DE4117733A1 - Cyclo-peptide(s) agonists to atrial natriuretic protein - Google Patents

Abstract

Cyclopeptides and their salts are atrial natriuretic protein (ANP) agonists derived from amino acid (aa) residues of hANP Arg(27)-Phe(8)-Gly(9)-Gly(10)-Arg(11)-Met(12)-Asp(13)-Arg(14)-Ile(15), generally named Bn-Cn-Dn-En-Fn-Gn-Hn-In-Kn respectively, and structural and functional equivs., wherein an An spacer gp. is attached to the free end of Bn and joins Bn with Kn thereby changing the structure resulting in binding of the cyclopeptide to ANP receptors. Also claimed is a process for the prodn. of the cyclopeptides by cyclisation of a linear sequence made by known methods and possible salt formation, and a pharmaceutical cpd. contg. a cyclopeptide as claimed. The part of An closest to Bn pref. contains an aromatic or cycloaliphatic residue and Bn, Cn, En, Fn, Gn, Hn, In and Kn are in the L-form.

Description

The invention relates to new cyclopeptides that natural and unnatural amino acid residues are built, their manufacture and their use as a medicine. The peptides are ANP agonists.

The new cyclopeptides are partial sequences, interlinked discontinuously Partial sequences, modified partial sequences and Analogs of the so-called "atrial natriuretic Factor ", ANF, or" atrial natriuretic peptide ", ANP.

ANP is mainly found in the muscle cells of the Auricles synthesized, there also as a prohormone saved and increased by the mechanical stimulus Wall tension released. It has a vasodilatory effect, hypotensive, diuretic and saluretic, increased glomerular filtration reduces the plasma volume and increases the hematocrit, lowers the Plasma renin activity and plasma aldosterone levels, has a spasmolytic effect on the smooth muscles of the Intestinal and broncholytic. The effect is over specific receptors mediated.

The in this description and the claims Abbreviations used follow the recommendations of ZUPAC-IUB Joint Commission of Biochemical Nomenclature (Eur. J. Biochem. 138, 9-37 (1984)). A few others Abbreviations have been added. It will be shown below some of these abbreviations are given:  

The term amino acid includes (if in the following text not expressly stated otherwise) natural and unnatural amino acids, both the D- as well as the L shape. The expression also includes "α-amino acid" also α, α-disubstituted Amino acids.

If an amino acid is specified without a prefix (e.g. Orn), this information stands for the L-form of the amino acid. The D-form is expressly stated (e.g. D-Orn).  

The invention relates to new cyclopeptides of the formula I.

and their pharmaceutically acceptable salts, wherein:

A is an amino acid residue of the formula

-NH- (CH₂) _n -CH (R) -CO- (II)

is where n is an integer from 1 to 11 and
R -NH (H) -C (O) -CH₂-X¹
-N (H) -C (O) -CH₂-O-X¹
-N (H) -C (O) -X¹
-N (H) -C (O) -CH = CH-X¹ or
Is -N (H) -C (O) -O-CH₂-X¹,
wherein X¹ (a) phenyl, (b) phenyl substituted by 1, 2 or 3 substituents (substituent (s): halogen, trifluoromethyl or nitro), (c) naphthyl, (d) benzo [b] thienyl, (e) pyridyl or (f) is pyrazinyl;
B is a covalent bond, Phe or an α-amino acid residue with one or two side chains, wherein the side chain (s) or one of the two side chains contains (contain) one, two, three or four N-containing group (s);
C is an α-amino acid residue with one or two alkyl side chains optionally containing -0-, the side chain (s) or one of the two side chains bearing one or more residues, the respective residue being cycloalkyl with 4, 5, 6 or 7 carbon atoms, phenyl, naphthyl substituted (e.g. NO ₂ ) phenyl or a 5- or 6-membered aromatic heterocycle, wherein 2 members are N or one member is N and one member is O or S, or one member is N, S or Is O and the remaining members are C, preferably thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, isoquinolyl, quinolyl, chromanyl, thiazolyl, oxazolyl, morpholinyl;
D and E are each independently Gly or an α-amino acid residue whose side chain (s) does not carry a functional group or
D and E together are an ω-amino acid residue -NH- (CH ₂ ) _2-11 -CO- or a peptide template;
F is an α-amino acid residue with one or two side chains, in which the side chain (s) or one of the two side chains contains (contain) one, two, three or four N-containing group (s);
G is an α-amino acid residue with one or two lipophilic side chains;
H is a covalent bond or an α-amino acid residue that does not contain a functional group or -COOH or -CONH ₂ as a functional group in the side chain (s);
I is an α-amino acid residue with one or two side chains, in which the side chain (s) or one of the two side chains contains (contain) one, two, three or four N-containing group (s);
K is an α-amino acid residue with one or two lipophilic side chains.

