TEMPLATE-FIXED BETA-HAIRPIN PEPTIDOMIMETICS THAT ARE LIGANDS FOR G-PROTEIN-COUPLED RECEPTORS (GPCRS) AND ARE MODULATORS OF TRANSCRIPTION FACTORS AND COACTIVATORS

Abstract

Template-fixed β-hairpin peptidomimetics of the general formula

##STR00001##

wherein Z is a template-fixed chain of 8 α-amino acid residues which, depending on their positions in the chain (counted starting from the N-terminal amino acid), are Gly or Pro or of certain types which, as the remaining symbols in the above formula, are defined in the description and the claims, and salts thereof, have agonizing or antagonizing activity against urotensin II or show inhibition of the STAT6/NCoA-1 interaction and can be used for preventing or treating diseases or disorders related to urotensin II, STAT6 and NCoA-1.

These β-hairpin peptidomimetics can be manufactured by a process which is based on a mixed solid- and solution phase synthetic strategy.

Claims

1. Compounds of the general formula ##STR00035## is a dipeptide residue made up of two different amino acid building blocks, the dipeptide being .sup.DPro-.sup.LPro, .sup.DSer-.sup.LPro or .sup.DGlu-.sup.LPro; and Z is a chain made up of 8 alpha-amino acid residues, the positions of said amino acid residues in said chain being counted starting from the N-terminal amino acid, in which a P1 residue is a residue of Asp; a P2 residue is a residue of Cys; a P3 residue is a residue of Phe, Tyr; a P4 residue is a residue of Trp, .sup.DTrp; a P5 residue is a residue of Lys, Orn; a P6 residue is a residue of Tyr; a P7 residue is a residue of Cys, a P8 residue is a residue of Cha, Leu, Val; and two Cys, which are present as the P2 and the P7 residues, being linked by a disulfide bridge formed by replacement of the two —SH groups in the two residues of Cys by one —S—S-group; in free form or in pharmaceutically acceptable salt form, and wherein the compounds have an agonistic activity (EC 50%) of <2 nm, a human plasma stability (T.sub.1/2) of 240 minutes, a hemolysis value at 100 μM of 0% and a cytotoxicity value (GI.sub.50) of >50 μM, and wherein Z is selected from the group consisting of: SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, and wherein the compounds showing inhibition of the STAT6/NCoA-1 interaction being useful for treating renal disease, diabetes, cardiovascular dysfunction, inflammation as well as allergic airways diseases like allergic rhinitis and asthma.

2. A compound of formula I according to claim 1 wherein the template is the dipeptide residue .sup.DPro-.sup.LPro and the P1 residue is the residue of Asp; the P2 residue is the residue of Cys; the P3 residue is the residue of Phe; the P4 residue is the residue of .sup.DTrp; the P5 residue is the residue of Orn; the P6 residue is the residue of Tyr; the P7 residue is the residue of Cys; and the P8 residue is the residue of Val; two Cys, which are present as the P2 and the P7 residues, being linked by a disulfide bridge formed by replacement of the two —SH groups in the two residues of Cys by one —S—S-group.

3. Enantiomers of compounds of the general formula ##STR00036## is a dipeptide residue made up of two different amino acid building blocks, the dipeptide being .sup.DPro-.sup.LPro, .sup.DSer-.sup.LPro or .sup.DGlu-.sup.LPro; and Z is a chain made up of 8 alpha-amino acid residues, the positions of said amino acid residues in said chain being counted starting from the N-terminal amino acid, in which a P1 residue is a residue of Asp; a P2 residue is a residue of Cys; a P3 residue is a residue of Phe, Tyr; a P4 residue is a residue of Trp, .sup.DTrp; a P5 residue is a residue of Lys, Orn; a P6 residue is a residue of Tyr; a P7 residue is a residue of Cys, a P8 residue is a residue of Cha, Leu, Val; and two Cys, which are present as the P2 and the P7 residues, being linked by a disulfide bridge formed by replacement of the two —SH groups in the two residues of Cys by one —S—S-group; in free form or in pharmaceutically acceptable salt form, and wherein the compounds have an agonistic activity (EC 50%) of <2 nm, a human plasma stability (T.sub.1/2) of 240 minutes, a hemolysis value at 100 μM of 0% and a cytotoxicity value (GI.sub.50) of >50 μM, and wherein Z is selected from the group consisting of: SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, and wherein the compounds showing inhibition of the STAT6/NCoA-1 interaction being useful for treating renal disease, diabetes, cardiovascular dysfunction, inflammation as well as allergic airways diseases like allergic rhinitis and asthma.

4. A pharmaceutical composition containing a compound according to any one of claims 1 to 3 and a pharmaceutically inert carrier.

5. Compositions according to claim 4 in a form suitable for oral, topical, transdermal, injection, buccal, transmucosal, pulmonary or inhalation administration.

6. Compositions according to claim 5 in a form of tablets, dragees, capsules, solutions, liquids, gels, plaster, creams, ointments, syrup, slurries, suspensions, spray, nebuliser or suppositories.

7. The use of compounds according to any one of claims 1 to 3 for the manufacture of a medicament for use as an agonist or antagonist of urotensin II or an inhibitor of the STAT6/NCoA-1 interaction.

8. The use according to claim 7 wherein said urotensin II agonizing or antagonizing or STAT6/NCoA-1 interaction inhibiting medicament is intended to be used in cases where renal disease is mediated or resulting from, or where diabetes is mediated or resulting from, or where cardiovascular dysfunction is mediated or resulting from, or where inflammation is mediated or resulting from urotensin II activity; or where allergic airways diseases like allergic rhinitis and asthma are mediated or resulting from the STAT6/NCoA-1 interaction.

