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Title:
INHIBITORS OF BLOOD COAGULATION FACTOR XIII
Document Type and Number:
WIPO Patent Application WO/2019/201432
Kind Code:
A1
Abstract:
The invention relates to a compound of general formula (I) as novel inhibitor ofblood coagulation factor XIII, methods for synthesisthereofand to use thereof for the prophylaxis ortreatment of diseases associated with blood coagulation factor XIII.

Inventors:
PASTERNACK RALF (DE)
HILS MARTIN (DE)
BÜCHOLD CHRISTIAN (DE)
Application Number:
PCT/EP2018/059798
Publication Date:
October 24, 2019
Filing Date:
April 17, 2018
Export Citation:
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Assignee:
ZEDIRA GMBH (DE)
International Classes:
A61K47/64; A61P7/02
Domestic Patent References:
WO2008055488A12008-05-15
WO2014012858A12014-01-23
WO2014090835A12014-06-19
WO2018122419A12018-07-05
WO2008055488A12008-05-15
Other References:
HEIL ANDREAS ET AL: "Differences in the inhibition of coagulation factor XIII-A from animal species revealed by Michael Acceptor- and thioimidazol based bloc", THROMBOSIS RESEARCH, vol. 131, no. 5, 13 March 2013 (2013-03-13), XP028539427, ISSN: 0049-3848, DOI: 10.1016/J.THROMRES.2013.02.008
WILDT BEREND VAN DER ET AL: "Strategies towards in vivo imaging of active transglutaminase type 2 using positron emission tomography", AMINO ACIDS, vol. 49, no. 3, 5 July 2016 (2016-07-05), pages 585 - 595, XP036181967, ISSN: 0939-4451, [retrieved on 20160705], DOI: 10.1007/S00726-016-2288-Y
LORAND L; JACOBSEN A., NATURE, vol. 195, 1962, pages 911 - 2
STIELER M ET AL., ANGEW CHEMIE INT ED, vol. 52, 2013, pages 11930 - 4
BOHM M ET AL., J MED CHEM, vol. 57, 2014, pages 10355 - 65
MUSZBEK L, PHYSIOL REV, vol. 91, 2011, pages 931 - 72
BYRNES JR ET AL., BLOOD, vol. 126, 2015, pages 1940 - 8
LORAND L, ARTERIOSCLER THROMB. VASE. BIOL., vol. 20, 2000, pages 2 - 9
GRIFFIN JH, NATURE, vol. 378, 1995, pages 337 - 8
WEITZ JI; FREDENBURGH JC., FRONT MED, 2017, pages 4
FINNEY S ET AL., BIOCHEM J, vol. 324, 1997, pages 797 - 805
LEIDY EM ET AL., THROMB RES, vol. 59, 1990, pages 15 - 26
SHEBUSKI RJ ET AL., BLOOD, vol. 75, 1990, pages 1455 - 9
NOVAKOVIC J ET AL., J PHARM BIOMED ANAL, vol. 38, 2005, pages 293 - 7
KOGEN H ET AL., J. AM. CHEM. SOC., vol. 122, 2000, pages 1842 - 1843
OERTEL K; HUNFELD A; SPECKER E; REIFF C; SEITZ R; PASTERNACK R; DODT J.: "A highly sensitive fluorometric assay for determination of human coagulation factor XIII in plasma", ANAL BIOCHEM, vol. 367, 2007, pages 152 - 8, XP022138785, DOI: doi:10.1016/j.ab.2007.05.011
LORAND L; LOCKRIDGE OM; CAMPBELL LK; MYHRMAN R; BRUNER-LORAND J.: "Transamidating enzymes", ANAL BIOCHEM, vol. 44, 1971, pages 221 - 31, XP024817477, DOI: doi:10.1016/0003-2697(71)90363-0
LAEMMLI UK: "Cleavage of structural proteins during the assembly of the head of bacteriophage T4", NATURE, vol. 227, 1970, pages 680 - 5, XP000568538, DOI: doi:10.1038/227680a0
LANG T; VON DEPKA M: "Possibilities and limitations of thrombelastometry/-graphy", HAMOSTASEOLOGIE, vol. 26, 2006, pages 20 - 9
Attorney, Agent or Firm:
ARTH, Hans-Lothar (DE)
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Claims:
Claims

1. A compound of the general formula (I):

wherein

R1 represents -H, -CH3, -C(CH3)3, -cyclo-C3H5, -cyclo-C4H7, -cyclo-C5H9, -cyclo-CeHn, -CH2-CH(CH3)2, -CH2CH3J -CH2CH2CH3J -CH2CH2CH2CH3J -CH2CH2CH2CH2CH3J -CH2CH2CH2CH2CH2CH3J -CH(CH3)2J -CH2-C(CH3)3J -CH2CH2SCH3J -CH2-cyclo-C3H5, -CH2-cyclo-C4H7, -CH2-cyclo-C5H9 or -CH2-cyclo-C6Hn;

R2 represents -A1-A2-A3-E, -A1-A2-A3-A4-E or -A1-A2-A3-A4-A5-E;

R represents

R4 represents -OR*, -NH2, -NHR# or -NR*R#;

R* and R# represent independently of each other -CH3, -CH2CH3, -CH(CH3)2, -CH2CH2CH3J -CH2CH(CH3)2J -C(CH3)3J -cyclo-C3H5, -cyclo-C4H7, -cyclo-C5H9, -cyclo-C6Hn, -CH2-cyclo-C3H5, -CH2-cyclo-C4H7, -CH2-cyclo-C5H9, -CHz-cyclo-CeH·,·,, -CH2-Ph, -CH2OCH3, -CH2OCH2CH3,

-CH2CH2OCH3, or -CH2CH2SCH3;

A1 represents

A2 - A5 represent independently of each other

R13 and R14 represent independently of each other: -H, -CH3, -CH2CH3, -C(CH3)3,

L1 - L8 represents independently of each other a covalent bond,

R5 - R12 and R15 - R23 represents independently of each other

-0-cyclo-C3H5, -OCH2-cyclo-C3H5, -0-C2H4-cyclo-C3H5, -CHO, -COCH3, -COCF3, -COC2H5, -COC3H7J -COCH(CH3)2J -COC(CH3)3J -COOH, -COOCHg, -COOC2H5, -COOC3H7J -COOCH(CH3)2J -COOC(CH3)3J -OOC-CH3, -OOC-CF3, -OOC-C2H5, -OOC-C3H7J -OOC-CH(CH3)2J -OOC-C(CH3)3J -NH2, -NHCH3, -NHC2H5, -NHC3H7J -NHCH(CH3)2J -NHC(CH3)3J -N(CH3)2J -N(C2H5)2, -N(C3H7)2J -N[CH(CH3)2]2J -N[C(CH3)3]2J -NHCOCHg, -NHCOCFg, -NHCOC2H5, -NHCOC3H7, -NHCOCH(CH3)2,

-NHCOC(CH3)3, -CONH2, -CONHCH3, -CONHC2H5, -CONHC3H7,

-CONHCH(CH3)2J -CONH-cyclo-C3H5, -CONHC(CH3)3, -CON(CH3)2, -CON(C2H5)2, -CON(C3H7)2J -CON[CH(CH3)2]2J -CON[C(CH3)3]2J -SO2NH2, -SO2NHCH3, -SO2NHC2H5, -S02NHC3H7, -S02NHCH(CH3)2,

-S02NH-cyclo-C3H5, -S02NHC(CH3)3, -S02N(CH3)2, -S02N(C2H5)2,

or R7 and R8 or R8 and R9 form together one of the following ring moieties; or E/Z-isomer, regiomer, diastereomer, enantiomer, a mixture of E/Z-isomers, a mixture of regiomers, a mixture of diastereomers, a mixture of enantiomers, prodrug, solvate, hydrate, or pharmaceutically acceptable salts thereof.

2. The compound according to Claim 1 , wherein the compound has any one of the formulae (11-1) to (II-3):

wherein R2, R3 and R4 have the meanings as defined in claim 1.

The compound according to Claim 1 or 2, wherein A2 is selected from

and/or A2 is preferably selected from

4. The compound according to any one of Claims 1 - 3, wherein -A1-A2- represents

5. The compound according to any one of Claims 1 - 4, wherein -A2-A3- represents

6. The compound according to any one of Claims 1 - 5, wherein R represents

and R7, R8 and R9 have the meanings as defined in Claim 1. 7. The compound according to any one of Claims 1 - 6, wherein the compound has any one of the formulae (MI-1 ) and (III-2):

wherein

E2 represents

R represents and

A1, A3, A4, A5, R4, R7, R8, R9 and E have the meanings as defined in Claim 1

8. The compound according to Claim 1 or 7 having any one of the formulae (IV-1) to

wherein

E2 represents -E, -A3-E, -A3-A4-E or -A3-A4-A5-E;

R4 represents -OCH3 or -OCH2CH3;

R7, R8 and R9 are independently of each other selected from -H, -Cl, -OH, -N02 or -COOH; and

A1, A3, A4, A5 and E have the meanings as defined in Claim 1.

9. The compound according to any one of Claims 1 - 8, wherein the compound has any one of the formulae (V-1) and (V-2):

wherein

E2 represents -E, -A3-E, -A3-A4-E or -A3-A4-A5-E;

R4 represents -OCH3 or -OCH2CH3;

A1 represents

10. The compound according to any one of Claims 1 - 7 having the formula (VI):

wherein

A1, A4, A5, R4, R7, R8, R9 and E have the meanings as defined in Claim 7.

11. The compound according to claim 1 selected from a group consisting of: compounds 1a, 1b, 2a, 2b, 3, 4, 5a, 5b, 6, 7a, 7b, 8a, 8b, 9a, 9b, 10a, 10b, 11a, 11b, 12a, 12b, 13a, 13b, 14a, 14b, 15a, 15b, 16a, 16b, 17a, 17b, 18a, 18b, 19a, 19b, 20a, 20b, 21a, 21b, 22a, 22b, 23a, 23b, 24a, 24b, 25a, 25b, 26a, 26b, 27, 28, 29, 30, 31a, 31b, 32a, 32b, 33a, 33b, 34a, 34b, 35a, 35b, 36a, 36b, 37a, and 37b,

12. The compound according to any one of claims 1 - 11 for use as a medicament, especially for use as a medicament in anticoagulation and FXIIIa inhibition.

13. A pharmaceutical composition comprising at least one compound according to any one of claims 1 - 11 as an active ingredient together with at least one pharmaceutically acceptable carrier, excipient and/or diluent.

14. The compound according to any one of claims 1 - 11 or the pharmaceutical composition according to claim 13 for use in the treatment or prophylaxis of coeliac disease, Duhring-Brocq-disease, gluten ataxia, tissue fibrosis, cystic fibrosis, kidney fibrosis and diabetic nephropathy, liver fibrosis, cataract, ichthyosis, acne, psoriasis, skin aging, candidosis, neurodegenerative disorders including Huntington’s disease, Parkinson’s disease and Alzheimer’s disease as well as atherosclerosis, thrombosis, thrombocytopenia and thrombopreventive indications and for use as anticoagulant in the treatment of sepsis, stroke, recurrent occlusion and acute care setting including acute kidney injury, acute lung injury and acute coronary syndrome.

15. A Method for producing compound according to claim 1 comprising:

Step (OB): providing a resin, suitable for solid-phase peptide synthesis (SPPS).

SPPS

resin

Step (1 B) (a): performing coupling reaction of the corresponding C-terminal amino acid building block PG4NH-A-OH.

(d) deprotecting the protecting group PG4 of a resulting compound after Step (a);

(e) repeating the steps (a) and (b) / times, wherein / is 1 -5, to obtain a resin bound intermediate compound (lllc).

PG4H- A1— A2 - A'— Q

lllc

Step (2B):cleavage from resin and deprotecting the protecting group PG4 to obtain an intermediate compound (llld).

H2— A1— A2 - A'-E

Mid

Step (3B): performing coupling reaction of the intermediate compound llld with an amino acid building block AO

to obtain a compound IVc.

Step (4B): deprotecting the protecting group PG5 and subsequent coupling reaction with protected amino acid la

la; to obtain compound

Step (5B): deprotecting the protecting group PG1 and subsequent coupling reaction with a N-terminal building block R3-C02H to produce the compound of the formula (I); or

Step (0C): providing a N-deprotected C-terminal building block H-E or H-A'-E. Step (1C): (a) performing coupling reaction of the corresponding C-terminal amino acid building block PG4NH-A'-OH or PG4NH-A' 1-OH.

(d) deprotecting the protecting group PG4 of a resulting compound after Step (a);

(e) repeating the steps (a) and (b) / times, wherein / is 1 -5 or 1 -4,

to obtain intermediate compound (llld).

H2 _A1 A2 A'-E ( md )

Step (3B): performing coupling reaction of the intermediate compound llld with an amino acid building block AO

R1

PG 'N CO2H

H AO

to obtain a compound IVc.

Step (4B): deprotecting the protecting group PG and subsequent coupling reaction with protected amino acid la

to obtain compound

Step (5B): deprotecting the protecting group PG1 and subsequent coupling reaction with a N-terminal building block R3-C02H to produce the compound of the formula (I).

Description:
Inhibitors of blood coagulation factor XIII

Description

The invention relates to novel inhibitors of blood coagulation factor XIII, methods for their synthesis and to their use for the prophylaxis or treatment of diseases associated with blood coagulation factor XIII.

