HK1006843B - Peptide antagonists of bradykinin - Google Patents

Description

The invention relates to new peptides with bradykinin antagonistic action and a method for their production.

Bradycinin antagonist peptides are described in WO 86/07263 in which, inter alia, L-Pro is replaced in position 7 of the bradycinin peptide hormone or other bradycinin analogues by a D-amino acid such as D-Phe, D-Thia, D-Pal, CDF, D-Nal, MDY, D-Phg, D-His, D-Trp, D-Tyr, D-hPhe, D-Val, D-Ala, D-His, D-Ile, D-Leu and DOMT.

The purpose of the invention is to find new effective peptides with bradykinin antagonistic activity.

This task is solved by the peptides of formula I. The following shall be added to the list of substances which are to be classified in Annex I to Regulation (EC) No 1907/2006: where E = Leu, Tbg or Cha and their physiologically compatible salts.

Unless otherwise stated, the abbreviation of an amino acid residue without a stereodecritor is the L-form abbreviation for the residue (see Schröder, Lübke, The Peptides, Volume I, New York 1965, pp. XXII-XXIII; Houben-Weyl, Methods of Organic Chemistry, Volumes XV/1 and 2, Stuttgart 1974), such as The following entries are added in column A. The following entries are added in column B. The following entries are added in column C. The following entries are added in column C. The following entries are added in column C. The following entries are added in column C. The following entries are added in column C.

The salts are in particular alkaline or alkaline earth salts, salts with physiologically compatible amines and salts with inorganic or organic acids such as HCl, HBr, H2SO4, H3PO4, maleic acid, fumaric acid, citric acid, tartaric acid, acetic acid.

Examples of formula I peptides are: H- ((D) -Arg-Arg-Pro-Hyp-Gly-Leu-Ser- ((D) -Tic-Oic-Arg-OH) - The name of the substance is not known. H- ((D) -Arg-Arg-Pro-Hyp-Gly-Cha-Ser- ((D) -Tic-Oic-Arg-OH) is the chemical formula for the chemical compound H- (O) -O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O- The following shall be added to the list of substances which are to be classified in Annex I to Regulation (EC) No 1907/2006:

The invention also relates to a process for the production of peptides of formula I characterized by the use of a (a) a C-terminal free carboxyl group fragment or its activated derivative is translated with a corresponding N-terminal free amino group fragment; or in the compound obtained under (a) or (b), where appropriate, separate one or more protective groups temporarily introduced to protect other functions and transfer the compounds of formula I thus obtained to their physiologically compatible salt, where appropriate.

Err1:Expecting ',' delimiter: line 1 column 813 (char 812)

Solid-phase synthesis begins at the C-terminal end of the peptide by coupling a protected amino acid to a corresponding resin. Such starting materials can be obtained by coupling a protected amino acid to a modified polystyrene or polyalamide resin with a chloromethyl, hydroxymethyl, benzhydrylamino ((BHA), methylbenzhydrylamino ((MBHA) group via an ester or amide bond. The resins used as carrier materials are commercially available. BHA and MBHA resins are usually used when the synthesized peptide contains a free carbamide group at the C-terminus. In case the peptide contains a secondary C-terminal, a corresponding chloromethyl or amide group should be used at the end.To preserve the tert-butyl protective groups of the amino acid side chain in the peptide, the synthesis with the Fmoc protective group for temporary blocking of the α-amino group of the amino acid can be carried out using the method described e.g. by R.C. Sheppard, J. Chem. Soc., Chem. Comm. 1982, 587, whereby the guanidine function of the arginine is protected by pyridinium perdichlorate protonation and the protective function of the other proteins functionalised in the amino acid side chain by catalytic transfer in sodium hydroxide (A. Felix et al. J. W. 13, 1974) or ammonium oxide (A. Felix et al. J. 13, 1984)The first is the use of a single-membered ring.

After splitting the amino group of the resin-coupled amino acid with an appropriate reagent, such as trifluoroacetic acid in methylene chloride in the case of the Boc protective group or a 20% solution of piperidine in dimethylformamide in the case of the Fmoc protective group, the subsequent protected amino acids are coupled in the desired order. The intermediate N-terminally protected peptide resins are unblocked by the prescribed reagents before binding to the subsequent amino acid derivative.

