DD146289A1 - PROCESS FOR THE PRODUCTION OF SOMATOSTATIN AND SOMATOSTATINANALOGA - Google Patents

Abstract

The invention relates to a process for the preparation of somatostatin and somatostatin analogs of the general formula I H-Ala-Gly-X-Lys-Asn-Phe-Y-Cys-Thr-Phe-Thr-Ser-X-OH wherein X is cysteine or selenocysteine and Y stands for L- or D-Trp. These compounds can be used for the treatment of acromegaly, angiopathy and diabetes mellitus as well as for diagnostic purposes.

Description

-a- 21 5 85

Title of the invention

Process for the preparation of somatostatin and somatostatic analogs

Field of application of the invention

The invention relates to a process for the preparation of somatostatin and its analogs. In particular, the invention relates to a process for the preparation of diseleno-D

Trp -Somatostatin and in the synthesis of intermediates used.

The somatostatin or its analogues prepared according to the invention can be used for clinical pharmacology with regard to the treatment of acromegaly, angiopathy and diabetes mellitus as well as for diagnostic purposes.

Characteristic of the known technical solutions

It is already known that somatostatin and its analogs are both conventionally / "See, for example, D. Sarantakis and Wc A. Mekinley, Biochem., Biophys., Cornmun., 54, (1973) HU Immer et al., HeIv. Chim. Acta £ 7, 730 (1974) _7 ^as well as on the polymeric support / "Cf. For example, J. Eivier, J. Amer. Chem. Soc. 6, 2986 (1974) j

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Brazeau et al., Biochem. Biophys. Res. Commun. 6, 1202 (1974) J. Rivier et al., CE Acad. Sci 276, 2737 (1973) 5 D. Sarantakis et al., Biochem. Biophys. Res. Commun. 55, 538 (1973) 5 D · Sarantakis et al., Biochem. Biophys. Res. Commun. 72S ₅ 336 (1976) _7 can be prepared by various methods. ·

Many of the somatostatin syntheses described so far in the literature use solid-phase technology. However, this method is not without concern applicable to the presentation of longer chain peptides. Owing to non-quantitative turnover rates in each synthesis cycle, core and failure sequences can result, resulting in substantial complication in the purification of the final product. Moreover, due to the high excess of acylating moiety in each reaction cycle, the solid-phase technique requires maximum protection of the sidechain functions with sufficiently high stability over the repetitive deblocking steps to release the c6 amino function. At the end of the synthesis, these side-chain protective groups can be partially removed again only under relatively drastic conditions, whereby damage to the molecule and, associated therewith, a reduction in the biological activity can not be ruled out.

On the other hand, conventional process technology offers the possibility of controllable synthesis (intermediate product characterization and purification). Which in many cases is chosen in conventional syntheses

7 ft main interface between Phe and Trp has the advantage that aoidolytic deblocking steps at the trp can lead to surface reactions / "" cf. For example, M. Louw et al ·, Hoppe-Seyler's Z. Physiol ^1. Chem. 2§S »1643 (1978); W. Bauer and J. Pless, Peptides 1975 Proc. IV Amer. Pept · Symp._7 ct. R. Welk and I. lieienhofer, Ann. Arbor, Michigan 48106 p. 341_7

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Object of the invention

The object of the invention is to disclose a method which has the following advantages:

Subdivision of the tetradecapeptide sequence of somatostatin into suitable fragments whose general use is also possible for the synthesis of analogues with little preparative effort. In the scope of the present invention, in particular, a process for the synthesis of

Diseleno-D-Trp -Somatostatin,

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Explanation of the essence of the invention

The invention has for its object to provide a method for the preparation of somatostatin and its analogs using fragment condensation. According to the invention, this object is achieved by 1 · shifting the main interface between Phe ⁺ '

7 and Phe, eliminating the need for direct acidolysis on tryptophan in position 8 (reduction of side reactions),

2. the subdivision of the tetradecapeptide sequence of somatostatin into suitable fragments according to the invention, which allows a synthesis of somatostatin analogues with little preparative effort,

3. the preferred application of the active ester method of representing the partial fragments, thus enabling the general application of the tactic of minimal protection of the side chain functions,

solved.

The method according to the invention / allows a simple synthesis of the potentially pharmacologically significant

Diseleno-D-Trp-somatostatin compound that could not be previously displayed in any other way, e.g. B. with the aim of investigating the importance of the disulfide bridge of somatostatin for its biological activity.

The compounds represented by the process according to the invention are represented by the formula (I)

I i

H-Ala-Gly-X-Lys-Asn-Phe-Phe-Y-Lys-Thr-Phe-Thr-Ser-X-OH (i)

in

wherein X is cysteine or selenocysteine and Y has the meaning L-Trp or D-Trp.

- {-) Abbreviations for amino acid and peptide derivatives, cf. JUPAG-IUB Commission on Biochemical Nomenclature, J. Biol. Chem. 247, 977 (1972)

A preferred compound of the invention is represented by formula (I) wherein X is selenocysteine and Y has the meaning of D-Trp.

The compounds of formula (i) wherein X and Y are as previously defined are prepared by a process characterized by reacting the protected peptide of formula (II)

S-Ala-Gly-X (T) -Lys (U) -Asn-Phe-Phe-Y-Lys ()) -Thr ~ Phe-Thr-V (II)

into the two main fragments of the formula (III) S-Ala-Gly-X (T) -Lys (U) -Asn-Phe-W (III)

and (IV) H-Phe-Y-Lys (U) -Thr.Phe-Thr-Ser-X (T) -V (iv)

whose combination preferably results after hydrazinolysis of the peptide of formula III via the acid method, but also after alkaline hydrolysis using OCCI / additive method, the compounds of formula II, in which

S for tert. Butyloxycarbonyl, benzyloxycarbonyl,

p-methoxybenzyloxycarbonyl- T for benzyl; p methoxybenzyl U for benzyloxycarbonyl, p-chlorobenzyloxycarbonyl;

o-chlorobenzyloxycarbonyl V for benzyl · -; p-nitrobenzyl ester and W for methyl? Ethyl-; benzyl ester

stand.

The two main fragments of the formulas III and IV (sequence C and sequence L), wherein S, T, U, V, W, X, Y are as defined above, can be obtained for formula III (sequence C) by linking the partial fragments of the sequences A and B and for formula IV (sequence L) by linking the sub-questions

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Elements of the sequences D, E, P "or, D, E, G and X in different order according to the usual methods of peptide chemistry (E. Wünsch, synthesis of peptides in Houben-Weyl Bd. 1 ^ / 11, methods of org Chemistry (Ed. E. Müller) Georg Thieme Verlag, Stuttgart 1974) according to the general synthesis scheme 1 build.

