DE2311099A1 - PSYCHOPHARMACOLOGICALLY ACTIVE PEPTIDES AND PEPTIDE DERIVATIVES - Google Patents

Description

AKZO NV
IJssellaan 82, Arnhem, The Netherlands

concerning
Psychopharmacologically active peptides and peptide derivatives

The invention relates to peptides and peptide derivatives with valuable psychophannacological properties. From European Journal of Pharmacology 2, 14 (1967) it is known that certain peptide fragments of the natural adrenocorticotropic Hormones (ACTH) prevent the extinction of the acquired evasive reaction. Especially the peptide with the amino acid sequence ACTH 1-10 was found to be effective in this regard. In addition, it has been shown that the first three Amino acids (Ser-Tyr-Ser) could even be completely omitted without a substantial loss of activity. Of the Article ends with the conclusion that a peptide with the amino acid sequence 4-10 of ACTH, namely H-Ket-GIu-His-Phe-Arg (or Lys) -Trp-Gly-OH is the shortest peptide and maybe the key sequence for said activity.

309837/1164

- 2 - 1A-42 653

The peptide with amino acid sequence 4-10 of ACTH shows but not only the psychopharmacological mentioned above Activity, but also a slight MSH activity, as is common with such fragments of ACTH. Although the Effect of a low dose of an MSH-active peptide on Is not yet known to people, investigations have been carried out to find peptides that have the same psychopharmacological activity, but none or at least one decreased MSH activity.

In an older proposal it is indicated that the amino acid L-Met with the terminal N atom of the 4-10 ACTH peptide can be replaced by the amino acid or acid groups D-Met, L- or D-Met (- # 0), L - or D-Met (- * Op), Desamino-Met, Desamino-Met (-? 0), Desamino-Met - (^ O ₂ ) or the group H ₂ NBC-

0 in the B is a branched or unbranched alkylene group with 1-6 carbon atoms such as a glycyl, valyl, alanyl, ß-Alanyl or K-Me) -alanyl group means without a Loss of activity occurs. The modification (from L-Met) to D-Met, methionyl sulfoxide, methionyl sulfone, desamino-methionyl-de8amino-methionyl sulfoxide, Desamino-methionylsulf on and ß-alanyl even increase the mentioned psychopharmacological Activity.

Surprisingly, it has now shown that a Replacement of the peptide residue Trp-Gly-OH with a terminal carbon atom the original peptide 4-10 ACTH by L-Phe-OH, L-Phe-Gly-OH or a (N-phenylalkyl) amino group to one substantial increase in the inhibition of the extinction of the acquired evasive reaction results in comparison with 4-10 ACTH.

- 3 -309 837/1164

- 3 - 1A-42 653

The present invention therefore relates to the production and use of peptides and peptide derivatives of the general formula

A-L-Glu (X) -L-His-L-Phe-L-Arg (or L-Lys) -Y,

in the A: HL- or D-Met, HL- or D-Met (-> 0), HL- or D-Met (-X) ₂ ), Deaamino-Met, Deaamino-Met (- # 0), Desamino -Met (- »0 ₂ ) or the group HpN-B-CO-, in which B • is a branched or non-branched alkylene group

with 1-6 carbon atoms iat, X is the group OH or NH ₂ and

Y is the group L-Phe-OH, L-Phe-Gly-OH or an (N-phenylalkyl) amino group the formula- -NH-AIk- / \,

in the alk a branched or non-branched alkylene group with 1-6 carbon atoms and R is a Hydrogen or halogen atom or a hydroxy or is a lower alkyl or alkoxy group, the Alkyl group contains 1-4 carbon atoms, mean

as well as their functional derivatives.

By replacing the peptide residue Trp-Gly-OH with terminal! C atom of the original 4-10 AGTH peptide by groups indicated by Y, the obtained paychopharmacological activity becomes approximately three times stronger. This activity can be further increased by replacing the amino acid residue L-Met with a terminal N atom (of the original 4-10 ACTH peptide) by another suitable group, especially ß-Ala, D-Met, L- or D-Met ( - »0), L- or D-Met (-» O ^, Desamino-Met or the corresponding sulfoxide or sulfone.

The peptides and peptide derivatives according to the invention are produced by a method as is customarily described in US Pat

_ 4 309837/1164

- 4 - 1A-42 653

Peptide chemistry is applied. The method, as they are commonly used to Herateilung the compounds of the invention can be summarized as follows:

1. Condensation of a compound (amino acid, peptide) with a free carboxyl group and protected further reactive groups with a compound (amino acid, peptide or amine) with a free amino group and protected other reactive groups in the presence of a condensation agent ;

2. Condensation of a compound (amino acid, peptide) with an activated carboxyl group and optionally protected other reactive groups with a compound (amino acid, peptide, amine) with a free NHp group, if appropriate

un <y protected other reactive groups;

3 condensation of a compound (amino acid, peptide) with a free carboxyl group ^ ιηψ protected other reactive groups with a compound (amino acid, peptide, amine) with an activated amino group and optionally protected other reactive groups,

whereupon the protecting groups are removed if necessary.

Activation of the carboxyl group can be achieved, for example, by converting the carboxyl group into an acid halide, azide, anhydride or imidazolide group or an activated ester group such as the N-hydroxysuccinimide ester group and the p-nitrophenyl ester group.

The amino group can be activated by conversion to a phosphite amide or by the "phosphorus-azo" method.

_ 5 309 837/116 4

- 5 - 1A-42 653

The usual procedures for the condensation reactions given above are: the carbodiimide process, the azide process, the mixed anhydride process and the Activated ester method (E. Schröder and K. Lübke in "The Peptides", Vol. I, 1965 (Academic Press)). Furthermore can Merrifield's "Fixed Phaeen Method" (J. Am. Chem. Soc. 85 ?, 2149 (1963)) for the production of the peptides and peptide derivatives according to the invention.

The reactive groups that are not allowed to take part in the condensation reaction take effect protected by the so-called protecting groups, which are easy can be removed again, e.g. by hydrolysis or reduction. For example, a carboxyl group can be effectively protected are esterified with methanol, ethanol, tert.-butanol, Benzyl alcohol or p-nitrobenzyl alcohol or by conversion into an amide. However, this last-named protective group can are very difficult to remove, so it is advisable to use this group only to remove the carboxyl group of the C-terminated amino acid in the last peptide or to protect the ^ carboxyl group of glutamic acid. In this case the peptide synthesis leads directly to the amide of the peptides of the general formula I.

Groups which are suitable for the effective protection of an amino group are usually acid groups, e.g. an acid group, which is derived from an aliphatic, aromatic

- 6 309837/1164

- 6 - "1A-42 653

see, araliphatic or heterocyclic carboxylic acid, such as acetic acid, benzoic acid, pyridinecarboxylic acid or an acid group which is derived from carbonic acid, such as an ethoxycarbonyl, benzyloxycarbonyl, tert-butyloxycarbonyl or ρ- methyloxybenzyloxyoarbonyl group or an acid group which is derived from a sulfonic acid like the benz oil sulfonyl or p-toluenesulfonyl group. However, other groups can also be used, such as substituted or unsubstituted aryl or aralkyl groups, for example benzyl and triphenylmethyl groups or groups such as the ortho-nitro-phenylsulfenyl or 2-benzoyl-1-methylvinyl group.

It is highly recommended to also visit the guanidino group of the Arginine, the S-amino group of lysine and the imidazole group of histidine, although this protection is not necessarily necessary is. Favorable protective groups in this context are a tert-butyl oxy carbonyl or a tosyl group for the C-amino group of lysine, a nitro group for the guanidine group of arginine and a benzyl, dinitrophenyl or trityl group for the imidazole group of histidine.

The protective groups can be split off by various customary methods, depending on the nature of the respective Group, e.g. with trifluoroacetic acid or by mild Reduction, e.g. with hydrogen and a catalyst such as ^ Palladium or with HBr in glacial acetic acid.

Peptides according to the invention which, as a group with terminal! Nitrogen is a methionyl sulfoxide or desaminomethionyl sulfoxide group can be prepared from the corresponding Met or Desamino-Met peptide using an an known mild oxidation processes, e.g. with dilute hydrogen peroxide or a peracid. Such a one

- 7 3098 * 37/1164

- 7 - 1A-42 653

Oxidation leads to a mixture of the S and R sulfoxides

(= d- or 1-sulfoxide), which in the usual way in the individual Diastereomers can be split.

