PROCESSES FOR THE PREPARATION OF OXYTOCIN ANALOGUES

Abstract

Provided herein is a process for the preparation of oxytocin receptor agonists that have the potential to be used for the treatment of neurological disorders.

Claims

1. A process for the preparation of a compound of formula I ##STR00019## wherein R.sup.1 is hydrogen or C.sub.1-7-alkyl and R.sup.2 is hydrogen or C.sub.1-7-alkyl; or R.sup.1 and R.sup.2 together with the nitrogen and the carbon atom to which they are attached form a 5-membered heterocyle which is optionally substituted with hydroxy or halogen; and R.sup.3 is C.sub.1-7-alkyl or an enantiomer and/or optical isomer thereof, comprising treating a resin bound peptide precursor of the formula II ##STR00020## wherein R.sup.1, R.sup.2 and R.sup.3 are as above and R.sup.4 is a hydroxy protecting group; R.sup.5 is Fmoc; R.sup.6 is allyl, t-butyl, 1-adamantyl, 4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]amino}benzyl or phenylisopropyl; R.sup.7 is an amide protecting group; and R.sup.8 is an amide protecting group or an enantiomer and/or optical isomer thereof, either according to method a) or b): a) wherein in case of R.sup.6 being allyl or 4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]amino}benzyl a.sup.1) the allyl group or the 4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]amino}benzyl group R.sup.6 is cleaved, in a subsequent step a.sup.2) the Fmoc group R.sup.5 is cleaved, thereafter a.sup.3) ring cyclization is effected on the resin, in a further step a.sup.4) global deprotection and cleavage from the resin is effected, and optionally a.sup.5) the oxytocin analogue of formula I so obtained is purified and isolated; b) wherein in case of R.sup.6 being t-butyl, 1-adamantyl or phenylisopropyl; b.sup.1) the Fmoc group R.sup.5 is cleaved, thereafter b.sup.2) global deprotection and cleavage from the resin is effected, in a further step b.sup.3) ring cyclization is effected in solution, then optionally b.sup.4) the oxytocin analogue of formula I so obtained is isolated and purified.

2. The process of claim 1, wherein the compound of formula I is further defined as a compound of formula Ia ##STR00021## wherein R.sup.1, R.sup.2 and R.sup.3are as above and wherein the resin bound peptide precursor of formula II has the formula ##STR00022## wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7 and R.sup.8 are as above.

3. The process of claim 1, wherein R.sup.1 is hydrogen or C.sub.1-4-alkyl and R.sup.2 is hydrogen or C.sub.1-4-alkyl; or R.sup.1 and R.sup.2 together with the nitrogen and the carbon atom to which they are attached form a pyrrolidine ring of proline which is optionally substituted with hydroxy or halogen; R.sup.3 stands for n-butyl or i-butyl; R.sup.4 is t-butyl, allyl, trityl, 2-chlorotrityl, t-butyloxycarbonyl, t-butyldiphenylsilyl or t-butyldimethylsilyl; R.sup.5 is Fmoc; R.sup.6 is allyl, 1-adamantyl, 4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]amino}benzyl, phenylisopropyl or t-butyl; R.sup.7 is trityl, 2-chlorotrityl, 4-methyltrityl; and R.sup.8 is trityl, 2-chlorotrityl, 4-methyltrityl.

4. The process of claim 1, wherein R.sup.1 is hydrogen or methyl and R.sup.2 is hydrogen or R.sup.1 and R.sup.2 together with the nitrogen and the carbon atom to which they are attached forms pyrrolidine ring of proline which is optionally substituted with hydroxy or fluorine; R.sup.3 stands for n-butyl or i-butyl; R.sup.4 is t-butyl; R.sup.5 is Fmoc; R.sup.6 is allyl; R.sup.7 is trityl; and R.sup.8 is trityl.

5. The process of claim 1, wherein the resin bound peptide precursor of formula II is prepared on the resin by repeated Fmoc cleavage and coupling of the respective Fmoc protected amino acids.

6. The process of claim 5, wherein the Fmoc cleavage is performed with a solution of piperidine or 4-methyl-piperidine in a suitable organic solvent.

7. The process of claim 5, wherein the Fmoc coupling is performed using a coupling agent selected from the group consisting of benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), bromotripyrrolidinophosphonium hexafluorophosphate (PyBroP), hydroxybenzotriazole (HOBt) and N,N-diisopropylcarbodiimide (DIC), N,N,N,N-tetramethyl-O-(benzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HBTU), O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HATU), O-(6-chlorobenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HCTU), (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), tetramethylfluoroformamidinium hexafluorophosphate (TFFH), 2-hydroxy-pyridine (HOPy) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) in the presence of an organic amine base and a suitable organic solvent.

8. The process of claim 7, wherein the resin is a 4-[(2,4-Dimethoxyphenyl)Fmoc-aminomethyl]phenoxyacetamido methyl resin.

9. The process of claim 1 wherein, in the steps a.sup.1) the allyl group or the 4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]amino}benzyl group R.sup.6 is cleaved in the presence of a palladium or a rhodium compound or of hydrazine; a.sup.2) the Fmoc group R.sup.5 is cleaved with a solution of piperidine or 4-methyl-piperidine in a suitable organic solvent; a.sup.3) ring cyclization is effected on the resin, using a cyclization agent selected from benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), N,N,N,N-tetramethyl-O-(1H-benzotriazol-1-yl)uranium hexafluorophosphate (HBTU), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), O-(6-chlorobenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HCTU), (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), 2-hydroxy-pyridine (HOPy) or 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) in the presence of an organic amine base; a.sup.4) global deprotection and cleavage from the resin is effected in the presence of trifluoroacetic acid and a suitable scavenger such as thioanisole, anisole, phenol, triisopropylsilane, triethylsilane, ethanedithiol or dithiothreitol; a.sup.5) optionally, the oxytocin analogue of formula I so obtained is purified and isolated.

