CH644352A5 - Process for the preparation of peptides - Google Patents

Description

The invention relates to a process for the preparation of peptides and their derivatives; these compounds have interesting physiological properties and can be made up into pharmaceutical preparations and as such in the

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Human and veterinary medicine are used.

The invention relates to a process for the preparation of peptides and their derivatives which show activity as morphine antagonists. As is generally assumed and also used in the present case, a morphine antagonist is understood to mean a compound whose biological activity mimics that of the natural alkaloid.

The pharmacological properties and therapeutic uses of morphine are documented in detail in the literature, for which on "The Pharmacological Basis of Therapeutics", published by L.S. Goodman and A. Gilman, published by The MacMillan Company, New York, 3rd edition (1965), particularly Chapter 15, pages 247 to 266, and "Martindale: The Extra Pharmacopoeia", edited by N.W. Blacow, published by The Pharmaceutical Press, London, 26th edition (1972), in particular pages 1100 to 1106. However, it is known (compare LS Goodman et al., Ie, Chapter 16) that repeated administration of morphine to the recipient leads to generation of addiction to the drug and getting used to its effects, as well as its manifestation by the appearance of withdrawal symptoms when the Administration is interrupted, leads. The search for a compound that has the activity spectrum of morphine without having its disadvantages has been the goal of many years of research. The invention now provides a method for producing new peptides and their derivatives, i.e. their salts (Q- to C4-) alkyl esters, amides, N- (Cr to C5-) alkylamides and the corresponding acid addition salts, which show an activity as morphine antagonist in both in vitro and in vivo experiments.

The invention relates to a process for the preparation of peptides as claimed in claim 1. Preferred embodiments are mentioned in claims 2 to 25.

The abbreviations used for amino acids and their radicals correspond to those commonly used in the relevant technology and can be found, for example, in the publication in Biochemistry, 11 (1972) 1726. In the foregoing and below, all information relates to the L configuration of chiral amino acids and their radicals, unless stated otherwise.

The disclaimers in the patent claims are based on the publications in Helv. Chim. Acta, 47/2 (1964), pages 417/418 and in Nature, 258 (1975), pages 577/579.

An example of radicals X8 and X9 is: L-lysyl.

If R is (C 1 -C 4-) alkyl, the alkyl radical is in particular one having 1 to 2 carbon atoms.

Of the (Cr to C4) alkyl esters of the peptides of the formula I, for. B. to name the methyl, ethyl and tert-butyl ester.

A subclass of amides and N- (Cr to C5-) alkylamides of the peptides of Formula I can be represented by the following formula:

/ '

R- (X2) n-X3-X4-X5-X6-X7- (X8) p- (X9) q-N ^

R6

whereinX2, X3, X4, X5, X6, X7, X8, X9, n, p, q and R have the meanings given for formula I and R5 and R6 together with the nitrogen atom to which they are attached are an amino or N - (Cr to C4) alkylamino group.

In the case of the acid addition salts of the peptides of the formula I and of their derivatives, the base is to be regarded as the seat of the activity, so that the acid fraction is of minor importance, although pharmacologically and pharmaceutically acceptable residues are preferred for therapeutic purposes. Examples of suitable acids are:

a) mineral acids, e.g. B. hydrochloric acid, hydrobromic acid, phosphoric acid, methaphosphoric acid, nitric acid and sulfuric acid;

b) organic acids, e.g. Tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, gulonic acid, succinic acid and arylsulfonic acids, e.g. p-toluenesulfonic acid.

The pharmaceutically and pharmacologically acceptable acid addition salts, as well as those salts that are not equally acceptable, such as the salts of hydrogen fluoride * and perchloric acid, are important for the isolation and purification of the bases, and moreover the unacceptable salts can be used to prepare the acceptable ones Salts can be used using known techniques. Such peptides and their derivatives, which have a plurality of free amino groups, can be obtained in the form of mono- or polyacid addition salts or as mixed salts of a large number of acids.

In the same way, the nature of the cation is of minor importance in the case of the salts of the peptides in which the peptide is present as a carboxylate anion together with a cation, although the cation is preferably pharmacologically and pharmaceutically acceptable for pharmaceutical purposes. Examples of suitable cations are sodium and potassium.

The properties of the peptides of the formula I and of their derivatives as morphine antagonists are to be shown in the following, it being pointed out that this illustration is only for illustration and is not intended to be a restriction.

A) In vitro:

1. Inhibition of neural evoked contractions of the isolated vasferferens in mice, in studies using the method of Hughes et al. [Brain Research 88 (1975) 296] using pulsations at 0.1 Hz, the inhibition being removed by the known narcotic antagonist naxolone (1-N-allyl-7,8-dihydro-14-hydroxy-normorphinone).

2. Reduction of electrically induced contractions of isolated guinea pig intestine when used for stimulation by the method of Paton [Brit. J. Pharmacol., 12 (1957) 119-127]. [Each segment of the intestine was piled up by means of the anode and suspended, with a weight of 2 to 3 g being loaded. Stimulation parameters: frequency 0.1 Hz; Duration 0.4ms; Voltage (supramaximal) 30 to 40 V; the contractions were transferred isotonic).

B) in vivo:

1. The compounds showed analgesic activity, e.g. were they effective in reducing phenylbenzoquinone-induced ventricular fibrillation in mice when modified according to the Hendershot et al. [J. Pharm, exp. Therap., 125 (1959) 237] were investigated, the compounds being administered by means of intracerebroventricular injection. This reduction was canceled by Naxolon.

2. The compound shows an anti-tussive effect, e.g. B. when examining guinea pigs according to the method of Boura et al. [Brit. J. Pharmacol., 39 (1970) 225].

3. The compounds are effective against diarrhea, for example they reduce the diarrhea caused by castor oil in rats.

The following compounds can be mentioned as subclasses of the peptides of the formula I and their derivatives, in which:

1. n zero;

2. p and q both zero;

3. X3 L-tyrosyl;

4. X4 D-alanine;

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5. X5 glycyl;

6. X6 L-phenylalanyl;

7. X7 the rest of D-leucine or D-methionine.

As a further subclass, those peptides and their derivatives can be mentioned which have the formula I "

R-X3-X4-X5-X6-X7- (X8) p- (X9) q-R1 (I ")

correspond. In this formula:

R, R1, p and q the definition given in formula I;

X3 L-tyrosyl, O4 -acetyl-L-tyrosyl or N-methyl-L-tyrosyl;

X4 glycyl, L-alanyl, a-methyl-alanyl or D-alanyl;

X5 glycyl, sarcosyl or L-asparaginyl;

Xe L-phenylalanyl, L-tyrosyl or L-4-chlorophenylalanyl;

X7 L-leucyl, D-leucyl, L-methionyl, D-methionyl-L-Norleu

cyl, L-threonyl or D-ß-homoleucyl;

Xs L-threonyl, D-threonyl, L-phenylalanyl, L-tyrosyl or

L-lysyl; and X9 glycyl or L-lysyl.

As a further subclass, those peptides and their derivatives can be mentioned which have the formula

R-X ^ D-Ala-Gly-L-Phe-X ^ R1

wherein

R and R1 have the meaning given for formula I; X3 L-tyrosyl or N-methyl-L-tyrosyl; and X7 represent D-leucyl or D-methionyl.

The peptides of the formula I and their derivatives can be prepared by methods known for the preparation of compounds having an analogous structure. For example, they can be prepared by stepwise coupling of suitable amino acids, either by using the classic methods of peptide synthesis or solid-phase reactions, or by first producing subunits and then coupling them together.

Such reactions can be carried out, for example, by activating the carboxyl group of the incoming amino acid and protecting the amino and carboxyl groups not participating in the reaction. Such techniques are common in peptide chemistry. Details of suitable activation and protection (masking) methods and suitable reaction conditions both for the coupling reactions and for the removal of the protective groups while suppressing racemization as far as possible can be found in the references listed below. The literature quotes are of course only for explanation.

a) Published British Patent Nos. 1042487, 1048086 and 1281383.

b) Schröder and Lüebke, "The Peptides" (Academic Press) (1965).

c) Bellean and Malek, J. Am. Chem. Soc. 90 (1968) 165.

d) Tilak, Tetrahedron Letters (1970), 849.

e) Beyerman, Helv. Chim. Acta., 56 (1973) 1729.

f) Stewart and Young, "Solid Phase Peptide Synthesis" (W.H. Freeman and Co.) (1969).

