up in water, extracted repeatedly with ether and the organic phase was dried over magnesium sulfate and concentrated in vacuo. Due to impurities, the product was purified by silica gel chromatography. The purified fractions were crystallized from ether. A total of 1.9 g of the desired product was obtained.

(b) N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Cha-Pro-NH-CH ₂ -2- (4C (SCH 3) NH) -thiaz hydroiodide:

N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Cha-Pro-NH-CH ₂ -2- (4CSNH ₂ ) -thiaz (1.7 g, 2.67 mmol) together with 3.7 ml of methyl iodide in 30 ml of methylene chloride were stirred

-101 -

Overnight at room temperature, then concentrated in vacuo under mild conditions and used in the subsequent reaction as a crude product (2.08 g, max 2.67 mmmol).

c) N- (t-BuO 2 C-CH 2) -N-Boc- (D) -Cha-Pro-NH-CH ₂ -2- (4-C (NH ₂ ) NH) thiaz hydroacetate:

N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Cha-Pro-NH-CH ₂ -2- (4C (SCH ₃ ) NH) thiaz hydroiodide (2.08 g, max 2, 67 mmol) was dissolved in 20 ml acetonitrile, 0.6 g (8.01 mmol) ammonium acetate was added and the mixture was stirred for 1.5 hours at 40-50 ° C. The solvent was removed in vacuo on a rotary evaporator and the residue was taken up in methylene chloride, the insoluble excess of ammonium acetate was removed by filtration, the methylene chloride solution was concentrated, the residue was taken up in ether and the desired product precipitated with n-hexane as an amorphous solid. The crude product (2.1 g) was dissolved in 20 ml of methanol and converted to the corresponding acetate using an acetate ion exchanger (3.7 g, Fluka, > Product No. 0Q4Q2).

c) HOOC-CH2- (D) -Cha-Pro-NH-CH2-2- (4-am) -thiazide hydrochloride

N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Cha-Pro-NH-CH ₂ -2- (4C (NH ₂ ) NH) -thiaz x CH ₃ COOH (2.0 g , max 2.67 mmol) were heated for 4 hours at 40-50 ° C in a mixture of 10 mL of dioxane and 20 mL of 5N aqueous hydrochloric acid, then the mixture was repeatedly extracted with methylene chloride and the aqueous phase was concentrated slightly in vacuo and then dried freezing.

-102 · · · · · · · · · · · · · · · · · · · · · · · ·

1.4 g of HOOC-CH ₂ - (D) -Cha-Pro-NHCH ₂ -2- (4am) -thiaz dihydrochloride were obtained as a white amorphous solid. FAB-MS (M + H &lt; + &gt;): 465

Example 2: N- (hydroxycarbonylmethylene) - (D) -cyclohexylalanylprolyl- [4- (2-amidino) thienylmethyl] amide hydroacetate:

a) N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Cha-Pro-NH-CH ₂ -4- (2-CN) -thiof

Starting with N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -ChaPro-OH (6.35 g, 13.17 mmol) and 4-H ₂ N-CH ₂ -thiof- 2-CN (2.3 g, 13.17 mmol) was coupled analogously to Example 1 to give N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) Cha-Pro-NH- CH ₂ -4- (2-CN) -thiophene. After purification by chromatography, 6.95 g of the desired product is obtained.

b) N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Cha-Pro-NH, CH ₂ -4- (2-CSNH ₂ ) thioph

N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Cha-Pro-NH-CH ₂ -4- (2-CN) thioph (6.95 g, 11.53 mmol) was Dissolved at 40 m! pyridine and 7 ml of triethylamine, the solution was saturated with hydrogen sulfide at 0-5 ° C (green solution) and allowed to stand at room temperature over the weekend. After concentrating in vacuo at 35 ° C / 35 mbar, the yellow oily residue was taken up in 200 ml of ether and washed four times each with 20 ml of 20% sodium hydrogen sulphate solution, twice with 20 ml of saturated sodium bicarbonate solution and 20 ml of water each time and the organic phase was dried over sodium sulfate and concentrated in vacuo. 6.74 g was obtained as a yellow solid foam.

4 · 4

-103-

c) N- (t-BuO 2 C-CH 2) -N-Boc- (P) -Cha-Pro-NH-CH ₂ -4- (2-C (SCH ₃ ) NH) thiof hydroiodide

Crude product N- (t-BuO ₂ C-CH 2) -N-Boc- (D) -Cha-ProNHCH ₂ -4- (2-CSNH ₂ ) -thiof (6.74 g, 10.58 mmol) was added to 65 ml of methylene chloride, then 9.01 g (4.0 ml, 63.5 mmol) of methyl iodide was added and the mixture was allowed to stand overnight at room temperature. It was then concentrated in vacuo under mild conditions to give 8.36 g of a yellow solid foam.

d) N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Cha-Pro-NH-CH 2 -4- (2-C (NH 2) NH) thiof hydroiodide

Crude product N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Cha-Pro-NHCH ₂ -4- (2-C (SCH ₃ ) NH) -thiof hydroiodide (8.36 g) 10.58 mmol) together with 16.3 g (21.16 mmol) of a 10% solution. of ammonium acetate in methanol was stirred overnight at room temperature. Since the precursor did not react completely, an additional 1.63g of a 10% ammonium acetate solution was added and the mixture was stirred overnight. After evaporation of the solvent in a rotary evaporator under vacuum, the residue was taken up in methylene chloride, the undissolved excess ammonium acetate was removed by filtration, and the liquid was concentrated again in vacuo to give 7.12 g of the desired product as a yellow solid foam.

e) HOOC-CH ₂ - (D) -Cha-Pro-NH-CH 2 -4- (2-am) -thiof hydroacetate

Crude product N- (t-BuO ₂ C-CH 2) -N-Boc- (D) -Cha-Pro-NHCH ₂ -4- (2-C (NH ₂ ) NH) -thiof hydroiodide, obtained · *

-104 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · in 100 mL of methylene chloride, 24.5 mL of ethereal hydrochloric acid (approx. 5 N) was added and the mixture was stirred overnight at room temperature. The resulting suspension was concentrated in vacuo and co-distilled twice with methylene chloride and the residue was converted into the acetate salt using an acetate ion exchanger (Fluka, Product No. 00402) to give 4.92 g of the compound. 2.5 g of this were purified by MPLC (RP-18, acetonitrile / water) and the fractions were freeze-dried. 1.23 g of the target substance was obtained as an amorphous white solid.

FAB-MS (M + H &lt; + &gt;): 464

Example 3:

N- (hydroxycarbonylmethylene) - (D) -cyclohexylalanylpipecolinic acid [5- (2-amidino) thienylmethyl] amide hydroacetate:

) _H-Pic-NH-CH2 -5- (2-CN) -thiophene

Boc-Pic-OH (10.1 g, 44.05 mmol) and 5-H ₂ N-CH ₂ -thiof-2-CN hydrochloride (8.54 g, 48.88 mmol) were dissolved in dichloromethane (150 mL) ) and ethyl diisopropylamine (53.2 mL, 311.08 mmol) and a 50% solution of propanephosphonic anhydride in ethyl acetate (46 mL, 217 mmol) was added at 0 ° C. The reaction mixture was stirred for 1 hour at 0 ° C and 1 hour at room temperature, then diluted with dichloromethane and washed with 20% sodium hydrogen sulfate solution (4x), sodium bicarbonate solution (3x) and saturated sodium chloride solution (1x). After drying over sodium sulfate and removing the desiccant by filtration, the solvent was

-1 05 • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·. · Distilled under the vacuum pump. · • ováno est odd est est ováno est odd ováno ováno ováno ováno odd odd To remove the Boc group, the residue (18.41 g) was treated with 200 mL of isopropanol and 50 mL of a 6.8 N isopropanolic hydrochloride solution and stirred at room temperature overnight. The mixture was then evaporated to dryness and co-distilled twice with dichloromethane and the residue was extracted by stirring with ether. 12.7 g of the desired product were obtained as a light brown powder.

b) HOOC-CH ₂ - (D) -Cha-Pic-NH-CH ₂ -5- (2-am) thiof hydroacetate

This compound was prepared by coupling two structural blocks of N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Cha-OH and H-Pic-N H-CH ₂ -5- (2-CN) -thioph analogous to Example 2a). The reaction to obtain HOOC-CH ₂ - (D) -Cha-Pic-NHCH ₂ -5- (2-am) -thiophenoacetate as the final product was conducted analogously to Example 2b) to

d), FAB-MS (M + H &lt; + &gt;): 478

Example 4: N- (Hydroxycarbonylmethylene) - (D) -cyclohexylglycylprolyl- [2- (4-amidino) thienylmethyl] amide hydrochloride

a) HOOC-CH 2 - (D) -Chg-Pyr-NH-CH 2 -4- (2-am) -thiophide hydrochloride

This compound was prepared in several steps analogous to Examples 2 and 3 with Boc-Pyr-OH, 4-H ₂ N-CH ₂ thiof-2-CN x HCl and N- (t-BuO ₂ C-CH ₂ ) -N- Boc- (D) -Chg-OH as starting materials.

b) HOOC-CH ₂ - (D) -Chg-Pro-NH-CH ₂ -4- (2-am) -thiof dihydrochloride

-1061.1 g (2.11 mmol) of _HOOC-CH2 - (D) Chg-Pyr-NH-CH ₂ -4- (2am) thiophene dihydrochloride were dissolved in a mixture of 30 ml water and 10 ml of glacial acetic acid, 0.5 g of 10% palladium on charcoal was added and the mixture was hydrogenated for 8 hours at room temperature under a slightly elevated pressure. After replacement of the catalyst, hydrogenation was continued for 8 hours, then the catalyst was filtered off with suction, the mixture was filtered through Celite® and the aqueous-organic phase was then freeze-dried. 0.86 g of the desired product was obtained as a white amorphous solid.

FAB-MS (M + H &lt; + &gt;): 450

As an alternative to the described procedure, Boc-proline can be used instead of Boc- (L) -3,4-dehydroproline, which means that the hydrogenation step can be omitted.

Example 5: N- (hydroxycarbonylmethylene) - (D) -cyclohexylglycylprolyl- [2- (4-amidino) thiazolyl methyl] amide

This compound can be prepared analogously to Example 1 with N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Chg-Pro-OH and 2-H ₂ NCH ₂ -thiaz-4-CSNH ₂ as starting materials.

Example 6: N- (hydroxycarbonylmethylene) - (D) -cyclohexylalanylprolyl- [4- (2-amidino) thienylmethyl] amide:

This compound can be prepared analogously to Example 2 with N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Cha-Pro-OH and 4-H ₂ Nfc ·

-107 • fcfcfc

CH 2 -thiof-2-CN as starting materials or analogously to Example 4 by hydrogenation of HOOC-CH ₂ - (D) -Cha-Pyr-NH-CH 2 -4- (2-am) -thiof.