As mentioned above, the term includes Amino acid natural and unnatural amino acids. The unnatural amino acids are appropriate, provided are not expressly specified in in relation to their molecular weight or the length of the Side chains, on the order of natural Amino acids.

As salts come in particular those with physiologically tolerated inorganic or organic acids, such as. B. HCl, HBr, H ₂ SO ₄ , H ₃ PO ₄ , maleic acid, fumaric acid, citric acid, tartaric acid, acetic acid in question.

The chirality centers in the new peptides can each have R, S or R, S configuration.

Cyclopeptides or their salts, in which
A is an amino acid residue of formula II, wherein n is 1, 2, 3 or 4, preferably 1 or 4, and / or
B, F and I independently of one another Arg, D-Arg, Lys, D-Lys, Orn, D-Orn, Homo-Arg, D-Homo-Arg, Dap, D-Dap, 4-Amino-Phe, Ctr, D -Ctr, Arg (NO ₂ ), D-Arg (NO ₂ ) or
Are Phe, in particular in which B, F and I are independently Arg, D-Arg, Lys, D-Lys, Orn or Ctr, and / or,
C Phe, D-Phe, 4-NO ₂ -Phe, Cha, D-Cha, Ser (Bzl), D-Ser (Bzl), Tyr, D-Tyr, Tyr (Bzl), D-Tyr (Bzl), Nal, D-Nal, Thi, D-Thi, Asp (Bzl), D-Asp (Bzl), His or D-His, in particular where C is Phe, Cha, Tyr, Nal, Tyr (Bzl) or 4- Is NO ₂ -Phe, and / or
D and E are independently Ala, Gly, Phe, Val, Ile, Leu or Nle or their D-form, especially where D is D-Ala, Gly, Phe or D-Phe, and / or E Gly, Ala or Phe is, and / or
D and E together are an ω-amino acid residue of the formula -NH- (CH ₂ ) _2-5 -CO-, or
D and E together are Btu, Clg, Thc or Trc or their D forms, in particular D-Btu, and / or
G and K are, independently of one another, Ile, D-Ile, Met, D-Met, Nle, D-Nle, Leu, D-Leu, Val, D-Val, Cha, D-Cha, Cle or Aib, in particular where G and K are independently of one another Ile, Met, Nle or Leu, and / or
H HOOC- (CH ₂ ) _1-4 -CH (NH ₂ ) -CO-, the D-forms of which are Gly, Ala, D-Ala, Asn, D-Asn, Phe or D-Phe, in particular where I Asp, Glu or Gly is.

Cyclopeptides are preferred in which X ¹ in the group R (a) phenyl, (b) phenyl substituted by 1 or 2 substituents (CF ₃ , F, Cl, Br or NO ₂ ), (c) 1 or 2-naphthyl, ( d) 3-benzo [b] thienyl, (e) 2-pyridyl or (f) 2-pyrazinyl.

Of particular note are cyclopeptides and their salts, in which

A is as defined above,
B Arg,
Lys,
Phe or
Orn is;
C Phe,
Cha or
Ser (Bzl) is;
D is D-Ala;
E is Gly; or
D and E together
L-Clg or
Are D-Clg;
F Arg or
Lys is;
G Ile or
Nle is;
H is Asp;
I is Arg; and
K Ile is.