For temporary blocking of the Jj- amino group served the tert. Butyloxycarbonyl radical whose acidolytic cleavage with the aid of HCl / glacial acetic acid, HCl / dioxane, formic acid, 0.12 U HCl / formic acid, trifluoroacetic acid, trifluoroacetic acid / CHoClo (1: 1) preferably with HCl / dioxane due to the free hydroxyl functions of Thr and Ser took place.

To build up the partial fragment A, B, D, E, F, G, the usual in peptide chemistry knotting methods - DCCI, DCCI / additives, mixed anhydride, activated esters, azide, acid chloride - were used. Preferably, the structure was carried out by the method of activated esters, wherein predominantly the corresponding pentafluorophenyl and N-Hydroxysuocinimidester be used.

At the level of the dipeptide Asn-Phe to build sequence B, the use of Z-Asn-oPfp compared to the Boc derivative resulted in about 60 <& higher yield.

The linking of the partial fragments to the two main fragments (sequence C and L) was preferably carried out by using the DCCI / additive method, the acid coupling and the method of mixed anhydrides.

The deblocking reaction subsequent to the fragment condensation (Sequence C and Sequence L) to represent the protected tetradecapeptides of Formula II (Sequence M), i. H. the conversion of the protected mercapto or seleno, amino and carboxyl groups into free groups was carried out by known methods by treatment with sodium / liquid ammonia, hydrogen fluoride / anisole or boron tris (trifluoroacetate) / thioanisole.

The oxidation of the mercapto or seleno groups in positions 3 and 14 to the disulfide or diselenide bridge can be carried out by means of oxygen or potassium ferricyanide. Subsequently, the end products are purified on Sephadex G-25 F by gel and partition chromatography.

embodiments

The invention will be explained below with reference to exemplary embodiments

 The following abbreviations are used:

Amino Acid Symbols According to JUPAC-JUiB Commission on Biochemical Nomenclature, Sec stands for selenocysteine

AcOH acetic acid Boc tert. butyloxycarbonyl BTFA Bortris (trifluoroacetate) Bzl benzyl Caibe isobutylchloroformate DC TLC, - chromatogra.phic DCCI N, N ^I -dicyclohexylcarbodiimide DMP dimethylformamide EE Essigester HOBt H-hydroxybenzotriazole HV Hoohvakuum i. Vak. in a vacuum LM solvent Mb zl p-methoxybenzyl MeOH methanol OBzl benzyl ester OMe Methylester ORB p-Hitrobenzylester OHSU N-hydroxysuccinimide OPfp pentafluorophenyl PA ' petroleum ether ET room temperature ECO triethylamine TFA trifluoroacetic

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TEF tetrahydrofuran

Z is benzyloxycarbonyl

Z (oCl) o-chlorobenzyloxycarbonyl

Z (pCl) p-chlorobenzyloxycarbonyl

Method Δ: Misohanhydride Method Method B: DCCI / HOBt Method Method C: Method of Activated Ester C I: Coupling with U-acyl-amino acid

H-hydroxysuccinimide ester C II: Coupling with H-acyl-amino acid

pentafluorophenyl ester

Method D: Honzel-Rudinger variant of the azide method Method E: Alkaline hydrolysis of the peptide esters

For general instructions on coupling methods A to D see E, Wünsch, Synthesis of peptides in Houben-Y / eyl Bd. 1J5 / II Methods of organ. Chemistry (Ed. E. Müller), Georg Thieme Verlag, Stuttgart 1974.

By "usual work-up" is meant:

After completion of the coupling reaction using the methods A to D, the respective crude product is taken up in ethyl acetate and the solution washed successively twice with water (NaCl saturated), three times with 10 #iger citric acid solution, three times with water, three times with saturated sodium bicarbonate solution and three times with water , The organic phase is dried over Na ^ SCK and the crude product isolated by evaporation of the solvent in vacuo.

In the case of peptides which do not dissolve in ethyl acetate, the oily residue remaining after the solvent has been removed by evaporation is triturated with water or ethyl acetate to give the pest product, which is then washed on the frit with Y / water, 10% citric acid solution, water, saturated sodium bicarbonate solution and washed again with water.

It is finally crystallized from a suitable solvent or solvent mixture and the product is dried i. Vak. over phosphorus pentoxide. Deviations from this general rule are noted.

The following Laufelittelsysteme (in volumes) for thin layer chromatography on silica gel Pertig plates (Silufol ÜV 254, CSSE) were used:

2-butanol-formic acid-water 75 + 13.5 + 1 1-butanol-acetic acid-water I-butanol-acetic acid-water 1-butanol-pyridine-acetic acid

Water 40 + 10 + 10 + 20

Chloroform-methanol ethyl acetate-methanol 2 ~ propanol ~ 25 # ammonia water 70 + 10 + 10

In the amino acid analyzes, the values for tryptophan and cysteine were not determined, the amino acid marked with 'represents the reference value.

B A W 1 B e W 2 B e W W B P e C M e M P A W

75 η , 5 + 60 π + 20 80 ^ + 20 40 η + 10 70 η 50 η 13 h 20 r'20 h 10 20 r 50

ίο

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Example I - / "Sec ^ * ¹ *, D-Trp ⁸ ^ - Somatostatin

1.1 ,. Z-Asn-Phe-OMe (method G II)

4.32 g (10 mmol) of Z-Asn ^ -OPfp and 2.37 g (11 mmol) H-Phe-OMe ^ HCl in 40 ml of dioxane was added at 0 ⁰ C under stirring with 1.54 ml (11 mmol) TlS added. The reaction mixture was stirred for 0.5 h at 10 ⁰ C and over power at ST, then the solvent i. Vacuum distilled off. The oily backbone was triturated with water to give the solid, which was thoroughly washed with water, hot isopropanol, and methanol. After recrystallization from methanol, a TLC-uniform product was isolated.

yield * • »76 C C S - (88 fo . Th.) , CM 9 , 83 pp: 199 C 61st 09 199.5 ⁰ C , 42) 9 , 81 S + ' 61, in ⁰ (c = 1, AcOH) N I6, > 2 ^H 25 BAW, BEW2, DC: uniform , 82 IT ₃ O ₆ (427 ₃ 77 H 5.90 Ber. H 5.94 Gef.

H-Asn-Phe-OMe · HCl

The removal of the Z-protecting group of I ₁ .1 .. succeeded by catalytic hydrogenation using palladium black catalyst. For this purpose, 3.2 g (7.5 mmol) of Z-Asn-Phe-OMe were dissolved in 100 ml of methanol-acetic acid (1: 1) and dissolved in the presence of palladium black and 1.25 ml of 6W HCl (7.5 mmol). Kach ended carbon dioxide evolution (absence of ^w barium carbonate reaction ^t! ) Was filtered off from the catalyst and the filtrate i. Vak. concentrated to dryness. Each dissolving the oily residue in a little MeOH, the product could be precipitated by the addition of ether.

