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WO1994006453A1 - Bradykinin antagonists containing aliphatic amino acids in the 5-position - Google Patents

Bradykinin antagonists containing aliphatic amino acids in the 5-position Download PDF

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Publication number
WO1994006453A1
WO1994006453A1 PCT/US1993/008220 US9308220W WO9406453A1 WO 1994006453 A1 WO1994006453 A1 WO 1994006453A1 US 9308220 W US9308220 W US 9308220W WO 9406453 A1 WO9406453 A1 WO 9406453A1
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Prior art keywords
arg
bradykinin
aliphatic
pro
cpg
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PCT/US1993/008220
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French (fr)
Inventor
John M. Stewart
Lajos Gera
Ved Srivastava
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Stewart John M
Lajos Gera
Ved Srivastava
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Application filed by Stewart John M, Lajos Gera, Ved Srivastava filed Critical Stewart John M
Priority to AU50985/93A priority Critical patent/AU5098593A/en
Publication of WO1994006453A1 publication Critical patent/WO1994006453A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/18—Kallidins; Bradykinins; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to novel biologically active peptides which act as antagonists of the biological activities of bradykinin and its homologs and congeners, their pharmaceutically acceptable salts, and their application as therapeutic agents. More particularly, the invention pertains to modified bradykinin antagonist peptides that do not contain any aromatic amino acids or at least no aromatic residue in the 5-position and preferably also none in the 7- and 8-positions. Heretofore, all bradykinin antagonist peptides have had an aromatic or substituted aromatic amino acid residue at the position analogous to that of position 5 of bradykinin.
  • the bradykinin antagonist peptides of the present invention contain aliphatic, alicyclic, aliphatic heterocyclic or substituted alicyclic or aliphatic heterocyclic amino acid residues at that position.
  • Bradykinin (usually abbreviated BK) has the amino acid sequence:
  • bradykinin Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (BK) position : 1 2 3 4 5 6 7 8 9
  • Lys-BK also called kallidin
  • Met-Lys-BK Met-Lys-BK.
  • humans also produce the analog in which the 3-proline is replaced by trans- 4-hydroxyproline.
  • Bradykinin and its physiologically important related peptides kallidin (Lys-bradykinin) and Met-Lys-bradykinin, exhibit physiological actions which qualify them as mediators of inflammatory reactions, hypotensive states, and pain. Bradykinin is overproduced in pathological conditions such as septic shock (Robinson et al. , Am. J. Med.. 5_9.:61, 1975) and hemorrhagic (Hirsch et al . , J. Surg. Res. , 37:147, 1974), anaphylaxis
  • bradykinin The production of bradykinin from the plasma results in pain at the site of the pathological condition, and the overproduction intensifies the pain directly or via stimulation by bradykinin of the activation of the arachidonic acid pathway which produces prostaglandins and leukotrienes, the more distal and actual mediators of inflammation.
  • Literature references describing these actions of bradykinin and related peptides are found in Handbook of Experimental Pharmacology, Vol. 25, Springer-Verlag, 1970 and VOl . 25 Supplement, 1979 and in Stewart, in "Mediators of the Inflammatory Process", Henson and Murphy, eds. , Elsevier, 1989.
  • Bradykinin as discussed, has been found to be produced in inflammatory reactions in the intestine provoking contraction of smooth muscle and secretion of fluid and ions.
  • the existence of specific bradykinin receptors in the mucosal lining of the intestine and intestinal smooth muscle is demonstrated by Manning et al . in Nature (229 :256- 259, 1982) showing the influence of bradykinin in very low concentrations upon fluid and ion secretion.
  • bradykinin and associated pain in angina has been studied and reported by Kimura et al . in American Heart Journal ( 5.:635-647, 1973) and by Staszewska-Barczak et al. in Cardiovascular Research ( ⁇ j) :314-327, 1976) .
  • the reported action of bradykinin and prostaglandins acting in concert are the natural stimulus for excitation of the sensory receptors signalling the pain of myocardial ischaemia.
  • Bradykinin and bradykinin-related kinins are not only produced by the animal but may also be injected as a result of stings and bites. It is known that insects such as hornets and wasps inject bradykinin related peptides which also cause pain, swelling and inflammation.
  • bradykinin which is essential for the development of useful tools for diagnostic use, and for the development of therapeutic agents aimed at alleviating the intense pain and other symptoms caused by the production and overproduction of bradykinin, was severely hindered by the lack of specific sequence-related competitive antagonists of bradykinin until the discovery of the first effective bradykinin antagonists by Vavrek and Stewart in 1985. (Peptides. £.161-164, 1985; U.S. Patent 4,693,993, September 15, 1987) .
  • bradykinin In all those peptide antagonists of bradykinin, the proline residue at position 7 of bradykinin was replaced by a D-aromatic acid residue, usually D-phenylalanine or D-thienylalanine.
  • a D-aromatic acid residue usually D-phenylalanine or D-thienylalanine.
  • many modifications of the original bradykinin antagonists have been described, but all effective antagonists have had an aromatic amino acid residue at position 5 and a D-aromatic residue at position 7.
  • the residue at position 8 was usually an aromatic amino acid, although in certain antagonists, the 5-7-8 arrangement was aromatic-D-aromatic-aliphatic. More recently, effective bradykinin antagonists have been described in which the 5-7-8 arrangement is aromatic-D-aliphatic-aliphatic (R.J. Vavrek and J.M. Stewart in "Kinins 1991", H. Fritz, ed. , Agents and Actions Supplement, 1992; D.J. Kyle et al., J. Med. Chem.. 34_:2649-2653, 1991) .
  • the prior art has not described effective bradykinin antagonists having the 5-7-8 arrangement of aliphatic-D-aliphatic-aliphatic or aliphatic, D- aromatic, aliphatic. Effective, potent bradykinin antagonists having this type of structure are the subject of this invention.