Example 7: N- (hydroxycarbonylmethylene) - (D) -cyclohexylalanylprolyl- [5- (2-amidino-3,4-dimethyl) thienylmethyl] amide:

With the ingestion of compounds 5-H2N-CH2- (3,4-Me ₂₎ -thiophene-2CONH2 and N- (t-Bu02C-CH2) -N-Boc- (D) Cha-Pro-OH as starting materials can be said compound converted analogously to Example 2a) to the compound N- (t-BuO 2 C-CH 2) -N-Boc- (D) -Cha-ProNH-CH 2 -5- (2-CONH ₂ -3,4-Me 2) thiof. After dehydration of the amide with trifluoroacetic anhydride and addition of the nitrile functionality with diisopropylethylamine in methylene chloride, the amidine functionality can be analogous to Example 2 and then deprotected.

Example 8a: N- (hydroxycarbonylmethylene) - (D) -cycloheptylglycyl prolyl- [4- (2-amidino) thienylmethyl] amide hydroacetate:

Example 8b: N- (hydroxycarbonylmethylene) - (L) -cycloheptylglycyl prolyl- [4- (2-amidino) thienylmethyl] amide hydroacetate

Using 4-H ₂ N-CH 2 -thiof-2-CN, Boc-PyrOH and N- (t-BuO 2 C-CH 2) -N-Boc- (D, L) -Cheg-OH as starting materials, the desired compounds prepared analogously to Example 3, hydrogenated in a final step analogously to Example 4, and then separated by MPLC (RP 18, acetonitrile / water). If, in addition to Boc-Pyr-αH, Boc-Pro-OH is used in the synthesis, the hydrogenation step can be omitted.

• · a · · a

-108 • ·

Example 9a: N- (hydroxycarbonylmethylene) - (D) -cyclopentylglycyl prolyl- [4- (2-amidino) thienylmethyl] amide hydroacetate:

Example 9b: N- (hydroxycarbonylmethylene) - (L) -cyclopentylglycyl prolyl- [4- (2-amidino) thienylmethyl] amide hydroacetate

Using 4-H ₂ N-CH ₂ -thiof-2-CN, Boc-PyrOH and N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D, L) -Cpg-OH as starting materials For example, the compounds of interest may be prepared analogously to Example 3, hydrogenated in a final step analogously to Example 4, and then separated by MPLC (RP 18, acetonitrile / water). If Boc-Pyr-aH is used in the synthesis as well as Boc-ProOH, the hydrogenation step can be omitted

Example 10: N- (hydroxycarbonylmethylene) - (D) -cyclohexylalanylprolyl- [4- (2-amidino) thiazolylmethyl] amide

This compound can be prepared analogously to Example 1 with N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Cha-Pro-OH and 4-H ₂ NCH ₂ -thiaz-2-CSNH ₂ as starting materials.

Example 11: N- (hydroxycarbonylmethylene) - (D) -cyclohexylglycylprolyl- [4- (2-amidino) thiazolylmethyl ethyl] amide

This compound can be prepared analogously to Example 1 with N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Chg-Pro-OH and 4-H ₂ NCH ₂ -thiaz-2-CSNH ₂ as starting materials.

Example 12: N- (hydroxy carbonyl methyl) - (D) -cyclohexylalanylprolyl 5- (3-amidino) isoxazolylmethyl] amide:

-109

Using the compounds N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -ChaPro-OH and 5-H ₂ N-CH ₂ -isox-3-CONH ₂ as starting materials, the following may be mentioned: the compound was converted analogously to Example 2a) to N (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Cha-Pro-NH-CH ₂ -5- (3-CONH ₂ ) -isox. After dehydration of the amide to form a nitrile functionality using acid anhydride, trifluoroacetic anhydride and diisopropylethylamine in methylene chloride, an amidine functionality can be formed analogously to Example 20 by reaction with ammonia and acetylcysteine followed by deprotection.

Example 13: N- [5- (3-amidino) thienyl] methyl] -1- [N- (hydroxycarbonylmethylene) - (D) -cyclohexylglycyl] azetidine-2-carboxamide

a) N- [5- (3-cyano) thienylmethyl] -1- [N-t-butoxycarbonyl) - (D) -cyclohexylglycyl] azetidine-2-carboxamide

5.9 g (45.8 mmol) of diisopropylethylamine and 11.5 ml (14.9 mmol) of a 50% solution of propanephosphonic anhydride in ethyl acetate were added dropwise at -5 ° C to the solution.

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-amino ethyl-3-cyanothiophene hydrochloride in 40 ml of methylene chloride. Stirring was continued for 2 hours during which the temperature exceeded 10 ° C. The organic phase was washed with water, 5% sodium bicarbonate solution and 5% citric acid solution, dried over sodium sulfate and evaporated to dryness. Purification of the residue by column chromatography (silica gel, eluent: ethyl acetate) gave

4.5 g (85% of theory) of a white, amorphous powder,

-110 FAB-MS: 461 (M + H &lt; + &gt;).

b) N- [5- (3-cyano) thienyl methyl] -1- [N- (t-butoxycarbonylmethylene) (D) -cyclohexylglycyl] azetidine-2-carboxamide

4.5 g (3.8 mmol) of the above compound was dissolved in 70 mL of isopropanol, 12.3 mL of 4N hydrochloric acid in dioxane was added and the mixture was allowed to stand overnight at room temperature. After distilling off the solvent, the residue ₍ 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) remained. This was dissolved in 50 ml of methylene chloride and 10 ml of acetonitrile and the mixture was allowed to stand for 24 hours after addition of 2.1 g (16 mmol) of diisopropylethylamine and 1.5 g (7.6 mmol) of t-butyl bromoacetate. hours at room temperature.

The organic phase was washed twice with 5% citric acid, 5% sodium bicarbonate and water each time, and dried over sodium sulfate, and then the solvent was distilled off. Yield: 3.3 g (92% of theory) of a light yellowish oil.

FAB-MS: 475 (M + H &lt; + &gt;).

c) N- [5- (3-amidino) thienylmethyl] -1- [N- (hydroxycarbonylmethylene) - (D) -cyclohexylglycyl] azetidine-2-carboxamide

The product was converted to an amidine analogous to Example 2. The resulting crude amidine contained substantial

-111 amount of secondary products and had to be purified by column chromatography (silica gel, eluent: methylene chloride: methanol: acetic acid = 24: 6: 1.5).

0.9 g of white, amorphous powder was isolated. The Boc protecting groups and the t-butyl ester group were removed by allowing the powder to stand for 12 hours in 3N hydrochloric acid. After the final removal of hydrochloric acid by distillation with toluene addition, the hydrochloride residue was converted to betaine chromatography on a silica gel column with methanol / 25% ammonia (50 / 2.5) to yield 0.45 g of white amorphous powder, FAB-MS: 463 (M + H) +).

with eluent system. It was

Example 14: N- (hydroxycarbonylmethylene) - (D) -cyclohexylalanyl prolyl- [5- (3-amidino) furylmethyl] amide hydroacetate

a) Prolyl- [5- (3-cyano) furylmethyl] amide hydrochloride

3.05 g (14 mmol) of Boc-Pro-OH and 6.11 g (47.3 mmol) of ethyldiisopropylamine were added at room temperature to a suspension of 2.5 g (15.8 mmol) of 5-aminomethyl-3-cyanofuran hydrochloride in 50 ml. ml of dichloromethane. 15.8 ml (74.5 mmol) of a 50% solution of propanephosphonic anhydride in ethyl acetate was added dropwise at 5 ° C with gentle cooling. After stirring for 30 minutes at room temperature, the mixture was diluted with ethyl acetate and washed three times with 5% citric acid solution, three times with saturated bicarbonate solution and once with saturated sodium chloride solution. After drying over sodium sulfate and removal of the desiccant by filtration, the solvent was removed

-112βββ e β distilled with vacuum pump. 4.3 g (86%) of a pale yellow oil were obtained and used directly in the next reaction.

The oil obtained in (a) (4.3 g, 13.5 mmol) was dissolved in 40 mL of ethyl acetate to remove the Boc group and was saturated with hydrogen chloride at 0 ° C. The reaction mixture was stirred overnight and evaporated to dryness in a rotary evaporator. 3.4 g (99%) of the title compound were obtained.

(b) N- (tert-butoxycarbonylmethylene) - (N-Boc) - (D) -cyclohexylanylprolyl- [5- (3-cyano) phenylmethyl] methyl

2.58 g (6.7 mmol) of t-BuO ₂ C-CH ₂ -Boc- (D) -Cha-OH and 1.7 g (6.7 mmol) of prolyl- [5- (3-cyano) furylmethyl] The hydrochloride amide was suspended in 20 mL of dichloromethane and 3.45 g (26.8 mmol) of ethyldiisopropylamine was added. The reaction mixture was cooled to approximately 5 ° C and then 6.7 ml of a 50% solution of propanephosphonic anhydride in ethyl acetate was added dropwise, during which the solution turned clear. After stirring overnight at room temperature, the mixture was diluted with ethyl acetate and then washed three times with 20% sodium hydrogen sulphate solution, saturated sodium bicarbonate solution and once saturated sodium chloride solution. After drying with sodium sulfate, the desiccant was removed and the solvent was then distilled off with a vacuum pump. 3.7 g of the expected product are obtained in the form of an oil.

c) N- (tert-butoxycarbonylmethylene) - (N-Boc) - (D) -cyclohexyl and lprolyl- [5- (3-aminothiocarbonyl) furyl methyl jamide]

-1134 · 4 4

4,444 4

004 43 4 0 4

4 4 4 4

The product obtained under b) was dissolved in pyridine (30 ml) and triethylamine (15 ml). The reaction mixture was saturated with hydrogen sulfide at room temperature and stirred overnight at room temperature. Excess hydrogen sulfide was removed with nitrogen and the reaction mixture was poured onto 300 ml of ice-cold 5% sodium bisulfate solution. The mixture was extracted three times with ethyl acetate and the combined organic phases were washed once more with 5% sodium bisulfate solution. After drying with sodium sulfate, the solvent was distilled off with a vacuum pump. The resulting crude product (3.3 g) was used in the next step without further purification.

d) N- (tert-butoxycarbonylmethylene) - (N-Boc) - (D) -cyclohexyl and lpropyl 1- [5- (3-S-methyliminothiocarbonyl) furyl methyl] amide ropes hydroiodide

The crude product obtained in (c) was dissolved in 50 ml of acetone and 8.3 g (58.7 mmol) of methyl iodide was added. After stirring overnight at room temperature, the solvent was distilled off with a vacuum pump. The residue was dissolved in a little ethyl acetate and the solution was added dropwise to diisopropyl ether, during which a precipitate formed, which was filtered off with suction and washed with diisopropyl ether. Drying at room temperature under vacuum gave 3.3 g of a solid foam.

e) N- (tert-butoxycarbonylmethylene) - (N-Boc) - (D) -cyclohexylalanylprolyl- [5- (3-amidino) furylmethyl] amide hydroacetate;

-1140 · 0000 ··· 0 * 0 · 0 0 0 00 «e β es * 0e 0

0 · 0 0 00 0 00 00 00 00

The crude product obtained in (d) (3.3 g, 4.3 mmol) was dissolved in 40 mL of acetonitrile, 0.99 g (12.9 mmol) of ammonium acetate was added, and the mixture was stirred for two hours at 40 ° C. . The solvent was then distilled off with a vacuum pump, the residue was taken up in diethyl ether, the salts were filtered off with suction and the filtrate was concentrated. The crude product was purified by reverse phase HPLC (acetonitrile / water and acetic acid buffering). 991 mg of a yellow solid foam was obtained.

f) N- (hydroxycarbonylmethyl) - (D) -cyclohexyl alaninylpoly- [5- (3-amidino) furylmethyl] amide hydroacetate ml of 1N aqueous hydrochloric acid solution was added to the product obtained according to e) (991 mg, 1.68 mmol); After stirring for three hours at 45 ° C, the mixture was diluted with water and the resulting mixture was freeze-dried. The resulting crude product was dissolved in methanol and converted to the acetic acid salt on an ion exchanger (Fluka, Product No. 00402). 484 mg of the desired product was obtained.