When coupling S- or R-sulfoxide (d- or 1-sulfoxide) of methionine or deaminomethionine with the peptide H-GIu (X) -His-Phe-Arg (or Lys) -Y, where X and Y are the ones given above
The individual enantiomers can also be obtained directly.

The peptides according to the invention which have a methionyl sulfone (Met-K) ₂ ) or desaminomethionyl sulfone (desamino-Met + opi group as a residue with a terminal nitrogen) can be used on
most conveniently made by a known per se
Oxidation of the corresponding Met- or Desamino-Met-Peptide, e.g. with H ₂ O ₂ or a peracid

309837/1164

- 8 - 1A-42 653

among functional derivatives of the peptides according to the invention and Peptide derivatives are to be understood as follows:

1. Pharmaceutically suitable acid addition salts of the peptides and peptide derivatives;

2 · peptides or peptide derivatives, "in which one or more free amino groups are substituted by an acyl group which is derived from an aliphatic carboxylic acid with 1-6 carbon atoms such as an acetyl group;

3 · unsubstituted amides or amides substituted by alkyl groups with 1-6 carbon atoms of the peptides according to the invention and peptide derivatives which have a free carboxyl group, such as an N (CH) ₂ or N (C ₂ Hc) ₂ group;

4 esters of the peptides according to the invention which are derived from aliphatic or araliphatic alcohols with 1-18 carbon atoms, particularly lower aliphatic alcohols with 1-6 carbon atoms, such as methanol, ethanol, butanol, pentanol, or ' Cyclohexanol, and lower araliphatic alcohols with 7-10 C atoms, such as benzyl alcohol, phenylethy! Alcohol, phenylpropy! Alcohol or Cinnamy! alcohol;

5 · Metal complexes that have been formed by bringing the peptides or peptide derivatives together with a little soluble salt, hydroxide or oxide of a metal, preferably zinc, or preparations that have been manufactured are by combining the peptides according to the invention with organic, mostly polymeric substances such as gelatin, Polyphloretin phosphate or polyglutamic acid to achieve a prolonged effectiveness

The acid addition salts are obtained by reacting the compounds according to the invention with a pharmaceutically suitable one organic or inorganic acid such as HCl phosphoric acid, acetic acid, maleic acid, tartaric acid or citric acid.

309837/1164

- 9. - 1A-42

As mentioned briefly above, have the inventive Compounds and their functional derivatives are valuable psychopharmacological activities. The inventive Compounds inhibit the erasure of the acquired

Evasive reaction. This means that they can be used generally as anti-depressant agents. In particular, they can be used to treat certain mental disorders, whereby a stimulation of the Mental response (mental perfomance) is desired, as in certain types of neurosis and in old age (Senility).

The peptides according to the invention and the derivatives given above can be administered orally, parenterally or intranasally. The peptides are preferably used as an injection preparation, for which purpose they are dissolved, suspended or emulsified in a suitable liquid. But in admixture with suitable auxiliaries and fillers can also be prepared in a form which is suitable for oral administration _f such as pills, tablets or dragees. The peptides according to the invention can also be administered in the form of suppositories or sprays.

The peptides are preferably administered in daily doses of 0.001 to 1 mg / kg body weight, depending on the Activity and the form in which they are administered. Particularly favorable preparations are obtained when the peptides according to the invention are prepared in a form in which they have a longer effectiveness, e.g. incorporated in gelatine, B5-Lyph - loretinphosphat or preferably

309837/1 164 -1 ° -

- 10 - 1A-42653 ^!

in the form of metal complexes. These metal complexes can be obtained by treating the peptides with poorly soluble Metal salts, metal hydroxides or metal oxides brings together. Are as poorly soluble metal salts commonly the metal phosphates, metal pyrophosphates and metal polyphosphates are used.

Metals that can be used for this process are the metals that belong to the b groups of the periodic table belong, e.g. cobalt, nickel, copper, iron and preferably zinc as well as the metals that belong to the main groups of the periodic table and can form complexes, such as magnesium and aluminum.

The production of these metal complexes takes place in the usual way. They can be obtained by adding of peptides and a sparingly soluble metal salt, metal hydroxides or metal oxide to an aqueous medium. They can also be obtained by adding an alkaline one Medium to an aqueous solution of the peptide and an insoluble metal salt, whereby an insoluble peptide-metal hydroxide complex arises. In addition, the metal complexes can be obtained by adding the peptide, one soluble metal salt and a soluble salt to an aqueous, preferably alkaline medium, wherein the insoluble peptide-metal salt complex in situ.

The metal complexes can be used immediately as suspensions or, for example, lyophilized and then resuspended will.

Biological effectiveness

a) Elimination of the acquired evasive reaction. Male white rats, weighing approximately 15o g, were made with the help of the so-called pole jumping test

. - 11 309837/1164

- 11 - 1A-42

conditioned. The conditioned stimulus was a light over the cage for 5 seconds to the unconditioned stimulus of a shock the lattice floor of the cage followed. On three consecutive days, 10 tests per day were carried out with an average interval of 60 seconds. After this learning period, the triggering in studies of 10 individual experiments examined. All those animals that reacted 8 or more times in the first attempt at extinction, were with the substance to be examined or a Treated placebo. Subsequently, extermination tests of 10 individual tests each of 2 and 4 hours were carried out carried out after treatment of the animals with the substance to be examined

In the following table the results with the known peptide 4-10 ACTH are compared with some according to the invention Peptides.

- 12-

309837/1164

iA-42 653

dose 1. Under 2 .. Under 3 · · Unt he {it calculates comparison Peptide, ug per search search also tmf / in view of the animal Isameness (4-10 fl-Met-Glu-His-Phe- S.C. O h 2 h 4 h 'ACTH = 1)
i Arg-Trp-GIy-OH j
1
J (4-10 ACTH) 100 8th 8th 7! H-Met-Glu-Hia-Phe- 30_, 8th 3; 1 Arg-Phe-Gly-OH 30th 8th 8th 7! H-Met (4 O) -GIu-HiS- 10 8th 5 4; 3 Phe-Arg-Phe-Gly-OK 10 -J 9 7 ^! ß-Ala-Glu-His-Phe- 3 9 9 4 ^j > 10 Lya-Phe-OH 30th 9 9 8th 10 8th 8th 7! H- ^ iet-Glu-Hi s-Phe- 3 9 ö 4 i 10 Lys-Amf 30th 9 β i 10 9 7th 5! > 3

309337/1 164

- 13

23 ^ 1099

3 - 1E-42

For the description, examples and claims the following applies:

I. If no optical configuration is mentioned, the L-shape is meant.

II. The following abbreviations are used for protecting groups or activation groups,

Z = benzyl oxycarbonyl

Boo = tert-butyl oxycarbonyl

'tBii = tert-butyl

Me = methyl

ONP = p-nitrophen \ ^r lo \ y

Su = succinimide

III. For the L \ ;? xuv;.? .- n: U, tei and H .- ^ genzien. the following abbreviations are used.

Bz = benzene

'EtOH = ethanol

Bu = But of oil

Py = PyridiiA

Ac or HAc = acetic acid

Wa = water

Am = amyl alcohol

iPro = isopropanol

DMF = dimethylformamide

THF = tetrahydrofuran

DCCI = dicyclohexylcarbodiimide

DCHU = dioyolohexylurea

TAA = triethylamine

TFA = trifluoroacetic acid

Fo = formic acid

308837/1164

2311093

1-42 653

IV. Applied for the amino acid rest:

Mead

Mead (- »O)

Mead (d ^ O)

Met (i ^ G)

MetbO ₂ )

GIu (X)

GIn (X)

His

'Phe

Arg

Lys

GIy

VaI

AIa

ß-Ala

15 AIa / earth ¹ ': e follow abbreviations

■ Mt " ν ιonyl Vi?". * .- ^ nylst; Ifoxid (Raoe.ti.rjT .;

= Me

.:.:. 7.r.yi (&; m , · ..r; y'j ("" ~ 3vl foxii -

. "i, n ^ I a al fön

• i,:. Vl (X = OH) = Gin

^; JyI - ■.-1. Gl a η vl

- GI

AJ. ·.

rlixl; xnvl

V. Abbreviations for other remainders:

PPA = (Iv'prjenylpropyl) amino group

PEA = (N-Pr; enylethyl) amino group

HPEA = (N - p-hydroxyphenylethyl) amino-

group

~ (i> ^T "I-Phenylisopropyl) affiinogr .: '^<r;; e

Amf Deeamino- ^ iet

Deeaniino-Met (-> 0) Deaainino-Met (- * 0 _o (derived from Amfetamine)

^ Desaminomethionyl (= / ^ methyl-thiobutyryl) group

= Deaminoniethionyl-suj.foxide = Desiioiinomethionyl-sulfone.

