Inhibitors of blood coagulation factor XIII

Abstract

The invention relates to a compound of general formula (I) as novel inhibitor of blood coagulation factor XIII, methods for synthesis thereof and to use thereof for the prophylaxis or treatment of diseases associated with blood coagulation factor XIII. ##STR00001##

Claims

1. A compound of the general formula (I): ##STR00212## wherein R.sup.1 represents —H, —CH.sub.3, —C(CH.sub.3).sub.3, -cyclo-C.sub.3H.sub.5, -cyclo-C.sub.4H.sub.7, -cyclo-C.sub.5H.sub.9, -cyclo-C.sub.6H.sub.11, —CH.sub.2—CH(CH.sub.3).sub.2, —CH.sub.2CH.sub.3, —CH.sub.2CH.sub.2CH.sub.3, —CH.sub.2CH.sub.2CH.sub.2CH.sub.3, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3, —CH(CH.sub.3).sub.2, —CH.sub.2—C(CH.sub.3).sub.3, —CH.sub.2CH.sub.2SCH.sub.3, —CH.sub.2-cyclo-C.sub.3H.sub.5, —CH.sub.2-cyclo-C.sub.4H.sub.7, —CH.sub.2-cyclo-C.sub.5H.sub.9 or —CH.sub.2-cyclo-C.sub.6H.sub.11; R.sup.2 represents -A.sup.1-A.sup.2-A.sup.3-E, -A.sup.1-A.sup.2-A.sup.3-A.sup.4-E or -A.sup.1-A.sup.2-A.sup.3-A.sup.4-A.sup.5-E; R.sup.3 represents ##STR00213## ##STR00214## R.sup.4 represents —OR*, —NH.sub.2, —NHR.sup.# or —NR*R.sup.#; R* and R.sup.# represent independently of each other —CH.sub.3, —CH.sub.2CH.sub.3, —CH(CH.sub.3).sub.2, —CH.sub.2CH.sub.2CH.sub.3, —CH.sub.2CH(CH.sub.3).sub.2, —C(CH.sub.3).sub.3, -cyclo-C.sub.3H.sub.5, -cyclo-C.sub.4H.sub.7, -cyclo-C.sub.5H.sub.9, -cyclo-C.sub.6H.sub.11, —CH.sub.2-cyclo-C.sub.3H.sub.5, —CH.sub.2-cyclo-C.sub.4H.sub.7, —CH.sub.2-cyclo-C.sub.5H.sub.9, —CH.sub.2-cyclo-C.sub.6H.sub.11, —CH.sub.2-Ph, —CH.sub.2OCH.sub.3, —CH.sub.2OCH.sub.2CH.sub.3, —CH.sub.2CH.sub.2OCH.sub.3, or —CH.sub.2CH.sub.2SCH.sub.3; A.sup.1 represents ##STR00215## ##STR00216## A.sup.2-A.sup.5 represent independently of each other ##STR00217## ##STR00218## ##STR00219## ##STR00220## ##STR00221## ##STR00222## ##STR00223## E represents: —OR.sup.13, —NR.sup.13R.sup.14, —NHSO.sub.2R.sup.13, —O—L.sub.1-R.sup.13, —O—L.sub.1-O—R.sup.13, —NH—L.sub.1-O—R.sup.13, —NH—L.sub.1-NR.sup.13R.sup.14, —NHSO.sub.2—L.sub.1-R.sup.13, ##STR00224## R.sup.13 and R.sup.14 represent independently of each other: —H, —CH.sub.3, —CH.sub.2CH.sub.3, —C(CH.sub.3).sub.3, —CH.sub.2CH.sub.2CH.sub.3, —CH(CH.sub.3).sub.2, —CH.sub.2CH.sub.2CH.sub.2CH.sub.3, —CH.sub.2CH(CH.sub.3).sub.2, —CH(CH.sub.3)CH.sub.2CH.sub.3, ##STR00225## ##STR00226## R.sup.N, R.sup.N1 and R.sup.N2 represents independently of each other —H, —CH.sub.3, —C.sub.2H.sub.5, —C.sub.3H.sub.7, —CH(CH.sub.3).sub.2, —C.sub.4H.sub.9, —CH.sub.2—CH(CH.sub.3).sub.2, —CH(CH.sub.3)—C.sub.2H.sub.5, —C(CH.sub.3).sub.3, -cyclo-C.sub.3H.sub.5, —CH.sub.2-cyclo-C.sub.3H.sub.5, —CH.sub.2F, —CHF.sub.2, —CF.sub.3, —CH.sub.2Cl, —CH.sub.2Br, —CH.sub.2l, —CH.sub.2—CH.sub.2F, —CH.sub.2—CHF.sub.2, —CH.sub.2—CF.sub.3, —CH.sub.2—CH.sub.2Cl, —CH.sub.2—CH.sub.2Br, —CH.sub.2—CH.sub.2l, —CH.sub.2—CH═CH.sub.2, —CH.sub.2—C≡CH, —CHO, —COCH.sub.3, —COC.sub.2H.sub.5, —COC.sub.3H.sub.7, —COCH(CH.sub.3).sub.2, —COC(CH.sub.3).sub.3, —COOCH.sub.3, —COOC.sub.2H.sub.5, —COOC.sub.3H.sub.7, —COOCH(CH.sub.3).sub.2, or —COOC(CH.sub.3).sub.3; L.sup.1-L.sup.8 represents independently of each other a covalent bond, —CH.sub.2—, —CH(CH.sub.3)—, —CH(CH.sub.3).sub.2—, —CO—, —SO—, —SO.sub.2—, ##STR00227## ##STR00228## R.sup.5—R.sup.12, R.sup.4′—R.sup.7′, and R.sup.15—R.sup.23 represents independently of each other —H, —F, —Cl, —Br, —I, —OH, —CN, —NO.sub.2, —CH.sub.3, —C.sub.2H.sub.5, —C.sub.3H.sub.7, —CH(CH.sub.3).sub.2, —C.sub.4H.sub.9, —CH.sub.2—CH(CH.sub.3).sub.2, —CH(CH.sub.3)—C.sub.2H.sub.5, —C(CH.sub.3).sub.3, -cyclo-C.sub.3H.sub.5, —CH.sub.2-cyclo-C.sub.3H.sub.5, —CH.sub.2F, —CHF.sub.2, —CF.sub.3, —CH.sub.2Cl, —CH.sub.2Br, —CH.sub.2l, —CH.sub.2—CH.sub.2F, —CH.sub.2—CHF.sub.2, —CH.sub.2—CF.sub.3, —CH.sub.2—CH.sub.2Cl, —CH.sub.2—CH.sub.2Br, —CH.sub.2—CH.sub.2l, —OCH.sub.3, —OC.sub.2H.sub.5, —OC.sub.3H.sub.7, —OCH(CH.sub.3).sub.2, —OC(CH.sub.3).sub.3, —OC.sub.4H.sub.9, —OCHF.sub.2, —OCF.sub.3, —OCH.sub.2CF.sub.3, —OC.sub.2F.sub.5, —OCH.sub.2OCH.sub.3, —O-cyclo-C.sub.3H.sub.5, —OCH.sub.2-cyclo-C.sub.3H.sub.5, —O—C.sub.2H.sub.4-cyclo-C.sub.3H.sub.5, —CHO, —COCH.sub.3, —COCF.sub.3, —COC.sub.2H.sub.5, —COC.sub.3H.sub.7, —COCH(CH.sub.3).sub.2, —COC(CH.sub.3).sub.3, —COOH, —COOCH.sub.3, —COOC.sub.2H.sub.5, —COOC.sub.3H.sub.7, —COOCH(CH.sub.3).sub.2, —COOC(CH.sub.3).sub.3, —OOC—CH.sub.3, —OOC—CF.sub.3, —OOC—C.sub.2H.sub.5, —OOC—C.sub.3H.sub.7, —OOC—CH(CH.sub.3).sub.2, —OOC—C(CH.sub.3).sub.3, —NH.sub.2, —NHCH.sub.3, —NHC.sub.2H.sub.5, —NHC.sub.3H.sub.7, —NHCH(CH.sub.3).sub.2, —NHC(CH.sub.3).sub.3, —N(CH.sub.3).sub.2, —N(C.sub.2H.sub.5).sub.2, —N(C.sub.3H.sub.7).sub.2, —N[CH(CH.sub.3).sub.2].sub.2, —N[C(CH.sub.3).sub.3].sub.2, —NHCOCH.sub.3, —NHCOCF.sub.3, —NHCOC.sub.2H.sub.5, —NHCOC.sub.3H.sub.7, —NHCOCH(CH.sub.3).sub.2, —NHCOC(CH.sub.3).sub.3, —CONH.sub.2, —CONHCH.sub.3, —CONHC.sub.2H.sub.5, —CONHC.sub.3H.sub.7, —CONHCH(CH.sub.3).sub.2, —CONH-cyclo-C.sub.3H.sub.5, —CONHC(CH.sub.3).sub.3, —CON(CH.sub.3).sub.2, —CON(C.sub.2H.sub.5).sub.2, —CON(C.sub.3H.sub.7).sub.2, —CON[CH(CH.sub.3).sub.2].sub.2, —CON[C(CH.sub.3).sub.3].sub.2, —SO.sub.2NH.sub.2, —SO.sub.2NHCH.sub.3, —SO.sub.2NHC.sub.2H.sub.5, —SO.sub.2NHC.sub.3H.sub.7, —SO.sub.2NHCH(CH.sub.3).sub.2, —SO.sub.2NH-cyclo-C.sub.3H.sub.5, —SO.sub.2NHC(CH.sub.3).sub.3, —SO.sub.2N(CH.sub.3).sub.2, —SO.sub.2N(C.sub.2H.sub.5).sub.2, —SO.sub.2N(C.sub.3H.sub.7).sub.2, —SO.sub.2N[CH(CH.sub.3).sub.2].sub.2, —SO.sub.2N[C(CH.sub.3).sub.3].sub.2, —NHSO.sub.2CH.sub.3, —NHSO.sub.2CF.sub.3, —NHSO.sub.2C.sub.2H.sub.5, —NHSO.sub.2C.sub.3H.sub.7, —NHSO.sub.2CH(CH.sub.3).sub.2, —NHSO.sub.2C(CH.sub.3).sub.3, —CH═CH.sub.2, —CH.sub.2—CH═CH.sub.2, —C(CH.sub.3)═CH.sub.2, —CH═CH—CH.sub.3, —C≡CH, —C≡C—CH.sub.3, —CH.sub.2—C≡CH, -Ph, —O-Ph, or —O—CH.sub.2-Ph, ##STR00229## or R.sup.7 and R.sup.8 or R.sup.8 and R.sup.9 form together one of the following ring moieties; ##STR00230## 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 (II-1) to (II-3): ##STR00231## wherein R.sup.2, R.sup.3 and R.sup.4 have the meanings as defined in claim 1.