Background of the invention

Substantial effort has been dedicated to the development of drugs targeting coagulation factors or platelet activation in order to prevent and treat venous thromboembolism, acute coronary syndrome or for reducing the risk of stroke in patients with atrial fibrillation. Coagulation factor XIII (FXIII, F13) is a promising but yet widely untapped and challenging target for drug development (LORAND L, JACOBSEN A., Nature 1962; 195: 911-2; Stieler M, et al ., Angew Chemie Int Ed 2013; 52: 11930— 4; Bohm M, et al., J Med Chem 2014; 57: 10355-65.) Whereas all the other enzymes within the coagulation cascade are serine proteases, the FXIII-A subunit belongs to the transglutaminase family (EC 2.3.2.13: protein-glutamine y-glutamyltransferase) consisting of eight human isoenzymes (FXIII-A and TG1-TG7). The most characteristic catalytic function for transglutaminases is the formation of isopeptide bonds between the side chains of susceptible protein bound glutamine and lysine residues in a tightly controlled manner.

FXIII plays a key role in clot formation, maturation and composition (Muszbek L,et al., Physiol Rev 2011 ; 91 : 931-72; Byrnes JR, et al. Blood 2015, 126, 1940-8).

FXIII recognizes fibrin as substrate and covalently cross-links fibrin g-chains and, in an ordered sequence, fibrin a-chains providing mechanical stability to the fibrin fibers. In parallel, enzymatic incorporation of anti-fibrinolytic proteins such as a 2 -antiplasmin renders the clot biochemically stable. In blood, the non-covalent FXIII-A2B2 heterotetramer (pFXIII) is bound to fibrinogen. During activation thrombin cleaves the N-terminal activation peptide from the FXIII-A subunits. Subsequent binding of calcium ions promotes dissociation of the carrier B-subunits yielding active FXIIIa.

The clot-modulating function and the positioning downstream of thrombin make FXIII a promising target for drug development (Lorand L, Arterioscler Thromb. Vase. Biol., 2000; 20: 2-9.). Specific inhibitors targeting FXIII would not interfere with thrombin generation, fibrin formation or with platelet activation. Blocking thrombin - directly or via upstream FXa - by the currently available anticoagulants is characterized by an enhanced bleeding risk, thus excluding many patients from beneficial treatment (Griffin JH, Nature, 1995; 378: 337-8.). Considering this well-known medical need, novel therapeutic approaches with minimal or no bleeding risk are desperately needed (Weitz

J I, Fredenburgh JC., Front Med 2017; 4.). Even if the development of FXIII inhibitors might provide one such option, noticeably few FXIII-blockers have been identified or synthesized so far.

Finney et al. reported that the 66 amino acid polypeptide“tridegin” from the salivary gland of the giant Amazon leech Haementeria ghilianii is a potent FXIII inhibitor (Finney S, et al., Biochem J, 1997; 324 ( Pt 3: 797-805.). Further, in the late 1980s, a series of small molecules irreversibly inhibiting FXIIIa were explored in animal models of thrombosis in the presence of t-PA facilitating increased clot lysis in vivo (Leidy EM, et al., Thromb Res, 1990; 59: 15-26; Shebuski RJ, et al., Blood 1990; 75: 1455-9.). Due to the lack of selectivity and potency along with short plasma half-lives of only a few minutes, these inhibitors were solely considered as pharmacological tools but not as prospective drug candidates. The pharmacokinetic profile of an irreversibly acting inhibitor carrying a thiadiazole warhead was studied in rabbits in order to support and facilitate the design and selection of drug candidates (Novakovic J, et al., J Pharm Biomed Anal 2005; 38: 293-7.). Further, medicinal chemists reported cyclopropenone derivatives from fungi and synthetic analogues as potent FXIIIa inhibitors (Kogen FI, et al., J. Am. Chem. Soc. 2000, 122, 1842-1843.). In both cases, from a drug discovery perspective, the low potency of the compounds disqualifies them for further development. In accordance with this assumption no (pre)clinical studies have been reported.

Potent, drug-like FXI I l-inhibitors are a prerequisite that is still lacking for further exploring the therapeutic concept.

The objective of the present invention is to provide novel, most probably irreversible inhibitors of blood coagulation factor XIII and methods for the synthesis therof as well as their use for the prophylaxis and treatment of diseases associated with blood coagulation factor XIII.

Said objective is solved by the technical teachings of the independent claims. Further advantageous embodiments, aspects and details of the invention are evident from the dependent claims, the description and the examples.

Surprisingly, it was found that certain peptidomimetic inhibitors carrying a Michael- acceptor warhead are potent and selective inhibitors of FXIIIa. The patent application discloses preferred structures characterized by the position 2 towards the C-terminus of the warhead. It was found that exactly at that position constrained peptide-mimetic amino acids are preferred to obtain potent and/or selective FXI I la-blockers. The peptide-mimetic backbone positions the soft electrophilic warhead in perfect orientation to the active site cysteine. The mechanism-based inhibitors efficiently inactivate FXIIIa even if the intrinsic reactivity of the Michael-acceptor warhead is low - a prerequisite to avoid side effects and toxicity.

Thus, the present invention relates to a compound of the general formula (I):

wherein

R 1 represents -H, -CH 3 , -C(CH 3 ) 3 , -cyclo-C 3 H 5 , -cyclo-C 4 H 7 , -cyclo-C 5 H 9 ,

-cyclo-CeHn, -CH 2 -CH(CH 3 ) 2 , -CH 2 CH 3J -CH 2 CH 2 CH 3J -CH 2 CH 2 CH 2 CH 3J -CH 2 CH 2 CH 2 CH 2 CH 3J -CH 2 CH 2 CH 2 CH 2 CH 2 CH 3J -CH(CH 3 ) 2J -CH 2 -C(CH 3 ) 3J — CH 2 CH 2 SCH 3J — CH 2 — cyclo-C 3 H5, — CH 2 — cyclo-C 4 H7, — CH 2 — cyclo-CsHg or — CH 2 — cyclo-CeHn;

R 2 represents -A 1 -A 2 -A 3 -E, -A 1 -A 2 -A 3 -A 4 -E or -A 1 -A 2 -A 3 -A 4 -A 5 -E;

R represents

R 4 represents -OR * , -NH 2 , -NHR # or -NR * R # ;

R* and R # represent independently of each other -CH 3 , -CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 CH 2 CH 3J -CH 2 CH(CH 3 ) 2J -C(CH 3 ) 3J -cyclo-C 3 H 5 ,

-cyclo-C 4 H 7 , -cyclo-C 5 H 9 , -cyclo-C 6 Hn, -CH 2 -cyclo-C 3 H 5 , -CH 2 -cyclo-C 4 H 7 , -CH 2 -cyclo-C 5 H 9 , -CHz-cyclo-CeH·,·,, -CH 2 -Ph, -CH 2 OCH 3 , -CH 2 OCH 2 CH 3 , -CH 2 CH 2 OCH 3 , or -CH 2 CH 2 SCH 3 ;

A 2 - A 5 represent independently of each other

L 1 - L 8 represents independently of each other a covalent bond,

R 5 - R 12 and R 15 - R 23 represents independently of each other

-0-cyclo-C 3 H 5 , -OCH 2 -cyclo-C 3 H 5 , -0-C 2 H 4 -cyclo-C 3 H 5 , -CHO, -COCH 3 ,

-COCF 3 , -COC 2 H 5 , -COC 3 H 7J -COCH(CH 3 ) 2J -COC(CH 3 ) 3J

-COOH, -COOCHg, -COOC 2 H 5 , -COOC 3 H 7J -COOCH(CH 3 ) 2J -COOC(CH 3 ) 3J -OOC-CH 3 , -OOC-CF 3 , -OOC-C 2 H 5 , -OOC-C 3 H 7J -OOC-CH(CH 3 ) 2J -OOC-C(CH 3 ) 3J -NH 2 , -NHCH 3 , -NHC 2 H 5 , -NHC 3 H 7J -NHCH(CH 3 ) 2J

-NHC(CH 3 ) 3J -N(CH 3 ) 2J -N(C 2 H 5 ) 2 , -N(C 3 H 7 ) 2J -N[CH(CH 3 ) 2 ] 2J -N[C(CH 3 ) 3 ] 2J

-NHCOCHg, -NHCOCFg, -NHCOC 2 H 5 , -NHCOC 3 H 7 , -NHCOCH(CH 3 ) 2 ,

-NHCOC(CH 3 ) 3 , -CONH 2 , -CONHCH 3 , -CONHC 2 H 5 , -CONHC 3 H 7 ,

-CONHCH(CH 3 ) 2J -CONH-cyclo-C 3 H 5 , -CONHC(CH 3 ) 3 , -CON(CH 3 ) 2 , -CON(C 2 H 5 ) 2 , -CON(C 3 H 7 ) 2J -CON[CH(CH 3 ) 2 ] 2J -CON[C(CH 3 ) 3 ] 2J -SO 2 NH 2 ,

-SO 2 NHCH 3 , -SO 2 NHC 2 H 5 , -S0 2 NHC 3 H 7 , -S0 2 NHCH(CH 3 ) 2 ,

-S0 2 NH-cyclo-C 3 H 5 , -S0 2 NHC(CH 3 ) 3 , -S0 2 N(CH 3 ) 2 , -S0 2 N(C 2 H 5 ) 2 ,

or R 7 and R 8 or R 8 and R 9 form together one of the following ring moieties; or E/Z-isomer, regiomer, diastereomer, enantiomer, a mixture of E/Z-isomers, a mixture of regiomers, a mixture of diastereomers, a mixture of enantiomers, prodrug, solvate, hydrate, or pharmaceutically acceptable salts thereof.

Surprisingly it was found that good factor XIII or FXIIIa inhibitors, both terms are used synonymously herein, have to be irreversible inhibitors with certain peptidic or peptidomimetic backbones. Reversible inhibitor have been proven to be less efficient in comparison to the irreversible inhibitors disclosed herein which in addition are very specific for FXIIIa and consequently show a minimum of side effects in case certain structural requirements are fulfilled. Further, irreversible acting inhibitors avoid the potential rebound of FXIII activity. One specific requirement is that the peptidic or peptidomimetic backbone has to have a certain length of at least 5 amino acids including amino acid derivatives and peptidomimetics as disclosed herein (cf. Ref. 6 and Ref. 7). Consequently, is R 2 = -A 1 -A 2 -A 3 -E a pentapentide results and with R 2 = -A 1 -A 2 -A 3 -A 4 -E a hexapeptide and with R 2 = -A 1 -A 2 -A 3 -A 4 -A 5 -E a septapeptide is obtained. Preferred are hexapeptides with R 2 = -A 1 -A 2 -A 3 -A 4 -E as well as septapeptides with -A 1 -A 2 -A 3 -A 4 -A 5 -E. In addition to this required minimum length of the peptidic or peptidomimetic backbone the two amino acids attached c-terminal to the amino acid with the Michael system are very important. In general formula (I) the amino acid attached c-terminal to the Michael system bearing amino acid is drawn as , wherein the substituent R 1 is limited to a small group of alkyl and cycloalkyl residues and methionine. Ionic or polar groups or aromatic groups or quite bulky groups with more than 7, preferably with more than 6 carbon atoms are found to decrease the inhibitor activity drastically. Also the stereochemistry of the substituent R 1 is important as shown in general formual (I). Especially important is the amino acid connected to the amino acid with R 1 which is the second amino acid C-terminal to the amino acid with the Michael acceptor system (warhead). This amino acid or amino acid derivative is referred to herein as A 1 . A 1 must be a cyclic amino acid, namely a proline or proline derivative as disclosed herein. The stereochemistry of A 1 is also very important and must be (S) or L in case of proline and in case of a proline derivative the same spatial orientation as (S)-proline is required (cf. Ref. 4). Although the stereogenic center of the piperidine ring of A 1 in reference example 4 has (S) configuration, it has in comparison to the proline moiety the other spatial orientation and it was found to be inactive as factor XIII inhibitor. Moreover stereogenic center in the proline moiety A 1 must not have a further substituent, i.e. one hydrogen atom must be present at the stereogenic center of the proline moiety (cf. Ref. 5). In the reference example 5 a methyl group is present at the stereogenic center and that makes the compound completely inactive as factor XIII inhibitor. Moreover it has to be stressed that a pyridinone moiety is not a proline derivative and a pyridinone moiety as A 1 leads also to compounds inactive as factor XIII inhibitors (cf. Ref. 1 - Ref. 3). Therefore there is a tight structural relationship (SAR: Structure - Activity - Relationship) between the inhibitory activity in regard to FXIIIa and the structural features of the inhibitors disclosed herein. Such a SAR was not evident from the state of the art and is somehow surprising.