309S37 / 116

2311039

- 15 - 1A-42 653

Production of the starting substances

A. Preparation of Boo- Met-Glu ( OtBu ) - His-N ₀ H _g

, ^ - 2

1) Boc-Met-Glu (OtBu) -HiS-OMe

10.52 g of Boc-Met-N "H, in 75 cm ^{5 of} DMF were removed to ⁰ ° C.

3
cools and then 23.6 cm 3.4 η HCl in THF was added and at -2O ⁰ C 5.85 cm ⁵ (43.3 mmol) isoamyl nitrite * The mixture was ^{stirred for 7 min. At -2O 0} C and then to a A solution of 17.05 g of H-Glu (OtBu) -His-OMe.2 HCl in 50 cm ^{5 of} dimethylformamide was added. Sufficient triethylamine was then added to adjust the resulting pH / eft of the mixture to 6.9. The mixture was ^{stirred at 0} ° C. for 3 days. The resulting triethylamine.HCl was then filtered off and the filtrate was evaporated to dryness. The residue was dissolved in 150 cm of ethyl acetate / v / water. The aqueous layer was separated and the ethyl acetate layer was washed twice with water. Then the aqueous layers were combined and extracted again with ethyl acetate (2 × 25 cm). The ethyl acetate layers were dried and then the solution was evaporated to about -100 cm and put ^{away at 0} ° C.
Mp 138-142 ⁰ C.
Rf in Bu: Ac: Wa (4: 1: 1) = 0.59

2) Boo-Met-Glu ( OtBu) -HiS-N ₂ H ₃

3.7 g of the methyl ester described above were dissolved in "70 cm" of methanol and then 3.7 cm ^{5 of} hydrazine hydrate were added. The mixture was stirred at room temperature for 5 hours. The solution was evaporated to dryness, stirred with water and dried.
Rf in Am: iPro: Wa (10: 4: 5) = 0.39

- 16 -

309837/1164

- 16 - 1A - 42 653

B. Preparation of Boo-D-Het-Glu (Ot Bu) -His-Ν , -, Κ-1) Boo-D-Met-Glu (OtBu) -His-OMe

10.52 g of Boc-D-Met-NH in 75 ce ⁵ DMF were reduced to ⁰ ° C.

3
cooled and then cm '3.4 η hydrochloric acid in THF was added at -20 ⁰ C and 23.6 cm 5.85 isoamyl nitrite. The mixture was stirred for 7 min and then 17 _} O5 g of H-GIu (OtBu) -HIs- OMe. 2 HCl in 50 cm DMF was added and the pH was adjusted to 6.9 with triethylamine. The mixture was ^{stirred for 3 days at 0} ° C. and filtered and the filtrate was evaporated to dryness in vacuo. The residue was taken up in i ^ C cm ethyl acetate / water and washed with water. The organic phase was dried and then evaporated to 100 cm and put ^{away at 0} ° C. The obtained crystals were dried.

Rf in Bu: Ac: Wa (4: 1: 1) = 0.63 on SiO ₃ . Pp: 69-71 ⁰ C.

2) Boo-D-Met-Glu (OtBu) -

3.2 g (5.45 mmol) of the methyl ester indicated above • 5 3

were dissolved in 60 cm of methanol and then 3.7 cm of hydrazine hydrate were added. The mixture was stirred at room temperature for 5 h , then the methanol was distilled off in vacuo and the residue was stirred with water. After drying, the hydrazide was processed further immediately. Rf in Am: iPro: Wa (10: 4: 5) = 0.37 (SiO ₂ ).

C. Manufacture of

Boc-Val-Glu (0tBu) -Hi8-0Me

Boo-Val-OH * 3126 g (15 mmol) / were in 20 carols of methylene chloride

dissolved and then added 1.73 g of N-hydroxysuccinimide. The mixture was cooled to -2O ⁰ C and then 3.09 g DCCI in 20 cm chilled methylene chloride was added and the

309837/1184 - 17 -

2311089

- 17 - 1A-42 653

resulting solution for 1 h at -20 ⁰ C and then stirred for 20 h "at + 2O ⁰ C.

The resulting DCHU was filtered and then added

PiItrat evaporated to dryness and the residue in 30 cm DMP solved. Then 7.33 g of Z-GIu (OtBu) -HiS-OMe (Kappler HeIv. 44, 1991, 1961) and 1.4 g of 1 (# palladium on carbon added. Oxygen was then passed through the solution for 5 hours and the solution was then stirred for 1 hour and filtered and evaporated the piltrate to dryness.

The residue was dissolved in aqueous ethyl acetate and washed with water, sodium bicarbonate and water. The organic Phase was dried and then the ethyl acetate was evaporated in vacuo. The residue was made from ethyl acetate / petroleum ether

Yield: 3.95 &; Mp 117 -. 119 ⁰ C
Rf in Bz: EtOE (8: 2) = 0

2) j ₃

3.73 g of the above-mentioned methyl ester were dissolved in 85 cm of methanol and then 3.72 g of hydrazine hydrate were added. The mixture was stirred for 7 hours at room temperature, then the solution was evaporated to dryness and the residue was triturated with ether.
Hf in Am: iPro: Wa (10: 4: 5) = 0.33

D. Manufacture of Boo-ß-Ala-

1) Boo-ß-Ala-Glu (OtBu) -His-OMe

3.78 g of βoc-β-Ala-OH were dissolved in methylene chloride. The solution was ^{cooled to 0} ° C. and then 2.3 g of H-hydroxyeuooinimide were added. The Gemisoh was further on

- 18 309837/1164

- 16 - 1A-42 653

-22 ⁰ C cooled and then C 1) 2 g DCGI added. The mixture was heated at -22 ⁰ O ₁ 3 r> for 30 min. Stirred at 0 ^{° C. for} 3 and 12 h at room temperature. The resulting precipitate (DCHU) was filtered off, the filtrate was evaporated to dryness and the residue was taken up in dimethylformamide. 9.77 g of Z-GIu (OtBu) - His-GKe were added to this mixture and. also 1.5 g of palladium (10>) on carbon οίε catalyst. Hydrogen gas was then passed through the mixture for 6 hours , and the mixture was then stirred for 12 hours. The Kcrai / sator vjur'ie filtered off and the filtrate to the T'rookene eirg ^; "Jsmpli." The residue was taken up in ethyl acetate and ai: .- solution was successively with sodium bicarbonate solution (S ^) and '. ^ S ^ er. The Ät'.iyl- aoetatphase was dried and ■ • ri, ^. Hr. ⁱ .eßfcr-i 'lie solution sur dryness and the -ftück ^' - aud aua Athyleoetat / petroleum ether (1: 1) umkristallisiört.
Pp: 93-95 ⁰ G.

Rf in Bz: EtOH (8: 2) = 0.25 on Si '., ..

2) Boo-ß-Ala-Glu (OtBu) -His-N ₂ F ..

3 g of the Hethyiestera obtained under 1) were dissolved in 60 cm of methanol and then 3 enr 'hydrazine hydrate was added. The mixture was stirred at room temperature for 6.5 hours, then the solution was evaporated to dryness and the residue was triturated with dry ether. The substance was used immediately for further conversion;> λ. Rf in Am: iPro: V / a (10: 4: 5) = 0.42 on SiO ₉ .

E. Preparation of Boo-G l.y-Glu (0t.bu) -Hi3

ⁿⁱ "" ■ ^J —'-- ■ ■ -—

KH

1) BoC-GIy-GIu (OtEu) -HiS-OMe

In the same way as described under C.1), Boo-Gly-Glu (OtBu) -His-OMe was prepared by reacting Boo-Gly-OH with H-GIu (OtBu) -HIs-OMe. Mp 103-108 ⁰ C. Rf in Bz: EtOH (8: 2) = 0.43 on SiO _2.