FAB-MS (M + H &lt; + &gt;): 446

Example 15: N- (hydroxycarbonylmethylene) - (D) -cyclohexylglycylprolyl- [5- (3-amidino) furylmethyl] amide hydroacetate:

FAB-MS (M + H &lt; + &gt;): 434

This compound can be prepared analogously to Example 14 using N (tert-butoxycarbonylmethylene) - (N-Boc) - (D) -cyclohexylglycine instead of N- (tert-butoxycarbonylmethylene) - (N-Boc) - (D) cyclohexylalanine.

-115-

Example 16: N- (hydroxycarbonylmethylene) - (D) cyclohexylalanylprolyl- [5- (2-amidino-1-methyl) pyrrolylmethyl] amide:

a) N-Boc-N- (tert-butyloxycarbonyl methylene) - (D) cyclohexylalanylproline (2.5 g, 5.18 mmol) was dissolved in dry methylene chloride (30 mL), cooled to -10 ° C and at this temperature N-ethyldiisopropylamine (3.9 mL, 22.27 mmol) was added. After stirring for 5 minutes, a solution of 5-aminomethyl-1-methylpyrrole-2-carbonitrile (0.7 g, 5.18 mmol) in methylene chloride (15 mL) was added. Then a 50% solution of propanephosphonic anhydride in ethyl acetate (4.6 mL, 6.21 mmol) was added dropwise over 20 minutes. After stirring for 90 minutes at -10 ° C to 0 ° C, the mixture was diluted with methylene chloride and washed 2x with saturated sodium bicarbonate solution (15 ml each), 2x 5% citric acid solution (15 ml each) and 1x saturated solution sodium chloride (15 mL). After drying over sodium sulfate, the mixture was concentrated in vacuo and the crude product was purified by chromatography (silica gel, methylene chloride: methanol = 95: 5). Yield: 2.3 g (74%).

(b)

The product obtained under a) (2.3 g, 3.83 mmol) was dissolved in a mixture of dry methylene chloride and methanol (1: 1, 50 mL), hydrochloride (664 mg, hydroxylamine 9.56 mmol) and Nethyldiisopropylamine ( 4 mL, 23.0 mmol) and the mixture was stirred for 7 hours at 40 ° C and then for 48 hours at room temperature. The solvent was distilled off in vacuo, water was added to the residue, and the mixture was stirred for 2 hours

-116 acidified to pH 5 with acetic acid. The aqueous solution was extracted with methylene chloride (2x) and ethyl acetate (1x). The combined organic phases were dried over sodium sulfate and the solvent was distilled off in vacuo. The crude product was purified by chromatography (silica gel, methylene chloride: methanol = 95: 5). Yield:

1.6 g (white foam, 66%), FAB-MS (M + H &lt; + &gt;): 633.

1. The product obtained under b) (1.6 g, 2.53 mmol) was dissolved in dry methanol (35 mL), acetic acid (0.3 mL, 5.06 mmol) and Raney nickel (84 mg) were added. ) and the mixture was hydrogenated at 50 ° C under a hydrogen pressure of 100 kPa (2.5 hours). After cooling, the catalyst was removed by filtration through Celite® and the filtrate was concentrated in vacuo. Yield: 1.7 g (white foam, 99%), FAB-MS (M + H +): 617.

c) The product obtained under c) (1.7 g, 2.50 mmol) was dissolved in dry methylene chloride (50 mL). The solution was cooled to 0 ° C and saturated dry HCl gas. After stirring for 2 hours, the solvent was distilled off in vacuo and the crude product was purified by chromatography (RP 18, acetonitrile = 1: 9 with addition of 0.1% acetic acid). Yield: 760 mg (57%), m.p. 184-185 ° C, FAB-MS (M + H +); 461.

Example 17: N- (hydroxycarbonylmethylene) - (D) -cyclohexylalanylprolyl- [2- (4-amidino-1-methyl) pyrrolomethyl) amide was prepared analogously to Example 16, FAB-MS (M + H +); 461.

JU ·,.

-117 • 5 · ·

Example 18: N- (hydroxycarbonylmethylene) - (D) -cyclohexylalanylprolyl- [4- (2-amidino-1-methyl) pyrrolylmethyl) amide can be prepared analogously to Example 16.

Example 19: N- (tert-butoxycarbonylmethylene) - (N-Boc) - (D) -cyclohexylalanylprolyl- [2- (4-amido) oxazolylmethyl] amide hydrochloride can be prepared analogously to Example 1 with starting materials N (t-BuO 2 C-). CH ₂ -N-Boc- (D) -Cha-Pro-OH and 2-aminomethyl-4-thiocarboxamidoxazole.

Example 20: N- (hydroxycarbonylmethylene) - (D) -cyclohexylalanylprolyl- [5- (3-amidino-1-methyl) pyrazolylmethyl] amide hydrochloride:

a) N- (tert-Butoxycarbonylmethylene) - (N-Boc) - (D) -cyclohexylalanylprolyl- [5- (3-amido-1-methyl) pyrazolylmethyl] amide

N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Cha Pro-OH (1.25 g, 2.59 mmol) was taken up in dichloromethane (30 mL). Diisopropylethylamine (1.95 mL, 11.16 mmol) was added dropwise at -10 ° C. A solution of 1-methyl-5-aminomethyl-pyrazole-3-carboxamide (0.4 g, 2.59 mmol) in tetrahydrofuran (20 mL) was then added. After stirring for 5 minutes, a 50% solution of propanephosphonic anhydride ethyl acetate (2.36 mL, 3.11 mmol) as well as dichloromethane (5 mL) was added dropwise over 5 minutes. After stirring for 45 minutes at 0 ° C, the mixture was warmed to room temperature for 12 hours. The solvent was removed in a rotary evaporator, the residue was taken up in dichloromethane and the mixture was washed twice with saturated sodium bicarbonate solution, 2x with 5% citric acid solution and once with saturated sodium chloride solution. After drying with sodium sulfate, the solvent was

-118 • · ί * removed in a rotary evaporator. The crude product was purified by chromatography (RP-18, acetonitrile, water). Yield: 220 mg (14%).

FAB-MS (M + H &lt; + &gt;); 619.

b) N- (tert-butoxycarbonylmethylene) - (N-Boc) - (D) -cyclohexylalanylprolyl- [5- (3-cyano-1-methyl) pyrazolylmethyl] amide

The product obtained under a) (220 mg, 0.36 mmol) was dissolved in dichloromethane (15 mL) and diisopropylethylamine (0.17 mL, 0.96 mmol) was added at -10 ° C. After stirring for 5 minutes, a solution of trifluoroacetic anhydride (0.057 mL, 0.41 mmol) in dichloromethane (1 mL) was added dropwise. After 1 hour at 0 ° C, the mixture was diluted with dichloromethane and washed 2x with saturated sodium bicarbonate solution, 2x with 5% citric acid solution and 1x with saturated sodium chloride solution. After drying over sodium sulfate, the solvent was removed on a rotary evaporator. Yield: 180 mg (84%).

c) N- (tert-butoxycarbonylmethylene) - (N-Boc) - (D) -cyclohexylalanyl prolyl- [5- (3-amidino-1-methyl) pyrazolylmethyl] amide hydroacetate

The product obtained under b) (180 mg, 0.3 mmol) was dissolved in methanol (1 mL) and acetylcysteine (52.8 mg, 0.32 mmol) was added. Ammonia was then passed through the mixture at 35 ° C until the reaction was complete. The solvent was removed on a rotary evaporator and the crude product was converted to acetate using an ion exchanger (acetate on a polymeric substrate, Fluka 00402). The crude product was purified by chromatography (RP-18, acetonitrile, water). Yield: 50 mg (16%). FAB-MS (M + H &lt; + &gt;): 618.

-119 • ··· ····

d) N- (hydroxycarbonylmethylene) - (D) -cyclohexyl alanylproly 1- [5- (3-amidino-1-methyl) pyrazolylmethyl] amide hydrochloride

The product obtained in (c) (50 mg, 0.081 mmol) was dissolved in dichloromethane (5 mL) and 5M hydrochloric acid in diethyl ether (0.147 mL) was added. After stirring for 12 hours at room temperature, the solvent was removed on a rotary evaporator and the product was taken up in water and lyophilized. Yield: 40 mg (92%). FAB-MS (M + H &lt; + &gt;): 462.

Example 21: N- (hydroxycarbonylmethylene) - (D) -cyclohexylalanylprolyl- [5- (3-amidino) -1,2,4-oxadiazolylmethyl] amide hydrochloride

This compound can be prepared analogously to Example 20 using t-BuO ₂ C-CH ₂ - (Boc) - (D) -Cha-Pro-OH and 5 amino-3-cyano-1,2,4-oxadiazole as starting materials.

Example 22: N- (hydroxycarbonylmethylene) - (D) -cyclohexylglycylprolyl- [5- (2-amidino-3-methyl) thienylmethyl] amide:

This compound was synthesized from the starting materials of the compounds N- (t-BuO ₂ C-CH ₂ ) -N-Boc (D) -Chg-Pro-OH and 5 aminomethyl-3-methylthiophene-2-carbonitrile hydrochloride using procedures described in Examples 2a) -e).

FAB-MS (M + H &lt; + &gt;): 464

Example 23: N- (hydroxycarbonylmethylene) - (D) -cyclohexylalanylprolyl- [5- (2-amidino-3-methyl) thienylmethyl] amide:

This compound was synthesized from the starting materials N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Cha-Pro-OH and 5-aminomethyl-3.

-1200 0 0 0 0 · 0 0 0 0 0 0 0 · · 0 0 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

methylthiophene-2-carbonitrile hydrochloride according to the procedures described in Examples 2a) -e).

FAB-MS (M + H &lt; + &gt;): 478.