  • the structures of effective bradykinin antagonist peptides described in the prior art can be summarized as follows:
  • bradykinin antagonists are also known in which other residues are used, such as:
  • This invention relates to modification of the structures of known bradykinin antagonist peptides to replace the aromatic amino acid residue at position 5 of the bradykinin antagonist structure with an aliphatic, alicyclic, or substituted alicyclic amino acid residue.
  • bradykinin analogs were previously synthesized as potential antagonists having an aliphatic amino acid residue at position 5, but always in combination with a position 7-8 substitution that was
  • bradykinin antagonist peptides of the present invention may be characterized by Formula 3 :
  • Formula 3 X--A--B--C--D--E--F--G--H--J--K--Z position number 0 1 2 3 4 5 6 7 8 9
  • X is an aromatic, aliphatic, or urethane-type acylating group, a single amino acid of the D- or L- configuration, or a di- or poly-peptide containing amino acids of the D- or L- configuration, or a combination of these
  • A is D-Arg or another basic or neutral aromatic, aliphatic, heterocyclic or alicyclic amino acid of the D- or L- configuration
  • B is Arg or another basic or neutral aromatic, aliphatic, heterocyclic or alicyclic amino acid of the D- or L- configuration
  • C is Pro, Hyp, or another basic or neutral aromatic, aliphatic, heterocyclic, or alicyclic amino acid of the D- or L- configuration
  • D is Pro, Hyp, or another basic or neutral aromatic, aliphatic, heterocyclic, or alicyclic amino acid of the D- or L- configuration
  • E is Gly or another basic or neutral aromatic, aliphatic, heterocyclic, or alicyclic amino acid of the D- or L- configuration
  • F is Cpg or another aliphatic, aliphatic heterocyclic, or alicyclic amino acid of the D- or L- configuration
  • G is preferably Ser or Cys of the D- or L- configuration or another aromatic, aliphatic, heterocyclic, or alicyclic amino acid of the D- or L- configuration
  • H is D-Cpg or another aliphatic, aliphatic heterocyclic, or alicyclic amino acid of the
  • J is Cpg or another aliphatic, aliphatic heterocyclic, or alicyclic amino acid of the D- or L- configuration
  • K is Arg or another basic or neutral aromatic, aliphatic, heterocyclic or alicyclic amino acid of the D- or L- configuration
  • Z is the carboxy-terminal carboxyl group or a carboxy-terminal extension composed of an amino acid of the D- or L-configuration or a peptide composed of amino acids of the D- or L-configuration.
  • Salts of bradykinin antagonist peptides of Formula 3 include salts with HCl, TFA, HOAc, as well as other pharmaceutically acceptable salts. Suitable substitutions in the various positions of all-aliphatic bradykinin antagonists peptides of Formula 3 may be represented as follows (alignment of the residues in a particular row does not imply nor limit to a given peptide sequence) :
  • bradykinin antagonist peptides of the present invention may be more exactly represented by Formula 4 :
  • a preferred compound of Formula 4 is the following peptide:
  • bradykinin antagonist peptides of the present invention by SPPS may be carried out manually (see Stewart & Young) or by use of the Beckman Model 990, Biosearch Model 9500 or other automatic peptide synthesizers, and involves use of standard procedures, defined as follows:
  • PROCEDURE A DCC coupling reaction:
  • PROCEDURE B DIC coupling:
  • PROCEDURE D TFA deprotection and neutralization: (Stewart & Young p. 76) .
  • the deprotection reagent is TFA:DCM (1:3) , containing 1 mg/ml indole. It is used for 30 minutes, following a prewash.
  • the neutralization reagent is 10% TEA in DCM, prepared fresh and used twice for one minute.
  • PROCEDURE F HF cleavage and deblocking: (Stewart & Young p. 85) .
  • PROCEDURE G PURIFICATION OF PEPTIDES:
  • the peptides may be purified by CCD for 100 transfers in the appropriate system, as determined by preliminary k estimation:
  • Chlorine-tolidine and Sakaguchi spray reagents may be used.
  • PROCEDURE J Paper electrophoresis (ELEC) :
  • ELEC may be done in buffers of pH 2.8 and 5.0 as described in Stewart & Young. Chlorine-tolidine and Sakaguchi spray reagents may be used.
  • Preparative HPLC may be carried out on large-pore reversed phase C4 or C8 columns in a gradient of 0.1% TFA in H20 to 0.08% TFA in acetonitrile. Detection may be by UV at 214 nm. Analytical HPLC may be carried out in the same system and in a gradient of acetonitrile in 0.25M triethylammonium phosphate, pH 6.5.
  • PROCEDURE L MASS spectroscopy
  • Peptides may be checked for- the correct molecular mass by FAB mass spectroscopy.
  • PROCEDURE M Amino acid analysis (AAA) :
  • Peptides may be hydrolyzed in 6N HCl and analyzed as described in Stewart &. Young, pp. 109-
  • the 990 synthesizer vessel is loaded with 1.66g of Boc-Arg(Tos) -OHMR (0.24 mmol/g substitution; 0.4 mmol total) .
  • Boc-L-Cpg is coupled by Procedure C. Coupling is checked for completeness with the Kaiser test, and is repeated if necessary.
  • Boc-D-Cpg and Boc-Ser(Bzl) are next coupled in order using Procedure C.
  • the peptide-resin is divided into 4 parts, and synthesis was continued on the 0.1 mmol scale, using BOP procedure C. The peptide-resin is deprotected by Procedure E and dried.
  • the peptide is cleaved from the resin and deblocked by Procedure F.
  • the peptide is purified by CCD using Procedure G and System A.
  • the purified peptide is checked for purity by TLC (Procedure H) , ELEC (Procedure I) and HPLC (Procedure K) , and characterized by AAA (Procedure M) and MASS (Procedure L) .