- 19 300037/1184

- 19 - 1A-42 653

2) Boc-Gly-Glu (OtBu) -Hio-N ₂ H

By reacting this substance with hydrazine hydrate, as described under A.2), Boc-Gly-Glu (OtBu) -HiS-N ₂ H was prepared.
Rf in Am: iPro: Wa (10: 4: 5) = 0.32

F. In the same way as described under G. and D. manufactured:

1) BoC-AIa-GIu (OtBu) -HiS-N ₂ H ₃ ;

Rf = 0.33 (Am: iPro: Wa = 10: 4: 5)

2) Boc-K-Ke) Ala-Glu (OtBu) -HiS-N ₃ H ₃ ; Hf = 0.31 "(Am: iPro: Wa = 10: 4: 5).

3) Desamino-Ket-Giu (OtBu) -His-N ₂ H ₃ ; Rf = 0.32 (Am: iPro: Wa = 10: 4: 5).

G. Preparation of Boo-Met-Gln-His-N pH

In the same way as described under C.1), the amino acid derivative Boc-Ket-OH was coupled to H-GIn-His-CMe, which was obtained from the corresponding Z-protected peptide (HeIv. 44, 476, 1961) by hydrogenation with IO7O Palladium on charcoal. The protected peptide ester Boc-Met-Gln-His-OMe was obtained, which was converted directly into the corresponding hydrazide by the method described under C.2) *
Rf in Am: iPro: Wa (10: 4: 5) = 0.28

H. Preparation of H-Phe-Arg-Phe-Gly-OH.HAo

1) Boo-Phe-Gly-OBzl

To a solution of 14.5 g of Boc-Phe-OH in 135 cnr methylene chloride was added 9.93 g of H-Gly-OBzl.HCl with stirring

- 20 309837 / 116A

20-1A-42 653

admitted. The suspension was cooled to -5 ° C. and 7.1 cm of triethylamine was then added. The mixture was stirred for 5 minutes, then sufficient triethylamine was added to adjust the pH of the mixture to 7 to 7.2 (approximately 3 cm). Then, the temperature of the mixture was reduced to ⁵ Methylenohlorid -22 ⁰ C and then added 10.1 g (49.3 mmol) of DCCI in 55 cm. The mixture was for 1 h at -2O ⁰ C and stirred for 12 h at room temperature and then allowed to stand for some time in the refrigerator. Then, the precipitation (DCHU) was filtered off and the FiItrat with NatriumbicarbonatIosung ^(5;? O), water, 0.1 HCl η ₁ water again and saturated NaCl solution. The organic phase was then dried and evaporated to dryness. The residue was recrystallized from ethyl acetate.
Mp: 130-132 ° C.
Hf in Bz: EtOH (9: 1) = 0.86 on SiO ₂ .

2) H-Phe-Gly-OBzl.HCl

12.5 g of the peptide obtained under 1) were in a mixture of 85 cm ethyl acetate, 33 cm nitromethane and 45 cm Dissolved methylene chloride. 54 cm of 5.66 η HCl in ethyl acetate were then added at room temperature. Gut became that Mixture stirred and evaporated to half its volume, whereupon dry ether (250 cnr) was added before gout became. The resulting white oil was separated from the liquid solution and stirred with ether.

3) Boo-Arg (N0 _o ) -Phe-Gly-0Bzl

· 3

10.03 g of the above oil were in 100 cm

Dissolved dioxane and then gradually 4.3 cm of triethyl

■ 5
amine, 2.2 lm of glacial acetic acid and 9.75% by weight of butyl oxy carbonyl (nitro) arginine acetoxime were added. The mixture was 40 h at

- 21 -

309837/1164

- 21 - 1A-42 653

Stirred tree temperature and then "to dryness" in a vacuum

evaporated. The residue was then dissolved in 150 cm of ethyl acetate . added and successively with 0.1 η ilCl, water, one

5 $ sodium bicarbonate solution, water and one saturated Washed NaCl solution. The organic layer was dried and evaporated and the residue recrystallized from dioxane.

Pp about 66 ⁰ C (conversion into a diokes oil).

Rf in Bz: EtOH (9: 1) "= 0.35

4) H-Arg (NO ₉ ) -Phe-Gly-OBzl.HCl

12.5 g of the peptide obtained under 3) were in 85 cm

•• 3 "

Ethyl acetate and 40 cm nitromethane dissolved. Then 35.5 era 5.7 η HCl in ethyl acetate was added and then the Mixture further treated as described under 2). Rf in Am: Py: Wa (5; 3; 2) = 0.66 on SiO,

'2 *

5) Boo-Phe-Arg {NO ₂ ) "Phe-Gly-0B31

To a solution of 9.14 g of the peptides obtained under 4) in 35 cm ⁵ cm of dimethylformamide, 3.42 of triethylamine was added at ⁰ C O, and then the solution was cooled to -20 ⁰ C. This solution was immediately added to a solution of 6.8 g Boo-Phe-ONP in 75 cm of dimethylformamide, which was also cooled to -20 ⁰ C, added. Then, the mixture was 1 h at -20 ⁰ C and 20 h at! Stirred at room temperature. The excess of the active ester was then removed by stirring for two hours in the presence of 147 mg of 2-dimethylaminoethylamine. The solvent was removed in vacuo and the oily residue was taken up in a mixture of ethyl acetate (200 μm) and water (35 cm). The ethyl acetate layer was washed successively with 0.1 HCl, water, a 5 # strength potassium carbonate solution, water and a saturated NaCl solution. The solid substance formed during the entire process was collected and dried in vacuo. The ethyl acetate sole was made with

- 22 309837/1164

- 22 - '1A-42 653

Aether filled up to a volume of about 1 liter, whereby further pesticide crystallized out.

Pp: 175-177 ⁰ C.

Hf in Bz- .EtOH (9: 1) = 0.32 on SiC ₂ -

6) H-Phe-Arg (NO ₂ ) -Phe-Gly-OBzl.TPA

10.4 g of the protected peptides diester obtained in 5) was dissolved with stirring in 36 cm 9Obiger trifluoroacetic acid (TFA), which had previously been cooled to approximately 1O ⁰ G. The reaction mixture was allowed to cook for 1.5 hours and then added to 400 cm of ether with vigorous stirring. After 2.5 h, the resulting gel-like substance was filtered off in vacuo and washed with ether. Rf in Bu: Py: Ac: tfa (4: 3/4: 1/4: 1) - 0.78

7) H-Phe-Arg-Phe-Gly-OH.ac et at

9.9 g of the peptide obtained under 6) were dissolved in 500 cm of 90% acetic acid. 1.8 g of 107 palladium on carbon were then added to the solution, and hydrogen was then passed through for 48 h while guiding. The catalyst was filtered off and washed with glacial acetic acid. The filtrate was evaporated to dryness in vacuo, giving a colorless oil which crystallized out ^{at 0} ° G after standing under ether for 24 hours. Hf in Bu: Py: Ac: Wa (4: 3/4: 1/4: 1) = 0.27 on SiO ₂

K. Manufacture of H-Phe-Lys (Boc) - (N-phenylpropyl) atnide

1) Z-Lya (BQd) -PPA

10.33 g (20.6 mmol) of Z-Lys (Boc) -ONP were dissolved in 80 cm ^{5 of} methylene chloride at approximately ⁰ ° G. Then 2.7 g of 3-phenylpropylamine were added to the solution and the mixture was then 1 _? Stirred for 5 h at ⁰ G and 18 h at room temperature . The solvent was evaporated and the squat was dissolved in 200 cm ^ Ätnylaoetat. The ethyl acetate solution was

- 23 -

303837/1164

- 23 - 1A-42 653

washed successively with an "! Oxygenated sodium carbonate solution, a 30% NaCl solution, 0.1 η HCl and a 30% NaGl solution . The ethyl acetate layer was then dried and evaporated to a volume of approximately 80 cm. Then sufficient ether was added until a turbidity arose and then the mixture is stored in the refrigerator for 2 h, the resulting precipitate was filtered off, mp., 78 - 79 ⁰ C.
Rf in Bz: EtOH (9: 1) = 0.53 on SiO ₂ -

2) H-Lys (Boc) -PPA

8.75 g of the compound obtained under 1) were dissolved in 120 cnr of methanol, to which 1.2 g of 10 $ palladium on carbon were added. With stirring, hydrogen was passed through the solution for 3.5 hours and then the catalyst was filtered off. The filtrate was evaporated to dryness to give a practically colorless oil which was used immediately for further reactions.
Rf in Am: Fo: Wa (7: 2: 1) = 0.58 on SiO ₂ .