Preferably, the present invention relates to a compound of the general formula (I), wherein

R 1 represents -C(CH 3 ) 3 , -cyclo-C 3 H 5 , -cyclo-C 4 H 7 , -cyclo-C 5 H 9 , -cyclo-C 6 Hn, -CH 2 -CH(CH 3 ) 2 , -CH 2 CH 2 CH 2 CH 3 , -CH 2 CH 2 CH 2 CH 2 CH 3 , -CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 , -CH 2 -C(CH 3 ) 3J -CH 2 CH 2 SCH 3J -CH 2 -cyclo-C 3 H 5 , -CH 2 -cyclo-C 4 H 7 , -CH 2 -cyclo- C 5 H 9 or -CH 2 -cyclo-C 6 Hn;

R 2 represents -A 1 -A 2 -A 3 -E, -A 1 -A 2 -A 3 -A 4 -E or -A 1 -A 2 -A 3 -A 4 -A 5 -E;

R 4 represents -OR * , -NH 2 , -NHR # or -NR * R # ;

R * and R # represent independently of each other -CH 3 -CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 CH 2 CH 3 , -CH 2 CH(CH 3 ) 2 , -C(CH 3 ) 3J -cyclo-C 3 H 5 ,

-cyclo-C 4 H 7 , -cyclo-C 5 H 9 , -cyclo-C 6 Hn, -CH 2 -cyclo-C 3 H 5 , CH 2 -cyclo-C 4 H 7 ,

— CH— cyclo-CsHg, — GH— cyclo-CeH-ii, — GH— Ph, — CH 2 0CH 3 , -CH 2 OCH 2 CH 3 ,

-CH 2 CH 2 OCH 3 ;

A 2 - A 5 represent independently of each other

L 1 - L 8 represents independently of each other a covalent bond,

R 5 - R 12 and R 15 - R 23 represents independently of each other

-0-cyclo-C 3 H 5 , -OCH 2 -cyclo-C 3 H 5 , -0-C 2 H 4 -cyclo-C 3 H 5 , -CHO, -COCH 3 ,

-COCF 3 , -COC 2 H 5 , -COC 3 H 7J -COCH(CH 3 ) 2J -COC(CH 3 ) 3J

-COOH, -COOCH 3 , -COOC 2 H 5 , -COOC 3 H 7J -COOCH(CH 3 ) 2J -COOC(CH 3 ) 3J -OOC-CH 3 , -OOC-CF 3 , -OOC-C 2 H 5 , -OOC-C 3 H 7J -OOC-CH(CH 3 ) 2J -OOC-C(CH 3 ) 3J -NH 2 , -NHCH 3 , -NHC 2 H 5 , -NHC 3 H 7J -NHCH(CH 3 ) 2J

-NHC(CH 3 ) 3J -N(CH 3 ) 2J -N(C 2 H 5 ) 2 , -N(C 3 H 7 ) 2J -N[CH(CH 3 ) 2 ] 2J -N[C(CH 3 ) 3 ] 2J

-NHCOCH 3 , -NHCOCF 3 , -NHCOC 2 H 5 , -NHCOC 3 H 7J -NHCOCH(CH 3 ) 2J -NHCOC(CH 3 ) 3J -CONH 2 , -CONHCH 3 , -CONHC 2 H 5 , -CONHC 3 H 7 ,

-CONHCH(CH 3 ) 2J -CONH-cyclo-C 3 H 5 , -CONHC(CH 3 ) 3 , -CON(CH 3 ) 2 ,

-CON(C 2 H 5 ) 2 , -CON(C 3 H 7 ) 2J -CON[CH(CH 3 ) 2 ] 2J -CON[C(CH 3 ) 3 ] 2J -SO 2 NH 2 ,

-SO 2 NHCH 3 , -SO 2 NHC 2 H 5 , -S0 2 NHC 3 H 7 , -S0 2 NHCH(CH 3 ) 2 ,

-S0 2 NH-cyclo-C 3 H 5 , -S0 2 NHC(CH 3 ) 3 , -S0 2 N(CH 3 ) 2 , -S0 2 N(C 2 H 5 ) 2 ,

-S0 2 N(C 3 H 7 ) 2 , -S0 2 N[CH(CH 3 ) 2 ] 2 , -S0 2 N[C(CH 3 ) 3 ] 2 , -NHSO 2 CH 3 ,

-NHSO 2 CF 3 , -NHSO 2 C 2 H 5 , -NHS0 2 C 3 H 7 , -NHS0 2 CH(CH 3 ) 2 ,

-NHS0 2 C(CH 3 ) 3 , -Ph, -O-Ph, or -0-CH 2 -Ph; or E/Z-isomer, diastereomer, enantiomer, a mixture of E/Z-isomers, a mixture of diastereomers, a mixture of enantiomers, prodrug, solvate, hydrate, or pharmaceutically acceptable salts thereof.

For all general formula disclosed herein unless otherwise defined, the amino acid residues A 2 - A 5 preferably represent independently of each other

Preferably, R 1 represents -C(CH 3 ) 3 , -cyclo-C 3 H 5 , -cyclo-C 4 H 7 , -cyclo-C 5 H 9 , -cyclo-CeHn, -CH 2 CH 2 CH 3 , -CH 2 CH 2 CH 2 CH 3J -CH 2 CH 2 CH 2 CH 2 CH 3J -CH 2 CH 2 CH 2 CH 2 CH 2 CH 3J -CH 2 -C(CH 3 ) 3J -CH 2 -cyclo-C 3 H 5 , -CH 2 -cyclo-C 4 H 7 , -CH 2 -cyclo-C 5 H 9 , -CH 2 -cyclo-C 6 Hn, -CH 2 CH 2 SCH 3 ; more preferably, R 1 represents -C(CH 3 ) 3 , -cyclo-C 3 H 5 , -CH 2 CH 2 CH 2 CH 3 , -CH 2 -C(CH 3 ) 3 , or

-CH 2 -cyclo-C 3 H 5 ; and most preferably R 1 represents -C(CH 3 ) 3 , -CH 2 CH 2 CH 2 CH 3 or -CH 2 -cyclo-C 3 H 5 .

Therefore, preferred are compounds of any one of the formulae (11-1 ) to (II-3):

wherein R 2 , R 3 , and R 4 have the same meanings as defined in formula (I).

Moreover, in any one of the formulae (I), and (11-1) - (II-3), A 1 is preferably selected from

and/or A 2 is preferably selected from:

More preferably, in any one of the formulae (I), and (11-1) - (II-3), A 1 represents:

More preferably, in any one of the formulae (I), and (11-1) - (II-3), A 2 represents

Most preferably, in any one of the formulae (I), and (11-1) - (II-3), A represents

Also preferred is that in the formulae (I), (11-1) - (II-3) the residue -A 1 -A 2 - represents

Preferably, in any one of the formulae (I), and (11-1 ) - (II-3), A 3 represents

More preferably, in any one of the formulae (I), and (11-1 ) - (II-3), -A 2 -A 3 - represents

Preferably, in any one of the formulae (I), and (11-1) - (II-3), R 3 represents

and R 7 , R 8 , and R 9 have the same meanings as defined herein or in formula (I),

more preferably, R 3 represents

More preferably, the present invention is directed to a compound of any one of the formulae (MI-1 ) and (III-2):

wherein

E represents

R 3 , represents

A 1 , A 3 , A 4 , A 5 , R 4 , R 7 , R 8 , R 9 and E have the same meanings as defined herein. Still more preferably, the present invention is directed to a compound of any one of the formulae (IV-1) - (IV-4):

wherein

E 2 represents -E, -A 3 -E, -A 3 -A 4 -E, or -A 3 -A 4 -A 5 -E;

R 4 represents -OCH 3 or -OC 2 H 5 ;

R 7 , R 8 , and R 9 represent independently of each other -H, -Cl, -OH, -N0 2 or -C0 2 H; and A 1 , A 3 , A 4 , A 5 , and E have the same meanings as defined herein.

Still more preferred are compounds of any one of the formulae (V-1) and (V-2):

wherein

E 2 represents -E, -A 3 -E, -A 3 -A 4 -E, or -A 3 -A 4 -A 5 -E;

R 4 represents -OCH 3 or -OC 2 H 5 ;

A 1 represents

Still more preferred is a compound of a formula (VI):

A 1 , A 4 , A 5 , R 4 , R 7 , R 8 , R 9 and E have the same meanings as defined herein or more preferably as defined in formula (V-1).

Preferably, in any general formula disclosed herein, R 3 is selected from the group consisting of:

and more preferably R 3 prepresents

Preferably, R 4 , R 5 and R 6 represent independently of each other: -H, -F, -Cl, -Br, -I, -CH 3> -CH2CH3, -CH(CH 3 ) 2 , -cyclo-CaHs, -OCH 3 , -CF 3> -OCF 3 , -OH, -CN, -COCH 3 , -CO2H, -C0 2 Me, -OCOCH 3 , -NH 2 , -NHCH 3 , -N(CH 3 ) 2> -NHCOCH 3 , -NHCOCF 3 , -NHS0 2 CH 3 , -NHS0 2 CF 3J -SCH 3J -S0 2 CH 3J -S0 2 CF 3J -S0 2 NH 2J -S0 2 NHCH 3J or -S0 2 N(CH 3 ) 2 ;

Preferably, E is selected from a C terminal group consisting of:

Also preferred are the following compounds selected from the group consisting of:

wo 2019/201432

32

PCT/EP2018/059798

A further aspect of the present invention relates to the production of compound of the formula (I). As shown in Scheme 1 , a method for producing the compound of the present invention comprises:

Step (0): providing a protected amino acid la

la;

Step 1 : (a) performing coupling reaction of the protected amino acid la with an amino acid building block lla lla;

(b) deprotecting an amino protecting group PG 2 of a resulting compound after Step (a); to obtain an intermediate compound 3a Ilia;

Step 2: (a) performing coupling reaction of the intermediate compound Ilia of Step 1 with a corresponding C-terminal amino acid building block H 2 -A'-OPG 3 ;

(b) deprotecting the protecting group PG 3 of a resulting compound after Step (a); (c) repeating the steps (a) and (b) / times, wherein / is 1 -5,

to obtain an intermediate compound IVa

Step 3: performing coupling reaction of the intermediate compound IVa of Step 2 with a corresponding C-terminal building block H-E;

to obtain an intermediate compound Va

Step 4: (a) deprotecting the protecting group PG 1 of the intermediate compound Va;

(b) performing coupling reaction of a resulting compound after Step (a) with a N- terminal building block R 3 -C0 2 H to produce the compound of the formula (I).

Scheme 1

Ste 1

Step 2 repeating the following steps:

i) coupling reaction with C-terminal amino acid building blocks H 2 A'-OPG 3 i times

(i =1 - 5) ii) deprotection of PG 3

Step 3

coupling reation with

C i l b ildi bl k

Step 4

i) deprotection of PG 1

ii) coupling reaction

with N-terminal building block

R 3 -C0 2 H

As shown in Scheme 2, an alternative method for producing the compound of the present invention comprises: Step (0): providing a protected amino acid Mb

Step 1A: (a) performing coupling reaction of the protected amino acid Mb with a N- terminal building block R 3 -C0 2 H,

(b) deprotecting an amino protecting group PG 4 of a resulting compound after Step (a), to obtain an intermediate compound Mb

Step 2A: (a) performing coupling reaction of the intermediate compound Mb with an amino acid building block Ma

(b) deprotecting an amino protecting group PG 2 of a resulting compound after Step (a); to obtain an intermediate compound Mlb

Step 3A: (a) performing coupling reaction of the intermediate compound Mlb with a corresponding C-terminal amino acid building block H 2 -A'-OPG 3 ;

(b) deprotecting the protecting group PG 3 of a resulting compound after Step (a);

(c) repeating the steps (a) and (b) / times, wherein / is 1 -5,

to obtain an intermediate compound IVb

Step 4A: performing coupling reaction of the intermediate compound IVb with a corresponding C-terminal building block H-E to produce the compound of the formula (I)·

Scheme 2

Step 1A

i) coupling reactionwith

N-terminal building block

R 3 -C0 2 H

ii) deprotection of PG 4

Step 2A

i) coupling reaction with amino acid building

Step 3A

repeating the following steps: O lla i) coupling reaction with C-terminal

ii) deprotection of PG 2 amino acid building blocks H 2 A'-OPG 3

G

As shown in Scheme 3, an alternative method for producing the compound of the present invention comprises:

Step (OB): providing a resin, suitable for solid-phase peptide synthesis (SPPS).

SPPS Q

resin

Step (1 B) (a): performing coupling reaction of the corresponding C-terminal amino acid buliding block PG4NH-A'-OH.

(b) deprotecting the protecting group PG4 of a resulting compound after Step (a);

(c) repeating the steps (a) and (b) / times, wherein / is 1 -5,

to obtain a resin bound intermediate compound (lllc).

PG 4 H-A 1 — A 2 — Q

lllc

Step (2B):cleavage from resin and deprotecting the protecting group PG4 to obtain an intermediate compound (llld).

H 2— A 1 — A 2 - A'-E

llld

Step (3B): performing coupling reaction of the intermediate compound llld with an amino acid building block AO

to obtain a compound IVc.

Step (4B): deprotecting the protecting group PG 5 and subsequent coupling reaction with protected amino acid la

la; to obtain compound Vc

Step (5B): deprotecting the protecting group PG 1 and subsequent coupling reaction with a N-terminal building block R 3 -C0 2 H to produce the compound of the formula (I). Scheme 3 repeating the following steps:

i) coupling reaction with C-terminal

amino acid building blocks PG 4 NH-A'-OH

SPPS O PG 4 H-A 1 — A 2 - A'— Q

ii) deprotection of PG 4

resin

lllc

i times

(i =1 - 5) Cleavage from resin

li ti ith 2

H AO

As shown in Scheme 4, an alternative method for producing the compound of the present invention comprises:

Step (0C): providing a N-deprotected C-terminal building block H-E or H-A'-E. Step (1C): (a) performing coupling reaction of the corresponding C-terminal amino acid buliding block PG 4 NH-A i -OH or PG 4 NH-A M -OH.

(b) deprotecting the protecting group PG 4 of a resulting compound after Step (a);

(c) repeating the steps (a) and (b) / times, wherein / is 1 -5 or 1 -4,

to obtain intermediate compound (llld).

Step (3B): performing coupling reaction of the intermediate compound llld with an amino acid building block AO

to obtain a compound IVc.

Step (4B): deprotecting the protecting group PG 5 and subsequent coupling reaction with protected amino acid la

la; to obtain compound Vc

Step (5B): deprotecting the protecting group PG 1 and subsequent coupling reaction with a N-terminal building block R 3 -C0 2 H to produce the compound of the formula (I). Scheme 4

H-E i times repeating th

i) coupling reaction with C-terminal H -A 1 — A : 2 - A D i'— E amino acid

ii) deprotec

H-A'-E

(i =1 - 4) R 1

coupling reaction

PG: A CO 2 H with amino acid building block

AO

i) deprotection of PG'

ii) coupling reaction

with protected

The above-described methods in schemes 3 and 4 may be also combined as follows: A method for producing the compound of the present invention comprises:

Step (OB): providing a resin, suitable for solid-phase peptide synthesis (SPPS).