Yield: 2.35 g (95 K> Th.) R <J% ° = + 13.37 ° Coal, IMF)

DC: uniform in BM, BEW1, CE, PAW

O ₄ (329.76)

Ber. C 50.99 H 6.11 N 12.74 Gef. C 50.81 H 6.15 Ir "12.65

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1.3. Boc-Lys / "z (oCl) _7-Asn-Phe-0Me (Method C II)

3.49 g (6 mmol) of Boc-Lys / Z (oCl) J7-0Pfp and 2.08 g (6.3 mmol) of H-Asn-Phe-OMe HCl were dissolved in 20 ml of DMP and, after cooling to 0 ⁰ C with 0.88 ml (6.3 mmol) TÄA, left for 0.5 h at 0 ⁰ C and at RT overnight. The usual work-up by washing on the Pritte and crystallization from Me0H / H ₂ 0 or MeOH provided the desired peptide.

Yield: 3.56 g (86 # d, Th). Pp: 159-161 ⁰ C.

βoj ^ = -3.72 ° (c = 1, DMP)

C ₃₃ H ₄₄ ClU ₅ O ₉ (690.15)

Ber. C 57.43 H 6.43 N 10.15 Gef. C 57.12 H 6.48 N 10.02

1.4. H-Lys / "" z (oCl) _7-Asn-.Phe-0Me .HCl

3.04 g (4.4 mmol) of Boc-Lys / "z (oCl) _7-Asn-Phe-0Me were dissolved in 10 ml of 3.8 N HCl / dioxane After 30 minutes, the solvent was partially evaporated on a rotary evaporator and etherified with ether and the product was filtered off, washed with ether and dried over PPs and KOH.

Yield: 2.76 g (quantitative) Pp: 215-218 ⁰ C.

DC: uniform in BAW, BEW2, CM

C ₂₈ H ₃₇ Cl ₂ Ii ₅ O ₇ (626.50)

Ber. C 53.68 H 5.95 II 11.18 Gef. C 53.40 H 6.02 U 10.95

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Boc-Sec (Bzl) -NH-in,

2.22 g (4.5 mmol) of Boc-Seo (Bzl) -ONB, prepared according to Theodoroponlos et al., Were dissolved in 7.5 ml of MeOH-DMI ¹ (2: 1) and mixed with 1.45 ml of hydrazine Hydrate added. After 24 h at ET, the reaction solution was colored very yellow. The product was precipitated with water, filtered with suction and washed thoroughly with water and ether, whereby it decolorized.

After crystallization from MeOH, a DC uniform product was obtained in the form of fine needles.

Yield: 1.39 g (83% of theory.). Pp: 130-132 ⁰ C.

ßcj ^ = -6.68 ° (c = 1, DMP) TLC: uniform in BAW, BEW2, CM

C ₁₅ H ₂₃ N ₃ O ₃ Se (372.32)

Ber. C 48.39 H 6.23 N 11.29 Gef. C 48.42 H 6.54 N 11.02

Boc-Sec (Bzl) -Lys / ~ Z (oCl) _7-Asn-Phe-0Me (Method D)

745 mg (2 mlol) Boc-Sec (Bzl) -NHNH ₂ were dissolved in 10 ml DMP, cooled to -20 ⁰ C and treated successively with 1.9 ml of 4.2 W HCl / dioxane (8 mmol HCl) and 0, 25 ml (2.1 mmol) of tert-butylnitrite are added. After 15 min at -15 ⁰ C 1.19 g (1.9 mmol) of 1.4. and 0.266 ml (1.9 mmol) TÄA dissolved in 5 ml DMP was added to the reaction mixture. To neutralize the hydrochloric acid, an additional 1.12 ml (8 mmol) of TÄA was added dropwise (avoiding an excess of base - ^ p _H -Contro He!). The re

p _H

action solution was further stirred for 1 h at -15 ⁰ C, then slowly warmed a.uf 0 ⁰ C and left for a further 20 h at this temperature.

After completion of the reaction, the DMP was evaporated in the HV, the oily residue triturated with water to the pest product and by Yifasehen on the Pritte as

ι * 215 85 9

worked up usual. After recrystallization from DMF / EA ether (1: 1), 1.5 g (1.61 mmol = 85 % of theory ) of R.I.S. are isolated, but still small amounts of starting compound 1 ^ .4. contained. For further purification, the tetrapeptide was dissolved in a few MP, precipitated with a hot solution of MeOH-H ₂ O (1: 2) and, after filtration, washed several times with warm water, EA and ether. Finally, the product was taken up again in a little DMF and precipitated with a mixture of EE and MeOH (1: 1).

Yield: 0.87 g (. 49 # d Th.) Mp: 195-196 ⁰ C.

£ (jl ° = -4.0 ° (c = 1, DMP) DG: uniform in BAW, BEW2, GM

C ₄₃ H ₅₅ ClN ₆ O ₁₀ Se (930.31)

Ber. C 55.51 H 5.96 N 9.03 Gef. C 55.19 H 5.82 N 8.79

1.8. Boc-Ala-Gly-Sec (Bzl) -Iys ^ f "Z (OCL) _7-Asn-Phe-0ME

(Method B)

222 mg (0.9 mmol) Boc-Ala-Gly-OH, 676 mg (0.78 mmol) 1.7. and 116 mg (0.86 mmol) HOBt was dissolved in 20 ml of DMF, the solution was cooled to 5 ⁰ C, and gave them sequentially with 1 (0.78 mmol) of N-methylmorpholine and 177 mg (0.86 mmol) of DCCI (dissolved in 2 ml of DMF). After 2 h at -5 ⁰ C and 12 h at ET was filtered off from the incurred dicyclohexylurea, the solvent Vak. evaporated and the oily residue triturated with ethyl acetate to the solid. Frit washing and crystallization on DMF / ether and DMF / EE yielded the desired peptide.

Yield: 712 mg (86 # d Th.). Mp: 194-200 ⁰ C (dec.)

£ = -13.37 ° (o = 1, B ») DCi uniform in BAW, BPEW

C ₄₈ H ₆₃ ClIT ₈ O ₁₂ Se (1058.44)

Ber. C 54.47 H 6.00 N 10.59 Gef. C 54.30 H 6.36 N 10.52

I. £. Boc-.Ala- & ly-Sec (Bzl) -Iys / ~Z (oCl) _> _7 ~ Asn-Phe-MIM ₂ 635 mg (0.6 mmol) I..8. were dissolved in 5 ml of DMP, treated at 0 ⁰ C with 4 ml of hydrazine hydrate, was allowed for 20 min at 0 ⁰ C and 2 days at ET. The product was then precipitated with water, filtered off, washed thoroughly on the Pritte with water, methanol and ether and over H ₂ SO ₄ i. Yak. dried.