Example 24: N- (hydroxycarbonylmethylene) - (D) -cyclohexylalanyl prolyl- [5- (3-amino-1-methyl) triazylmethyl] amide:

This compound was synthesized from the starting materials N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Cha-Pyr-OH and 5-aminomethyl-1-methyl-1H- [1,2,4] triazole-3-carboxamide. Initially, procedures similar to those described in Examples 20a) -d) were used to obtain N- (hydroxycarbonylmethylene) - (D) -cyclohexylalanyldehydroprolyl- [5- (3-amino-1-methyl) tri]. azy I methyl iamide. This compound was converted to the desired compound by the procedure used in Example 4b). FAB-MS (M + H &lt; + &gt;): 463

Example 25: N- (hydroxycarbonylmethylene) - (D) -cyclohexylglycylprolyl- [5- (3-amidino-1-methyl) -thiazole methylmethyl]:

This compound was synthesized from the starting materials N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Chg-Pyr-OH and 5-aminomethyl-1-methyl-1H- [1,2,4] triazole-3-carboxamide. The procedures described in Examples 20a) -d) were used first to obtain N (hydroxycarbonylmethyl) - (D) -cyclohexylglycidylhydroproyl- [5- (3-amidino-1-methyl) triazylmethyl] amide. This compound was converted to the desired compound using the procedure described in Example 4b).

FAB-MS (M + H &lt; + &gt;): 449

Example 26: N- (hydroxycarbonylmethylene) - (D) -cyclohexylalanylprolyl- [5- (3-amidino-4-chloro) thienylmethyl] amide:

-121 • ···

This compound was synthesized from the starting materials N- (t-BuO 2 C-CH 2) -N-Boc- (D) -Cha-Pro-OH and 5-aminomethyl-4-chlorothiophene-3-thiocarboxamide hydrochloride by the procedures used in Examples 1 and ) -d).

ESI-MS (M + H &lt; + &gt;): 498

Example 27: N- (ethoxycarbonylmethylene) - (D) -cyclohexylalanylprolyl- [5- (3-amidino-4-chloro) thienylmethyl] amide:

This compound was synthesized by passing hydrogen chloride through a solution of 100 mg (0.186 mmol) of N- (hydroxycarbonylmethylene) - (D) -cyclohexyl-1- [5 (3-amidino-4-) methyl) thienyl] methylamide (see Example 26 above) in 10 mL of ethanol cooled to 0 ° C and stirring was continued at room temperature for five hours. The mixture was concentrated and distilled three times each time with a small amount of toluene to remove the residue of hydrogen chloride: The residue (89 mg, 85%) was dissolved in ethanol and converted to the corresponding acetate using an acetate ion exchanger (Fluka, Product No. 00402).

FAB-MS (M + H &lt; + &gt;): 506

Example 28: N- (hydroxycarbonylmethylene) - (D) -cyclohexylanylprolyl-1- [5- (3-amidino-4-methyl) thienylmethyl] amide:

This compound was synthesized from the starting materials N- (t-BuO 2 C-CH 2) -N-Boc- (D) -Cha-Pro-OH and 5-aminomethyl-4-methylthiophene-3-thiocarboxamide hydrochloride by the procedures used in Example 1a) -d).

ESI-MS (M + H &lt; + &gt;): 478

F.

L • ···

-122 • · · ·

Example 29: N- (ethoxycarbonylmethylene) - (D) -cyclohexyl alanylprolyl- [5- (3-amidino-4-methyl) thienylmethyl] amide:

This compound was synthesized by passing hydrogen chloride through a solution of 100 mg (0.186 mmol) of N- (hydroxycarbonylmethyl) - (D) -cyclohexyl-alanylpolyl- [5 (3-amidino-4-methyl)] until saturation. thienyl] methylamide (see Example 28 above) in 10 mL of ethanol cooled to 0 ° C, and stirring was continued at room temperature for five hours. The mixture was concentrated and distilled three times, each time with a small amount of toluene to remove residual hydrogen chloride. The residue (89 mg, 85%) was dissolved in ethanol and converted to the corresponding acetate using an acetate ion exchanger (Fluka, Product No. 00402).

FAB-MS (M + H &lt; + &gt;): 506

Example 30: N- (hydroxycarbonylmethylene) - (D) -cyclohexylalanylprolyl- [5- (2-amidino-3-chloro) thienylmethyl] amide

This compound can be prepared by the following reaction procedure: coupling of N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -ChaPro-OH with 5-H ₂ N-CH ₂ - (2-CN-3) -CI) -thiof to give N- (t-BuO ₂ CCH ₂ ) -N-Boc- (D) -Cha-Pro-NH-CH ₂ -5- (2-CN-3-CI) -thiof, forming amidine followed by deprotection as described in Example 2.

Example 31: N- (hydroxycarbonylmethylene) - (D) -cyclohexylglycylprolyl- [5- (2-amidino-3-chloro) thienylmethyl] amide

This compound can be prepared by the following reaction procedure: coupling of N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Chg

··· ··· ·· · »· SW \ _Λ ··· ····· · · · t

- 1 / J - · ···········································

Pro-OH with 5-H ₂ N-CH ₂ - (2-CN-3-CI) -thioph to give N- (t-BuO ₂ CCH ₂ ) -N-Boc- (D) -Cha-Pro-NH- CH ₂ -5- (2-CN-3-Cl) -thioph, amidine formation and subsequent deprotection as described in Example 2.

Example 31: N- (hydroxycarbonylmethylene) - (D) -cyclohexylglycylproly | - [5- (2-amidino-3-chloro) thienylmethyl] amide

This compound can be prepared by the following reaction procedure: coupling of N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Chg Pro-OH with 5-H ₂ N-CH 2 - (2-CN-3-CI ) -thiof to give N- (t-BuO ₂ CCH ₂ ) -N-Boc- (D) -Chg-Pro-NH-CH 2 -5- (2-CN-3-Cl) -thiof, forming an amidine and subsequent deprotection as described in Example 2.

Example 32: N- (methoxycarbonylmethylene) - (D) -cyclohexylalanylprolyl- [4- (2-amidino) thienylmethyl] amide

This compound can be systetized from N- (t-BuO ₂ CCH ₂ ) -N-Boc- (D) -Cha-Pro-NH-CH ₂ -4- (2-am) -thiof by deprotection and transesterification (HCl in methanol at room temperature).

Example 33: N- (methoxycarbonylmethyl en) - (D) -cyclohexylglycylprolyl [4- (2-amidino) thienylmethyl] amide

This compound can be systetized from N- (t-BuO ₂ CCH ₂ ) -N-Boc- (D) -Chg-Pro-NH-CH 2 -4- (2-am) -thiof by deprotection and transesterification (HCl in methanol at room temperature).

• · c · • «· ···

-194- · * ·· · · * · ··· ·· *

I A * · · Τ # # »# # # # # # # # # #

Example 34: N- (methoxycarbonylmethylene) - (D) -cyclohexylalanylazetidine-2-carboxylic acid 4- (2-amidino) thienylmethylamide

This compound can be systetized from N- (t-BuO ₂ CCH ₂ ) -N-Boc- (D) -Cha-Aze-NH-CH 2 -4- (2-am) -thiof by deprotection and transesterification (HCl in methanol) at room temperature).

Example 35: N- (methoxycarbonylmethylene) - (D) -cyclohexylglycylazetidine-2-carboxylic acid 2- (4-amidino) thienylmethylamide

This compound can be synthesized from the N- (t-BuO2CCH ₂₎ -N-Boc- (D) Chg-Aze-NH-CH2-4- (2-am) -thiophene deprotection and transesterification (HCl in methanol at room temperature).

Example 36: N- (methoxycarbonylmethylene) - (D) -cyclohexylalanylprolyl- [2- (4-amidino) thiazolylmethyl] amide

This compound can be systetized from N- (t-BuO ₂ CCH ₂ ) -N-Boc- (D) -Cha-Pro-NH-CH ₂ -2- (4-ann) -thiaz by deprotection and transesterification (HCl in methanol at room temperature).

Example 37: N- (methoxycarbonylmethylene) - (D) -cyclohexylglycyl prolyl- [2- (4-amidino) thiazolylmethyl] amide

This compound can be grafted from N- (t-BuO2CCH2) -N-Boc- (D) -Chg-Pro-NH-CH2-2- (4-am) -thiaz by deprotection and transesterification (HCl in methanol at temperature rooms).

125 Example 38: N- (methoxycarbonylmethylene) - (D) -cyclohexylalanylazetidine-2-carboxylic acid 2- (4-amidino) thiazolylmethylamide

This compound can be systetized from N- (t-BuO ₂ CCH ₂ ) -N-Boc- (D) -Cha-Aze-NH-CH ₂ -2- (4-am) -thiaz by deprotection and transesterification (HCl) in methanol at room temperature).

Example 39: N- (methoxycarbonyl-methylene) - (D) -cyclohexylglycylazetidine-2-carboxylic acid 2- (4-amidino) thiazolylmethylamide

This compound can be systetized from N- (t-BuO ₂ CCH ₂ ) -N-Boc- (D) -Chg-Aze-NH-CH ₂ -2- (4-am) -thiaz by deprotection and transesterification (HCl) in methanol at room temperature).

Example 40: N- (Hydroxycarbonylmethylene) - (D) -cyclo hexyl alanylprolyl- [4- (2-hydroxyamidino) thienylmethyl] amide

This compound can be synthesized by reacting (tBuO ₂ C-CH ₂ -) - (Boc) - (D) -Cha-Pro-NH-CH ₂ - (2-CN) -4-thiophene with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) followed by deprotection (HCl in dichloromethane at room temperature).

Example 41: N- (methoxycarbonylmethylene) - (D) -cyclohexylalanylprolyl- [4- (2-hydroxyamidino) thienyl methyl] iodide

This compound can be synthesized by the reaction of (tBuO ₂ C-CH ₂ -) - (Boc) - (D) -Cha-Pro-NH-CH ₂ - (2-CN) -4-thiophene.

• hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) followed by deprotection and transesterification (HCl in methanol at room temperature).

Example 42: N- (ethoxycarbonylmethylene) - (D) -cyclohexylalanylprolyl- [4- (2-hydroxyamidino) thienylmethyl] amide

This compound can be synthesized by reacting (t-BuO ₂ C-CH ₂ -) (Boc) - (D) -Cha-Pro-NH-CH 2 - (2-CN) -4-thioph with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) followed by deprotection and transesterification (HCl in ethanol at room temperature).

Example 43: N- (hydroxycarbonylmethylene) - (D) -cyclohexylglycylprolyl- [4- (2-hydroxyamidino) thienylmethyl] amide

This compound can be synthesized by reacting (tBuO ₂ C-CH ₂ -) - (Boc) - (D) -Chg-Pro-NH-CH ₂ - (2-CN) -4-thiophene with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) followed by deprotection (HCl in dichloromethane at room temperature).

Example 44: N- (methoxycarbonyimethylene) - (D) -cyclohexylglycylprolyl- [4- (2-hydroxyamidino) thienylmethyl] amide

This compound can be synthesized by reacting (tBuO ₂ C-CH ₂ -) - (Boc) - (D) -Chg-Pro-NH-CH ₂ - (2-CN) -4-thiophene with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) followed by deprotection and transesterification (HCl in methanol at room temperature).

- 127 Example 45: N- (ethoxycarbonylmethylene) - (D) -cyclohexylglycylprolyl- [4- (2-hydroxyamidino) thienylmethyl jam id

This compound can be synthesized by reacting (tBuO ₂ C-CH ₂ -) - (Boc) - (D) -Chg-Pro-NH-CH ₂ - (2-CN) -4-thiophene with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) followed by deprotection and transesterification (HCl in ethanol at room temperature).