SPPS

resin Step (1 B) (a): performing coupling reaction of the corresponding C-terminal amino acid buliding block PG4NH-A-OH.

(b) deprotecting the protecting group PG4 of a resulting compound after Step (a);

(c) repeating the steps (a) and (b) / times, wherein / is 1 -5,

to obtain a resin bound intermediate compound (lllc).

lllc

Step (2B):cleavage from resin and deprotecting the protecting group PG4 to obtain an intermediate compound (llld).

H 2— A 1 — A 2 - A'-E

Mid

Step (3B): performing coupling reaction of the intermediate compound llld with an amino acid building block AO

to obtain a compound IVc.

Step (4B): deprotecting the protecting group PG 5 and subsequent coupling reaction with protected amino acid la

la; to obtain compound Vc

Step (5B): deprotecting the protecting group PG 1 and subsequent coupling reaction with a N-terminal building block R 3 -C0 2 H to produce the compound of the formula (I);

Or Step (0C): providing a N-deprotected C-terminal building block H-E or H-A'-E.

Step (1C): (a) performing coupling reaction of the corresponding C-terminal amino acid buliding block PG 4 NH-A-OH or PG 4 NH-A M -OH.

(b) deprotecting the protecting group PG 4 of a resulting compound after Step (a);

(c) repeating the steps (a) and (b) / times, wherein / is 1 -5 or 1 -4,

to obtain intermediate compound (llld).

Step (3B): performing coupling reaction of the intermediate compound llld with an amino acid building block A0

R 1

PG 'N CO 2 H

H A0

to obtain a compound IVc.

Step (4B): deprotecting the protecting group PG 5 and subsequent coupling reaction with protected amino acid la

la; to obtain compound Vc

Step (5B): deprotecting the protecting group PG 1 and subsequent coupling reaction with a N-terminal building block R 3 -C0 2 H to produce the compound of the formula (I).

Herein, A' represenst one of A 1 , A 2 , A 3 , A 4 , and A 5 . H 2 -A'-OPG 3 means amino acid having A' (one of A 1 - A 5 ) backbone and unprotected free amino (H 2 N-) group and carboxyl moiety protected by PG 3 group. The term“protecting groups” as used herein refers to commonly used protection groups in organic synthesis, preferably for amino and carboxyl groups. PG 1 and PG 5 are suitable protecting group for amino group. PG 2 , PG 3 and PG 4 are suitable protecting groups for carboxyl groups. Prerfably, PG 1 may be selected from the group consisting of or comprising: acetyl, benzoyl, benzyloxycarbonyl (Cbz), tert-butylcarbonyl, tert- butyloxycarbonyl (Boc), and fluorenylmethylenoxy group (Fmoc). PG 2 , PG 3 and PG 4 may be selected from the group consisting of or comprising: methoxy, ethoxy, isobutoxy, tert-butoxy, benzyloxy; preferably, tert-butoxy group. For the coupling reaction performed in Steps 1 -4, 1A - 4A, 1 B, 1 C, 3B - 5B, the coupling reaction as used herein refers to commonly used in peptide synthesis. For perfoming the coupling reaction, firstly carboxylic acid group is activated by introducing an activating group and promote the coupling reaction with amino group of amino acid building block. The activating group of carboxylic acid may be introduced by a separate reaction or in situ reaction. Prerfably, the activating group may be selected from the group consisting of or comprising: halides such as -F, -Br, -Cl, -I, anhydride group such as -OCOCFI3, N-oxy-benzotriazol group and N-oxy-succinimide. Preferably, the activating group is introduced in situ and it is well-known in peptide chemistry. Any of the following coupling reagent can be used to introduce activating group: BOP, PyBOP, AOP, PyAOP, TBTU, EEDQ, Polyphosphoric Acid (PPA), DPPA, HATU, HOBt, HOAt, DCC, EDCI, BOP-CI, TFFH, Brop, PyBrop, and CIP.

The warhear was synthesized following the route shown in Scheme 5.

Scheme 5: Synthesis of Michael acceptor warhead lb

Boc O The pharmaceutically acceptable salts of the compound of the present invention may be formed with organic or inorganic acids or bases. Examples of suitable acids for such acid addition salt formation are hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, oxalic acid, malonic acid, salicylic acid, p- aminosalicylic acid, malic acid, fumaric acid, succinic acid, ascorbic acid, maleic acid, sulfonic acid, phosphonic acid, perchloric acid, nitric acid, formic acid, propionic acid, gluconic acid, lactic acid, tartaric acid, hydroxymaleic acid, pyruvic acid, phenylacetic acid, benzoic acid, p-aminobenzoic acid, p-hydroxybenzoic acid, methanesulfonic acid, ethanesulfonic acid, nitrous acid, hydroxyethanesulfonic acid, ethylenesulfonic acid, p- toluenesulfonic acid, naphthylsulfonic acid, sulfanilic acid, camphorsulfonic acid, china acid, mandelic acid, o-methylmandelic acid, hydrogen-benzenesulfonic acid, picric acid, adipic acid, d-o-tolyltartaric acid, tartronic acid, (o, m, p)-toluic acid, naphthylamine sulfonic acid, trifluoroacetic acid, and other mineral or carboxylic acids well known to those skilled in the art. The salts are prepared by contacting the free base form with a sufficient amount of the desired acid to produce a salt in the conventional manner.

In the case the inventive compounds bear acidic groups, salts could also be formed with inorganic or organic bases. Examples for suitable inorganic or organic bases are, for example, NaOH, KOH, NH 4 OH, tetraalkylammonium hydroxide, lysine or arginine and the like. Salts may be prepared in a conventional manner using methods well known in the art, for example by treatment of a solution of the compound of the general formula (I) with a solution of an acid, selected out of the group mentioned above.

Therefore another aspect of the present invention relates to compound according to the general formula (I) for use as medicament as well as use thereof in medicine. Especially preferred is the use in anticoagulation and as an inhibitor of transglutaminases, in particular factor XIII.

The compounds according to general formula (I) described herein are especially suitable for the treatment and prophylaxis of diseases associated with and/or caused by transglutaminases, in particular factor XIII.

Therefore, another aspect of the present invention is the use of the inventive compounds of the general formula (I) for the treatment or prophylaxis of cardiovascular diseases, atherosclerosis, thrombosis, autoimmune diseases, neurodegenerative diseases, fibrotic disorders, dermatological diseases, wound healing and inflammatory diseases. In particular, the compound of any one of the formulae (I), (11-1 )— (II-2), (MI-1 )— (MI-2), (IV- 1)-(IV-4), (V-1)-(V-2) and (VI) is useful for the treatment or prophylaxis of coeliac disease, Duhring-Brocq-disease, gluten ataxia, tissue fibrosis, cystic fibrosis, kidney fibrosis and diabetic nephropathy, liver fibrosis, cataract, ichthyosis, acne, psoriasis, skin aging, candidosis, neurodegenerative disorders including Huntington’s disease, Parkinson’s disease and Alzheimer’s disease as well as atherosclerosis, thrombosis, thrombocytopenia and thrombopreventive indications and for use as anticoagulant in the treatment of sepsis, stroke, recurrent occlusion and acute care setting including acute kidney injury, acute lung injury and acute coronary syndrome.

Preferred potential indications for the compounds of the present invention mainly include thrombopreventive indications in groups of risk patients showing permanent plasmatic clotting activation. These groups include patients suffering from tumour diseases and, first and foremost, patients who need to undergo regular haemodialysis therapy. Preferred is anticoagulation using FXI I l-inhibitors in patients with a high risk for bleeding and/or side-effects like heparin induced thrombocytopenia.

Further indications include older, multimorbid patients suffering from cardiac arrest and/or dysrhythmia, which are frequently associated with discrete vascular clotting diseases as well as progressive chronic renal diseases. Due to the unique mode-of- action, the compounds are preferred anticoagulants in the acute care setting like acute kidney injury, acute lung injury and acute coronary syndrome. Further indications include sepsis, stroke, and recurrent occlusion also in combination with plasminogen activators (e.g. tPA and uPA). However the most important indications are the prevention and treatment of atherosclerosis and thrombosis.

Due to the possible inactivation of other transglutaminase isoenzymes at least of some compounds also treatment or prophylaxis of, coeliac disease, Duhring-Brocq-disease, gluten ataxia, tissue fibrosis, cystic fibrosis, kidney fibrosis and diabetic nephropathy, liver fibrosis, cataract, ichthyosis, acne, psoriasis, skin aging and candidosis is claimed. Further blood brain barrier permeable compounds may be used for the treatment of neurodegenerative disorders including Huntington’s disease, Parkinson’s disease and Alzheimer’s disease.

The term ..transglutaminase dependent diseases" comprises all diseases, dysfunctions or other impairments of the health, which are caused by or in connection with a dysfunction, perturbance or hyperactivity of transglutaminases in the body. Alternatively, it might be of benefit for certain at risk patients to prophylactically block a transglutaminase like FXI 11 e.g. in thrombophilic patients. The particular suitability of the inventive compounds of the general formula (I) is connected to the sterical and electronical properties which result from the molecule structure. The electrophilic warhead group appears to be an essential unit of the irreversible transglutaminase inhibitors, and especially in combination with the certain peptidomimetic backbone with certain kinds of amino acids at defined positions (like the position of the conformational ly constrained proline or the unnatural proline-based amino acids called herein proline derivatives or proline analogues) results in potent transglutaminase inhibitors, especially blood coagulation factor XIII and transglutaminase 2. Selectivity is obtained by implementing said components at selected positions within the backbone.

The pharmaceutical compositions according to the present invention comprise at least one compound according to the present invention. Preferably, the pharmaceutical compositions according to the present invention comprise at least one compound according to the present invention as an active ingredient together with at least one pharmaceutically acceptable (i.e. non-toxic) carrier, excipient and/or diluent.

The Pharmaceutical composition according to the present invention is useful for the treatment or prophylaxis of coeliac disease, Duhring-Brocq-disease, gluten ataxia, tissue fibrosis, cystic fibrosis, kidney fibrosis and diabetic nephropathy, liver fibrosis, cataract, ichthyosis, acne, psoriasis, skin aging, candidosis, neurodegenerative disorders including Huntington’s disease, Parkinson’s disease and Alzheimer’s disease as well as atherosclerosis, thrombosis, thrombocytopenia and thrombopreventive indications and for use as anticoagulant in the treatment of sepsis, stroke, recurrent occlusion and acute care setting including acute kidney injury, acute lung injury and acute coronary syndrome.

The present invention also includes pharmaceutical preparations for parenteral application, including dermal, intradermal, intragastral, intracutaneous, intravasal, intravenous, intramuscular, intraperitoneal, intranasal, intravaginal, intrabuccal, percutan, rectal, subcutaneous, sublingual, topical, or transdermal application, which preparations in addition to typical vehicles and/or diluents contain at least one compound according to the present invention and/or a pharmaceutical acceptable salt thereof as active ingredient.

Liquid form preparations include solutions, suspensions, and emulsions. As an example, there may be mentioned water or water/propylene glycol solutions for parenteral injections or addition of sweeteners and opacifiers for oral solutions, suspensions, and emulsions. Liquid form preparations may also include solutions for intranasal administration. Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be present in combination with a pharmaceutically acceptable carrier such as an inert, compressed gas, e.g. nitrogen.

Description of the Figures

Figure 1 :

A: Gel permeation chromatography of hydrolyzed fibrin-clots. Inhibition of cFXIII by compound 5 (dashed line) during fibrin clotting resulted in a shift of the main peak towards lower molecular weight products compared to control without inhibitor (solid line). Void volume (V 0 ) and total volume (V t ) of the GPC column as well as the apparent molecular mass of the (x)FDPs are indicated.

B: SDS-PAGE and Western blot analysis of human fibrinogen (FGN) and the main peak fractions (xFDPs/FDPs) of the gel permeation chromatography. Samples were analyzed under non-reducing conditions. SDS-PAGE revealed either the most characteristic“D-dimer” band or the“D-domain” degradation products (Fragments D). The monoclonal antibody against the fibrin g-chain showed a pattern similar to the Coomassie staining while the novel “DD-XLink-mab” (Zedira, A076) specifically recognized the isopeptide bond in the D-dimer product.

Figure 2 shows the thromboelastogram of whole human blood spiked with compound 5. Graphs represent concentrations [mM] of 0, 0.63, 2.5, 20 mM from high MCF [mm] to low.

Figure 3: two graphs (A) and (B) show the reduction of maximum clot firmness compared to inhibitor-free control (MCFc) and the increase in clot lysis at 60 minutes compared to control (LI60c).

Figure 4:

A: Experimental schedule of the rabbit model of venous stasis and reperfusion. B.S.: Blood sample; BL: Baseline; TEG: Thromboelastography.

B: The in vivo experiment proofs significantly higher flow rates after compound 5 infusion compared to PBS control animals. Data is depicted as mean ± S.E.M. (n=6-7) C: The area under the curve of flow rate is in accordance to the vein flow rate. In the compound 5 group the mean areas under the curve (AUC) of the jugular flow rate normalized to baseline between time points 35 and 135 minutes are significantly higher. Data is depicted as mean ± S.E.M. (n=6-7).