Cyclopeptides or are particularly preferred their salts, in which

A is an aminoalkanoic acid residue of the formula
-NH- (CH₂) _n -CH (R) -CO-, wherein
n is 1 or 4,
R -NH (H) -C (O) -CH₂-X¹
-N (H) -C (O) -CH₂-O-X¹
-N (H) -C (O) -X¹
-N (H) - (O) -CH = CH-X¹ or
Is -N (H) -C (O) -O-CH₂-X¹,
wherein X¹ (a) phenyl, (b) phenyl substituted by 1 or 2 substituents, (c) 1- or 2-naphthyl, (d) 3-benzo [b] tienyl, (e) 2-pyridyl or (f) 2 -Pyrazinyl;
B, F and I are independently Arg, D-Arg, Lys, D-Lys, Orn or Ctr;
C is Phe, Cha, Tyr, Nal, Tyr (Bzl) or 4-NO₂-Phe;
D is D-Ala, Gly, Phe or D-Phe;
E is Gly, Ala or Phe; or
D and E together are D-Btu, L-Clg or D-Clg;
G and K are independently Ile, Met, Nle or Leu; and
H is Asp, Glu or Gly;
especially those in which
A is -NH- (CH₂) ₄-CH (R) -CO-, wherein
R is as defined above and
-BCDEFGHIK- the chain
-Arg-Cha-D-Ala-Gly-Arg-Ile-Asp-Arg-Ile-.

The compounds according to the invention are ANP agonists. They show one like the natural ANP

• - Specific and affine binding to ANP receptors
• - diuretic and saluretic properties
• - hypotensive effect  
• - hematocrit increase
• - Increasing the plasma level of cyclic GMP (GMP: probably the intracellular messenger ("second messenger "), which is in the plasma after ANP administration has more evidence.)
• - Increase in glomerular filtration
• - vasodilatory effect
• - broncholytic effect
• - spasmolytic effect on smooth muscles, especially on the intestine.

These properties of the compounds according to the invention are tested in detail as follows:

Binding to ANP receptors

Binding to ANP receptors from zona glomerulosa Bovine adrenal glands are made using the method by Bürgisser et al. (Biochem. Biophys. Res. Commun. 133, 1201 (1985)), modified after Bürgisser (2nd World Congress on Biologically Active Atrial Peptides, May 16-21, New York Am. Soc. Hypertens., Abstr. B181, P. 209 (1987)) with a commercial available kit from ANAWA, Wangen, Switzerland, certainly.

Lowering blood pressure, diuretic / saluretic effect, Hematocrit increase, cyclo-GMP increase

The examinations are anesthetized (Nembutal®) spontaneously hypertensive rats (Ivanovas) performed. The trachea is cannulated. Blood pressure is measured from the carotid artery Pressure-voltage converter (Statham) on a recorder (Watanabe Multicorder) registered. The heart rate is the number of pulse waves per unit of time  calculated. The substance application takes place through a cannulated V. jugularis. With a little one Abdominal incision cannulates the bladder and urine caught. The urine volume becomes gravimetric certainly. Sodium and potassium measured by flame photometry, chloride through Electrotitration. The hematocrit is derived from the arterial blood measured. The cyclic GMP will from the arterial blood with a commercial available radioimmunoassay (IBL, Hamburg) certainly.

Influence on glomerular filtration

The glomerular filtration rate is on anesthetized dog by determining the Inulin clearance according to standard procedures Führ et al., (Klin. Wschr. 33, 727 (1955)).

Vascular relaxation

The vasodilatory effect in analogy to ANP is determined using a modified method by Faison et al. (Eur. J. Pharmacol. 102, 169 (1984)). The rabbit breast aorta is contracted by a supramaximal serotonin concentration. 15 minutes after adding the test substance, the serotonin contraction is measured in comparison to the solvent control. The EC _{50 is} determined graphically from several doses.

Broncholytic effect

According to the method of Konzett and Rössler (Arch. exper. Path. Pharmacol. 195, 71 (1940)) Antagonization of histamine bronchospasm after i.v. Administration of the test substance examined.  

Spasmolytic effect on the chicken rectum

According to the method of Currie et al. (Science, 221/4605, 71 (1983)) is the spasmolytic effect against carbachol co-traction on the chicken rectum certainly.

The compounds according to the invention can be administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally, by inhalation, transdermally, preferably promoted by iontophoresis or enhancers known from the literature, and orally. In the whole animal of different species, the doses are a significant reduction in blood pressure (<20 mm Hg) and / or diuresis (+ 300%) or Saluresis (+ 300%) and an increase in hematocrit (+ 3%) and the cyclic GMP ( + 200%) between 1 µg / kg and 50 mg / kg. The doses for a broncholytic effect on guinea pigs are of the same order of magnitude. The binding to the receptors of the ANP in zona-glomerulosa cells of bovine adrenal glands takes place with an IC ₅₀ between 1.10 ^-10 and 1.10 ^-5 mol / l. Vascular relaxation effect on rabbit aortic rings in vitro occurs with an EC ₅₀ between 1.10 ^-9 and 1.10 ^-4 mol / l. The concentration for a spasmolytic effect on the chicken rectum is of the same order of magnitude.