Yield: 612 mg (97 # d Th.). Pp: 218-225 ⁰ C (dec.) GG ^ = -20.33 ° (ο * 1 DMP) DC: uniformly in BAW, BEW2, BPEW

Amino acid analysis:

AIa (I) ⁺ 1.00 GIy (I) 0.96 Lys (i) 1.00 Asp (0.94 O Phe (O 1, 10

I.JhO.Boc-Thr-Phe-Olie (Method Cl)

3.23 g (15 mmol) of H-Phe-OMe-HCl were dissolved in 30 ml of DMP and 30 ml of THP and treated with 4.74 g (15 mmol) of Boc-Thr-ONSu. After everything had cleared, were added at 0 ⁰ C 2.1 ml (15 mmol) TÄS and the solution stirred for 3 h at 0 to 5 ⁰ C and another 48 h at ET. The product was worked up as usual and recrystallized from EE / PÄ and MeOH / H ₂ O.

Yield: 5.16 g (90.5% of theory.). Pp: 89-90 ⁰ C

² ^ = + 5.6.8 ° (c = 1, DMP) DC: uniform in BAW, BEWI, CE

C 19 ^H 28 ^Ν 2 ⁽ ) ₆ (380 , 42) H 7, 36 Ber. C 59 , 98 H 7, 42 7, 17 Gef. 59 99 H 7, 55

I.1i..H-Thr-Phe-0Me'HCl

The Boc group was removed with 4N HCl ¹ / dioxane (1 h at ET) split off and the resulting hydrochloride is precipitated with ether, filtered and recrystallized from THP / ether.

Yield: 0 C 96 % - d. th ( , , DMP ) pp: C 148 - 149 IF ⁰ C (316 , 78) C ° u \ +34, 42 ° 6 C = 1 j H 8.84 ^C 14 H ₂₁ C 6 2 ° 4 H 8.68 Ber. 53.08 H , 68 Gef. 53,30 H , 71st

, Boc-Lys / ~ Z (pCl) J ^r -Thr-Phe-OMe (Method C II)

5.81 g (10 mmol) of Boc-Lys / ~ Z (pClXJT-OPfρ were dissolved in 20 mL of THP and at 0 ⁰ C with 3.48 g (11 mmol) I.1J_. And 1.54 ml (11 mmol The product obtained after work-up and recrystallization from EE / PÄ or EE in 97% yield was uniform in TLC, but showed a pale yellowish color, and repeated crystallization from MeOH / H ₂ O gave a snow-white precipitate.

Yield: 6.26 (92.5% of theory.). Pp: 93-96 ⁰ C

βζβ *. = -4.56 ° (o-1, DMP) DC: uniform in BATi, BEW2, CM

C ₃₃ H ₄₅ ClIT ₄ O ₉ (677.17)

Ber. C 58.53 K 6.70 ΪΓ 8.27. Found C 58.33 H 6.60 N 7.88

17 21 585 9

1.12 x H-Lys / Z (pCl) _7-Thr-Phe-0Me · HCl

To cleave the Boc group, 5.9 g (8.71 mmol) of 1.12. dissolved in 25 ml of 4.2 N HCl / dioxane, the LM after 30 min i. Vak. evaporated and the product quantitatively precipitated with ether. Crystallization from MeOH / ether provided the desired peptide hydrochloride in 92% yield.

Ppi 185 -192 ⁰ C (dec.) ^ ^ J ^ - + 11.16 ° (c = 1, DMP) TLC: uniform in BAW, BEW2, PAW

Oc · Μ · Boc-Phe-D-Trp-OMe (Method A)

To a solution of 2.65 g (10 mmol) of Boc-Phe-OH, 1.4 ml (10 mmol) of TÄA and 1.3 ml (10 mmol) of Ca, BA in 30 ml of THP was added after an activation time of 6 min at -15 ⁰ C a solution of 2.54 g (10 mmol) of HD-Trp-0ME «HCl and 1.4 ml of ECO in 20 mL THP. Conventional work-up and recrystallization from EE led to the DC uniform product.

Yield: 4.28 g (. 92 $ of theory). Pp: 128.5 to 129 ⁰ C.

CoQ ² ^ = + 3.00 ° (c = 1, DMP) DC: uniform in BAW, -BEW 2, CM C ₂₆ H ₃₁ N ₃ O ₅ (465.53)

Ber. C 67.08 H 6.71 N 9.03 Gef. C 66.89 H 6.70 Έ 8.86

Boc-Phe-D-Trp-HHIHg

2.8 g (6 mmol) 1.14. were dissolved in 5 ml of DMP and 10 ml of MeOH. At 0 ° C was added 3 ml of hydrazine hydrate and left the reaction mixture for a further 24 h at 0 ⁰ C in the dark, wherein the peptide hydrazide crystallized out. By Zuga.be of water, it was completely precipitated, washed with water and ether, recrystallized from DMP / MeOH and HV

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dried over H ₂ SO ₄ .

Yield: 2.65 g (95% of theory.). Pp: 224-225 ⁰ C.

/ PQ ^ - = + 17.83 ° (0 * 1, BMP) TLC: uniform in BAW, BEW2, CM C ₂₅ H ₃₁ N ₅ O ₄ (465.54)

Ber. C 64.49 H 6.71 N 15.04 Gef. C 64.88 H 6.85 N 15.04

-Thr-Phe-OMe (Method D)

1.86 g (4 mmol) I.1J5. were dissolved in 20 ml of DMP, cooled to -20 ⁰ C and 4 ml (16 mmol) of 4N HCl / dioxane and 0.52 ml (4.4 mmol) of tert-butyl nitrite were added. After 15 min at -20 ⁰ C Kühren gave firstly 2.24 ml (16 mmol) of TIA and then a solution of 2.45 g (4 mmol) · 1-12 and 0.56 ml (4 mmol) in TIA 8 ml of DMP and left after brief stirring at -20 ⁰ C overnight at 0 ⁰ C. Subsequently, the LM was distilled off in the HV, the oily residue precipitated with water, washed on the pritte as usual and recrystallized from DMP / EE.

Yield: 2.75 g (68% of theory.). Pp: 131-133 ⁰ C.

Γ € 7ΐ ° = + 3.72 ° (ο = 1, DMP)

DC: uniform in BAW, BEW2, BPEW, amino acid analysis:

Phe (2) ⁺ 2.0 Lys (i) 1.2 Thr (i) 1.0

C ₅₃ H ₆₄ ClN ₇ O ₁₁ (1010.55)

Ber. C 62.99 H 6.38 N 9.70 Gef. C 62.38 H 6.49 N 9.45

19 21 5 85 9

1.12 · Boc-Phe-D-Trp-Lys / "Z (pCl) J7-Thr-Phe-0H (Method E)

For saponification, 5 g (4.95 mmol) I.IjS. dissolved in 50 ml of dioxane and 10 ml of DMP and treated with 6 ml in NaOH, the reaction time was 12 h. Thereafter, the LM i. Vak. stripped off and the oily residue at 0 ⁰ C with 10 #iger citric acid solution triturated to the pest product (crude product was no longer soluble in EE). After aspirating over a pritte, the peptide was washed with water, EA, a little MeOH and ether and recrystallized from DMP / EA.

