WO1993017701A1

WO1993017701A1 - Endothelin receptor-binding peptides

Info

Publication number: WO1993017701A1
Application number: PCT/US1993/002195
Authority: WO
Inventors: David H. Coy; Simon J. Hocart; Wojciech J. Rossowski
Original assignee: The Administrators Of The Tulane Educational Fund
Priority date: 1992-03-12
Filing date: 1993-03-10
Publication date: 1993-09-16

Abstract

Cyclic pentapeptides of the formula: cyclo (D-Trp-D-Asp-X-D-Val-Leu), in which X is an α-amino acid with a conformationally restricted side chain, the side chain containing 4-16 carbons, inclusive; or pharmaceutically acceptable salts thereof. Also disclosed are methods of preparing and using such peptides.

Description

E DOTHELIN RECEPTOR-BINDING PEPTIDES BACKGROUND OF THE INVENTION

Endothelin is a potent vasoconstrictor peptide of the following formula (see Yanagisawa M. , et al. Nature 332:411 [1988]) :

H-Cys-Ser-Cys-Ser-Ser-Leu-Met-Asp-Lys-Glu-Cys-Val- Tyr-Phe-Cys-His-Leu-Asp-Ile-Ile-Trp-OH

As an endogenous peptide, endothelin, by controlling liberation of various substances, directly or indirectly induces sustained contraction of vascular or non-vascular smooth muscles.

Excess production or excess secretion of endothelin is believed to be one of pathogeneses for nephritis, asthma, acute renal failure, cyclosporin- induced renal failure myocardial infarction, angina pectoris, atherosclerosis, cardiac graft rejection, restenosis, disseminated intravascular coagulation, cerebral infarction and cerebral vasospasm. Furthermore, it has been suggested that endothelin serves as an important mediator involved in diseases such as endotoxin shock, endotoxin-induced renal failure or hypertension.

Accordingly, an objective of the present invention relates to therapeutics for the treatment of the above- mentioned various diseases by providing potent endothelin receptor-binding peptides.

SUMMARY OF THE INVENTION

In one aspect, the present invention features a series of novel cyclic pentapeptides of the formula: cyclo (D-Trp-D-Asp-X-D-Val-Leu) (I) or pharmaceutically acceptable salts thereof. In formula (I) , X is an «-amino acid with a conformationally restricted side chain and the side chain contains 4-16 carbons, inclusi e. The symbol Trp, Asp, X or the like herein stands for an a ino acid residue, -NH-CH(R)-CO-, where R denotes the side chain (or identifying group) of an amino acid or its residue, e.g., R is -CH₂COOH for Asp. Also, where the amino acid residue is optically active, it is the L- form configuration that is intended unless D-form is expressly designated.

Note that the N-terminus is at the left and the C- terminus at the right in accordance with the conventional representation of a polypeptide chain. A short line between two amino acid residues indicates a peptide bond. The prefix "cyclo" indicates the presence of a peptide bond between the N-terminus and C-terminus. A cyclic peptide herein refers to a peptide chain with such a structural feature.

What is meant by "a conformationally restricted side chain" herein is a side chain which is less flexible and cannot rotate as freely as that of a natural amino acid (except Pro or hydroxy-Pro) . Such conformational constraint can be achieved either by tethering the side chain to the NH group of the peptide bond to form a ring structure or ring structures, thus preventing rotating and bending of the side group. Alternatively, the flexibility of a side chain can be lowered as a result of steric hindrance.

Pharmaceutically acceptable salts of the peptides of the invention include, but are limited to, sodium salt, potassium salt and ammonium salt.

Preferred embodiments of the invention are the cyclic pentapeptides of formula (I) , in which the side chain and C^ and if of X form 1-3, inclusive, separate or fused rings with the ^ and ϊf^~ being members of one of the rings or pharmaceutically acceptable salts thereof. Particularly preferred pentapeptides of the invention are those in which the rings are fused; for example, X is a D- or L- isomer of Tec, Tip, Oic, or Tic (see below for full names and structures) .

In another aspect, the invention also includes cyclic pentapeptides of the formula: cyclo (D-Trp-D-X-Y-D-Val-Leu) (II) or pharmaceutically acceptable salts thereof. In formula (II) , X is Ser or Thr and Y is an ∞-amino acid with a conformationally restricted side chain.