Since the receptor binding of the individual according to the invention Connections as well as those of ANP in potency well with the biological effects correlated is from identity the mechanism of action between the natural occurring peptides and those described here  Connections. This means that more can be used for the natural peptide ANP described biological Properties that are not described in detail here are expected from the compounds of the invention will.

The magnitudes of the effects of the invention Connections are comparable to those of ANP. The essential advantage of the compounds according to the invention is their much smaller molecular size. From the State of the art could not be concluded that the connections with so much less Molecular size Effect data in a satisfactory size exhibit. Due to the smaller molecular size, is the synthesis of the compounds of the invention much easier and therefore cheaper than that Synthesis of ANP or of ANP derivatives with large Molecular weights similar to that of ANP. The Bioavailability of the compounds according to the invention (especially with transdermal administration) much larger than ANP and ANP-like Derivatives. The compounds according to the invention contain unlike ANP, no disulfide bridges, which means that their metabolic stability to ANP and ANP-like Derivatives is improved.

Due to the spectrum of effects, the Compounds according to the invention as antihypertensives, as Hypotensives, as diuretics, to promote Blood circulation (vasodilatory effect), e.g. B. at vascular insufficiency and for the treatment of Heart failure, coronary insufficiency, cerebrovascular Insufficiency, renal failure, acute kidney failure as well as edema of any genesis, e.g. B. brain edema, also in  Cirrhosis of the liver, also as spasmolytics for everyone smooth muscular organs, especially gastrointestinal tract including gallbladder and urinary organs and as Broncholytics are used.

They can also be used to diagnose the listed Clinical pictures and for the examination of the listed Organ systems are used. In addition, they can as an aid to production and cleaning (Affinity chromatography) of antibodies or Receptor preparations as well as in immunological Test procedures (e.g. RIA, ELISA) and receptor Binding tests (e.g. radioreceptor assay) as specific and selective ligands are used.

The invention therefore also relates to the use of Compounds of general formula I as medicinal products and pharmaceutical preparations containing these compounds contain. Use in humans is preferred.

For parenteral administration, the inventive Connections possibly with the usual substances such as solubilizers, emulsifiers or others Excipients in solution, suspension or emulsion brought. As a solvent such. B. in question: water, physiological saline or alcohols, e.g. B. Ethanol, propanediol or glycerin, sugar solutions such as Glucose or mannitol solutions or a mixture from different solvents. In addition, the Connections through implants, e.g. B. made of polylactide, Polyglycolid or polyhydroxybutyric acid applied will. Other application options are intranasal application, inhalation application  (Piezo device, dosing aerosol, powder inhaler) that transdermal application (plaster preparation, cream, Ointment, gel, passive patch, the effect of "Enhancer" for penetration and / or through a electric field (iontophoresis) can be increased), oral application (tablets, capsules, drag'es Etc.).

The invention also relates to the use of Compounds of general formula I as components and Intermediates in the biochemical, biotechnical and immunological processes (generation of antibodies, affinity chromatography, RIA, ELISA, Radioreceptor assay).

The compounds according to the invention can be prepared by generally known methods in peptide chemistry. Such processes are described in Houben-Weyl, Methods of Organic Chemistry, Vol. 15/2. Production according to solid phase peptide synthesis (ZBG Barany, RB Merrifield in The Peptides-Analysis, Synthesis, Biology, Vol. 2, 2-284 (1980), Academic Press, New York or RC Sheppard, Int. J. Pept. Prot Res. 21, 118 (1983)) or equivalent known methods. The amino protecting groups used are those described in Houben-Weyl, Methods of Organic Chemistry, Vol. 15/1. Urethane protecting groups such as. B. the fluorenylmethoxycarbonyl or the tert-butyloxycarbonyl group applied. In order to prevent side reactions, any groups present in the side chains of the amino acids are generally additionally protected by suitable protective groups (see, for example, Houben-Weyl, Vol. 15/1 or TW Greene, Protective Groups in Organic Synthesis). Arg (NO ₂ ), Arg (di-Z), Arg (Pmc), Arg (Mtr), Tyr (tBu), Tyr (Bzl), Tyr (2,6-Di-Cl-Bzl), Ser are used (tBu), Ser (Bzl), Asp (tBu), Asp (Bzl), Glu (tBu), Glu (Bzl), His (Trt), His (Bum), Lys (Boc), Lys (Z), Z -Lys, Orn (Boc), Dap (Boc), Homo-Arg (Mtr), Homo-Arg (Pmc), Homo-Arg (NO ₂ ).