3) Z-Phe-Lys ( Boc) -PPA

6.39 g of the protected amino acid derivative obtained in 2) were dissolved in 68 cm of dimethylformamide and closing a solution of 7.61 g Z-Phe-ONP in 20 cm j dimethylformamide. The mixture was stirred for 20 h at room temperature , then the solvent was evaporated off in vacuo and the residue was dissolved in 170 cm of ethyl acetate and | successively with a 5% potassium carbonate solution, a 30% HaCl solution, 0.1 η HCl and a 30% NaCl solution; washed. The ethyl acetate layer was then dried _{over Na P} SO and evaporated to approximately 100 cm. The solution was stored in the refrigerator for 3 days, during which the peptide completely crystallized out.
Rf: ^{134-136 0} C. Rf in Bz: EtOH (8: 2) = 0.72 on SiO ₂ - i

309837/1164 " ²⁴ "

- 24 - 1A-42 653

• 4) H-Phe-Lys (Boc) -P? A

9.07 g of the peptide derivative obtained under 3) were in 3 -ζ

300 cm of dimethylformamide dissolved, to which 4 cm 4 η HCl and 1.5 g 10/6 palladium on charcoal had been added. Hydrogen was passed through the solution with stirring for 3.5 hours and then the catalyst is filtered off and the filtrate evaporated to Trookene to give a practically colorless oil. Rf in Bu: Ac: Wa (4: 1: 1) = 0.63

L. In the manner described in Example K. were prepared

1) H-Phe-Lys (3oc) -Amf (the starting amine of this synthesis is L-amphetamine)

Rf in Bu: Ac: Wa (4: 1: 1) = 0.60 on SiO ₂

2) H-Phe-Lys (Boc) -PEA (the starting amine is phenylethylamine) Rf in Bu: Ac: Wa (4: 1: 1) = 0.65 on SiO ₂

3) H-Phe-Lys (Boc) -HPSA (the starting amine is p-hydroxyphenylethylamine) Rf = 0.57

M. Preparation of H-Phe-Lys (Bo o) -Phe derivatives

1) Z-Phe-Lys (Boc) -Phe-OMe

4.24 g of H-Lys (Boc) -Phe-OMe were dissolved in 25 cm of dimethylformamide and then 4.77 g (11.4 mmol) of Z-Phe-ONP were added to the solution. The mixture was stirred for approximately 24 hours and then the solvent was evaporated in vacuo. The residue was dissolved in a mixture of 120 µm ethyl acetate and 30 cm water and the ethyl acetate phase was then washed successively with 0.1 HCl, water, a sodium carbonate solution (5 $) and v / ater. The ethyl acetate was distilled off and the Äüokstand from ethyl acetate, to which a small amount of petroleum ether had been added, recrystallized.
Rf in Bz: EtOH (8: 2) = 0.76 on SiO ₃ . - 25 -

309837/1164

- 25 - 1A-42 653

2) H-Phe-Lys (Boo) -Phe-OMe

1.9 g of the peptides obtained under 1) were dissolved in 50 cm of methanol, to which 0.5 g of 10 ^ palladium on carbon were added. Hydrogen gas was then passed through the solution for 3 hours and then the catalyst was filtered off and the piltrate evaporated to Trookene, Rf in Bz: BtOH (8: 2) = 0.35 on SiO ₃ .

3) H-Phe-Lys (Boo) -Phe-NH ₂

500 mg of the ester obtained under 1) were dissolved in methanol and then the solution was saturated with ammonia. The mixture was stirred for 24 hours. The Z-protected peptide amide crystallized out of the solution.

The protective group Z was removed from this amide in the manner described under ?).

Rf in Bz: EtOH (8: 2) = 0.23 on SiO ₂ -

4) H-Phe-Lye (Boc) -Fhe-OH

0.5 g of the peptide ester obtained under 2) were in 6 cm of methanol dissolved and. then 1 eq. NaOH added.

The mixture was stirred for 1 h and then slightly

acid
acids to precipitate the tripeptide /.

Rf in Bz: BtOH (8: 2) = 0.18 on SiO ₂ .

5) H-Phe-Lys (Boc) -Phe-0 (CH ₂ ) ₃ ~ C ^

. 1 g of Z-Phe-Lys (Boo) -Phe-OH, which had been obtained by saponification of the corresponding methyl ester described under 1) by the method described under 4), was dissolved in 15 cor of DMF, whereupon 1.1 eq. Phenylpropyl bromide and 1.1 eq. Dioyolohexylamine was added to the solution. After stirring for two days, the suspension obtained was cooled to ⁰ ° C. and filtered off. The filtrate was evaporated

-26- 309837/1164

- 26 - 1A-42 653

and then add aqueous ethyl ac et-at to the residue . The mixture was then washed with 0.1 η HCl _t water, ^{0 L} A sodium bicarbonate and water and then the ethyl acetate phase was dried and evaporated. The residue obtained was partly freed from the protective groups with the aid of the procedure described under ?.) (Removal of group Z). Rf in Bz: EtOH (8: 2) = 0.40 on SiO ₀ .

it

N. H-Phe-Arg-Phe derivatives

1) Z-Phe-Arg ( NO ₅ ) -Phe-OMe

2.1 g of Z-Phe-ONP in 25 om I ^ ■ · '■' were au a- "Loounf of 2.3 g of H-ATg (NO ^) - PhS-OMe. HBr (Pp : ö3 ° Zc, rs.) _t which had been cooled to 0 ⁰ C, and 0.73 cm ^y 1 riäthylaiiiin added. the reaction mixture was 2 h "at C vnä ⁰ C. Stirred for 20 h at 20 ^J C and then evaporated and dw FiItrat stirred three times with dry ether. The residue was recrystallized from methane chloride / ether (2: 1). Rf in Bz: EtOH (8: 2) = 0.68 on SiC,

2) Z-Phe-Arg (NO ₀ ) -Phe-NH _o or: ~ Phe-Arg (HO ,,) - Phe-N!

Cc. c-

CTG was added to a solution of 0.8 g of tripeptide according to 1) in 30 cm methanol at about dry ammonia (saturated solution), or about ¹ O equiv. Dimethylamine added.

After 20 hours of stirring at 0 ⁰ G, the temperature was raised to room temperature and the suspension filtered to einstundigem stirring.

Rf in Bz: EtOH (8: 2) = 0.53 on SiO ^ for the unisubstituted amide Rf in Bz: BtOH (8: 2) = 0.62 on SiO ₂ for the dimethylamide.

30S837 / 1U4

- 27 - 1-42. 653

3) Z-Phe-Arg (N0 _o ) -Phe-0H

Tri χ

1.28 g of the / peptide ester naoh 1) were in 2 cm .1 η

Dissolved sodium hydroxide and 10 cm of methanol. As one When a clear solution was obtained, the mixture was stirred for a further 15 min and then acidified to a pH of 3 with hydrochloric acid.

The resulting precipitate was filtered off and washed with water washed.

Rf in Bz: EtOH (8: 2) = 0.43 on

4) Removal of the protecting groups

0.5 g of tripeptides 1, 2 or 3 were dissolved in 25 cm '90% acetic acid. Then 1C $ palladium on charcoal was added and the mixture was then hydrogenated for 2 days.

The resulting black suspension was filtered and then the filtrate was evaporated to dryness in vacuo and the residue was taken up in water.

The piltrate was filtered again and then lyophilized

1. H-Phe-Arg-Phe-OMe.acetate Rf * = 0.25

2. H-Phe-Arg ~ Phe-Nii ₂ .acetate Rf * = 0.18

3. H-Phe-Arg-Phe-N (CH ₃ ) ₂ .acetate Rf * = 0.22

4. HrPhe-Arg-Phe-OH.aoetat Rf * = 0.12

*) Rf in Bu: Py: Ac: Wa (4: 3/4: 1/4: 1)

P. H-Phe-Lys (Boc) -Phe-Gly-derivatives 1)

12 g of Z-Phe-Ly s (Boo) -OMe were dissolved in 30 cm of methanol and 6 cm of hydrazine hydrate . The solution was left to stand for 24 hours and then stirred at ⁰ ° C. for 2 hours. The resulting precipitate was filtered off, washed with cold methanol and dried. Rf in BztEtOH (8: 2) = 0.12

- 28

309837/1164

- 28 - 1A-42 653

2) Z-Phe-Lys (Boc) -Phe-Gly-OBzl

5 | 42 g of the hydrazide given above were dissolved in 20 cm of DMP. The solution was ^{cooled to 0} ° C. and then 2 eq. Hydrochloric acid / THF was added and the mixture cooled to -2O ⁰ C. Then 1.35 cm isoamyl nitrite were added and the mixture is then stirred for 7 min at -2O ⁰ C and zn a solution of 3.5 g H-Phe-Gly-OBzl.HCl (H.2) and 4.2 cm ³ of triethylamine (pH 7) given. The mixture was allowed to stand for 70 h at ⁰ ° C and then distilled off the solvent and / added water of .Rückstand in ethyl acetate. Then the organic phase was washed with 0.1 η HCl, sodium bicarbonate (5 #) and water. The ethyl acetate layer was dried over sodium sulfate and then the ethyl acetate was distilled off in vacuo.
Hf in Bz: EtOH (8: 2) = 0.71 (SiO ₂ ).