10. The process of claim 1 wherein, in the steps b.sup.1) the Fmoc group R.sup.5 is cleaved with a solution of piperidine or 4-methyl-piperidine in a suitable organic solvent; b.sup.2) global deprotection and cleavage from the resin is effected in the presence of trifluoroacetic acid and a suitable scavenger such as thioanisole, anisole, phenol, triisopropylsilane, triethylsilane, ethanedithiol or dithiothreitol; b.sup.3) ring cyclization is effected in solution using a cyclization agent selected from benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), N,N,N,N-tetramethyl-O-(1H-benzotriazol-1-yl)uranium hexafluorophosphate (HBTU), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), O-(6-chlorobenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HCTU), (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), 2-hydroxy-pyridine (HOPy) or 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) in the presence of an organic amine base; b.sup.4) optionally the oxytocin analogue of formula I so obtained is isolated and purified.

11. The process of claim 9, wherein the organic amine base is selected from pyridine, imidazole, N,N-diisopropylethyl amine, triethylamine, N-methylmorpholine, N,N-dimethyl-4-aminopyridine, 1,8-Diazabicyclo[5.4.0]undec-7-ene or 1,4-diazabicyclo[2.2.2]octane.

12. The process of claim 1, wherein the compound of formula I is further defined as a compound of formula Ia ##STR00023## wherein R.sup.1 is hydrogen or C.sub.1-4-alkyl and R.sup.2 is hydrogen or C.sub.1-4-alkyl; or R.sup.1 and R.sup.2 together with the nitrogen and the carbon atom to which they are attached form a pyrrolidine ring of proline which is optionally substituted with hydroxy or halogen; R.sup.3 is n-butyl or i-butyl or a corresponding enantiomer and/or optical isomer thereof, comprising treating a resin bound peptide precursor of formula II ##STR00024## wherein R.sup.1, R.sup.2 and R.sup.3 are as above and R.sup.4 is t-butyl, allyl, trityl, 2-chlorotrityl, t-butyloxycarbonyl, t-butyldiphenylsilyl or t-butyldimethylsilyl; R.sup.5 is Fmoc; R.sup.6 is allyl, t-butyl, 1-adamantyl or phenylisopropyl; R.sup.7 is trityl, 2-chlorotrityl, 4-methyltrityl; and R.sup.8 is trityl, 2-chlorotrityl, 4-methyltrityl according to the method: b.sup.1) the Fmoc group R.sup.5 is cleaved with a solution of piperidine or 4-methyl-piperdine in a suitable organic solvent; b.sup.2) global deprotection and cleavage from the resin is effected in the presence of trifluoroacetic acid and a suitable scavenger such as thioanisole, anisole, phenol, triisopropylsilane, triethylsilane, ethanedithiol or dithiothreitol; b.sup.3) ring cyclization is effected in solution using a cyclization agent selected from benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), N,N,N,N-tetramethyl-O-(1H-benzotriazol-1-yl)uranium hexafluorophosphate (HBTU), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), O-(6-chlorobenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HCTU), (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), 2-hydroxy-pyridine (HOPy) or 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) in the presence of an organic amine base; b.sup.4) optionally the oxytocin analogue of formula I so obtained is isolated and purified.

13. The process of claim 10, wherein the organic amine base is selected from pyridine, imidazole, N,N-diisopropylethyl amine, triethylamine, N-methylmorpholine, N,N-dimethyl-4-aminopyridine, 1,8-Diazabicyclo[5.4.0]undec-7-ene or 1,4-diazabicyclo[2.2.2]octane.

Description

EXAMPLES

[0114] Abbreviations:

[0115] SPPS=Solid-phase peptide synthesis, PL-Rink resin=4-[(2,4-Dimethoxyphenyl)Fmoc-aminomethyl]phenoxyacetamido methyl resin from Agilent Technology (PL1467-4749: 0.32 mmol/g 75-150-10.sup.6m; PL1467-4799: 0.55 mmol/g 75-150-10.sup.6m; PL1467-4689: 0.96 mmol/g 150-300-10.sup.6m), Fmoc=9-Fluorenylmethoxycarbonyl, Gly=Glycine, Leu=Leucine, Glu(OAll)=Allyl-protected glutamic acid, Glu(tBu)=tert Butyl-protected glutamic acid, Asn(Trt)=Trityl-protected asparagine, Gln(Trt)=Trityl-protected glutamine, Ile=Isoleucine, Tyr(tBu)=tert Butyl-protected tyrosine, Sar=N-methylglycine, Pro=Proline, Nle=Norleucine, DMF=N,N-Dimethylformamide, HOBt=1-Hydroxybenzotriazole, HOPy=2-hyxroxy-pyridine, DIC=N,N-Diisopropylcarbodiimide, NEP=N-Ethylpyrrolidone, PyBOP=(Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, DIPEA=Diisopropylethyl amine, MeOH=Methanol, CH.sub.2Cl.sub.2=Dichloromethane, MTBE=Methyl tert-butyl ether, MeTHF=2-Methyltetrahydrofuran, TFA=Trifluoroacetic acid, MeCN=Acetonitrile, PyAOP=(7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, HBTU=N,N,N,N-Tetramethyl-O-(1H-benzotriazol-1-yl)uranium hexafluorophosphate, HATU=1-[Bis(dimethylamino) methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate, HCTU=O-(6-Chlorobenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate, COMU=(1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate, DMTMM=4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, NMP=1-Methyl-2-pyrrolidinone, DMSO=Dimethyl sulfoxide, DMI=1,3-Dimethyl-2-imidazolidinone, DMPU=1,3-Dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone, NMM=N-Methylmorpholine, DMAP=N,N-Dimethyl-4-aminopyridine, DIPEA=N,N-Diisopropylethylamine, DBU=1,8-Diazabicyclo[5.4.0]undec-7-ene, DABCO=1,4-Diazabicyclo[2.2.2]octane.