9. A process for the manufacture of compounds according to claim 1 which process comprises (a) coupling an appropriately functionalized solid support with an appropriately N-protected derivative of that amino acid which in the desired end-product is in positions 3, 4 or 5, any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (b) removing the N-protecting group from the product thus obtained; (c) coupling the product thus obtained with an appropriately N-protected derivative of that amino acid which in the desired end-product is one position nearer the N-terminal amino acid residue, any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (d) removing the N-protecting group from the product thus obtained; (e) repeating steps (c) and (d) until the N-terminal amino acid residue has been introduced; (f) coupling the product thus obtained with a compound of the general formula ##STR00037## is as defined in claim 1 and X is an N-protecting group or, alternatively, (fa) coupling the product obtained in step (e) with an appropriately N-protected derivative of an amino acid of the general formula
HOOC-B-H  III or
HOOC-A-H  IV wherein B and A are as defined in claim 1, any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (fb) removing the N-protecting group from the product thus obtained; and (fc) coupling the product thus obtained with an appropriately N-protected derivative of an amino acid of the above general formula IV or formula
HOOC-B3-H  V wherein B3 is as defined in claim 1 and, respectively, formula III, any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (g) removing the N-protecting group from the product obtained in step (f) or (fc); (h) coupling the product thus obtained with an appropriately N-protected derivative of that amino acid which in the desired end-product is in position, 8 any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (i) removing the N-protecting group from the product thus obtained; (j) coupling the product thus obtained with an appropriately N-protected derivative of that amino acid which in the desired end-product is one position farther away from position 8, any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (k) removing the N-protecting group from the product thus obtained; (l) repeating steps (j) and (k) until all amino acid residues have been introduced; (m) if desired, selectively deprotecting one or several protected functional group(s) present in the molecule and appropriately substituting the reactive group(s) thus liberated; (n) if desired, forming an interstrand linkage between side-chains of appropriate amino acid residues at positions 2 and 7; (o) detaching the product thus obtained from the solid support; (p) cyclizing the product cleaved from the solid support; (q) removing any protecting groups present on functional groups of any members of the chain of amino acid residues and, if desired, any protecting group(s) which may in addition be present in the molecule; and (r) if desired, converting the product thus obtained into a pharmaceutically acceptable salt or converting a pharmaceutically acceptable, or unacceptable, salt thus obtained into the corresponding free compound of formula I or into a different, pharmaceutically acceptable, salt.

10. A process for the manufacture of compounds according to claim 1 which process comprises (a′) coupling an appropriately functionalized solid support with a compound of the general formula ##STR00038## is as defined in claim 1 and X is an N-protecting group or, alternatively, (a′a) coupling said appropriately functionalized solid support with an appropriately N-protected derivative of an amino acid of the general formula
HOOC-B-H  III or
HOOC-A-H  IV wherein B and A are as defined in claim 1, any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (a′b) removing the N-protecting group from the product thus obtained; and (a′c) coupling the product thus obtained with an appropriately N-protected derivative of an amino acid of the above general formula IV or formula
HOOC-B3-H  V wherein B3 is as defined in claim 1 and, respectively, formula III, any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (b′) removing the N-protecting group from the product obtained in step (a′) or (a′c) (c′) coupling the product thus obtained with an appropriately N-protected derivative of that amino acid which in the desired end-product is in position 8, any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (d′) removing the N-protecting group from the product thus obtained; (e′) coupling the product thus obtained with an appropriately N-protected derivative of that amino acid which in the desired end-product is one position farther away from position 8, any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (f′) removing the N-protecting group from the product thus obtained; (g′) repeating steps (e′) and (f′) until all amino acid residues have been introduced; (h′) if desired, selectively deprotecting one or several protected functional group(s) present in the molecule and appropriately substituting the reactive group(s) thus liberated; (i′) if desired forming an interstrand linkage between side-chains of appropriate amino acid residues at opposite positions 2 and 7; (j′) detaching the product thus obtained from the solid support; (k′) cyclizing the product cleaved from the solid support; (l′) removing any protecting groups present on functional groups of any members of the chain of amino acid residues and, if desired, any protecting group(s) which may in addition be present in the molecule; and (m′) if desired, converting the product thus obtained into a pharmaceutically acceptable salt or converting a pharmaceutically acceptable, or unacceptable, salt thus obtained into the corresponding free compound of formula I or into a different, pharmaceutically acceptable, salt.