Example 46: N- (hydroxycarbonylmethylene) - (D) -cyclohexylglycylazetidine-2-carboxylic acid 4- (2-hydroxyamidino) thienylmethyl] amide

This compound can be synthesized by reacting (tBuO ₂ C-CH ₂ -) - (Boc) - (D) -Chg-Aze-NH-CH ₂ - (2-CN) -4-thiophene with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) followed by deprotection (HCl in dichloromethane at room temperature).

Example 47: N- (methoxycarbonylmethylene) - (D) -cyclohexylglycylazetidine-2-carboxylic acid [4- (2-hydroxyamidino) thienylmethyl] amide

This compound can be synthesized by reacting (tBuO ₂ C-CH ₂ -) - (Boc) - (D) -Chg-Aze-NH-CH ₂ - (2-CN) -4-thiophene with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) followed by deprotection and transesterification (HCl in methanol at room temperature).

Example 48: N- (ethoxycarbonylmethylene) - (D) -cyclohexyl-glyclazetidine-2-carboxylic acid [4- (2-hydroxyamidino) thienylmethyl] amide

«HsL ·

This compound can be synthesized by reacting (tBuO ₂ C-CH ₂ -) - (Boc) - (D) -Chg-Aze-NH-CH ₂ - (2-CN) -4-thiophene with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) followed by deprotection and transesterification (HCl in ethanol at room temperature).

Example 49: N- (hydroxycarbonylmethyl) - (D) -cyclohexylalanylazetidine-2-carboxylic acid [4- (2-hydroxyamidino) thienylmethyl] amide

This compound can be synthesized by reacting (tBuO ₂ C-CH ₂ -) - (Boc) -T- (D) -Chg-Aze-NH-CH ₂ - (2-CN) -4-thiophene with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) followed by deprotection (HCl in dichloromethane at room temperature)

Example 50: N- (methoxycarbonylmethylene) - (D) -cyclohexylalanylazetidine-2-carboxylic acid [4- (2-hydroxyamidino) thienylmethyl] amide

This compound can be synthesized by reacting (tBuO ₂ C-CH ₂ -) - (Boc) - (D) -Cha-Aze-NH-CH ₂ - (2-CN) -4-thiophene with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) followed by deprotection and transesterification (HCl in methanol at room temperature).

Example 51: N- (ethoxycarbonylmethylene) - (D) -cyclohexylalanylazetidine-2-carboxylic acid [4- (2-hydroxyamidino) thienylmethyl] amide

- 129., · »· i J J J J i. . · »·· ·. ·· ®

.. »»

This compound can be synthesized by reacting (tBuO ₂ C-CH ₂ -) - (Boc) - (D) -Cha-Aze-NH-CH ₂ - (2-CN) -4-thiophene with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) followed by deprotection and transesterification (HCl in ethanol at room temperature).

Example 52: N- (hydroxycarbonylmethylene) - (D) -cyclohexylalanyl prolyl- [2- (4-hydroxyamidino) thiazolylmethyl] amide

This compound can be synthesized by reacting (tBuO ₂ C-CH ₂ -) - (Boc) - (D) -Cha-Pro-NH-CH ₂ - (4-CN) -2-thiaz with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) followed by deprotection (HCl in dichloromethane at room temperature).

Example 53: N- (methoxycarbonylmethylene) - (D) -cyclohexylalanylprolyl- [2- (4-hydroxyamidino) thiazolylmethyl] amide

This compound can be synthesized by reacting (tBuO ₂ C-CH ₂ -) - (Boc) - (D) -Cha-Pro-NH-CH ₂ - (4-CN) -2-thiaz with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) followed by deprotection and transesterification (HCl in methanol at room temperature).

Example 54: N- (ethoxycarbonylmethylene) - (D) -cyclohexylalanylprolyl- [2- (4-hydroxyamidino) thiazolylmethyl] amide

This compound can be synthesized by reacting (tBuO ₂ C-CH ₂ -) - (Boc) - (D) -Cha-Pro-NH-CH ₂ - (4-CN) -2-thiaz with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) followed by deprotection and transesterification (HCl in ethanol at room temperature).

• ·

- 130 9 · ’· ·

Φ · · »* * <

• 4 · 4 · 9 · 9 · 9 · 9 · 9 · 9

Example 55: N- (hydroxycarbonylmethylene) - (D) -cyclohexy Iglycylprolyl- [2- (4-hydroxyamidino) thiazolyl methyl] amide

This compound can be synthesized by reacting (tBuO ₂ C-CH ₂ -) - (Boc) - (D) -Chg-Pro-NH-CH ₂ - (4-CN) -2-thiaz with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) followed by deprotection (HCl in dichloromethane at room temperature).

Example 56: N- (methoxycarbonylmethylene) - (D) -cyclohexylglycylprolyl- [2- (4-hydroxyamidino) thiazolylmethyl] amide

This compound can be synthesized by reacting (t-BuO ₂ C-CH ₂ -) - (Boc) - (D) -Chg-Pro-NH-CH ₂ - (4-CN) -2-thiaz with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) followed by deprotection and transesterification (HCl in methanol at room temperature).

Example 57: N '- (ethoxycarbonylmethylene) - (D) -cyclohexylglycyl · prolyl- [2- (4-hydroxyamidino) thiazolylmethyl] amide

This compound can be synthesized by reacting (tBuO ₂ C-CH ₂ -) - (Boc) - (D) -Chg-Pro-NH-CH ₂ - (4-CN) -2-thiaz with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) followed by deprotection and transesterification (HCl in ethanol at room temperature).

Example 58: N- (hydroxycarbonylmethylene) - (D) -cyclohexylglycylazetidine-2-carboxylic acid [2- (4-hydroxyamidino) thiazolylmethyl] amide

- 131 -

This compound can be synthesized by reacting (tBuO ₂ C-CH ₂ -) - (Boc) - (D) -Chg-Aze-NH-CH ₂ - (4-CN) -2-thiaz with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) followed by deprotection (HCl in dichloromethane at room temperature).

Example 59: [2- (4-Hydroxyamidino) thiazolylmethyl] amide

N- (2-methoxycarbonylmethylene) - (D) -cyclohexylglycylazetide of 2-carboxylic acid

This compound can be synthesized by reacting (tBuO ₂ C-CH 2 -) - (Boc) - (D) -Chg-Aze-NH-CH ₂ - (4-CN) -2-thiaz with hydroxylamine hydrochloride (methanol, diisopropylethylamine, temperature followed by deprotection and transesterification (HCl in methanol at room temperature).

Example 60: N- (ethoxycarbonylmethylene) - (D) -cyclohexylglycylazetidine-2-carboxylic acid [2- (4-hydroxyamidino) thiazolyrmethyl] amide

This compound can be synthesized by reacting (tBuO 2 C-CH 2 -) - (Boc) - (D) -Chg-Aze-NH-CH ₂ - (4-CN) -2-thiaz with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) and subsequent deprotection (HCl in ethanol at room temperature).

Example 61: N- (hydroxycarbonylmethylene) - (D) -cyclohexylalanylazetidine-2-carboxylic acid [2- (4-hydroxyamidino) thiazolylmethyl] amide

This compound can be synthesized by the reaction of (tBuO ₂ C-CH 2 -) - (Boc) - (D) -Cha-Aze-NH-CH ₂ - (4-CN) -2-thiaz

Hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) followed by deprotection (HCl in dichloromethane at room temperature).

Example 62: [2- (4-Hydroxyamidino) thiazolylmethyl] amide

N- (methoxycarbonylethylene) - (D) -cyclohexylalanylazetine-2-carboxylic acid

This compound can be synthesized by reacting (tBuO ₂ C-CH ₂ -) - (Boc) - (D) -Cha-Aze'-NH-CH ₂ - (4-CN) -2-thiazole with hydroxylamine hydrochloride (methanol) , diisopropylethylamine, room temperature) followed by deprotection and transesterification (HCl in methanol at room temperature).

Example 63: N (Ethoxycarbonylmethylene) - (D) -cyclohexylalanylazetidine-2-carboxylic acid [2- (4-hydroxyamidino) thiazolylmethyl] amide

This compound can be synthesized by reacting (tBuO ₂ C-CH ₂ -) - (Boc) - (D) -Cha-Aze-NH-CH ₂ - (4-CN) -2-thiaz with hydroxylamine hydrochloride (methanol, diisopropylethylamine, room temperature) followed by deprotection and transesterification (HCl in ethanol at room temperature).

Example 64: N (Hydroxycarbonylmethylene) - (R) -cyclohexylalanyl- (3S) -2,3,4,5-tetrahydropyridazine-3-carboxylic acid [4- (2-amidino) thienylmethyl] amide hydrochloride

(a) N-Boc-N- (tert-butoxycarbonylmethylene) - (D) -cyclohexylalanyl (3S) -2,3,4,5-tetrahydro-razine-3-carboxylic acid

3.3 g (25.8 mmol) of diisopropylethylamine and 1.5 g

- 133 (12.3 mmol) dimethylaminopyridine was added at -5 ° C to a solution of 9.95 g (25.8 mmol) of N-Boc-N (tert-butoxycarbonylmethylene) - (D) -cyclohexylalan 7.4 g (25.8 mmol) of (S) -4-benzyl-3 - [(S) -2,3,4,5-tetrahydro-3-pyridazinyl] -2-oxazolidinone (Y. Nakamura, C. Shin, Chem. Lett. 1991, 1953) and 7.4 g (38.7 mmol) of EDC hydrochloride in 80 mL of CH 2 Cl 2 and the mixture was stirred for 2 hours at -5 ° C and for 12 hours at room temperature. The reaction solution was diluted with 200 mL of ether, washed with 5% citric acid solution, 5% NaHCO ₃ solution and water, dried, the solvent was stripped off and the residue was purified by column chromatography (methylene chloride / acetone, 50 / 2.5). 4.0 g (24% of th.) Of a yellowish oil were obtained, FAB-MS (M + H +): 655. The oil was dissolved in 80 mL of THF and 27 mL of water, and then gradually added dropwise at 0 ° C 2. 8 ml of 30% H2O2 and 12.6 ml of 1N NaOH and stirring was continued for 2.5 hours. After addition of 15 g of saturated aqueous Na ₂ S ₂ O ₃ solution, the mixture was extracted with ether and the alkaline phase was removed and acidified with 1M KHSO 4 solution. After repeated extraction with ether, drying and solvent removal by distillation, 2.2 g of a white amorphous powder was obtained.

b) [2- (4am) thienyl methyl] -amide hydrochloride salt of _HOOC-CH2 - (D) -Cha (3S) -2,3,4,5-tetrahydropyridazin-3-carboxylic acid

0.7 g (1.4 mmol) of the above acid and 0.3 g of 2-aminomethyl-4-aminothiophene dihydrochloride were suspended in 4 ml of DMF. After addition of 0.145 g (1.44 mmol) of N-methylmorpholine at 0 ° C, almost complete dissolution occurred and 0.475 g (1.45 mmol) of O-

sa

134 [(cyanoethoxycarbonylmethylene) amino] -N, N, Ν ', N'tetramethyluronium tetrafluoroborate (TOTU) and 0.14 g of N-methylmorpholine. The reaction mixture was stirred for 3 hours at 0 ° C under a nitrogen atmosphere and then most of the DMF was distilled off at a bath temperature of 35 ° C and a pressure of about 1 mbar. The residue was purified by column chromatography (eluent: CFhCl / MeOH, 45/5, 0.7 parts of 50% acetic acid was added before the end). 0.75 g of a slightly yellow amorphous powder was obtained.