  • TLC Process H
  • ELEC Protectedure I
  • HPLC Procedure K
  • AAA Procedure M
  • MASS Procedure L
  • bradykinin antagonists are assayed on isolated rat uterus in natural or induced estrus and on guinea pig ileum, according to the commonly accepted assay methods for bradykinin and related kinins as described by Trautschold (Handbook of Expt. Pharmacol., Vol. 25, Springer Verlag, pp. 53- 55, 1969) for inhibition of the myotropic activity of bradykinin.
  • the inhibition potencies are determined according to the commonly accepted manner described by Schild for antagonists of biologically active compounds (Br. J. Pharmacol.. 2..189, 1947), and expressed as pA values. In the assays, a dose- response curve is determined for the reference substance bradykinin.
  • the dose of bradykinin which produces a half-maximal contraction of tissue is the ED dose.
  • An amount of bradykinin equivalent to twice the ED dose is administered to the tissue 30 seconds after the start of incubation of the tissue with a dose of antagonist.
  • Doses of antagonist are increased in the protocol until the dose of antagonist is found that causes the tissue response to a double ED 50 dose of bradykinin in the presence of antagonist to equal the response of an EDb_ n (J dose of bradykinin without antagonist.
  • the pA value represents the negative logarithm of the molar concentration of antagonist necessary to reduce the response of a double ED dose of bradykinin to that of an ED dose without antagonist.
  • a change of one unit of pA value represents an order of magnitude change in potency.
  • the negative log of the dose of BK that half-maximal contraction of the tissues, is commonly known as the pD value.
  • the pD value for bradykinin is 7.9 on the rat uterus and 7.4 on the guinea pig ileum.
  • bradykinin antagonists The m vivo effects of bradykinin antagonists on blood pressure in the anesthetized rat are determined according to the assay described by Roblero, Ryan and Stewart (Res. Commun. Pathol. Pharmacol. , 6.:207, 1973) .
  • the antagonists produce inhibition of the bradykinin response when injected as a bolus admixture of bradykinin plus antagonist by either the ia or iv route of administration, or when administered as an infusion. Potencies for the antagonist in this assay are not reported quantitatively but rather are indicated qualitatively. The results of tests on compounds of the various Examples are reported in Table I.
  • Antagonist potency is given as percent of BK potency.
  • Antagonist potency is given as pA foi and is underlined.
  • blood pressure assay ⁇ indicates unquantitated antagonist potency.
  • the antagonists of the invention may be used in the form of conventional pharmaceutical compositions comprising the antagonist and a pharmaceutically acceptable carrier. Such compositions may be adapted for topical, oral, aerosolized, intramuscular, subcutaneous or intravenous administration.
  • the amount of antagonist present in such compositions will range from, for example, about 0.001 to 90.0% by weight depending on the application and mode of administration although more or less of the active component may be used.
  • Conventional dosages will vary considerably on the basis of the intended application and mode of administration, e.g. 0.1 to 100 micrograms per kg body weight per minute are contemplated for use in the context of the septic shock.

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Abstract

Bradykinin antagonist peptides which have an aliphatic or alicyclic or aliphatic heterocyclic residue in the 5-position and preferably in the 8- and 7-positions.

Description

BRADYKININ ANTAGONISTS CONTAINING ALIPHATIC AMINO ACIDS IN THE 5-POSITION
BACKGROUND OF THE INVENTION The invention described herein was made in the course of work under a grant or award from the Department of Health, Education and Welfare.
Field of the Invention
The invention relates to novel biologically active peptides which act as antagonists of the biological activities of bradykinin and its homologs and congeners, their pharmaceutically acceptable salts, and their application as therapeutic agents. More particularly, the invention pertains to modified bradykinin antagonist peptides that do not contain any aromatic amino acids or at least no aromatic residue in the 5-position and preferably also none in the 7- and 8-positions. Heretofore, all bradykinin antagonist peptides have had an aromatic or substituted aromatic amino acid residue at the position analogous to that of position 5 of bradykinin. The bradykinin antagonist peptides of the present invention contain aliphatic, alicyclic, aliphatic heterocyclic or substituted alicyclic or aliphatic heterocyclic amino acid residues at that position.
Description of the Prior Art
Bradykinin (usually abbreviated BK) has the amino acid sequence:
Formula 1 : Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (BK) position : 1 2 3 4 5 6 7 8 9 Two homologs of bradykinin are also produced naturally in the human: Lys-BK (also called kallidin) and Met-Lys-BK. In some inflammatory conditions, humans also produce the analog in which the 3-proline is replaced by trans- 4-hydroxyproline.
Bradykinin, and its physiologically important related peptides kallidin (Lys-bradykinin) and Met-Lys-bradykinin, exhibit physiological actions which qualify them as mediators of inflammatory reactions, hypotensive states, and pain. Bradykinin is overproduced in pathological conditions such as septic shock (Robinson et al. , Am. J. Med.. 5_9.:61, 1975) and hemorrhagic (Hirsch et al . , J. Surg. Res. , 37:147, 1974), anaphylaxis
(Collier and James, J. Phvsiol. , 16JD:15P, 1966), arthritis (Jasani et al., Am. Rheum. Pis. , 28 :497, 1969; Hamberg et al . , Agents Actions, 8.:50, 1978; Sharma et al. , Arch. Int. Pharmacodvn, 262 :279, 1983), rhinitis (Proud et al. , J. Clin. Invest. ,
7^:1678, 1983; Naclerio et al. , Clin. Res.. 33 :613A. 1985), asthma (Christiansen et al., J. Clin. Invest. , 79_:188-197 (1987), inflammatory bowel disease (Zeitlin and Smith, Gut, 4.:133-138, 1973), and certain other conditions including acute pancreatitis, post-gastrectomy dumping syndrome, carcinoid syndrome, migraine, and angioneurotic edema (Lese, Handb. Exp. Pharmacol .. 50/1:464-522, 1978) . The production of bradykinin from the plasma results in pain at the site of the pathological condition, and the overproduction intensifies the pain directly or via stimulation by bradykinin of the activation of the arachidonic acid pathway which produces prostaglandins and leukotrienes, the more distal and actual mediators of inflammation. Literature references describing these actions of bradykinin and related peptides are found in Handbook of Experimental Pharmacology, Vol. 25, Springer-Verlag, 1970 and VOl . 25 Supplement, 1979 and in Stewart, in "Mediators of the Inflammatory Process", Henson and Murphy, eds. , Elsevier, 1989. Bradykinin, as discussed, has been found to be produced in inflammatory reactions in the intestine provoking contraction of smooth muscle and secretion of fluid and ions. The existence of specific bradykinin receptors in the mucosal lining of the intestine and intestinal smooth muscle is demonstrated by Manning et al . in Nature (229 :256- 259, 1982) showing the influence of bradykinin in very low concentrations upon fluid and ion secretion.