3. The compound according to claim 1, wherein A.sup.2 is selected from: ##STR00232## ##STR00233## and/or A.sup.2 is selected from: ##STR00234##

4. The compound according to claim 1, wherein -A.sup.1-A.sup.2- represents ##STR00235## ##STR00236## ##STR00237## ##STR00238##

5. The compound according to claim 1, wherein -A.sup.2-A.sup.3- represents ##STR00239##

6. The compound according to claim 1, wherein R.sup.3 represents ##STR00240## and R.sup.7, R.sup.8 and R.sup.9 have the meanings as defined in claim 1.

7. The compound according to claim 1 of the general formula (III): ##STR00241## wherein E.sup.2 represents -E, -A.sup.3-E, -A.sup.3-A.sup.4-E, or -A.sup.3-A.sup.4-A.sup.5-E; and A.sup.1, A.sup.3, A.sup.4, A.sup.5, R.sup.1, R.sup.3, R.sup.4, 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 (IV-4): ##STR00242## wherein E.sup.2 represents -E, -A.sup.3-E, -A.sup.3-A.sup.4-E or -A.sup.3-A.sup.4-A.sup.5-E; R.sup.4 represents —OCH.sub.3 or —OCH.sub.2CH.sub.3; R.sup.7, R.sup.8 and R.sup.9 are independently of each other selected from —H, —Cl, —OH, —NO.sub.2 or —COOH; and A.sup.1, A.sup.3, A.sup.4, A.sup.5 and E have the meanings as defined in claim 1.

9. The compound according to claim 1, wherein the compound has any one of the formulae (V-1) and (V-2): ##STR00243## wherein E.sup.2 represents -E, -A.sup.3-E, -A.sup.3-A.sup.4-E or -A.sup.3-A.sup.4-A.sup.5-E; R.sup.4 represents —OCH.sub.3 or —OCH.sub.2CH.sub.3; A.sup.1 represents ##STR00244## ##STR00245## and A.sup.3, A.sup.4, A.sup.5 and E have the meanings as defined in claim 1.

