Abstract:
The substitution of the L-Pro at the 7-position with D-Phe or D-Tic and substitution of the L-Phe at the 8-position with hydroxyproline ethers and thioethers of the peptide hormone bradykinin and other additional substituted analogs of bradykinin converts bradykinin agonists into bradykinin antagonists. The invention further includes additional modifications at other positions within the novel 7- and 8-position modified bradykinin antagonists, which increase enzyme resistance, antagonist potency, and/or specificity of the new bradykinin antagonists. The analogs produced are useful in treating conditions and diseases of a mammal and human in which an excess of bradykinin or related kinins are produced or injected as by insect bites.

Description:
This application is a continuation of U.S. patent application Ser. No. 07/866,385, filed Apr. 14, 1992, abandoned, the entire contents of which are hereby incorporated, and which in turn is a continuation-in-part of U.S. patent application Ser. No. 07/687,950, filed Apr. 19, 1991 abandoned. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to compounds which are bradykinin receptor antagonists, pharmaceutical compositions and methods for using these compounds to antagonize the effects of bradykinin in mammals, including humans. More particularly, the invention relates to the substitution of the L-Pro at position 7 with D-Phe or D-Tic and substitution of the L-Phe at position 8 with hydroxyproline ether or thioether compounds and its L-configuration intermediate product which convert bradykinin agonists into antagonists and also includes additional modifications at other positions within the 7- and 8-position modified bradykinin antagonist which confer increased antagonist potency, resistance to enzymatic degradation and/or tissue specificity on the D-amino acid-containing bradykinin sequence. 
     2. Description of the Prior Art 
     Bradykinin (BK) is a nonapeptide generated as a result of the activity of kallikreins, a group of proteolytic enzymes present in most tissues and body fluids, on kininogens. Once released, kinins produce many physiological responses, including pain and hyperanalgesia by stimulating C- and A-fibers in the periphery. There is also considerable evidence that kinins contribute to the inflammatory response. 
     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, anaphylaxis, rhinitis, asthma, inflammatory bowel disease, and certain other conditions including acute pancreatitis, post-gastrectomy dumping syndrome, carcinoid syndrome, migraine, and angioneurotic edema. 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 bradykinin-induced activation of the arachidonic acid pathway which produces prostaglandins and leukotrienes, the more distal and actual mediators of inflammation. 
     In addition to its analgesic and proinflammatory effects, bradykinin is a vasodilator. Because of its ability to lower blood pressure, bradykinin has been implicated in the pathogenesis of several shock syndromes, particularly septic or endotoxic shock. Bradykinin is also a potent bronchoconstrictor in animals and asthmatic subjects and it has been implicated as a contributor to the pathogenesis of airway inflammatory conditions such as allergic asthma and rhinitis. 
     Thus a bradykinin inhibitor or bradykinin receptor antagonist is expected to possess a number of desirable biological effects in the treatment, for example, of inflammation, septic shock, asthma, burn pain, rhinitis, and allergy. 
     The search for understanding 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 caused by the production and overproduction of bradykinin, has been hindered by the lack of specific sequence-related competitive antagonists of bradykinin. 
     Several non-peptide, non-specific and non-selective antagonists of one or more of the biological activities of bradykinin have been described among compounds as diverse as analgesics and anti-inflammatory substances, which act via the prostaglandin system and not directly on bradykinin biological receptors. These are antihistamines; bradykinin-antibodies; benzodiazepine derivatives; high molecular weight ethylene oxide polymers; gallic acid esters; and serotonin inhibitors. None of these compounds or classes of compounds specifically inhibit bradykinin. 
     Heptyl esters of various amino acid-containing substances, such as single basic amino acids, the dipeptide Phe-Gly and of analogs of C- terminal peptide fragments of bradykinin (i.e., Pro-Phe-Arg) have been reported as anti-bradykinin substances. When tested in bradykinin assay systems, they prove to be weak partial agonists/antagonists, depending on the dose, with little specificity for inhibiting bradykinin action. 
     Preparations of damaged vascular tissue have been reported to respond to bradykinin analogs which lack the C-terminal arginine residue, but not to bradykinin itself, and analogs of these des-Arg(9)-bradykinins have been developed as antagonists for the non-physiological activity of bradykinin. These antagonists have no significant bradykinin-like agonist effects, nor any antagonist effect on any of the physiologically significant kinin-responding systems. Furthermore, several bradykinin analogs containing the O-methyl ether of Tyr residues at positions 5 and/or 8 have been reported to produce mixed agonist/antagonist activity on isolated uteri of galactosemic rats, but not on normal rats. 
     Other changes in the bradykinin molecule have been additions of amino acids at the N-terminal end which affect the rate of enzymatic degradation of bradykinin in vivo. 
     It has been reported that the half life of bradykinin in the systemic circulation is less than 30 seconds. Bradykinin appears to be completely destroyed (98-99% destruction) on a single passage through the pulmonary circulation as determined in an anesthetized rat by measuring the depressor effects of an agonist following intra-aortic (IA) (bypassing the pulmonary circulation) and intravenous (IV) administration. Resistance of bradykinin agonists to pulmonary kininase destruction in vivo also appears promoted by addition of single (i.e., D-Arg-, DLys-, Lys-) and double (DLys-Lys-) basic amino acid residues to the N-terminal of the bradykinin sequence. The addition of the dipeptide Lys-Lys to the N-terminal of bradykinin agonists has been reported to confer complete resistance to in vivo destruction on initial passage through the pulmonary circulation. 
     Several research groups have prepared bradykinin receptor antagonists. Stewart and Vavrek in U.S. Pat. No. 4,801,613, (which reference is incorporated in its entirety herein) disclose a series of bradykinin antagonists wherein the L-Pro at the 7-position of the peptide hormone bradykinin or other substituted analogs of bradykinin is substituted with an aromatic amino acid of the D-configuration which converts bradykinin agonists into bradykinin antagonists. The analogs produced are useful in treating conditions and diseases of a mammal and human in which an excess of bradykinin or related kinins are produced or injected as by insect bites into the body. The specific L-Pro substitutions are selected from the group consisting of D-Nal, D-PNF, D-Phe, D-Tyr, D-Pal, D-OMT, D-Thi, D-Ala, D-Trp, D-His, D-Homo-Phe, D-Phe, pCl-D-Phe (CDF), D-Phg, D-Val, D-Ile, D-Leu, and MDY. 
     In U.S. Pat. No. 4,693,993, also to Stewart and Vavrek, additional L-Pro substitution materials are disclosed. 
     Abandoned U.S. application Ser. No. 07/687,959, discloses and claims additional Bradykinin agonist materials. 
     U.S. Pat. No. 4,242,329 to Claeson et al. disclose the formation of Bradykinin-inhibiting tripeptide derivatives. A process for producing said tripeptide derivatives by synthesis and purification methods which are known in the peptide chemistry is also disclosed as well as pharmaceutical preparations comprising the tripeptide derivative. 
     Published European Patent Application No. 0 413 277 A1 discloses bradykinin antagonists as having natural or synthetic amino acids including ring-constrained heterocyclic amino acids (e.g., spiro(bicycl[2.2.1]heptan)-2,3-pyrrolidin-5-carboxylic acid) wherein the peptides were prepared using standard solid phase FMOC technology. This publication also discloses that the G position, namely position 8, may be a fragment of a heterocyclic ring system, whereby the preferred substituents on the heterocycles are: pyrrolidinyl-2-carboxylic acid, piperidinyl-2-carboxylic acid, 1,2,3-4-tetrahydroisoquinolinyl-3-carboxylic acid, cis-and-trans-deca-hydroisoquinolinyl-4-carboxylic acid, cis-exo, trans-octahydroiso-quinolinyl-2-carboxylic acid, cis-endo, cis-exo, trans-octahydrocyclopenta-[b]-pyrrolyl-2-carboxylic acid or hydroxy-prolinyl-2-carboxylic acid. 
     SUMMARY OF THE INVENTION 
     The present invention resides in the discovery that the novel compounds identified below, are potent bradykinin receptor antagonists. The compounds are useful in the treatment of various diseases including inflammatory disorders, asthma, septic shock and burn pain. Included in the invention are pharmaceutical compositions containing the inventive compounds and methods of using the compounds as bradykinin receptor antagonists. 
     More particularly, the invention relates to the modification of the sequence of the mammalian peptide hormone bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) and pharmaceutically acceptable salts thereof, at the L-Pro residue at position 7 and the L-Phe residue at position 8 in a unique manner which produces sequence-related analogues that act as specific and competitive inhibitors of the biological activities of bradykinin. The invention specifically relates to the substitution of the L-Pro at position 7 with D-Phe or D-Tic and at position 8 with a material having the formula:                           
     wherein R is selected from the group consisting of C 1 -C 6  alkyl, substituted C 1 -C 6  alkyl, C 2 -C 8  alkenyl, C 3 -C 8  cycloalkyl, C 3 -C 8  cycloalkyl substituted C 1 -C 6  alkyl, an aryl group, a substituted aryl group, an arylalkyl group, and a group of the formula R 1 NHC(o)— where R 1  is C 1 -C 6  alkyl or aryl, and where X is sulfur or oxygen; 
     and pharmaceutically acceptable salts thereof. The * can be either “D” or “L” at the carbon indicated, with the “L” being preferred. 
     More specifically, the invention relates to the formation of peptides having the formula: 
     