3) H-Phe-Lys (Boc) -Phe-Gly-OH

1 g of the peptide obtained under 2) was dissolved in 20 cm of methanol / water (1: 1). Then 10 ^ palladium on carbon was added and then hydrogen was passed through the mixture for 5 hours. The mixture was filtered through Hyflo and then the filtrate was evaporated and the residue was stirred in dry ether.
Rf in Bu: Ac: Wa (4: 1: 1) = 0.43 (SiO ₂ ).

- 29 -

309837/1164

- 29 - 1A-42 653

example 1 Production of Η-Met-GIu-Hls-Phe-Arg-Phe-Gly-OH

A) Boc-Met-Glu (OtBu) -His-Phe-Arg-Phe-Gly-OH

8.39 g of Boo-Met-Glu (OtBu) -HiS-N ₃ H ₃ (A.2) were dissolved in 80 cm ^{3 of} dimethylformamide. To this solution was 26.2 cm ³ 1.6 η stirred HC1 / THP ^"0 C was added at O under fiühren and then isoamyl nitrite at 1.88 onr ~ 20 ^a 0. Then, the mixture was 20 minutes at -2O ⁰ C and then gradually 5.8 cm triethylamine, 10 cm dimethylformamide and a solution of 8.14 g H-Phe-Arg-Fhe-Gly-OH.acetat (H.7) in 90 om ³ dimethylformamide, which with triethylamine on one pH was titrated from added. by £ ugabe of extracellular triethylamine the pH of the whole mixture was adjusted to 7. the Gsuisch was stirred for 30 inin at -20 ⁰ C and then kept irr refrigerator. at certain times has been the The pH was measured and, if necessary, adjusted to 7. After 48 h, the precipitate formed (triethylammonium chloride) was filtered off and the filtrate was evaporated to dryness in vacuo. The residue was taken up in 450% ethyl acetate. The mixture was cooled, a greenish- white matter crystallized, which was centrifuged off d was washed successively with ethyl acetate, ether, 0.07 η HCl and water and dried ashließelioh. Rf in Bu: Py: Ao: Wa (4: 0.75: 0.25: 1) = 0.41 on SiO ₂ .

B) H-Met-Glu-His-Phe-Arg-Phe-Gly-OH.acetate

3.71 g (3.42 mmol) of the protected heptapeptide, which had been obtained according to A), were dissolved in 40 cm 9 iger trifluoroacetic acid and stirred for 2 h at room temperature. Then the mixture was poured into 350 omr ether with stirring.

- 30 -301837/1164

- 30 - '1A-42 653

After stirring was filtered de ⁷ * resulting white precipitate and washed with ether. After drying , the substance was dissolved in a mixture of butanol and water (1: 1) and then 2 g of Dowex X θ ion exchanger in the acetate form were added. The mixture was then stirred for 30 minutes and then another 2 g of Dowex was added. The ion exchanger was then filtered off and the liter was lyophilized. Rf in Bu: Py: Ao: Wa (2: 0.75: 0.25: 1) = 0.30 on SiO ₂ . Finally, the product obtained was subjected to purification according to the countercurrent principle. System Eha: Ac: Wa = 4: 1: 5 * amino acid composition: His 1.04

Arg 1.00 GIu 1.00 GIy ι.00 Met O ₁ 98 Phe 1.99

Example 2

Production of A ~ Glu-His-Phe-Arg ~ Phe-Gly-peptides

Condensation of the peptide H-Phe-Arg-Phe-Gly-OH.HAc (H.7), with one of the peptides according to B.2, G.2, D.2, F.2 and P.3 according to the example 1 "resulted in the following peptide acetates after removal of the protective groups:

1. H-D-Met-Glu-Hi s-Phe-Arg-Phe-Gly-OH

2. H-Val-Glu-His-Phe-Arg-Phe-Gly-OH

3. H-β-Ala-Glu-His-Phe-Arg-Phe-Giy-OH

4. H - (* .- Me) Ala-Glu-His-Phe-Arg-Phe-Gly-OH

5. Deaamino-Met-Glu-His-Phe-Arg-Phe-Gl y-OH

"* Ef in Bu: Py; Ac: Wa (2: 3/4: 1/4: 1)

3QS837 / 1164

Rf * 0.31 Rf * 0.28 Rf * 0.28 Rf * 0.30 Rf * 0.33

- 31 - 1A-42 653

Example 3

A-Glu-His-Phe-Lys-Phe-Gly-peptide (A = Met, Desamino-Met or

GIy; X = OH or NHg)

1) Boc-Met-Glu (OtBü) -His-Phe-Lys (Boo) -Phe-Gly-OH

2.1 g of Boc-Met-GIu (OtBu) -HiS-N ₂ H ₃ (A.2) were dissolved according to the method described in Example 1 A) and converted into the azide with isoamyl nitrite and then with H-Phe-Lys (Boc) -Phe-Gly-OH (P.3) condensed. The reaction mixture obtained was ^{stirred at 0} ° C. for 70 h and then poured into water, whereupon the precipitate formed was dissolved in ethyl acetate / ethanol (1: 1). The solution was slowly added to ether. The precipitate was filtered off.
Rf in Bu: Py: Ac: Wa (4: 3/4: 1/4: 1) = 0.54

2) In the same way were obtained:

a) Boc-D-Met-Glu (OtBu) -His-Phe-Lys (Boc) -Phe-Gly-OH Condensation of peptide B.2 with peptide P.3;

b) Desamino-Ket-Giu (0tBu) -His-Phe-Ly8 (Boo) -Phe-Gly-OH by condensation of the peptide P.3 with the peptide P.3;

o) Boc-Met-Gln-His-Phe-Lys (Boc) -Phe-Gly-OH by condensation of peptide G with peptide P.3;

d) BoO-GIy-GIu (OtBu) -HiS-PhS-LyS (BOc) -PhO-GIy-OH . Condensation of the peptide E.2 with the peptide P.3

3) Removal of the protective groups from the peptides according to 1) and 2) The peptides were dissolved in 90% TFA and stirred for 1 h at room temperature. The solvent was distilled off and the residue was taken up in water and lyophilized. After drying over solid KOH, the residue was dissolved in tBu: Wa (1: 1). Dowex X θ in the acetate form was added

- 32 309837 / 116A

- 32 - 1A-42 653

and then the mixture was stirred for about 1.5 hours. The ion exchanger was then filtered off and the filtrate was lyophilized. The acetates were obtained from:

Peptides: Rf *

a) H-Met-Glu-His-Phe-Lys-Phe-Gly-OH 0.27

b) H-D-Met-Glu-His-Phe-Lys-Phe-Gly-OH 0.28 o) Desamino-Met-Glu-His-Phe-Lys-Phe-Gly-OH. 0.31

d) H-Met-Gln-Hia-Phe-Lys-Phe-Gly-OH 0.29

e) H-Gly-Glu-His-Phe-Lys-Phe-Gly-OH 0.25

* Rf in Bu: Py: Ac: Wa (2: 3/4: 1/4: 1)

Example 4

Production of A-Glu-His-Phe-Arg-Phe derivatives

1.a. Boo-Met-Glu (OtBu) -His-Phe-Arg-Phe-OH

1.17 g of Boc-Met-Glu (OtBu) -HiS-N ₂ H ₃ (A.2) were dissolved in 20 cm of DMP. This solution was cooled and then 3 cm ⁵ 2 η HCl in THF at ^{0 0} G and 0.27 cm ⁵ isoamyl nitrite at -20 ⁰ G and the mixture was stirred for 7 min. Then 1.58 g of the peptide H-Phe-Arg-Phe-OH.acetat (N.4.4) were added and the pH was adjusted to 7 with TAA.