10. The compound according to claim 7 having the formula (VI): ##STR00246## wherein E.sup.1 represents -E, -A.sup.4-E or -A.sup.4-A.sup.5-E; R.sup.3 represents ##STR00247## and A.sup.1, A.sup.4, A.sup.5, R.sup.4, R.sup.7, R.sup.8, R.sup.9 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, 37b, 38, 39a, 39b, 40, 41, 42a, 42b, 43, 44, 45a, and 45b.

12. A pharmaceutical composition comprising at least one compound according to claim 1 as an active ingredient together with at least one pharmaceutically acceptable carrier, excipient and/or diluent.

13. A Method for producing compound according to claim 1 comprising: Step (0B): providing a resin, suitable for solid-phase peptide synthesis (SPPS); ##STR00248## Step (1B) (a): performing coupling reaction of the corresponding C-terminal amino acid building block PG.sup.4NH-A.sup.i-OH; (b) deprotecting the protecting group PG.sup.4 of a resulting compound after Step (a); (c) repeating the steps (a) and (b)/times, wherein i is 1-5, to obtain a resin bound intermediate compound (IIIc); ##STR00249## Step (2B): cleavage from resin and deprotecting the protecting group PG.sup.4 to obtain an intermediate compound (IIId);
H.sub.2-A.sup.1-A.sup.2----A.sup.i-E   IIId Step (3B): performing coupling reaction of the intermediate compound IIId with an amino acid building block A0 ##STR00250## to obtain a compound IVc; ##STR00251## Step (4B): deprotecting the protecting group PG.sup.5 and subsequent coupling reaction with protected amino acid Ia ##STR00252## to obtain compound Vc ##STR00253## Step (5B): deprotecting the protecting group PG.sup.1 and subsequent coupling reaction with a N-terminal building block R.sup.3—CO.sub.2H to produce the compound of the formula (I); or Step (0C): providing a N-deprotected C-terminal building block H-E or H-A.sup.i-E; Step (1C): (a) performing coupling reaction of the corresponding C-terminal amino acid building block PG.sup.4NH-A.sup.i-OH or PG.sup.4NH-A.sup.i-1-OH; (b) deprotecting the protecting group PG.sup.4 of a resulting compound after Step (a); (c) repeating the steps (a) and (b) i times, wherein i is 1-5 or 1-4, to obtain intermediate compound (IIId);
H.sub.2-A.sup.1-A.sup.2----A.sup.i-E  (IIId) Step (3B): performing coupling reaction of the intermediate compound IIId with an amino acid building block A0 ##STR00254## to obtain a compound IVc; ##STR00255## Step (4B): deprotecting the protecting group PG.sup.5 and subsequent coupling reaction with protected amino acid 1a ##STR00256## to obtain compound Vc ##STR00257## Step (5B): deprotecting the protecting group PG.sup.1 and subsequent coupling reaction with a N-terminal building block R.sup.3—CO.sub.2H to produce the compound of the formula (I).

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1:

(2) 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.sub.0) and total volume (V.sub.t) of the GPC column as well as the apparent molecular mass of the (x)FDPs are indicated.

(3) 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 γ-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.

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

(5) FIG. 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).

(6) FIG. 4:

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

(8) 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)

(9) 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).

(10) 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).

(11) 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

(12) 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 HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate PyAOP (7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate

CHEMICAL EXAMPLES

(13) 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 ZED788

(14) ##STR00131##

(15) 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%), NaHCO.sub.3 solution (10%) and brine. The organic phase was dried over Na.sub.2SO.sub.4, filtered and the solvent was evaporated. The raw product was used without further purification.

(16) Yield: 13.4 g, >100%; ESI-MS: 318.3 [M+H].sup.+

Preparation of Compound ZED720

(17) ##STR00132##

(18) 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%), NaHCO.sub.3 solution (10%) and brine. The organic phase was dried over Na.sub.2SO.sub.4, filtered and the solvent was evaporated. The raw product was used without further purification.

(19) Yield: 13.7 g, 83%

(20) ESI-MS: 418.3 [M+H].sup.+

Preparation of Compound ZED721

(21) ##STR00133##

(22) 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 (1M 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.sub.2SO.sub.4, filtered and concentrated to dryness. The raw product was used without further purification.

(23) Yield: 13.3 g, >100%

(24) ESI-MS: 388.3 [M+H].sup.+

Preparation of Compound ZED755

(25) ##STR00134##

(26) 13.3 g of ZED721 (˜32.8 mmol) were dissolved in 60 ml of benzene and 11.2 g of (carbmethoxymethylene)-triphenylphosphorane (1 eq) was added portionwise. After stirring overnight, the solvent was evaporated. The residue was purified by flash chromatography.

(27) Yield: 12.0 g, 83%

(28) ESI-MS: 444.3 [M+H].sup.+

Preparation of compound Ib

(29) ##STR00135##

(30) 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.sub.2SO.sub.4, filtered and the solvent was evaporated. The residue was purified by flash chromatography.

(31) Yield: 5.89 g, 76%

(32) ESI-MS: 288.3 [M+H].sup.+

Preparation of Compound Ic

(33) ##STR00136##

(34) The synthesis of Ic was performed according to Ib, using (carbethoxymethylene)-triphenylphosphorane in step 4.

(35) Yield: 3.27 g, 59% (last step)

(36) ESI-MS: 302.3 [M+H].sup.+

Preparation of Compound Id

(37) ##STR00137##

(38) The synthesis of Id was performed according to Ib, using triphenylphosphonium carbamoylmethylide in step 4.

(39) Yield: 623 mg, 36% (last step)

(40) ESI-MS: 273.3 [M+H].sup.+

(41) Preparation of Compound Ie

(42) ##STR00138##

(43) The synthesis of Ie was performed according to Ib, using N-methyl-triphenylphosphonium carbamoylmethylide in step 4.

(44) Yield: 241 mg, 32% (last step)

(45) ESI-MS: 287.3 [M+H].sup.+

Preparation of Compound ZED3478

(46) ##STR00139##

(47) ZED3478 was synthesized by standard Fmoc solid-phase peptide chemistry (reactions in DMF, coupling with TBTU/HOBt/DIPEA, deprotection with piperidine) using 0.41 g (0.28 mmol) Rink Amide MBHA 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.