       
         N-A-B-C-D-E-F-G-H-I-J-Cn 
       
     
     wherein N is hydrogen; 
     A and B are independently selected from the group consisting of L-Arg, D-Arg, D-Gln, L-Gln, D-Asn, L-Asn, N-ε-acetyl-D-lysine, ε-acetyl-L-lysine, N G -p-tosyl-Arg, N G -nitro-Arg, Lys-Lys, acetyl-D-Arg, L-Citrulline, L-Lys, D-Lys, and Sar; 
     C and D are a direct bond or are independently selected from the group consisting of Pro, dehydropro, 4Hyp, Tic, Aoc, L-azetidine-2-carboxylic acid, Eac, Gly, Thz, Oic, Aib, and Ala; 
     E is a direct bond or is selected from the group consisting of Gly, Ala, Thr, and Ser; 
     F is selected from the group consisting of Phe, Thi, Leu, Ile, Tic, Oic, homoPhe, phenylGly, β-cyclohexylalanine, Nal, and Val; 
     G is a direct bond or is selected from the group consisting of Ser, Thr, 4Hyp, Gly, Val, and Ala; 
     H is selected from the group consisting of D-Phe and D-Tic; 
     I is a compound having the formula:                           
     wherein R is selected from the group consisting of C 1 -C 6  alkyl, substituted C 1 -C 6  alkyl, C 2 -C 8  alkenyl, C 3 -C 8  cycloalkyl, C 3 -C 8  cycloalkyl substituted C 1 -C 6  alkyl, an aryl group, a substituted aryl group, an arylalkyl group, and a group of the formula R 1 NHC(o)— where R 1  is C 1 -C 6  alkyl or aryl, and wherein X is sulfur or oxygen; 
     J is selected from the group consisting of Arg, Orn, Asn, Gln, N-ε-acetyl-Lys, N-8-acetyl-Orn, and Lys; 
     Cn is a hydroxyl group or a C-terminal extension selected from the group consisting of an amide, alkoxy group, an acidic, basic or neutral aliphatic, aromatic, a cyclic amino acid residue of the D- or L-configuration and a peptide extension composed of D- or L-amino acids; and pharmaceutically acceptable salts thereof. 
     A particularly preferred material is a peptide wherein: 
     N is hydrogen; 
     A and B are independently selected from the group consisting of L-Arg, D-Arg, Lys-Lys, and Lys; 
     C and D are independently selected from the group consisting of Pro, dehydroPro, and 4Hyp; 
     E is Gly; 
     F is selected from the group consisting of Phe, Thi, Leu, and β-cyclohexylalanine; 
     G is a direct bond or is selected from the group consisting of Ser, and Thr; 
     H is selected from the group consisting of D-Phe and D-Tic; 
     I is a compound having the formula:                           
     wherein R is selected from the group consisting of C 1 -C 6  alkyl, substituted C 1 -C 6  alkyl, C 2 -C 8  alkenyl, C 3 -C 8  cycloalkyl, C 3 -C 8  cycloalkyl substituted C 1 -C 6  alkyl, an aryl group, a substituted aryl group, an arylalkyl group, and a group of the formula R 1 NHC(o)— where R 1  is C 1 -C 6  alkyl or aryl, and where X is sulfur or oxygen; 
     J is selected from the group consisting of Arg and Lys; 
     Cn is a hydroxyl group; 
     and pharmaceutically acceptable salts thereof. 
     Another preferred material is a peptide wherein: 
     N is hydrogen; 
     A is D-Arg; 
     B is Arg; 
     C is Pro; 
     D is selected from the group consisting of Pro and 4Hyp; 
     E is Gly; 
     F is selected from the group consisting of Phe, Leu, and Thi; 
     G is a direct bond or is Ser; 
     H is selected from the group consisting of D-Phe and D-Tic; 
     I is a compound having the formula:                           
     wherein R is selected from the group consisting of methyl, ethyl, propyl, isobutyl, cyclohexylmethyl, alkyl, prenyl, methallyl, benzyl, phenyl, nitrophenyl, and phenylcarbamoyl, and X is sulfur or oxygen; 
     J is Arg; 
     Cn is a hydroxyl group; 
     and pharmaceutically acceptable salts thereof. 
     The invention may also include some of the intermediate compounds in the L-stereochemical configuration having the formula:                           
     wherein R is selected from the group consisting of C 1 -C 6  alkyl, substituted C 1 -C 6  alkyl, C 2 -C 8  alkenyl, C 3 -C 8  cycloalkyl, C 3 -C 8  cycloalkyl substituted C 1 -C 6  alkyl, an aryl group, a substituted aryl group, an arylalkyl group, and a group of the formula R 1 NHC(o)— where R 1  is C 1 -C 6  alkyl or aryl, and wherein X is either sulfur or oxygen; 
     R 2  is C 1 -C 6  alkyl, C 2 -C 8  alkenyl, aryl, or arylalkyl; 
     R 3  is H or a suitable amine protecting group. 
     Preferred peptides according to the invention include the following nonlimiting materials: 
     4Hyp Pro Alkyl Ethers 
     D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(Hydroxyproline trans methyl ether)-Arg 
     D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(Hydroxyproline trans ethyl ether)-Arg 
     D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(Hydroxyproline trans propyl ether)-Arg 
     D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline trans methyl ether)-Arg 
     D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline trans ethyl ether)-Arg 
     D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline trans propyl ether)-Arg 
     D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(Hydroxyproline cis propyl ether)-Arg 
     D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(Hydroxyproline cis methyl ether)-Arg 
     D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(Hydroxyproline cis ethyl ether)-Arg 
     D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline cis ethyl ether)-Arg 
     D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Phe(Hydroxyproline trans methyl ether)-Arg 
     D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Phe(Hydroxyproline trans propyl ether)-Arg 
     Pro-Pro Alkyl Ethers 
     D-Arg-Arg-Pro-Pro-Gly-Thi-Ser-D-Tic-(Hydroxyproline trans methyl ether)-Arg 
     D-Arg-Arg-Pro-Pro-Gly-Thi-Ser-D-Tic-(Hydroxyproline trans ethyl ether)-Arg 
     D-Arg-Arg-Pro-Pro-Gly-Thi-Ser-D-Tic-(Hydroxyproline trans propyl ether)-Arg 
     D-Arg-Arg-Pro-Pro-Gly-Phe-Ser-D-Tic-(Hydroxyproline trans methyl ether)-Arg 
     D-Arg-Arg-Pro-Pro-Gly-Phe-Ser-D-Tic-(Hydroxyproline trans ethyl ether)-Arg 
     D-Arg-Arg-Pro-Pro-Gly-Phe-Ser-D-Tic-(Hydroxyproline trans propyl ether)-Arg 
     Thioalkyl Ethers 
     D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(4-trans thiomethylproline)-Arg 
     D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(4-trans thioethylproline)-Arg 
     D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(4-trans thiopropylproline)-Arg 
     D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(4-trans thiopropylproline)-Arg 
     D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(4-trans thiomethylproline)-Arg 
     D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(4-trans thioethylproline)-Arg 
     Pro 4Hyp Carbamoyl Ethers 
     D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(Hydroxyproline trans phenyl carbamoyl)-Arg 
     D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline trans phenyl carbamoyl)-Arg 
     Aryl Ethers and Substituted Aryl Ethers 
     D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline cis 2-nitrophenyl ether)-Arg 
     D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline cis 4-nitrophenyl ether)-Arg 
     D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline cis phenyl thioether)-Arg 
     D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline cis phenyl ether)-Arg 
     Thiophenyl Ethers 
     D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(4-trans thiophenylproline)-Arg 
     D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(4-trans thiophenylproline)-Arg 
     Allyl Ethers 
     D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(Hydroxyproline trans allyl ether)-Arg 
     D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline trans allyl ether)-Arg 
     Cycloalkyl Substituted Alkyl Ethers 
     D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline cis cyclohexylmethyl ether)-Arg 
     Another embodiment of the invention involves a pharmaceutical composition useful as a bradykinin receptor antagonist comprising a pharmaceutical carrier and an effective amount of the novel bradykinin-type peptide. The invention also involves a process for antagonizing bradykinin receptor activity in mammals which comprises: administering to a subject an effective amount of the novel compound to antagonize bradykinin receptor activity. 
     A further embodiment involves a pharmaceutical preparation for treating local pain and inflammation from burns, wounds, cuts, rashes and other such trauma, and pathological conditions caused by the production of bradykinin or related kinins by an animal which comprises administering an effective amount of the novel bradykinin type peptide sufficient to antagonize bradykinin with a suitable pharmaceutical carrier. Another aspect of this invention involves a process for treating local pain and inflammation which comprises administering an effective amount of the pharmaceutical preparation to an animal in need thereof. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The present compounds which are bradykinin receptor antagonists have the following formula: 
     