Yield: 3.82 g (77.5 % of theory ), mp: 140.5 - 142 ^° C.

/ "^ 7ß ° - + 8.67 ° (c = 1, DMP) DCj uniform in BAW, BEW2, CM

C ₅₂ H ₆₂ ClN ₇ O ₁₁ (996.53)

Ber. C, 62.67, H, 6.27, N, 9.84, C, 62.43, H, 6.39, N, 9.62

JE.JjB. Boc-The-Sec-OMe (Method C II)

1.93 g (5 mmol) Boc-Thr OPfp were suspended in 10 ml THP together with 0.85 g (5.5 mmol) H-Ser-OMe-HCl. After cooling to 0 ⁰ C is first admitted to 0.7 ml (5 mmol) and, after 30 minutes, again 0.07 ml (0.5 mmol) of ECO, the solution was stirred an additional hour at 0 ⁰ C and overnight at RT and worked up as usual. The product (oil) uniform in BAW, BEW2 and CM DC was obtained in 95% yield (1j52 g) and was immediately transferred to its hydrochoride without intermediate characterization

19.01 ;. H-Thr-Ser-OMe "HCl

The oily product Ι · 1_8 · could be deacylated by reaction with 10 equivalents of 4 U HCl / dioxane and converted into its hydrochloride. After 30 min at RT, the LM i. Vak. distilled off and the

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Residue crystallized by addition of ether, resulting in a highly hygroscopic product.

Yield: 85 # d. th

L% jl ° - + 3.69 ° (o-1, IMP) BC: uniform in BAW, BEW2, CM

Boc-Phe-Trp-Lys-Il / 'Z ^ Cl ^ Thr-Phe-Thr-Ser-OMe

(Method B)

At 4.19 g (4.2 BiMoI) 1.17. ¹ »* 1 S (5.5 mmol) 1.1 ^. and 0.62 g (4.6 mmol) in 30 ml of MP was added at -5 ⁰ C 0.61 ml (5.5 mmol) IT ~ methylmorpholine and 0.95 g (4.6 mmol) OCCI (dissolved HOBt in 2 ml of IMF). The mixture was stirred for 2 h at 0 ⁰ C and laugh over ET and worked as usual by washing on the Pritte on. The product was then redissolved in a little OMF and precipitated with EA (1: 1), filtered and washed with ether, EA and a little MeOH.

Yield: 4.6 β (91 10 Ber. # d. Th. ) Ly s Thr Ser fp: 147 - 149 10 Gef. ⁰ C 1 2 1 ßü ² v - -1 < DO ⁰ (o C ₆₀ H ₇₆ ClH = 1, DMF) 1.2 1.9 1.0 Amino acid analysis: , 52 Phe ⁺ 98.73) , 34 2 2.0 9O ₁₅ (11 Ber. Ή Gef. N

1.21 · p / (p) _7

(Method E)

4.44 g (3.7 mmol) 1.20. were dissolved in 10 ml of DIF and 20 ml of MeOH and saponified by addition of a total of 4.2 ml of 1 N ETaOH within 12 h. The resulting lemon yellow solution was i. Yak. concentrated and the

oily residue was triturated EB to the pest product, with the EE turning very yellow. After filtration and thorough washing with citric acid solution, HoO, EE and ether, the mixture was crystallized from MeOH-DMF (3: 1) / H ₂ O.

Yield: 3.6 g (. 82% of theory). Pp: 165-170 ⁰ C (dec.) / JkJl ⁰ -. + 2.35 ° (c = 1, MP) DO: uniform in BAW, BEW2

Amino acid analysis: Ber. Phe ⁺ (1 Ly s Thr Ser Gef. 2 1 2 1 ^C 60 ^H 76 ^C: 2.0 1.2 1.9 1.0 10.52 198 73) Ber. N 10.34 Gef. N

1.2J _- . Boc-Phe-D-Trp-Lys / '^ zCpCl -Thr-Phe-Thr-Ser-OH

(Method E)

4.44 g (3.7 mmol) 1.20. were dissolved in 10 ml of EMF and 20 ml of MeOH and saponified by addition of a total of 4.2 ml of 1 H NaOH over the course of 12 h The resulting lemon yellow solution was concentrated in vacuo and the oily residue was triturated with EA to give the pest product the EE turned very yellow and filtered and washed thoroughly with citric acid solution, HpO, EE and ether, to crystallize from MeOH-DMP (3: 1) / H ₂ O.

Yield: 3.6 g (82 <fo of theory.). Pp: 165-170 ⁰ C (dec ") {PGF = + 2.35 ° (ο * 1 DMP) DC: uniformly in BAW, BE /? 2 amino acid analysis: Phe

Ber. 2

Gef. 2,0

Lys ⁺ Thr Ser 1 2 1 1.0 1.0

22 21 5 85 9

Ber. N 10.64 Gef. N 10.12

1.22. H.Sec (Bzl) -ONB »HCl

The Boc-Schutζ group of Boc-Sec (Bzl) -ONB was cleaved with 4N HCl / dioxane (30 min at 5 ⁰ C). After precipitation with ether, the hydrochloride was isolated quantitatively. The product was then recrystallized from CH ₂ Cl ₂ or THP. Yield: 91 # d. th

Pp: 87-90 ⁰ C

^ = -2.84 ° (c = 1, DMP) TLC: uniform in AC, BEW2, BPEW, CM C ₁₇ H ₁₉ N ₂ O ₄ SeCl (429.76)

Ber. C 47.51 H 4.46 N 6.52 Gef. C 47.73 H 4.60 N 6.39

, Boc-Phe-D-Trp-Lys / ~Z (pCl) _i J7-Thr-Phe-Thr-Ser-Sec (Bzl) -ONB (Method B)

2.6 g (2.2 mmol) 1.21, 1.5 g (3.5 mmol) H-Sec (Bzl) -ONB-HCl and 3.24 mg (2.4 mmol) HOBt in 25 ml DMP were treated with 0.39 ml (3.5 mmol) of N-methylmorpholine and 495 mg DCCI was added (in 3 ml of DMP) at -5 ⁰ C. Was allowed to stir 1 h at -5 ⁰ C, overnight at +4 ⁰ C and then for 4 hours at ET. After filtering off the Dicyclohexylharnstoffs the DMP was subtracted in the HV, the oily residue taken up in EA and washed with 10% citric acid and NaCl solution. Since the product already partially precipitated on shaking out the EE phase with NaHCO 3 solution, the precipitate was filtered off and the filtrate was eliminated by evaporation. Vak. concentrated. The remaining oil was rubbed with NaHCOo solution to the pesticide and combined with the already extracted product. It was then washed on the praise with NaHCO 3 solution, water and ether. On-

23 21585

Finally, 1. was dissolved in a little DMP, distilled off again in the HV, resulting in an oily residue which dissolved well in EE and crystallized upon addition of P1.