For the synthesis of the compounds of general formula I after the solid phase synthesis known resins are on Suitable for polystyrene, polyacrylamide and polyether. For the synthesis of cyclopeptides it is necessary to use such anchor groups in the split Deliver peptide carboxylic acids. It is special advantageous to use anchor groups in which the Cleavage takes place under conditions so mild that possibly existing side chain protection groups stay. When using the Fmoc strategy, there are Anchor groups: the 2-methoxy-4-alkoxybenzyl alcohol Group (M. Mergler et. Al., Proceedings of the 10th American Peptide Symposium 1987, St. Louis, p. 259, G. R. Marshall ed Escom, Leiden (1988), the Hydroxycrotonoylamidomethyl group (H. Kunz, B. Dombo, Appl. Chem. Int. Ed. Engl. 27, 711 (1988)) or the Trialkoxybenzhydryl alcohol group (H. Rink, P. Sieber, Peptides 1988, p. 139, G. Jung, E. Bayer Eds., W. de Gruyter, Berlin 1989).

The amino protective group is split off in the case of Boc group with trifluoroacetic acid in dichloromethane or in the case of the Fmoc group, preferably with organic bases, especially amines such. B. piperidine or morpholine in DMF  or N-methylpyrrolidone (NMP). Usual concentrations are 20 to 50% of the base in the solvent, the Response times are between 10 and 120 minutes. Splitting in two steps is preferred carried out, the first reaction time about 3 Minutes. The resin is then briefly with Washed solvent. Another closes Elimination with 20% piperidine in DMF, after which the Solution is suctioned off and the resin carefully Solvent is washed. Preferred solvents for these washing steps are DMF, NMP, dichloromethane, Trichloromethane, methanol, ethanol, isopropanol, water and tetrahydrofuran. After complete removal of the Piperidins becomes the one required for the next coupling Fmoc amino acid coupled. This cycle will run through one after the other until the desired one Resin-bound peptide has emerged.

Those known in peptide chemistry can be used for coupling Methods (see Houben-Weyl, methods of organic chemistry, Vol. 15/2) can be applied. Preferably used Carbodiimides such as e.g. B. dicyclohexylcarbodiimide, Diisopropylcarbodiimide, ethyl (3-dimethylaminopropyl) - carbodiimide or O-benzotriazol-1-yl-tetramethyl uronium hexafluorophosphate or tetrafluoroborate (R. Knorr et al., THL 30, 1927 (1989)) or benzotriazol-1-yl-oxy tris (dimethylamino) phosphonium hexafluorophosphate (B. Castro et al., THL 1975, 1219). By adding 1-hydroxybenzotriazole (HOBt) or 3-Hydroxy-4-oxo-3,4-dihydrobenzotriazine (HOObt) can optionally suppresses the racemization or the Reaction speed can be increased. The Amino acids Asn and Gln are preferred in the form of their N-protected p-nitrophenyl ester coupled. The  Couplings are usually 2- to 5-fold Excess of N-protected amino acid and Coupling reagent in solvents such as dichloromethane, Dimethylformamide, N-methylpyrrolidone (NMP) or mixtures carried out from these. The course of the Coupling reaction is determined by the Kaiser test (E. Kaiser u. a., anal. biochem. 34, 595 (1970)) or by the TNBS test tracked. If incomplete acylation is determined, the clutch is up to Completeness repeated. The solid phase synthesis can both manually and automatically using a Peptide synthesizers are done.