Depending on the reaction conditions, the peptides of the formula I and their derivatives are obtained in the form of the free bases or the acid addition salts, or in the case of the peptides themselves, the salts. The acid addition salts can be converted into the free bases or into the salts of other acids, while the bases can be converted into acid addition salts. The methods used are generally known. Similarly, the peptides can be converted into their salts and vice versa, the salts into the peptides or into other salts, which is also known from 644352

ten methods can happen.

The peptides of formula I and their derivatives are produced according to the invention by using a compound of formula II

R-Y'-OH (II),

wherein

R has the meaning given above; and Y1 is a partial sequence which contains at least one amino acid residue and is identical to the corresponding N-terminal partial sequence in formula I,

with a compound of formula III

H - Y2 - R1 (III),

in which R1 has the meaning given above and Y2 corresponds to the remaining part of the product to be formed, the compounds of the formulas II and III being optionally activated and / or intermediately protected and the product obtained optionally in the free base, a salt or an acid addition salt transferred.

For the preparation of peptides of the formula I and their derivatives, in which X7 is methionyl (D or L), the compound of the formula II preferably corresponds to the N-terminal fragment thereof and has either 1. the methionyl group X7 in the C-terminal position ( in this case the radical (X8) p is located in the N-terminal position in the case of the compound of the formula III or 2. the radical X6 in the C-terminal position (in this case the compound of the formula III has the methionyl group X7 in the N-terminal position); Because of the general usefulness, the former option is preferred.

It is obvious to a person skilled in the art that the L-arginyl residues can not only be introduced into the peptide chain in the manner described above, but also in situ in the already joined chain or a subunit thereof, by guanidation of an L-ornithyl Residues can be formed using a reagent such as l-guanyl-3,5-dimethylpyrazole.

It should also be mentioned that other conversions to be carried out in situ are also possible for the peptides of the formula I and their derivatives. For example, the amides and N- (Cr to C5-alkylamides can be obtained by reacting a peptide (Ci to C4-) alkyl ester, e.g. the methyl ester, with ammonia, a heterocyclic base or a mono- (Cr to C5- The peptide (Cr to C4-) alkyl esters can be obtained from the peptides by conventional esterification reactions, the (Q to C4-) alkyl esters can in turn be converted into the peptides by saponification. Existing hydroxy substituents can of course be converted into alkoxy or Benzyloxy groups can be converted using the corresponding diazoalkanes, for example a methoxy group is obtained with diazomethane Existing benzyloxy and alkanoyloxy substituents can be removed leaving the hydroxyl groups, for example by hydrogenolysis in methanol using a catalyst, for example 10% palladium Charcoal, or can occur through alkaline hydrolysis converted to alkanoyloxy groups by standard methods. These methods are generally customary in peptide chemistry and can be carried out analogously to the methods mentioned in the literature references mentioned; There you will also find detailed information on the reaction conditions to be used and suitable methods for introducing and removing protective groups.

Because of their already mentioned effectiveness as morphine antagonists, the peptides of formula I and their

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Derivatives for the treatment of mammals are used both in humanais and in veterinary medicine, provided that it is the treatment of conditions which make the use of agents with a morphine-like effect appear appropriate. Specific areas of application are mentioned below by way of example:

1. Relief of pain (analgesia), e.g. B. for pain caused by smooth muscle spasms, such as kidney and biliary colic; Pain in diseases leading to death such as cancer; post-operative pain and birth pain.

2. immobilization, e.g. B. in pre-anesthetic medication; Reassurance; Falling asleep, especially if the insomnia is caused by pain or cough, and relief from anxiety in general.

3. Suppression of cough.

4. Relief from shortness of breath, e.g. caused by acute failure of the left ventricle or by pulmonary edema.

5. Delay in defecation, e.g. B. after an ileostomy or colostomy, or in the treatment of diarrhea and dysentery.

6. inducing euphoria and treating depression, e.g. B. in connection with pain relief in diseases leading to death such as cancer.

In all of these uses, the amount of peptide or peptide derivative, hereinafter referred to as the active component, depends on the mode of administration and the severity of the condition to be treated and is ultimately at the discretion of the doctor or veterinarian. In general, however, the dose is in the range from 0.025 mg to 40 mg, preferably from 0.025 mg to 4.0 mg and in particular from 0.25 to 400 mg per kg of body weight of the mammal treated, all dosages being based on the peptide base be calculated.

The active components can be administered in any manner appropriate to the condition being treated, e.g. oral, rectal, nasal, topical (buccal), vaginal or parenteral such as subcutaneous, intramuscular or intravenous. The preferred administration form in the individual case depends on the condition to be treated, for example administration to the spine can be advantageous to relieve birth pain.

Although it is possible to administer the active component as a raw chemical, it is preferable to offer it in the form of a pharmaceutical preparation.

The formulations for both human and veterinary use contain an active component together with one or more acceptable excipients, and optionally with other therapeutic ingredients. Acceptable means that the carrier must be compatible with the other components of the preparation and does not cause any damage to the recipient; it is desirable that the formulations contain no oxidizing agents or other substances with which peptides are incompatible.

The formulations can be brought into a form suitable for oral, rectal, nasal, topical (buccal), vaginal or parenteral (including subcutaneous, intramuscular and intravenous) administration, the form of administration depending on the active component used and the condition to be treated in individual cases . The preparations are expediently offered in the form of single doses and can be prepared by methods known in pharmacy. All methods include the combination of the active component with the carrier, which is composed of one or more excipients. In general, the formulations are prepared by uniformly and intimately mixing the active component with a liquid or finely divided carrier or both and then.

if necessary, bring the product into the appropriate shape.

Formulations suitable for oral administration can be in the form of discrete units such as capsules, sachets or tablets which contain a certain amount of the active component; as powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or offered as a liquid oil-in-water or water-in-oil emulsion. In addition, the active component can be offered as a bolus, latwerge or paste.

Tablets can be made by compressing or molding, optionally with one or more adjuvants. Pressed tablets can be made by free-flowing the active component, e.g. B. as a powder or granules, optionally together with a binder, lubricant, inert diluent, surface-active agent or dispersant, mixed and pressed in a suitable machine. Molded tablets can be made by molding a mixture of the powdered component moistened with an inert liquid diluent in a suitable machine.

Formulations suitable for rectal administration can be offered as suppositories with the commonly used carriers such as cocoa butter, while suitable formulations for nasal administration are offered in nasal drops which contain the active component in an aqueous or oily solution.

Formulations suitable for topical use in the mouth can be in the form of lozenges which contain the active component in a tasty base, e.g. B. sucrose and gum arabic or tragacanth, and pastilles which contain the active component in an inert base such as gelatin and glycerol or sucrose and gum arabic are present.

Preparations suitable for vaginal administration can be in the form of a pessary, cream, paste or spray and, in addition to the active component, contain carriers known to be suitable.

Preparations suitable for parenteral administration expediently contain sterile aqueous solutions of the active component and are preferably isotonic with the blood of the recipient. Formulations of this type are expediently prepared by dissolving the active component in solid form in water and sterilizing the solution obtained and making it isotonic with the blood of the recipient. These preparations can be in single or multiple dose containers, e.g. B. in sealed ampoules or vials.

Preparations suitable for nasal administration can also be made up in the form of powders with a solid carrier. These preparations contain a coarse-grained powder with a particle size of, for example, 20 to 500 microns and are administered in the manner in which snuff is taken, i.e. H. by inhaling quickly through the nasal passages from a powder container held just under the nose.

Of course, other components such as diluents, buffers, flavors, binders, surface-active agents, thickeners, lubricants, preservatives including antioxidants and similar substances can be added to the preparations in addition to the ingredients mentioned above.

If the preparations for use in human or veterinary medicine are offered in the form of single doses, these single doses expediently contain the active component in an amount of from 0.125 to 2 g, preferably from 1.25 to 200 mg and in particular from 12 to 5 jxg to 20 mg; all weights are calculated with reference to the peptide base.