Comparison Example

[0116] A comparative experiment was run for the preparation of

[0117] c[Gly-Tyr-Ile-Gln-Asn-Glu]-Gly-Leu-Gly-NH.sub.2 (1)

[0118] in analogy to the synthesis description of the WO2014/095773 (Solid phase cyclization) and as outlined in scheme 1 below:

##STR00008##

Synthesis performance has been measured based on the yield and the ratio of product (1) to the dimer-by product of the formula shown in scheme 2 below:

##STR00009##

[0119] a) Fmoc-Cleavage:

[0120] A SPPS reactor (100 mL; peptide synthesizer CS136XT ex CSBio) was charged with PL-Rink resin (load. 0.55 mmol/g, 5.00 g, 2.75 mmol) and 20% piperidine in DMF (50.0 mL). The mixture was then stirred at 25 C. for 10 min. After draining the solvent, another portion of 20% piperidine in DMF (50.0 mL) was added and the mixture was stirred at 25 C. for 30 min. After draining the solvent, the resultant resin was washed with DMF (850.0 mL) to yield deFmoc-PL-Rink resin.

[0121] b) Coupling with Fmoc-AA-Derivatives:

[0122] To deFmoc-PL-Rink resin, a solution of Fmoc-Gly-OH in 0.35M HOBt/DMF (32.0 mL, 11.2 mmol), 0.92M DIC in DMF (16.0 mL, 14.7 mmol) and 10% pyridine in DMF (16.0 mL, 19.8 mmol) were added and stirred at 25 C. for 3 h. After draining the solvent, the resultant resin was washed with DMF (450.0 mL) to yield Fmoc-Gly-resin.

[0123] Fmoc-Cleavage and Fmoc-AA-derivative coupling steps were repeated 8 times employing instead of Fmoc-Gly-OH, the following Fmoc-amino acid-derivatives: Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Glu(OAll)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH to yield Fmoc-Gly-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Glu(OAll)-Gly-Leu-Gly-resin. A sample was cleaved from the resin (vide below) to confirm the correct mass. MS (m/z): 1211.8 (M+H).sup.+

[0124] c) Fmoc-Cleavage:

[0125] Fmoc-Cleavage of the terminal Gly was conducted as described above to yield H-Gly-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Glu(OAll)-Gly-Leu-Gly-resin. A sample was cleaved from the resin (vehicle below) to confirm the correct mass. MS (m/z): 989.8 (M+H).sup.+

[0126] d) Allyl-Cleavage:

[0127] To H-Gly-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Glu(OAll)-Gly-Leu-Gly-resin, a solution of tetrakis triphenylphosphine palladium (159 mg, 0.138 mmol) and phenylsilane (3.40 mL, 27.6 mmol) in CH.sub.2Cl.sub.2 (50.0 mL) was added and stirred at 25 C. for 30 min. After draining the solvent, this step was repeated once more and washed with DMF (250.0 mL). A solution of sodium dithiocarbamate (250 mg) and DIPEA (0.250 mL) in DMF (50.0 mL) was added and the mixture was stirred at 25 C. for 15 min. After draining the solvent, this step was repeated once more. After draining the solvent, the resultant resin was washed with DMF (450.0 mL) to yield H-Gly-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Glu-Gly-Leu-Gly-resin. A sample was cleaved from the resin (vehicle below) to confirm the correct mass. MS (m/z): 949.7 (M+H).sup.+

[0128] e) Cyclization on Resin:

[0129] A solution of PyBOP (2.36 g, 4.54 mmol) and DIPEA (2.40 mL, 13.8 mmol) in NEP (60.0 mL) was added to H-Gly-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Glu-Gly-Leu-Gly-resin and the mixture was stirred at 25 C. for 4 h. After draining the solvent, the resultant resin was washed with DMF (450.0 mL), CH.sub.2Cl.sub.2 (350.0 mL) and MeOH (350.0 mL). The resin was dried at 10 mbar at 25 C. for 1 day to afford c[Gly-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Glu]-Gly-Leu-Gly-resin (8.60 g).

[0130] f) Global Deprotection and Resin Cleavage:

[0131] To a precooled (10-15 C.) solution of triisopropylsilane (2.80 mL) in TFA (40.0 mL) and water (10.0 mL), c[Gly-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Glu]-Gly-Leu-Gly-resin (8.60 g) was added and stirred at 25 C. for 3 h. The resin was filtered off and the filtrate was concentrated in vacuo. The residue was added to MTBE (100 mL) and the mixture was stirred at 25 C. for 15 h. The mixture was filtered and the cake was washed with MTBE (50.0 mL) followed by drying to afford crude c[Gly-Tyr-Ile-Gln-Asn-Glu]-Gly-Leu-Gly-NH.sub.2 1 (2.01 g, assay 11.3 wt %, total 9% yield) as a white solid with 15.9% purity (HPLC area-%, HPLC method cf. Example 1). The ratio of 1/dimer was 8.5.