Yield: 3.03 g (88% of theory). Pp: 1.51-154 ⁰ C (dec.) PJl ⁰ -4.7 ° (oi, DMP) BC: uniform in BAW, BEW2, BPlW

₁ O ₁₈ Se (1559.98)

Ber. C 58.51 H 5.82 IT 9.88 Gef. C 57.87 H 5.96 Έ 9.72

1.24. H ~ Phe ~ I) -Trp-Lys / XpCl) J7-Thr-Phe-Thr-Ser-Sec (Bzl) -ONB "TPA

For cleavage of the Boc-G-group, 1.3 g (0.83 mmol) 1.23 ,. with the addition of 0.94 g (8 mmol) of indole and 0.87 ml (8 mmol) of anisole dissolved in 6 ml of methylene chloride and 15 ml of TPA. After 15 min at 0 ⁰ C and 30 min at RT, the LM i. Vak. distilled off and the oily residue taken up three times in MeOH and again concentrated on a rotary evaporator. The product was recrystallized from MeOH / ether, was thoroughly washed with ether and dried in the HV over P ₂ O ₅ and KOH.

Uniform in BAW: 122 ⁰ C (dec.) / P £ 7 ^ ° = -12.66 ° (o = 1, DMP) DC - 117: Yield: 1.02 g (78% of theory..) Pp BEW2, BPEW amino acid analysis: Phe Lys ⁺ Thr Ser

Ber. 2 1 2 Gef. 2.04 1.00 1.98 0.93

215 85 9

1.25. Boc-Ala-Gly-Sec (Bzl) -Lys / "z (oCl) _- 7-Asn-Phe-Phe-D-Trp-Lys /""Z (pCl) _7-2iir-Phe-Thr-SeivSec ( Bzl) -0HB

(Method D)

Preparation of hexapeptide azide from 582 mg (0.55 mmol) I..9. 0.60 ml (2.2 mmol) 3,7 Ii HCl / dioxane and 78.5 frl (0.66 mmol) of tert-butyl nitrite in 10 ml DMF was neutralized at -20 ⁰ C and 308 J ^ 1 TIA and followed by a solution of 866 mg (0.55 mmol) of 24 and 77 ^, 1 (0.55 mmol) TiA added in 3 ml of DMI. ¹ After a reaction time of 2 h at -25 ⁰ C, 24 h at ^{0 0} C and 5 h at RT, the DMP was partially distilled off in the HV. From the residue, the crude product was precipitated with EE and PA * and then worked up by washing on the frit.

It was then boiled with MeOH (

Removal of an impurity - according to DC I.2_4.) And washed thoroughly with MeOH, EE and ether.

Yield: 860 mg (63 # d Th.). Pp: 195-220 ⁰ C (dec.) Ssöfi £ = -17.34 ° (C = VDMF) TLC uniformly in BAW, BEW2, BPEW

Amino acid analysis:

AIa ⁺ GIy Ber. 1 1 Gef. 1.00 0.98

Lys Asp Phe Thr Ser 2 1 3 2 1 2.17 1.05 3.21 1.82 0.91

01.26 ,. H-Ala-Gly-Sec-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-

Thr-Ser-Sec-OH (Diseleno-D-Trp ⁸ -SS)

For the removal of the protective groups, the protected Tetradecapeptid 1.2J ^. first with boron tris (trifluoroacetate) with addition of thioanisole (protected peptide: thioanisole: BTI ¹ A = 1:50:50) and then treated with sodium in liquid ammonia: 746 mg (0.3 mmol) 1.2 ^. and 1.76 ml (15 mmol) of thioanisole were dissolved at 0 ⁰ C in 15 ml TFA. To this was added 30 ml (15 mmol) 0.5 M BTFA dissolved in 15 ml TFA.

To this was added 30 ml (15 mmol) of 0.5M BTPA in TPA and the mixture was shaken for 1 h at RT. Subsequently, i. Vak. evaporated, the residue was suspended several times in a little MeOH and in each case i. Vak. concentrated.

Ether was added to the oily residue, triturated to the pesticide and centrifuged. The ether was carefully poured off and the product i. Vak. dried. 690 mg of this partially already debected tetradecapeptide were then suspended in 50 ml of liquid ammonia (freshly distilled over sodium) and mixed with intensive stirring with metallic sodium in small portions until the persistent blue coloration. The peptide was completely dissolved. After decolorization of the solution by addition of the equivalent amount of ammonium chloride, the ammonia was evaporated and the last residues removed in the HV. This resulted in a slightly reddish pest product. The deblocked peptide was dissolved in 10 ml of AoOK and diluted with 1.5 l of water. The solution was then adjusted to a p _H value of 6.5 with 10% UH 2 OH and stirred in the dark over a period of 72 h at RT, continuously drawing air through the solution. The solution was then concentrated after filtering off polymeric material on an Amberlite ISC-50 column (3.5 x 15 cm) at a plating rate of 30 ml / h. The peptide was eluted with the solvent system pyridine-AcOH-E ^ O (30: 4: 66) and the individual fractions (10 ml each) of DC were examined for their peptide content. The prills containing the product were combined and the eluent evaporated i. Vak. The residue was dissolved in 2M AcOH (separating a small amount of an insoluble material that was not stained in the TLC) and lyophilized to isolate about 600 mg of a highly hygroscopic yellowish pesticide.

²⁶ 215 85 9

To remove remaining impurities, the peptide was dissolved in 20 ml of 1M AcOH and extracted four times with EA, the aqueous phase was largely decolorized. It was diluted with water and lyophilized again.

The resulting crude product was dissolved in 4 ml of 2M AcOH and chromatographed on a Sephadex C-25 F column (3.0 x 95 cm) with 2M AcOH as eluant. One collected fractions of 7.5 ml each, whose absorbance at 254 nm was measured.

DO: uniform in BPEW (tail formation) Hj ₁ : 0.43

ß0l ° = -26.76 °

Fractions 30-46 gave, after lyophilization, a product uniformly thin in BPEW, chromatographically uniform (Rp: 0.43).

Yield: 82 rag

PJ ^ = -26.76 ° (c = 0.5, 1M AcOH) amino acid analysis:

Ala Gly Sec Ly s Asp Phe Thr Ser

Ber. 112 2 13 2

Gef. ^ ₁ O <\ 8 4 ₄ SO ^ 3.0 A ₁ SO

27 215859

Example II - / ~ D-Trp J - somatostatin

II .1 ,. Boc-Cys (Mbzl) -Lys ^ f "Z (oCl) _7-Asn-Phe-0Me (Method A)

1.71 g (5 mmol) of Boc-Cys (MBzl) -OH in 20 ml THP were added at -15 ⁰ C 0.7 ml (5 mmol) of ECO and 0.65 ml (5 mmol) of CAIBE and after activation with a solution of 3.13 g (5 mmol ^ä ) and 0.7 ml of TÄA in 10 ml of THP and 10 ml of DMF. The reaction solution was stirred for 0.5 h at -15 ⁰ C and overnight at ET, worked up as usual and then recrystallized from DMP / EE.