       
         N-A-B-C-D-E-F-G-H-I-J-Cn 
       
     
     wherein N is hydrogen; 
     A and B are independently selected from the group consisting of L-Arg, D-Arg, D-Gln, L-Gln, D-Asn, L-Asn, N-ε-acetyl-D-lysine, ε-acetyl-L-lysine, N G -p-tosyl-Arg, N G -nitro-Arg, Lys-Lys, acetyl-D-Arg, L-citrulline, L-Lys, D-Lys, and Sar; 
     C and D are a direct bond or are independently selected from the group consisting of Pro, dehydropro, 4Hyp, Tic, Aoc, L-azetidine-2-carboxylic acid, Eac, Gly, Thz, Oic, Aib, and Ala; 
     E is a direct bond or is selected from the group consisting of Gly, Ala, Thr, and Ser; 
     F is selected from the group consisting of Phe, Thi, Leu, Ile, Tic, Oic, homophe, phenylGly, β-cyclohexylalahine, Nal, and Val; 
     G is a direct bond or is selected from the group consisting of Ser, Thr, 4Hyp, Gly, Val, and Ala; 
     H is selected from the group consisting of D-Phe and D-Tic; 
     I is a compound having the formula:                           
     wherein R is selected from the group consisting of C 1 -C 6  lower alkyl, substituted C 1 -C 6  alkyl, C 2 -C 8  alkenyl, C 3 -C 8  cycloalkyl, C 3 -C 8 cycloalkyl substituted C 1 -C 6  alkyl, an aryl group, a substituted aryl group, an arylalkyl group, and a group of the formula R′NHC(o)— where R′ is C 1 -C 6  alkyl or aryl and wherein X is sulfur or oxygen; 
     J is selected from the group consisting of Arg, Orn, Asn, Gin, N-ε-acetyl-Lys, N-δ-acetyl-Orn, and Lys; 
     Cn is a hydroxyl group a C-terminal extension selected from the group consisting of an amide, alkoxy group, an acidic, basic or neutral aliphatic aromatic, a cyclic amino acid residue of the D- or L-configuration and a peptide extension composed of D-or L-amino acids; and pharmaceutically acceptable salts thereof. 
     Formula 2 
     Preferred compounds are those in which: 
     N is hydrogen; 
     A and B are independently selected from the group consisting of L-Arg, D-Arg, Lys-Lys, and Lys; 
     C and D are independently selected from the group consisting of Pro, dehydropro, and 4Hyp; 
     E is Gly; 
     F is selected from the group consisting of Phe, Thi, Leu, and β-cyclohexylalanine; 
     G is a direct bond or is selected from the group consisting of Ser, and Thr; 
     H is selected from the group consisting of D-Phe and D-Tic; 
     I is a compound having the formula:                           
     wherein R is selected from the group consisting of C 1 -C 6  alkyl, substituted C 1 -C 6  alkyl, C 2 -C 8  alkenyl, C 3 -C 8  cycloalkyl, C 3 -C 8  substituted C 1 -C 6  alkyl, an aryl group, a substituted aryl group, an arylalkyl group and X is either sulfur or oxygen; 
     J is selected from the group consisting of Arg and Lys; 
     Cn is hydroxyl; 
     and pharmaceutically acceptable salts thereof. 
     Formula 3 
     Most preferred are compounds wherein: 
     N is hydrogen; 
     A is D-Arg; 
     B is Arg; 
     C is Pro; 
     D is selected from the group consisting of Pro and 4Hyp; 
     E is Gly; 
     F is selected from the group consisting of Phe, Leu, and Thi; 
     G is a direct bond or is Ser; 
     H is selected from the group consisting of D-Tic and D-Phe; 
     I is a compound having the formula:                           
     wherein R is selected from the group consisting of methyl, ethyl, propyl, isobutyl, cyclohexylmethyl, allyl, prenyl, methallyl, benzyl, phenyl, nitrophenyl, phenylcarbamoyl, and X is sulfur or oxygen; 
     J is Arg; 
     Cn is a hydroxyl group; and pharmaceutically acceptable salts thereof. 
     Formula 4 
     The inventive compositions also include the following preferred formulations: 
     A is D-Arg; 
     B is Arg; 
     C and D are selected from the group consisting of Pro and 4Hyp; 
     E is Gly; 
     F is Phe; 
     G is Ser; 
     H is a D-Tic; 
     I is a compound of the following formula:                           
     wherein R is selected from the group consisting of C 1 -C 6  alkyl, substituted C 1 -C 6  alkyl, C 1 -C 8  alkenyl, C 3 -C 8  cycloalkyl, C 3 -C 8  substituted C 1 -C 6  alkyl, an aryl group, a substituted aryl group, an arylalkyl group and a group of the formula R 1 NHC(0)-where R 1  is C 1 -C 6  alkyl or aryl, and X is either sulfur or oxygen; 
     J is Arg; 
     and pharmaceutically acceptable salts thereof. 
     Formula 5 
     Another preferred formulation is when: 
     A is D-Arg; 
     B is Arg; 
     C and D are selected from the group consisting of Pro and 4Hyp; 
     E is Gly; 
     F is Phe; 
     G is Ser; 
     H is D-Phe; 
     I is a compound having the formula:                           
     wherein R is selected from the group consisting of methyl, ethyl, propyl, phenyl, and nitrophenyl and X is sulfur or oxygen; 
     J is Arg; 
     and pharmaceutically acceptable salts thereof. 
     Formula 6 
     Another preferred formulation is when: 
     A is D-Arg; 
     B is Arg; 
     C and D are selected from the group consisting of Pro and 4Hyp; 
     E is Gly; 
     F is Phe; 
     G is Ser; 
     H is D-Tic; 
     I is a compound having the formula:                           
     wherein R is selected from the group consisting of methyl, ethyl, propyl, phenyl, and nitrophenyl and X is sulfur or oxygen; 
     J is Arg; 
     and pharmaceutically acceptable salts thereof. 
     Formula 7 
     Another preferred formulation is when: 
     A is D-Arg; 
     B is Arg; 
     C and D are selected from the group consisting of Pro and 4Hyp; 
     E is Gly; 
     F is Phe; 
     G is a direct bond; 
     H is selected from the group consisting of D-Phe and D-Tic; 
     I is a compound having the formula:                           
     wherein R is selected from the group consisting of methyl, ethyl, propyl, phenyl, and nitrophenyl and X is sulfur or oxygen; 
     J is Arg; and pharmaceutically acceptable salts thereof. 
     Formula 8 
     Another preferred formulation is when: 
     A is D-Arg; 
     B is Arg; 
     C and D are selected from the group consisting of Pro and 4Hyp; 
     E is Gly; 
     F is Phe; 
     G is Ser; 
     H is D-Tic; 
     I is a compound having the formula: 
     wherein R is methyl and X is oxygen; 
     J is Arg; 
     and pharmaceutically acceptable salts thereof. 
     Formula 9 
     Another preferred formulation is when: 
     A is D-Arg; 
     B is Arg; 
     C and D are selected from the group consisting of Pro and 4Hyp; 
     E is Gly; 
     F is Phe; 
     G is Ser; 
     H is D-Tic; 
     I is a compound having the formula:                           
     wherein R is ethyl and X is oxygen; 
     J is Arg; 
     and pharmaceutically acceptable salts thereof. 
     Formula 10 
     Another preferred formulation is when: 
     A is D-Arg; 
     B is Arg; 
     C and D are selected from the group consisting of Pro and 4Hyp; 
     E is Gly; 
     F is Phe; 
     G is Ser; 
     H is D-Tic; 
     I is a compound having the formula:                           
     wherein R is propyl and X is oxygen; 
     J is Arg; 
     and pharmaceutically acceptable salts thereof. 
     Formula 11 
     Another preferred formulation is when: 
     A is D-Arg; 
     B is Arg; 
     C and D are selected from the group consisting of Pro and 4Hyp; 
     E is Gly; 
     F is Phe; 
     G is Ser; 
     H is D-Tic; 
     I is a compound having the formula:                           
     wherein R is phenyl and X is oxygen; 
     J is Arg; 
     and pharmaceutically acceptable salts thereof. 
     Formula 12 
     Another preferred formulation is when: 
     A is D-Arg; 
     B is Arg; 
     C and D are selected from the group consisting of Pro and 4Hyp; 
     E is Gly; 
     F is Phe; 
     G is Ser; 
     H is D-Tic; 
     I is a compound having the formula:                           
     wherein R is allyl and X is oxygen; 
     J is Arg; 
     and pharmaceutically acceptable salts thereof. 
     Formula 13 
     Another preferred formulation is when: 
     A is D-Arg; 
     B is Arg; 
     C and D are selected from the group consisting of Pro and 4Hyp; 
     E is Gly; 
     F is Phe; 
     G is Ser; 
     H is D-Tic; 
     I is a compound having the formula:                           
     wherein R is methyl and X is sulfur; 
     J is Arg; 
     and pharmaceutically acceptable salts thereof. 
     Formula 14 
     Another preferred formulation is when: 
     A is D-Arg; 
     B is Arg; 
     C and D are selected from the group consisting of Pro and 4Hyp; 
     E is Gly; 
     F is Phe; 
     G is Ser; 
     H is D-Tic; 
     I is a compound having the formula:                           
     wherein R is ethyl and X is sulfur; 
     J is Arg; 
     and pharmaceutically acceptable salts thereof. 
     Formula 15 
     Another preferred formulation is when: 
     A is D-Arg; 
     B is Arg; 
     C and D are selected from the group consisting of Pro and 4Hyp; 
     E is Gly; 
     F is Phe; 
     G is Ser; 
     H is D-Tic; 
     I is a compound having the formula:                           
     wherein R is propyl and X is sulfur; 
     J is Arg; 
     and pharmaceutically acceptable salts thereof. 
     As used in the specification and claims, “alkyl” is a paraffinic hydrocarbon group which may be derived from an alkane by dropping one hydrogen from the formula such as methyl, ethyl, propyl, isopropyl, butyl, and so forth; “substituted alkyl” is a branched alkyl such as methyl butyl; “aryl” is an aromatic ring compound such as benzene, phenyl, naphthyl; “substituted aryl” is a substituted aromatic ring such as nitro substitution, and halogen substitution; and “aralkyl” is an aryl being attached through an alkyl chain, straight or branched, containing from one through six carbons, such as a phenylpropyl group. A “direct bond” is a bond which replaces a particular amino acid compound which may also be between adjacent amino acid and indicated to be absent by the term “molecule”. The phrase “a suitable amino protecting group is a group, such as BOC (t-butyloxycarbonyl-) protecting group which protects the amine moiety from reaction and which can be removed under mild conditions so as not to affect the rest of the molecule. 
     Exemplary Boc protected amino acids include the following nonlimiting materials: 
     N-Boc-L-cis-4-methoxyproline 
     N-Boc-L-cis-4-ethoxyproline 
     N-Boc-L-cis-4-(n-propoxy)proline 
     N-Boc-L-cis-4-phenylthioproline 
     N-Boc-L-trans-4-methoxyproline 
     N-Boc-L-trans-4-ethoxyproline 
     N-Boc-L-trans-4-(n-propoxy)proline 
     N-Boc-L-trans-4-cyclohexylmethoxyproline 
     N-Boc-L-trans-4-phenylthioproline 
     N-Boc-L-cis-4-(4-nitrophenyloxy)proline 
     N-Boc-L-cis-4-(2-nitrophenyloxy)proline 
     Definitions of the amino acid abbreviations used herein are as follows: 
     Arg is arginine; Ala is alanine; Aib is 2 aminoisobutyric acid, Aoc is (S,S,S)-2-azabicyclo[3.3.0]octane-3-carboxylic acid; Asn is asparagine; Eac is E-aminocarproic acid; Gln is glutamine; Gly is glycine; Ile is iscleucine; Leu is leucine; Lys is lysine; Met is methionine; Nal is beta-2-naphthylalanine; Orn is ornithine; Pro is proline; dehydropro is dehydroproline; homoPhe is homophenylalanine; 4Hyp is 4-hydroxyproline; hydroxyproline is 4-hydroxyproline; Ser is serine; Thi is beta-2-thienylalanine; Thr is threonine; Thz is thiazolidine-4-carboxylic acid; Phe is phenylalanine; Sar is sarcosine; Tic is tetrahydroisoquinoline-3-carboxylic acid; Oic is (2S, 3aS, 7aS)-octahydro-1H-indole-2-carboxylic acid; Val is valine. Furthermore, prenyl is a 3-methyl-2 butenyl radical. 
     Aoc can be prepared by the method of V. Teetz, R. Geiger and H. Gaul,  Tetrahedron Lett . (1984) 4479. Tic can be prepared by the method of K. Hayashi, Y. Ozaki, K. Nunami and N. Yoneda,  Chem. Pharm. Bull  (1983) 31,312. 
     All amino acids residues, except Gly and Sar, described in the specification are of the L-configuration unless otherwise specified. The H position must always be the D-configuration whereas the I position may be either in the D- or L-configuration with the L-preferred. The symbols and abbreviations used for amino acids, their derivatives and protecting groups, and peptides and their salts are those customarily used in peptide chemistry. (See  Biochem. J ., (1972) 126, 773, which Journal reference is hereby incorporated by reference). 
     Table I shows the general location of the amino acid groups as used herein. 
     