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The following examples serve to illustrate the invention, but without restricting it. All temperatures are given in ° C.

Experimental section The following abbreviations have been used throughout:

HOBT

DCCI

DCU

NMM

DMF

Pr

Pr20 pe

EtOAc

1-hydroxybenzotriazole

Dicyclohexylcarbodiimide

Dicyclohexylurea

N-methylmorpholine

Dimethylformamide

Isopropanol diisopropyl ether petroleum ether ethyl acetate d) the coupling reactions were carried out for 24 h in a cold room at + 4 ° C;

e) after the coupling, the product obtained was purified by 5 washing with acid and base to avoid unreacted

Remove reactants;

f) the alkaline saponification was carried out in aqueous methanol using an autotitrator at pH 11.5 to 12.0 with n-NaOH;

g) benzyloxycarbonyl protecting groups were removed by hydrogynolysis in methanol / acetic acid with 10% palladium on charcoal;

h) the acetate salts resulting from the hydrogenolysis were converted into the corresponding hydrochlorides by adding methanolic hydrogen chloride;

i) existing benzyl protective groups were removed by hydrogeolysis in methanol with 10% palladium on charcoal;

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Z benzoyloxycarbonyl

Bu tert-butyl

BOC tert-butyloxycarbonyl

Bzl benzyl j) existing tert-butyl and tert-butyloxycarbonyl protective groups were removed with n-hydrochloric acid in acetic acid in the presence of anisole as a rinsing agent. The cleavage reaction was allowed to proceed for 60 to 90 min;

The peptides were analyzed by thin layer chromatography on Merck silica gel plates using the following solvent systems:

k) existing OBu protective groups on the alcoholic functions of threonine and serine were removed with trifluoroacetic acid, which contained 10% water; the cleavage reaction was allowed to proceed for 90 min;

1. methyl ethyl ketone;

2. n-butanol: acetic acid: water (3: 1: 1);

3. chloroform: methanol: 32% acetic acid (12: 9: 4);

4. Chloroform: methanol: 0.880 ammonia (12: 9: 4);

5. Ethyl acetate: n-butanol: acetic acid: water (1: 1: 1: 1);

6. Chloroform: methanol (8: 1).

35 1) The peptides obtained as the final product were isolated in the form of their hydrochlorides and lyophilized from aqueous solution.

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Production Example 1 (starting compounds) BOC.Tyr.Gly.Gly.Phe.OH

Unless otherwise stated, all amino acids used showed the L configuration. The optical rotations were determined with an automatic polarimeter «Bendix NPL». The amino acids in the peptide hydrolyzates (the hydrolysis was carried out with 6n.HCl for 24 h at 110 ° C in sealed evacuated tubes) was determined using a “Beckman Spinco amino acid analyzer” model 120C or with a Rank Chromostak amino acid analyzer.

This product was made according to the scheme shown in Table 1. In Table 1, the various protecting groups have the following identities:

Z: benzyloxycarbonyl see Me: methyl

BOC: tert-butyloxycarbonyl

The following general methods were used to synthesize the peptides:

a) The coupling was carried out in DMF, DCCI serving as a coupling agent;

b) amino acid ester hydrochlorides were converted to the free esters by adding a tertiary base, either triethylamine or N-methylmorpholine;

c) if the fragment condensation contains a peptide which contains an optically active amino acid with a terminal carboxy group, e.g. B. BOC.Tyr.D-Ala.Gly.Phe.OH, HOBT was added during the coupling reaction;

55 All coupling reactions were carried out in dimethylformamide using dicyclohexylcarbodiimide, the reaction mixtures being stirred at 4 ° C. for at least 24 hours. The peptide methyl esters (1), (5) were saponified in solution (methanol / water 3: 1 v / v) with the addition of aqueous 60 2N sodium hydroxide solution at pH 11.5 (pH stat). The protective group Z was cleaved from the protected tripeptide (3) by hydrogenolysis in methanol in the presence of 10% palladium on charcoal as a catalyst.

65 The characteristic data are shown in Table 2, in which Rf relates to thin layer chromatography using Merck silica gel plates and the solvent systems indicated.

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Table 1

Tyr

Gly

Gly

Phe

BOC.Tyr.OH

BOC.Tyr

BOC.Tyr

Z.Gly.OH H.Gly.OMe (1)

Z .Gly Z.Gly Z.Gly H. Gly

- Gly

- Gly

(2)

-Gly.OMe Gly.OH

(3)

(4)

(5)

Gly Gly

Gly

(6)

Gly

H.Phe.OMe - Phe.OMe —Phe.OMe

Phe.OMe

—Phe.OH

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Production Example 2 (starting compounds) BOC.Tyr.Gly.Gly.Phe.OH

The compound (4) was prepared using the method described in the preceding preparation example

Benzyl group) as shown in Table 3, coupled and gave the protected tetrapeptide (9). This tetrapeptide was saponified in the manner described in Preparation 1 to give Compound (10); from this group Bzl was converted by hydrogenolysis in methanol in the presence of 10% palla

and with a fully protected tyrosine (Bzl means a dium on charcoal as a catalyst.

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Table 2

Rf *

Mp ° C

Crystallization from solvent

Elemental analysis

(1)

0.65a

66-67

Ethyl acetate / petroleum ether

C13Hi6N205 calculated:

C 55.7; H 5.7; N 10.0%

aqueous ethanol found:

C 55.8; H 5.9; N 10.3%

(2)

0.59a

180

QJH14N2O5 calculated:

C 54.1; H 5.3; N 10.5%

found:

C 54.3; H 5.6; N 10.4%

(3)

0.40b

58

Ethyl acetate / diisopropyl ether

C22H25N3O6 calculated:

C 61.8; H 5.9; N 9.8%

found:

C 61.6; H 5.8; N 9.6%

(4)

(H.CI)

0.77c

182.3

Methanol / isopropanol

C14H20CIN3O4 calculated:

C 51.0; H 6.1; N 12.8%

found:

C 51.3; H 6.3; N 12.6%

(5)

0.33b

(6)

0.8a

* Solvent

(a) n-butanol: acetic acid: water (3: 1: 1)

(b) methyl ethyl ketone

(c) Chloroform: methanol: 32% acetic acid (12: 9: 4)

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Table 3

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BOC.

BOC.

BOC. BOC.

Tyr OBzl t

Tyr.OH OBzl

I.

Tyr

OBzl

I.

Tyr

Tyr

Gly

H-Gly

Gly

Phe

(4)

Gly-

Gly Gly

(9)

(10)

(6)

Gly

Gly

Gly-Gly-

Phe.OMe

Phe.OMe

Phe.OH Phe.OH

Rf *

Mp and crystallization / solvent

Elemental analysis

(9)

0.83a

0.34b

(10)

0.70a

138.2 ° C

calculates C24H4oN408:

C 64.56; H 6.33; N)

3.86%

0.06b

Ethyl acetate found C24H4oN408:

C 64.42; H 6.46; N!

3.55%

* Solvent

(a) n-butanol: acetic acid: water (3: 1: 1)

(b) methyl ethyl ketone

* Solvent (a) n-butanol: acetic acid: water (3: 1: 1) (b) methyl ethyl ketone

The characteristic data for intermediates (9) and (10) are shown in Table 3, where Rf refers to thin layer chromatography using Merck silica gel and the solvent systems indicated.

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Examples 1 to 3 peptides of the formula: H.Tyr.Gly.Gly.Phe.X7.OH

Peptides corresponding to this formula were obtained by condensation of the protected tetrapeptides BOC.Tyr.Gly. Gly .Phe-OH (preparation examples 1 and 2) with suitable amino acid derivatives with a protected carboxyl group of the formula H-X7-OR.