Preferably, the side chain and * and N* of Y in formula (II) form 1-3, inclusive, separate or fused rings with the C" and N* being members of one of the rings. For example, Y can be a D- or L- isomer of Pro, hydroxy-Pro, Tec, Tip, Oic, or Tic. Hydroxy-Pro herein refers to any of 2-hydroxy-Pro, 3-hydroxy-Pro, 4-hydroxy-Pro, and 5- hydroxy-Pro; 4-hydroxy-Pro is preferred.

Also included in the present invention is a solid- phase chemical synthetic method for preparing a cyclic pentapeptide having the formula of cyclo (D-Trp-D-Asp-X- D-Val-Leu) , wherein X is an «-amino acid with a conformationally restricted side chain, the side chain containing 4-16 carbons, inclusive, which synthetic method comprises the steps of:

(a) linking a protected Leu to a solid phase via its carboxyl group and thereafter deprotecting the amino group of the Leu;

(b) coupling to the Leu a protected D-Val via its carboxyl group and thereafter deprotecting the amino group of the Val;

(c) coupling to the D-Val a protected X via its carboxyl group and thereafter deprotecting the amino group of the X;

(d) coupling to the X a protected D-Asp via its «- carboxyl group and thereafter deprotecting the amino group of the Asp; (e) coupling to the D-Asp a protected D-Trp via its carboxyl group;

(f) cleaving the pentapeptide thus formed from the solid phase; and (g) forming a peptide bond between the Leu and the D-Trp.

In the above-described synthetic method, the side chain and ( and i of X may form 1-3, inclusive, separate or fused rings with the * and i being members of one of the rings. For example, X can be a D- or L-isomer of Tec, Tip, Oic, or Tic.

Another synthetic method of the invention is an improvement in preparing a cyclic peptide from a linear peptide of 3-10 amino acid residues, inclusive, with an N-terminal residue or a residue adjacent thereto having a carboxyl group-containing side chain by forming a peptide bond between the «*-amino group of the Ν-terminal residue and the «-carboxyl group of the C-terminal residue of the linear peptide, the improvement being forming the peptide bond with the carboxyl group unprotected. Three cyclic pentapeptides which can be prepared by this improved method are cyclo (D-Trp-D-Asp-Tic-D-Val-Leu) cyclo (D- Trp-D-Asp-Tic-D-Val-Leu) , cyclo (D-Trp-D-Asp-Tip-D-Val- Leu) . In a preferred embodiment, the N-terminal residue is Asp and the linear peptide may contain 3-7, or preferably 4-6 amino acid residues, inclusive. In another preferred embodiment, the Ν-terminal residue does not have a carboxyl-group containing side chain and the residue adjacent to the Ν-terminal residue has a carboxyl-group containing side chain, such as CH₂-COOH or CH₂-CH₂-COOH. Note that the method can be used to link the C- and N-termini of a linear peptide which either (1) has an Asp side chain in its N terminal residue, the residue adjacent thereto, or both, or (2) has a Glu side chain only in the position adjacent to the N-terminus, but not in the N-terminus.

A further aspect of the invention is a method of treating hypertension, nephritis, acute renal failure, cyclosporin-induced renal failure, myocardial infarction, angina pectoris, cerebral infarction, cerebral vasospasm, asthma, atherosclerosis, cardiac graft rejection, restenosis, endotoxin shock, endotoxin-induced multiple organ failure or disseminated intravascular coagulation in a mammalian subject, which method includes administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a cyclic pentapeptide having the formula of cyclo (D-Trp-D-Asp-X- D-Val-Leu) , wherein X is an «-amino acid with a conformationally restricted side chain, the side chain containing 4-16 carbons, inclusive.

The terms "pharmaceutical composition" and "therapeutically effective amount" will be defined or exemplified in detailed description of the preferred embodiments under the heading "USE". Infra .

For the pentapeptide used in the above-described therapeutic method, the side chain and C and if of X form 1-3, inclusive, separate or fused rings with the c and if being members of one of the rings. For example, X can be a D- or L-isomer of Tec, Tip, Oic, or Tic.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Abbreviations and Structures The names of the compounds denoted by unusual abbreviations are set forth below, followed by their structures:

Tec: 1,2,3,4-tetrahydro-2-carboline-3-carboxylic acid Tic: 1,2,3,4-tetrahydro-2-carboline-3-yl-carboxylic acid

Tip: 4,5,6,7-tetrahydro-lH-imidazo[c]pyridine-6- carboxylic acid Oic: (3aS,7aS)-octahydroindol-2-yl-carboxylic acid