TABLE-US-00001 TABLE 1 Total Purity of crude Cyclization yield cyclic peptide Ratio of Example method (%) (HPLC area-%) 1/dimer Comparison Solid phase 9 15.9 8.5 1 (invention) Solid phase 38 62.7 21.9 2 (invention) Liquid phase 31 56.6 15.1

Example 1

Solid Phase Cyclization

[0132] c[Gly-Tyr-Ile-Gln-Asn-Glu]-Gly-Leu-Gly-NH.sub.2 (1)

##STR00010##

[0133] a) Fmoc-Cleavage:

[0134] A SPPS reactor (100 mL; peptide synthesizer CS136XT ex CSBio) was charged with PL-Rink resin (load. 0.55 mmol/g, 5.00 g, 2.75 mmol) and 20% piperidine in DMF (50.0 mL). The mixture was then stirred at 25 C. for 10 min. After draining the solvent, another portion of 20% piperidine in DMF (50.0 mL) was added and the mixture was stirred at 25 C. for 30 min. After draining the solvent, the resultant resin was washed with DMF (850.0 mL) to yield deFmoc-PL-Rink resin.

[0135] b) Coupling with Fmoc-AA-Derivatives:

[0136] To deFmoc-PL-Rink resin, a solution of Fmoc-Gly-OH in 0.35M HOBt/DMF (32.0 mL, 11.2 mmol), 0.92M DIC in DMF (16.0 mL, 14.7 mmol) and 10% pyridine in DMF (16.0 mL, 19.8 mmol) were added and stirred at 25 C. for 3 h. After draining the solvent, the resultant resin was washed with DMF (450.0 mL) to yield Fmoc-Gly-resin.

[0137] Fmoc-Cleavage and Fmoc-AA-derivative coupling steps were repeated 8 times employing instead of Fmoc-Gly-OH, the following Fmoc-amino acid-derivatives: Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Glu(OAll)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH to yield X (Fmoc-Gly-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Glu(OAll)-Gly-Leu-Gly-resin). A sample was cleaved from the resin (vide below) to confirm the correct mass. MS (m/z): 1211.8 (M+H).sup.+

[0138] Allyl-Cleavage:

[0139] To X (Fmoc-Gly-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Glu(OAll)-Gly-Leu-Gly-resin), a solution of tetrakis triphenylphosphine palladium (159 mg, 0.138 mmol) and phenylsilane (3.40 mL, 27.6 mmol) in CH.sub.2Cl.sub.2 (50.0 mL) was added and stirred at 25 C. for 30 min. After draining the solvent, this step was repeated once more and washed with DMF (250.0 mL). A solution of sodium dithiocarbamate (250 mg) and DIPEA (0.250 mL) in DMF (50.0 mL) was added and the mixture was stirred at 25 C. for 15 min. After draining the solvent, this step was repeated once more. After draining the solvent, the resultant resin was washed with DMF (450.0 mL) to yield Fmoc-Gly-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Glu-Gly-Leu-Gly-resin. A sample was cleaved from the resin (vehicle below) to confirm the correct mass. MS (m/z): 1171.8 (M+H).sup.+

[0140] d) Fmoc-Cleavage:

[0141] Fmoc-Cleavage of the terminal Gly was conducted as described above to yield H-Gly-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Glu-Gly-Leu-Gly-resin. A sample was cleaved from the resin (vehicle below) to confirm the correct mass. MS (m/z): 949.7 (M+H).sup.+

[0142] e) Cyclization on Resin:

[0143] A solution of PyBOP (2.36 g, 4.54 mmol) and DIPEA (2.40 mL, 13.8 mmol) in NEP (60.0 mL) was added to (H-Gly-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Glu-Gly-Leu-Gly-resin and the mixture was stirred at 25 C. for 4 h. After draining the solvent, the resultant resin was washed with DMF (450.0 mL), CH.sub.2Cl.sub.2 (350.0 mL) and MeOH (350.0 mL). The resin was dried at 10 mbar at 25 C. for 1 day to afford c[Gly-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Glu]-Gly-Leu-Gly-resin (9.17 g).

[0144] f) Global Deprotection and Resin Cleavage:

[0145] To a precooled (10-15 C.) solution of triisopropylsilane (2.50 mL) in TFA (40.0 mL) and water (10.0 mL), c[Gly-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Glu]-Gly-Leu-Gly-resin (9.17 g) was added and stirred at 25 C. for 3 h. The resin was filtered off and the filtrate was concentrated in vacuo. The residue was added to MTBE (100 mL) and the mixture was stirred at 25 C. for 15 h. The mixture was filtered and the cake was washed with MTBE (50.0 mL) followed by drying to afford crude c[Gly-Tyr-Ile-Gln-Asn-Glu]-Gly-Leu-Gly-NH.sub.2 1 (2.39 g, assay 40.9 wt %, total 38% yield) as a white solid with 62.7% purity (HPLC area-%, HPLC method: Aquity UPLC BEH130 C18 column, 1502.1 mm; mobile phase, A: 0.05% TFA in water, B: 0.05% TFA in MeCN; flow: 0.13 mL/min for 40 min, 0.25 mL/min for 15 min; isocratic 90/10 (A/B) for 3 min, gradient from 90/10 (A/B) to 62/38 (A/B) within 37 min, gradient from 62/38 (A/B) to 10/90 (A/B) within 5 min, isocratic 10/90 (A/B) for 10 min. Temp: 60 C., UV:214 nm). The ratio of 1/dimer was 21.9.