(48) Yield: 124 mg, 85%; ESI-MS: 407.3 [M+H].sup.+

Preparation of Compound ZED3480

(49) ##STR00140##

(50) 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 μl (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%), NaHCO.sub.3 solution (10%) and brine. The organic phase was dried over Na.sub.2SO.sub.4, filtered and the solvent was evaporated. The raw product was used without further purification.

(51) Yield: 119 mg, 80%; ESI-MS: 620.5 [M+H].sup.+

Preparation of Compound ZED3481

(52) ##STR00141##

(53) 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) Ib, 72.2 mg (1 eq) HATU and 64.6 μl (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%), NaHCO.sub.3 solution (10%) and brine. The organic phase was dried over Na.sub.2SO.sub.4, filtered and the solvent was evaporated. The raw product was used without further purification.

(54) Yield: 86 mg, 57%; ESI-MS: 789.6 [M+H].sup.+

Example 1-1. Preparation of Compounds 1a/b

(55) ##STR00142##

(56) 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 μl (2 eq) DIPEA were added and the reaction was stirred at room temperature overnight. The solvent was evaporated and the residue was purified by HPLC.

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

(58) ESI-MS: 882.5 [M+H].sup.+

Example 1-2. Preparation of Compounds 2a/b

(59) ##STR00143##

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

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

(62) ESI-MS: 880.5 [M+H].sup.+

Example 1-3. Preparation of Compound 3

(63) ##STR00144##

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

(65) Yield: 39 mg, 49%

(66) ESI-MS: 839.5 [M+H].sup.+

Example 1-4. Preparation of Compound 4

(67) ##STR00145##

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

(69) Yield: 18 mg, 26%

(70) ESI-MS: 882.5 [M+H].sup.+

Example 1-5. Preparation of Compounds 5a/b

(71) ##STR00146##

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

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

(74) ESI-MS: 838.6 [M+H].sup.+

Example 1-6. Preparation of Compound 6

(75) ##STR00147##

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

(77) Yield: 12 mg, 18%

(78) ESI-MS: 855.5 [M+H].sup.+

Example 1-7. Preparation of Compounds 7a/b

(79) ##STR00148##

(80) The synthesis of example 1-7 was performed according to example 1-1, using the corresponding building block Ic.