D: The Thrombus wet weight is significantly reduced in the compound 5 group. Thrombus wet weight was determined after 135 min of infusion. Data is depicted as dot plot with mean ± S.E.M. (n=6-7, p=0.0265).

E: Most importantly the template skin bleeding time is not influenced. Template skin bleeding time was determined after 60 min of infusion. No difference between PBS and compound 5 was observed. Maximal observation time was pre-defined at 300 seconds. Data is depicted as dot plot with mean ± S.E.M. (n=7-8).

Examples

Following abbreviations used in the examples have the following meaning.

DMAP: 4-(Dimethylamino)-pyridine

TEA: Triethylamine

DMF: Dimethylformamide

DIPEA: N-Ethyldiisopropylamine

TFA: Trifluoroacetic acid

EtOAc Ethyl acetate

FIATU 1 -[Bis(dimethylamino)methylene]-1 FI-1 ,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate

PyAOP (7-Azabenzotriazol-1 -yloxy)tripyrrolidinophosphonium

hexafluorophosphate

Chemical Examples

The following examples are intended to illustrate the invention with selected compounds without limiting the protecting scope of the present intellectual property right on these concrete examples. It is clear for a person skilled in the art that analogous compounds and compounds produced according to analogous synthetic ways fall under the protecting scope of the present intellectual property right.

Preparation of compound

(S)-1 -ferf-butyl 5-methyl 2-(ferf-butoxycarbonylamino)pentanedioate

IcM ll dl “(Jl l i lU ld . '-'15G I27 ’6

Exact Mass: 317,18

Molecular Weight: 317,38

12.0 g of Boc-L-Glu-OtBu (39.6 mmol) and 7.09 g of cesium carbonate (21.8 mmol, 0.55 eq) were suspended in 100 ml of DMF and stirred for 1 h at room temperature. 2.47 ml iodomethane (39.6 mmol) we added and the mixture was stirred at room temperature over night. The solvent was evaporated and the residue was dissolved in ethyl acetate and washed with twice with each citric acid solution (10 %), NaFiC0 3 solution (10 %) and brine. The organic phase was dried over Na 2 S0 4 , filtered and the solvent was evaporated. The raw product was used without further purification.

Yield: 13.4 g, >100 %; ESI-MS: 318.3 [M+H] + Preparation of compound

Boc O

(S)-1 -ferf-butyl 5-methyl 2-(bis(fe/'f-butoxycarbonyl)amino)pentanedioate

Chemical Formula: C20H35NO8

Exact Mass: 417,24

Molecular Weight: 417,49

13.4 g of ZED788 (-39,6 mmol) and 986 mg of N,N-dimethyl-4-aminopyridine (DMAP) were dissolved in 30 ml of acetonitrile. 17.6 g of di-tert-butyl bicarbonate (77.1 mmol) in 100 ml of acetonitrile was added and the solution was stirred at room temperature over night. The solvent was evaporated and the residue was dissolved in ethyl acetate and washed with twice with each citric acid solution (10 %), NaHC0 3 solution (10 %) and brine. The organic phase was dried over Na 2 S0 4 , filtered and the solvent was evaporated. The raw product was used without further purification.

Yield: 13.7 g, 83 %

ESI-MS: 418.3 [M+H] +

Preparation of compound ZED721

Boc

Boc O

(S)-ferf-butyl 2-(bis(fe/7-butoxycarbonyl)amino)-5-oxopentanoate

Chemical Formula: C.19H33NO7

Exact Mass: 387,23

Molecular Weight: 387,47

13.7 g of ZED720 (32.8 mmol) were dissolved in 200 ml of dry diethyl ether and cooled to -78°C under argon atmosphere. 36.1 ml of diisobutylaluminum hydride (1 M in hexane) were added dropwise and the solution was stirred for 30 min at -78° C before being quenched with potassium sodium tartrate (Rochelle salt) solution. The organic layer was separated, dried over Na 2 S0 4 , filtered and concentrated to dryness. The raw product was used without further purification. Yield: 13.3 g, >100 %

ESI-MS: 388.3 [M+H] +

Preparation of compound ZED755

Boc O

(S,E)-7-ferf-butyl 1 -methyl 6-(bis(ferf-butoxycarbonyl)amino)hept-2-enedioate

Chemical Formula: C 22 H 37 N0 8

Exact Mass: 443,25

Molecular Weight: 443,53

13.3 g of ZED721 (-32.8 mmol) were dissolved in 60 ml of benzene and 1 1 .2 g of (carbmethoxymethylene)-triphenylphosphorane (1 eq) was added portionwise. After stirring overnight, the solvent was evaporated. The residue was purified by flash chromatography.

Yield: 12.0 g, 83 %

ESI-MS: 444.3 [M+H] +

Preparation of compound lb

(S,E)-2-(ferf-butoxycarbonylamino)-7-methoxy-7-oxohept-5-eno ic acid

Chemical Formula: C 13 H 2i N0 6

Exact Mass: 287,14

Molecular Weight: 287,31

12.0 g of ZED755 (27.1 mmol) are dissolved in 100 ml DCM/TFA (1 :1 ) and stirred at room temperature for 1 h. The solvent was evaporated and the residue was dissolved in 100 ml DMF and 9.23 ml DIPEA (2 eq). 7.15 g of N-(tert- Butoxycarbonyloxy)succinimide were added and the reaction was stirred at room temperature overnight. The solvent was evaporated and the residue was dissolved in ethyl acetate and washed with twice with each citric acid solution (10 %) and brine. The organic phase was dried over Na 2 S0 4 , filtered and the solvent was evaporated. The residue was purified by flash chromatography.

Yield: 5.89 g, 76 %

ESI-MS: 288.3 [M+H] +

Preparation of compound lc

(S,E)-2-(ferf-butoxycarbonylamino)-7-ethoxy-7-oxohept-5-enoi c acid

Chemical Formula: C 14 H 23 N0 6

Exact Mass: 301 ,15

Molecular Weight: 301 ,34

The synthesis of lc was performed according to lb, using (carbethoxymethylene)- triphenylphosphorane in step 4.

Yield: 3.27 g, 59 % (last step)

ESI-MS: 302.3 [M+H] +

Preparation of compound Id

(S,E)-7-amino-2-(ferf-butoxycarbonylamino)-7-oxohept-5-enoic acid

Chemical Formula: C 12 H 2 oN 2 0 5

Exact Mass: 272,14

Molecular Weight: 272,30

The synthesis of Id was performed according to lb, using triphenylphosphonium carbamoylmethylide in step 4.

Yield: 623 mg, 36 % (last step)

ESI-MS: 273.3 [M+H] + Preparation of compound le

(S,E)-2-(ferf-butoxycarbonylamino)-7-(methylamino)-7-oxohept -5-enoic acid

Chemical Formula: C-13H22N2O5

Exact Mass: 286,15

Molecular Weight: 286,32

The synthesis of le was performed according to lb, using N-methyl- triphenylphosphonium carbamoylmethylide in step 4.

Yield: 241 mg, 32 % (last step)

ESI-MS: 287.3 [M+H] +

Preparation of compound

(S)-/V-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)- 1 -cyclohexyl-2-oxoethyl)-4-oxopyrrolidine-2-carboxamide

Chemical Formula: C2-1 H34N4O4

Exact Mass: 406,26

Molecular Weight: 406,52

ZED3478 was synthesized by standard Fmoc solid-phase peptide chemistry (reactions in DMF, coupling with TBTU/FIOBt/DIPEA, deprotection with piperidine) using 0.41 g (0.28 mmol) Rink Amide MBFIA resin as starting material. Coupling of N-alpha-(9- Fluorenylmethyloxycarbonyl)-L-cyclohexylglycine (twice), followed by (S)-N-Boc-4- oxopyrrolidine-2-carboxylic acid led to Boc-protected resin bound“Boc-ZED3478- resin”. Subsequently, ZED3478 was cleaved from the resin (using 95% TFA / 2.5 % water / 2.5 % triisopropylsilane). The solution was reduced and the raw product (TFA salt) was precipitated from diethyl ether.

Yield: 124 mg, 85 %; ESI-MS: 407.3 [M+H] +

Preparation of compound ZED3480

ferf-butyl (S)-1 -((S)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 -cyclohexyl- 2-oxoethylcarbamoyl)-4-oxopyrrolidin-1-yl)-3,3-dimethyl-1-ox obutan-2-ylcarbamate

Chemical Formula: C32H53N5O7

Exact Mass: 619,39

Molecular Weight: 619,79

124 mg (0.24 mmol) of ZED3478 were dissolved in 5 ml DMF. 55.5 mg (1 eq) Boc-L- tert-leucine, 91.3 mg (1 eq) HATU and 81.6 mI (2 eq) DIPEA were added and the reaction was stirred at room temperature overnight. The solvent was evaporated and the residue was dissolved in ethyl acetate and washed with twice with each citric acid solution (10 %), NaHC0 3 solution (10 %) and brine. The organic phase was dried over Na 2 S0 4 , filtered and the solvent was evaporated. The raw product was used without further purification.

Yield: 119 mg, 80 %; ESI-MS: 620.5 [M+H] +

Preparation of compound ZED3481

(S,E)-methyl 7-((S)-1 -((S)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 -cyclohexyl- 2-oxoethylcarbamoyl)-4-oxopyrrolidin-1-yl)-3,3-dimethyl-1-ox obutan-2-ylamino)-6-(ferf- butoxycarbonylamino)-7-oxohept-2-enoate

Chemical Formula: C 40 H 64 N 6 O 10

Exact Mass: 788,47

Molecular Weight: 788,97

119 mg (0.19 mmol) of ZED3480 were dissolved in 6 ml DCM/TFA (1 :1 ) and stirred at room temperature for 1 h. The solvent was evaporated and the residue was dissolved in 5 ml DMF. 54.6 mg (1 eq) lb, 72.2 mg (1 eq) FIATU and 64.6 mI (2 eq) DIPEA were added and the reaction was stirred at room temperature overnight. The solvent was evaporated and the residue was dissolved in ethyl acetate and washed with twice with each citric acid solution (10 %), NaFiC0 3 solution (10 %) and brine. The organic phase was dried over Na 2 S0 4 , filtered and the solvent was evaporated. The raw product was used without further purification.

Yield: 86 mg, 57 %; ESI-MS: 789.6 [M+H] + Example 1-1. Preparation of compounds 1a/b:

2-((S,E)-1 -((S)-1 -((S)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 -cyclohexyl- 2-oxoethylcarbamoyl)-4-oxopyrrolidin-1 -yl)-3,3-dimethyl-1 -oxobutan-2-ylamino)-7- methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)-4/5-nitrobenzoic acid

Chemical Formula: C^HsgNyO·^

Exact Mass: 881 ,42

Molecular Weight: 881 ,97

86 mg (0.11 mmol) of ZED3481 were dissolved in 5 ml DCM/TFA (1 :1 ) and stirred at room temperature for 1 h. The solvent was evaporated and the residue was dissolved in 3 ml DMF. 19.6 mg (1 eq) 4-nitrophthalic anhydride and 37 pi (2 eq) DIPEA were added and the reaction was stirred at room temperature overnight. The solvent was evaporated and the residue was purified by FIPLC.

Yield: 26 mg, 27 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 882.5 [M+H] +

Example 1-2. Preparation of compounds 2a/b:

2-((S,E)-1 -((S)-1 -((S)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 - cyclohexyl-2-oxoethylcarbamoyl)-4-oxopyrrolidin-1 -yl)-3-cyclopropyl-1 - oxopropan-2-ylamino)-7-methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)-4/5- nitrobenzoic acid

Chemical Formula: C43H57N7O-13

Exact Mass: 879,40

Molecular Weight: 879,95

The synthesis of example 1 -2 was performed according to example 1 -1 , using the corresponding amino acids.

Yield: 21 mg, 36 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 880.5 [M+H] + Example 1-3. Preparation of compound 3:

3-((S,E)-1 -((S)-1 -((S)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 -cyclohexyl- 2-oxoethylcarbamoyl)-4-oxopyrrolidin-1-yl)-3,3-dimethyl-1-ox obutan-2-ylamino)-7- methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)pyrazine-2-carboxylic acid

Chemical Formula: C 41 H 58 N 8 0 11

Exact Mass: 838,42

Molecular Weight: 838,95

The synthesis of example 1 -3 was performed according to example 1 -1 , using the corresponding anhydride.

Yield: 39 mg, 49 %

ESI-MS: 839.5 [M+H] +

Example 1-4. Preparation of compound 4:

3-((S,E)-1 -((S)-1 -((S)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 -cyclohexyl- 2-oxoethylcarbamoyl)-4-oxopyrrolidin-1-yl)-3,3-dimethyl-1-ox obutan-2-ylamino)-7- methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)-5-nitrobenzoic acid

Chemical Formula: C43H59N70 1 3

Exact Mass: 881 ,42

Molecular Weight: 881 ,97

The synthesis of example 1 -4 was performed according to example 1 -1 , using the corresponding carboxylic acid.

Yield: 18 mg, 26 %

ESI-MS: 882.5 [M+H] + Example 1 -5. Preparation of compounds 5a/b:

3/4-((S,E)-1 -((S)-1 -((S)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 -cyclohexyl- 2-oxoethylcarbamoyl)-4-oxopyrrolidin-1 -yl)-3,3-dimethyl-1 -oxobutan-2-ylamino)-7- methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)iso/nicotinic acid

Chemical Formula: C42H59N7O-1 -1

Exact Mass: 837,43

Molecular Weight: 837,96

The synthesis of example 1 -5 was performed according to example 1 -1 , using the corresponding anhydride.