The production of bradykinin and associated pain in angina has been studied and reported by Kimura et al . in American Heart Journal ( 5.:635-647, 1973) and by Staszewska-Barczak et al. in Cardiovascular Research (πj) :314-327, 1976) . The reported action of bradykinin and prostaglandins acting in concert are the natural stimulus for excitation of the sensory receptors signalling the pain of myocardial ischaemia.
Bradykinin and bradykinin-related kinins are not only produced by the animal but may also be injected as a result of stings and bites. It is known that insects such as hornets and wasps inject bradykinin related peptides which also cause pain, swelling and inflammation.
The search for understanding of the mechanism of action of bradykinin, which is essential for the development of useful tools for diagnostic use, and for the development of therapeutic agents aimed at alleviating the intense pain and other symptoms caused by the production and overproduction of bradykinin, was severely hindered by the lack of specific sequence-related competitive antagonists of bradykinin until the discovery of the first effective bradykinin antagonists by Vavrek and Stewart in 1985. (Peptides. £.161-164, 1985; U.S. Patent 4,693,993, September 15, 1987) . In all those peptide antagonists of bradykinin, the proline residue at position 7 of bradykinin was replaced by a D-aromatic acid residue, usually D-phenylalanine or D-thienylalanine. In the references cited and in many other publications since (reviewed by J.M. Stewart and R.J. Vavrek in R.M. Burch, ed. , "Bradykinin Antagonists", Pergamon, 1990), many modifications of the original bradykinin antagonists have been described, but all effective antagonists have had an aromatic amino acid residue at position 5 and a D-aromatic residue at position 7. The residue at position 8 was usually an aromatic amino acid, although in certain antagonists, the 5-7-8 arrangement was aromatic-D-aromatic-aliphatic. More recently, effective bradykinin antagonists have been described in which the 5-7-8 arrangement is aromatic-D-aliphatic-aliphatic (R.J. Vavrek and J.M. Stewart in "Kinins 1991", H. Fritz, ed. , Agents and Actions Supplement, 1992; D.J. Kyle et al., J. Med. Chem.. 34_:2649-2653, 1991) . The prior art has not described effective bradykinin antagonists having the 5-7-8 arrangement of aliphatic-D-aliphatic-aliphatic or aliphatic, D- aromatic, aliphatic. Effective, potent bradykinin antagonists having this type of structure are the subject of this invention. The structures of effective bradykinin antagonist peptides described in the prior art can be summarized as follows:
Formula 2: X-Basic-Basic-Pro-Pro-Gly-Arom-Ser-DArom-Arom-Basic position: 0 1 2 3 4 5 6 7 8 9
A typical embodiment might be:
H-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DPhe-Thi-Arg position: 0 1 2 3 4 5 6 7 8 9
Useful bradykinin antagonists are also known in which other residues are used, such as:
Figure imgf000007_0001
1972) and unless prefixed with D are all of the L- configuration (except glycine and MP4, which are not optically active) .
Abbreviations used for unnatural amino acids in bradykinin analogs: Alg Allylglycine
Azt Azetine-carboxylic acid CDF p-Chloro-D-Phe
Chg CyclohexylGly ( - Aminocyclohexaneacetic acid) Cpg CyclopentylGly (α-Aminocyclopentaneacetic acid) (Cpg-ps-Arg) Pseudo (CH2NH) cyclopentylGly-Arg Dhp 3,4-dehydro-Pro FDF p-fluoro-DPhe Hyp trans-4-hydroxy-Pro HypTE hydroxyproline- trans-thiophenyl ether Inip Isonipecotic acid MDY O-Methyl-DTyr MP3 2, 3-Methanoproline (2-Azabicyclo-
(3,1,0) -hexane-1-carboxylic acid) MP4 2,4-Methanoptroline (2-Azabicyclo- (2,1, ) hexane-1-carboxylic acid) Nal β-2-Napthyl-Ala Nle Norleucine NMF N-MethylPhe Oic Octahydroindole-2-carboxylic acid OMT O-Methyl-Tyr Pal β-3-Pyridyl-Ala PCF p-Chloro-Phe Pip Pipecolic acid ("homo-Pro") Pop trans-4-PropoxyPro Thi β-2-Thienyl-Ala Thz Thiazolidine-4-carboxylic acid Tic 1,2,3,4-Tetrahydroisoquinoline-3- carboxylic acid
Abbreviations used for acylating groups (X in Formula 2)
Aaa- Adamantaneacetyl-
Ac- Acetyl-
Bz- Benzoyl Cha- Cyclohexaneacetyl-
Cpa- Cyclopentaneacetyl-
Dhq Dihydroquinuclidine-3-carboxyl-
Nba- Norbormaneacetyl-
Pba- Phenylbutyryl- Ppa- Phenylpropionyl-
Tba- t-Butylacetyl-
SUMMARY OF THE INVENTION
This invention relates to modification of the structures of known bradykinin antagonist peptides to replace the aromatic amino acid residue at position 5 of the bradykinin antagonist structure with an aliphatic, alicyclic, or substituted alicyclic amino acid residue. Several bradykinin analogs were previously synthesized as potential antagonists having an aliphatic amino acid residue at position 5, but always in combination with a position 7-8 substitution that was
D-aromatic-aromatic or D-aromatic-aliphatic. Those analogs (described in Stewart and Vavrek "Chemistry of bradykinin B2 antagonists" in R. M. Burch, ed. : "Bradykinin Antagonists," Pergamon, 1990) had a proline residue at position 5, and were devoid of antagonist activity; in certain cases they possessed weak agonist activity. Thus all the prior art teaches that the amino acid residue in position 5 must be aromatic in nature for antagonist activity. The bradykinin antagonist peptides of the present invention may be characterized by Formula 3 :