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

(82) ESI-MS: 896.6 [M+H].sup.+

Example 1-8. Preparation of Compounds 8a/b

(83) ##STR00149##

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

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

(86) ESI-MS: 852.6 [M+H].sup.+

Example 1-9. Preparation of Compounds 9a/b

(87) ##STR00150##

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

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

(90) ESI-MS: 884.6 [M+H].sup.+

Example 1-10. Preparation of Compounds 10a/b

(91) ##STR00151##

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

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

(94) ESI-MS: 960.6 [M+H].sup.+

Example 1-11. Preparation of Compounds 11a/b

(95) ##STR00152##

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

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

(98) ESI-MS: 960.6 [M+H].sup.+

Example 1-12. Preparation of Compounds 12a/b

(99) ##STR00153##

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

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

(102) ESI-MS: 974.6 [M+H].sup.+

Example 1-13. Preparation of Compounds 13a/b

(103) ##STR00154##

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

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

(106) ESI-MS: 854.5 [M+H].sup.+

Example 1-14. Preparation of Compounds 14a/b

(107) ##STR00155##

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

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

(110) ESI-MS: 882.5 [M+H].sup.+

Example 1-15. Preparation of Compounds 15a/b

(111) ##STR00156##

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

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

(114) ESI-MS: 823.6 [M+H].sup.+

Example 1-16. Preparation of Compounds 16a/b

(115) ##STR00157##

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

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

(118) ESI-MS: 882.6 [M+H].sup.+

Example 1-17. Preparation of Compounds 17a/b

(119) ##STR00158##

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

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

(122) ESI-MS: 837.6 [M+H].sup.+

Example 1-18. Preparation of Compounds 18a/b

(123) ##STR00159##

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

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

(126) ESI-MS: 908.6 [M+H].sup.+

Example 1-19. Preparation of Compounds 19a/b

(127) ##STR00160##

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

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

(130) ESI-MS: 922.7 [M+H].sup.+

Example 1-20. Preparation of Compounds 20a/b

(131) ##STR00161##

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

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

(134) ESI-MS: 886.6 [M+H].sup.+

Example 1-21. Preparation of Compounds 21a/b

(135) ##STR00162##

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

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

(138) ESI-MS: 886.6 [M+H].sup.+

Example 1-22. Preparation of Compounds 22a/b

(139) ##STR00163##

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

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

(142) ESI-MS: 866.6 [M+H].sup.+

Example 1-23. Preparation of Compounds 23a/b

(143) ##STR00164##

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

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

(146) ESI-MS: 904.5 [M+H].sup.+

Example 1-24. Preparation of Compounds 24a/b

(147) ##STR00165##

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

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

(150) ESI-MS: 880.6 [M+H].sup.+

Example 1-25. Preparation of Compounds 25a/b

(151) ##STR00166##

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

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

(154) ESI-MS: 884.6 [M+H].sup.+

Example 1-26. Preparation of Compounds 26a/b

(155) ##STR00167##

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

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

(158) ESI-MS: 870.5 [M+H].sup.+

Example 1-27. Preparation of Compound 27

(159) ##STR00168##

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

(161) Yield: 79 mg, 52%

(162) ESI-MS: 824.6 [M+H].sup.+

Example 1-28. Preparation of Compound 28

(163) ##STR00169##

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

(165) Yield: 26 mg, 32%

(166) ESI-MS: 881.6 [M+H].sup.+

Example 1-29. Preparation of Compound 29

(167) ##STR00170##

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

(169) Yield: 15 mg, 27%

(170) ESI-MS: 931.6 [M+H].sup.+

Example 1-30. Preparation of Compound 30

(171) ##STR00171##

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

(173) Yield: 36 mg, 52%

(174) ESI-MS: 990.7 [M+H].sup.+

Example 1-31. Preparation of Compounds 31a/b

(175) ##STR00172##

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

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

(178) ESI-MS: 972.7 [M+H].sup.+

Example 1-32. Preparation of Compounds 32a/b

(179) ##STR00173##

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

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

(182) ESI-MS: 1107.8 [M+H].sup.+

Example 1-33. Preparation of Compounds 33a/b

(183) ##STR00174##

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

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

(186) ESI-MS: 1145.5 [M+Na].sup.+

Example 1-34. Preparation of Compounds 34a/b

(187) ##STR00175##

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

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

(190) ESI-MS: 839.4 [M+H].sup.+

Example 1-35. Preparation of Compounds 35a/b

(191) ##STR00176##

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

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

(194) ESI-MS: 853.6 [M+H].sup.+

Example 1-36. Preparation of Compounds 36a/b

(195) ##STR00177##

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

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

(198) ESI-MS: 852.6 [M+H].sup.+

Example 1-37. Preparation of Compounds 37a/b

(199) ##STR00178##

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

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

(202) ESI-MS: 856.5 [M+H].sup.+

Example 1-38

1.1 Preparation of Compound Ref. 08

(203) ##STR00179##

(204) The backbone tetrapeptide H-Val-Pro-Leu-Chg-Nhfe was built by standard Fmoc solid-phase peptide chemistry according to compound ZED3478, using the corresponding amino acids. The Michael acceptor (ethyl acrylate) was directly coupled according to compound ZED3481, using compound Ic. Finally, compound Ref. 08 was synthesized according to Example 1-1, using Cbz-Cl.

(205) Yield: 86 mg, 61%

(206) ESI-MS: 783.6 [M+H].sup.+

1.2 Preparation of Compound 38

(207) ##STR00180##
The synthesis of compound 38 was performed according to compound Ref. 06, using 4,5-dichlorophthalic anhydride instead of Cbz-Cl in the final step.

(208) Yield: 36 mg, 51%

(209) ESI-MS: 865.5 [M+H].sup.+

Example 1-39

1. Preparation of Compound Ref. 09

(210) ##STR00181##

(211) The synthesis of compound Ref. 09 was performed according to compound Ref. 08, using the corresponding amino acids for the synthesis of the backbone tetrapeptide by standard Fmoc solid-phase peptide chemistry.

(212) Yield: 93 mg, 57%

(213) ESI-MS: 812.6 [M+H].sup.+

2. Preparation of Compound 39a/39b (Regioisomers)

(214) ##STR00182##

(215) The synthesis of compound 39 (39a/39b) was performed according to compound Ref. 09, using the corresponding amino acids for the buildup of the backbone tetrapeptide and 3,4-pyridinedicarboxylic anhydride instead of Cbz-Cl in the final step.

(216) Yield: 41 mg, 36%, ratio of regioisomers: approximately 1:1

(217) ESI-MS: 812.6 [M+H].sup.+

Example 1-40

1. Preparation of Compound Ref. 10

(218) ##STR00183##

(219) The backbone tripeptide Fmoc-Val-Pro-Chg-OH was built by standard Fmoc solid-phase peptide chemistry according to compound ZED3478, using 2-chlorotrityl resin and the corresponding amino acids. After cleavage from the resin (using 95% TFA/2.5% water/2.5% triisopropylsilane), C-terminal coupling of isopentylamine was performed according to compound ZED3480 with HATU/DIPEA. Subsequently the Fmoc protecting group was removed with piperidine in DMF and the Michael acceptor (ethyl acrylate) was coupled according to compound ZED3481, using compound Ic. Finally, compound Ref. 10 was synthesized according to Example 1-1, using Cbz-Cl.

(220) Yield: 72 mg, 45%

(221) ESI-MS: 740.6 [M+H].sup.+

2. Preparation of Compound 40

(222) ##STR00184##

(223) The synthesis of compound 40 was performed according to compound Ref. 10, using the corresponding amino acids for the buildup of the backbone tripeptide but 5-nitroisophthalic acid (coupling according to compound ZED3480 with HATU/DIPEA) instead of Cbz-Cl in the final step.

(224) Yield: 56 mg, 62%

(225) ESI-MS: 827.7 [M+H].sup.+

Example 1-41

1. Preparation of Compound Ref. 11

(226) ##STR00185##

(227) The synthesis of compound Ref. 11 was performed according to compound Ref. 08, using the corresponding amino acids for the synthesis of the backbone tetrapeptide and 2-Thiophenecarboxylic acid (coupling according to compound ZED3480 with HATU/DIPEA) instead of Cbz-Cl in the final step.

(228) Yield: 69 mg, 56%

(229) ESI-MS: 788.5 [M+H].sup.+

2. Preparation of Compound 41

(230) ##STR00186##

(231) The synthesis of compound 41 was performed according to compound Ref. 11, using the corresponding amino acids for the buildup of the backbone tetrapeptide.

(232) Yield: 45 mg, 61%

(233) ESI-MS: 771.7 [M+H].sup.+

Example 1-42

1. Preparation of Compound Ref. 12

(234) ##STR00187##

(235) The synthesis of compound Ref. 12 was performed according to compound Ref. 08, using the corresponding amino acids for the solid phase chemistry yielding the backbone tetrapeptide.

(236) Yield: 78 mg, 52%

(237) ESI-MS: 823.8 [M+H].sup.+

2. Preparation of Compound 42a/42b (Regioisomers)

(238) ##STR00188##

(239) The synthesis of compound 42a/42b was performed according to compound Ref. 12, using the corresponding amino acids for the buildup of the backbone tetrapeptide and 4-nitrophthalic anhydride instead of Cbz-Cl in the final step.

(240) Yield: 32 mg, 37%, ratio of regioisomers: approximately 1:1

(241) ESI-MS: 868.7 [M+H].sup.+

Example 1-43

1. Preparation of Compound Ref. 13

(242) ##STR00189##

(243) The backbone pentapeptide H-Nle-Nle-Leu-Pro-Trp-OH was built by standard Fmoc solid-phase peptide chemistry according to compound ZED3478, using 2-chlorotrityl resin and the corresponding amino acids. The Michael acceptor (methyl acrylate) was directly coupled according to compound ZED3481, using compound Ib. Finally, compound Ref. 13 was synthesized according to compound ZED3480 by coupling Ac-(D)-Asp(OtBu)-OH with HATU/DIPEA followed by cleavage of the tort-butyl ester with TFA in DCM.

(244) Yield: 142 mg, 65%

(245) ESI-MS: 967.8 [M+H].sup.+

2. Preparation of Compound Ref. 14

(246) ##STR00190##

(247) The synthesis of compound Ref. 14 was performed according to Ref. 13, using the corresponding amino acids for the buildup of the backbone tetrapeptide.