Yield: 1.03 g, 48 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 838.6 [M+H] +

Example 1-6. Preparation of compound 6:

(S,E)-methyl 7-((S)-1 -((S)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)- 1 -cyclohexyl-2-oxoethylcarbamoyl)-4-oxopyrrolidin-1 -yl)-3,3-dimethyl-1 - oxobutan-2-ylamino)-6-(6-hydroxy-5-nitronicotinamido)-7-oxoh ept-2-enoate

Chemical Formula: C 41 H5 8 N 8 0 12

Exact Mass: 854,42

Molecular Weight: 854,95

The synthesis of example 1 -6 was performed according to example 1 -1 , using the corresponding carboxylic acid.

Yield: 12 mg, 18 %

ESI-MS: 855.5 [M+H] +

Example 1-7. Preparation of compounds 7a/b:

2-((S,E)-1 -((S)-1 -((S)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 -cyclohexyl- 2-oxoethylcarbamoyl)-4-oxopyrrolidin-1 -yl)-3,3-dimethyl-1 -oxobutan-2-ylamino)-7-ethoxy- 1 ,7-dioxohept-5-en-2-ylcarbamoyl)-4/5-nitrobenzoic acid

Chemical Formula: C 44 H 61 N 7 0 13

Exact Mass: 895,43

Molecular Weight: 895,99

The synthesis of example 1 -7 was performed according to example 1 -1 , using the corresponding building block lc.

Yield: 63 mg, 52 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 896.6 [M+H] +

Example 1-8. Preparation of compounds 8a/b:

3/4-((S,E)-1 -((S)-1 -((S)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 - cyclohexyl-2-oxoethylcarbamoyl)-4-oxopyrrolidin-1 -yl)-3,3-dimethyl-1 -oxobutan-2- ylamino)-7-ethoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)iso/nicotinic acid

Chemical Formula: C^He-i NyO-n

Exact Mass: 851 ,44

Molecular Weight: 851 ,98

The synthesis of example 1 -8 was performed according to example 1 -1 , using the corresponding anhydride and building block lc.

Yield: 46 mg, 49 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 852.6 [M+H] +

Example 1-9. Preparation of compounds 9a/b:

2-((S,E)-1 -((S)-1 -((2S,4R)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 - cyclohexyl-2-oxoethylcarbamoyl)-4-hydroxypyrrolidin-1-yl)-3, 3-dimethyl-1 -oxobutan-2- ylamino)-7-methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)-4/5-nitrobenzoic acid

Chemical Formula: C 4 3H 61 N70 13

Exact Mass: 883,43

Molecular Weight: 883,98

The synthesis of example 1 -9 was performed according to example 1 -1 , using the corresponding amino acids.

Yield: 79 mg, 56 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 884.6 [M+H] +

Example 1-10. Preparation of compounds 10a/b:

2-((S,E)-1 -((S)-1 -((2S,4S)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 - cyclohexyl-2-oxoethylcarbamoyl)-4-phenoxypyrrolidin-1-yl)-3, 3-dimethyl-1-oxobutan-2- ylamino)-7-methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)-4/5-nitrobenzoic acid

Chemical Formula: C49H 6 5N70 13

Exact Mass: 959,46

Molecular Weight: 960,08

The synthesis of example 1 -10 was performed according to example 1 -1 , using the corresponding amino acids.

Yield: 21 mg, 32 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 960.6 [M+H] +

Example 1-11. Preparation of compounds 11a/b:

2-((S,E)-1 -((S)-1 -((2S,4R)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 - cyclohexyl-2-oxoethylcarbamoyl)-4-phenoxypyrrolidin-1-yl)-3, 3-dimethyl-1-oxobutan-2- ylamino)-7-methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)-4/5-nitrobenzoic acid

Chemical Formula: C 49 H 6 5N70 13

Exact Mass: 959,46

Molecular Weight: 960,08

The synthesis of example 1 -11 was performed according to example 1 -1 , using the corresponding amino acids.

Yield: 36 mg, 43 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 960.6 [M+H] +

Example 1-12. Preparation of compounds 12a/b:

2-((S,E)-1-((S)-1-((2S,4R)-2-((S)-2-((S)-2-amino-1-cyclohexy l-2-oxoethylamino)-1- cyclohexyl-2-oxoethylcarbamoyl)-4-(benzyloxy)pyrrolidin-1-yl )-3,3-dimethyl-1-oxobutan- 2-ylamino)-7-methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)-4/5-nitrobenzoic acid

Chemical Formula: C 50 H 6 7N7O 13

Exact Mass: 973,48

Molecular Weight: 974,11

The synthesis of example 1 -12 was performed according to example 1 -1 , using the corresponding amino acids.

Yield: 13 mg, 23 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 974.6 [M+H] +

Example 1-13. Preparation of compounds 13a/b:

2-((S,E)-1 -((S)-1 -((S)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 -cyclohexyl- 2-oxoethylcarbamoyl)azetidin-1 -yl)-3,3-dimethyl-1 -oxobutan-2-ylamino)-7-methoxy-1 ,7- dioxohept-5-en-2-ylcarbamoyl)-4/5-nitrobenzoic acid

Chemical Formula: C42H59N7O-12

Exact Mass: 853,42

Molecular Weight: 853,96

The synthesis of example 1 -13 was performed according to example 1 -1 , using the corresponding amino acids.

Yield: 36 mg, 56 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 854.5 [M+H] +

Example 1-14. Preparation of compounds 14a/b:

2-((S,E)-1 -((S)-1 -((R)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 -cyclohexyl- 2-oxoethylcarbamoyl)piperidin-1 -yl)-3,3-dimethyl-1 -oxobutan-2-ylamino)-7-methoxy-1 ,7- dioxohept-5-en-2-ylcarbamoyl)-4/5-nitrobenzoic acid

Chemical Formula: C 4 4H 6 3N70 1 2

Exact Mass: 881 ,45

Molecular Weight: 882,01

The synthesis of example 1 -14 was performed according to example 1 -1 , using the corresponding amino acids.

Yield: 26 mg, 41 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 882.5 [M+H] +

Example 1-15. Preparation of compounds 15a/b:

3/4-((S,£)-7-amino-1 -((S)-1 -((S)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2- oxoethylamino)-1 -cyclohexyl-2-oxoethylcarbamoyl)-4-oxopyrrolidin-1 -yl)-3,3- dimethyl-1 -oxobutan-2-ylamino)-1 ,7-dioxohept-5-en-2-ylcarbamoyl)iso/nicotinic acid

Chemical Formula: C 41 H 58 N 8 O 10

Exact Mass: 822,43

Molecular Weight: 822,95

The synthesis of example 1 -15 was performed according to example 1 -1 , using the corresponding anhydride and building block Id.

Yield: 18 mg, 26 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 823.6 [M+H] +

Example 1-16. Preparation of compounds 16a/b:

2-((S,E)-1 -((S)-1 -((R)-3-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 -cyclohexyl- 2-oxoethylcarbamoyl)piperidin-1 -yl)-3,3-dimethyl-1 -oxobutan-2-ylamino)-7-methoxy-1 ,7- dioxohept-5-en-2-ylcarbamoyl)-4/5-nitrobenzoic acid

Chemical Formula: C 44 H 63 N70 12

Exact Mass: 881 ,45

Molecular Weight: 882,01

The synthesis of example 1 -16 was performed according to example 1 -1 , using the corresponding amino acids.

Yield: 39 mg, 56 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 882.6 [M+H] +

Example 1-17. Preparation of compounds 17a/b:

3/4-((S,E)-1 -((S)-1 -((S)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 -cyclohexyl- 2-oxoethylcarbamoyl)-4-oxopyrrolidin-1 -yl)-3,3-dimethyl-1 -oxobutan-2-ylamino)-7- (methylamino)-l ,7-dioxohept-5-en-2-ylcarbamoyl)iso/nicotinic acid

Chemical Formula: C 42 H 60 N 8 O 10

Exact Mass: 836,44

Molecular Weight: 836,97

The synthesis of example 1 -17 was performed according to example 1 -1 , using the corresponding anhydride and building block le.

Yield: 9 mg, 23 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 837.6 [M+H] +

Example 1-18. Preparation of compounds 18a/b:

2-((S,E)-1 -((S)-1 -((1 R,2S,5S)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 -cyclohexyl- 2-oxoethylcarbamoyl)-6,6-dimethyl-3-azabicyclo[3.1 .0]hexan-3-yl)-3,3-dimethyl-1 -oxobutan-2- ylamino)-7-methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)-4/5-nitrobenzoic acid

Chemical Formula: C^HesNyO^

Exact Mass: 907,47

Molecular Weight: 908,05

The synthesis of example 1 -18 was performed according to example 1 -1 , using the corresponding amino acids.

Yield: 17 mg, 31 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 908.6 [M+H] +

Example 1-19. Preparation of compounds 19a/b:

2-((2S,E)-1 -((2S)-1 -((2S)-2-((R)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 -cyclohexyl- 2-oxoethylcarbamoyl)octahydro-1 H-indol-1 -yl)-3,3-dimethyl-1 -oxobutan-2-ylamino)-7- methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)-4/5-nitrobenzoic acid

Chemical Formula: C^HeyNyO-ip

Exact Mass: 921 ,48

Molecular Weight: 922,07

The synthesis of example 1 -19 was performed according to example 1 -1 , using the corresponding amino acids.

Yield: 25 mg, 36 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 922.7 [M+H] +

Example 1-20. Preparation of compounds 20a/b:

2-((S,E)-1 -((S)-1 -((2S,4R)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 - cyclohexyl-2-oxoethylcarbamoyl)-4-fluoropyrrolidin-1 -yl)-3,3-dimethyl-1 -oxobutan-2- ylamino)-7-methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)-4/5-nitrobenzoic acid

Chemical Formula: C 43 H 6 oFN70 12

Exact Mass: 885,43

Molecular Weight: 885,97

The synthesis of example 1 -20 was performed according to example 1 -1 , using the corresponding amino acids.

Yield: 12 mg, 29 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 886.6 [M+H] +

Example 1-21. Preparation of compounds 21a/b:

2-((S,E)-1 -((S)-1 -((2S,4S)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 - cyclohexyl-2-oxoethylcarbamoyl)-4-fluoropyrrolidin-1 -yl)-3,3-dimethyl-1 -oxobutan-2- ylamino)-7-methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)-4/5-nitrobenzoic acid

Chemical Formula: C43H 6 oFN70 1 2

Exact Mass: 885,43

Molecular Weight: 885,97

The synthesis of example 1 -21 was performed according to example 1 -1 , using the corresponding amino acids.

Yield: 19 mg, 36 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 886.6 [M+H] +

Example 1-22. Preparation of compounds 22a/b:

2-((S,E)-1 -((S)-1 -((S)-2-((R)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 -cyclohexyl- 2-oxoethylcarbamoyl)-2,5-dihydro-1 /-/-pyrrol-1 -yl)-3,3-dimethyl-1 -oxobutan-2-ylamino)-7- methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)-4/5-nitrobenzoic acid

Chemical Formula: C43H59N7O.12

Exact Mass: 865,42

Molecular Weight: 865,97

The synthesis of example 1 -22 was performed according to example 1 -1 , using the corresponding amino acids.

Yield: 36 mg, 53 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 866.6 [M+H] +

Example 1-23. Preparation of compounds 23a/b:

2-((S,E)-1 -((S)-T((S)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 -cyclohexyl- 2-oxoethylcarbamoyl)-4,4-difluoropyrrolidin-1-yl)-3,3-dimeth yl-1-oxobutan-2-ylamino)-7- methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)-4/5-nitrobenzoic acid

Chemical Formula: C^HsgFpNyO-ip

Exact Mass: 903,42

Molecular Weight: 903,97

The synthesis of example 1 -23 was performed according to example 1 -1 , using the corresponding amino acids.

Yield: 14 mg, 26 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 904.5 [M+H] +

Example 1-24. Preparation of compounds 24a/b:

2-((S,E)-1 -((S)-1 -((S)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 - cyclohexyl-2-oxoethylcarbamoyl)-4-methylenepyrrolidin-1-yl)- 3,3-dimethyl-1-oxobutan- 2-ylamino)-7-methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)-4/5-nitrobenzoic acid

Chemical Formula: C 44 H 61 N 7 0 12

Exact Mass: 879,44

Molecular Weight: 879,99

The synthesis of example 1 -24 was performed according to example 1 -1 , using the corresponding amino acids.

Yield: 23 mg, 32 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 880.6 [M+H] +

Example 1-25. Preparation of compounds 25a/b:

2-((S,E)-1 -((S)-1 -((R)-3-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 - cyclohexyl-2-oxoethylcarbamoyl)morpholino)-3,3-dimethyl-1 -oxobutan-2-ylamino)- 7-methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)-4/5-nitrobenzoic acid

Chemical Formula: C 43 H 61 N70 13

Exact Mass: 883,43

Molecular Weight: 883,98

The synthesis of example 1 -25 was performed according to example 1 -1 , using the corresponding amino acids.

Yield: 39 mg, 53 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 884.6 [M+H] +

Example 1-26. Preparation of compounds 26a/b:

2-((S,E)-1 -((S)-1 -((S)-4-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 -cyclohexyl- 2-oxoethylcarbamoyl)oxazolidin-3-yl)-3,3-dimethyl-1 -oxobutan-2-ylamino)-7-methoxy-1 ,7- dioxohept-5-en-2-ylcarbamoyl)-4/5-nitrobenzoic acid

Chemical Formula: C 42 H 59 N 7 0 13

Exact Mass: 869,42

Molecular Weight: 869,96

The synthesis of example 1 -26 was performed according to example 1 -1 , using the corresponding amino acids.