11. A process for the manufacture of compounds according to claim 3 which process comprises (a) coupling an appropriately functionalized solid support with an appropriately N-protected derivative of that amino acid which in the desired end-product is in positions 3, 4 or 5, any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (b) removing the N-protecting group from the product thus obtained; (c) coupling the product thus obtained with an appropriately N-protected derivative of that amino acid which in the desired end-product is one position nearer the N-terminal amino acid residue, any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (d) removing the N-protecting group from the product thus obtained; (e) repeating steps (c) and (d) until the N-terminal amino acid residue has been introduced; (f) coupling the product thus obtained with a compound of the general formula ##STR00039## is as defined in claim 3 and X is an N-protecting group or, alternatively, (fa) coupling the product obtained in step (e) with an appropriately N-protected derivative of an amino acid of the general formula
HOOC-B-H  III or
HOOC-A-H  IV wherein B and A are as defined in claim 3, any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (fb) removing the N-protecting group from the product thus obtained; and (fc) coupling the product thus obtained with an appropriately N-protected derivative of an amino acid of the above general formula IV or formula
HOOC-B3-H  V wherein B3 is as defined in claim 3 and, respectively, formula III, any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (g) removing the N-protecting group from the product obtained in step (f) or (fc); (h) coupling the product thus obtained with an appropriately N-protected derivative of that amino acid which in the desired end-product is in position, 8 any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (i) removing the N-protecting group from the product thus obtained; (j) coupling the product thus obtained with an appropriately N-protected derivative of that amino acid which in the desired end-product is one position farther away from position 8, any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (k) removing the N-protecting group from the product thus obtained; (l) repeating steps (j) and (k) until all amino acid residues have been introduced; (m) if desired, selectively deprotecting one or several protected functional group(s) present in the molecule and appropriately substituting the reactive group(s) thus liberated; (n) if desired, forming an interstrand linkage between side-chains of appropriate amino acid residues at positions 2 and 7; (o) detaching the product thus obtained from the solid support; (p) cyclizing the product cleaved from the solid support; (q) removing any protecting groups present on functional groups of any members of the chain of amino acid residues and, if desired, any protecting group(s) which may in addition be present in the molecule; and (r) if desired, converting the product thus obtained into a pharmaceutically acceptable salt or converting a pharmaceutically acceptable, or unacceptable, salt thus obtained into the corresponding free compound of formula I or into a different, pharmaceutically acceptable, salt.

12. A process for the manufacture of compounds according to claim 3 which process comprises (a′) coupling an appropriately functionalized solid support with a compound of the general formula ##STR00040## is as defined in claim 1 and X is an N-protecting group or, alternatively, (a′a) coupling said appropriately functionalized solid support with an appropriately N-protected derivative of an amino acid of the general formula
HOOC-B-H  III or
HOOC-A-H  IV wherein B and A are as defined in claim 3, any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (a′b) removing the N-protecting group from the product thus obtained; and (a′c) coupling the product thus obtained with an appropriately N-protected derivative of an amino acid of the above general formula IV or formula
HOOC-B3-H  V wherein B3 is as defined in claim 3 and, respectively, formula III, any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (b′) removing the N-protecting group from the product obtained in step (a′) or (a′c) (c′) coupling the product thus obtained with an appropriately N-protected derivative of that amino acid which in the desired end-product is in position 8, any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (d′) removing the N-protecting group from the product thus obtained; (e′) coupling the product thus obtained with an appropriately N-protected derivative of that amino acid which in the desired end-product is one position farther away from position 8, any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (f′) removing the N-protecting group from the product thus obtained; (g′) repeating steps (e′) and (f′) until all amino acid residues have been introduced; (h′) if desired, selectively deprotecting one or several protected functional group(s) present in the molecule and appropriately substituting the reactive group(s) thus liberated; (i′) if desired forming an interstrand linkage between side-chains of appropriate amino acid residues at opposite positions 2 and 7; (j′) detaching the product thus obtained from the solid support; (k′) cyclizing the product cleaved from the solid support; (l′) removing any protecting groups present on functional groups of any members of the chain of amino acid residues and, if desired, any protecting group(s) which may in addition be present in the molecule; and (m′) if desired, converting the product thus obtained into a pharmaceutically acceptable salt or converting a pharmaceutically acceptable, or unacceptable, salt thus obtained into the corresponding free compound of formula I or into a different, pharmaceutically acceptable, salt.

13. A method for treating a disease in a patient, comprising: administering the pharmaceutical composition according to claim 4 to a patient in need thereof, and wherein the disease to be treated is renal disease, diabetes, cardiovascular dysfunction, inflammation as well as allergic airways diseases.

14. The method according to claim 13, wherein the allergic airways diseases are allergic rhinitis and asthma.

15. The compounds of claim 1, wherein the compounds show inhibition of the STAT6/NCoA-1 interaction being useful for treating renal disease, diabetes, cardiovascular dysfunction, inflammation as well as allergic airways diseases like allergic rhinitis and asthma.

Description

EXAMPLES

1. Peptide Synthesis

Coupling of the First Protected Amino Acid Residue to the Resin

[0929] 1 g (1.4 mMol) of 2-chlorotritylchloride resin (1.4 mMol/g; Barlos et al. Tetrahedron Lett. 1989, 30, 3943-3946) was filled into a dried flask. The resin was suspended in CH.sub.2Cl.sub.2 (5 ml) and allowed to swell at room temperature under constant shaking for 30 min. A solution of 0.98 mMol (0.7 eq) of the first suitably protected amino acid residue (see below) in CH.sub.2Cl.sub.2 (5 ml) completed by 960 μl (4 eq) of diisopropylethylamine (DIEA) was added. After shaking the reaction mixture for 4 hours at 25° C. the resin was filtered and washed successively with CH.sub.2Cl.sub.2 (1×), DMF (1×) and CH.sub.2Cl.sub.2 (1×). A solution of CH.sub.2C12/MeOH/DIEA (17/2/1, 10 ml) was added to the resin and the suspension was shaken for 30 min. After filtration the resin was washed in the following order with CH.sub.2Cl.sub.2 (1×), DMF (1×), CH.sub.2Cl.sub.2 (1×), MeOH (1×), CH.sub.2Cl.sub.2 (1×), MeOH (1×), CH.sub.2Cl.sub.2 (2×), Et.sub.2O (2×) and dried under vacuum for 6 hours.

[0930] Loading was typically 0.6-0.7 mMol/g.

[0931] The following preloaded resins were prepared: Fmoc-ProO-chlorotritylresin, Fmoc-4Hyp2(tBu)O-chlorotritylresin, Fmoc-OicO-chlorotritylresin, and Fmoc-4Mpl(Trt)O-chloro-tritylresin.