       
         
               
               
             
           
               
                 TABLE I 
               
               
                   
               
             
             
               
                 N - A - B - C - D - E - F - G - H - I - J - Cn 
                 (formula) 
               
               
                        Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg 
                 Bradykinin 
               
               
                         1   2   3   4   5   6   7   8   9 
                 (position 
               
               
                   
                 number) 
               
               
                   
               
             
          
         
       
     
     The synthesis of the peptides of this invention including derivation, activation, and coupling of protected amino acid residues, and their purification, and the analytical methods for determining identity and purity are included in the general body of knowledge of peptide chemistry, as described in Houben Weyl “ Methoden der Organischen Chemie ” (1974) Vol. 16, parts I &amp; II for solution-phase synthesis, and in  Solid Phase Pentide Synthesis  (1984) by Stewart and Young for synthesis by the solid-phase method of Merrifield. 
     Any chemist skilled in the art of peptide synthesis can synthesize the peptides of this invention by standard solution methods or by manual or automated solid phase methods. 
     The appropriate hydroxyproline substituents used in the 8-position are prepared by the process described in the Examples and depicted in the sequences shown below. The starting materials are commercially available and/or can be prepared by known procedures. Both the cis and trans stereoisomers can be prepared by these means and are within the scope of the present invention. 
     In Scheme II M represents sodium, potassium and other useable salts such as alkaline earth metals and alkali metals and X is oxygen or sulfur.                           
     Alternately, they also can be prepared by the method of Scheme II from commercially available starting materials.                           
     The preparation of compounds for administration in pharmaceutical preparations may be performed in a variety of well known methods known to those skilled in the art. Appropriate pharmaceutically acceptable salts within the scope of the invention are those derived from mineral acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid and sulfuric acid; and organic acids such as tartaric acid, fumaric acid, lactic acid, oxalic acid, ethylsulfonic acid, citric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like, giving the hydrochloride, sulfate, phosphate, nitrate, methanesulfonate, tartrate, benzenesulfonate, p-toluensulfonate, and the like, salt, respectively. 
     The compounds of the invention may contain an asymmetric carbon atom. Thus, the invention includes the individual stereoisomers, and the mixtures thereo. The individual isomers may be prepared or isolated by methods known in the art. 
     Therapeutic applications of the novel bradykinin antagonists include not only treatment for the production of bradykinin or related kinins by the animal but also the injection of bradykinin related peptides into an animal as a result of bites and stings. Topical application alone or in combination with subcutaneous utilization of the bradykinin antagonists of the invention can be employed to treat the effects of bradykinin-related peptides causing pain, inflammation, and swelling. 
     The therapeutic use of bradykinin antagonists of this invention for other traumatic inflammatory or pathological conditions which are known to be mediated by bradykinin or exacerbated by an overproduction of bradykinin can also be achieved. These conditions include local trauma such as wounds, burns and rashes, angina, arthritis, asthma, allergies, rhinitis, shock, inflammatory bowel disease, low blood pressure, and systemic treatment of pain and inflammation. 
     In parenteral administration of the novel compounds and compositions of the invention the compounds may be formulated in aqueous injection solutions which may contain antioxidants, buffers, bacteriostats, etc. Extemporaneous injection solutions may be prepared from sterile pills, granules or tablets which may contain diluents, dispersing and surface active agents, binders and lubricants which materials are all well known to the ordinary skilled artisan. 
     In the case of oral administration, fine powders or granules of the compound may be formulated with diluents and dispersing and surface active agents, and may be prepared in water or in a syrup, in capsules or cachets in the dry state or in a non-aqueous suspension, where a suspending agent may be included. The compounds may also be administered in tablet form along with optional binders and lubricants, or in a suspension in water or syrup or an oil or in a water/oil emulsion and may include flavoring, preserving, suspending, thickening and emulsifying agents. The granules or tablets for oral administration may be coated and other pharmaceutically acceptable agents and formulations may be utilized which are all known to those skilled in the pharmaceutical art. 
     Solid or liquid carriers can also be used. Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Liquid carriers include syrup, peanut oil, olive oil, saline, and water. Ointments and creams are prepared using various well known hydrophilic and hydrophobic bases. Topical reservoirs suitably are prepared using known polymeric materials such as various acrylic-based polymers selected to provide desired release characteristics. Suppositories are prepared from standard bases such as polyethylene glycol and cocoa butter. 
     The method of treatment according to this invention comprises administering internally or topically to a subject an effective amount of the active compound. Doses of active compounds in the inventive method and pharmaceutical compositions containing same are an efficacious, nontoxic quantity selected from the range of 0.01 to 100 mg/kg of active compound, preferably 0.1 to 50 mg/kg. Persons skilled in the art using routine clinical testing are able to determine optimum doses for the particular ailment being treated. The desired dose is administered to a subject from 1 to 6 or more times daily, orally, rectally, parenterally, topically, or by inhalation. 
     The efficacy of the inventive compounds of this invention as bradykinin receptor antagonists can be determined using the bradykinin binding and tissue assays described herein. The results of these assays demonstrate that the novel compounds are potent, selective bradykinin receptor antagonists. 
    