(248) Yield: 107 mg, 59%

(249) ESI-MS: 781.7 [M+H].sup.+

3. Preparation of Compound 43

(250) ##STR00191##

(251) The synthesis of compound 43 was performed according to Ref. 14 using the corresponding amino acids for the synthesis of the backbone tetrapeptide. N-terminal capping was obtained by 5-nitroisophthalic acid (coupling according to compound ZED3480 with HATU/DIPEA) instead of Ac-D-Asp(OtBu)-OH in the final step.

(252) Yield: 65 mg, 57%

(253) ESI-MS: 813.6 [M+H].sup.+

Example 1-44

1. Preparation of Compound Ref. 15

(254) ##STR00192##

(255) The synthesis of compound Ref. 15 was performed according to Ref. 13, using the corresponding amino acids for the buildup of the backbone pentapeptide and (S)—N-Boc-4-oxopyrrolidine-2-carboxylic acid (coupling according to compound ZED3480 with HATU/DIPEA) instead of Ac-D-Asp(OtBu)-OH in the final step. The Boc protecting group is cleaved with TFA in DCM accordingly.

(256) Yield: 127 mg, 57%

(257) ESI-MS: 921.8 [M+H].sup.+

2. Preparation of Compound Ref. 16

(258) ##STR00193##

(259) The synthesis of compound Ref. 16 was performed according to Ref. 15, using the corresponding amino acids for the buildup of the backbone tetrapeptide.

(260) Yield: 93 mg, 47%

(261) ESI-MS: 735.6 [M+H].sup.+

3. Preparation of Compound 44

(262) ##STR00194##

(263) The synthesis of compound 44 was performed according to Ref. 14 using the corresponding amino acids for the buildup of the backbone tetrapeptide.

(264) Yield: 78 mg, 59%

(265) ESI-MS: 733.6 [M+H].sup.+

Example 1-45

1. Preparation of Compound Ref. 17 (ZED1265)

(266) ##STR00195##

(267) The backbone decapeptide H-Glu-Gln-Val-Ser-Pro-Leu-Thr-Leu-Lys(Alloc)-OH was built by standard Fmoc solid-phase peptide chemistry according to compound ZED3478, using 2-chlorotrityl resin and the corresponding (side chain protected) amino acids. The Michael acceptor (methyl acrylate) was directly coupled according to compound ZED3481, using compound Ib. Finally, compound ZED1265 was synthesized according to compound ZED3480 by coupling Ac-Asn-OH with HATU/DIPEA followed by cleavage of the alloc protecting group with Tetrakis(triphenylphosphine)palladium(0) in DCM.

(268) Yield: 177 mg, 36%

(269) ESI-MS: 1453.2 [M+H].sup.+

2. Preparation of Compound Ref. 18 (ZED1246)

(270) ##STR00196##

(271) The synthesis of compound ZED1246 was performed according to ZED1265, using the corresponding amino acids for the buildup of the backbone nonapeptide.

(272) Yield: 158 mg, 39%

(273) ESI-MS: 1325.1 [M+H].sup.+

3. Preparation of Compound Ref. 19 (ZED1274)

(274) ##STR00197##

(275) The synthesis of compound ZED1274 was performed according to ZED1265, using the corresponding amino acids for the buildup of the backbone octapeptide.

(276) Yield: 132 mg, 45%

(277) ESI-MS: 1212.0 [M+H].sup.+

4. Preparation of Compound Ref. 20 (ZED1282)

(278) ##STR00198##

(279) The synthesis of compound ZED1282 was performed according to ZED1265, using the corresponding amino acids for the buildup of the backbone heptapeptide.

(280) Yield: 165 mg, 61% ESI-MS: 1098.8 [M+H].sup.+

5. Preparation of Compound Ref. 21 (ZED1283)

(281) ##STR00199##

(282) The synthesis of compound ZED1283 was performed according to ZED1265, using the corresponding amino acids for the buildup of the backbone hexapeptide.

(283) Yield: 127 mg, 47%

(284) ESI-MS: 997.8 [M+H].sup.+

Example 1-46: Preparation of Compound 45a/45b (Regioisomers)

(285) ##STR00200##

(286) The synthesis of compound 45a/45b was performed according to Ref. 11 using the corresponding amino acids for the buildup of the backbone tetrapeptide and capping by 3,4-pyridinedicarboxylic anhydride instead of Ac-(D)-Asp(OtBu)-OH in the final step.

(287) Yield: 77 mg, 32%, ratio of regioisomers: approximately 1:1

(288) ESI-MS: 799.6 [M+H].sup.+

Example 1-47

Reference Example 1 (Ref. 1)

(289) ##STR00201##

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

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

(292) ESI-MS: 910.5 [M+H].sup.+

Reference Example 2 (Ref. 2)

(293) ##STR00202##

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

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

(296) ESI-MS: 1023.5 [M+H].sup.+

Reference Example 3 (Ref. 3)

(297) ##STR00203##

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

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

(300) ESI-MS: 926.5 [M+H].sup.+

Reference Example 4 (Ref. 4)

(301) ##STR00204##

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

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

(304) ESI-MS: 882.6 [M+H].sup.+

Reference Example 5 (Ref. 5)

(305) ##STR00205##

(306) The synthesis of Ref. 5 was performed according to example 1-1, using the corresponding anhydride and amino acids.

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

(308) ESI-MS: 868.6 [M+H].sup.+

Reference Compound 6 (Ref. 6) Disclosed in WO 2008055488 A1 as Compound 38

(309) ##STR00206##

Reference Compound 7 (Ref. 7) Disclosed in WO 2008055488 A1 as Compound 4.1

(310) ##STR00207##

Reference Compound 22 (Ref. 22-ZED1301): Compound 4 of WO2014/090835

(311) ##STR00208##

Reference Compound 23 (Ref. 23-ZED1390): Compound 7 of WO2014/090835

(312) ##STR00209##

Reference Compound 24 (Ref. 24-ZED1251) Disclosed in Thrombosis Research, 2013, 131, 0214-6222

(313) ##STR00210##

Reference compound 25 (Ref. 25-ZED1227) disclosed in Amino Adds, 2017, 49, 585-595

(314) ##STR00211##

BIOLOGICAL EXAMPLES

Example 2-1: FXIII Activity Assays

(315) A: Isopeptidase Assay for Estimating Inhibitor Potency

(316) FXIIIa activity has been determined using substrate A101 (Zedira GmbH, Darmstadt, Germany), which is based on the N-terminal dodecapeptide of α.sub.2-antipiasmin. 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, HunfekJ 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.)