Tyr

BOC.Tyr

BOC

h »y *

HrTyr

Gly

Gly Gly Gly

Gly

Gly Gly Gly

Phe. X;

Pne.OH H

Phe

Phs

yj

, 0R

X7

.OR

X7

.OH

-OR expediently represents one of the residues -OMe; -OEt; Hydrochloric acid in acetic acid, are released; with -OBzl or

-OBu '; -OBzl or -0Bzl (p-N02), the selection of the last -OBzl (p-N02) protected carboxyl group being able to do this

In the end, hydrogenolysis can be released from the chosen type of cleavage of the protective group.

depends. From a carboxyl- <> 5 protected with -OMe or -OEt

group can be released by alkaline saponification stage A

the ; from a carboxyl group protected with -OBU1 General method for the production of BOC.Tyr. Gly. Gly,

this by mild acidolysis, e.g. with trifluoroacetic acid or n- Phe.X7.OR. X7 means Gly, Ala, Val. R means Me.

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0.92 mmol BOC.Tyr.Gly.Gly.Phe. OH, 0.92 mmol HCl.H.X7.OMe (e.g. glycine methyl ester hydrochloride) and 1.84 mmol HOBT were dissolved in 5 ml DMF and 0.2 mmol NMM was added. The mixture was cooled to -10 ° C, after which 0.92 mmol DCCI was added. The solution was allowed to stand until it slowly warmed to room temperature and stirred overnight. The DCU was filtered, washed with a little DMF and the filtrate evaporated in vacuo. The residue was dissolved in ethyl acetate and successively with 5% aqueous NaHCÓ3; H20; 5% citric acid and water washed. The solution was dried over magnesium sulfate, evaporated and the residue crystallized from a suitable solvent.

X7 mp (crystallization / solvent)

Rf lo-fè

Elemental analysis

Amino acid analysis

Gly 108-110 ° C

MeOH / EtOAc / pe

Ala 115-117 ° C EtOAc

0.973 0.954 0.855

0.873 0.954 0.915

+ 1.37 ° C

(C = 0.5, MeOH)

-8.81 ° C

(C = 0.5 in MeOH)

C30H39N5O9. Hjo; found:

C31H41N5O9: found:

C 57.05; H 6.50; N 11.09% C 57.02; H 6.62; N 10.64%

C 59.32; H 6.58; N 11.16% C 59.19; H 7.06; N 10.74%

Gly (3.08) Tyr (1.00) Phe (1.05)

Gly (1.98) Ala (1.09) Tyr (1.00) Phe (1.03)

Val

150-151 ° C Pr / EtOAc / Pr-iO

0.973 0.955

-6.08 ° C

(C = 0.5 in MeOH)

C33H45N5O9: C 60.44; H 6.92; N 10.68% found: C 59.94; H 7.02; N 10.37%

Gly (1.97) Val (0.94) Tyr (1.00) Phe (0.999)

Level B

General method of making BOC.Tyr.Gly.Gly.-Phe.X7.OH. X7 means Gly, Ala, Val.

The protected pentapeptide BOC.Tyr. Gly. Gly.Phe.X7.OMe was dissolved in 6 ml of methanol, then 3 ml of water 35 was added and the pH was kept at 11.5 to 12.0 with n-NaOH until the theoretical amount of alkali had been added. The solution was concentrated in vacuo to the methanol.

to remove, and then diluted with water. Traces of insoluble material were filtered off; extract the solution with ethyl acetate to remove any remaining esters. The pH of the aqueous phase was then adjusted to 3 using 0.7 M citric acid solution. The precipitated peptide was extracted with ethyl acetate, the peptide dissolving in the ethyl acetate. The extract was washed with water, dried over MgS04 and concentrated in vacuo

X7

Rf

Elemental analysis

Gly

Amorphous, with petroleum ether

Ethyl acetate precipitated

0.843; 0.744; 0.835

C29H37N5O9. H20: found:

C 56.40; H 6.32; N 11.34% C 56.33; H 6.41; N 11.07%

Ala

Amorphous, precipitated with ethyl acetate from Pr20

0.803; 0.664; 0.875

Ç30H39N5O9. H20: found:

C 57.05; H 6.50; N 11.09% C 57.20; H 6.43; N 10.67%

Val

Amorphous, precipitated with petroleum ether from ethyl acetate

0.883; 0.854; 0.935

C32H43N5O9: found:

C 59.89, H 6.75% C 59.98, H 6.50%

Level C

General method of making H.Tyr. Gly. Gly. Phe.X7.OH.HCl. X7 means Gly, Ala, Val.

To 100 mg of the pentapeptide BOC.Tyr. Gly. Gly. Phe.X7. OH 5 ml 1, 0N hydrochloric acid in acetic acid and 1 ml anisole were added. After the mixture was stirred at room temperature for 90 minutes, the solvents were removed in vacuo 65. An amorphous solid was obtained by triturating the residue with dry ether. The product was dissolved in water, filtered and lyophilized to give the hydrochloride salt of the peptide as a white solid.

11

644352

Example - X7

Rf

[afè

Elemental analysis

Amino acid analysis

Gly (HCl)

Ala (HCl)

Val (HCl)

amorphous (lyophilized)

amorphous (lyophilized)

amorphous (lyophilized)

0.823 0.634 0.605

0.503 0.584 0.675

0.802 0.674 0.795

+ 27.9 ° C

(C = 0.18, in MeOH)

+ 22.3 ° C

(C = 0.2, in MeOH)

+ 31.7 ° C

(C = 0.1, in MeOH)

C23H29N507HC1.H20: found:

C 50.97; H 5.91; N 12.93% C 51.09; H 6.19; N 12.54%

C, 4H31N507.HC1.H20: C 51.85; H 6.12; N 12.60% found: C 52.14; H 6.20; N 12.10%

Q7H35N5O7.HCl.2h-2O:

found:

C 52.80; H 6.52; N 11.40% C 52.89; H 6.45; N 11.26%

Gly (3.02) Tyr (1.00) Phe (0.99)

Gly (1.95) Ala (1.01) Tyr (1.00) Phe (0.98)

Gly (2.1) Val (1.02) Tyr (1.00) Phe (1.05)

The following peptides were prepared with the characteristic data given, standard methods used in peptide chemistry being used, analogous to those described in the preceding examples and preparation examples. According to the agreement, C-terminal derivatives are identified as follows:

- OMe: methyl ester

- NH:: amide

- NHEt: ethyl amide

In Example 10, BOC.Typ.Gly.Gly.Tyr.Leu.OBuin methanol / methylene dichloride, which contained a few drops of boron trifluoride etherate, was dissolved. Then an excess of an ethereal solution of diazomethane was added and the solution was left to stand at room temperature until its reaction with Pauly's reagent was negative. The solution was evaporated and the BOC and Bu protective groups removed from the remaining solid using n-hydrochloric acid in acetic acid in the presence of anisole. The peptide hydrochloride was described as a colorless "

Amorphous powder isolated from aqueous solution after lyophilization. In Example 21, the peptide 20 obtained as a product was purified by chromatography on a silica gel column (Merck) by elution with n-butanol: acetic acid: water (3: 1: 1). The Pauly-positive fractions with Rf: 0.442 were combined, concentrated in vacuo and the residue was lyophilized from aqueous solution. The peptide base of Example 105 was prepared by reduction of the compound H.Tyr.Gly.Gly.Phe.Leu.OMe, using lithium aluminum hydride or other equivalent agents as commonly known as reducing agents. are used.

The peptide bases of Examples 111 and 112 were prepared by (a) 30 preparing Z-leucinamide from: the protected amino acid;

(b) converting the amide to nitrii (phosphorus oxychloride);

(c) preparing the protected leucine tetrazole by reacting the nitrile with hydrochloric acid; (d) Removing the protective group of the leucylamino function by hydrogenolysis

35 (10% palladium on charcoal); and (e) collecting the pentapeptide obtained in a conventional manner.