This powder was dissolved in 5 mL of CH 2 Cl 2 and 10 mL of trifluoroacetic acid and the solution was allowed to stand overnight at room temperature. After addition of 30 mL of toluene, the mixture was concentrated in vacuo and the residue treated with ether and then converted to betaine on a silica gel column (eluent: MeOH / 25% strength NH ₃ , 50/2). The betaine was dissolved in 20 ml of water, the pH was adjusted to 4.5 with 1N HCl and lyophilized. 0.36 g of amorphous powder was obtained, FABMAS (M + H +): 476.

Example 65: N (Hydroxycarbonylmethylene) - (R) -cyclohexylalanyl- (3S) -pyrazolidine-3-carboxylic acid [4- (2-amidinothienylmethyl) -amide hydrochloride 8.36 g (21.7 mmol) of N-Boc-N- (t) -butoxycarbonylmethylene) - (D) -cyclohexylalanine and 5 g (21.7 mmol) of methyl (3S) -1-tert-butoxycarbonylpyrazoline-3-carboxylate (HO Kim, C. Lum, MS Lee (1997), THL) 38 (28), 4935) was dissolved in 60 mL of CH 2 Cl 2, while stirring at -8 ° C, 6.1 g (31.8 mmol) of EDC.HCl was added, and after another 20 minutes 4.0 g (31 mmol) was added.

-135diisopropylethylamine. The mixture was stirred for 40 minutes, then 0.8 g of DMAP was added and the mixture was allowed to stand at room temperature for 2 days. After addition of 200 ml of ether, the mixture was washed with 5% citric acid, 5% NaHCO 3 solution and water, and after drying the ether was distilled off. After purification by column chromatography (eluent: CH ₂ Cl ₂ / acetone, 50/2), 8.2 g (63% of theory) of white amorphous powder was isolated.

Hydrolysis: 8.0 g (13.4 mmol) of the ester was dissolved in 60 mL of dioxane and at 10 ° C were added 12 mL of water and 15 mL of 1N NaOH. After 1.5 hours, the pH was adjusted to 8 with TN HCl, the dioxane was distilled off and the residue was diluted with 250 mL of water and extracted with ether. The pH of the aqueous phase was adjusted to 2.5 with 1N KHSO4 solution and the separated acid was extracted with ether. After stripping off the ether, 7.7 g of amorphous acid remained. The sample was recrystallized from a water-saturated n-hexane system, having a melting point of 115-120 ° C and a rotation angle of [α] D ₂₀ + 112.4 ° C (CHCl 3, c = 1).

Coupling with 4-aminomethyl-2-amidinothiophene dihydrochloride was carried out as described in Example 64, step b). After purification by column chromatography (eluent: CH ₂ Cl ₂ / MeOH / 50% acetic acid, 40/10 / 0.7), 4 g of N-Boc-N acetate [4- (2-amidino) thienylmethyl] amide was obtained. butoxycarbonylmethylene) - (R) -cyclohexylanyl- (3S) pyrazolidine-3-carboxylic acid, starting from 4.15 g of the above acid.

Deprotection: The above compound was dissolved in 12 mL dioxane, 20 mL TN HCl was added and the mixture was heated at 75 ° C for 4.5 hours. The solution was diluted with 50 mL of water, the pH was adjusted to 4 with

136 ion exchangers (3-A4 resin, BioRad) and water were distilled off. The residue was dissolved in isopropanol and the hydrochloride was precipitated by addition of ether. After purification by column chromatography (eluent: CH ₂ Cl ₂ / MeOH / 50% acetic acid, 35/15/7), the residue was dissolved in water, the pH was adjusted to 4 with 1N HCl and the sample was lyophilized. 1.6 g of amorphous hydrochloride were obtained, FAB-MS (M + H +): 465.

The procedures described in Example 65 were used in the following examples:

Example 66: N (Hydroxycarbonylmethylene) - (R) -hexylalanyl- (3R) -pyrazoline-3-carboxylic acid [4- (2-amidino) thienylmethyl] amide hydrochloride

White amorphous powder. FAB-MS (M + H &lt; + &gt;): 465

Example 67: N (Hydroxycarbonylmethylene) - (R) -cyclohexylglycyl- (3R) -pyrazoline-3-carboxylic acid [4- (2-amidino) -thienylmethyl] amide hydrochloride

White amorphous powder. FAB-MS (M + H &lt; + &gt;): 451

Example 68: N- (Hydroxycarbonylmethyl) - (R) -cyclohexyl-1- (3S) -pyrazolidine-3-carboxylic acid [4- (2-amidino) -thienylmethyl] -amide hydrochloride

White amorphous powder. FAB-MS (M + H &lt; + &gt;): 451

Example 69: N (Hydroxycarbonylmethyl) - (R) -hexylglycyl (-) - thiazoline-2-carboxylic acid [4- (2-amidino) thienylmethyl] amide hydrochloride

Starting material: methyl (-) - thiazolidine-2-carboxylate [R.L. Johnson, E.E. Smissman (1978), J. Med. Chem. 21, 165]

Example 70: N- (hydroxycarbonylmethylene) - (R) -cyclohexylalanyl (-) - thiazolidine-2-carboxylic acid [4- (2-amidino) thienylmethyl] amide

White amorphous powder. FAB-MS (M + H &lt; + &gt;): 482

Example 71: N (Hydroxycarbonylmethylene) - (R) -cyclohexylalanyl- (L) octahydroindole-2-carboxylic acid [4- (2-amidino) thienylmethyl] amide hydrochloride

White amorphous powder. FAB-MS (M + H &lt; + &gt;): 518

Example 72: N (Hydroxycarbonylmethylene) - (R) -cyclohexylglycyl- (L) octahydroindole-2-carboxylic acid [4- (2-amidino) thienylmethyl] amide hydrochloride

White amorphous powder, FAB-MS (M + H +): 504

Example 73: N (hydroxycarbonylmethylene) - (D) -cyclohexylalanyl- (45) -5,5-dimethylthiazoline-4-carboxylic acid [2- (4-amidino) thienylmethyl] amide

This compound can be prepared by the following reactions: coupling of N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Cha-OH with (S) -5,5 Me ₂ -thz-4-OMe- [ J. Samanen et al. (1990), Int. J. Peptide Protein Res. 35, 501 (1990)] to give N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Cha (2,2-Me ₂ -thz-4) -OMe by alkaline hydrolysis of the methyl ester,

Φ

- 138 • · · · 0 0 e β 0 0 0 β ® 0 • 00000 00 •. · 0 0 0 · -W 0 0

0 ··· 00 00 00 00 00 by coupling the resulting acid with H ₂ N-CH ₂ - (2-CN) -2-thioph to give N- (t-BuO ₂ C-CH 2) -N-Boc- (D) -Cha- (2,2-Me ₂ -thz-4) -NH-CH ₂ -2- (4-CN) -thiof, amidine formation and subsequent deprotection as described in Example 2b-e. FAB-MS (M + H &lt; + &gt;):505; mp 184-7 ° C (decomposition)

Example 74: N- (hydroxycarbonylmethylene) - (D) -cyclohexylglycyl-5,5-dimethylthiazolidine-4-carboxylic acid [4- (2-amidino) thienylmethyl] amide

This compound can be prepared by the following reactions: coupling of N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Chg-OH with 5,5Me ₂ -thz-4-OMe- to give N- ( t-BuQ ₂ C-CH ₂ ) -N-Boc- (D) -Chg- (2,2Me ₂ -thz-4) -OMe, by alkaline hydrolysis of the methyl ester, by coupling the resulting acid with H ₂ N-CH ₂ - (2 -CN-4-thioph to give

N- (t-BuO ₂ C-CH ₂ ) -N-Boc- (D) -Chg- (5,5-Me ₂ -thz-4) -NH-CH ₂ -4- (2CN) -thiof, forming amidine followed by deprotection according to the methods described in Example 2b-e. FAB-MS (M + H): 491 mp 164-166 ° ^C (decomposition)

Examples 75-90

The following compounds can be synthesized from the corresponding A-B-D- and E-F- units using the procedures described in Example 14 of WO98 / 06741:

75. HOOC-CH ₂ - (D) - (p-OMe-Phe) -Pro-NH-CH ₂ -2- (4-am) -thioph

76. HOOC-CH ₂ - (D) - (p-OMe-m-Cl-Phe) -Pro-NH-CH ₂ -2- (4-am) -thioph

77. HOOC-CH ₂ - (D) - (p-OMe-Phe) -Pro-NH-CH ₂ -5- (2-am-3-Me) -thioph

78. HOOC-CH ₂ - (D) - (p-CF ₃ -Phe) -Pro-NH-CH ₂ -5- (2-am-3-Me) -thioph

79. HOOC-CH ₂ - (D) - (p-Cl-m-Cl-Phe) -Pro-NH-CH ₂ -5- (2-am-3-Me) thioph

• «

80. HOOC-CH ₂ - (D) - (p-CF ₃ -Phe) -Pro-NH-CH ₂ -2- (4-am) -thioph

81. HOOC-CH ₂ - (D) -Cha-Pro-NH-CH ₂ -2- (4-am-5-Me) -thiaz

82. HOOC-CH2- (D) -Chg-Pro-NH-CH2-2- (4-am-5.Me) -thiaz

83. HOOC-CH 2 - (D) - (p-OMe-Phe) -Pro-NH-CH ₂ -2- (4-am-5-Me) -thiaz

84. HOOC-CH ₂ - (D) - (p-O 1 / le-Phe) -Pro-NH-CH ₂ -2- (4-am) -thiaz

85. HOOC-CH ₂ - (D) - (p-CF ₃ -Phe) -Pro-NH-CH ₂ -2- (4-am) -thiaz

86. HOOC-CH ₂ - (D) - (p-CF ₃ -Phe) -Pro-NH-CH ₂ -2- (4-am-5-Me) -thiaz

87. HOOC-CH ₂ - (D) - (β-iPr-m-Me-Phe) -Pro-NH-CH ₂ -2- (4-am) -thiaz

88. HOOC-CH2- (D) - (p-OMe-m-Cl-Phe) -Prp-NH-CH2-2- (4-am) -thiaz

89. HOOC-CH2- (D) - (p-Cl-m-Cl-Phe) -Pro-NH-CH2-2- (4-am) -thiaz

90. HOOC-CH2- (D) - (p-Cl-m-Cl-Phe) -Pro-NH-CH2-2- (5-am-4-Me) thiazole

Example 91

The following compound can be synthesized from the corresponding E-unit and N- (t-BuO ₂ C-TCH ₂ ) -N-Boc- (D) -Cha-ProOH as described in Example 20.