[0932] The synthesis was carried out employing a Syro-peptide synthesizer (MultiSynTech) using 24-96 reaction vessel. In each vessel 0.04 mMol of the above resin were placed and the resin was swollen in CH.sub.2Cl.sub.2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out:

TABLE-US-00002 Step Reagent Time 1 DMF, wash and swell 2 × 1 min 2 20% piperidine/DMF 1 × 5 min, 1 × 15 min 3 DMF, wash 5 × 1 min  .sup. 4a 5 eq Fmoc amino acid/DMF + 5 eq HCTU/DMF, 10 eq DIEA 1 × 60 min 5 DMF, wash 3 × 1 min

[0933] Step 4a was repeated once.

[0934] If not indicated otherwise, the final coupling of an amino acid is followed by an Fmoc deprotection by applying steps 1-3 of the above described reaction cycle.

[0935] The following Fmoc-protected amino acid derivative had to be synthesized before its usage in the linear peptide synthesis described above.

Synthesis of N-Fmoc-protected L-6-chlorotryptophan

[0936] (modified procedure following E. Atherton, R. Sheppard, Solid phase peptide synthesis. A practical approach, IRL Press, Oxford, 1989, page 49).

Cyclization and Work Up of Backbone Cyclized Peptides

Cleavage of the Fully Protected Peptide Fragment

[0937] After completion of the synthesis, the resin (0.04 mMol) was suspended in 1 ml (0.13 mMol, 3.4 eq) of 1% TFA in CH.sub.2Cl.sub.2 (v/v) for 3 minutes, filtered, and the filtrate was neutralized with 1 ml (0.58 mMol, 14.5 eq) of 10% DIEA in CH.sub.2Cl.sub.2 (v/v). This procedure was repeated three times to ensure completion of the cleavage. The filtrate was evaporated to dryness and a sample of the product was fully deprotected by using a cleavage mixture containing 95% trifluoroacetic acid (TFA), 2.5% water and 2.5% triisopropylsilane (TIS) to be analyzed by reverse phase-HPLC (column C.sub.18) and ESI-MS to monitor the efficiency of the linear peptide synthesis.

Cyclization of the Linear Peptide

[0938] The fully protected linear peptide (0.04 mMol) was dissolved in DMF (4 Mol/ml). Then 30.4 mg (0.08 mMol, 2 eq) of HATU, 10.9 mg (0.08 mMol, 2 eq) of HOAt and 28 μl (0.16 mMol, 4 eq) DIEA were added, and the mixture was vortexed at 25° C. for 16 hours and subsequently concentrated under high vacuum. The residue was partitioned between CH.sub.2Cl.sub.2 and H.sub.2O/CH.sub.3CN (90/10; v/v). The CH.sub.2Cl.sub.2 phase was evaporated to yield the fully protected cyclic peptide.

Fully Deprotecting the Cyclic Peptide

[0939] The cyclic peptide obtained was dissolved in 3 ml of the cleavage mixture containing 82.5% trifluoroacetic acid (TFA), 5% water, 5% thioanisole, 5% phenol and 2.5% ethandithiole (EDT). The mixture was allowed to stand at 25° C. for 2.5 hours and thereafter concentrated under vacuum. After precipitation of the cyclic fully deprotected peptide in diethylether (Et.sub.2O) at 0° C. the solid was washed twice with Et.sub.2O and dried. Cyclic peptides without designed β-strand linkages were purified by reverse phase HPLC, cyclic peptides arranged for additional β-strand linkages were processed as described below.

Formation of Disulfide β-Strand Linkage and Purification

[0940] After deprotection, the crude peptide was dissolved in 9 ml of 5% AcOH (buffered with NaHCO.sub.3 to pH 5-6). 0.5 ml DMSO were added and the solution was shaken overnight. Following evaporation the residue was purified by preparative reverse phase HPLC.

Analytical Method 1a:

[0941] Analytical HPLC retention times (RT, in minutes) were determined using an Acquity UPLC BEH C18 1.7 μm column with the following solvents A (H.sub.2O/CH.sub.3CN, 95/5 [v/v], +0.1% TFA) and B (CH.sub.3CN+0.09% TFA) and the gradient: 0 min: 99% A, 1% B; 0.2 min: 99% A, 1% B; 4 min: 5% A, 95% B; 4.2 min: 5% A, 95% B; 4.25 min: 99% A, 1% B; 5.0 min: 99% A, 1% B.

Analytical Method 1b:

[0942] Analytical HPLC retention times (RT, in minutes) were determined using an Acquity UPLC BEH C18 1.7 am column with the following solvents A (H.sub.2O+0.1% TFA) and B (CH.sub.3CN+0.09% TFA) and the gradient: 0 min: 95% A, 5% B; 0.2 min: 95% A, 5% B; 4 min: 5% A, 95% B; 4.2 min: 5% A, 95% B; 4.25 min: 95% A, 5% B; 5.0 min: 95% A, 5% B.

Analytical Method 2:

[0943] Analytical HPLC retention times (RT, in minutes) were determined using an Acquity UPLC BEH C18 1.7 am column with the following solvents A (H.sub.2O/CH.sub.3CN, 95/5 [v/v], +0.1% TFA) and B (CH.sub.3CN+0.09% TFA) and the gradient: 0 min: 99% A, 1% B; 0.2 min: 99% A, 1% B; 4 min: 35% A, 65% B; 4.05 min: 5% A, 95% B; 4.20 min: 5% A, 95% B; 4.25 min: 99% A, 1% B; 4.5 min: 99% A, 1% B.