Yield: 56 mg, 59 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 870.5 [M+H] +

Example 1-27. Preparation of compound 27:

5-((S,E)-1 -((S)-1 -((S)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 - cyclohexyl-2-oxoethylcarbamoyl)pyrrolidin-1 -yl)-3,3-dimethyl-1 -oxobutan-2- ylamino)-7-methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)nicotinic acid

Chemical Formula: C 42 H 61 N7O 10

Exact Mass: 823,45

Molecular Weight: 823,97

The synthesis of example 1 -27 was performed according to example 1 -1 , using the corresponding carboxylic acid and amino acids.

Yield: 79 mg, 52 %

ESI-MS: 824.6 [M+H] +

Example 1-28. Preparation of compound 28:

(S,E)-methyl 7-((S)-1 -((1 S,3aR,6aS)-1 -((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)- 1 -cyclohexyl-2-oxoethylcarbamoyl)hexahydrocyclopenta[c]pyrrol -2(1 /-/)-yl)-3,3-dimethyl-1 - oxobutan-2-ylamino)-6-(6-hydroxy-5-nitronicotinamido)-7-oxoh ept-2-enoate

Chemical Formula: C 44 H 64 N 8 0- | -i

Exact Mass: 880,47

Molecular Weight: 881 ,03

The synthesis of example 1 -28 was performed according to example 1 -1 , using the corresponding carboxylic acid and amino acids.

Yield: 26 mg, 32 %

ESI-MS: 881.6 [M+H] +

Example 1-29. Preparation of compound 29:

2-((S,E)-1 -((S)-1 -((1 R,2S,5S)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 - cyclohexyl-2-oxoethylcarbamoyl)-6,6-dimethyl-3-azabicyclo[3. 1 0]hexan-3-yl)-3,3-dimethyl-1 - oxobutan-2-ylamino)-7-methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)-4,5-dichlorobenzoic acid

Chemical Formula: C4 6 H 64 CI 2 N 6 O 10

Exact Mass: 930,41

Molecular Weight: 931 ,94

The synthesis of example 1 -29 was performed according to example 1 -1 , using the corresponding anhydride and amino acids.

Yield: 15 mg, 27 %

ESI-MS: 931.6 [M+H] +

Example 1-30. Preparation of compound 30:

3-((2S,£)-1 -((2S)-1 -((2S)-2-((2S)-1 -((2S)-2-(1 -(2,6-dimethylphenoxy)propan-2-ylcarbamoyl)-2- methylpyrrolidin-1 -yl)-4-methyl-1 -oxopentan-2-ylcarbamoyl)-4-oxopyrrolidin-1 -yl)-3, 3-dimethyl- 1 -oxobutan-2-ylamino)-7-methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)-5-nitrobenzoic acid

Chemical Formula: CsoHgyNyO^

Exact Mass: 989,47

Molecular Weight: 990,1 1

The synthesis of example 1 -30 was performed according to scheme 4, using the corresponding carboxylic acid, amine (H-E) and amino acids.

Yield: 36 mg, 52 %

ESI-MS: 990.7 [M+H] +

Example 1-31. Preparation of compounds 31a/b:

3/4-((S,£)-1 -((S)-1 -((S)-2-((S)-1 -cyclohexyl-2-((S)-2-((R)-1 -(2,6-dimethylphenoxy)propan-2- ylcarbamoyl)-2-methylpyrrolidin-1 -yl)-2-oxoethylcarbamoyl)-4-oxopyrrolidin-1 -yl)-3, 3-dimethyl- 1 -oxobutan-2-ylamino)-7-methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl) iso/nicotinic acid

Chemical Formula: C 51 H 6 9N70 12

Exact Mass: 971 ,50

Molecular Weight: 972,13

The synthesis of example 1 -31 was performed according to scheme 4, using the corresponding anhydride, amine (H-E) and amino acids.

Yield: 26 mg, 48 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 972.7 [M+H] +

Example 1-32. Preparation of compounds 32a/b:

3/4-((S,E)-1 -((S)-1 -((S)-2-((S)-2-((S)-2-((S)-1 -((S)-2-carbamoyl-2-methylpyrrolidin-1 -yl)-3-(1 H-indol-3-yl)-1 - oxopropan-2-ylcarbamoyl)-2-methylpyrrolidin-1 -yl)-1 -cyclohexyl-2-oxoethylcarbamoyl)-4-oxopyrrolidin-1 -yl)- 3,3-dimethyl-1 -oxobutan-2-ylamino)-7-methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)iso/nicotinic acid

Chemical Formula: C57H74N 10O-13

Exact Mass: 1 106,54

Molecular Weight: 1 107,26

The synthesis of example 1 -32 was performed according to example 1 -1 , using the corresponding anhydride and amino acids.

Yield: 56 mg, 53 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 1107.8 [M+H] + Example 1 -33. Preparation of compounds 33a/b:

2-((S,E)-1 -((S)-1 -((S)-2-(1 -(2-((S)-1 -((S)-2-carbamoylpyrrolidin-1 -yl)-3-(1 H-indol-3-yl)-1 -oxopropan- 2-ylamino)-2-oxoethyl)-2-oxo-1 ,2-dihydropyridin-3-ylcarbamoyl)pyrrolidin-1 -yl)-1 -oxohexan-2- ylamino)-7-methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)-4/5-nitrobenzoic acid

Chemical Formula: C 5 oH 58 N 10 0 14

Exact Mass: 1022,41

Molecular Weight: 1023,05

The synthesis of example 1 -33 was performed according to example 1 -1 , using the corresponding anhydride and amino acids.

Yield: 32 mg, 65 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 1145.5 [M+Na] +

Example 1-34. Preparation of compounds 34a/b:

3/4-((S,E)-1 -((S)-1 -((S)-2-((S)-2-((S)-carboxy(cyclohexyl)methylamino)-1 -cyclohexyl- 2-oxoethylcarbamoyl)-4-oxopyrrolidin-1 -yl)-3,3-dimethyl-1 -oxobutan-2-ylamino)-7- methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)iso/nicotinic acid

Chemical Formula: C 42 H 58 N 6 0 12

Exact Mass: 838,41

Molecular Weight: 838,94

The synthesis of example 1 -34 was performed according to example 1 -1 , using the corresponding anhydride, amino acids and Wang resin.

Yield: 15 mg, 24 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 839.4 [M+H] +

Example 1-35. Preparation of compounds 35a/b:

3/4-((S,E)-1 -((S)-1 -((S)-2-((S)-1 -cyclohexyl-2-((S)-1 -cyclohexyl-2-methoxy-2-oxoethylamino)- 2-oxoethylcarbamoyl)-4-oxopyrrolidin-1 -yl)-3,3-dimethyl-1 -oxobutan-2-ylamino)-7-methoxy- 1 ,7-dioxohept-5-en-2-ylcarbamoyl)iso/nicotinic acid

Chemical Formula: C 43 H 6 oN 6 0 12

Exact Mass: 852,43

Molecular Weight: 852,97

The synthesis of example 1 -35 was performed according to scheme 4, using the corresponding anhydride and amino acids.

Yield: 24 mg, 41 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 853.6 [M+H] +

Example 1 -36. Preparation of compounds 36a/b:

3/4-((S,E)-1 -((S)-1 -((S)-2-((S)-1 -cyclohexyl-2-((S)-1 -cyclohexyl-2-(methylamino)-2- oxoethylamino)-2-oxoethylcarbamoyl)-4-oxopyrrolidin-1 -yl)-3,3-dimethyl-1 -oxobutan- 2-ylamino)-7-methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)iso/nicotinic acid

Chemical Formula: C^He-i NyO-n

Exact Mass: 851 ,44

Molecular Weight: 851 ,98

The synthesis of example 1 -36 was performed according to example 1 -1 , using the corresponding anhydride, amino acids and Methyl Indole AM resin.

Yield: 36 mg, 32 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 852.6 [M+H] +

Example 1-37. Preparation of compounds 37a/b:

4-((S,E)-1 -((S)-1 -((S)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 -cyclohexyl-2- oxoethylcarbamoyl)-4-oxopyrrolidin-1 -yl)-4-(methylthio)-1 -oxobutan-2-ylamino)-7-methoxy- 1 ,7-dioxohept-5-en-2-ylcarbamoyl)nicotinic acid

Chemical Formula: C 41 HsyNyO S

Exact Mass: 855,38

Molecular Weight: 856,00

The synthesis of example 1 -37 was performed according to example 1 -1 , using the corresponding anhydride and amino acids.

Yield: 31 mg, 42 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 856.5 [M+H] +

Reference example 1 (Ref.1):

2-((S,E)-1-(1-(2-((S)-2-((S)-1-((S)-2-carbamoylpyrrolidin-1- yl)-3-(1 /-/-indol-3-yl)-1-oxopropan- 2-ylcarbamoyl)pyrrolidin-1-yl)-2-oxoethyl)-2-oxo-1 ,2-dihydropyridin-3-ylamino)-7-methoxy- 1 ,7-dioxohept-5-en-2-ylcarbamoyl)-4/5-nitrobenzoic acid

Chemical Formula: C 44 H 47 N 9 0- |3

Exact Mass: 909,33

Molecular Weight: 909,90

The synthesis of Ref.1 was performed according to example 1 -1 , using the corresponding anhydride and amino acids.

Yield: 12 mg, 26 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 910.5 [M+H] +

Reference example 2 (Ref .2):

2-((S,E)-1 -((S)-1 -(1 -(2-((S)-2-((S)-1 -((S)-2-carbamoylpyrrolidin-1 -yl)-3-(1 H-indol-3-yl)-1 - oxopropan-2-ylcarbamoyl)pyrrolidin-1 -yl)-2-oxoethyl)-2-oxo-1 ,2-dihydropyridin-3-ylamino)-1 - oxohexan-2-ylamino)-7-methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)-4/5-nitrobenzoic acid

Chemical Formula: C5 0 H 58 N 10 O 14

Exact Mass: 1022,41

Molecular Weight: 1023,05

The synthesis of Ref.2 was performed according to example 1 -1 , using the corresponding anhydride and amino acids.

Yield: 16 mg, 23 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 1023.5 [M+H] +

Reference example 3 (Ref.3)

2-((S,E)-1 -((S)-1 -(1 -(2-((S)-1 -((S)-2-carbamoylpyrrolidin-1 -yl)-3-(1 /-/-indol-3-yl)-1 -oxopropan- 2-ylamino)-2-oxoethyl)-2-oxo-1 ,2-dihydropyridin-3-ylamino)-1 -oxohexan-2-ylamino)-7- methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)-4/5-nitrobenzoic acid

Chemical Formula: C45H5.i N9O.i3

Exact Mass: 925,36

Molecular Weight: 925,94

The synthesis of Ref.3 was performed according to example 1 -1 , using the corresponding anhydride and amino acids.

Yield: 11 mg, 19 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 926.5 [M+H] +

Reference example 4 (Ref .4):

2-((S,E)-1 -((S)-1 -((S)-3-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 -cyclohexyl- 2-oxoethylcarbamoyl)piperidin-1 -yl)-3,3-dimethyl-1 -oxobutan-2-ylamino)-7-methoxy-1 ,7- dioxohept-5-en-2-ylcarbamoyl)-4/5-nitrobenzoic acid

Chemical Formula: C 4 4H 6 3N70 1 2

Exact Mass: 881 ,45

Molecular Weight: 882,01

The synthesis of Ref.4 was performed according to example 1 -1 , using the corresponding anhydride and amino acids.

Yield: 79 mg, 58 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 882.6 [M+H] +

Reference example 5 (Ref.5):

2-((S,E)-1 -((S)-1 -((S)-2-((S)-2-((S)-2-amino-1 -cyclohexyl-2-oxoethylamino)-1 - cyclohexyl-2-oxoethylcarbamoyl)-2-methylpyrrolidin-1 -yl)-1 -oxopentan-2-ylamino)- 7-methoxy-1 ,7-dioxohept-5-en-2-ylcarbamoyl)-4/5-nitrobenzoic acid

Chemical Formula: C 4 3H 61 N70 12

Exact Mass: 867,44

Molecular Weight: 867,98 The synthesis of Ref.5 was performed according to example 1 -1 , using the corresponding anhydride and amino acids.

Yield: 41 mg, 56 %, ratio of regioisomers: approximately 1 :1

ESI-MS: 868.6 [M+H] + Biological Examples

Example 2-1 : FXIII activity assays

A: Isopeptidase assay for estimating inhibitor potency

FXIIIa activity has been determined using substrate A101 (Zedira GmbH, Darmstadt, Germany), which is based on the N-terminal dodecapeptide of a 2 -antiplasmin. FXIIIa catalyzes by its isopeptidase activity the release of dark quencher dinitrophenyl at the original substrate glutamine position resulting in fluorescence increase (based on the N-terminal 2-aminobenzoyl fluorescent dye) (Oertel K, Hunfeld A, Specker E, Reiff C, Seitz R, Pasternack R, Dodt J. A highly sensitive fluorometric assay for determination of human coagulation factor XIII in plasma. Anal Biochem 2007; 367: 152-8.)

Briefly, 12 pl_ recombinant cFXIII-A2 (T027, Zedira GmbH, Darmstadt, Germany) or FXIII-A2B2 derived from human plasma (T007) (25 pg/mL) and 3 pl_ human a-thrombin (0.5 U/mL, T056, Zedira) were mixed with 270 mI_ assay buffer (50 mM Tris-HCI, 10 mM CaCI 2 , 150 mM NaCI, 5.56 mM glycine methyl ester, 5 mM DTT, pH 7.5) containing 55 mM A101 substrate. The mixture was incubated for 20 min at room temperature to activate FXIII. 15 pL of inhibitor solution (serial dilution from 1.25 mM to 1.25 nM) dissolved in DMSO/assay buffer were added, mixed and the kinetic measurement started after 3 min. Fluorescence emission was monitored at 418 nm (l bc = 313 nm) and 37 °C for 30 min using a CLARIOstar fluorescence micro plate reader (BMG Labtech, Ortenberg, Germany). For measurements without inhibitor, 15 pL of assay buffer / 2% (v/v) DMSO were added. All measurements were performed in triplicate. The respective IC 50 values were calculated by non-linear regression using the MARS software package (BMG Labtech).