The mixture was ^{then stirred at 0} ° C. for 70 h and then the solvent was evaporated off in vacuo and the residue was taken up in ethyl acetate and washed with water. The organic layer was evaporated to dryness and the residue was recrystallized from ethyl acetate. (Rf in Bz: EtOH (8: 2) = 0.20).

1.b. Boc-Met-Glu (OtBu) -His-Phe-Arg-Phe derivatives

By condensation of Boc-Met-Glu (Otüu) -His-N ₂ H ₇ (A.2) with H-Phe-Arg-Phe-NH ₂ (N.4.2), H-Phe-Arg-Phe-N ( CH) _g (N.4.3)

- 33 309837/1164

- 33 - 1A-42 653

or H-Phe-Arg-Phe-ΟΜθ (N.4.1) according to the one described under a) Procedure one obtained:

Boc-Met-Glu (OtBu) -His-Phe-Arg-Phe-NH ₂ Rf = 0.29 Boc-Met-Glu (OtBu) -His-Phe-Arg-Phe-N (CH ₃ ) ₂ Hf = 0 .32 Boc-Met-Glu (OtBu) -His-Phe-Arg-Phe-OMe Rf = 0.37 (Rf in Bz: EtOH = 8: 2).

1.O. Boo-A-Glu {OtBu) -His-Phe-Arg-Phe-OH (A = VaI or AIa)

By condensation of BoC-VaI-GIu (OtBu) -HiS-N ₂ H ₅ (C.2) or BoO-AIa-GIu (OtBu) -HiS-N ₂ H ₃ (P.1) with H-Phe-Arg -Phe-OH (N.4.4) after the method described above one obtained: Boc-Val-Glu (OtBu) -His-Phe-Arg-Phe-OH and Boo-Ala-Glu (OtBu) -His-Phe-Arg -Phe-OH.

1.d. Desamino-Ket-Glu (OtBu) -His-Phe-Arg-Phe-OH

Condensation of desamino-Met-Glu (OtBu) -HIs-N ₂ H ₅ (F.3) with H-Phe-Arg-Phe-OH (N.4.4) by the azide method described under a) gave the protected peptide desamino -Met-Glu (OtBu) -His-Phe-Arg-Phe-OH.

2. Removal of the protecting groups

250 mg of the peptides prepared in 1) was added 90 $ strength Trifluoresoigsäure and stirred for 1 h at 2O ⁰ C in 10 cm. The solvent was distilled off and then the residue was taken up in water and lyophilized. After drying over solid potassium hydroxide, the residue was dissolved in t-butanol: water (1: 1) and the solution was stirred with Dowex X θ in the acetate form to replace the trifluoroacetate group with the acetate group. When the pH of the solution became 4 or 5, the ion exchanger was filtered off and the filtrate was lyophilized. After countercurrent distribution, the acetates were obtained from:

- 34 -309837/1164

231 1099 1A-42 653 Rf * 0.22 0.27 0.29 0.33 0.21 0.21 0.26

Η-Met-GIu-Hi s-Phe-Arg-Phe-OH H ^ let -GIu-Hi s-Phe -Arg-Phe -NH, H-Met-Glu-His-Phe-Arg-Phe-I Η-Met-GIu-Hi s-Phe-Arg-Phe-OMe H-Val-Glu-His-Phe-Arg-Phe-OH H-AIa-Glu-His-Phe-Arg-Phe-OH Desamino-Met-Glu-His-Phe-Arg-Phe-OH * Rf in Bu: Py: Ac: v / ä (4: 3/4: 1/4: 1).

Example 5

Synthesis of A - Glu-His-Phe-Lys-Phe ~ deriva th

1 .a. Boc-D-Met-Glu (OtBu) -His-Phe-I, ys (lioc) -Phe derivatives

1.17 g of Boc-D-Met-Glu (OtBu) -BIs - N ₀ H ^ (B.2) - was dissolved in 20 cm ^: DMF. The solution was cooled and then 3 cm .2 η HCl in THP at ⁰ ° C and (2 "7 cm ³ of isoamyl nitrite at -2O ⁰ C and then the Geri / ^h";: ~ in i-RU. nrt. Then 3 mMcl of the; -: .. '■■ ■ ".-. ■ :::: ■■. 3 ui M. 2, K. 3, M.4 or M.5 prepared Pep ti α β Ur-Phe- ;;;: -. Üoo) -? H <i - 'lers vate) added and the pH-'z / ert sit TAa s-ii 7.5 a _L eztellx . I ^ aoh 70h stirring at C ^; J .1 ^ c ' ^: ic *? £ cz ±; .c ■ ii vacuum distilled off and the * n ^.; arose: tj ι >' ■ '..]; ■ ι - ·:>. γ : · ^ ·.: ...: ^ enonüne η and washed with water.. Dom; was. ' .. · Or, ", ^ -π λ ε ·:;. ■ '. ν Sc: i. ^ ht evaporated to dryness. The üücKsr-a "!" Jr ^r -; r .--. 3 further processing purely fenug. On dieee - ■ '"or ^ :; c ..! ■ ~ tn:

Boo-D ~ Met-Glu (OtBu) -hi.-.- F ^; : - ': -J - <;s' r. ^: . ^ - τ 0; · .. = O _; ti 2

Boo-D-Ket-Glu (OttJu) -riis-i λ ■, - :. · - .. ^ ^: ■ ', 0> 5C Boo-D-Met-Glu (0 + 3u) ~ Hin—: : - ^r . ;; r ^? -;

<-, i Π if \ <■ ' C-,

ar j - ^1. *. ··. - * 'Rf in Bu: Py: Ao: wa (A: 5 / A: 1/4: 1

309837/1184

- 35 - 1A-42

1.b. When using the 3 peptides Boc-ß-Ala-Glu (0tBu) -His-N ₂ H (D.2) instead of BoO-D-MOt-GIu (OtBu) -HiS-N ₂ H-, Boo-ß- Ala-Glu (OtBu) -His-Phe-Lys (Boc) -Phe-OH Rf = 0.42.

Ie When using the peptide derivative Desamino-Met-Glu (OtBu) -HiS-N ₂ H ₅ (P.3) instead of Boc-D-Met-GIu (OtBu) -HiS-N ₂ H ₃ in the method according to a) one received:
Desamino-Met-Glu (OtBu) -His-Phe-Lys (Boc) -Phe-OH
Desamino-Met-Glu (0tBu) -His-Ph9-Lys (Boc) -Phe-0Me
Desamino-Met-Glu (OtBu) -His-Phe-Lys (Boc) -Pho-NHg
Dosamino-Met-Glu (OtBu) -His-Phe-Lys (Boc)

2. Removal of the protecting groups

On the in Example 4-2. In the manner described, the peptides obtained under 1. were freed from the protective groups. The acetates of the following peptides were obtained:

Peptide: Rf *)

H-D-Met-Glu-His-Phe-Lys-Phe-OH 0.21

H-D-Met-Glu-His-Phe-Lya-Phe-OMe 0.30

H-D-Met-Glu-His-Phe-Lya-Phe-NHg 0.24

HD-Met-Glu-His-Phe-Lys-Phe-O (CH ₂ J ₅ -G ₆ H ₅ 0.33

H-β-Ala-Glu-His-Phe-Lys-Phe-OH 0.20

Desamino-Ket-Glu-His-Phe-Lys-Phe-OH 0.25

Desamino-Met-Glu-His-Phe-Lys-Phe-OKe 0.36

Desamino-Met-GIu-His-PhO-LyS-PhB-NH ₂ 0.29

Deeamino-Met-Glu-His-Phe-Lys-Phe-O (CH ₀ ) -C 1 Hc 0.38

"^ Rf in Bu: Py: Ao: Wa (4: 3/4: 1/4: 1) on

- 36 -

309837/1164

- 36 - 1A-42 653

Example 6

Synthesis of A-Glu-His-Phe-Lys-phenylalkylamides (X = OH or

NH ₂ )

According to the method described in Example 1 a , one of the peptide hydrazides prepared under A to G was converted into the azide and treated with H-Phe-Lys (3oc) -PPA (K.4), H-Phe-Lys (Boc) - Amf. (L.1), H-Phe-Lys (Boc) -PEA (L.2) or H-Fhe-Lya (3oc) -HPEA (L.3) condensed.