The linear peptides thus synthesized are then according to suitable, known in the literature Process cyclized. Can be used as cyclization reagents those used for the coupling of the amino acids or also pentafluorophenyl ester / DMAP or Diphenylphosphoryl azide (DPPA) find application. For the synthesis of the compounds of general formula I is preferably the solid phase synthesis after the Fmoc strategy using the 2Methoxybenzyloxybenzyl ester anchor preferred. To the Structure of the sequences are used as coupling methods DCC / HOBt, the DIC / HOBt or the TBTU process prefers. After building up the sequences on the Harz N-terminal Fmoc group cleaved as usual, the resin after removing the piperidine thoroughly with dichloromethane washed and then with to cleave the peptide a 1% solution of trifluoroacetic acid in Treated dichloromethane. The remain Preserved side chain protecting groups of the peptides. To The peptides are distilled off with the solvent a cyclization reagent is preferred  Diphenylphosphoryl azide, to the corresponding Cyclopeptides implemented. Then still existing side chain protection groups by appropriate Removal reagents removed. Are preferred Trifluoroacetic acid / scavenger mixtures. As a scavenger come substances such as B. anisole, thioanisole, cresol, Thiocresol, ethanedithiol, water or the like as well Mixtures of these scavengers are preferred for use. The Peptides are then used in peptide chemistry usual methods worked up and cleaned.

The raw products obtained are cleaned by means of Gel chromatography z. B. on Sephadex G25 (MR <1400) or G15 (MR <1400) with 1% or 5% acetic acid. If further purification is necessary using preparative RP-HPLC with methanol or Acetonitrile-water gradient with the addition of 1 to 2% Trifluoroacetic acid.

Cation exchangers can also be used for cleaning Sephadex or polystyrene base can be used.

The cleaning is preferably carried out by reversed phase HPLC using water / acetonitrile gradients with Addition of 0.1 to 0.2% trifluoroacetic acid.

The compounds of general formula I are by means of RP-HPLC checked for purity. It becomes one at a time Amino acid analysis on the ion exchanger (LKB) and on Gas chromatograph on chiral column for additional Racemization control carried out. Furthermore 13C NMR spectra (Bruker 400 MHz) as well  FAB mass spectra (Finnigan MAT 90) recorded. The Sequence determination takes place by means of gas phase sequencing after tryptic cleavage.

The following examples illustrate the synthesis of the Compounds according to the invention without the invention is restricted to this.

Example 1

The peptide synthesis was carried out with a peptide synthesizer Advanced ChemTech ACT200 using the Fmoc strategy using a modified Control program. The 50 ml shaker reactor was charged with 1 g 2-methoxybenzyl ester resin from Bachem, Switzerland was loaded with 0.5 mmol Fmoc-isoleucine. The following amino acid derivatives were used: Fmoc-Arg (Mtr) -OH, Fmoc-Asp (tBu) -OH, Fmoc-Ile-OH, Fmoc-Gly-OH, Fmoc-D-Ala-OH, Fmoc-Cha-OH and 2-pyridylacetyl-Lys (Fmoc) -OH. The clutches were using 3 equivalents each Fmoc amino acid, 1-hydroxybenzotriazole and Dicyclohexylcarbodiimid carried out (coupling time 40 Minutes). After the TNBS test was carried out at incomplete acylation under the coupling Use the same reagents and excesses repeated. When the acylation was complete, the next synthesis cycle started. The spin-off of the Fmoc protective groups were in each case with 20% piperidine DMF (once 3 minutes, once 15 minutes) performed.  

Between the reactions, the resin was washed 10 times with DMF. After building up the linear sequence 2-pyridylacetyl-Lys-Arg (Mtr) -Cha-D-Ala-Gly-Arg (Mtr) - Ile-Asp (tBu) -Arg (Mtr) -Ile- on the polymeric support, the resin became thorough washed with dichloromethane and then treated 5 times with 20 ml of a 1% solution of trifluoroacetic acid in dichloromethane for a maximum of 10 minutes at room temperature (until the resin turns an intense purple color). The solutions were combined and concentrated in vacuo. The residue is triturated with ether, the ether is decanted off, the peptide is dried in a stream of nitrogen and taken up in 130 ml of DMF, the pH is adjusted to about 8.5 with triethylamine, the solution is cooled to -20 ° C. and 0.2 g (0.75 mmol) diphenylphosphoryl azide added. It was left for 48 hours at -20 ° C, 48 hours at 4 ° C. The pH was kept at 8.5 with triethylamine. Then DMF was removed in vacuo, the residue was triturated twice with ether, the ether was decanted off and the residue was dried with a stream of nitrogen. The side chain protecting groups were cleaved with trifluoroacetic acid / anisole (90/10) for 24 hours at room temperature. The solution was concentrated in vacuo, the residue was digested with ether and dried. The crude peptide was passed through a Dynamax C18, 3 µ column (10 × 2.14 cm) using a gradient of A: water / acetonitrile / trifluoroacetic acid 95/5 / 0.2 and B: ditto 20/80/0, 2 from 10% B to 90% B in 11 minutes, flow 10 ml / min, retention time 8.2 minutes. After freeze-drying, an amorphous colorless powder was obtained.
FAB-MS (M + H) + = 1338.6.