Any activation reagent used in peptide synthesis, see e.g. Houben-Weyl, methods of organic chemistry, band 15/2, can be used as a coupling reagent, but in particular carbodiimides such as N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide or N-ethyl-N'- ((3-dimethylaminopropyl) carbodiimide. The coupling can be achieved directly by adding amino acid derivative to the activation reagent and, if necessary, by giving a separation inhibitor such as 1-hydroxyhydriazol (HOBt) (W. King, R. King, Geigenetics, 1973-703) or 3-hydroxyhydriazol (HOBt) or 3-hydroxyhydriazol (HARO-44-E) in a solution of copper or ammonium, or by giving the chemical HOBT (HARO-40) as a chemical reagent suitable for the activation of the active substance or compound, or the active substance, or the active substance, or the active substance, or the active substance, or the active substance, or the active substance, or the active substance, or the active substance, or the active substance, or the active substance, or the active substance, or the active substance, or the active substance, or the active substance, or the active substance or the active substance, or the active substance, or the active substance or the active substance, or the active substance or the active substance or the active substance, the active substance or the active substance or the active substance or the active substance or the active substance.

The coupling or activation of the amino acid derivatives with any of the above activation reactions may be carried out in dimethylformamide, N-methylpyrrolidone or methyl chloride or a mixture of the above solvents. The activated amlno acid derivative is usually used in an excess of 1.5 to 4 times. In cases where incomplete coupling occurs, the coupling reaction is repeated without first de-blocking the α-amino group of the peptide harse to the next amino acid necessary for coupling.

The successful course of the coupling reaction can be verified by the ninhydrin reaction, as described e.g. by E. Kaiser et al. Anal. Biochem. 34 595 (1970). The synthesis can also be carried out automatically, e.g. by a peptide synthesizer model 430A of Fa. Applied Biosystems, using either the synthesis programmes provided by the device manufacturer or those created by the user himself.

After the peptide has been synthesised in the above manner, the peptide can be separated from the resin by reagents such as liquid hydrogen fluoride (preferably in the case of peptides produced by the Boc method) or trifluoric acid (preferably in the case of peptides synthesised by the Fmoc method), which not only dissolve the peptide but also the other side chain protective groups of the amino acid derivatives. This gives the peptide in the free form, apart from the use of BHA and MBHA sulphides, whereas the BHA and MBHA sulphides respectively, whereby the solution of the phenols with fluoride or methanol is obtained by the addition of methanol, the peptide is further diluted by the dissolved acid. The peptide is then treated in a single solution, such as Tethyl ethanol, EP282 or Tethyl ethanol, followed by two other substances, such as ethyl ethanol, EP282 or ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl ethyl

If the tert-butyl or benzyl side chain protecting groups of the peptides are to be preserved, the peptide synthesized from a specially modified carrier resin with 1% trifluoroacetic acid shall be cleaved into methylene chloride, as described e.g. in R.C. Sheppard J. Chem. Soc., Chem. Comm. 1982, 587.

The modified carrier resin described by Sheppard is also used for the synthesis of peptides with a C-terminal carbamoyl group or an ω-amino or ω-guanidinoalkyl group.

A further method for the production of peptides with an ω-amino alkyl group is described in EP-A 264 802.

The peptides of the present invention have been synthesized preferably using the solid phase technique following two general protection group tactics:

The synthesis was performed with an automatic peptide synthesizer model 430 A from Fa. Applied Biosystems using Boc or Fmoc protecting groups to temporarily block the α-amino group.

The use of the Boc protection group used the synthesis cycles preprogrammed by the manufacturer of the device.

The peptide with a free carboxyl group at the C-terminal end was synthesized from a 4- ((hydroxymethyl) phenylacetamidomethyl polystyrene resin (R.B. Merrifield, J. Org. Chem. 43, 2845 (1978)) functionalised with the corresponding boc amino acid by Fa. Applied Biosystems. The activation reagents were N,N'-dicyclohexylcarbodiamide or N,N'-diisopropylcarbodiamide. Activation was carried out as a symmetric anhydride, HOBt ester or HOObt ester in CH2Cl2, CH2Cl2 - DMF mixtures or NMP. 2-4 equivalents of activated amino acid derivative were used for the coupling.

When the FMOC protective group was used to temporarily protect the α-aminogroups, the synthesis with the automatic peptide synthesizer Model 430A of Fa. Applied Biosystems was carried out using its own synthesis programs. The synthesis was carried out on a p-benzyloxybenzyl alcohol resin (S. Wang, J.Am.Chem.Soc. 95, 1328 (1973)) of Fa. Bachem which had been tested with the corresponding amino acid by the known method (E. Atherton et al. J.C.S.Chem. Comm. 1981, 336). The activation of the amino derivatives described as copper group or HOO-resin was repeated by the products supplied directly to the equipment by ZF Piper.