Tec:

Tic:

Tip :

Oic:

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

SYNTHESIS A detailed description regarding the synthesis of two cyclic pentapeptides of the present invention, cyclo (D-Trp-D-Asp-Tcc-D-Val-Leu) , cyclo (D-Trp-D-Asp-Tic-D- Val-Leu) , cyclo (D-Trp-D-Asp-Tip-D-Val-Leu) , and cyclo (D-Trp-D-Ser-Pro-D-Val-Leu) is set forth below. Other peptides of the invention can be prepared by making appropriate modifications, within the ability of someone of ordinary skill in this field, of the synthetic methods disclosed herein. See Coy, D.H. et al. J. Biol. Chem. 266:16441-16447 (1991).

Synthesis of cyclo (D-Trp-D-Asp-Tcc-D-Val-Leu)

Step 1: Preparation of Boc-D-Trp-O-benzyl-D-Asp- Tcc-D-Val-Leu-O-Merrifield resin Tert-butyloxycarbonyl("Boc")-Leu-O-polystyrene resin (1.25 g, 0.5 mmole) was first placed in the reaction vessel of an Advanced ChemTech peptide synthesizer which was programmed to perform successively each step of the following wash-reaction cycle: (a) methylene chloride; (b) 33% trifluoroacetic acid in methylene chloride (2 times for 1 and 25 min each) ; (c) methylene chloride; (d) ethanol; (e) methylene chloride; and (f) 10% triethylamine in chloroform.

The neutralized resin was stirred with Boc-D-Val and diisopropylcarbodiimide (1.5 mmole each) in methylene chloride for 1 hr and the resulting amino acid resin was then cycled through steps (a) to (f) in the above wash- reaction program. The following amino acids (1.5 mmole) were then coupled successively by the same procedure: Boc-Tcc, Boc-o-benzyl-D-Asp, and Boc-D-Trp. After washing and drying, the completed resin weighed 1.59 g. Step 2: Preparation of cyclo (D-Trp-D-Asp-Tcc-D-

Val-Leu) The resin prepared following Step 1 (1.74 g, 0.5 mmole) was mixed with anisole (5 ml) , dithiothreitol (100 mg) and anhydrous hydrogen fluoride (35 ml) at 0°C and stirred for 15 min. Excess hydrogen fluoride was evaporated rapidly under a stream of dry nitrogen and free peptide precipitated and washed with ether. The crude peptide was then dissolved in a minimum volume of 50% acetic acid and eluted on a column (2.5 x 100 cm) of Sephadex G-25 using the same solvent. Fractions containing a major component by UV absorption and Ehrlich reaction upon thin layer chromatography ("TLC") are then pooled, evaporated to an oil.

The material was dissolved in 200 ml of dimethyl ormamide and treated with 2.0 equivalents of BOP reagent [benzotriazole-1-yl-oxy-tris(dimethylamino)- phosphonium hexafluorophosphate] and 4 equivalents of diisopropylethylamine for 1 hr after which time complete cyclization was indicated by thin layer chromatography. Dimethylformamide was removed from the reaction mixture under vacuum and the residual oil was dissolved in 50% acetic acid. Fractions containing the desired peptide as indicated by TLC and high performance liquid chromatography ("HPLC") were pooled and lyophilized. At all stages of this synthesis, care must be exercised to avoid thermal decomposition of the Tec residue.

The resulting white powder was shown to be homogeneous by both TLC and HPLC and weighed 44 mg after drying. The structure of the peptide thus prepared was confirmed by amino acid analysis of acid hydrolysates and FAB-MS.

Synthesis of cyclo (D-Trp-D-Asp-Tic-D-Val-Leu) Step 1: Preparation of Boc-D-Trp-O-benzyl-D-Asp- Tic-D-Val-Leu-O-Merrifield resin Boc-Leu-O-polystyrene resin (1.25 g, 0.5 mmole) was first placed in the reaction vessel of an Advanced ChemTech peptide synthesizer which was programmed to perform successively each step of the following wash- reaction cycle: (a) methylene chloride; (b) 33% trifluoroacetic acid in methylene chloride (2 times for 1 and 25 min each) ; (c) methylene chloride; (d) ethanol; (e) methylene chloride; and (f) 10% triethylamine in chloroform.