HOOC-CH ₂ - (D) -Cha-Pro-NH-CH ₂ -3- (5-am-1-Me) -pyraz

Example 92

HOOC-CH ₂ - (D) -Cha-Thz-4 NH-CH ₂ -5- (2-am) -thioph was synthesized from Boc-Thz-4-OH, 5-H ₂ N-CH ₂ -thioph- 2-CN and N- (t-BuO ₂ C-CH ₂ ) -N-Boc (D) Cha-Pro-OH by a method similar to that described in Example 3.

Pharmacological tests

Example A

E

- 140 Chromogen test for kallikrein inhibitors

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

Deisenhofen, Germany)

Substrate: Chromozyme GK (No. 709875, Boehringer,

Mannheim, Germany)

Buffer: 20 mM Tris (HCl pH = 8.50)

Experimental methodology:

A chromogenic assay to determine the activity of kallikrein is performed on microplates. To 93 µl of buffer add 2 μΙ of a solution of the mixture to which DMSO is added and the mixture thus obtained at a final concentration of 0.01 units / ml of kallikrein. Incubation was carried out for 10 minutes at 20-25 ° C. The test itself is started by adding 100 μΙ of substrate (500 pmol / l final concentration). After another 30 min incubation, light absorption at 405 nm was measured with a photometer.

Example B

Time required for action on thrombin

Reagents: Thrombin reagent (Product No. 126 594, Boehringer, Mannheim, Germany)

Preparation of citrate plasma:

parts of human venous blood obtained from the vein of cephalica are mixed with one part of sodium citrate solution (0.11 mol / l). This mixture is then centrifuged. Plasma can be stored at -20 ° C.

Experimental methodology:

μΙ of test substance solution and 50 μΙ of citrate plasma are incubated for 2 minutes at 37 ° C (CL8, balloon type,

4 4 · · 4 4 ·

- 141 4 4 · 4 4 4 4 4 4 4 4 4 4 4 4 4 4

4 4 4

44

Bender & Hobein, Munich, FRG). 100 µl of thrombin reagent (37 ° C) is then added. The time after which the fibrin clot is formed is determined.

Example C

Chromogen test thrombin inhibitors

Reagents: Thrombin from human plasma (No. T-8885, Sigma, Deisenhofen, Germany)

Substrate: H-D-Phe-Pip-Arg-pNA-2HCI (S-2238, Chromogenix, Molndahl, Sweden)

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

Experimental methodology:

The chromogenic assay is performed on microtiter plates. To 250 μΙ of thrombin buffer (final concentration 0.1 NIH units / ml), add 10 μΙ of a solution of the test substance in DMSO and incubate for 5 minutes at 20 to 28 ° C. The test itself is started by adding 50 μΙ of substrate solution in buffer (final concentration 100 pmol / l), incubating the mixture at 28 ° C and after 5 minutes the reaction is terminated with 50 μΙ citric acid (concentration 35%). Absorbance is measured at 405/630 nm.

Example D

Platelet aggregation in plasma, platelet rich

Reagents: Human plasma thrombin (No. T-8885, Sigma, Deisenhofen, Germany)

- 142 44 *

5 · • ··· ·

• * 4 • 4 4

4 4 4

44 '• 4

4 4 4

4 «4

4 44

Preparation of plasma enriched with citrate and platelets:

Venous blood is taken from the cephalica vein from healthy individuals who are not receiving any medications. This blood is mixed 9: 1 with 0.13 molar sodium citrate. Platelet-rich plasma (PRP) is prepared by centrifugation at 250 x g (10 minutes at room temperature). Platelet-poor plasma (PPP) is prepared by centrifugation at 3600 x g for 20 minutes. PRP and PPP can be stored for 3 hours at room temperature in closed PE containers. Platelet concentrations are measured with a hematocytometer and should be between 2.5 and 2.8-10 ® / ml.

Experimental methodology:

Platelet aggregation is measured turbidimetrically at 37 ° C (PAP 4, Biodata Corporation, Horsham, PA, USA). Prior to the addition of thrombin, 215.6 µl of PRP is incubated for 3 minutes with 2.2 μΙ of the test substance and the mixture is then stirred for another 2 minutes at 1000 rpm. Use of a final concentration of 0.15 NIH units / ml, 2.2 µl of thrombin in solution results in maximal aggregation effect at 37 ° C / 1000 rpm. The inhibitory effect of the test substances is determined by comparing the rate at which thrombin aggregates (propensity to clot) are formed without the test substance with the rate of thrombin aggregation with the addition of the test substance in several different concentrations.

Claims (13)