Removal of the protective groups from the peptides obtained in this way with 90% TFA and subsequent exchange with acetate using Dowex X 8 in the acetate form gave the acetates of the following peptide derivatives: Peptide:

H-Met-Glu-Hi s-Phe-Ly s-PPA H-De3amino-Met-Glu-His-Phe-Lys-Amf H-ß-Ala-Glu-His-Phe-Lys-PPA H-Met-Glu-His-Phe-Lys-Amf H-Val-Glu-His-Phe-Lys-PEA H-Ala-Glu-Hi s-Phe-Lys-PEA H-Met-Gln-His-Phe-Lys-HPEA

^x) Rf in Bu: Py: Ac: Wa (2: 3/4: 1/4: 1).

Example 7

Sulfoxides

1. 45 mg of the peptide H-Met-Gla-His-Fhe-Arg-Phe-OH (example 4.2) were dissolved in 4 cm acetic acid and then 15 μl 30 $ H ₂ O _{2 were} added. The mixture was stirred for 1 hour at approximately 20 ° C. and then a suspension of 20 ml of Tlatinum black in 2.5 cm of glacial acetic acid was added and stirred for 30 minutes. The mixture was then filtered. ü äas ^ iltrat; ..:. "akuum

- 37 3CS837 / 116A

Starting substances _Rf x) A.2 - 0.40 F.3 ι 0.45 D.2 -i 0.38 A. 2 Ή 0.39 C.2 π 0.43 F.1 η 0.42 G H 0.41 ι- K. 4 h L.1 (- K. 4 ι- L.1 ι- L.2 h L.2 h 1.3

- 37 - 1A-42 653

evaporated to dryness. The residue was in 10 cm tBu: Wa picked up and lyophilized.

Rf of the obtained peptide:. Η-Met (- »O) -Glu-His-Phe-Arg-Phe-OH in Bu: Py: Ac: Wa (4: 3/4: 1/4: 1) = 0.20 on SiO ₉ .

2. In the same way, the following peptide sulfoxides were made obtain:

H-Met (-> O) -Glu-His-Phe-Lys-PPA Rf = 0.36

Η-Met hO ) -GIu-Hi s-Phe-Arg-Phe-Gly-OH Rf = 0.28

H-D-Me t (- * O) -Glu-His-Phe-Lys-Phe-OH Rf = 0.19

Desamino-Met (^ 0) -GIu-His-Phe-Lys-Amf. Rf = 0.41

Desamino-Met (-> 0) -Gl u-His-Phe-Arg-Phe-OMe Rf = 0.34

Example 8

Sulfones

1. 175 mg of the peptide H-D-Met-Glu-His-Phe-Lys-Phe-OH

(Example 5-2) were in a mixture of 0.5 cm of water,

3 3

0.1 cm 4 η fluoric acid, 0.02 cm 0.5 m ammonium molybdate dissolved and then 0.06 cm 30 / ό HpOp added.

The mixture was stirred for 2 h at a temperature of about 10 ⁰ G and then added Dowex X 8 in the acetate form. After stirring for 30 minutes, the ion exchanger was filtered off and the filtrate was lyophilized.

Rf in Bu: Ac: Wa (4: 1: 1) of the peptide: HD-Met (* O ₂ ) -GIu-His-Phe-Lys-Phe-OH = 0.22 on SiO ₂ -

2. The following sulfones were prepared in the same way: Rf * ^ H-Met (■ * O ₂ ) -Glu-His-Phe-Arg-Phe-Gly-OH 0.29 Desamino-Met (- * 0 ₂ ) -Glu-His-Phe-Lys-Phe-Gly-OH 0.29 Desamino-Met (- * 0 ₂ ) -Glu-His-Phe-Lys-Phe-OMe O ₁ 38

- 38 309837/1164

- 38 - 1A-42 653

Η-Met (- »O ₂ ) -Glu-His-Phe-Lys-PPA 0.38

H-Desamino-Met (- * 0 ₂ ) -Glu-His-Phe ~ Lys-Amf. 0.43

Rf in Bu: Ao: v / a (4: 1: 1)

Example 9 Zinc complexes

1.5 cm of a solution of zinc chloride containing 50 mg of zinc / cm were added to a solution of 31.5 mg of Na ₂ HPO..2 H ₂ O in 30 cm of distilled water. The ent-standing during the addition of precipitation of zinc phosphate was dissolved by adding 4 η HCl. Then, 175 mg of NaCl and 0.5 g of benzyl alcohol were added to the mixture, and then 50 1 of the hexapeptide HL-Met-L-Glu-L-His-L-Phe-L-Lys-L-Phe-OH in the mixture mg solved. Sufficient 1 η of sodium hydroxide was then added until a pH of 8.5 was obtained and the volume was then made up to 50 cm with distilled water. 1 cm suspension contained

3 mg hexapeptide
1.5 mg zinc
0.63 mg Na ₂ HPO ₄ .2 H ₂ O
3.5 mg NaCl
10 mg benzyl alcohol

Example 10

, Production of Η-Met (1, - »O) -Glu-Hi5-Phe-Arp-Phe-OH Boo-L-Met (l, · * O) -OH was produced by reacting Boo-azide with HL- Met (1 → 0) -OH (J. Biol Chem 169, 477 (1j47)..) · Boc-L-Met (I ₁ ^ O) -OH: mp 68 ⁰ C; / _ \ / ^ ° = -58 ° (c = 1, DHP). The K-protected amino acid was converted to the corresponding N-hydroxysuccinimide ester by treatment with DCCI and HOSu.

- 39 309837/1164

- 39 - 1A-42 653

The active ester obtained was immediately used for a coupling reaction with H-Glu (OtBu) -His-Phe-Arg-Phe-OH (obtained by condensation of Z-Glu (OtBu) -His-azide with H-Phe-Arg-Phe-OH and subsequent hydrogenation of group Z) in the presence of N-ethylmorpholine.

The resulting peptide derivative Boc-L-Met (l, - * 0) -GIu (OtBu) -His-Phe-Arg-Phe-OH, Rf in Bz: EtOH (8: 2) = 0.18 on SiO ₂ was on freed from the protective group in the manner described above with the aid of TFA and converted into the acetate. HL-Met (1, - * O) -Glu-His-Phe-Arg-Phe-OH. ac et at: Rf in Bu: Py: Ac: Wa (4: 3/4: 1/4: 1) = 0.28.

The peptide sulfoxide HL-Met (d, ^ O) -Glu-His-Phe-Arg-Phe-OH.acetat was prepared in the same way, starting from Boc-L-Met (d, - * O) -OH ₁ mp 135 ° C

Claims

^62XXI 309837 / 11S4

Claims (5)

Claims before 1 .J peptides of the general formula A-L-GIu (X) -L-His-L-Phe-L-Arg (or L-Lys) -Y in the A: H-L-Met; H-D-Met; H-L-Met (- »O); H-D-I-Iet (-X)); HL-Met (4O ₂ ); HD-Met (-> O ₂ ); Desamino-het; Desamino-Met (-> 0), Desamino-Met (-> 0 _? ) Or the group HpJw-B-CO, in which ß is a branched or unbranched alkylene group with 1-6 carbon atoms, X is the group OH or NH ₂ and Y the group L-Phe-OH, L-Phe-Gly-CK or a (N-phenylalkyl) amino group of the * 'ormel -NH-AIk- /'"X , in the alk a branched or unbranched alkylene group having 1-6 carbon atoms and R represents a hydrogen or halogen atom or a hydroxy group, a lower alkyl group with 1-4 carbon atoms or a lower ^ alkoxy group with 1-4 carbon atoms, mean or the functional derivatives of these peptides and peptide derivatives. 2. Peptide according to claim 1, characterized in that Y is the group L-Phe-OH. 309837/1164 1A-42 653 3 _V peptide according to claim 1 or 2, characterized in that A denotes the group ß-Ala, D-Met, Desaiaino-Met or the sulfoxide or sulfone of L- or D-Met or Desamino-Met ". 4. Peptides according to claim 1 to 3, characterized in that they are in the form of the metal complexes are present. . 5. Process for the preparation of the peptides according to claim 1 to 3ι, characterized in that the required peptide fragments are coupled together in the correct order and then optionally the protective groups are removed and the peptides thus obtained are optionally converted into the functional derivatives and / or into the corresponding peptide sulfoxides or Peptide sulfone oxidizes if the radical A with a terminal nitrogen atom denotes the group Met or desamino-Met. 62m «* · *? / 1Τ. · 4