Analytical Method 3:

[0944] Analytical HPLC retention times (RT, in minutes) were determined using a zorbax Eclipsed XDB C18 column with the following solvents A (H.sub.2O+0.1% TFA) and B (CH.sub.3CN+0.1% TFA) and the gradient: 0 min: 60% A, 40% B; 21 min: 10% A, 90% B; 27 min: 100% B.

Analytical Method 4:

[0945] Analytical HPLC retention times (RT, in minutes) were determined using a Laubscher Labs Interchrom 218QTP54 C18, 250×4.6 mm, 5 m, 300 A with the following solvents A (H.sub.2O+0.1% TFA) and B (CH.sub.3CN+0.1% TFA) and the gradient: 0 min: 70% A, 30% B; 16.7 min: 100% B.

[0946] Examples 1, 4 and 6-27 are shown in Table 1. The peptides were synthesized starting with the amino acid Pro which was grafted to the resin. Starting resin was Fmoc-ProO-chlorotrityl resin, which was prepared as described above. The linear peptide was synthesized on solid support according to the procedure described above in the following sequence: Resin-Pro-.sup.DPro-P8-P7-P6-P5-P4-P3-P2-P1. Following a final Fmoc deprotection as described above, the peptide was cleaved from the resin, cyclized, deprotected and purified as indicated above.

[0947] For Ex. 4 HPLC-retention times (minutes) were determined using the gradient method 1b, for Ex. 6, 9-10, 15-16 and 26-27 the gradient method 1a was applied; for Ex. 7-8, 11-12 and 19-25 HPLC-retention times (minutes) were determined using the gradient method 3 and finally for Ex. 1, 13-14 and 17-18 HPLC-retention times were identified by using method 4 as described above.

[0948] Example 2 is shown in Table 1. The peptide was synthesized starting with the amino acid Hyp which was grafted to the resin. Starting resin was Fmoc-4Hyp2(tBu)O-chlorotrityl resin, which was prepared as described above. The linear peptide was synthesized on solid support according to the procedure described above in the following sequence: Resin-Hyp-.sup.DPro-P8-P7-P6-P5-P4-P3-P2-P1. Following a final Fmoc deprotection as described above, the peptide was cleaved from the resin, cyclized, deprotected and purified as indicated above.

[0949] HPLC-retention times (minutes) was determined using the gradient method 1 as described above.

[0950] Example 3 is shown in Table 1. The peptide was synthesized starting with the amino acid Oic which was grafted to the resin. Starting resin was Fmoc-OicO-chlorotrityl resin, which was prepared as described above. The linear peptide was synthesized on solid support according to the procedure described above in the following sequence: Resin-Oic-.sup.DPro-P8-P7-P6-P5-P4-P3-P2-P1. Following a final Fmoc deprotection as described above, the peptide was cleaved from the resin, cyclized, deprotected and purified as indicated above.

[0951] HPLC-retention time (minutes) was determined using the gradient method 1 as described above.

[0952] Example 5 is shown in Table 1. The peptide was synthesized starting with the amino acid Mp1 which was grafted to the resin. Starting resin was Fmoc-4Mp1(Trt)O-chlorotrityl resin, which was prepared as described above. The linear peptide was synthesized on solid support according to the procedure described above in the following sequence: Resin-4Mp1-.sup.DPro-P8-P7-P6-P5-P4-P3-P2-P1. Following a final Fmoc deprotection as described above, the peptide was cleaved from the resin, cyclized, deprotected and purified as indicated above.

[0953] HPLC-retention time (minutes) was determined using the gradient method 1 as described above

[0954] Example 28 is shown in Table 1. The peptide was synthesized starting with the amino acid Pro which was grafted to the resin. Starting resin was Fmoc-ProO-chlorotrityl resin, which was prepared as described above. The linear peptide was synthesized on solid support according to the procedure described above in the following sequence: Resin-Pro-.sup.DSer-P8-P7-P6-P5-P4-P3-P2-P1. Following a final Fmoc deprotection as described above, the peptide was cleaved from the resin, cyclized, deprotected and after formation of the disulfide β-strand linkage purified as indicated above.

[0955] HPLC-retention time (minutes) was determined using the gradient method 2 as described above

[0956] Example 29 is shown in Table 1. The peptide was synthesized starting with the amino acid Pro which was grafted to the resin. Starting resin was Fmoc-ProO-chlorotrityl resin, which was prepared as described above. The linear peptide was synthesized on solid support according to the procedure described above in the following sequence: Resin-Pro-.sup.DHyp-P8-P7-P6-P5-P4-P3-P2-P1. Following a final Fmoc deprotection as described above, the peptide was cleaved from the resin, cyclized, deprotected and after formation of the disulfide 3-strand linkage purified as indicated above.

[0957] HPLC-retention time (minutes) was determined using the gradient method 2 as described above

[0958] Example 30 is shown in Table 1. The peptide was synthesized starting with the amino acid Pro which was grafted to the resin. Starting resin was Fmoc-ProO-chlorotrityl resin, which was prepared as described above. The linear peptide was synthesized on solid support according to the procedure described above in the following sequence: Resin-Pro-.sup.DGlu-P8-P7-P6-P5-P4-P3-P2-P1. Following a final Fmoc deprotection as described above, the peptide was cleaved from the resin, cyclized, deprotected and after formation of the disulfide β-strand linkage purified as indicated above.

[0959] HPLC-retention time (minutes) was determined using the gradient method 2 as described above

[0960] Examples 31-35 are shown in Table 1. The peptides were synthesized starting with the amino acid Pro which was grafted to the resin. Starting resin was Fmoc-ProO-chlorotrityl resin, which was prepared as described above. The linear peptides were synthesized on solid support according to the procedure described above in the following sequence: Resin-Pro-.sup.DPro-P8-P7-P6-P5-P4-P3-P2-P1. Following a final Fmoc deprotection as described above, the peptides were cleaved from the resin, cyclized, deprotected and after formation of the disulfide β-strand linkage purified as indicated above.