The inhibition of FXIIIa from animal species was performed accordingly using 36 pg/mL mouse FXIII-A2 (T061 , Zedira), 27 pg/mL rat FXIII-A 2 (T065), 11 pg/mL pig FXIII-A 2 (T066), 32 pg/mL dog FXIII-A 2 (T062), and 22 pg/mL cynomolgus cFXIII-A 2 (T161 ), all produced recombinantly.

B: Transamidation assay for determining inhibitor selectivity

The most relevant off-targets are the transglutaminase isoenzymes especially tissue transglutaminase (TG2) because the enzyme is ubiquitously expressed throughout the human body. To determine selectivity, the fluorescence increase upon transglutaminase-catalyzed incorporation of dansylcadaverine into the universal transglutaminase substrate N,N-dimethylcasein was used (Lorand L, Lockridge OM, Campbell LK, Myhrman R, Bruner-Lorand J. Transamidating enzymes. Anal Biochem 1971 ; 44: 221-31.). Briefly, 15 pL of recombinant transglutaminase enzyme[5] [15 pg/mL hTG1 (T035, Zedira), 69 pg/mL hTG2 (T022), 29 pg/mL hTG6 (T021 ), 18 pg/mL hTG7 (T011 )] were mixed with 270 pL assay buffer containing dansylcadaverine and N,N-dimethyl casein. In the case of FXIII, 12 pL cFXIII (25 pg/mL) and 3 pL human a-thrombin (0.5 U/mL, T056, Zedira) were mixed with 270 pL assay buffer. The mixture was incubated for 20 min at room temperature to activate FXIII. In the case of TG3 78 pg hTG3 (T024) were activated using 14 pg dispase II (Roche, Mannheim, Germany) in the presence of 1.4 mM CaCI 2 and incubated for 30 min at 25°C. The activated hTG3 was subsequently assayed as described above. 15 pL of inhibitor solution dissolved in DMSO/assay buffer were added, mixed and the kinetic measurement started after 3 min. Fluorescence emission was continuously monitored for 30 min at 500 nm (l bc = 330 nm) and 37 °C using the CLARIOstar fluorescence plate reader. All measurements were performed in triplicate. The respective IC 50 values were calculated by non-linear regression using the MARS software package (BMG Labtech, Ortenberg, Germany).

The following tables summarize the inhibition data of compound 5 (=5a/b) against human plasma derived FXIII-A2B2 and the recombinant cellular form (FXIII-A 2 ). In addition, the inhibition of cFXIII from mouse, rat, rabbit, dog, pig and cynomolgus is shown using different assays.

Table 1

Isopeptidase Transamidation

Assay Assay

Transglutaminas

IC 50 [nM] I C50 [n M]

e Species

pFXIII human 10 ± 0.1 4 ± 0.4

cFXIII human 16 ± 0.6 24 ± 1.5

cFXIII mouse 19 ± 0.6 15 ± 1.2

cFXIII rat 8 ± 0.1 17 ± 0.6

cFXIII rabbit 20 ± 1 .0 7 ± 1.7

cFXIII dog 28 ± 0.6 24 ± 0.6

cFXIII pig 365 ± 8.0 16 ± 1.5

cFXIII cynomolgus 15 ± 0.1 19 ± 0.6

Table 2. The selectivity against human transglutaminases iso-enzymes.

Transamidation

Assay Selectivity

Transglutaminas IC 50 [n Ml e

cFXIII 24 ±1.5 1

TG1 11035 ± 1003 463

TG2 445 ±20 19

TG3 66511 ±4544 2791

TG6 17 ±0.6 0.7

TG7 1330 ± 102 56

Table 3 inhibitory activity of the inventive compounds against FXIII and TG2 selectivity for selected compounds

Compounds a/b means 1 to 1 mixutere of two regiomers

Reference compound 6 (Ref.6) disclosed in WO 2008055488 A1 as compound 38.

Reference compound 7 (Ref.7) disclosed in WO 2008055488 A1 as compound 4.1.

Example 2-2: Inhibition of fibrin cross-linking and size exclusion chromatography of fibrin degradation products

For the in vitro preparation of fibrin clots, HSA-free human fibrinogen (2.5 mg/mL, FIB3, Enzyme Research Laboratories, South Bend, IN, USA), diluted in 20 mM Tris-HCI, 300 mM NaCI, pH 7.4, was mixed with cFXIII (10 pg/mL, T027, Zedira), and 5 mM CaCI 2 . Prior to the addition of 50 U human thrombin (T053, Zedira), either DMSO (2.4% v/v) or the inventive compound as inhibitor (10 mM final concentration dissolved in DMSO) were added to the mixture. To allow completion of fibrin cross-linking, incubation was performed at 37°C for 16 h. Subsequently, recombinant human plasmin (0.2 mg/mL, P012, Zedira) was added to the mixture and incubated at 37°C for 1 h to solubilize the fibrin clots. Separation of cross-linked and non-cross-linked fibrin degradation products (xFDPs / FDPs, respectively) was performed using a Sephacryl S-200 column (CV = 120 mL, GE Healthcare, Uppsala, Sweden) equilibrated in 20 mM Tris-HCI, 500 mM NaCI, pH 7.4.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli (Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5.). Briefly, samples were mixed with non-reducing 5 c SDS-PAGE loading buffer (128 mM Tris- HCI at pH 6.8, 30% glycerol, 10% SDS, and 0.05% bromophenol blue), and loaded on a 10% polyacrylamide gel. Separation was performed at 200 V for 40 min. Gels were stained with Coomassie Brilliant Blue R-250. Electro-blotting was performed in a Trans- Blot SD semi-dry transfer cell (Bio-Rad, Hercules, CA, U.S.A.) at 20 V for 80 min. After blotting, the nitrocellulose membranes were pre-soaked in 48 mM Tris, 39 mM glycine, 1.3 mM SDS, and 20% (v/v) methanol. Residual binding sites were blocked in 5% skimmed milk powder in TBS-T [10 mM Tris, 150 mM NaCI, and 0.05% (v/v) Tween 20 at pH 8.0] for 60 min. The membrane was washed in TBS-T wash buffer and incubated for 1 h. with primary antibody (diluted 10,000-fold in TBS-T). After washing for 3 x 5 min in TBS-T, anti-mouse IgG (Sigma-Aldrich, Schnelldorf, Germany) conjugated to alkaline phosphatase and diluted 10,000-fold in TBS-T buffer was added to the membranes followed by 1 h incubation. The membranes were placed in detection reagent (100-fold dilution of AP color reagent in color development buffer, Bio-Rad, Hercules, CA, U.S.A. ). Excess detection reagent was drained off, and the staining reaction was stopped with 20 mM Tris-HCI and 5 mM EDTA at pH 8.0. All steps were performed at room temperature on a shaker (Figure 1 ).

Example 2-3: Thromboelastometry (TEM) Thromboelastometry is a visco-elastic method for the assessment of blood coagulation (Lang T, von Depka M. Possibilities and limitations of thrombelastometry/-graphy. Hamostaseologie 2006; 26: S20-9.). Clotting time (CT), clot formation time (CFT), maximum clot firmness (MCF) and lysis index at 60 min (LI60) were obtained using fresh whole blood in the ROTEM delta device according to the manufacturer’s instructions.

The potency of the inventive compounds as inhibitors (serial dilution covering 20.0 mM to 0.08 mM final concentration) in the presence of 0.02 pg/mL tissue plasminogen activator (t-PA; P016, Zedira) was investigated. Briefly, 20 pL star-TEM ® (0.2 mol/L CaCI 2 ), 20 pL r ex-TEM (recombinant tissue factor, phospholipids, heparin inhibitor), 10 pL inhibitor stock solution (720 pM - 2.88 pM), combined with 10 pL t-PA stock solution (0.72 pg/mL) to yield concentrations of 360 pM - 1.44 pM in 7.2% DMSO/PBS with 0.36 pg/mL t-PA and 300 pL fresh citrated whole blood (from healthy consenting donors) were mixed in a disposable cuvette. As a control the inhibitor stock solution was replaced by 3.6 % DMSO / 0.36 pg/mL t-PA in PBS.

The figure 2 shows the thromboelastogram of whole human blood spiked with compound 5 at serial dilution 20 pM - 0.63 pM compared to control in the presence of 0.02% t-PA

In Figure 3, two graphs (A) and (B) show the reduction of maximum clot firmness compared to inhibitor-free control (MCFc) and the increase in clot lysis at 60 minutes compared to control (LI60c).

The inhibitor has no influence on the clotting time (CT) indicating that the compound does not interfere with other coagulation factors leaving the coagulation cascade, the fibrin formation, and the platelet activation untapped.

Example 2-4: anti-coagulation in a rabbit model of venous stasis and reperfusion

Purpose bred animals were identified upon arrival in the test facility according to the respective guidelines. Male New Zealand White rabbits (2-3 kg) were anesthetized for the duration of the procedure. The rabbit’s right jugular vein was exposed and any collateral veins to the venous stasis segment were ligated. In order to prevent embolization, approximately 4 cm of a 10 cm long polyester suture thread was inserted from upstream into the lumen of the designated stasis segment prior to the ligations to allow thrombus formation around the thread. An ultrasound probe was placed perivascularly on the right jugular vein downstream to the venous stasis segment, and blood flow was recorded continuously (3 mm probe, Transonic Systems Inc, Ithaca, USA). Blood samples were taken from the right femoral artery (1 mL in 150 mM sodium citrate) as shown below. Test compound 5 (n=7) or negative control (n=6) were administered at the selected concentration and flow rate through an i.v. bolus or infusion via the right femoral vein as visualized below. The negative control animals received 2 x PBS / 5% glucose containing in mM: NaCI 273.8, NaH 2 P0 4 x 2 H 2 0 14.2, KCI 5.4 and KH 2 P0 4 2.9, pH 7.4 ± 0.05 / 5% (w/v) glucose. This solution was administered at the same volume, via the same route and at the same flow rate as the test compound 5 (identical formulation). Fifteen min (counting from the end of the injection) after the slow bolus injection (approx. 60 s injection time) of test article or vehicle, the right jugular vein was clamped, starting with the downstream clamp, followed 10 s later by the upstream one. 150 pl_ of blood were collected from the femoral artery and supplemented with 45 mI_ of 0.25 M calcium chloride. Next, 25 mI_ of human a-thrombin (2.5 U/mL, Sigma-Aldrich) was added to the blood mixture to induce coagulation. Immediately after mixing the blood, the clotting blood was administered in the isolated part of the right jugular vein. After a venous stasis period of 15 min, the vessel clamps were removed to restore blood flow from the jugular vein. The test article was infused during this venous stasis period. Blood flow was recorded with the transonic flow probe for a period of two h while the test article was infused at the selected concentration (see Table 4). 2 h after reperfusion, the venous stasis segment was removed, opened longitudinally and emptied into a petri dish containing 5% sodium citrate solution. Any existing thrombi were removed and blotted on a filter paper. The thrombi were measured, weighed and the appearance was judged.

A bleeding time was performed 30 min after beginning of reperfusion using an ITC Surgicutt™ Bleeding Time Device (International Technidyne SU50I via Fisher Scientific, Ottawa, Canada). Bleeding time was assessed with a filter paper by carefully collecting blood from the wound rim until no red staining of the filter paper could be observed. For each measure, a different non-stained part of the filter paper was used. The maximum bleeding time was defined as 300 s.

For each blood sample time point, plasma samples were generated using 150 mM sodium citrate as anticoagulant. Samples were stored at -20°C until further analysis: determination of compound 5 concentration by HPLC and determination of residual FXIII activity after thrombin activation using the isopeptidase assay described above. In addition, one blood sample was taken at 60 min after the test article administration for thromboelastography (TEG). The TEG 5000 traces were recorded on the fresh whole blood sample for a period of 60 min according to the manufacturer. The read-outs are similar to the thromboelastometry and key parameters were combined to give the coagulation index (Cl). Subsequent to the observation period of about 150 min after reperfusion, the animals were euthanized following an intracardiac blood draw by administering an overdose of pentobarbital.

Statistical Analysis Unpaired Student’s t-tests were performed on all experimental conditions, comparing the values obtained from rabbits injected with the test article to the values obtained from the negative control rabbits. Statistical significance is indicated if p<0.05 compared to negative control animals. For each group, data is expressed as the mean ± S.E.M.

Experimental schedule of the rabbit model of venous stasis and reperfusion is briefly presented in Figure 4A. Table 4. Intravenous Injection Parameters

The in vivo experiment proofs significantly higher flow rates after compound 5 infusion compared to PBS control animals (Figure 4B). The area under the curve of flow rate is in accordance to the vein flow rate. In the compound 5 group the mean areas under the curve (AUC) of the jugular flow rate normalized to baseline between time points 35 and 135 minutes are significantly higher (Figure 4C). The Thrombus wet weight is significantly reduced in the compound 5 group. Thrombus wet weight was determined after 135 min of infusion (Figure 4D). Most importantly the template skin bleeding time is not influenced. Template skin bleeding time was determined after 60 min of infusion. No difference between PBS and compound 5 was observed. Maximal observation time was pre-defined at 300 seconds (Figure 4E).