[0146] Retention time: 23.2 min (c[Gly-Tyr-Ile-Gln-Asn-Glu]-Gly-Leu-Gly-NH.sub.2), 18.8 min (H-Gly-Tyr-Ile-Gln-Asn-Glu-Gly-Leu-Gly-NH.sub.2), 26.1 min (dimer)

[0147] g) Purification and Isolation:

[0148] Crude c[Gly-Tyr-Ile-Gln-Asn-Glu]-Gly-Leu-Gly-NH.sub.2 was dissolved in water-MeCN (10-1) and filtered. The filtrate was diluted with the same volume of water. The solution was purified by preparative HPLC on a Kromasil-C18-100 column (25080 mm, 10 um particle size, A: 0.1% TFA-water, B: MeCN; flow: 300 mL/min; isocratic 95/5 (A/B) for 2 min, gradient from 95/5 (A/B) to 80/20 (A/B) within 1 min, gradient from 80/20 (A/B) to 77/23 (A/B) within 17 min, gradient from 77/23 (A/B) to 10/90 (A/B) within 1 min, isocratic 10/90 (A/B) for 7 min, gradient from 10/90 (A/B) to 95/5 (A/B) within 1 min, isocratic 95/5 (A/B) for 6 min. The fractions were collected and lyophilized to yield pure c[Gly-Tyr-Ile-Gln-Asn-Glu]-Gly-Leu-Gly-NH.sub.2 1 (0.708 g) as a white powder with 99.2% purity (HPLC area-%, HPLC method cf. Example 1). No dimer was observed in pure 1. MS (m/z): 931.0 (M+H).sup.+

Example 2

Solution Phase Cyclization

[0149] c[Gly-Tyr-Ile-Gln-Asn-Glu]-Gly-Leu-Gly-NH.sub.2 (1)

##STR00011##

[0150] a) Fmoc-Cleavage:

[0151] A SPPS reactor (100 mL) was charged with PL-Rink resin (load. 0.55 mmol/g, 5.00 g, 2.75 mmol) and 20% piperidine in DMF (50 mL). The mixture was then stirred at 25 C. for 10 min. After draining the solvent, another portion of 20% piperidine in DMF (50.0 mL) was added and the mixture was stirred at 25 C. for 30 min. After draining the solvent, the resultant resin was washed with DMF (850.0 mL) to yield deFmoc-PL-Rink-resin.

[0152] b) Coupling of Fmoc-AA-Derivatives:

[0153] To deFmoc-PL-Rink-resin, a solution of Fmoc-Gly-OH in 0.35M HOBt/DMF (32.0 mL, 11.2 mmol), 0.92M DIC in DMF (16.0 mL, 14.7 mmol) and 10% pyridine in DMF (16.0 mL, 19.8 mmol) were added and stirred at 25 C. for 3 h. After draining the solvent, the resultant resin was washed with DMF (450.0 mL) to yield Fmoc-Gly-resin.

[0154] Fmoc-Cleavage and Fmoc-AA-derivative coupling steps were repeated 8 times employing instead of Fmoc-Gly-OH, the following Fmoc-amino acid-derivatives: Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH to yield X (Fmoc-Gly-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Glu(tBu)-Pro-Leu-Gly-resin). A sample was cleaved from the resin (vide below) to confirm the correct mass. MS (m/z): 1171.8 (M+H).sup.+

[0155] c) Fmoc-Cleavage:

[0156] Fmoc-Cleavage of the terminal Gly was conducted as described above. After draining the solvent, the resultant resin was washed with DMF (850.0 mL), CH.sub.2Cl.sub.2 (350.0 mL) and MeOH (350.0 mL). The resin was dried at 10 mbar at 25 C. for 1 day to afford to yield H-Gly-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Glu(tBu)-Gly-Leu-Gly-resin (10.8 g).

[0157] d) Global Deprotection and Resin Cleavage:

[0158] To a precooled (10-15 C.) solution of triisopropylsilane (2.50 mL) in TFA (40.0 mL) and water (10.0 mL), H-Gly-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Glu(tBu)-Gly-Leu-Gly-resin (10.8 g) was added and stirred at 25 C. for 3 h. The resin was filtered off and the filtrate was concentrated in vacuo. The residue was added to MeTHF (100 mL) and the mixture was stirred at 25 C. for 15 h. The mixture was filtered and the cake was washed with MeTHF (50.0 mL) followed by drying to afford LP1 (H-Gly-Tyr-Ile-Gln-Asn-Glu-Gly-Leu-Gly-NH.sub.2) (3.60 g) as a white solid with 67.4% purity (HPLC area-%, HPLC method cf. example 1). MS (m/z): 949.7 (M+H).sup.+

[0159] e) Cyclization in Solution:

[0160] To a mixture of LP1 (H-Gly-Tyr-Ile-Gln-Asn-Glu-Gly-Leu-Gly-NH.sub.2) (3.50 g) in NEP (60.0 mL) and DIPEA (3.13 mL, 18.4 mmol) was added PyBOP (1.92 g, 3.69 mmol) and stirred at 25 C. for 1 h. To complete conversion, another portion of PyBOP (0.960 g, 1.84 mmol) was added and stirred at the same temperature for 1 h. The resultant mixture was added to a solution of MTBE/MeTHF solution (400 mL/100 mL) and stirred at 25 C. for 15 h. The mixture was filtered and the cake was washed with MTBE (50.0 mL) followed by drying to afford crude c[Gly-Tyr-Leu-Gln-Asn-Glu]-Gly-Leu-Gly-NH.sub.2 1(4.30 g, assay 18.0 wt %, total 31% yield) as a white solid with 56.6% purity (HPLC area-%, HPLC method cf. example 1). The ratio of 1/dimer was 15.1.