[0961] HPLC-retention times (minutes) were determined using the gradient method 2 as described above

TABLE-US-00003 TABLE 1 Examples (Ex.) Ex. Seq ID P1 P2 P3 P4 P5 P6 P7 P8 Template Purity %.sup.a) [M + H] RT 1 SEQ ID NO: 1 Phe Glu Trp(6Cl) Leu Ala Trp Glu Phe .sup.DPro.sup.LPro 95 1338.7 12.20 2 SEQ ID NO: 2 Phe Glu Trp Leu Ala Trp Glu Phe .sup.DPro.sup.L4Hyp2 90 1319.9 3.20 3 SEQ ID NO: 3 Phe Glu Trp Leu Ala Trp Glu Trp .sup.DPro.sup.LOic 95 1357.7 3.71 4 SEQ ID NO: 4 Phe Glu Trp Leu .sup.DAla Trp Glu Phe .sup.DPro.sup.LPro 95 1303.7 3.49 5 SEQ ID NO: 5 Phe Thr Trp(6Cl) Leu Ala Trp Glu Phe .sup.DPro.sup.L4Mp1 85 1369.9 3.65 6 SEQ ID NO: 6 Phe Glu Trp(6Cl) Leu Ala OctG Glu Phe .sup.DPro.sup.LPro 90 1320.9 4.24 7 SEQ ID NO: 7 Phe Glu Trp Gly Ala Trp Glu Phe .sup.DPro.sup.LPro 95 1247.7 12.70 8 SEQ ID NO: 8 Phe Glu Trp Leu Gly Trp Glu Phe .sup.DPro.sup.LPro 95 1289.8 14.70 9 SEQ ID NO: 9 Phe Glu Trp Leu Ala Trp Glu Glu(cHx) .sup.DPro.sup.LPro 90 1402.0 3.82 10 SEQ ID NO: 10 Phe Glu Trp Leu Ala Trp Glu Ile .sup.DPro.sup.LPro 60 1269.7 3.48 11 SEQ ID NO: 11 Phe Glu Trp Tyr Ala Trp Glu Phe .sup.DPro.sup.LPro 95 1354.7 12.90 12 SEQ ID NO: 12 Phe Glu Trp Leu Tyr Trp Glu Phe .sup.DPro.sup.LPro 92 1396.8 15.10 13 SEQ ID NO: 13 Phe Glu Trp(6Cl) Leu Asp Trp Ala Phe .sup.DPro.sup.LPro 95 1323.5 11.60 14 SEQ ID NO: 14 Phe Ala Trp(6Cl) Leu Asp Trp Glu Phe .sup.DPro.sup.LPro 95 1325.6 12.10 15 SEQ ID NO: 15 Phe Glu Trp(6Cl) Cys Ala Trp Glu Phe .sup.DPro.sup.LPro 50 1327.6 3.48 16 SEQ ID NO: 16 Phe Glu Trp Leu Asp Trp Glu Met .sup.DPro.sup.LPro 80 1331.9 3.25 17 SEQ ID NO: 17 Phe Glu Ala Leu Asp Trp Glu Phe .sup.DPro.sup.LPro 95 1332.6 10.00 18 SEQ ID NO: 18 Phe Glu Trp(6Cl) Leu Asp Ala Glu Phe .sup.DPro.sup.LPro 95 1266.5 10.30 19 SEQ ID NO: 19 Phe Glu Trp Leu Asp Trp Glu Phe .sup.DPro.sup.LPro 95 1347.7 13.80 20 SEQ ID NO: 20 Phe Glu Trp Leu Ala Trp Glu Glu .sup.DPro.sup.LPro 95 1285.8 9.40 21 SEQ ID NO: 21 Phe Glu Trp Leu Ala Trp Tyr Phe .sup.DPro.sup.LPro 95 1337.9 16.00 22 SEQ ID NO: 22 Phe Leu Trp Leu Ala Trp Glu Phe .sup.DPro.sup.LPro 95 1287.7 16.20 23 SEQ ID NO: 23 Glu Glu Trp Leu Ala Trp Glu Phe .sup.DPro.sup.LPro 95 1285.7 9.30 24 SEQ ID NO: 24 Phe Glu Trp Leu Ala Trp Leu Phe .sup.DPro.sup.LPro 95 1287.9 16.70 25 SEQ ID NO: 25 Cha Glu Trp Leu Ala Trp Glu Phe .sup.DPro.sup.LPro 90 1309.8 17.20 26 SEQ ID NO: 26 Cha Glu Trp Leu Ala Trp Glu Cha .sup.DPro.sup.LPro 80 1315.4 4.00 27 SEQ ID NO: 27 Met Glu Trp Leu Asp Trp Glu Phe .sup.DPro.sup.LPro 80 1331.9 4.29 28 SEQ ID NO: 28 Asp Cys Phe Trp Lys Tyr Cys Leu .sup.DSer.sup.LPro 95 1243.0 3.12 29 SEQ ID NO: 29 Asp Cys Phe Trp Lys Tyr Cys Leu .sup.D4Hyp2.sup.LPro 95 1267.8 3.05 30 SEQ ID NO: 30 Asp Cys Phe Trp Lys Tyr Cys Leu .sup.DGlu.sup.LPro 90 1283.5 3.10 31 SEQ ID NO: 31 Asp Cys Phe Trp Lys Tyr Cys Val .sup.DPro.sup.LPro 90 1237.3 3.11 32 SEQ ID NO: 32 Asp Cys Phe .sup.DTrp Orn Tyr Cys Val .sup.DPro.sup.LPro 90 1223.4 3.06 33 SEQ ID NO: 33 Asp Cys Tyr Trp Lys Tyr Cys Leu .sup.DPro.sup.LPro 90 1267.5 2.95 34 SEQ ID NO: 34 Asp Cys Phe .sup.DTrp Lys Tyr Cys Val .sup.DPro.sup.LPro 85 1237.5 3.07 35 SEQ ID NO: 35 Asp Cys Phe Trp Lys Tyr Cys Cha .sup.DPro.sup.LPro 90 1292.8 3.60 Cys in pos. 2 and 7 in Ex. 28-35 form a disulfide bridge, .sup.a)%-purity of compounds after prep. HPLC.