The neutralized resin was stirred with Boc-D-Val and diisopropylearbodiimide (1.5 mmole each) in methylene chloride for 1 hr and the resulting amino acid resin was then cycled through steps (a) to (f) in the above wash- reaction program. The following amino acids (1.5 mmole) were then coupled successively by the same procedure: Boc-Tic, Boc-O-benzyl-D-Asp, and Boc-D-Trp. After washing and drying, the completed resin weighed 1.62 g. Step 2: Preparation of cyclo (D-Trp-D-Asp-Tic-D- Val-Leu) The resin prepared following Step 1 (1.65 g. 0.5 mmole) was mixed with anisole (5 ml) , dithiothreitol (100 mg) and anhydrous hydrogen fluoride (35 ml) at 0°C and stirred for 15 min. Excess hydrogen fluoride was evaporated rapidly under a stream of dry nitrogen and free peptide precipitated and washed with ether. The crude peptide was then dissolved in a minimum volume of 50% acetic acid and eluted on a column (2.5 x 100 cm) of Sephadex G-25 using the same solvent. Fractions containing a major component by UV absorption and Ehrlich reaction upon TLC were then pooled, evaporated to an oil.

This material was dissolved in 100 ml of dimethylformamide and treated with 2.0 equivalents of BOP reagent and 4 equivalents of diisopropylethyla ine for 1 hr after which time complete cyclization was indicated by TLC. Dimethylformamide was removed from the reaction mixture under vacuum and the residual oil was dissolved in 50% acetic acid and eluted on a column (2.5 x 90 cm) of Sephadex G-15 with 50% acetic acid. Fractions containing the desired peptide as indicated by TLC and HPLC were pooled and lyophilized. The resulting white powder was demonstrated to be homogeneous by both TLC and HPLC and weighed 94 mg after drying.

The structure of the peptide thus prepared was confirmed by amino acid analysis of acid hydrolysates and FAB-MS.

Synthesis of cyclo (D-Trp-D-Asp-Tip-D-Val-Leu) Step l: Preparation of Boc-D-Trp-O-benzyl-D-Asp- Tip-Val-Leu-O-Merrifield resin Boc-Leu-O-polystyrene resin (1.25 g, 0.5 mmole) was first placed in the reaction vessel of an Advanced ChemTech peptide synthesizer which was programmed to perform successively each step of the following wash- reaction cycle: (a) methylene chloride; (b) 33% trifluoroacetic acid in methylene chloride (2 times for 1 and 25 min each) ; (c) methylene chloride; (d) ethanol; (e) methylene chloride; and (f) 10% triethylamine in chloroform. The neutralized resin was stirred with Boc-D-Val and diisopropylearbodiimide (1.5 mmole each) in methylene chloride for 1 hr and the resulting amino acid resin was then cycled through steps (a) to (f) in the above wash- reaction program. The following amino acids (1.5 mmole) were then coupled successively by the same procedure: Boc-D-Val, Bis-Boc-Tip, Boc-O-benzyl-D-Asp, and Boc-D- Trp. After washing and drying, the completed resin weighed 1.71 g. Step 2: Preparation of cyclo (D-Trp-D-Asp-Tip-D- Val-Leu)

The resin prepared following Step 1 (1.71 g, 0.5 mmole) was mixed with anisole (5 ml) , dithiothreitol (100 mg) and anhydrous hydrogen fluoride (35 ml) at 0°C and stirred for 15 min. Excess hydrogen fluoride was evaporated rapidly under a stream of dry nitrogen and free peptide precipitated and washed with ether. The crude peptide was then dissolved in a minimum volume of 50% acetic acid and eluted on a column (2.5 x 100 cm) of Sephadex G-25 using the same solvent. Fractions containing a major component by UV absorption and Ehrlich reaction upon TLC were then pooled, evaporated to an oil.

The material was dissolved in 100 ml of dimethylformamide and treated with 2.0 equivalents of BOP reagent and 4 equivalents of diisopropylethyla ine for 1 hr after which time complete cyclization was indicated by thin layer chromatography. Dimethylformamide was removed from the reaction mixture under vacuum and the residual oil was dissolved in 50% acetic acid and eluted on a column (2.5 x 90 cm) of Sephadex G-15 with 50% acetic acid. Fractions containing the desired peptide as indicated by TLC and HPLC were pooled and lyophilized. The resulting white powder was shown to be homogeneous by both TLC and HPLC and weighed 25 mg after drying.