Examples 2 to 11

Using X-Lys (Fmoc) -OH instead 2-Pyridylacetyl-Lys (Fmoc) -OH was as in Example 1 described a crude peptide obtained under Use of the described chromatography Conditions (10% to 90% B in 11 min, retention time see following table).

Claims (29)

1. Cyclopeptides of the formula and their pharmaceutically acceptable salts, wherein:
A is an amino acid residue of the formula-NH- (CH₂) _n -CH (R) -CO- (II), where n is an integer from 1 to 11 and
R -NH (H) -C (O) -CH₂-X¹
-N (H) -C (O) -CH₂-O-X¹
-N (H) -C (O) -X¹
-N (H) -C (O) -CH = CH-X¹ or
Is -N (H) -C (O) -O-CH₂-X¹,
wherein X¹ (a) phenyl, (b) phenyl substituted by 1, 2 or 3 substituents (substituent (s): halogen, trifluoromethyl or nitro), (c) naphthyl, (d) benzo [b] thienyl, (e) pyridyl or (f) is pyrazinyl;
B is a covalent bond, Phe or an α-amino acid residue with one or two side chains, wherein the side chain (s) or one of the two side chains contains (contain) one, two, three or four N-containing group (s);
C is an α-amino acid residue with one or two alkyl side chains, if desired, containing -O-, the side chain (s) or one of the two side chains bearing one or two residues, the respective residue being cycloalkyl with 4, 5, 6 or Is 7 carbon atoms, phenyl, naphthyl substituted (e.g. NO ₂ ) phenyl or a 5- or 6-membered aromatic heterocycle, wherein 2 members are H or one member is H and one member is O or S, or one member is H, S or Is O and the remaining members are C, preferably thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, isoquinolyl, quinolyl, chromanyl, thiazolyl, oxazolyl, morpholinyl;
D and E are each independently Gly or an α-amino acid residue whose side chain (s) does not carry a functional group or
D and E together are an ω-amino acid residue -NH- (CH ₂ ) _2-11 -CO- or a peptide template;
F is an α-amino acid residue with one or two side chains, in which the side chain (s) or one of the two side chains contains (contain) one, two, three or four N-containing group (s);
G is an α-amino acid residue with one or two lipophilic side chains;
H is a covalent bond or an α-amino acid residue that does not contain a functional group or -COOH or -CONH ₂ as a functional group in the side chain (s);
I is an α-amino acid residue with one or two side chains, in which the side chain (s) or one of the two side chains contains (contain) one, two, three or four N-containing group (s);
K is an α-amino acid residue with one or two lipophilic side chains. 2. Cyclopeptide according to claim 1, wherein A is a Amino acid residue of formula II, wherein n is 1, 2, 3 or 4, preferably 1 or 4. 3. Cyclopeptide according to claim 1 or 2, wherein X ^{1 is} phenyl or mono- or di-substituted phenyl. 4. Cyclopeptide according to claim 1 or 2, wherein X ^{1 is} 2-pyridyl. 5. Cyclopeptide according to claim 1 or 2, wherein X ^{1 is} 2-pyrazinyl. 6. Cyclopeptide according to claim 1 or 2, wherein X ^{1 is} 3-benzo [b] thienyl. 7. Cyclopeptide according to claim 1 or 2, wherein X ^{1 is} 1- or 2-naphthyl. 