    
     The following examples are illustrative of preferred embodiments of methods of preparation and compounds of the invention and are not to be construed as limiting the invention thereto. 
     EXAMPLE 1 
     This Example demonstrates the preparation of N-Boc-L-cis-4-methoxyproline by Scheme I. 
     To a stirred suspension of sodium hydride (3.38 g, 5 80%, 112 mmole) [washed with hexanes, 2×20 mL] in anhydrous dimethylformamide (60 mL) was added dropwise a solution of N-Boc-L-cis-4-hydroxyproline (10.0 g, 43.0 mmole) in anhydrous dimethylformamide (60 mL) at room temperature (22° C.) under argon. After 30 min, the suspension was treated with iodomethane (20.0 g, 146 mmole) and the resultant mixture was stirred at room temperature for 24 hours. Water (100 mL) and aqueous hydrochloric acid (1N) were added until the solution was acidic to the Congo red indicator. The aqueous solution was extracted with diethyl ether (3×250 mL), the combined extracts dried over sodium sulfate, and concentrated to an oil. The crude product was used directly in the next step without purification. 
     To a stirred solution of the crude product in methanol (30 mL) was added aqueous sodium hydroxide (25 mL, 3 N, 75 mmol) at room temperature (22° C.). After 18 hours, the reaction mixture was diluted with water (35 mL) and concentrated hydrochloric acid was added to adjust the mixture to pH 10. The mixture was extracted with diethyl ether (3×55 mL) and the organic layer was discarded. The aqueous layer was further acidified to the Congo red indicator endpoint and extracted with ethyl acetate (2×250 mL, 1×100 mL). Drying with sodium sulfate and concentration gave an oil. Addition of exane caused precipitation of the product. The solids were collected, washed with 50% ethyl acetate in hexane (20 mL), and dried in vacuo at room temperature to afford the desired product (7.75 g, overall yield 73.3%): mp 119.5-121.8° C. 
     EXAMPLE 2 
     This Example demonstrates the preparation of N-Boc-L-cis-4-n-propoxyproline according to Scheme I. 
     To a stirred suspension of sodium hydride (2.86 g, 80%, 95.5 mmole) [washed with anhydrous hexane (2×20 mL)] in anhydrous dimethylformamide (60 mL) was added dropwise a solution of N-Boc-L-cis-4-hydroxyproline (8.80 g, 37.8 mmole) in anhydrous dimethylformamide (60 mL) at room temperature (22° C.) under argon. After 30 min., a solution of allyl bromide (11.46 g, 94.7 mmole) in anhydrous dimethylformamide (35 mL) was added dropwise at room temperature. After 24 hours, water (100 mL) was added followed by aqueous hydrochloric acid (1 N) until the mixture was acidic (pH 3). The aqueous solution was extracted with diethyl ether (3×160 mL), the combined extracts were dried over sodium sulfate, and concentrated to an oil. The crude product was used directly in the next step without purification. 
     To a stirred solution of the crude product in methanol (30 mL) was added aqueous sodium hydroxide (3 N, 25 mL, 75 mmol) at room temperature. After 18 hours, the reaction mixture was diluted with water (35 mL) and concentrated hydrochloric acid was added to adjust the solution to pH 10. The solution was washed with diethyl ether (2×55 mL) and the combined organics were discarded. The aqueous layer was acidified to the Congo red indicator endpoint and extracted with ethyl acetate (3×180 mL). The combined organics were dried over sodium sulfate and concentrated to an oil (9.60 g). 
     A suspension of the above product and 5% platinum on activated carbon (0.74 g) in ethyl acetate (100 mL) was shaken under 35 psi of hydrogen at room temperature. After 6.5 hours, the catalyst was removed and washed with ethyl acetate. Concentration and flash chromatography (silica gel, 20% methanol in methylene chloride) gave the desired product (8.49 g, overall yield 86.2%) as an oil: IR (neat film) cm−1 3500-2550 (broad), 2972, 2933, 2877, 1748, 1707, 1478, 1400, 1367, 1164, 1100, 1007, 900, 856: 1H NMR (300 MHz, CDCl3) ppm 0.89 (t, 3H, J=7.2 Hz), 1.45 (2×s, 9H), 1.55 (q, 2H, J=7.2 Hz), 2.21 (m, 2H), 3.40 (m, 2H), 4.04 (t, 1H, J=3.3 Hz), 4.43 (m, 1H), 8.80 (s, 1H) 
     EXAMPLE 3 
     This Example demonstrates the preparation of N-Boc-L-trans-4-n-propoxyproline according to Scheme I. 
     To a stirred suspension of sodium hydride (1.68 g, 80%, 56.0 mmole) [washed with anhydrous hexane, (2×20 mL)] in anhydrous dimethylformamide (30 mL) was added dropwise a solution of N-Boc-L-trans-4-hydroxyproline (5.0 g, 21.5 mmole) in anhydrous dimethylformamide (35 mL) at room temperature (22° C.) under argon. After 30 min, a solution of allyl bromide (5.73 g, 47.4 mmole) in anhydrous dimethylformamide (35 mL) was added dropwise at room temperature. After 24 hours, the mixture was diluted with water (10 mL) and acidified with aqueous hydrochloric acid (5 N) to pH 3. The aqueous solution was extracted with diethyl ether (2×75 mL), and with ethyl acetate (2×75 mL). The combined extracts were washed with water (2×100 mL), with brine (70 mL), and dried over sodium sulfate. Concentration gave an oil. 
     The crude product was used directly in the next step without purification. 
     A suspension of the crude product (6.0 g) and 5% platinum on activated carbon (0.97 g) in ethyl acetate (65 mL) was shaken under 35 psi of hydrogen at room temperature (22° C.). After 6.5 hours, the catalyst was removed and washed with ethyl acetate. Concentration and flash chromatography (silica gel, gradient elution with ethyl acetate in hexane (1:1) to ethyl acetate) gave N-Boc-L-trans-4-n-propoxyproline propyl ester (2.90 g) as an oil. 
     To a stirred solution of N-Boc-L-trans-4-hydroxyproline propyl ether propyl ester (2.90 g, 9.16 mmole) in ethanol (10 mL) was added aqueous sodium hydroxide (12 mL, 3 N, 36 mmol) at room temperature. After 4 hours, the reaction mixture was acidified to the Congo red indicator endpoint with aqueous hydrochloric cid (3 N), the reaction mixture was saturated with sodium chloride, and extracted with diethyl ether (4×45 mL). The combined organics were dried over sodium sulfate and concentrated to an oil. Flash chromatography (silica gel, methylene chloride: methanol: acetic acid 90:8:2) gave the desired product (2.50 g, overall. yield 42.5%) as an oil:  1 H NMR (300 MHz, CDCl3) ppm 0.91(t, 3H, J=7.5); 1.45 (2×s, 9H), 1.56 (m, 2H), 2, 28 (t, 1H, J=6.6 Hz), 2.37 (m, 1H), 3.40 (m, 2H), 3.55 (m, 2H), 4.06 (q, 1H, J=4.5 Hz), 4.38 (m, 1H), 10.54 (s, 1H). 
     EXAMPLE 4 
     This Example demonstrates the preparation of N-Boc-L-cis-4-ethoxyproline. 
     To a stirred suspension of sodium hydride (1.94 g 80% 64.8 mmol) in anhydrous dimethylformamide (100 mL) was added in small portions N-Boc-L-cis-4-hydroxyproline (6.0 g 26 mmol) at room temperature under argon. After 30 min the suspension was treated with iodoethane (5.20 mL 54.5 mmol) at room temperature. After 27 hours, the reaction mixture was acidified with aqueous hydrochloric acid solution at the Congo red indicator endpoint and saturated brine, dried over sodium sulfate, and concentrated to an oil which was used directly in the next step without purification. 
     To a stirred solution of the product in methanol (25 mL) was added aqueous sodium hydroxide solution (20 mL, 3N, 60 mmol) at room temperature. After stirring 6 hours, water was added and the mixture extracted with diethyl ether (2×20 mL). The organic extracts were discarded. The aqueous layer was acidified to the Congo red indicator endpoint and extracted with ethyl acetate (3×100 mL). The combined extracts were dried over sodium sulfate. Flash chromatography (silica gel, 15% methanol in dichloromethane) gave the desired product (5.50 g, 68.4%) as a solid: mp 53-56.2° C.; IR (KBr) cm −1  3500-2500, 1723, 1622, 1434, 1250, 1095, 897, 848, 769;  1 H NMR (300 MHz, CDCl 3 )ppm 1.18 (t, 3 H, J=6.6Hz), 1.46 (s, 9 H), 2.32 (m, 2H), 3.51 (m, 4H), 4.06 (m, 1H), 4.37 (m, 1H), 8.20 (s, 1H). 
     Dicyclohexylamine salt (recrystallized from heptane): mp 161-162.4° C.; [α] 21.5   D =−33.5 (c=0.98, methanol). Anal. Calcd for C 24 H 44 N 2 O 5  (440.62 g/mol): C, 65.42;; H, 10.07; N, 6.36. Found: C, 65.32; H, 10.05; N, 20 6.37. 
     EXAMPLE 5 
     This example demonstrates the preparation of N-Boc-L-trans-4-methoxyproline. 
     A stirred suspension of sodium hydride (1.43 g, 80%, 47.7 mmol) [washed twice with hexanes] in a mixture of anhydrous N,N-dimethylformamide (15 mL) and anhydrous tetrahydrofuran (40 mL) at 5° C. under argon was added N-Boc-L-trans-4-hydroxyproline (5.00 g, 21.6 mmol). When the gas evolution had subsided (ca. 10 min), iodomethane (3.40 mL, 54.1 mmol) was added at 5° C. After 24 hours at room temperature, the suspension was diluted with water (30 mL) and acidified to the Congo red indicator endpoint with aqueous hydrochloric acid (1N) and extracted with ethyl acetate (4×100 mL). The combined organics were washed with aqueous sodium thiosulfate, with water, with brine, and dried (magnesium sulfate) Concentration gave a yellow oil which was used in the next step without purification. 
     To a stirred solution of the oil in water (25 mL) and 2-propanol (8 mL) was added aqueous potassium hydroxide (16.5 mL, 2.0 N, 33 mmol). After 5 days at room temperature, the mixture was diluted with water (10 mL) and extracted with diethyl ether (2×50 mL). The combined organics were back-extracted with half-saturated aqueous potassium bicarbonate (20 mL) and discarded. The combined aqueous layers were cooled to 5° C., acidified to pH 4 with citric acid, saturated with sodium chloride, and extracted with ethyl acetate (4×50 mL). The combined ethyl acetate extracts were dried (sodium sulfate) and concentrated to an oil. Flash chromatography (silica gel, 91:8:1 chloroform: methanol: acetic acid) followed by extensive drying in vacuo gave the desired product as a slightly yellow syrup (4.90 g, 92% overall)): IR (KBr) cm −1  2977, 2933, 1746 (sh), 1697, 1417, 1368, 1254, 1162, 1098:  1 H NMR (300 MHz, CDCl 3 ) ppm 1.42 &amp; 1.47 (2×s, 9H total), 2.10 (m, 1H), 2.24 (m, 1H), 3.33 (s, 3H), 3.60 (m, 2H), 4.00 (m, 1H), 4.30 &amp; 4.42 (2×m, 1H total), 10.27 (br s, 1H). 
     Dicyclohexylammonium salt (recrystallized from n-heptane): mp 126-128° C.; [α] 22.5   D =−30.5 (c=1.02, methanol). Anal. for C 23 H 42 N 2 O 5  (426.60 g/mol): C, 64.76; H, 9.92; N, 6.57. Found: C, 64.68; H, 9.96; N, 6.53. 
     Cyclohexylammonium salt (recrystallized from ethyl acetate): mp 155-158° C.; {α] 22.5   D =−38.7 (c=1.01, methanol). Anal, Calcd for C 17 H 32 N 2 O 5  (344.45 g/mol): C, 59.28; H, 9.36; N, 8.13. Found C, 59.02; H, 9.38; N, 8.09. 
     EXAMPLE 6 
     This Example demonstrates the preparation of N-Boc-D-trans-4-phenylthioproline according to Scheme II. 
     To a stirred suspension of hexane washed sodium hydride (3.06 g, 80%, 38.1 mmol) in anhydrous tetrahydrofuran (95 mL) was added dropwise thiophenol (4.50 mL, 43.7 mmol) at room temperature (22° C.) under argon. After 1 hour, the mixture was treated with N-Boc-D-cis-4-(p-toluenesulfonyloxy)proline (5.00 g, 12.5 mmol) at room temperature. The resultant mixture was heated to reflux for 8 hours. After cooling to room temperature, the mixture was acidified to the Congo red indicator endpoint with aqueous hydrochloric acid. The solution was extracted with ethyl acetate (4×80 mL) and the combined extracts were dried over sodium sulfate. Concentration gave an oil which was used directly in the next step without purification. 
     To a stirred solution of the crude N-Boc-D-trans-4-phenylthioproline methyl ester in methanol (20 mL) at room temperature was added a solution of sodium hydroxide (3N, 18 mL). After two days at room temperature, water (30 mL) was added and the mixture was extracted with diethyl ether (3×45 mL). The combined organics were discarded and the aqueous layer was acidified with aqueous hydrochloric acid (5 N) to the Congo red indicator endpoint. The aqueous layer was extracted with ethyl acetate (3×110 mL) and the combined extracts were dried over sodium sulfate. Concentration followed by flash chromatography (silica gel, methylene chloride/methanol/acetic acid 90:8:2) gave N-Doc-D-trans-4-phenylthio-proline (3.64 g, 79.3%) as an oil: IR (neat film) cm −1  3300-2500, 1749, 1702, 1583 (w), 1415, 1398, 1368, 1164, 743;  1 H NMR (300 MHz, CDCl 3 ) ppm 1.45 &amp; 1.48 (2×s, 9H), 2.31 (m, 1H), 3.44 (m, 1H), 3.76 (m, 2H), 4.43 (m, 1H), 7.37 (m, 3H), 7.42 (m, 2H), 9.77 (s, 1H), 
     EXAMPLE 7 
     This Example demonstrates the preparation of N-Boc-L-trans-4-ethoxyproline. 