The structure of the peptide was confirmed by amino acid analysis of acid hydrolysates and FAB-MS. Synthesis of cyclo (D-Trp-D-Ser-Pro-D-Val-Leu) Step 1: Preparation of Boc-D-Trp-O-benzyl-D-Ser- Pro-D-Val-Leu-O-Merrifield resin Boc-Leucine-O-polystyrene resin (1.25 g, 0.5 mmole) was placed in the reaction vessel of an Advanced ChemTech peptide synthesizer programmed to perform the following wash-reaction cycle: (a) methylene chloride; (b) 33% trifluoroacetic acid in methylene chloride (2 times for 1 and 25 min each) ; (c) methylene chloride; (d) ethanol; (e) methylene chloride; and (f) 10% triethylamine in chloroform.

The neutralized resin was stirred with Boc-Leu and diisopropylearbodiimide (1.5 mmole each) in methylene chloride for 1 hr and the resulting amino acid resin was then cycled through steps (a) to (f) in the above wash- reaction program. The following amino acids (1.5 mmole) were then coupled successively by the same procedure: Boc-D-Val, Pro, Boc-O-benzyl-D-Ser, and Boc-D-Trp. After washing and drying, the completed resin weighed 1.65 g. Step 2: Preparation of cyclo (D-Trp-D-Ser-Pro-D-

Val-Leu) The resin prepared following Step 1 (1.75 g, 0.5 mmole) was mixed with anisole (5 ml) , dithiothreitol (100 mg) and anhydrous hydrogen fluoride (35 ml) at 0°C and stirred for 15 min. Excess hydrogen fluoride was evaporated rapidly under a stream of dry nitrogen and free peptide precipitated and washed with ether. The crude peptide was then dissolved in a minimum volume of 50% acetic acid and eluted on a column (2.5 x 100 cm) of Sephadex G-25 using the same solvent. Fractions containing a major component by UV absorption and Ehrlich reaction upon TLC were then pooled, evaporated to an oil.

This material was dissolved in 100 ml of dimethylformamide and treated with 2.0 equivalents of BOP reagent and 4 equivalents of diisopropylethylamine for 1 hr after which time complete cyclization was indicated by TLC. Dimethylformamide was removed from the reaction mixture under vacuum and the residual oil was dissolved in 50% acetic acid and eluted on a column (2.5 x 90 cm) of Sephadex G-15 with 50% acetic acid. Fractions containing the desired peptide as indicated by TLC and HPLC were pooled and lyophilized. The resulting white powder was homogeneous by TLC and HPLC and weighed 65 mg after drying. The structure of the peptide was confirmed by amino acid analysis of acid hydrolysates and FAB-MS.

Note that according to the protocols set forth above for preparing cyclo (D-Trp-D-Asp-Tcc-D-Val-Leu) , cyclo (D-Trp-D-Asp-Tic-D-Val-Leu) and cyclo (D-Trp-D-Asp- Tip-D-Val-Leu) , the /3-carboxyl group of Asp was not protected during the coupling reaction to produce a cyclic pentapeptide from its linear counterpart. Such a method is a deviation from the conventional approach and is preferred since the tedious steps of separately deprotecting a carboxyl group in the side chain are omitted.

Other cyclic pentapeptides (either with or without a carboxyl group-containing side chain in its N-terminal residue or adjacent thereto) of the invention, e.g., cyclo (D-Trp-D-Asp-Oic-D-Val-Leu) , can be readily prepared in an analogous fashion by a person of ordinary skill in the art.

BIOLOGICAL ACTIVITIES To test the biological activities of the compounds of the invention, two types of assays were performed on Cyclo (D-Trp-D-Ser-Pro-D-Val-Leu) , Cyclo (D-Trp-D-Asp- Tcc-D-Val-Leu) and Cyclo (D-Trp-D-Asp-Tic-D-Val-Leu) and cyclo (D-Trp-D-Asp-Tip-D-Val-Leu) . As described below, the tested compounds were effective in abolish endothelin-induced contractions on strips- of rat uterus smooth muscle. Note that both of the assays used are well known in the art and can be used by a person of ordinary skill in the art to screen suspected compounds without undue experimentation.

Assay on binding to endothelin receptor Procedures

Evaluation of the binding activity of the suspected peptides with respect to the endothelin receptor was performed using radioligand binding assays. See Martin, E.R. et al. J. Biol. Chem. 265:14044-14049 (1990) , which is incorporated herein by reference.