[0161] f) Purification and Isolation:

[0162] Crude c[Gly-Tyr-Leu-Gln-Asn-Glu]-Gly-Leu-Gly-NH.sub.2 was dissolved in water-MeCN (10-1) and filtered off undissolved material. The filtrate was diluted with the same volume of water. The solution was purified by preparative HPLC on a Kromasil-C18-100 column (25080 mm, 10 um particle size, A: 0.1% TFA-water, B: MeCN; flow: 300 mL/min; isocratic 95/5 (A/B) for 2 min, gradient from 95/5 (A/B) to 80/20 (A/B) within 1 min, gradient from 80/20 (A/B) to 77/23 (A/B) within 17 min, gradient from 77/23 (A/B) to 10/90 (A/B) within 1 min, isocratic 10/90 (A/B) for 7 min, gradient from 10/90 (A/B) to 95/5 (A/B) within 1 min, isocratic 95/5 (A/B) for 6 min. The fractions were collected and lyophilized to yield pure c[Gly-Tyr-Leu-Gln-Asn-Glu]-Gly-Leu-Gly-NH.sub.2 1 (444 mg) as a white powder with 99.7% purity (HPLC area-%, HPLC method cf. Example 1). No dimer was present in pure 1. MS (m/z): 931.0

Example 3 a-g

Optimization of Coupling Reagents

[0163] c[Gly-Tyr-Ile-Gln-Asn-Glu]-Gly-Leu-Gly-NH.sub.2 (1)

[0164] In an analogous manner to Example 2, the cyclizations were performed employing the coupling reagents as listed in Table 2.

TABLE-US-00002 TABLE 2 Yield in Purity in reaction reaction Coupling mixture mixture Ratio of Example reagent (%) (HPLC area-%) 1/dimer 3a PyBOP 63 54.9 13.4 3b PyAOP 63 52.7 23.2 3c HBTU 45 51.0 12.4 3d HATU 51 47.1 10.6 3e HCTU 55 10.6 13.8 3f COMU 26 11.5 28.0 3g DMTMM 17 18.5 34.9

Example 4 a-g

Optimization of Solvents

[0165] c[Gly-Tyr-Ile-Gln-Asn-Glu]-Gly-Leu-Gly-NH.sub.2 (1)

[0166] In an analogous manner to Example 2, the cyclizations were performed employing the solvents as listed in Table 3.

TABLE-US-00003 TABLE 3 Yield in Purity in reaction reaction Solvent mixture mixture Ratio of Example (Concentration) (%) (HPLC area-%) 1/dimer 4a NEP (5 mM) 63 60.3 30.4 4b NEP (80 mM) 63 54.9 13.4 4c NMP (80 mM) 44 46.9 13.3 4d DMSO (80 mM) 51 52.8 13.8 4e DMF (80 mM) 35 44.8 8.9 4f DMI (80 mM) 38 44.5 11.3 4g DMPU (80 mM) 35 45.3 17.7

Example 5 a-g

Optimization of Bases

[0167] c[Gly-Tyr-Ile-Gln-Asn-Glu]-Gly-Leu-Gly-NH.sub.2 (1)

[0168] In an analogous manner to Example 2, the cyclizations were performed employing the bases as listed in Table 4.

TABLE-US-00004 TABLE 4 Yield in Purity in reaction reaction mixture mixture Ratio of Example Base (%) (HPLC area-%) 1/dimer 5a Imidazole 63 55.7 10.6 5b NMM 68 57.7 22.0 5c DABCO 47 46.4 15.4 5d DMAP 43 47.8 13.6 5e DIPEA 63 54.9 13.4 5f DBU 25 27.0 19.4 5g NMM, 0 C. 66 54.5 20.0

Example 6 a-d

Comparison of the Resin Loading/Amino Acid Equivalent

[0169] c[Gly-Tyr-Ile-Gln-Asn-Glu]-Pro-Leu-Gly-NH.sub.2 (2)

##STR00012##

[0170] In an analogous manner to Example 2, pure cyclic peptide 2 was synthesized employing the Fmoc-AA-acids: Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH

[0171] Scale of synthesis: 9.60 mmol (load: see example 6a-d; resin 30.0 g)

[0172] Yield: 40% (after purification)

[0173] Purity: 98.2% (HPLC area-%, HPLC method cf. example 1)

[0174] Retention time: 29.8 min (HPLC method cf. Example 1)

[0175] MS (m/z): 971.5 (M+H).sup.+

[0176] Purity and yield of the linear peptide intermediate LP2 (H-Gly-Tyr-Ile-Gln-Asn-Glu-Pro-Leu-Gly-NH.sub.2) was determined employing the resin loadings/amino acid equivalents as listed in Table 5.