Formula 3: X--A--B--C--D--E--F--G--H--J--K--Z position number 0 1 2 3 4 5 6 7 8 9 wherein X is an aromatic, aliphatic, or urethane-type acylating group, a single amino acid of the D- or L- configuration, or a di- or poly-peptide containing amino acids of the D- or L- configuration, or a combination of these, A is D-Arg or another basic or neutral aromatic, aliphatic, heterocyclic or alicyclic amino acid of the D- or L- configuration,
B is Arg or another basic or neutral aromatic, aliphatic, heterocyclic or alicyclic amino acid of the D- or L- configuration,
C is Pro, Hyp, or another basic or neutral aromatic, aliphatic, heterocyclic, or alicyclic amino acid of the D- or L- configuration, D is Pro, Hyp, or another basic or neutral aromatic, aliphatic, heterocyclic, or alicyclic amino acid of the D- or L- configuration,
E is Gly or another basic or neutral aromatic, aliphatic, heterocyclic, or alicyclic amino acid of the D- or L- configuration,
F is Cpg or another aliphatic, aliphatic heterocyclic, or alicyclic amino acid of the D- or L- configuration, G is preferably Ser or Cys of the D- or L- configuration or another aromatic, aliphatic, heterocyclic, or alicyclic amino acid of the D- or L- configuration,
H is D-Cpg or another aliphatic, aliphatic heterocyclic, or alicyclic amino acid of the
D-configuration, or a D-aromatic amino acid or aromatic ether of trans-hydroxyproline,
J is Cpg or another aliphatic, aliphatic heterocyclic, or alicyclic amino acid of the D- or L- configuration,
K is Arg or another basic or neutral aromatic, aliphatic, heterocyclic or alicyclic amino acid of the D- or L- configuration,
Z is the carboxy-terminal carboxyl group or a carboxy-terminal extension composed of an amino acid of the D- or L-configuration or a peptide composed of amino acids of the D- or L-configuration.
Salts of bradykinin antagonist peptides of Formula 3 include salts with HCl, TFA, HOAc, as well as other pharmaceutically acceptable salts. Suitable substitutions in the various positions of all-aliphatic bradykinin antagonists peptides of Formula 3 may be represented as follows (alignment of the residues in a particular row does not imply nor limit to a given peptide sequence) :
10
15
20
Figure imgf000012_0001
DHypTE
A principal subset of the bradykinin antagonist peptides of the present invention may be more exactly represented by Formula 4 :
Formula 4 :
X-Basic-Basic-Pro-Pro-Gly-Aliph-Ser-DAliph-Aliph-Basic position:
wherein "Aliph" is an aliphatic or alicyclic or aliphatic heterocyclic amino acid residue and "Basic" is a basic amino acid residue. A preferred compound of Formula 4 is the following peptide:
Formula 5: H-DArg-Arg-Pro-Hyp-Gly-Cpg-Ser-DCpg-Cpg-Arg position:
DETAILED DESCRIPTION
The description of peptide synthesis methods uses several abbreviations for standard solvents, reagents and procedures, defined as follows:
BOP Benzotriazolyloxy-tris-
(dimethylamino)phosphonium hexafluorophosphate
BuOH Butanol
DCC Dicyclohexylcarbodiimide DCM Dichloromethane
DIC Diisopropylcarbodiimide
DIEA Diisopropylethyl amine
DMF Dimethylformamide
HOAc Acetic acid HOBT 1-Hydroxybenzotriazole hydrate
OHMR Hydroxymethylpolystyrene resin for peptide synthesis, 1% crosslinked.
TEA Triethyl amine
TFA Trifluoroacetic acid
The following abbreviations for blocking groups used in synthesis are:
Boc t-Butyloxycarbonyl Tos p-Toluenesulfonyl Bzl Benzyl ether
The following abbreviations for standard techniques are used:
AAA Amino acid analysis (Stewart & Young p. 108) CCD Countercurrent distribution (Stewart & Young p. 96) ELEC Paper electrophoresis (Stewart & Young p. 117) HPLC High performance liquid chromatography
(Stewart & Young, p. 100) Kaiser test Ninhydrin test for completeness of coupling reactions (Stewart & Young, p. 105)
SPPS Solid phase peptide synthesis TLC Thin-layer chromatography
(Stewart & Young, p. 103) The synthesis of the peptides of general Formula 3 including preparation of appropriate amino acid derivatives, their activation and coupling to form peptides and methods for purification of peptides and determination of their purity are included in the general body of knowledge of peptide chemistry, as described in Houben-Weyl, "Methoden der Organische Chemie", Vol. 16, partes I & II (1974) for solution-phase synthesis, and in "Solid Phase Peptide Synthesis" by Stewart and Young (1984) for synthesis by the solid-phase method of Merrifield. Any chemist skilled in the art of peptide synthesis can synthesize the peptides of general formula 3 by standard solution methods or by manual or automated solid-phase methods.
Synthesis of the bradykinin antagonist peptides of the present invention by SPPS may be carried out manually (see Stewart & Young) or by use of the Beckman Model 990, Biosearch Model 9500 or other automatic peptide synthesizers, and involves use of standard procedures, defined as follows:
PROCEDURE A: DCC coupling reaction:
(As defined in Stewart & Young, p 76ff) . A 2.5-fold excess of Boc amino acids is used in the Model 990 synthesizer. Completeness of coupling may be determined by use of the Kaiser reagent.