More specifically, the compounds were tested to determine their ability to displace [I¹²⁵] endothelin bound to the endothelin receptor. Evaluating the competitive displacement binding data, the preferred compounds are those which exhibit a high affinity (i.e., low IC₅₀ value) for the endothelin receptor. A10 smooth muscle cells were cultured in Dulbecco's modified Eagle's medium (DMEM) without antibiotics and supplemented with 10% (vol/vol) fetal calf serum. The incubation atmosphere consisted of 10% CO₂-90% humidified air at 37°C. Male Sprague-Dawley rats (200-300 g) were obtained from Taconic Farms and maintained in our animal facilities under a 12 hr light-dark cycle. Food and water were available ad libitum. Crude membranes were prepared by homogenization of the AlO cells or rat cerebral cortical tissue in 20 ml of ice-cold 50 mM Tris-HCl (Buffer A) with a Brinkman Polytron (setting 6, 15 sec) . Buffer was added to obtain a final volume of 40 ml, and the homogenate was centrifuged in a Sorval SS-34 rotor at 39,000 g for 10 min at 0-4°C. The resulting supernatant was decanted and discarded. The pellet was rehomogenized in ice-cold buffer, diluted, and centrifuged as before. The final pellet was resuspended in the 50 mM Tris-HCl, containing 0.1 mg/ml bacitracin, and 0.1% BSA (Buffer B) , and held on ice for the receptor binding assay.

For assay, aliquots (0.4 ml) were incubated with 0.05 nM [¹²⁵I]endothelin (-2000 Ci/mmole) and Buffer B, with and without 0.05 ml of unlabeled competing peptide or drugs. After a 60 min incubation (25°C) , the bound [¹²⁵I]endothelin was separated from the free [¹²⁵I]endothelin by rapid filtration through Whatmen GF/B glass-fiber filters. The filters were then washed three times with 5 ml aliquots of ice-cold Buffer A, and the bound radioactivity trapped on the filters was counted by gamma spectrometry. Specific binding was defined as the total [¹²⁵I]endothelin bound minus that bound in the presence of 1 μM unlabeled endothelin.

Results

The results of the assay are summarized in Table 1. All three tested peptides demonstrated great affinity for binding to endothelin receptor, BIM-36020 being the most potent with an IC₅₀ value as low as 16.2 n .

TABLE 1 Endothelin Receptor Binding Assay

Structure Code Number IC

Cyclo (D-Trp-D-Ser-Pro-D-Val-Leu) 147

Cyclo (D-Trp-D-Asp-Tcc-D-Val-Leu) BIM-36020 16.2

Cyclo (D-Trp-D-Asp-Tic-D-Val-Leu) BIM-36022 45.0

* IC₅₀ unit in nm

Assay on inhibition of endothelin-induced contractions Procedures Experiments were performed to test the ability of Cyclo (D-Trp-D-Ser-Pro-D-Val-Leu) , Cyclo (D-Trp-D-Asp- Tcc-D-Val-Leu) , Cyclo (D-Trp-D-Asp-Tic-D-Val-Leu) , and cyclo (D-Trp-D-Asp-Tip-D-Val-Leu)^' to abolish the contractile effects of endothelin itself on strips of rat uterus smooth muscle, which is a standard system for assaying binding activity of compounds to endothelin receptor. See ozuka, M. et al. Biochem. Biophys. Res. Commun. 159:317-323 (1989), which is incorporated herein by reference. Briefly, virgin female Sprague-Dawley rats which had been pre-treated with estrogen were killed by decapitation and their uteruses removed. Segments of the uteruses were then cleaned and attached longitudinally to a transducer (Narco Biosystems Myograph transducer F-60) and placed in a siliconized 25 ml organ bath (Harvard Instruments) containing oxygenated Munsick buffer at 37°C. Endothelin-l was used as the standard contracting agent (1 x 10^~9 M) alone and preceded by various doses of the suspected cyclic pentapeptides of the invention in order to calculate their IC₅₀ values.

Results

As clearly shown in Table 2, three of the tested peptides of the present invention were effective in inhibiting endothelin-induced contractions. BIM-36020 was shown to be the most potent, followed by BIM-36022. Interestingly, Cyclo (D-Trp-D-Asp-Tip-D-Val-Leu) demonstrated potent agonistic activity. An endothelin agonist can be coupled to a radioactive or fluorescent label and subjected to binding to cells or tissue extracts under various conditions, thus enabling one to obtain information regarding the structure and pharmacology of the endothelin receptor.