2. Biological Methods

2.1. Preparation of the Peptides

[0962] Lyophilized peptides were weighed on a Microbalance (Mettler MT5) and dissolved in sterile water to a final concentration of 1 mM unless stated otherwise. Stock solutions were kept at +4° C., light protected.

2.2. Cell Culture

[0963] Mouse pre-B cells were cultured in RPMI1640 plus 5% FBS, antibiotic/antimycotic, non essential amino acid, 50 μM β-mercaptoethanol and 1 mM natrium pyruvate. HELA cells were maintained in RPMI1640 plus 10% FBS, pen/strept and 2 mM L-glutamine. Cos-7 cells were grown in DMEM medium with 4500 mg/mL glucose supplemented with 10% FCS, pen/strept and 2 mM L-glutamine. All cell lines were grown at 37° C. at 5% CO.sub.2. Cell media, media supplements, PBS-buffer, HEPES, antibiotic/antimycotic, pen/strept, non essential amino acid, L-glutamine, β-mercaptoethanol and sera were purchased from Gibco (Pailsey, UK). All fine chemicals were supplied by Merck (Darmstadt, Germany).

2.3. Ca.SUP.2+− Assay: UTR2 Receptor-Agonizing and Antagonizing Activity of the Peptides

[0964] The mouse pre-B cell line 300-19 was stably transfected with the cDNA encoding the human UTR2 receptor (GenBank Acc# NM_018949), and expression was confirmed with a positive calcium signal in response to human urotensin (Sigma Aldrich). Increases in intracellular calcium were monitored using a Flexstation 384 (Molecular Devices, Sunnyvale, Calif.). The cells were batch loaded with the Calcium 4 Assay kit (Molecular Devices) in assay buffer (Hanks Balanced salt solution, HBSS, 20 mM HEPES, pH 7.4, 0.1% BSA) for 1 h at room temperature and labeled cells were dispensed into either black 96 well or 384 well assay plates (Greiner). Calcium mobilization induced by urotensin or test compounds was measured in the Flexstation 384 (excitation, 485 nm; emission, 525 nm) for 70 seconds. Agonist activity was determined by direct addition of ligand or peptides, while antagonists were identified by spiking the cells with test compounds prior to urotensin addition. A dose response curve (compound concentration versus % maximum response for urotensin) was determined for each active agonist and antagonist and was fitted to a four parameter logistic equation using SoftmaxPro 4.8 (Molecular Devices), from which EC50% and IC50% values were calculated.

2.4. Fluorescence Polarization Assay: NCoA-1 Binding Affinities of Peptides

[0965] A fluorescence polarization (FP) assay was established to determine the NCoA-1 binding affinities of the peptides (M. Seitz, L. T. Maillard, D. Obrecht, J. A. Robinson, ChemBioChem 2008, 9, 1318) starting with the K.sub.D-determination of the FluoSTAT6 (N-terminal fluorescein-labeled STAT6 794-814 peptide)—NCoA-1 complex: Solutions containing FluoSTAT6 (1 M final concentration) and NCoA-1 (0-14 M final concentration) were prepared in HEPES buffer (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.1% BSA) and dispensed in a black 96 well plate (Greiner). The mixtures were shaken thoroughly. Fluorescence polarization was measured on a SPECTRAmax M5 spectrometer (Molecular device) following 5-30 min incubation at room temperature. FluoSTAT6 was excited at 490 nm and emission polarization was detected at 525 nm. 40 intensity measurements were collected for each well, 20 at horizontal position of the dynamic polarizer, 20 at parallel position. As the fraction of FluoSTAT6 bound to NCoA-1 is correlated to the fluorescence polarization (F.sub.P), after normalization, the fraction of bound FluoSTAT6 (B) was determined, and the K.sub.D was calculated according to B=[(1+P/R+K.sub.D/R)−((1+P/R+K.sub.D/R).sup.2−4P/R).sup.0.5]/2, wherein P is the total probe concentration, R the total protein concentration and K.sub.D the dissociation constant (M. H. Roehrl, J. Y. Wang, G. Wagner, Biochemistry 2004, 3, 16056).

[0966] To determine the K.sub.i values of potential inhibitors of the STAT6/NCoA-1 interaction (competition fluorescence polarization) the compounds (dilution series from 0 to 100 μM final concentration) dissolved in HEPES buffer (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.1% BSA) were dispensed in a black 96 well plate (Greiner) and FluoSTAT6 (200 nM final concentration) in HEPES buffer was added. The mixture was completed by a HEPES buffer solution of NCoA-1 (1 μM final concentration) and processed as described above. STAT6Y (C-terminal tyrosine extended STAT6 794-814 peptide) was used as a positive control. As the total fluorescence intensity of FluoSTAT6 remains similar for all samples, the fraction of peptide bound to NCoA-1 is correlated to the fluorescence polarization.