The peptides of the invention, individually or in combination, have a bradykinin antagonistic effect which can be tested in various models (see Handbook of Exp. Pharmacol. Vol. 25, Springer Verlag, 1970, pp. 53-55), e.g. on isolated rat uterus, guinea pig oil or guinea pig pulmonary artery.

For the testing of the peptides of the invention on the pulmonary artery isolated, guinea pigs (Dunkin Hartley) weighing 400 to 450 g are killed by neck slapping.

The chest is opened and the pulmonary artery is carefully prepared, the surrounding tissue is carefully removed and the pulmonary artery is cut at a 45° angle.

The vascular strip, 2.5 cm long and 3-4 mm wide, is fixed in a 10 ml organ bath filled with Ringer' s solution. Other

Zusammensetzung der Lösung in mmol/l
NaCl	154
KCl	5,6
	1,9
	2,4
Glukose	5,0

The solution is permeated with 95% O2 and 5% CO2 and heated to 37°C. The pH is 7.4 and the load on the vascular strip is 1.0 g.

The isometric contraction changes are recorded with a lever and a Hugo Sachs RF modem and recorded on a compensation recorder (BEC, Goerz Metrawatt SE 460).

After 1 hour of equilibration, the test is started. Once the vascular strips have reached their maximum sensitivity to 2 x 10-7 mol/ l bradycinin - bradycinin causes vascular strips to contract - the peptides in doses 5 x 10-8 - 1 x 10-5 mol/ l are allowed to act for 10 minutes and after re-administration of bradycinin, the decrease in bradycinin effect is compared to the control.

For partial agonist effects, the peptides are used in doses 1 x 10-5 - 1 x 10-3 mol/ l.

The IC50 values of the peptides of the invention calculated from the dose curves are given in Table 1. Other Tabelle 1

Verbindung
H-(D)-Arg-Arg-Pro-Hyp-Gly-Leu-Ser-(D)-Tic-Oic-Arg-OH
H-(D)-Arg-Arg-Pro-Hyp-Gly-Cha-Ser-(D)-Tic-Oic-Arg-OH
H-(D)-Arg-Arg-Pro-Hyp-Gly-Tbg-Ser-(D)-Tic-Oic-Arg-OH

The therapeutic use of the peptides of the invention includes all pathological conditions mediated, induced or supported by bradycinin and bradycinin-related peptides, including, but not limited to, trauma such as wounds, burns, rashes, erythema, edema, angina, arthritis, asthma, allergies, rhinitis, shock, inflammation, low blood pressure, pain, itching and altered sperm motility.

The invention therefore also concerns the use of formula I peptides as medicinal products and pharmaceutical preparations containing these compounds.

Pharmaceutical preparations contain an active substance of formula I, alone or in combination, together with an inorganic or organic pharmaceutically usable carrier.

The dosage of the active substance depends on the warm-blooded species, body weight, age and the type of application.

The pharmaceutical preparations of the present invention are manufactured by known solvent, mixing, granulation or dressing processes.

For oral application or application to the mucous membranes, the active substances are mixed with the usual additives such as carriers, stabilisers or inert diluents and are put into appropriate forms, such as tablets, dragees, capsules, aqueous, alcoholic or oily suspensions or aqueous, alcoholic or oily solutions, by the usual methods. The inert carriers may be used, for example, rubber arabic, magnesia, magnesium carbonate, potassium phosphate, milk sugar, glucose, magnesium stearyl arate or starch, in particular corn starch. The preparation may be carried out in both dry and wet form. The preparation may be carried out as a vegetable or vegetable oil or in solvents, such as vegetable oil, sunflower oil and animal starch.

A preparation for topical use may be available as an aqueous or oily solution, lotion, emulsion or gel, ointment or grease ointment or, if possible, in a spray form, where the adhesion may be improved by the addition of a polymer, if necessary.

For intranasal use, the compounds are mixed with the usual additives such as stabilisers or inert diluents and are then brought to suitable dosage forms, such as aqueous, alcoholic or oily suspensions or aqueous, alcoholic or oily solutions, by the usual methods.

For inhalation use, nebulizer or compressed gas packaging using inert carrier gases may be used.