Example No. Connection

Rf

[a] d (in methanol)

4th

5 (a) 5 (b)

6

7

10th

11

12

13 "14

15

16

17th

18th

19th

20th

21st

22

23

24th

25th

26

H.Tyr.D-Ala.Gly.Phe.Leu.OH HCl H.Tyr .D-Ala.Gly.Phe.Met. OH HCl H.Tyr .D-Ala.Gly.Phe.Met. OMe H.Tyr.D-Ala.Ala.Phe.Leu.OH HCl. H.Tyr.Gly.Gly.Tyr.Leu.OH HCl H.Phe.Gly.Gly.Tyr.Leu.OH HCl Me

H.Tyr. Gly. Gly. Phe. Leu. OH HCl Me Me

I I

H.Tyr.Gly.Gly.Tyr.Leu.OH H.Tyr.Gly .D-Ala.Phe.Leu. OH HCl H.Tyr. Ala.D-Ala.Phe.Leu.OH HCl H .Tyr. Gly. Gly. Phe. Met. Thr. OH HCl H.Tyr .D-Ala.Gly.Phe.Met.Thr. OH HCl H.Arg.Tyr.Gly.Gly.Phe.Leu.OH 2HC1 H.Tyr. Ala.D-Ala.Phe.Met.OMe HCl H.Tyr. Gly.D-Ala.Phe.Met. OMe HCl H.Tyr.Ala.D-Ala.Phe.Met. OH HCl H.Tyr.Gly .D-Ala.Phe.Met. OH HCl H.Arg.Tyr.Gly.Gly.Phe.Leu.Thr.OH 2HC1 H.Tyr.D-Ala-Gly.Phe.Pro.OH HCl H.Tyr.D-Ala.D-Ala.Phe. Leu.OH HCl H.Tyr.D-Ala.Gly.Leu.Leu.OH HCl H.Tyr.Ile.Asn.Met.Leu.OH H.Tyr.Ala.Gly.Phe.Leu.OH HCl H.Tyr .Gly.Gly.Phe.Nle.OH HCl

0.632

+ 19.8 ° (c = 0.5)

0.622;

0.603

+ 17.2 ° (c = 0.5)

0.672 0.703

+ 10.4 ° (c = 0.5)

0.652;

0.564

+ 1.68 ° (c = 0.5)

0.472;

0.503

+ 24.4 ° (c = 0.54)

0.552;

0.574;

+ 26.45 ° (c = 0.54)

©

Lh OO

K>

0.624

+ 21.77 ° (c = 0.5)

0.642

0.923

+ 21.0 ° (c = 0.51)

0.602

0.903;

0.634

+ 36.67 ° (c = l)

0.602

0.943;

0.554

+ 6.43 ° (c = l)

0.72 *

0.633;

0.604; 0.605

0.10 '

0.492;

0.684

+ 12.46 ° (c = 0.52)

0.382

0.614

+ 17.7 ° (c = 0.52)

0.582

0.973;

0.884

-3.66 ° (c = 0.2)

0.612

0.983;

0.854

+ 30.19 ° (c = 0.2)

0.582

0.543;

0.614

+ 4.22 ° (c = l)

0.502

0.553;

0.564

+ 34.19 ° (c = l)

0.162

0.323;

0.514

+ 1.63 ° (c = 0.5)

0.442

-1.52 ° (c = 0.51)

0.682

0.933;

0.934

+ 43.71 ° (c = l)

0.682

0.743;

0.884

-4.43 ° (c = 0.5)

0.572

0.834

-13.5 ° (c = 0.2)

0.552

0.843;

0.864

-1.35 ° (c = l)

0.582

0.853;

0.864

+ 24.44 ° (c = 0.4)

644352

Example No. Connection

12

Rf

[a] n (in methanol)

27th

H.Tvr.D-Leu.Gly.Phe.Leu.OH HCl

0.85 '

0.862;

0.793

28

H.Tyr. Ala. Ala.Phe.Leu.OH HCl

0.76 '

0.622;

0.743

29

H.Tyr.Gly.Pro.Phe.Leu.OH HCl

0.76 '

0.682;

0.753

30th

H.Tyr.D-Ala.Gly.D-Phe.Leu.OH HCl

0.572

0.863

31

H.Tyr. Gly.Gly.Phe.D-Leu. OH HCl

0J11

0.492;

0.793

32

H.Tyr.Gly.Gly.D-Phe.Leu.OH HCl

0.512

0.853;

0.484

33

H.Tyr. Gly. Gly.Phe.Ile.OH HCl

0.761

0.852;

0.653

34

H.Tyr.D-Ala.Gly.C-Phenylglycyl.Leu.OH HCl

0.642

0.853

35

H.Phe.D-Ala.Gly.Phe.Leu.OH HCl

0.662

0.933

36

H.Tyr.D-Ala.Sar.Phe.Leu.OMe HCl

0.642

0.904

37

H.Tyr. Gly .Ala.Phe.Leu.OH HCl

0.622

0.863;

0.634

38

H.Tyr.D-Ala. Gly.Phe.Thr. OH HCl

0.502

0.514

39

H.Tyr.D-Ala.Asn.Phe.Leu.OH HCl

0.55 '

0.893;

0.694

40

H.Tyr.D-Ala.Gly.Phe.D-Leu.OMe HCl

0.632

0.924

41

H.Tyr.D-Ala.Gly.Phe.D-Leu.OH HCl

0.572

0.933;

O

CO

+ 17.3 ° (c = 0.2) -33.6 ° (c = 0.6) -30.0 ° (c = 0.5) + 27.2 ° (c = 0.5) +28 .7 ° (c = 0.4) + 24.5 ° (c = 0.52) + 22.7 ° (c = 0.6) + 43.7 ° (c = 0.48) +21.2 ° (c = 0.52) + 3.69 ° (c = 0.4) -12.07 ° (c = l) + 19.8 ° (c = 0.51) + 6.26 ° (c = 0.49) + 39.6 ° (c = 0.51) + 31.5 ° (c = 0.53)

42

H.Tyr. D-Ala. Gly .Phe.Leu. OH HCl Ac

0.602; 0.943; 0.854

+ 14.6 ° (c = 0.5)

-Ty r- = 04'-acetyltyrosyl

43

H.Tyr.D-Ala.Sar.Phe.Leu.OH HCl

0.582

0.854

+ 9.89 °

c =

= 1)

44

H.Tyr.D-Ala.Gly.Phe.Leu.Thr.OH HCl

0.552

0.933;

0.724

+ 5.13 °

C:

= 0.5)

45

H.MeTyr.Gly.Gly.Phe.Leu.OH HCl

0.572

0.643;

0.644

+ 25.5 °

c =

= 0.5)

46

H.Tyr.D-Ala.Gly.Phe.Nle.OH HCl

0.622

0.684

+ 23.2 °

c =

= 1.0)

47

H .Tyr. Gly. S ar. Phe. Leu. OMe HCl

0.562

0.703;

0.904

+ 10.2 °

c =

= 0.4)

48

H.D-Tyr.D-Ala.Gly.Phe.Leu.OH HCl

0.552

0.683

-20.7 °

c =

= 0.5)

49

H.D-Tyr.D-Ala.Gly.Phe.D-Leu.OH HCl

0.572

0.623;

0.504

-4.51 °

c =

= 0.5)

50

H.Tyr.Gly.Sar.Phe.Leu.OH HCl

0.592

0.803;

0.684

+ 17.1 °

c =

= 1.0)

51

H.Tyr.D-Ala.Gly.Phe.D-Leu.Thr. OH HCl

0.512

0.663;

0.554

+ 51.5 °

c =

= 0.52)

52

H.Tyr.D-Ala.Gly.Phe.D-Met.OH HCl

0.552

0.673;

0.584; 0.825

+ 31.0 °

c =

= 0.51)

53

H.Tyr.Pro.Gly.Phe.Leu.OH HCl

0.923

0.764;

0.665

-8.6 ° (c =

0.2)

54

H.Tyr.D-Ala.Gly.Phe.D-Met.Thr.OH HCl

0.612

0.763

+ 47.8 °

c =

= 0.51)

55

H.Tyr.Sar.Gly.Phe.Leu.OH HCl

0.723

0.914;

0.542

+ 20.8 °

c =

= 1.0)

56

H.Me.Tyr.D-Ala.Gly.Phe.Leu.OH

0.904

0.853;

0.522

57

H.Tyr.GIy.D-Pro.Phe.Leu.OH HCl

0.894

0.823;

0.592

+ 55.0 °

c =

= 1.0)

58

H.Tyr.D-Ala.D-Pro.Phe.Leu.OH sodium salt

0.744

0.813;