(317) Briefly, 12 μL recombinant cFXIII-A2 (T027, Zedira GmbH, Darmstadt, Germany) or FXIII-A.sub.2B.sub.2 derived from human plasma (T007) (25 μg/mL) and 3 μL human α-thrombin (0.5 U/mL, T056, Zedira) were mixed with 270 μL assay buffer (50 mM Tris-HCl, 10 mM CaCl.sub.2), 150 mM NaCl, 5.56 mM glycine methyl ester, 5 mM DTT, pH 7.5) containing 55 μM A101 substrate. The mixture was incubated for 20 min at room temperature to activate FXIII. 15 μL of inhibitor solution (serial dilution from 1.25 μM 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 (λ.sub.ex=313 nm) and 37° C. for 30 min using a CLARIOstar fluorescence micro plate reader (BMG Labtech, Ortenberg, Germany). For measurements without inhibitor, 15 μL of assay buffer/2% (v/v) DMSO were added. All measurements were performed in triplicate. The respective IC.sub.50 values were calculated by non-linear regression using the MARS software package (BMG Labtech).

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

(319) B: Transamidation Assay for Determining Inhibitor Selectivity

(320) 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 O M, Campbell L K, Myhrman R, Bruner-Lorand J. Transamidating enzymes. Anal Biochem 1971; 44: 221-31.).

(321) Briefly, 15 μL of recombinant transglutaminase enzyme[5] [15 μg/mL hTG1 (T035, Zedira), 69 μg/mL hTG2 (T022), 29 μg/mL hTG6 (T021), 18 μg/mL hTG7 (T011)] were mixed with 270 μL assay buffer containing dansylcadaverine and N,N-dimethyl casein. In the case of FXIII, 12 μL cFXIII (25 μg/mL) and 3 μL human α-thrombin (0.5 U/mL, T056, Zedira) were mixed with 270 μL assay buffer. The mixture was incubated for 20 min at room temperature to activate FXIII. In the case of TG3 78 μg hTG3 (T024) were activated using 14 μg dispase II (Roche, Mannheim, Germany) in the presence of 1.4 mM CaCl.sub.2) and incubated for 30 min at 25° C. The activated hTG3 was subsequently assayed as described above. 15 μL 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 (λ.sub.ex=330 nm) and 37° C. using the CLARIOstar fluorescence plate reader. All measurements were performed in triplicate. The respective IC.sub.50 values were calculated by non-linear regression using the MARS software package (BMG Labtech, Ortenberg, Germany).

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

(323) TABLE-US-00002 TABLE 1 Isopeptidase Transamidation Transglutaminase Species Assay IC.sub.50 [nM] Assay IC.sub.50 [nM] 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

(324) TABLE-US-00003 TABLE 2 The selectivity against human transglutaminases iso-enzymes. Transamidation Transglutaminase Assay IC.sub.50 [nM] Selectivity cFXIII .sup. 24 ± 1.5 1 TG1 11035 ± 1003 463 TG2 445 ± 20 19 TG3 66511 ± 4544 2791 TG6 .sup. 17 ± 0.6 0.7 TG7 1330 ± 102 56

(325) TABLE-US-00004 TABLE 3 inhibitory activity of the inventive compounds against FXIII and TG2 selectivity for selected compounds (isopeptidase assay) Compound IC.sub.50 [nM] FXIII IC.sub.50 [nM] TG2 selectivity  1a/b 11 75 6.8  2a/b 16 69 4.3  3 20 16 0.8  4 13 14 1.1  5a/b 16 67 4.2  6 17 12 0.7  7a/b 21 610 29.0   8a/b 21 417 19.9   9a/b 139 501 3.6 10a/b 42 38 0.9 11a/b 70 178 2.5 12a/b 51 257 5.0 13a/b 96 221 2.3 14a/b 26 59 2.3 15a/b 69 483 7.0 16a/b 106 204 1.9 17a/b 41 295 7.2 18a/b 510 239 0.5 19a/b 141 298 2.1 20a/b 156 218 1.4 21a/b 59 100 1.7 22a/b 42 56 1.3 23a/b 91 160 1.8 24a/b 18 63 3.5 25a/b 20 35 1.8 26a/b 43 98 2.3 27 27 64 2.4 28 36 41 1.1 29 24 32 1.3 30 15 16 1.1 31a/b 10 27 2.7 32a/b 12 72 6.0 33a/b 2138 7980 3.7 34a/b 123 356 2.7 35a/b 86 198 2.3 36a/b 41 168 4.1 37a/b 69 324 4.7 Ref. 1 >10000 7040 — Ref. 2 >10000 >10000 — Ref. 3 >10000 >10000 — Ref. 4 >10000 7039 — Ref. 5 9762 3462 0.4 Ref. 6 >10000 48 — Ref. 7 >10000 32 — *Compounds a/b means 1 to 1 mixutere of two regiomers

(326) TABLE-US-00005 TABLE 4 inhibitory activity of the inventive compounds against FXIII and TG2 selectivity of prior art compounds Compound FXIII IC.sub.50 [nM] TG2 IC.sub.50 [nM] Selectivity Ref. 08 3648 36 0.01 38 47 760 16.17  Ref. 09 5690 26 0.00 39a/b 39 698 17.90  Ref. 10 8183 176 0.02 40 81 848 10.47  Ref. 11 5361 253 0.05 42a/b 127 1207 9.50 Ref. 12 6771 345 0.05 42a/b 56 679 12.13  Ref. 13 465 2800 6.02 Ref. 14 >10.000 2150 — 43 158 856 5.42 Ref. 15 226 117 0.52 Ref. 16 >10.000 196 — 44 141 798 5.66 Ref. 17 420 227 0.54 Ref. 18 995 341 0.34 Ref. 19 4975 370 0.07 Ref .20 8475 381 0.04 Ref. 21 >10.000 421 — 45a/b 35 705 20.14  Ref. 22 110 2919 26.54  Ref. 23 56 102 1.82 Ref. 24 306 169 0.55 Ref. 25 >10.000 45 —

Example 2-2: Inhibition of Fibrin Cross-Linking and Size Exclusion Chromatography of Fibrin Degradation Products

(327) For the in vitro preparation of fibrin clots, HSA-free human fibrinogen (2.5 mg/mL, FIB3, Enzyme Research Laboratories, South Bend, Ind., USA), diluted in 20 mM Tris-HCl, 300 mM NaCl, pH 7.4, was mixed with cFXIII (10 μg/mL, T027, Zedira), and 5 mM CaCl.sub.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 μM 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-HCl, 500 mM NaCl, pH 7.4.