TABLE-US-00005 TABLE 5 Loading of Amino Purity of Yield of resin acid crude LP2 crude LP2 Example (mmol/g) (eq.) (HPLC area-%) (%) 6a 0.32 4 79.0 90 6b 0.55 4 83.3 116 6c 0.55 2 78.5 108 6d 0.96 4 82.7 96

Example 7

[0177] c[Gly-Tyr-Ile-Gln-Asn-Glu]-Pro-Leu-Gly-NH.sub.2 (2)

[0178] Example 7 was performed in an analogous manner to Example 2, with the exception that the cyclizations were performed employing N-methylmorpholine as base.

[0179] a) Fmoc-Cleavage:

[0180] A SPPS reactor (250 mL; peptide synthesizer CS536XT ex CSBio) was charged with PL-Rink resin (load. 0.55 mmol/g, 10.0 g, 5.50 mmol) and 20% piperidine in DMF (100 mL). The mixture was then stirred at 25 C. for 10 min. After draining the solvent, another portion of 20% piperidine in DMF (100 mL) was added and the mixture was stirred at 25 C. for 30 min. After draining the solvent, the resultant resin was washed with DMF (8100 mL) to yield deFmoc-PL-Rink-resin.

[0181] b) Coupling of Fmoc-AA-Derivatives:

[0182] To deFmoc-PL-Rink-resin, a solution of Fmoc-Gly-OH in 0.35M HOBt/DMF (64.0 mL, 22.4 mmol), 0.92M DIC in DMF (32.0 mL, 29.4 mmol) and 10% pyridine in DMF (32.0 mL, 39.6 mmol) were added and stirred at 25 C. for 3 h. After draining the solvent, the resultant resin was washed with DMF (4100 mL) to yield Fmoc-Gly-resin.

[0183] Fmoc-Cleavage and Fmoc-AA-derivative coupling steps were repeated 8 times employing instead of Fmoc-Gly-OH, the following Fmoc-amino acid-derivatives: Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH to yield Fmoc-Gly-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Glu(tBu)-Pro-Leu-Gly-resin. A sample was cleaved from the resin (vehicle below) to confirm the correct mass. MS (m/z): 1211.1 (M+H).sup.+

[0184] c) Fmoc-Cleavage:

[0185] Fmoc-Cleavage of the terminal Gly was conducted as described above. After draining the solvent, the resultant resin was washed with DMF (8100 mL), CH.sub.2Cl.sub.2 (3100 mL) and MeOH (3100 mL). The resin was dried under 10 mbar at 25 C. for 1 day to afford to yield H-Gly-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Glu(tBu)-Pro-Leu-Gly-resin (18.6 g). A sample was cleaved from the resin (vehicle below) to confirm the correct mass. MS (m/z): 989.7 (M+H).sup.+

[0186] d) Global Deprotection and Resin Cleavage:

[0187] To a precooled (10-15 C.) solution of triisopropylsilane (3.00 mL) in TFA (48.0 mL) and water (12.0 mL), H-Gly-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Glu(tBu)-Pro-Leu-Gly-resin (6.00 g) was added and stirred at 25 C. for 3 h. The resin was filtered off and the filtrate was concentrated in vacuo. The residue was added to MeTHF (120 mL) and the mixture was stirred at 25 C. for 15 h. The mixture was filtered and the cake was washed with MeTHF (60.0 mL) followed by drying to afford H-Gly-Tyr-Ile-Gln-Asn-Glu-Pro-Leu-Gly-NH.sub.2 LP2 (1.84 g) as a white solid with 87.3% purity (HPLC area-%, HPLC method cf. Example 1). Retention time: 23.9 min (HPLC method cf. Example 1); MS (m/z): 989.7 (M+H).sup.+

[0188] e) Cyclization in Solution:

[0189] To a mixture of H-Gly-Tyr-Ile-Gln-Asn-Glu-Pro-Leu-Gly-NH.sub.2 LP2 (300 mg) in N-ethylpyrrolidone (3.60 mL) and NMM (0.167 mL, 1.52 mmol) was added PyBOP (237 mg, 0.455 mmol) and stirred at 25 C. for 1 h. To complete conversion, another portion of PyBOP (47.4 mg, 0.0910 mmol) was added and stirred at the same temperature for 1 h. The resultant mixture was added to a solution of MTBE (24.0 mL) and MeTHF (6.00 mL), and then stirred at 25 C. for 15 h. The mixture was filtered and the cake was washed with MTBE (15.0 mL). The cake was dissolved in water/MeCN (10/1, 3.3 mL) and filtered off undissolved materials. The filtrate was lyophilized to afford crude c[Gly-Tyr-Leu-Gln-Asn-Glu]-Pro-Leu-Gly-NH.sub.2 2 (313 mg, assay 54.0 wt %, total 60% yield) as a white solid with 71.4% purity (HPLC area-%, HPLC method cf. Example 1).MS (m/z): 971.5 (M+H).sup.+

Example 8

[0190] c[Gly-Tyr-Ile-Gln-Asn-Glu]-Sar-Leu-Gly-NH.sub.2 (3)

##STR00013##

[0191] In an analogous manner to Example 2, pure cyclic peptide 3 was synthesized employing the Fmoc-AA-acids: Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Sar-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH

[0192] Scale of synthesis: 9.60 mmol (load. 0.32 mmol/g, resin 30.0 g)

[0193] Yield: 41% (after purification)

[0194] Purity: 98.9% (HPLC area-%, HPLC method cf. Example 1)