0.742

+ 43.2 °

c =

= 1.0)

59

H.MeTyr.Gly.Gly.Phe.Leu.OMe HCl

0.984

0.993;

0.482

+ 18.1 °

c =

= 0.4)

60

H.MeTyr.D-Ala.Gly.Phe.Leu.OMe HCl

0.914

0.893;

0.632

61

H.Tyr.D-MeAla.Gly.Phe.D-Leu.OMe HCl

0.914

0.853;

0.602;

+ 61.3 °

c =

= 0.39)

62

H.Tyr.D-MeAla.Gly.Phe.D-Leu.OH HCl

0.684

0.723;

0.622

+ 49.9 °

c =

= 0.45)

63

H.Tyr.D-Ala.Gly.Phe.Leu.OMe HCl

0.954

0.953;

0.632

+ 11.6 °

c =

= 0.44)

64

H. Tyr. D-Ala. Gly.Phe. D-Met.OMeHCl

0.944

0.933;

0.572

+ 37.8 °

c =

= 0.38)

65

H.D-Tyr.Gly.Gly.Phe.Leu.OH HCl

0.582

-39.0 °

C:

= 1.0)

66

H.MeTyr.D-Ala.Gly.Phe.D-Leu.OH HCl

0.552

0.813;

0.824

+ 40.9 °

c =

= 1)

67

H.MeTyr.D-Ala.Gly.Phe.D-Leu.OMe HCl

0.522

0.903;

0.924

+ 54.7 °

C-

= 1)

68

H.DOPA. Gly.Gly.Phe.Leu.OH HCl

0.492

0.853

+ 13.8 °

c =

= 0.4)

DOPA = 3,4-dihydroxyphenylalanyl

0.883;

69

H.Tyr.MeAla.Gly.Phe.D-Leu.OH HCl

0.512

0.794

+ 17 ° (c

= 1.0)

70

H.Tyr.D. Ala.Gly.Phe.D-Leu.D-Thr.OH HCl

0.502

+ 48.9 °

c =

= 0.5)

71

H.D-Tyr.Gly.Gly.Phe.Leu.OMe HCl

0.632

0.973;

0.934

-42.7 °

C:

= 0.1)

72

H.Tyr.MeAla.Gly.Phe.D-Leu.OMe HCl

0.592

+ 9.03 °

c:

= 0.2)

73

H.Tyr.D-Ala.Gly.Phe.D-Leu.NHEt HCl

0.612

0.934;

0.615

+ 46.5 °

c-

= 0.5)

74

H. MeTy r. D-Ala .Gly.Phe. D-Leu. NHEt HCl

0.582

0.923;

0.964

+ 50.0 °

c-

= 1)

75

Me-MeTyr. D-Ala. Gly .Phe.Leu. OH acetate

0.542

0.623;

0.664

+ 30.2 °

C:

= 0.4)

76

Me-MeTyr.Gly.Gly.Tyr.Leu.OH acetate

0.352

0.633;

0.874

+ 31.3 °

c

= 0.4)

77

Me-MeTyr.Gly.Ala.Phe.Leu.OH HCl

0.552

0.663;

0.804

+ 38.2 °

c

= 1.0)

78

Me-MeTyr.Gly.Gly.Phe.Leu.OH HCl

0.452

0.653;

0.584

+ 52.4 °

c-

= 1.0)

79

Me-MeTyr.D-Ala.Gly.Phe.Leu.OMe HCl

0.452

0.973;

0.974

80

Me-MeTyr.Gly.Gly.Phe.Leu.OMe HCl

0.382

0.923:

0.964

81

Me-MeTyr.D-Ala.Gly.Phe.D-Leu.OH HCl

0.402

0.863;

0.784

+ 62.4 °

c-

= 1.0)

82

Me-MeTyr. D-Ala. Gly.Phe.D-Leu.OMe HCl

0.412

0.753;

0.964

+ 66.4 °

c-

= 1.0)

83

Me-D-MeTyr. Gly. Gly. Phe. Leu. OH HCl

0.362

0.893;

0.604

-67.4 °

c

= 0.5)

84

Me-MeTyr.D-MeAla.Gly.Phe.D-Leu.OH HCl

0.332

0.763;

0.814

+ 58.4 °

c

= 0.2)

85

H.Tyr.Gly.Gly.Phe.Leu.Tyr.Gly.OH HCl

0.863

0.844

-4.79 °

c

= 0.1)

86

H.Tyr.D-Ala.Gly.His.Leu.OH 2HC1

0.262

0.473.

0.524

+ 20.8 °

c

= 1.0)

13 644352

Example No. Compound Rf [cc] d (in methanol)

87 H.Tyr. D-Ala. Gly.Phe.D-Leu.Phe.Gly.OH HCl 0.632 + 13.4 ° (c = 0.5)

88 H.Tyr.Gly.Gly.Phe.Met (O) .OH HCl 0.182; 0.563; 0.514 + 18.7 ° (c = 0.2)

89 H. Tyr. D-Ala. Gly. Phe. D-Leu. Lys. Lys. OH diacetate 0.443; 0.384 + 12.0 ° (c = 0.5)

90 H.Tyr.D-Trp.Gly.Phe.Leu.OH sodium salt 0.762; 0.843 -4.72 ° (c = 1.0)

Ac

I.