For intravenous, subcutaneous, epicutanal or intradermal application, the active substances or their physiologically compatible salts are dissolved, suspended or emulsified, if desired, with the pharmaceutically usual excipients, such as isotonic or pH adjusting agents, solvents, emulsifiers or other excipients.

Due to the short half-lives of some of the medicinal products described in body fluids, the use of injectable retardant preparations is appropriate, e.g. oily crystal suspensions, microcapsules, rods or implants, the latter of which may be composed of tissue-compatible polymers, in particular biodegradable polymers such as those based on polylactic acid-polyglycolic acid copolymers or human albumin.

A dosage range of 0.01 to 5 mg/ ml is appropriate for topical and inhaled applications, and 0.01 to 10 mg/ kg for systemic applications.

List of abbreviations:

The abbreviations used for amino acids are the three-letter code used in peptide chemistry as described in Europ. J. Biochem. 138, 9 (1984). Other abbreviations used are listed below.AcmAcetamidomethylε-Ahxε-AminohexanoylAoccis, endo-2-Azabicylo[3.3.0]octane-3-S-carbonylBoctert-butyloxycarbonylButter-ButylBzlbenzylCDFClor-D-phenyl-ChalcylsulphonylCyclohexanyl-Cylcylcylcylcylcylglycyl-Cylcylcyl-Cylcylcyl-Cylcylcylcyl-Cylcylcylcylcylcyl-Cylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcylcyl

The following examples are intended to illustrate the preferred methods of solid phase synthesis of the peptides of the invention, without limiting the invention to them.

The following amino acid derivatives were used: The following definitions shall be used for the purposes of the calculation of the CO2 savings of the product:

Example 1: H- ((D) -Arg-Arg-Pro-Hyp-Gly-Leu-Ser- ((D) -Tic-Oic-Arg-OH) - The name of the substance is not known.

was constructed in stages using a peptide synthesizer model 430 A from Fa. Applied Biosystems using the Fmoc method on a p-benzyloxybenzyl alcohol resin of Fa. Novabiochem esterified with Fmoc-Arg ((Mtr) -OH (load of approximately 0,5 mmol/g resin) using 1 g of the resin and synthesised using a synthesis programme modified for the Fmoc method.

The amino acids were pre-activated directly in the cartridges by dissolving in 4 ml DMF and adding 2 ml of a 0.55 mol/ l solution of diisopropylcarbodiimide in DMF. The HOObt esters of the other amino acids were dissolved in 6 ml of NMP and then coupled with the pre-activated amino acids in situ to the resin previously unblocked with 20% piperidine in DMF. After completion of the synthesis, the peptide was separated from the resin by trifluoric acid, using thioanisol and ethandithiol as a cation catcher, while simultaneously removing the side chain protecting groups. The residue obtained after removal of the trifluoric acid was repeatedly digested and centrifuged with acetic esters. The remaining residue was chromatographed to ®Sephadex LH 20 with 10% acetic acid. The recycled peptide containing fractions were combined and frozen. The number of employees is determined by the number of employees.

The following examples are produced by analogy with example 1.

Example 2: H- ((D) -Arg-Arg-Pro-Hyp-Gly-Cha-Ser- ((D) -Tic-Oic-Arg-OH) is the chemical formula for the chemical compound H- (O) -O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-

The following information is provided for the purpose of the calculation of the amount of the aid:

Example 3: The following shall be added to the list of substances which are to be classified in Annex I to Regulation (EC) No 1907/2006:

The following information is provided for the purpose of this Decision:

Claims (5)

1. A process for the preparation of a peptide of the formula I H-(D)-Arg-Arg-Pro-Hyp-Gly-E-Ser-(D)-Tic-Oic-Arg-OH   (I) where E is Leu, Tbg or Cha and the physiologically tolerated salts thereof, which comprises

   a) reacting a fragment with a C-terminal free carboxyl group or its activated derivative with a corresponding fragment with an N-terminal free amino group, or

   b) synthesizing the peptide stepwise,

   eliminating one or more protective groups temporarily introduced into the compound obtained as in (a) or (b) to protect other functions where appropriate, and converting the compound of the formula I which has been obtained in this way into its physiologically tolerated salt where appropriate.
2. A process for the preparation of a pharmaceutical composition containing a peptide of the formula I as claimed in claim 1, which comprises converting the latter and a vehicle into a suitable dosage form.