Yield: 3.56 g (78% of theory.). Mp: 194-196 ⁰ C ßdj ^ = -3.72 ° (c = 1, DMP) DC: uniformly in BAW, BEW2, CM

C ₄₄ H ₅₇ ClEr ₆ O ₁₁ S (913.44)

Ber. C 57.85 H 6.29 K 9.20 Gef. C 57.85 H 6.32 Ν 9.12

 *) H-Lys / ~ Z (oCl) _7-Asn-Phe-0Me »HCl (1.4)

II.2. H-Cys (MBzl) -Lys fZ ^ (OCl) JP-ASn-PiIe-OMe-HCl

The Boc cleavage of Ι, .Ι ,. was carried out with 3.8 N HCl / dioxane.

Yield: quantitative Pp: 177-187 ⁰ C (dec.) CQ ^ = + 8.67 ° (e = 1, DMF) DC: uniformly in BAW, BEW2, CM

C ₃₉ H ₅₀ Cl ₂ N ₆ O ₉ S (849.82)

Ber. C 55.12 H 5.93 Έ 9.89 Gef. C 54.91 H 5.94 N 9.82

₂₈ 21 5859

II. 3, Boc-Ala-Gly-Cys (Mbzl) -Lys / "Z (oCl) J7-Asn-Phe-0Me

(Method B)

For linking 0.66 g (2.7 mmol) of Boc-Ala-Gly-OH with 2.12 g (2.5 mmol) of II.sup.2. in 25 ml DMP 0.37 g (2.75 mmol) HOBt and at -15 ⁰ C successively 0.278 ml of N-methylmorpholine and 0.57 g (2.75 mmol) DCCI was added to the reaction solution (dissolved in 2 ml DMP) , The further steps were identical to those for I..8. described. The product was then washed thoroughly with EA and 50% aqueous methanol solution.

Yield: 2.2 g (85 fo of theory.). Pp: 197-200 ⁰ C (dec.)

D & fi £ = -13.37 ° (c = 1, DMP) TLC: uniform in BAY /, BEW2 amino acid analysis: AIa (I) ⁺ 1.0 Gly (1) 1.0

Lys (i) 1.0 Asp (i) 1.2

Phe (i) 1,2

C ₄₉ H ₆₅ ClN ₈ O ₁₃ S (1041.60)

B »C 56.50 H 6.29 N 10.76 Gef. C 56.09 H 6.38 N 10.34

II .4. Boo-Alar-Gly-Cys <MBzl) -Lys / f "Z ₍₂ 2.083 g (2 mmol) of II. 3. were dissolved in 8 ml of DMP and at 0 ° C with 1.16 ml (20 mlfcl) hydrazine hydrate was added. the mixture was left for 20 min at 0 ⁰ C and 2 days at RT. the mixture was then precipitated with water, filtered, washed on the Pritte thoroughly with water, MeOH and ether, and dried over HgSO ^ in HY.

Yield: 1.96 g (94 fo of theory.). Pp: 231-234 ⁰ C (dec.) SsQ ^ Ü ^a -27.01 ° (c-1 DMP) DC: uniformly in BAW, BEW2

29 21 5

Boc-Ser-Cys (Mbzl) -OBzl (Method C II)

According to the general procedure for C II, 1.86 g (5 mmol) of Boc-Ser-OPfp were admixed with 2.02 g (5.5 mmol) H-Cys (Mbzl) -OBzl.HCl and 0.77 ml (5 5 ml) of TÄA in 15 ml of THP and worked up as usual. Before recrystallization from EE / PÄ *, the product was dissolved in EE and heat treated with activated charcoal.

Yield: 2.0 G (77 ίο d. Th.) pp: 41 - 42 ⁰ C -37 , 14 ° (0-1 f DMP)

DC: uniform in BAW, BEY / 2, CM

^C 26 ^H 34 ^M 2 ° 7 ^S (518.62) Ber. C 60.21 H 6.61 H 5.40 Gef. C 59.78 H 6.44 H 5.26

II.6. H-Ser-Cys (Mbzl) ~ OBzl »HCl

For deacidification, 1.76 g (3.4 mmol) of II.J5 · 10 ml of 5.8% HCl / dioxane were dissolved and allowed to react for 30 min. The hydrochloride was recrystallized from MeOH / ether.

Yield: 1.45 g (94 S d · Th.) Pp: 100-120 ⁰ C (dec.) / Fi7v ° = -27.74 ° (c = 1, DMP)

C ₂₁ H ₂₇ Cll ₂ O ₅ S (454.98)

Ber. C 55.43 H 5.98 H 6.16 Gef. C 54.93 H 6.09 H 5.98

³⁰ 21 5859

II.χ, Boc-Thr-Ser-Cys (Mbzl) OBzl (Method C II)

1.04 g (2.7 mmol) of Boc-Thr-OPfp in 8 ml THP were added at 0 ⁰ C with 1.36 g (3 mmol) II.6. and 0.42 ml (3 mmol) TA'A and worked up after 0.5 h at 0 ⁰ C and 15 h at ET as usual. After crystallization from EE / PA, MeOH / H ₂ O and isopropanol / HgO / resulted in a highly hygroscopic, DC uniform product.

Yield: 1.3 g (78% of theory.). Pp: 45-50 ⁰ C (highly hygroscopic) SSCSs ^ = -21.32 ° (o '1, DMP) DC: uniformly in BAW, BEW2, CM

II.8. H-Thr-Ser-Cys (MBzl) -OBzl.HCl

After deacylation of 1.24 g (2 mmol) II x 2 x with 5 ml of 5.9 W HCl / dioxane, the product was washed thoroughly with ether and recrystallized from MeOH / ether. The DC uniform product proved to be highly hygroscopic, so that a closer characterization was not possible,

Yield: 0.95 g (85.5% of theory) TLC: uniform in BAW, BEW2, CM

II_. £ * Boo-Phe-D-Trp-Lys / "Z (pCl) J7-Thr-Phe-Thr-Ser-Cys (Mbzl) -OBzl (Method B)

To a solution of 1150 mg (1.15 mmol) Boc-Phe-D-Trp-Lys2f "2 (pCl) J7-Thr-Phe-OH 1.17.695 mg (1.25 mmol) II.8 and 169 mg (1.25 mmol) of HOBt in 20 ml DMP was added at -5 ⁰ C 0.14 ml (1.25 mmol of N-Methylniorpholin and 285 mg DCCI (1.25 mmol) - was dissolved in 2 ml DMP After a. Reaction time of 2 h at 0 ⁰ C and overnight at RT was filtered after addition of a few drops of glacial acetic acid and several hours of storage of the solution in the freezer chilling dicyclohexylurea and deducted the DMP in the HV

31 215 85 9

Residue was "dissolved" in EA (slightly oily, streaky solution from which the product precipitated immediately after addition of HoO) and the crude product precipitated with PÄ, filtered and washed as usual on the frit. Wax recrystallization from DMF / ether gave a DC uniform product.