91 H.Tyr .D-Ala.Gly.Phe.D-Leu.Tyr.OH 0.722; 0.675 + 35.6 ° (c = 0.5)

92 H.Tyr.D-Trp.Gly.Phe.D-Leu-OH HCl 0.762 + 16.58 ° (c = 2) *

94 H.Tyr.D-Ala.Gly.Phe (4Cl) .D-Leu.OH HCl 0.424A -4.56 ° (c = 0.2)

Me

"I

95 H.Tyr.Gly.Gly.Tyr.Leu.OH HCl 0.552; 0.933; 0.794 + 28.4 ° (c = 0.1 l)

96 H.Tyr.D-Ala.Gly.Phe. (4Cl) .D-Leu.OMe HCl 0.655 + 36.5 ° (c = 0.5)

97 H.Tyr. Gly. Gly.Phe.D-Leu. OMe HCl 0.525 + 40.0 ° (c = 0.5)

98 H-MeTyr.D-Ala.GIy.Phe.D-Leu.NH2 HCl 0.562 + 50.7 ° (c = l)

99 H.Tyr.Gly.Gly.Phe.D-Met.OMe HCl 0.623; 0.645 + 43.7 ° (c = 0.5)

100 H.Tyr.Gly.Gly.Phe.D-Met.OH HCl 0.552; 0.515 + 38.3 ° (c = 0.5)

101 H. MeT y r. D-Ala. Gly .Phe. D-Met. OMe HCl 0.622; 0.753; 0.924 + 59.4 ° (c = 0.12)

102 H.Tyr. Asn. Gly. Phe.D-Met.OH 0.522; 0.533A -11.8 ° (c = 0.52)

103 H.Tyr.Asn.Gly.Phe.D-Leu.OH 0.482; 0.583A -21.0 ° (c = 0.5)

104 Me-MeTyr. Me Ala. Gly. Phe .D-Leu. OH HCl 0.432; 0.483 + 13.8 ° (c = 0.1 l)

105 'H.Tyr. Gly. Gly .Phe. O-acetylleucinol HCl 0.813; 0.844; 0.685

-O-acetylleucinol =

-NH.CH (CH2.CH (CH3) 2) .CH2.O.CO.CH3

106 H.Tyr.Gly.Gly.Phe.leucinol HCl 0.813; 0.754; 0.685 -leucinol = -NH.CH (CH2.CH (CH3) 2) .CH2.OH

107 H.Tyr.Ala (aMe) .Gly.Phe.D-Leu.OH HCÏ 0.552; 0.773; 0.594 + 25.4 ° (c = 0.52) -Ala (aMe) - = -NH.C (CH3) 2.CO-

108 H.Tyr.Ala (aMe) .Gly.Phe.D-Leu.OMe HCl 0.702; 0.893; 0.954 + 28.5 ° (c = 0.5)

109 H.ßHomoTyr.Gly.Gly.Phe.Leu.OH HCl 0.532; 0.633; 0.524 -3.14 ° (c = 0.5)

110 H.Tyr.D-Ala.Gly.Phe.D-ß-HomoLeu.OH HCl 0.5s1; 0.823; 0.494 + 10.7 ° (c = 0.5)

111 H. Tyr. D-Ala. Gly. Phe. Leu-tetrazole HCl 0.602; 0.923; 0.964 + 48.2 ° (c = 0.1)

112 H.Tyr.D-Ala.Gly.Phe.D-Leu-tetrazole HCL 0.602; 0.903; 0.944 + 39.7 ° (c = 0.1 l)

ÇH (CH3) 2

ch2

-Leu-tetrazole = -NH-CH-C-NH

NN

\ //

N

113 H-DOPA.D-Ala.Gly.Phe.D-Leu-OH HCl 0.562; 0.723 + 29.9 ° (c = 0.1)

114 H.D-DOPA-D-Ala.Gly.Phe.D-Leu.OH HCl 0.582; 0.683 + 9.8 ° (c = 0.1 l)

115 H-MeTyr.D-Ala.Gly.Phe.D-Met-NHEt HCl 0.592; 0.843; 0.904 + 59.7 ° (c = l)

116 H.MeTyr.D-Ala.Gly.Phe.D-Met.OH HCl 0.522; 0.713; 0.694 + 34.7 ° (c = l)

117 H.MeTyr.D-Ala.Gly.Phe.D-Met.NH2 HCl 0.522; 0.723; 0.854 + 48.9 ° (c = l)

118 H.Phe (4Cl) .D-Ala.Gly.Phe.D-Leu.OH acetate 0.653A; 0.454A -0.78 ° (c = 0.2)

119 H.Tyr .D-Ala.Gly.Phe (4Cl) .D-Leu.NHEt HCl 0.632; 0.683A; 0.685 + 40.7 ° (c = 0.5)

Me

120 H.Tyr.Gly.Gly.Tyr.Leu.OMe HCl 0.622; 0.963; 0.156 + 18.9 ° (c = l)

* In glacial acetic acid

4A: chloroform: methanol: ammonia (density 0.880) (120: 90: 5)

* 3A: chloroform: methanol: 32% acetic acid (120: 90: 5)

* 3A: chloroform: methanol: 32% acetic acid (120: 90: 5) 4A: chloroform: methanol: ammonia (density 0.880) (120: 90: 5)

644352

14

Pharmaceutical preparations A. tablets (20 mg / tablet)

Compound of formula I.

Lactose

Cornstarch

gelatin

Magnesium stearate

20 mg 76 mg 10 mg 2 mg 2 mg

C. Pessary (5 mg / product) compound of formula I lactose

Povidone (polyvinylpyrrolidone) magnesium stearate

To prepare tablets, compound m of formula I is mixed with lactose and corn starch. Then the mass is granulated with the gelatin dissolved in water, the granules are dried and then the magnesium stearate is added. The mass obtained in this way is pressed into tablets which weigh 110 mg per tablet.

15

B. suppositories (5 mg / product) compound of the formula I suppository base (Massa Esterinum C) for supplement

The suppository base is melted at 40 ° C. and then the compound of the formula I, which is in the form of a fine powder, is added in portions and mixed until a homogeneous mixture is present. The mixture thus obtained is poured into suitable molds, 2 g being poured into each mold. Then let the mass harden.

Massa Esterinum C is a commercially available supplement mass consisting of a mixture of mono-, di- and triglycerides of saturated vegetable fatty acids. It is launched by Henkel International in Düsseldorf.

5 mg 400 mg 5 mg 5 mg

250 mg 100 g 20

25th

30th

The compound of the formula I is mixed with lactose; then the mass is granulated with a solution of povidone in 50% aqueous ethanol. The granules are dried, mixed with the magnesium stearate and pressed with a suitably shaped stamp; the weight of the pessary thus obtained is 415 mg.

D. Freeze-dried injection (100 mg / vial) compound of the formula 100 mg

Water for injection solutions,

to 2.0 ml

The compound of formula I is dissolved in the water for injection solutions; sterilize the solution by passing through a membrane filter with a pore size of 0.2 µm and collect the filtrate in a sterile container. The solution thus obtained is filled into sterile glass vials, 2 ml / vial, under aseptic conditions and freeze-dried. The vials are sealed with a sterile rubber seal that is secured with an aluminum seal.

The injection is reconstituted by adding an appropriate volume of water for injections or sterile saline before use.

In the preceding formulation examples, the weight of the compound of formula I is calculated in each case with reference to the peptide base.

M

Claims (25)