[0195] Retention time: 27.6 min (HPLC method cf. Example 1)

[0196] MS (m/z): 945.5 (M+H).sup.+

Example 9

[0197] c[Gly-Tyr-Ile-Gln-Asn-Glu]-Sar-Nle-Gly-NH.sub.2 (4)

##STR00014##

[0198] In an analogous manner to Example 2, pure cyclic peptide 4 was synthesized employing the Fmoc-AA-acids: Fmoc-Gly-OH, Fmoc-Nle-OH, Fmoc-Sar-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH

[0199] Scale of synthesis: 9.60 mmol (load. 0.32 mmol/g, resin 30.0 g)

[0200] Yield 41% (after purification)

[0201] Purity: 99.2% (HPLC area-%, HPLC method cf. Example 1)

[0202] Retention time: 25.9 min (HPLC method cf. Example 1)

[0203] MS (m/z): 945.5 (M+H).sup.+

Example 10

[0204] c[Gly-Tyr-Ile-Gln-Asn-Glu]-trans-4-fluoro-Pro-Leu-Gly-NH.sub.2 (5)

##STR00015##

[0205] In an analogous manner to Example 2, pure cyclic peptide 5 was synthesized employing the Fmoc-AA-acids: Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-trans-4-fluoro-Pro-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH

[0206] Scale of synthesis: 9.60 mmol (load. 0.32 mmol/g, resin 30.0 g)

[0207] Yield: 39% (after purification)

[0208] Purity: 98.8% purity (HPLC area-%, HPLC method cf. Example 1)

[0209] Retention time: 25.7 min (HPLC method cf. Example 1)

[0210] MS (m/z): 988.5 (M+H).sup.+

Example 11

[0211] c[Gly-Tyr-Ile-Gln-Asn-Glu]-trans-4-hydroxy-Pro-Leu-Gly-NH.sub.2 (6)

##STR00016##

[0212] In an analogous manner to Example 2, pure cyclic peptide 6 was synthesized employing the Fmoc-AA-acids: Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-trans-4-tertbutoxy-Pro-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH

[0213] Scale of synthesis: 9.60 mmol (load. 0.32 mmol/g, resin 30.0 g)

[0214] Yield: 22% (after purification)

[0215] Purity: 98.7% purity (HPLC area-%, HPLC method cf. Example 1)

[0216] Retention time: 23.3 min (HPLC method cf. Example 1)

[0217] MS (m/z): 987.5 (M+H).sup.+.

Example 12

Solid Phase Cyclization

[0218] c[Gly-Tyr-Ile-Gln-Asn-Glu]-trans-4-fluoro-Pro-Leu-Gly-NH.sub.2.sub._(5)

##STR00017##

[0219] In an analogous manner to Example 1 employing a CS536XT peptide synthesizer from CSBio, pure cyclic peptide 5 was synthesized employing the Fmoc-AA-acids: Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-trans-4-fluoro-Pro-OH, Fmoc-Glu(OAll)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH. Throughout the entire synthesis, for Fmoc-cleavage 10% 4-methyl-piperidine in DMF instead of 20% piperidine in DMF was used, and all amino acid couplings in the linear sequence were conducted employing HOPy instead of HOBt. In the final PyBOP promoted cyclization on resin step, 4-methylmorpholine instead of DIPEA was used as base and the cyclization was run in DMF instead of NEP as solvent. The preparative HPLC purification of crude c[Gly-Tyr-Ile-Gln-Asn-Glu]-trans-4-fluoro-Pro-Leu-Gly-NH.sub.2 was conducted on a Kromasil-C18-100 column (2504.6 mm, 10 um particle size, A: 20 mM NH4OAc pH5, B: MeCN; flow: 1 mL/min; isocratic 90/10 (A/B) for 1 min, gradient from 90/10 (A/B) to 80/20 (A/B) within 1 min, gradient from 80/20 (A/B) to 75/25 (A/B) within 10 min, gradient from 75/25 (A/B) to 10/90 (A/B) within 1 min, gradient from 10/90 (A/B) to for 5 min, gradient from 10/90 (A/B) to 90/10 (A/B) within 0.1 min, isocratic 90/10 (A/B) for 6.9 min. The collected fractions were diluted with water (1:1) and concentrated/desalted by loading on a conditioned (water/ACN 90/10) Kromasil C18-100-10 column (2504.6 mm) and eluated afterwards with water/ACN (1:1). The collected fractions (UV 280 nm, threshold 1000mAu) were rotatory evaporated to remove ACN and lyophilized afterwards to yield the pure peptide as a white lyo product

[0220] Scale of synthesis: 5.50 mmol (loading 0.55 mmol/g, resin 10.0 g)

[0221] Yield: 34% (after purification)

[0222] Purity: 98.8% purity (HPLC area-%, HPLC method cf. Example 1)

[0223] Retention time: 25.3 min (HPLC method cf. Example 1)

[0224] MS (m/z): 989.5 (M+H).sup.+

Example 13

Solid Phase Cyclization

[0225] c[Gly-Tyr-Ile-Gln-Asn-Glu]-trans-4-fluoro-Pro-Leu-Gly-NH.sub.2.sub._(5)

##STR00018##

[0226] In an analogous manner to Example 13, pure cyclic peptide 5 was synthesized employing HOBt instead of HOPy throughout the entire synthesis of the linear peptide on resin.

[0227] Scale of synthesis: 5.50 mmol (loading 0.55 mmol/g, resin 10.0 g)

[0228] Yield: 25% (after purification)