Yield: 1.45 g (84%> of theory.). Pp: - (dec. From 150 ⁰ C) 163 165 ⁰ C / ² OCj ^ = -7.44 ° (c = 1, IMi ¹⁾ DC: uniform in BAW, BEW2, CM

Amino acids- Analysis: Ber. Phe Lys Thr Ser Gef. 2 1 2 1 53 ^C1N 1 2.2 1.0 2.0 0.9 C ₇₇ H, H 6 0 ° 17 ^p (1498.1 3) Ber. C 61 , 73 H 6 , 26 Ii 9,35 Gef. C 61 , 03 , 41 IT 9.00

II. Ij). H-Phe ~ D-Trp-Lys / ~ Z (pCl) J7-Thr-Phe ~ Thr-Ser · Cys (Mbzl) -OBzl »HCl

1.39 g (0.94 mmol) of I 1 E 2 were dissolved in 20 ml of 0.12 H HCl / HC00H with the addition of 1 ml of mercaptoethanol, and the solution was left under Et 2 for 30 minutes under an argon atmosphere. The acid was then removed on a rotary evaporator and the residue was suspended twice each time in a little methanol and ether and evaporated again. The product, which crystallized after renewed addition of ether and was thoroughly etherified, was uniform in the eluent systems BAW, BEW2 and CM DE.

Yield: 1.23 g (91% of theory.). Pp: 120-140 ⁰ C (decomposition)

Boc-Ala-Gly-Cys ^ ^ bzl lys ^ 'XoClXJ'-Asn-Phe-Phe-I) -Trp-Lys-Z (pCl) -Thr-Phe-Thr-Ser-Cys (MBzl) -OBzl

(Method D)

871 mg (0.836 mmol) ΙΙ x 4. were dissolved in 10 ml of IMP, cooled to -20 ⁰ C and then treated with 0.7 ml (3.5 mmol) of 5 N HCl / dioxane and 119 al (1 mmol) of tert-butyl nitrite. Hach 20 min at -20 ⁰ C was neutralized with 0.49 ml (3.5 mmol) of ECO and then a solution of 120 mg (0.836 mmol) 11.1J). ^and 117 ^ 1 (0.836 mmol) TÄA in 5 ml DMP to the reaction. After a reaction time of 2 h at -15 ⁰ C, 24 h at 0 ⁰ C and 5 h at RT was worked up as usual, reprecipitated from DMP / ether and washed with EA, MeOH and ether. This gave a product that was almost uniform in the BAY /, BEW2 and BPEW DC systems.

Yield: 1.9 (90 ψ of the th.)

Pp: Zers, over a large temperature interval

£ ζ? 1 ° = -16.84 ° (0-1, DMP)

R _F : 0.74 (BAW)

0.75 (BEW2) 0.82 (BPEW)

Amino acid analysis:

AIa ⁺ Gly Lys Asp Phe Thr Ser Ber. 1 1 2 1 3 2 1 Gef. 1.00 0.97 2.12 1.04 3.00 1.96 0.74

Claims (4)

-χ- 215 8 5 9 ERPINDU Ή GS AISPRUCHE 1. Process for the preparation of somatostatin and somatostatin analogues of the general formula (I) H-Ala-Gly-X-Lys-Asn-Phe-Phe-Y-Lys-Thr-Phe-Thr-Ser-X-OH 1 2 3 4 5 6 7 8 9 10 11 12 13 H (D, in which X is cysteine or selenocysteine and Y can be L- or D-tryptophan, characterized in that hexapeptides of the general formula (III) S-Ala-Gly-X (T) -Lys (U) -Asn-Phe-W (III) in which X has the above meaning and S, T, U and W can be defined as follows, S = tert-butyloxycarbonyl; benzyloxycarbonyl; p-chloro- benzyloxycarbonyl; p-methoxybenzyloxycarbonyl T = benzyl; p-methoxybenzyl
U = benzyloxycarbonyl; p-chlorobenzyloxycarbonyl; o -chlorobenzyloxycarbonyl
W = methyl, ethyl or benzyl ester, subjecting them to a hydrazinolysis or alkaline hydrolysis and using the azide method or the DCCI / additive method or the mixed anhydride technique with protected octapeptides of the general formula (IV) H-Phe-Y-Lys (U) -Thr-Phe ~ Thr-Ser-X (T) -V (IV), in which X, Y, U, T are as defined above and V is benzyl- or p--tiproberyl, respectively, to give protected tetradecapeptides of the general formula (II) S-Ala-Gly-X (T) -Lys (U) -Asn-Phe-Phe-Y-Lys (U) -Thr-Phe-Thr-Ser-X (T) -V (II) - «- 215 85 9 with the abovementioned meanings for X, Y, S, T, U and V whose Schutζgruppen by treatment with hydrogen fluoride / anisole, boron tris (trifluoroacetate) / thioanisole or sodium / liquid. Cleaved ammonia and then from the resulting reduced compounds, the cyclic disulfides or diselenides of the general formula (I) are prepared by oxidation with atmospheric oxygen or with potassium ferricyanide. 2. A process for the preparation of somatostatin and soraatostatin analogs of general formula (I) according to item 1, characterized in that X is selenocysteine and Υ has the meaning of D-Trp, d. H. H-Ala-Gly-Sec-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Sec-OH 3. A process for the preparation of somatostatin and somatostatin analogs of general formula (I) according to item 1, characterized in that for the intermediate of formula (II) according to item 1 X for selenocysteine, Y has the meaning of D-Trp and S, T, U and V can be defined as in point 1, d. H. S-Ala-Gly-Sec (T) -Lys (U) -Asn-Phe-Phe-D-Trp-Lys (U) -Thr-Phe-Thr-Ser-Sec (T) -V (II) is. 4. A process for the preparation of somatostatin and somatostatin analogs of general formula (I) according to item 1, characterized in that for the intermediate of formula (HE) according to item 1 X for selenocysteine and S, T, U and W as in Point 1 are defined, d. H. S-Ala-Gly-Seo (T) -Lys (U) -Asn-Phe-W (III) is. A process for the preparation of somatostatin and somatostatin analogues of general formula (I) according to item 1, characterized in that for the intermediate of formula (IV) according to item 1 J is selenocysteine, Y has the meaning of D-Trp and U, T and V are as defined in point 1, d. H. H-Phe-D-Trp-Lys (U) -Thr-Phe-Thr-Ser-Sec (T) -V (IV).