644352 2. The method according to claim 1, characterized in that a (Q- to C4-) alkyl ester of formula I with ammonia or a mono- (Cr to C4) alkylamine in the corresponding amide or N- (Cr to C4-) alkylamide transferred. 2nd PATENT CLAIMS 1. Process for the preparation of a peptide of formula I. R- (X2) n-X3-X4-X5-X ^ X7- (XV (X®VRl (I), or their salts, (Cr to C4-) alkyl esters, amides, N- (Cr to Cs-) alkylamides and the corresponding acid addition salts, in which n is Ooderl; X2 L-arginyl; X3 D- or L-tyrosyl, D- or LN-methyltyrosyl, D- or L-3,4-dihydroxyphenylalanyl, L-04'-methyltyrosyl, L-O4 -acetyltyrosyl, L-phenylalanyl, L-4-chlorophenylalanyl or L -ß-homotyrosyl; X4 glycyl, sarcosyl, a-methylalanyl, D- or L-alanyl, D- or L-N-methylalanyl, L-asparaginyl, L-isoleucyl, L-pro-lyl, D-leucyl or D-tryptophyl; X3 glycyl, sarcosyl, D- or L-alanyl, D- or L-prolyl or L-asparaginyl; X6 D- or L-phenylalanyl, L-tyrosyl, L-04'-methyltyrosyl, L-4-chlorophenylalanyl, L-C-phenylglycyl, L-leucyl, L-methionyl or L-histidyl; X7 glycyl, D- or L-leucyl, D- or L-methionyl, L-alanyl, L-isoleucyl, L-norleucyl, L-valyl, L-methionyl sulfoxide, L-threonyl, L-prolyl or D-ß-homoleucyl ; Xs D- or L-threonyl, L-lysyl, L-phenylalanyl or L-tyrosyl; X9 glycyl or L-lysyl; R is hydrogen or (Cr to C4-) alkyl; R1 is the hydroxyl group of the 1-carboxyl group of the C-terminal amino acid residue or one of the following residues which replace these carboxyl groups: a group of the formula -CTLOR4, in which R4 represents hydrogen or alkanoyl, in which the alkyl residue has 1 to 4 C atoms, or the 5-tetrazolyl group; and p and q are zero or 1, except peptides of the formula R-L-Phe-L-Ile-Gly-L-Leu-L-Met-OH or H-X3-Gly-Gly-X6-X7-OH and their salts, esters, amides and N-alkylamides and the corresponding acid addition salts, in which R has the meaning given above, X7 L-leucyl or L-methionyl and either X3 L-tyrosyl and X6 L-phenylalanyl or X3 L-tyrosyl and X6 L-4-chlorophenylalanyl, characterized in that a compound of formula II R-Y'-OH (II), wherein R has the meaning given above; and Y1 is a partial sequence which contains at least one amino acid residue and is identical to the corresponding N-terminal partial sequence in formula I, with a compound of formula III H-Y2-R '(III), wherein Rl has the meaning given above and Y2 corresponds to the remaining part of the product to be formed, the compounds of the formulas II and III optionally being activated and / or protected intermediately and the product obtained being optionally in the free base, a salt or an acid addition salt transferred. 3rd 644352 or their salts, (Cr to C4-) alkyl esters, amides, N- (Cr to C5-) alkylamides and the corresponding acid addition salts, in which n is 0 or 1; X2 L-arginyl; X3 L-tyrosyl, L-N-methyltyrosyl, L-3,4-dihydroxyphenylalanyl, L-O4-methyltyrosyl, L-04'-acetyltyrosyl or L-phenylalanyl; X4 glycyl, sarcosyl, D- or L-alanyl, D- or L-N-methyl- alanyl, L-isoleucyl, L-prolyl or D-leucyl; X5 glycyl, sarcosyl, D- or L-alanyl, or D- or L-prolyl; X6 L-phenylalanyl, L-tyrosyl, L-O4-methyltyrosyl or L-C-phenylglycyl; X7 glycyl, L-leucyl, L-methionyl, L-alanyl, L-isoleucyl, L- Norleucyl, L-valyl or L-prolyl; X8 L-threonyl; and p 0 or 1 with the proviso that if n and p both represent zero and X3 represents L-tyrosyl, X4 glycyl, X5 glycyl or X6 L-phenylalanyl, then X7 represents an L-methionyl or L-leucyl, characterized in that to obtain a compound of the formula II ' H - Y1 - OH (II '), wherein Y1 is a partial sequence which contains at least one amino acid residue and is identical to the corresponding N-terminal partial sequence in formula I ', with a compound of formula III' H-Y2-OH (III '), in which Y2 corresponds to the remaining part of the product to be formed, reacting the compounds of the formulas II 'and III', if appropriate, and / or protecting them intermediately and converting the product obtained, if appropriate, into the free base, a salt or an acid addition salt. 3. The method according to claim 1, characterized in that a compound of formula I, wherein Rj is the hydroxyl radical of the 1-carboxyl group of the C-terminal amino acid residue, is esterified to the corresponding (Cr to C4) alkyl ester. 4. The method according to claim 1, characterized in that a (Q- to C4-) alkyl ester is saponified to a corresponding compound of formula I, wherein Ri is the hydroxyl group of the 1-carboxyl group of the C-terminal amino acid residue. 5 5. The method according to any one of claims 1 to 4 for the preparation of a peptide of formula I ' H- (X2) n-X3-X4-X5-X6-X7- (X8) p-OH (I ') or their salts, (Cr to C4-) alkyl esters, amide-N- (Q- to C5-) alkylamides and the corresponding acid addition salts, in which n is 0 or 1; X2 L-arginyl; X3 D- or L-tyrosyl, D- or L-N-methyltyrosyl, D- or L-3,4-dihydroxyphenylalanyl, L-O4-methyltyrosyl, L-O4-acetyltyrosyl or L-phenylalanyl; X4 glycyl, sarcosyl, D- or L-alanyl, D- or L-N-methyl- alanyl, L-isoleucyl, L-prolyl or D-leucyl; X3 glycyl, sarcosyl, D- or L-alanyl, D- or L-prolyl or L-asparaginyl; X6 D- or L-phenylalanyl, L-tyrosyl, L-04'-methyltyrosyl, L- C-phenylglycyl, L-leucyl or L-methionyl; X7 glycyl, D- or L-leucyl, D- or L-methionyl, L-alanyl, L-isoleucyl, L-norleucyl, L-valyl, L-threonyl or L-prolyl; X8 D- or L-threonyl; and p 0 or 1 mean, with the proviso that if n and p are both zero and X3 is L-tyrosyl, X4 glycyl, X3 glycyl and X6 are L-phenylalanyl, then X7 is not L-methionyl or L-leucyl, and with the exception of the compound of the formula H-L-Phe-L-Ile-Gly-L-Leu-L-Met-OH as well as its salts, esters, amide and N-alkylamide and the corresponding acid addition salts, characterized in that a compound of the formula II ' H - Y1 - OH (II '), wherein Y1 is a partial sequence which contains at least one amino acid residue and is identical to the corresponding N-terminal partial sequence in formula I ', with a compound of formula III' H-Y2-OH (III '), in which Y2 corresponds to the remaining part of the product to be formed, reacting the compounds of the formulas II 'and III', if appropriate, and / or protecting them intermediately and converting the product obtained, if appropriate, into the free base, a salt or an acid addition salt. 6. The method according to any one of claims 1 to 4 for the preparation of a peptide of formula I ' H- (X2) n-X3-X4-X5-X6-X7- (X8) p-OH (I ') 7. The method according to any one of claims 1 to 4 for the preparation of a peptide of the formula I " R-X3-X4-X5-X6-X7- (X8) p- (X9) q-R1 (I "), or their salts, (Cr to C4-) alkyl6esters, amides, N- (Cr to C5-) alkylamides and the corresponding acid addition salts, in which R, R1, p and q have the meanings given in claim 1, X3 L-tyrosyl, L-O4 -acetyltyrosyl or N-methyl-L-tyrosyl; X4 glycyl, L-alanyl, D-alanyl or a-methylalanyl; X5 glycyl, sarcosyl or L-asparaginyl; X6 L-phenylalanyl, L-tyrosyl or L-4-chlorophenylalanyl; X7 L-leucyl, D-leucyl, L-methionyl, D-methionyl, L-Norleu cyl, L-threonyl or D-ß-homoleucyl; and X8 and X9 have the meanings given in claim 1. 8. The method according to any one of claims 1 to 4 for the preparation of a peptide of the formula R-X ^ D-Ala-Gly-L-Phe-X ^ R1 or its salts, (Cr to C4-) alkyl esters, amide-N- (Cr to Q-) alkylamides and the corresponding acid addition salts, in which R and R1 have the meanings given in claim 1, X3 is L-tyrosyl or N-methyl-L-tyrosyl and X7 is D-leucyl or D-methionyl. 9. The method according to claim 1 for the preparation of a peptide of the formula R- (X2) n-X3-X4-X5-X6-X7- (X8) p- (X9) q-NC R6 or its acid addition salts, in which X2, X3, X4, X5, X6, X7, X8, X9, n, p, q and R have the meanings given in Claim 1 and R5, R6 and the nitrogen atom to which they are bonded together contain an amino or (Cr to C4) alkylamino group. 10. The method according to claim 1 or 4 for the preparation of a peptide of the formula I, in which R1 denotes the hydroxyl group of the 1-carboxyl group of the C-terminal amino acid residue. 10th 11. The method according to any one of claims 1 to 4 for the preparation of a peptide of formula I, wherein R is hydrogen. 12. The method according to any one of claims 1 to 4 for the preparation of a peptide of formula I, wherein n is zero. 13. The method according to any one of claims 1 to 4 for the preparation of a peptide of formula I, wherein p and q are both zero 14. The method according to any one of claims 1 to 4 for the preparation of a peptide of formula I, wherein X3 is L-tyrosyl. 15. The method according to any one of claims 1 to 4 for the preparation of a peptide of formula I, wherein X4 is the residue of a corresponding D-amino acid. 15 16. The method according to any one of claims 1 to 4 for the preparation of a peptide of formula I, wherein X4 is D-alanyl. 17. The method according to any one of claims 1 to 4 for the preparation of a peptide of formula I, wherein X5 is glycyl. 18. The method according to any one of claims 1 to 4 for the preparation of a peptide of formula I, wherein X6 is L-phenylalanyl. 19. The method according to any one of claims 1 to 4 for the preparation of a peptide of formula I, wherein X7 represents the residue of a corresponding D-amino acid. 20. The method according to any one of claims 1 to 4 for the preparation of a peptide of formula I, wherein X7 is D-leucyl or D-methionyl. 20th 25th 30th 35 40 45 50 55 60 65 21. The method according to claim 1 or 4 for the preparation of a peptide of the formula H-L-Tyr-D-Ala-Gly-L-Phe-D-Leu-OH, its salts or acid addition salts. 22. The method according to claim 1 or 4 for the preparation of a peptide of the formula H-L-Tyr-D-Ala-Gly-L-Phe (4Cl) -D-Leu-OH, its salts or its corresponding acid additiotis salts. 23. The method according to claim 1 or 2 for the preparation of a peptide of the formula H-L-Tyr-D-Ala-Gly-L-Phe-D-Leu-ethyl amide or its corresponding acid addition salts. 24. The method according to any one of claims 1 to 4 for the preparation of a physiologically or pharmaceutically acceptable salt or acid addition salt of a peptide of formula I. 25. The method according to any one of claims 1 to 4 for the preparation of the acid addition salt of a peptide of formula I with hydrochloric acid.