PROCEDURE B: DIC coupling:
In the Model 9500 synthesizer a 6-fold excess of Boc-amino acids is used with an equivalent of DIC. The solvent is DCM:DMF (1:1) . The resin is washed with the same solvent before and after coupling. PROCEDURE C: BOP coupling reaction (for hindered amino acids) : A 3-fold excess of Boc-amino acid over peptide-resin is mixed with an equivalent amount of BOP and 2 equivalents of DIEA in DMF. The peptide-resin is washed with DMF before and after the coupling reaction, and after coupling is then washed 2 times with methanol before continuing standard DCM washes . Completeness of coupling is checked by the Kaiser test.
PROCEDURE D: TFA deprotection and neutralization: (Stewart & Young p. 76) . The deprotection reagent is TFA:DCM (1:3) , containing 1 mg/ml indole. It is used for 30 minutes, following a prewash. The neutralization reagent is 10% TEA in DCM, prepared fresh and used twice for one minute.
PROCEDURE E: Terminal deprotection:
(As described by Stewart & Young, p. 79) .
PROCEDURE F: HF cleavage and deblocking: (Stewart & Young p. 85) .
PROCEDURE G: PURIFICATION OF PEPTIDES:
(Stewart & Young p. 96) . The peptides may be purified by CCD for 100 transfers in the appropriate system, as determined by preliminary k estimation:
A: n-BuOH:l% TFA for average antagonist peptides
B: n-BuOH:ethyl acetate:1% TFA (1:1:2) for more hydrophobic antagonist peptides. PROCEDURE H: TLC:
TLC may be carried out on silica gel plates with systems F (n-BuOH:HOAc:H20:pyridine =15:3:8:10) and I (n-BuOH:HOAc:H20=4 :1:1) . Chlorine-tolidine and Sakaguchi spray reagents may be used.
PROCEDURE J: Paper electrophoresis (ELEC) :
ELEC may be done in buffers of pH 2.8 and 5.0 as described in Stewart & Young. Chlorine-tolidine and Sakaguchi spray reagents may be used.
PROCEDURE K: HPLC:
Preparative HPLC may be carried out on large-pore reversed phase C4 or C8 columns in a gradient of 0.1% TFA in H20 to 0.08% TFA in acetonitrile. Detection may be by UV at 214 nm. Analytical HPLC may be carried out in the same system and in a gradient of acetonitrile in 0.25M triethylammonium phosphate, pH 6.5.
PROCEDURE L: MASS spectroscopy:
Peptides may be checked for- the correct molecular mass by FAB mass spectroscopy.
PROCEDURE M: Amino acid analysis (AAA) :
Peptides may be hydrolyzed in 6N HCl and analyzed as described in Stewart &. Young, pp. 109-
112, using a Beckman Model 6300 amino acid analyzer.
SYNTHESIS OF SPECIFIC EXAMPLES
The following examples are illustrative of compounds of this invention having general Formula 3 and are not intended to be limitative. All percentages and ratios are by weight when solids are involved and by volume when only liquids are involved.
EXAMPLE 1: DArg-Arg-Pro-Hyp-Gly-Cpg-Ser-DCpg-Cpg-Arg
The 990 synthesizer vessel is loaded with 1.66g of Boc-Arg(Tos) -OHMR (0.24 mmol/g substitution; 0.4 mmol total) . After deprotection and neutralization by Procedure D, Boc-L-Cpg is coupled by Procedure C. Coupling is checked for completeness with the Kaiser test, and is repeated if necessary. Boc-D-Cpg and Boc-Ser(Bzl) are next coupled in order using Procedure C. After deprotection by Procedure D, the peptide-resin is divided into 4 parts, and synthesis was continued on the 0.1 mmol scale, using BOP procedure C. The peptide-resin is deprotected by Procedure E and dried. The peptide is cleaved from the resin and deblocked by Procedure F. The peptide is purified by CCD using Procedure G and System A. The purified peptide is checked for purity by TLC (Procedure H) , ELEC (Procedure I) and HPLC (Procedure K) , and characterized by AAA (Procedure M) and MASS (Procedure L) . Using the same general procedures, the following peptides are synthesized, purified and characterized:
EXAMPLE 2: DArg-Arg-Pro-Hyp-Gly-Cpg-Ser-DChg-Oic-Arg
EXAMPLE 3: DArg-Arg-Pro-Hyp-Gly-Chg-Ser-DChg-Oic-Arg
EXAMPLE 4: DArg-Arg-Gly-Pro-Hyp-Cpg-Ser-DChg-Oic-Arg EXAMPLE 5: DArg-Arg-Pro-Hyp-Gly-Chg-Ser-DChg-Chg-Arg
EXAMPLE 6: DArg-Arg-Pro-Hyp-Gly-Cpg-Ser-DChg-Chg-Arg
EXAMPLE 7: DArg-Arg-Pro-Hyp-Gly-Leu-Ser-DChg-Chg-Arg