     To a stirred suspension of hexane washed scdium hydride (1.56 g, 80%, 51.9 mmol) in anhydrous tetrahydrofuran (100 mL) was added in small portions N-Boc-L-trans-4-hydroxyproline (6.00 g, 25.9 mmol) at room temperature and under argon. After 1 hour, the suspension was treated with iodoethane (4.15 mL, 51.9 mmol) at room temperature. The reaction mixture was heated to reflux for 3 hours then cooled to room temperature and stirred overnight. The reaction mixture was diluted with water and extracted with hexane (30 mL). The hexane extract was discarded. The aqueous layer was acidified with concentrated hydrochloric acid to the Congo red indicator endpoint and extracted with ethyl acetate (3×120 mL). The combined extracts were dried over sodium sulfate. Flash chromatography (silica gel, methanol: dichloromethane: acetic acid 10:90:1) gave the desired product (4.98 g, 74.2%) as a solid: mp 48.5-51.2° C.; IR (KBr) cm −1  3500-2600, 1738, 1640, 1434, 1367, 1244, 1172, 1100, 771;  1 H NMR (300 MHz, CDCl 3 ) ppm 1.20 (t, 3 H, J=6.9 Hz), 1.42 &amp; 1.48 (2×s, 9 H), 2.25 (m, 2H), 3.51 (m, 4H), 4.08 (m, 1H), 4.35 (t, 1/2H, J=7.8 Hz), 4.44 (m, 1/2H), 9.06 (s, 1H). 
     Dicyclohexylamine salt (recrystallized from heptane): mp 128.5-130.5° C.; [α] 21.5   D =−30.2 (c=1.02, methanol). Anal. Calcd for C 24 H 44 N 2 O 4  (440.62 g/mol): C, 65.42; H, 10.07; N, 6.36. Found C, 65.31; H, 10.02; N, 6.38. 
     EXAMPLE 8 
     This Example demonstrates the preparation of N-Boc-L-cis-4-phenylthioproline. 
     To a stirred suspension of hexane washed sodium hydride (1.61 g, 80%, 53.6 mmol) in anhydrous tetrahydrofuran (120 mL) was added thiophenol (6.30 mL, 61.3 mmol) dropwise at room temperature under argon. After 1 hour, the mixture was treated with N-Boc-L-trans-4-(p-toluenesolfonyloxy)proline methyl ester (5.00 g, 12.5 mmol) at room temperature. The resultant mixture was heated to reflux for 7.5 hours, then cooled to room temperature and stirred overnight. The mixture was acidified to the Congo red indicator endpoint with aqueous hydrochloric acid and the layers were separated. The aqueous layer was extracted with ethyl acetate (3×100 mL) and the combined organics were dried over sodium sulfate. Concentration gave an oil which was used directly in the next step without purification. 
     To a stirred solution of the crude N-Boc-L-cis-4-phenylthioproline methyl ester in methanol (25 mL) at 5° C. was added aqueous sodium hydroxide (15 mL, 3 N, 45 mmol). The mixture was allowed gradually to warm to room temperature. After 18 hours at room temperature, water was added and the mixture was extracted with hexane (2×25 mL). The combined organics were discarded and the aqueous layer was acidified with aqueous hydrochloric acid (5 N) to the Congo red indicator endpoint. The aqueous layer was extracted with ethyl acetate (3×120 mL) and the combined extracts dried over sodium sulfate. Concentration followed by flash chromatography (silica gel, dichloromethane/methanol/acetic acid 90:10:1) gave the desired product (5.60 g, 98.8%) as a white , hygroscopic solid: mp 76-79.5° C.; IR (neat film) cm −1 3500-2500, 1699 (br), 1584 (w), 1478, 1416, 1159, 743, 691; H NMR (300 MHz, CDCl 3 )ppm 1.42 &amp; 1.47 (2×s, 9H), 2.22 (m, 1H), 2.65 (m, 1H), 3.38 (m, 1H), 3.66 (m, H), 3.92 (m, 1H), 4.34 (m, 1H), 7.29 (m, 3H), 7.42 (d, 2H, J=6.6 Hz), 9.45 (s, 1H). 
     Dicyclohexylamine salt (recrystallized from acetonitrile): mp 168-169.2° C.; [α] 23   D =−44.2 (c=1.03, methanol). Anal. Calcd for C 28 H 44 N2O 4 S (504.73 g/mol): C, 66.63; H, 8.79; N, 5.55. Found C, 66.56; H, 8.82; H, 15 5.53. 
     EXAMPLE 9 
     This Example demonstrates the preparation of N-Boc-L-trans-4-phenylthioproline. 
     To a stirred suspension of hexane washed sodium hydride (1.35 g, 80%, 45.1 mmol) in anhydrous tetrahydrofuran (90 mL) was added thiophenol (3.90 mL, 38.0 mmol) Boc-L-cis-4-(p-toluenesulfonyloxy)proline methyl ester (10.0 g, 25.0 mmol) at room temperature. The resultant mixture was heated to reflux for 5 hours, then stirred at room temperature overnight. After 16 hours, the mixture was diluted with water and acidified to the Congo red indicator endpoint with aqueous hydrochloric acid (SN). The solution was extracted with thyl acetate (3×100 mL) and the combined extracts ried over sodium sulfate. Concentration gave an oil hich was used directly in the next step without purification. 
     To a stirred solution of the crude N-Boc-L-trans-4-phenylthioproline methyl ester in methanol (30 mL) at room temperature was added aqueous sodium hydroxide (18.0 mL, 3N, 54 mmol). After 18 hours, water was added and the mixture was extracted with diethyl ether (3×20 mL). The combined organics were discarded and the aqueous layer was acidified with aqueous hydrochloric acid (5 N) to the Congo red indicator endpoint. The aqueous layer was extracted with ethyl acetate (4×120 mL) and the combined extracts dried over sodium sulfate. Concentration gave a yellow oil which was used in the next step without purification. 
     The oil was dissolved in acetonitrile at room temperature and treated with cyclohexylamine (3.30 mL, 28.8 mmol). The precipitated solid was recrystallized in the same solvent. The crystalline product was dissolved in water and acidified with aqueous hydrochloric acid (5 N) to the Congo red indicator endpoint. The aqueous layer was extracted with ethyl acetate (3×120 mL) and the combined extracts dried over sodium sulfate. 
     Concentration gave N-Boc-L-trans-4-phenylthioproline (6.85 g, 85%) as a foam: IR (film) cm −1  3300-2500, 1749, 1702, 1583, 1415, 1398, 1368, 1164, 743;  1 H NMR (300 MHz, CDCl 3 ) ppm 1.45 &amp; 1.48 (2×s, 9H), 2.31 (m, 2H), 3.44 (m, 1H), 3.76 (m, 2H), 4.43 (m, 1 H), 7.36 (m, 3H), 7.42 (m, 2H), 9.77 (s, 1H); [α] 23.5   D =−26.9 (c 2.04, methanol). 
     Dicyclohexylamine salt (recrystallized from acetonitrile): mp 164.5-165.8° C.; [α] 23.5   D =−16.0 (c 1.00, methanol). Anal. Calcd for C 28 H 44 N 2 O 4 S (504.73 g/mol): C, 66.64; H, 8.79; N, 5.55. Found: C, 66.65; H, 8.81; H, 5.57. 
     Cyclohexylamine salt (recrystallized from acetonitrile): mp 170-172.5° C.; [α] 23.5   D =−18.5 (c 1.02, methanol). Anal. Calcd for C 22 H 34 N 2 O 4 S (422.58 g/mol): C, 62.53; H, 8.11; N, 6.63. Found: C, 62.52; H, 8.13; N, 6.62. 
     EXAMPLE 10 
     This Example demonstrates the preparation of (2S, 4R)-N-(tert-Butoxycarbonyl)-4-O-(phenylcarbamoyl)proline. 
     To a stirred solution of N-Boc-L-trans-hydroxyproline methyl ester (4.05 g, 16.5 mmol) and 4-dimethylaminopyridine (0.11 g, 0.89 mmol) in CHCl 3  (30 mL) was added phenyl isocyanate (1.82 mL, 16.7 mnol) at room temperature. After 21 hours, the mixture was washed with aqueous HCl (10 mL, 0.5 N) and dried (MgSO 4 ). Concentration and drying in vacuo gave (2S, 4R)-N-Boc-4-O-(phenylcarbamoyl)proline methyl ester (6.00 g, 100%) as white solids: mp 129-131° C. 
     The a stirred suspension of this ester (5.00 g, 14.4 mmol) in MeOH (20 mL) and water (5 mL) was added aqueous NaOH (5.0 mL, 3 N, 15 mmol). After 20 hours at room temperature, additional aqueous NaOH (1.0 mL, 3N, 3.0 mmol) was added. After an additional 4 hours, the mixture was extracted with EtOAc (3×30 mL). The organics were discarded and the aqueous layer was acidified to the Congo red indicator endpoint with concentrated HCl at 5° C. The mixture was saturated with NaCl and extracted with EtOAc (4×50 mL). The combined organics were dried (Na 2 SO 4 ) and concentrated to give white solids which were purified by flash chromatography (silica gel, 90:10:1 CH 2 Cl 2 :MeOH:HOAc) to afford the desired product (3.66 g, 76%) as white solids: mp 161-163° C.; IR (KBr) cm −1  3430, 3247, 1730, 1686, 1607, 1550, 1445, 1419, 1226, 1159, 1069, 753;  1 H NMR (300 MHz, CDCl 3 ) δ 1.44 &amp; 1.47 (2×s, 9H), 2.38 (m, 1H), 2.49 (m, 1H), 3.70 (m, 2H), 4.41 (m, 1H), 5.33 (m, 1H), 7.07 (m, 2H), 7.30 (m, 3H), 7.38 (br s, 1H), 9.67 (br s, 1H); [α] 22.5   D =−38.9 (c=1.05, MeOH). Anal. Calcd for C 17 H 22 N 2 O 6 . 0.75 H 2 O (363.88 g/mol): C, 56.11; H, 6.51; N, 7.70. Found: C, 56.20 &amp; 56.13; H, 6.49 &amp; 6.51; N, 7.75. 
     General Procedure for Synthesis of Nitrophenyl Ethers of Hydroxyproline 
     To a warm (40°-60° C.) solution of powdered KOH (8.69 g, 158 mmol) in absolute ethanol (240 mL) was added a solution of 2- or 4-nitrophenol in 120 mL of acetone. 
     The resulting suspension was stirred vigorously and heated to reflux. At reflux a solution of Boc-4Hyp(Tosyl)-OMe (30.0 g, 63.2 mmol) in 120 mL of acetone was added dropwise during one hour. The mixture was allowed to stir at reflux for the number of days given below for each specific compound. At the end of this time, the precipitate which had formed was filtered off and washed with acetone. The filtrate and washing were evaporated at reduced pressure and the residue from evaporation was diluted with 750 mL of water. The aqueous suspension was extracted three times with 200 mL portions of CH 2 Cl 2 . The extracts were combined and splashed three times with 200 mL portions of 5% aqueous NaOH. The CH 2 Cl 2  solution was washed once with 200 mL of saturated aqueous NaHCO 3 , washed with 200 mL of brine, dried over MgSO 4 , filtered, evaporated under reduced pressure, and stored in vacuo. 
     The compounds below were synthesized using the general procedure but with appropriately scaled amounts of reagents. 
     EXAMPLE 11 
     This Example demonstrates the preparation of N-Boc-L-cHyp(2-nitrophenyloxy)-OMe 
     From 2.0 g (3.0 mmol) of N-Boc-L-cHyp(Tosyloxy)-OMe and 2.5 equivalents each of KOH (0.42 g, 7.5 mmol) and 2-nitrophenol (1.04 g, 7.5 mmol) at reflux for 6 days was obtained 1.16 g (82%) of N-Boc-L-cHyp(2-nitrophenyloxy)-OMe as an oil after purification by column chromatography on silica gel with ethyl acetate. The purified nitrophenyl hydroxyproline ether was about 60% ethyl and 40% methyl ester, due to the long reaction time. The two esters were not separable. 
     IR(neat): v 3583, 3499, 3978, 1751, 1700, 16.07, 5.27, 1484, 1401, 1365, 1281, 1255, 1201, 1170, 1071, 1067, 905, 851, 771, 746, 661 Cm −1 . 
     NMR(CDCl 3 ): δ 1.214-1.281 (m, 2.5H), 1.415-1.455 (2s, 9H), 2.551-2.590 (m, 2H), 3.701-3.852 (m, 3.2H), 4.086-4.449 (m, 4H), 6.928-6.956 (d, 1H, J=8.30 Hz), 7.045 (m, 1H), 7.486-7.510 (m, 1H), 7.782-7.809 (d, 1H, J=8.06). 
     EXAMPLE 12 
     This Example demonstrates the preparation of N-Boc-L-cHyp(4-nitrophenyloxy)-OMe 
     Using N-Boc-L-tHyp(Tosyloxy)-OMe (5g, 10.5 mmol), powdered KOH (1.75 g, 26.5 mmol) and 4-nitrophenol (3.66 g, 26.3 mmol) after 4 days at reflux, 2.52 g (65%) of the corresponding 4-nitrophenyl ether was obtained by column chromatography (silica ge, ethyl acetate/hexane mixtures) and crystallization of the appropriate fractions from ethyl acetate/hexane (1:1). An alternate method is to triturate the oily crude and pentane several times [hexane doesn&#39;t work] and then add a very, small amount of ethyl acetate to solidify the product. The filtered, solid product may than be crystallized as above. Elemental analysis for C 17 H 22 N 2 O 7  (366.324 g/mmol): Calculated C, 55.74; H, 6.05; N, 7.65. Found C, 55.71; H, 6.05; N, 7.65. 
     IR(neat): V 3111, 3080, 2978, 1756, 1733, 1694, 1607, 1594, 1512, 1494, 1399, 1363, 1337, 1260, 1198, 1173, 1160, 1121, 1108, 1062, 962, 900, 890, 846, 751, 689, 648 cm −1 . 
     EXAMPLE 13 
     This Example demonstrates the preparation of N-Boc-D-tHyp(2-nitrophenyloxy)-OMe. 
     From N-Boc-D-cHyp(Tosyloxy)-OMe (5.27 mmol, 2.50 g), powdered KOH (13.2 mmol, 0.74 g), and 4-nitrophenol (13.2 mmol, 1.83 g), held at reflux for 6 days, the 4-nitrophenyl ether of D-trans-hydroxyproline methyl ester (0.39 g, 20%, m.p. 154-155° C.) was obtained after purification by column chromatography (silica gel, ethyl acetate/hexane mixtures). Impure fractions (1.14 g) accounted for the low yield relative to L-hydroxyproline derivatives above. Elemental analysis for C 17 H 22 N 2 O 7 : Calculated C, 55.74; H, 6.05; N, 7.65. Found: Calculated C, 55.71; H, 6.08; N, 7.69. 
     EXAMPLE 14 
     This Example demonstrates the preparation of N-Boc-D-tHyp(4-nitrophenyloxy)-OMe. 
     From N-Boc-D-cHyp(Tosyloxy)-OMe (5.27 mmol, 2.50 g), powdered KOH (13.2 mmol, 0.74 g), and 2-nitrophenol (13.2 mmol, 1.83 g), held at reflux for 6 days, the 2-nitrophenyl ether of D-trans-hydroxyproline methyl ester (0.29 g, 14%) was obtained. Purification by column chromatography (silica ge, ethyl acetate/hexane mixtures) yielded no pure fractions. All fractions bearing product were combined and purified in three equal portions using the chromatotron (4 mm silica gel plates) and a mixture of ethyl acetate/CH 2 Cl 2 /hexane (2:9:9:) which was found to effect separation on TLC plates. The mixture of ethyl acetate/CH 2 Cl 2  (2:9) will be referred to as mixture A or simply A. 