TABLE 2 Assay on Inhibition of Endothelin-Induced Contractions

Structure Code Number 1C-_Q / CEC^_Q )

Cy.clo (D-Trp-D-Ser-Pro-D-Val-Leu) 1 x 10 -5 Cyclo (D-Trp-D-Asp-Tcc-D-Val-Leu) BIM-36020 2 x 10^"7 Cyclo (D-Trp-D-Asp-Tic-D-Val-Leu) BIM-36022 1 x 10^"6

Cyclo (D-Trp-D-Asp-Tip-D-Val-Lue) 1 x 10 -6

(EC5₀;agonist)

ICJ-_Q unit in moles

USE

Cyclic pentapeptides of the invention can be used for the treatment hypertension, nephritis, acute renal failure, cyclosporin-induced renal failure, myocardial infarction, angina pectoris, cerebral infarction, cerebral vasospasm, asthma, atherosclerosis, cardiac

•graft rejection, restenosis, endotoxin shock, endotoxin- induced multiple organ failure or disseminated intravascular coagulation in a mammalian subject. While it is possible for a compound of the invention to be administered as the pure or substantially pure compound, it is preferable to present it as a pharmaceutical composition which is suitable for parenteral administration, oral administration or external administration. Such composition can be prepared by mixing the compound with solid or liquid excipient carriers known in this field.

Suitable pharmaceutical compositions include a liquid formulation such as an injection formulation, an inhalant formulation, a syrup formulation or an emulsion, a solid formulation such as tablets, capsules or granules, and an external drug such as an ointment or a suppository. Furthermore, these drug formulations may contain additives which are commonly employed, such as an adjuvant, a stabilizer, a wetting agent, an emulsifier, an absorption-promoting agent or a surfactant, as the case requires. As the additives, distilled water for injection, physiological saline, Ringer's solution, glucose, sugar syrup, gelatin, vegetable oil, cacao butter, ethylene glycol, hydroxypropyl cellulose, lactose, sucrose, corn starch, magnesium stearate and talc may be used.

The dose of the compound of the present invention for treating the above-mentioned diseases varies depending upon the manner of administration, the age and the body weight of the subject and the condition of the subject to be treated, and ultimately will be decided by the attending physician or veterinarian. Such amount of the active compound as determined by the attending physician or veterinarian is referred to herein as a "therapeutically effective amount". A typical administration method is oral administration or parenteral administration. The daily dose in the case of oral administration is typically in the range of from 0.1 to 100 mg/kg body weight, and the daily dose in the case of parenteral administration from 0.01 to 10 mg/kg body weight.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient(s) into association with the carrier which constitutes one or more accessory ingredients. In general, the formulations for tablets or powders are prepared by uniformly and intimately blending the active ingredient with finely divided solid carriers, and then, if necessary as in the case of tablets, forming the product into the desired shape and size. Formulations suitable for intravenous administration, on the other hand, conveniently comprise sterile aqueous solutions of the active ingredient(s) . Preferably, the solutions are isotonic with the blood of the subject to be treated. Such formulations may be conveniently prepared by dissolving solid active ingredient(s) in water to produce an aqueous solution, and rendering the solution sterile. The formulation may be presented in unit or multi-dose containers, for example, sealed ampoules or vials.

Other Embodiments The foregoing description has been limited to specific embodiments of this invention. It will be apparent, however, that variations and modifications may be made to the invention, with the attainment of some or all of the advantages of the invention. Such embodiments are also within the scope of the following claims.

What is claimed is:

Claims

1. A cyclic pentapeptide of the formula: cyclo (D-Trp-D-Asp-X-D-Val-Leu) , wherein X is an «-amino acid with a conformationally restricted side chain, said side chain containing 4-16 carbons, inclusive; or a pharmaceutically acceptable salt thereof.

2. The cyclic pentapeptide of claim 1, wherein said side chain and c and if of X form 1-3, inclusive, separate or fused rings with said and if being members of one of said rings; or a pharmaceutically acceptable salt thereof.

3. The cyclic pentapeptide of claim 2, wherein said rings are fused; or a pharmaceutically acceptable salt thereof.

4. The cyclic pentapeptide of claim 2, wherein X is a D- or L- isomer of Tec, Tip, Oic, or Tic; or a pharmaceutically acceptable salt thereof.