EXAMPLE 8: DArg-Arg-Pro-Hyp-Gly-Chg-Ser-Chg-Oic-Arg
EXAMPLE 9: DArg-Arg-Pro-Hyp-Gly-Cpg-Ser-DAlg-Oic-Arg
EXAMPLE 10: DArg-Arg-Pro-Hyp-Gly-Chg-Ser-DAlg-Oic-Arg
EXAMPLE 11: DArg-Arg-Pro-Hyp-Gly-Leu-Ser-DAlg-Oic-Arg
EXAMPLE 12: DArg-Arg-Pro-Hyp-Gly-Val-Ser-DAlg-Oic-Arg
EXAMPLE 13: DArg-Arg-Pro-Hyp-Gly-Ile-Ser-DAlg-Oic-Arg
EXAMPLE 14: DArg-Arg-Pro-Hyp-Gly-Chg-Ser-Alg-Oic-Arg
EXAMPLE 15: DArg-Arg-Pro-Hyp-Gly-Cpg-Ser-DCpg-Oic-Arg
EXAMPLE 16: DArg-Arg-Pro-Hyp-Gly-Oic-Ser-DCpg-Oic-Arg
EXAMPLE 17: DArg-Arg-Pro-Hyp-Gly-Cpg-Ser-MP3-Oic-Arg
EXAMPLE 18: DArg-Arg-Pro-Hyp-Gly-Cpg-Ser-MP3-Cpg-Arg
EXAMPLE 19: DArg-Arg-Pro-Hyp-Gly-MP3-Ser-DCpg-Cpg-Arg
EXAMPLE 20: DArg-Arg-MP3-Hyp-Gly-Cpg-Ser-DCpg-Cpg-Arg
EXAMPLE 21: DArg-Arg-Pro-MP3 -Gly-Cpg-Ser-DCpg-Cpg-Arg
EXAMPLE 22: DArg-Arg-Pro-Hyp-Gly-Cpg-Ser-MP4-Oic-Arg
EXAMPLE 23: DArg-Arg-Pro-Hyp-Gly-Cpg-Ser-MP4-Cpg-Arg EXAMPLE 24: DArg-Arg-Pro-Hyp-Gly-MP4-Ser-DCpg-Cpg-Arg
EXAMPLE 25: DArg-Arg-MP4-Hyp-Gly-Cpg-Ser-DCpg-Cpg-Arg
EXAMPLE 26: DArg-Arg-Pro-MP4-Gly-Cpg-Ser-DCpg-Cpg-Arg
EXAMPLE 27: DArg-Arg-Pro-Hyp-Gly-Cpg-Cys-DCpg-Cpg-Arg
EXAMPLE 28: DArg-Arg-Pro-Hyp-Gly-Chg-Cys-DTic-Cpg-Arg
EXAMPLE 29: DArg-Arg-Gly-Pro-Hyp-Cpg-Ser-DTic-Cpg-Arg
EXAMPLE 30: DArg-Arg-Pro-Hyp-Gly-Cpg-Ser-DPhe-Opg-Arg
EXAMPLE 31: DArg-Arg-Pro-Hyp-Gly-Cpg-Cys-DPhe-Cpg-Arg
EXAMPLE 32: DArg-Arg-Pro-Hyp-Gly-Cpg DPhe-Cpg-Arg
EXAMPLE 33: DArg-Arg-Gly-Pro-Hyp-Cpg DChg-Chg-Arg
EXAMPLE 34: Dhq-DArg-Arg-Pro-Hyp-Gly-Cpg-Ser-DCpg-Cpg-Arg
EXAMPLE 35: Dhq-DArg-Arg-Pro-Hyp-Gly-Cpg-Ser-DCpg-Oic-Arg
EXAMPLE 36: Dhq-DArg-Arg-Pro-Hyp-Gly-Cpg-Ser-DCpg-MP3-Arg
EXAMPLE 37: Dhq-DArg-Arg-Pro-Hyp-Gly-MP3-Ser-DCpg-Oic-Arg
EXAMPLE 38: Dhq-DArg-Arg-Pro-Hyp-Gly-Cpg-Ser-DCpg-MP4-Arg
EXAMPLE 39: Dhq-DArg-Arg-Pro-Hyp-Gly-MP4-Ser-DCpg-Oic-Arg
4
EXAMPLE 40:DArg-Arg-Pro-Hyp-Gly-Cpg-Ser-DCpg- (Cpg-ps-Arg)
EXAMPLE 41: DArg-Arg-Pro-Hyp-Gly-Cpg-Cys-DChg-Oic-Arg
The following examples are synthesized, purified and analyzed in a similar fashion except that Procedure A is used throughout for coupling. When the Model 9500 synthesizer is used, coupling Procedure B is used:
EXAMPLE 42: Arg-Pro-Hyp-Gly - He - Ser-DIle - Ile-Arg
EXAMPLE 43 :
DArg-Arg-Pro-Hyp-Gly - He - Ser-DIle - Ile-Arg
EXAMPLE 44 : Arg- Pro -Hyp -Gly - Leu- Ser-DIle- Ile-Arg
EXAMPLE 45 : DArg-Arg-Pro-Hyp-Gly - Leu- Ser-DIle- Ile-Arg
BRADYKININ ANTAGONIST ACTIVITY
The bradykinin antagonists are assayed on isolated rat uterus in natural or induced estrus and on guinea pig ileum, according to the commonly accepted assay methods for bradykinin and related kinins as described by Trautschold (Handbook of Expt. Pharmacol., Vol. 25, Springer Verlag, pp. 53- 55, 1969) for inhibition of the myotropic activity of bradykinin. The inhibition potencies, are determined according to the commonly accepted manner described by Schild for antagonists of biologically active compounds (Br. J. Pharmacol.. 2..189, 1947), and expressed as pA values. In the assays, a dose- response curve is determined for the reference substance bradykinin. The dose of bradykinin which produces a half-maximal contraction of tissue is the ED dose. An amount of bradykinin equivalent to twice the ED dose is administered to the tissue 30 seconds after the start of incubation of the tissue with a dose of antagonist. Doses of antagonist are increased in the protocol until the dose of antagonist is found that causes the tissue response to a double ED 50 dose of bradykinin in the presence of antagonist to equal the response of an EDb_n(J dose of bradykinin without antagonist. The pA value represents the negative logarithm of the molar concentration of antagonist necessary to reduce the response of a double ED dose of bradykinin to that of an ED dose without antagonist. A change of one unit of pA value represents an order of magnitude change in potency. For comparison, the negative log of the dose of BK, that half-maximal contraction of the tissues, is commonly known as the pD value. The pD value for bradykinin is 7.9 on the rat uterus and 7.4 on the guinea pig ileum.