     Chromatotron separation procedure: A sample of the impure product (1.0 g) was dissolved in A and applied to the chromatotron plate wetted and equilibrated with A/hexane (1:30) followed by a hexane wash. While monitoring the migration of the impurities (fast bands) vs. product (slowest band) by UV light, the amount of A was gradually increased in the mixtures by reducing the amount of hexane in increments of 5 parts. The volume of each mixture was 200 mL. Fractions of 5-10 mL were collected when the product band seemed to be eluting. When the ratio of A to hexane reached 1:10, 200 mL of 1:9 was applied and the plate was polarized with ethyl acetate/CH 2 Cl 2 /hexane (1:4:4). The fractions bearing pure product were concentrated to a yellow oil which solidified several days later after the side of the glass container was scratched. 
     Elemental analysis for C 17 H 22 N 2 O 7 : Calculated C, 55.74; H, 6.05; N, 7.65. Found: Calculated C, 55.67; H, 6.12; H, 7.50. 
     IR(neat): V 3114, 3083, 2978, 2933, 1749, 1700, 1609, 1594, 1515, 1497, 1401, 1368, 1342, 1257, 1209, 1162, 1129, 1113, 1072, 905, 849, 753, 692, 645, 609 cm −1 . 
     NMR(CDCl 3 : δ1.484-1.479 (2s, 9H), 2.518 (br s and m, 2H total), 3.672-3.886 (m, 5H), 4.451-4.488 (dd, 0.6H, J A =2.51 Hz, J B =8.48 Hz), 4.588 (t, 0.4H, J=8.56 Hz), 5.005-5.018 (m, 2H), 6.854-6.881 (d, 1H, J=8.31 Hz), 8.180-8.207 (d, 2H, J=8.08 Hz). 
     EXAMPLE 15 
     This Example demonstrates the preparation of N-Boc-(2S, 3aS, 7aS)-Octahydro-1H-indole-2-carboxylic Acid (Oic). 
     A mixture of (S)-indoline-2-carboxylic acid (10.01 g, 60.73 mmol) and 10% platinum on activated carbon (0.57 g) in aqueous hydrochloric acid (150 Ml, 1 N, 150 mmol) and ethanol (20 Ml) was shaken in a Parr bottle under 45 psi of hydrogen at room temperature (22° C.). After 20 hours, the mixture was filtered and the solids washed with methanol. The combined filtrates were concentrated to 50 Ml and the solution treated with sodium carbonate (9.65 g, 91 mmol) di-tert-butyl dicarbonate (17.5 Ml, 77.4 mmol) and 20 Ml of dioxane. The mixture was stirred for 18 hours, diluted with water (50 Ml), and the mixture extracted with ethyl ether (3×30 Ml). The aqueous layer was decolorized with charcoal, acidified to the Congo red indicator endpoint with concentrated hydrochloric acid, saturated with sodium chloride and extracted with ethyl acetate (5×50 Ml). The combined extracts were dried (sodium sulfate) and the solvent removed to give a white foam. Recrystallization from heptane gave the carboxylic acid (11.51 g, 70%) as white crystals: mp 129-131° C. 
     General Procedure for Automated Peptide Synthesis: 
     Preparation of D-Arg-Arg-Pro-4Hyp-Gly-Thi-D-Tic-(hydroxyproline cis propyl ether)-Arg 
     The peptide was synthesized employing t-Boc chemistry on a solid phase synthesizer (Milligen Biosearch 9600 Peptide Synthesizer). Boc-Arg(Tos)-PAM resin (Applied Biosystems) (PAM=phenylacetamidomethyl), 0.25 g, with a resin substitution of 0.62 mmol Arg/gram of resin, was placed in the reaction vessel and subjected to Procedure A for the coupling of Boc-Oic. Commercially available amino acids were purchased from Bachem Bioscience. Volumes of reagents and solvents were approximately 20 ml/gram of resin. 
     Procedure A: 
     1. Deprotection: Removal of the t-butyloxycarbonyl-protecting group (Boc) was achieved by treatment of the resin with deblocking reagent (trifluoroacetic acid (TFA)/anisole/dichloromethane(DCM) 45:2.5:52.5 v/v containing 1 mg/Ml of indole), two times for one minute and once for twenty minutes. The resin was then washed with DCM several times, followed by neutralization with base [10% diisopropylethylamine (DIEA) in DCM], three times for one minute. The resin was subsequently washed with DCM and dimethylformamide (DMF). 
     2. Coupling: All couplings and recouplings were mediated in the same manner. Boc-Oic (1.47 mmol, 0.4 M in DMF) was mixed with one equivalent of diisopropylcarbodiimide (DIPCDI) (1.47 mmol, 0.4 M in DCM) for a two minute activation period prior to coupling with the resin. The mixture was added to the reaction vessel containing the resin and mixed for two hours. Coupling efficiency of the amino acid to the growing peptide chain on the resin was checked. Incomplete coupling of an amino acid resulted in a recoupling step. Recoupling involved washing the resin-peptide three times for one minute with base followed by DCM and DMF. Amino acid activation with DIPCDI with addition to the peptide-resin was repeated and allowed to mix an additional two hours. After a successful coupling the peptide-resin was washed several times with DCM. 
     3. Capping: The growing peptide chain was capped on the α-amino group by acetylation with 1-acetylimidazole (0.3 M in DMF) at the end of each coupling or recoupling. The resin was washed three times with base followed by DCM and DMF. The resin was treated with capping reagent for 30 minutes and then washed with DMF. 
     Procedure B: 
     The N-terminal protecting group was removed by the following procedure: 
     Terminal deprotection: Following the capping of the final amino acid to be added to the growing peptide chain, the peptide-resin was treated with deblocking reagent (TFA/anisole/DCM) twice for one minute and once for 20 minutes. The resin was washed with DCM followed by methanol and then dried by a stream of inert gas. 
     The following amino acids were added to the growing peptide chain according to the listed programs: Boc-D-Phe (A), Boc-Ser(Bzl) (A), Boc-Thi (A), Boc-Gly (A), Boc-Hyp(Bzl) (A), Boc-Pro (A), Boc-Arg(Tos) (A), Boc-D-Arg(Tos) (A),(B). This yielded 0.481 g of protected peptide-resin as the TFA salt. 
     HF Cleavage: The peptide-resin (0.481 g) was suspended in 5 Ml of liquid anhydrous HF (ratio of 10 Ml HF/g resin) containing 0.48 Ml of anisole at −70° C. and stirred for 60 minutes at 0° C. The HF was removed by a stream of nitrogen gas followed by vacuum (water aspirator). The resin was washed three times with 30 Ml of ethyl ether and dried under high vacuum for 30 minutes. The peptide was extracted with distilled deionized water (200 Ml) and the solution was lyophilized to give 176 mg of crude deprotected peptide. 
     Purification: The peptide was purified on a reverse phase C-18 (2×25 cm) Vydac HPLC column using a gradient of 0.1% TFA/H2O and acetonitrile (0.1% TFA) to give 53 mg of purified deprotected peptide. 
     Analysis: Purified peptide was characterized by amino acid analysis and gave the following results: Arg, 2.9 (3.0); Ser, 0.92 (1.0); Thi, 1.09 (1.0); Gly, 1.0 (1.0). 
     The peptide was also characterized by mass spectrometry (JEOL HX110/110 FAB) [M+H] obsd 1308.7, [M+H] calcd 1308.6. 
     EXAMPLE 16 
     Using the method of Example 15, the peptide D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(hydroxyproline cis methyl ether)-Arg was prepared. Purified peptide was characterized by amino acid analysis and gave the following results: Arg, 3.38 (3.0); Ser, 0.84 (1.0); Thi, 1.14 (1.0); Gly, 1.0 (1.0). The peptide was also characterized by mass spectrometry (JEOL HX110/110 FAB) [M+H] obsd 1280.7, [M+H] calcd 1280.6 
     EXAMPLE 17 
     Using the method of Example 15, the peptide D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(Hydroxyproline trans propyl ether)-Arg was prepared from the appropriate amino acids. Purified peptide was characterized by amino acid analysis and by mass spectrometry (JEOL HX110/110 FAB). 
     EXAMPLE 18 
     Preparation of D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline cis 2-nitrophenyl ether)-Arg 
     Using the method of Example 15, the peptide D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline cis 2-nitrophenyl ether)-Arg was prepared. Purified peptide was characterized by amino acid analysis and gave the following results: Arg, 2.57 (3.0); Ser, 1.01 (1.0); Phe, 0.94 (1.0); Gly, 1.0 (1.0). The peptide was also characterized by mass spectrometry (JEOL HX110/110 FAB) [M+H] obsd 1381.77, [M+H] calcd 1381.7. 
     EXAMPLE 19 
     Preparation of D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline cis 4-nitrophenyl ether)-Arg. 
     Using the method of Example 15, the peptide D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline cis 4-nitrophenyl ether)-Arg was prepared. Purified peptide was characterized by amino acid analysis and gave the following results: Arg, 3.15 (3.0); Ser. 0.94 (1.0); Phe, 0.98 (1.0); Gly, 1.0 (1.0). The peptide was also characterized by mass spectrometry (JEOL HX110/110 FAB) [M+H] obsd 1381.98, [M+H] calcd 1381.7. 
     EXAMPLE 20 
     Preparation of D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline cis ethyl ether)-Arg. 
     Using the method of Example 15, the peptide D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline cis ethyl ether)-Arg was prepared. Purified peptide was characterized by amino acid analysis and gave the following results: Arg, 3.15 (3.0); Ser, 0.87 (1.0); Phe, 1.05 (1.0); Gly, 1.0 (1.0). The peptide was also characterized by mass spectrometry (JEOL HX110/110 FAB) [M+H] obsd 1288.17, [M+H] calcd 1288.68. 
     EXAMPLE 21 
     Preparation of D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline cis cyclohexylmethyl ether)-Arg. 
     Using the method of Example 15, the peptide D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline cis cyclohexylmethyl ether)-Arg was prepared. Purified peptide was characterized by amino acid analysis and gave the following results: Arg, 2.83 (3.0); Ser, 0.98 (1.0); Phe, 1.03 (1.0); Gly, 1.0 (1.0). The peptide was also characterized by mass spectrometry (JEOL HX110/110 FAB) [M+H] obsd 1357.08, [M+H] calcd 1356.7. 
     EXAMPLE 22 
     Preparation of D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline cis phenyl thioether)-Arg. 
     Using the method of Example 15, the peptide D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline cis phenyl thioether)-Arg was prepared. Purified peptide was characterized by amino acid analysis and gave the following results: Arg, 2.51 (3.0); Ser, 0.78 (1.0); Phe, 1.0 (1.0). The peptide was also characterized by mass spectrometry (JEOL HX110/10 FAB) [M+H] obsd 1352.73, [M+H] calcd 1352.7. 
     EXAMPLE 23 
     Preparation of D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline cis phenyl ether)-Arg. 
     Using the method of Example 15, the peptide D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline cis phenyl ether)-Arg was prepared. Purified peptide was characterized by amino acid analysis and gave the following results: Arg, 2.69 (3.0); Ser, 0.89 (1.0); Phe, 1.05 (1.0); Gly, 1.0 (1.0). The peptide was also characterized by mass spectrometry (JEOL HX110/110 FAB) [M+H] obsd 1336.79, [M+H] calcd 1336.7. 
     Bradykinin Binding Procedure 
     Binding of  3 H-Bradykinin was preformed using the method of D. C. Manning, R. Vavrek, J. M. Stewart, and S. H. Synder,  J. Pharmacol. Exp. Ther ., (1986),237 504. The tissues used in the binding assay were terminal ileum from male Hartley guinea pigs (150-350 g). After dissection, tissues were placed in 20 vol of ice-cold buffer A (25 Mm TES containing 0.2 g/L of 1,10-phenanthroline adjusted to Ph 6.8 with ammonium hydroxide) and homogenized using a Ploytron Tissumizer at setting 6 for 15 sec. The homogenate was centrifuged at 50,000×g for 10 min, the supernatant discarded, and the pellet resuspended in ice-cold buffer A by homogenization with the Polytron. Each tissue was homogenized and centrifuged three times. The final pellet was resuspended in buffer A containing bovine serum albumin (1 g/L) and Bacitracin (0.14 g/L) to a final volume of 170 Ml/g of the original tissue weight. The binding assay consisted of 1 Ml in 12×75 mm polypropylene tubes: 50 uL  3 H-bradykinin (20,000 dpm, ˜0.3 Nm in the final assay volume), 100 L displacing drug in buffer A, and 750 Ul tissue homogenate. Each tray contained tubes, to which no drug was added to measure maximum binding and tubes to which bradykinin (1 uM final concentration) had been added, to measure specific binding. Specific binding accounted for 96-98% total binding. Tubes were incubated for 90 min at ambient temperature. The assays were terminated by filtration over Whatman GF/B glass fiber filters that had been pretreated for 2 hours with polyethyleneimine (2 g/L) using a Brandel Tissue Harvester, followed by washing with 4×1 Ml aliquots of ice-cold 50 Mm Tris, Ph 7.4. Filters were dissolved in Ready-Safe Fluor (Beckman) for at least 90 min before quantitation by liquid scintillation spectrometry. Kd values were determined using saturation binding and analysis by EBDA [(G. A. MacPherson,  J. Pharmacol. Methods , (1985), 213)], followed by LIGAND [P. J. Munson, D. Rodbard,  Anal. Biochem ., (1980), 220]. Ki values were determined using competitive analysis followed by EBDA and LIGAND. The following test results were obtained. 
     