······ · * · »** * · ρν ΐοοο-ύίζ PATENT CLAIMS A compound of formula I A-B-D-E-F I wherein A, B, D, E and F have the following meanings: A: R ² AND R ¹ - (CH ₂ ) _n - C - (CH ₂ ) _n - wherein ^{r 3} m is 0, 1 or 2, n is 0, 1 or 2, R ¹ is HOOC-, C 1-6 -alkyl-OOC-, aryl-C 0-4 -alkyl-OOC or -OH, R ² is H, Ci-4-alkyl, or R ¹ - (CH ₂₎ m R ³ is H or Ci-4-alkyl, R ^{6 B} 'R ⁴ (R ⁷ -CR ⁸ ) p I I -N-C-CO: -R ⁵ wherein R ⁴ is H-, C 1-4 -alkyl- or R ¹ - (CH 2) _m - (wherein R ¹ and _m are as defined above), p is 0 or 1, R ⁵ is H- or C _1-4 -alkyl-, R ⁶ is H-, C 1-8 -alkyl-, 2-thienyl-, 3-thienyl-, 3-indolyl-, 4-imidazolyl-, 2-pyridyl-, 3-pyridyl-, 4-pyridyl-, phenyl-, which may carry up to three identical or different radicals from the group which includes C 4 alkyl, CF ₃ -, C 4 -alkoxy-, HO-, BnO- F- or Cl-, or _C3 -8 -cycloalkyl, which may carry up to four identical or different C 1-4 -alkyl radicals and / or wherein one or two CC single bonds in the ring may - 144 _. , may be replaced by a C = C double bond and / or the phenyl ring may be unfused, C ₇ -C ₁₂ -bicycloalkyl- or C 3-6 -tricycloalkyl- or R ⁴ and R ⁶ together may form an ethylene or propylene group, R ⁷ is H, C 1-6 -alkyl-, phenyl-, which may carry up to three identical or different radicals from the group consisting of C _1-4 -alkyl-, CF ₃ -, C 1-8 -alkyl-, C 3-8 -alkyl-, C 1-8 -alkyl-, C 3-8 -alkyl-, phenyl-, ₄ alkoxy, F or Cl, or C ₃ .8-cycloalkyl-, which can carry up to four identical or different C. _4- alkyl radicals, and R 8 is H or C 1. _4- alkyl wherein R ²⁰ is H, C 1-6 alkyl; _4- alkyl, Bn or BnO (CO) - and where the following is true: if D is II, III or XI, then E has the following meaning: _R 10 _R 9 R 11 Y - z wherein (a) where X = S, O, NH or NR ¹² , - 145 -. Y is -CR ¹³ =, -CH = and Z is -CR ¹⁴ = or Y is -CR ¹³ = and Z is -CH = or b) when X = NR ¹² , then Y is -CH = and - Z is -CH = or (c) where X = S, O or NH, Y is -CR ¹⁵ = and Z is -N = or Y is-N = and Z is -CR ¹⁵ = or dj when X = -NR ¹² - then Y is -N = a Z is -CR ¹⁶ =, -N = or Y is -CR ¹⁶ = and Z is N = and R ⁹ is H- or C 1-3 -alkyl-, R ¹⁰ is H- or Ci. _4- alkyl-, R ¹¹ is H- or Ci. _4- alkyl-, R ¹² is CH ₃ - or C ₂ H ₅ -, R ¹³ is Cl, CF 3 or C ₄ -alkyl- R ¹⁴ is Cl, CF ₃ - or C ₄ -alkyl- -146-. • * 4 ·· '·· ·· ·· R ¹⁵ is CF 3 - or C 1-4 -alkyl-, R ¹⁶ is H-, CF ₃ - or C _1-4 -alkyl- and κ has the meaning given above, or, if D is IV, VI, VII, VIII is: IX or X, then E has the following where X is O, S or -NR ¹⁷ and Y is N = a Z is -CR ¹⁶ = or-N = or Y is -CR ¹⁶ = a Z is-N = or Y is -CR ¹⁸ = a Z is -CR ¹⁹ = a f '1, κ, κ and R are as defined above, R ¹⁷ is H, CH _{3 -} or C ₂ H ₅ -, R ¹⁸ is H, Cl, CF ₃ -or C 4 -alkyl-, R ¹⁹ is H-, Cl-, CF ₃ - or C ₁ . _4- alkyl-, or when D is II, III, IV, VI, VII, VIII, IX, x or XI, E has the following meanings: R 10 n - c - or R 9 R 11 Z 147 where (a) if X = S, then Y is -CR ¹⁸ = a Z is -CR ¹⁹ = or Y is -CR ¹⁶ = a Z is -N = or b) when X = O or -NR ¹² -, then Y is -N =, -CR ¹⁶ = a Z is -N =, -CR ¹⁸ = nh _2, or a prodrug thereof, or a salt thereof with a physiologically acceptable acid. A compound of formula I according to claim 1, wherein A to E have the following meanings: A:, HOOC- (CH ₂₎ t (t = 1, 2 or 3), _{(H00C-CH2) 2} -CH · HOOC-CH ₂ -CH (COOH) -, HOOC-CH (Ci. ₄ alkyl 1 -, HOOC-C (C 1-4 -alkyl) ₂ LC-C 1-6 -alkyl-OOC-CH 3 -. B is R ⁶ R ⁴ (R ⁷ -CR ⁸ ) co ^; - 1/1 Q * · '·· * ’* ·· - I 4 O > ► • · p is 0 or 1, R 6 is H-, C 1-4 -alkyl- or HOOC- (CH ₂ ) m - (m = 1, 2 or 3), R ⁵ is H, methyl R ⁶ is H-, C 1-6. _8- 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 which includes CH ₃ -, CF ₃ -, CH ₃ O-, HO-, BnO-, F or C, or C ₃ .8 -cycloalkyl which may carry up to four methyl radicals, bicyclo [ 2.2.2] octyl-, bicyclo [2.2.1] heptyl-, adamantyl-, indanyl-, decalinyl-, R ⁷ is H, C 1-8 -alkyl-, phenyl-, which may carry up to three identical or different radicals from CH ₃ -, CF ₃ -, CH ₃ O-, F- or Cl-, or _C3-.8 cycloalkyl which may carry up to four methyl radicals, R ⁸ is H, C 1-4 -alkyl, wherein R ²⁰ is H, CH ₃ , Bn or BnO (CO) - and - 149 where the following applies: if D is II, III or XI, then E means: H where Η h y-z (a) where X = S, O or NR ¹⁷ , then Y is -CR ¹³ = or -CH = a Z is -CR ¹⁴ = or Y is -CR ¹³ = a Z is -CH = or b) when X = NR ¹² , then Y is -CH = a Z is -CH =, or c) when X = S, O or NH, then Y is -CR ¹⁵ = a Z is N = or Y is N = a Z is -CR ¹⁵ = or d) when X = NR ^12, Y is N = a Z is -CR ¹⁶ =, -N = or Y is -CR ¹⁶ = a Z is-N = • 4 '* · • · 4 9 - 150 -Z '; 449 · 4 44 4 · «'4' 4 4 4 · 4 4 4 · 4 · 3 * · 4 4 4 4 • 4 4 · 4 4 4 9 4 · ·· R ¹² is CH ₃ - or C ₂ H ₅ -, R ¹³ is Cl, CF ₃ - or C 4 alkyl-, R ¹⁴ is Cl, CF ₃ -or C 4 -alkyl-, R ¹⁵ is CF _{3 -} or C 1-4 -alkyl, R ¹⁶ is H-, CF ₃ - or C 1-4 -alkyl-, R ¹⁷ is H, CH _{3 -} or C ₂ H 5 R ²⁰ is as above, or if D is IV, VI, VII, VIII, IX or X, then E is: H where ^{HHY — 2} X is O, S or-NR ¹⁷ -a wherein Y is -N = a Z is -CR ¹⁶ = or -N = or Y is -CR ¹⁶ = a Z is -N = or Y is -CR ¹⁸ = a Z is -CR ¹⁹ = a , 17, 20 -R ¹⁶ , R ¹⁷ , R ²⁰ have the meanings given above, R ¹⁸ is H-, Cl-, CF ₃ - or C 1-4 -alkyl- a R ¹⁹ is H-, C 1-, CF ₃ -or C 1-4 -alkyl, or if D is II, III, IV, VI, VII, VIII, IX, X or XI, then E has the meanings: B I Ν - ΟΙ Η H or ^ x ·· φφφ -151 ···· ·· Where (a) if X = S, then Y is -CR ¹⁸ = a Z is -CR ¹⁹ = or Y is -CR ¹⁶ = a Z is -N = or b) - in the event that X = OY is -N = or -CR ¹⁶ = Z is -N = or -CR ¹⁸ = and R ^12, R ^16, R ^18, R ¹⁹ _or 'NR ^12, and R have the above-mentioned meanings, F: NH NH, or N-OH 'nh. and salts thereof with physiologically acceptable acids A compound of formula I according to one of claims 1 or 2, wherein A, B, D, E and F have the following meanings: A: HOOC-CH 2, HOOC-CH 2 -CH 2, HOOC-CH (CH ₃ ), HOOC-CH (C ₂ H ₅ ) R ^{6 B:} I R ⁴ (R-C-R ') _p - N - C --- COP is 0 or 1, R ⁴ is H-, CH ₃ R ⁵ is H-, CH ₃ -, R ⁶ is C 1-8 -alkyl-, C 3-8 -cycloalkyl-, which may carry up to four methyl radicals, 2-thienyl-, 3-indolyl-, 4-imidazolyl-, 2-pyridyl-, 3-pyridyl-, 4-pyridyl, phenyl, which may carry up to three identical or different radicals from CH ₃ -, CF ₃ -, CH ₃ O-, HO-, BnO-, F- or Cl-, bicyclo [2.2. 2] octyl, bicyclo [2.2.1] heptyl, adamantyl, indanyl, decainyl, with particular emphasis on cyclopentyl, cyclohexyl and cycloheptyl, R ⁷ is H, CH 3 -, R ⁸ is H, CH 3 -, wherein R ²⁰ is H, BnO (CO) - and where the following applies: if D is II, III or XI, then E means: • · ·· where X Y Yippee Yippee -S-and where -CH = a of Yippee or -cr ¹³ = Y Yippee -CR ¹³ = a of Yippee -CH = or Y Yippee -CR ¹⁵ = a OF Yippee or -N = Y Yippee -N = a of and Yippee -CR ^1S = R ¹³ is Cl ·, CF ₃ - or CH ₃ , r15 is CF ₃ - or CH ₃ - and R ²⁰ is as described above or if D is IV, VI, VII, VIII, IX or X, then E means: where X is S, and where Y is N = a Z is -CR ¹⁶ = or Y is -CR ¹⁶ = a Z is -N = 0 '0 0 0 0 · 0 '' 0 - 154 -. 0. • 0 0 0 '0 0' 0 0 ' 0 0 0 0 0 · 0 0> 0 0 0 0 ·· or ’ Y is -CR ¹³ = a Z is -CH = or Y is -CH = a Z is -CR ¹³ = or Y is -CH = a Z is -CH = a R @ ¹ , R @ ³ , R @ ²⁰ are as defined above, and R @ ¹⁶ is H--, CF @ ₃ - or CH @ _3, or if D is II, III, IV, VI, VII, VIII, IX, X or XI, then E is: where either a) when X = S, Y is-CH = a Z is -CR ¹⁸ = or Y is -CR ¹⁶ = and Z is -N = or Y is -CR ¹⁸ = a Z is -CH = or b) when X = O or NCH ₃ Y is -CH = and Z is -CR ¹⁶ = or Y is -CR ¹⁶ = a Z is -CH = or - 155 • ·· «• ♦ c) when X = -NR ¹² Y is -N = and Z is -CR ¹⁸ = R ¹² is CH _{3 -} or C ₂ H ₅ -a R ¹⁸ is H, Cl -, CF _{3 -} or CH ₃ -, and R ¹⁶ , R ²⁰ have the meanings given above F: N-OH - or ^NH 2 / h ₂ and their salts with physiologically acceptable acids. A compound of formula I, according to any one of claims 1 to 3, wherein A, B, D, E and F have the following meanings: A: HOOC-CH 2, HOOC-CH 2 -CH 2, HOOC-CH (CH ₃ ), HOOC-CH (C ₂ H ₅ ) ^B: I r ^{, r,} r '” -N-C-COI R ⁵ p is 0 or 1, R ⁴ is H-, R ⁵ is H-, R ⁶ is C 1-8 alkyl-, 2-thienyl, 3-indolyl, 4-imidazolyl, 2-pyridyl, 3pyridyl-, 4-pyridyl, C ₅ -8-cycloalkyl which may carry up to four methyl radicals, phenyl-, which may carry up to three identical or different radicals from the group consisting of CH ₃ -, CF ₃ -, CH ₃ O-, HO-, BnO-, F- or -156 • · · · · · CI-, bicyclo [2.2.2] octyl, bicyclo [2.2.1] heptyl, adamantyl, indanyl, decalinyl, with particular emphasis on cyclopentyl-, cyclohexyl- and cycloheptyl- where the following applies: if D is II, III or XI, then E means: where X is S and Y is -CR ¹³ = a Z is -CH = or Y is -CH = a Z is -CR ¹³ = or Y is -CR ¹⁵ = a Z is -N = H H H Y-z · · · · · · - 157 0 '0' 0 · · '' · 0 0.000 0 0 0 0 0 0 0 0 0 0 0 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 Υ is Ν = a Z is -CR = ^1S and R ¹³ is Cl-, CF ₃ -or CH ₃ -a R ¹⁵ is CF ₃ - or CH ₃ - or if D is IV, VI, VII, VIII, IX or X; then E means: H X is S and Y Yippee -N = of Yippee -cr ¹⁶ = or Y Yippee -cr ¹⁶ = of ¹ Yippee -N = or Y Yippee -CH = of Yippee or -cr ¹³ = Y Yippee -cr ¹³ = of Yippee or -CH = Y Yippee -CH = of Yippee -CH = and 13 z R is as defined above and R ¹⁶ is H, CF ₃ - or CH ₃ -, or - 158 · · 158 • 158 158 158 • • • • • • 4444 ·· • · '· 44 4 4 • 4 4 4 4-4 4 4 4 4 4 44 If D is II, III, IV, VI, VII, VIII, IX, X or XI, then E is where and) if X = with, Y is -CH = a OF is -CR ¹⁸ = or Y is -CR ¹⁸ = a of is -CH = or. Y It is -CR ¹⁶ =, and of or is -N = (b) if X = 0 or NCH ₃ Y is -CH = a of . is -CR ¹⁶ = or Y It is -CR ¹⁶ =, and of is -CH = or C) if X = nch ₃ Y is -N = a Z is -CR ¹⁶ = R ¹⁶ is as defined above and R ¹⁸ is H, Cl-CF ₃ - or CH ₃ -; -159 • · • ··· F: NH N-OH NH, or NH, and their salts with physiologically acceptable acids. 5. A compound comprising a structural element NH NH, wherein D and E are as defined in any one of claims 1 to 4, and wherein at least one of the radicals selected from the group consisting of hydrogen, a protecting group, an unsubstituted or substituted natural or unnatural amino acid, an unsubstituted or substituted carboxylic acid the acid, unsubstituted or substituted alkyl radical is attached to the nitrogen atom of structural block D. A compound comprising a structural element --E NH wherein E is as defined in any one of claims 1 to 4 and wherein at least one of the radicals selected from the group consisting of hydrogen, protecting group, unsubstituted or substituted natural or unnatural amino acid, unsubstituted or substituted carboxylic acid, unsubstituted or substituted the alkyl radical is attached to the nitrogen atom of the NR ⁹ group A compound comprising a structural element of the formula wherein Q is CH 3 or Cl, T is N CH 3, O or S and W is NCH 3 or S. A composition comprising, in addition to conventional adjuvants and excipients, a compound according to any one of claims 1 to 4 and a compound according to any one of claims 5, 6 or 7. Use of a compound of formula I according to any one of claims 1 to 4 or a compound according to any one of claims 5, 6 and 7 for the manufacture of a pharmaceutical composition for the treatment of: • diseases whose pathogenetic mechanism is directly or indirectly derived from the proteolytic effect of thrombin • diseases whose pathogenetic mechanism is derived from thrombin-dependent receptor activation and signaling • diseases associated with the stimulation or inhibition of gene expression in body cells • diseases derived • Thrombin-dependent changes in thrombin-dependent changes in contractility and permeability of epithelial cells. • Thrombin-dependent thromboembolic events. • Multiple intravascular coagulation. - 161 99 99 99 signs of early reocclusion and restenosis after PTCA, • thrombin-dependent proliferation of smooth muscle cells, accumulation of active thrombin in the CNS; • tumor growth and prevention of tumor cell adhesion and metastasis. Use of a compound of formula I according to any one of claims 1 to 4 or a compound according to any one of claims 5, 6 and 7 for the manufacture of a pharmaceutical composition for the treatment of: • diseases whose pathogenetic mechanism is directly or indirectly derived from the proteolytic effect of kininogenases, particularly kallikrein, inflammatory diseases such as asthma, pancreatitis, rhinitis, arthritis, urticaria and other internal diseases in which kallikrein is involved. A compound of formula I according to any one of claims 1 to 4 for coating surfaces. A compound of formula Va or Vb A-B-D-E-CN Va A - B - D - E - CSNH ₂ Vb, wherein A, B, D and E have the meanings given in any one of claims 1 to 4. Use of a compound according to any one of claims 1 to 7 for the manufacture of compositions which are useful as thrombin inhibitors.