5. The cyclic pentapeptide of claim 4, wherein X is Tec, Tic or Tip; or a pharmaceutically acceptable salt thereof.

6. A cyclic pentapeptide of the formula: cyclo (D-Trp-D-X-Y-D-Val-Leu) , wherein X is Ser or Thr and Y is an «-amino acid with a conformationally restricted side chain; or a pharmaceutically acceptable salt thereof.

7. The cyclic pentapeptide of claim 6, wherein said side chain and C⁰' and if of Y form 1-3, inclusive, separate or fused rings with said < and if being members of one of said rings; or a pharmaceutically acceptable salt thereof.

8. The cyclic pentapeptide of claim 7, wherein said rings are fused; or a pharmaceutically acceptable salt thereof.

9. The cyclic pentapeptide of claim 1 , wherein Y is a D- or L- isomer of Pro, hydroxy-Pro, Tec, Tip, Oic, or Tic; or a pharmaceutically acceptable salt thereof.

10. The cyclic pentapeptide of claim 9, wherein Y is Pro, hydroxy-Pro, Tec, Tip, Oic or Tic; or a pharmaceutically acceptable salt thereof.

11. A solid-phase synthetic method for preparing a cyclic pentapeptide having the formula of cyclo (D-Trp- D-Asp-X-D-Val-Leu) , wherein X is an «-amino acid with a conformationally restricted side chain, said side chain containing 4-16 carbons, inclusive, which synthetic method comprises the steps of:

(a) linking a protected Leu to a solid phase via its carboxyl group and thereafter deprotecting the amino group of said Leu;

(b) coupling to said Leu a protected D-Val via its carboxyl group and thereafter deprotecting the amino group of said Val;

(c) coupling to said D-Val a protected X via its carboxyl group and thereafter deprotecting the amino group of said X;

(d) coupling to said X a protected D-Asp via its «-carboxyl group and thereafter deprotecting the amino group of said Asp;

(e) coupling to said D-Asp a protected D-Trp via its carboxyl group; (f) cleaving said pentapeptide thus formed from the solid phase; and

(g) forming a peptide bond between said Leu and said D-Trp.

12. The method of claim 11, wherein said side chain and c and if of X form 1-3, inclusive, separate or fused rings with said and if being members of one of said rings.

13. The method of claim 12, wherein said rings are fused.

14. The method of claim 12, wherein X is a D- or L-isomer of Tec, Tip, Oic, or Tic.

15. An improved synthetic method for preparing a cyclic peptide from a linear peptide of 3-10 amino acid residues, inclusive, with an N-terminal residue or a residue adjacent thereto having a carboxyl group- containing side chain by forming a peptide bond between the «-amino group of the N-terminus and the «-carboxyl group of the C-terminus of said linear peptide, wherein the improvement comprises forming said peptide bond with said carboxyl group unprotected.

16. The method of claim 15, wherein said N- terminal residue is Asp.

17. The method of claim 16, wherein said linear peptide contains 3-7 amino acid residues, inclusive.

18.. The method of claim 17, wherein said linear peptide contains 4-6 amino acid residues, inclusive.

19. The method of claim 15, wherein said N- terminal residue does not have a carboxyl-group containing side chain and said residue adjacent to said N-terminal residue has a carboxyl-group containing side chain.

20. The method of claim 19, wherein said linear peptide contains 3-7 amino acid residues, inclusive.

21. The method of claim 20, wherein said linear peptide contains 4—6 amino acid residues, inclusive.

22. The method of claim 21, wherein said linear peptide contains 5 amino acid residues.

23. The method of claim 22, wherein said residue adjacent to said N-terminal residue is Asp or Glu.

24. A method of treating hypertension, nephritis, acute renal failure, cyclosporin-induced renal failure, myocardial infarction, angina pectoris, cerebral infarction, cerebral vasospasm, asthma, atherosclerosis, cardiac graft rejection, restenosis, endotoxin shock, endotoxin-induced multiple organ failure or disseminated intravascular coagulation in a mammalian subject, which method includes administering to said subject a pharmaceutical composition comprising a therapeutically effective amount of a cyclic pentapeptide having the formula of cyclo (D-Trp-D-Asp-X-D-Val-Leu) , wherein X is an «-amino acid with a conformationally restricted side chain, said side chain containing 4-16 carbons, inclusive.

25. The method of claim 24, wherein said side chain and ( and if of X form 1-3, inclusive, separate or fused rings with said < and if being members of one of said rings.

26. The method of claim 25, wherein said rings are fused.

27. The method of claim 25, wherein X is a D- or L-isomer of Tec, Tip, Oic, or Tic.