8. Cyclopeptide according to one of the preceding claims, wherein B, F and I independently of one another Arg, D-Arg, Lys, D-Lys, Orn, D-Orn, Homo-Arg, D-Homo-Arg, Dap, D-Dap , 4-Amino-Phe, Ctr, D-Ctr, Arg (NO ₂ ), D-Arg (NO ₂ ) or Phe. 9. Cyclopeptide according to claim 8, wherein B, F and Z independently of one another Arg, D-Arg, Lys, D-Lys, Orn or Ctr. 10. Cyclopeptide according to one of the preceding claims, wherein C Phe, D-Phe, 4-NO ₂ -Phe, Cha, D-Cha, Ser (Bzl), D-Ser (Bzl), Tyr, D-Tyr, Tyr ( Bzl), D-Tyr (Bzl), Nal, D-Nal, Thi, D-Thi, Asp (Bzl), D-Asp (Bzl), His or D-His. 11. Cyclopeptide according to claim 10, wherein C is Phe, Cha, Tyr, Nal, Tyr (Bzl) or 4-NO ₂ -Phe. 12. Cyclopeptide according to one of the preceding Claims wherein D and E are independent of each other Ala, Gly, Phe, Val, Ile, Leu or Nle or their D shape are. 13. Cyclopeptide according to claim 12, wherein D D-Ala, Gly, Phe or D-Phe is. 14. Cyclopeptide according to claim 12, wherein E Gly, Ala or phe is. 15. Cyclopeptide according to one of claims 1 to 11, wherein D and E together are an ω-amino acid residue of the formula -NH- (CH ₂ ) _2-5 -CO-. 16. Cyclopeptide according to one of claims 1 to 11, wherein D and E together Btu, Clg, Thc or Trc or their D-forms, preferably D-Btu. 17. Cyclopeptide according to one of the preceding Claims wherein G and K are independent of each other Ile, D-Ile, Met, D-Met, Nle, D-Nle, Leu, D-Leu, Val, D-Val, Cha, D-Cha, Cle or Aib. 18. Cyclopeptide according to claim 17, wherein G and K are independently Ile, Met, Nle or Leu. 19. Cyclopeptide according to any one of the preceding claims, wherein H HOOC- (CH ₂ ) _1-4 - CH (NH ₂ ) -CO-, their D-forms, Gly, Ala, D-Ala, Asn, D-Asn, Phe or D-Phe. 20. Cyclopeptide according to claim 19, wherein I Asp, Glu or Gly is.   21. Cyclopeptide or its salt according to claim 1, wherein
A is an aminoalkanoic acid residue of the formula
-NH- (CH₂) _n -CH (R) -CO-, wherein
n is 1 or 4,
R -NH (H) -C (O) -CH₂-X¹
-N (H) -C (O) -CH₂-O-X¹
-N (H) -C (O) -X¹
-N (H) -C (O) -CH = CH-X¹ or
Is -N (H) -C (O) -O-CH₂-X¹,
wherein X¹ (a) phenyl, (b) phenyl substituted by 1 or 2 substituents, (c) 1- or 2-naphthyl, (d) 3-benzo [b] thienyl, (e) 2-pyridyl or (f) 2 -Pyrazinyl;
B, F and I are independently Arg, D-Arg, Lys, D-Lys, Orn or Ctr;
C is Phe, Cha, Tyr, Nal, Tyr (Bzl) or 4-NO₂-Phe;
D is D-Ala, Gly, Phe or D-Phe;
E is Gly, Ala or Phe; or
D and E together are D-Btu, L-Clg or D-Clg;
G and K are independently Ile, Met, Nle or Leu;
H is Asp, Glu or Gly. 22. Cyclopeptide or its salt according to claim 21, wherein
A is -NH- (CH₂) ₄-CH (R) -CO-, wherein
R is as defined in claim 21 and
-BCDEFGHIK the chain
-Arg-Cha-D-Ala-Gly-Arg-Ile-Asp-Arg-Ile-. 23. A method for producing a cyclopeptide according to one of claims 1 to 22 or its salt, characterized in that after in the Peptide chemistry known linear methods Sequence of the peptide from the corresponding Builds up fragments and then these cyclized and, if desired, into the appropriate salt transferred. 24. Pharmaceutical preparation containing one A compound according to any one of claims 1 to 22. 25. Use of a connection according to one of the Claims 1 to 22 as an antihypertensive or hypotensive. 26. Using a connection according to one of the Claims 1 to 22 as a diuretic. 27. Use of a connection according to one of the Claims 1 to 22 as a vasodilator. 28. Use of a connection according to one of the Claims 1 to 22 as an antispasmodic. 29. Use of a connection according to one of the Claims 1 to 22 as a broncholytic.