The m vivo effects of bradykinin antagonists on blood pressure in the anesthetized rat are determined according to the assay described by Roblero, Ryan and Stewart (Res. Commun. Pathol. Pharmacol. , 6.:207, 1973) . The antagonists produce inhibition of the bradykinin response when injected as a bolus admixture of bradykinin plus antagonist by either the ia or iv route of administration, or when administered as an infusion. Potencies for the antagonist in this assay are not reported quantitatively but rather are indicated qualitatively. The results of tests on compounds of the various Examples are reported in Table I.
EXAMPLES OF BRADYKININ ANTAGONIST ACTIVITY TABLE I: BIOLOGICAL ACTIVITIES OF PEPTIDE EXAMPLES
Figure imgf000024_0001
Agonist potency is given as percent of BK potency. Antagonist potency is given as pA„ and is underlined. In blood pressure assayέ indicates unquantitated antagonist potency. THERAPEUTIC APPLICATIONS OF THE BRADYKININ ANTAGONISTS
The antagonists of the invention may be used in the form of conventional pharmaceutical compositions comprising the antagonist and a pharmaceutically acceptable carrier. Such compositions may be adapted for topical, oral, aerosolized, intramuscular, subcutaneous or intravenous administration. The amount of antagonist present in such compositions will range from, for example, about 0.001 to 90.0% by weight depending on the application and mode of administration although more or less of the active component may be used. Conventional dosages will vary considerably on the basis of the intended application and mode of administration, e.g. 0.1 to 100 micrograms per kg body weight per minute are contemplated for use in the context of the septic shock. Having described the invention above, the scope thereof is defined in the following claims wherein:

Claims

WHAT IS CLAIMED IS:
1. A bradykinin antagonist peptide having a non-aromatic residue in the 5-position.
2. A bradykinin antagonist peptide according to claim 1 wherein the non-aromatic residue is aliphatic, alicyclic, aliphatic heterocyclic or substituted aliphatic heterocyclic.
3. A bradykinin antagonist peptide as in claim 1 having non-aromatic residues in the 5- and 8-positions.
4. A bradykinin antagonist peptide according to claim 3 wherein the non-aromatic residue is aliphatic, alicyclic, aliphatic heterocyclic or substituted aliphatic heterocyclic.
5. A bradykinin antagonist peptide as in claim 3 which does not contain any aromatic substituent in the 5-, 7- and 8-positions.
6. A bradykinin antagonist peptide according to claim 5 wherein the non-aromatic residue is aliphatic, alicyclic, aliphatic heterocyclic or substituted aliphatic heterocyclic.
7. A bradykinin antagonist peptide according to claim 3 which contains an aliphatic, alicyclic or substituted alicyclic amino acid residue in the 5- and 8-positions and a D-aliphatic, D-alicyclic or D-aromatic residue in the 7-position.
8. A bradykinin antagonist according to claim 7 wherein a D-aliphatic or D-alicyclic substituent is in the 7-position.
9. A bradykinin antagonist peptide according to claim 1 which is represented by the formula:
H-DArg-Arg-Pro-Hyp-Gly-Cpg-Ser-DCpg-Cpg-Arg.
10. A pharmaceutical composition comprising, as the active ingredient, a peptide according to claim 1, and a pharmaceutically acceptable carrier.
11. A method of antagonizing bradykinin in a host which comprises administering to the host an effective amount of a peptide according to claim 1.
PCT/US1993/008220 1992-09-11 1993-09-08 Bradykinin antagonists containing aliphatic amino acids in the 5-position WO1994006453A1 (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2739553A1 (en) * 1995-10-06 1997-04-11 Oreal USE OF BRADYKININE ANTAGONISTS TO STIMULATE OR INDUCE HAIR GROWTH AND / OR STOP THE HAIR LOSS
EP0836853A1 (en) * 1996-10-14 1998-04-22 Hoechst Aktiengesellschaft Use of bradykinin antagonists for the manufacture of a medicament for treating Alzheimer's disease
US5834431A (en) * 1995-09-08 1998-11-10 Cortech, Inc. Des-Arg9 -BK antagonists
US5863901A (en) * 1996-03-27 1999-01-26 Hoechst Aktiengesellschaft Use of bradykinin antagonists for the production of pharmaceuticals for the treatment of chronic fibrogenetic liver disorders and acute liver disorders

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4693993A (en) * 1985-06-13 1987-09-15 Stewart John M Bradykinin antagonist peptides

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4693993A (en) * 1985-06-13 1987-09-15 Stewart John M Bradykinin antagonist peptides

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5834431A (en) * 1995-09-08 1998-11-10 Cortech, Inc. Des-Arg9 -BK antagonists
FR2739553A1 (en) * 1995-10-06 1997-04-11 Oreal USE OF BRADYKININE ANTAGONISTS TO STIMULATE OR INDUCE HAIR GROWTH AND / OR STOP THE HAIR LOSS
WO1997013493A1 (en) * 1995-10-06 1997-04-17 L'oreal Use of bradykinin antagonists for stimulating or inducing hair growth and/or arresting hair loss
US6468972B1 (en) 1995-10-06 2002-10-22 Societe L'oreal S.A. Method to promote, stimulate and/or delay hair loss by a brady kinin antagonist
US5863901A (en) * 1996-03-27 1999-01-26 Hoechst Aktiengesellschaft Use of bradykinin antagonists for the production of pharmaceuticals for the treatment of chronic fibrogenetic liver disorders and acute liver disorders
EP0836853A1 (en) * 1996-10-14 1998-04-22 Hoechst Aktiengesellschaft Use of bradykinin antagonists for the manufacture of a medicament for treating Alzheimer's disease
US6245736B1 (en) 1996-10-14 2001-06-12 Aventis Pharma Deutschland Gmbh Use of peptidic bradykinin antagonists for the treatment and prevention of Alzheimer's disease

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