[0967] Following normalization data were fitted with IGORpro Software® (WaveMetrics, Lake Oswego, Oreg., USA) to a sigmoid equation to determine IC.sub.50 values. The K.sub.i values were calculated from IC.sub.50 values according to the method described by Nikolovska-Coleska et al. Anal. Biochem., 2004, 332, 261).

2.5. Cytotoxicity Assay

[0968] The cytotoxicity of the peptides to HELA cells (Acc57) and COS-7 cells (CRL-1651) was determined using the MTT reduction assay. Briefly, the method was as follows: 7000 HELA cells/well and 4500 COS-7 cells/well were seeded and grown in 96-well microtiter plates for 24 h at 37° C. at 5% CO.sub.2. Thereafter, time zero (Tz) was determined by MTT reduction (see below). The supernatant of the remaining wells was discarded, and fresh medium and compounds in serial dilutions (12.5, 25 and 50 μM, triplicates) were pipetted into the wells. After incubation of the cells for 48 h at 37° C. at 5% CO.sub.2 the supernatant was discarded again and 100 μL MTT reagent (0.5 mg/mL in RPMI1640 and DMEM, respectively)/well was added. Following incubation at 37° C. for 2 h the media were aspirated and the cells were spiked (100 μl isopropanol/well). The absorbance of the solubilized formazan was measured at 595 nm (OD.sub.595peptide). For each concentration averages were calculated from triplicates. The percentage of growth was calculated as follows: (OD.sub.595peptide-OD.sub.595Tz-OD.sub.595Empty well)/(OD.sub.595Tz-OD.sub.595Empty well)×100%. The GI.sub.50 (Growth Inhibition) concentrations were calculated for each peptide by using a trend line function for the concentrations (50, 25, 12.5 and 0 μM), the corresponding percentages and the value 50, (=TREND (C.sub.50:C.sub.0,%.sub.50:%.sub.0,50).

2.6. Hemolysis

[0969] The peptides were tested for their hemolytic activity against human red blood cells (hRBC). Fresh hRBC were washed three times with phosphate buffered saline (PBS) and centrifuged for 10 min at 2000×g. Compounds (100 μM) were incubated with 20% hRBC (v/v) for 1 h at 37° C. The final erythrocyte concentration was approximately 0.9×10.sup.9 cells/mL. A value of 0% and 100% cell lyses, respectively, was determined by incubation of hRBC in the presence of PBS alone and 0.1% Triton X-100 in H.sub.2O, respectively. The samples were centrifuged, the supernatants were 20-fold diluted in PBS buffer and the optical densities (OD) were measured at 540 nm. The 100% lyses value (OD.sub.540H.sub.2O) gave an OD.sub.540 of approximately 1.3-1.8. Percent hemolysis was calculated as follows:

(OD.sub.540peptide/OD.sub.540H.sub.2O)×100%.

2.7. Plasma Stability

[0970] The stability of the peptides in human and mouse plasma was determined by applying the following method: 315 μl/deep well of freshly thawed human plasma (Basler Blutspende-dienst) and mouse plasma (Harlan Sera-Lab, UK), respectively, were spiked with 35 μl/well of compound in PBS (100 μM, triplicate) and incubated at 37° C. At t=0, 15, 30, 60, 120 and 240 min aliquots of 50 μl were transferred to filtration plate wells containing 150 μl/well of acetonitrile. Following shaking for 2 min the occurred suspensions were filtrated by vacuum and finally. 100 μl of each filtrate were transferred to a propylene microtiter plate, and analyzed by LC/MS as follows: Column: Waters, XBridge C18, mobile phases: (A) water+0.1% formic acid and (B) acetonitrile/water, 95/5 (v/v)+0.1% formic acid, gradient: 5%-100% (B) in 2 minutes, electrospray ionization, MRM detection (triple quadrupole). The peak areas were determined and triplicate values were averaged. The stability was expressed in percent of the initial value at t=0. (t.sub.x/t.sub.0×100). By using the TREND function of EXCEL (Microsoft Office 2003) T.sub.1/2 were determined.

TABLE-US-00004 TABLE 1 Ex. EC50% [nM], Urotensin II receptor 28 <2 29 <2 30 <2 31 <2 33 <2 34 <2

TABLE-US-00005 TABLE 2 Ex. IC50% [nM] ± SD, Urotensin II receptor 32 0.03 ± 0.01 35  0.2 ± 0.01

TABLE-US-00006 TABLE 3 Ex. K.sub.i [μM], peptide binding to NCoA-1 1 0.7 2 4.7 3 6.2 4 15 5 3.8 6 3.6 7 7.1 8 3.0 9 1.5 10 3.1 11 8.7 12 9.3 13 2.0 14 1.4 15 3.1 16 6.6 17 15.8 18 3.3 19 3.7 20 11.5 21 19.6 22 8.9 23 16.4 24 24.3 25 6.0 26 7.7 27 7.9

TABLE-US-00007 TABLE 4 Cytotoxicity Hemolysis Plasmastability Hela Cells Cos-7 Cells at 100 μM human pl. mouse pl. Ex. GI.sub.50 [μM] GI.sub.50 [μM] [%] T.sub.1/2 [min] T.sub.1/2 [min] 28 >50 >50 0 240 240 29 >50 >50 0 240 240 30 >50 >50 0 240 240 31 >50 >50 0 240 240 32 >50 >50 0 240 240 33 >50 >50 0 240 240 34 >50 >50 0 240 240 35 >50 >50 0 240 240