(328) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli (Laemmli U K. 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×SDS-PAGE loading buffer (128 mM Tris-HCl 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, Calif., 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 NaCl, 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 3×5 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, Calif., U.S.A.). Excess detection reagent was drained off, and the staining reaction was stopped with 20 mM Tris-HCl and 5 mM EDTA at pH 8.0. All steps were performed at room temperature on a shaker (FIG. 1).

Example 2-3: Thromboelastometry (TEM)

(329) 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.

(330) The potency of the inventive compounds as inhibitors (serial dilution covering 20.0 μM to 0.08 μM final concentration) in the presence of 0.02 μg/mL tissue plasminogen activator (t-PA; P016, Zedira) was investigated. Briefly, 20 μL star-TEM® (0.2 mol/L CaCl.sub.2)), 20 μL r ex-TEM® (recombinant tissue factor, phospholipids, heparin inhibitor), 10 μL inhibitor stock solution (720 μM-2.88 μM), combined with 10 μL t-PA stock solution (0.72 μg/mL) to yield concentrations of 360 μM-1.44 μM in 7.2% DMSO/PBS with 0.36 μg/mL t-PA and 300 μL 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 μg/mL t-PA in PBS.

(331) The FIG. 2 shows the thromboelastogram of whole human blood spiked with compound 5 at serial dilution 20 μM-0.63 μM compared to control in the presence of 0.02% t-PA

(332) In FIG. 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).

(333) 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.

(334) The potency of several published FXIII inhibitors (e.g. Ref. 22, Ref. 23, Ref. 24 as well as reference molecules Ref. 13, Ref. 14, Ref. 15, and Ref. 16) was evaluated using thromboelastometry. The detailed dose-dependent influence on key coagulation parameters of compound 5, as novel preferred compound, is shown in FIGS. 2, 3A and 3B. Compound 5 reduced the maximum clot firmness (MCFc, FIG. 3A) and increased the clot lysis at 60 minutes (LI60c, FIG. 3B) compared to control lacking the FXIII inhibitor. As detailed in the graphs 3A/3B, at a concentration of 2.5 μM, compound 5 lowers the clot firmness (MCFc) by 20% while increasing the lysis by 35% compared to control.

(335) The potency of Ref. 22, Ref. 23, Ref. 24 as well as reference molecules Ref. 13, Ref. 14, Ref. 15, and Ref. 16 were determined at fixed concentration of 2.5 μM in the same experimental setting. Spiked into whole human blood the compounds did not influence the clot parameters MCFc and LI60c. Therefore, the molecules were considered being not preferred, while missing the novel structural features claimed herein. In contrast, novel compounds 43, 44, and 45a/b (also spiked at 2.5 μM) showed efficacy in reducing clot firmness MCFc by 8%, 7%, and 17% (compare to FIG. 3A) while facilitating clot lysis to 14%, 11%, and 27% (LI60c, compare to FIG. 3B). Compounds 43, 44, and 45a/b are novel displaying the structural features claimed herein to design potent drug-like FXIII inhibitors.

(336) Further, the stability of compounds in plasma was determined as prerequisite for drug-likeness. Briefly, after spiking the chemical entities, the plasma was incubated at 37° C. At certain time points (e.g. at 15 and 120 minutes), 3 vol. cold MeOH were added to extract the compounds. After centrifugation, the supernatant was analyzed by HPLC. The half life was calculated based on the respective calibration curve. Compounds Ref. 22, Ref. 23, Ref. 24 as well as reference molecules Ref. 13, Ref. 14, Ref. 15, Ref. 16, Ref. 17, and Ref. 18 were unstable in plasma as indicated by plasma half life below 15 minutes. Therefore, they were considered being not suitable FXIII inhibitors due to the short half life in blood. These compounds are missing the novel structural features disclosed herein. In contrast, compounds 1a/b, 2a/b, 3, 4, 5a/b, 6, 7a/b, 8a/b, 9a/b, 10a/b, 11a/b, 12a/b, 13a/b, 14a/b, 15a/b, 16a/b, 17a/b, 18a/b, 19a/b, 20a/b, 21a/b, 22a/b, 23a/b, 24a/b, 25a/b, 26a/b, 27, 28, 29, 30, 31a/b, 32a/b, 33a/b, 34a/b, 35a/b, 36a/b, 37a/b, 38, 39a/b, 40, 41, 42a, 42b, 43, 44, and 45a/b were found to be stable in plasma over a period of 2 hours (>75% recovery each determined by HPLC).

(337) In contrast to the reference compounds exceeding a length of 7 amino acids, each inventive compound showed a plasma half-life exceeding 2 h so that the FXIII inhibitors discloses herein are sufficiently drug-like in contrast to the reference compounds having a half-life in blood of less than 15 minutes.

Example 2-4: Anti-Coagulation in a Rabbit Model of Venous Stasis and Reperfusion

(338) 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×PBS/5% glucose containing in mM: NaCl 273.8, NaH.sub.2PO.sub.4×2 H.sub.2O 14.2, KCl 5.4 and KH.sub.2PO.sub.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 μL of blood were collected from the femoral artery and supplemented with 45 μL of 0.25 M calcium chloride. Next, 25 μL of human α-thrombin (2.5 U/mL, Sigma-AkJrich) 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.

(339) 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.

(340) 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 (CI). 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.

(341) Statistical Analysis

(342) 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.

(343) Experimental schedule of the rabbit model of venous stasis and reperfusion is briefly presented in FIG. 4A.

(344) TABLE-US-00006 TABLE 4 Intravenous Injection Parameters Compound 5 Injection (=5a/b) Flow Total Time Concentration Volume Rate Dose Injection (min) (mg/mL) (mL) (mL/min) (mg) Bolus 1 4 8 8 32 Infusion 15 4 2 0.13 8 Infusion 135 (15 4 18 0.13 72 stasis + 120 post stasis)

(345) The in vivo experiment proofs significantly higher flow rates after compound 5 infusion compared to PBS control animals (FIG. 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 (FIG. 4C). The Thrombus wet weight is significantly reduced in the compound 5 group. Thrombus wet weight was determined after 135 min of infusion (FIG. 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 (FIG. 4E).