       
         
               
               
               
             
               
               
               
               
             
           
               
                   
               
               
                 Test 
                 Compound 
                 Ki (mM) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                  1) 
                 D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic- 
                 11.54 
                   
               
               
                   
                 (Hydroxyproline  trans  phenyl carbamoyl)-Arg 
               
               
                  2) 
                 D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic- 
                 1.83 
               
               
                   
                 (Hydroxyproline  cis  methyl ether)-Arg 
               
               
                  3) 
                 D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic- 
                 1.58 
               
               
                   
                 (Hydroxyproline  cis  propyl ether)-Arg 
               
               
                  4) 
                 D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic- 
                 4.85 
               
               
                   
                 (Hydroxyproline  trans  propyl ether)-Arg 
               
               
                  5) 
                 D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic- 
                 3.76 
               
               
                   
                 (Hydroxyproline cis 2-nitrophenyl 
                 +\−0.89 
               
               
                   
                 ether)-Arg 
               
               
                  6) 
                 D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic- 
                 3.98 
               
               
                   
                 (Hydroxyproline cis 4-nitrophenyl 
                 +\−1.5 
               
               
                   
                 ether)-Arg 
               
               
                  7) 
                 D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic- 
                 4.15 
               
               
                   
                 (Hydroxyproline cis ethyl ether)-Arg 
                 +\−0.58 
               
               
                  8) 
                 D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic- 
                 13.25 
               
               
                   
                 (Hydroxyproline cis cyclohexyl-methyl 
                 +\−1.55 
               
               
                   
                 ether)-Arg 
               
               
                  9) 
                 D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic- 
                 0.06 
               
               
                   
                 (Hydroxyproline cis phenyl thioether)-Arg 
                 +\−0.01 
               
               
                 10) 
                 D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic- 
                 2.51 
               
               
                   
                 (Hydroxyproline cis phenyl ether)-Arg 
                 +\−0.06 
               
               
                 11) 
                 D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic- 
                 5.44 
               
               
                   
                 (hydroxyproline trans ethyl ether)-Arg 
               
               
                   
               
             
          
         
       
     
     Determination of Bradykinin Antagonist Activity 
     This protocol was designed to identify compounds that possess antagonist activity at bradykinin receptors on intestinal (ileal longitudinal) smooth muscle. 
     Guinea pig intestine was removed and placed in a Petri dish containing Tyrodes solution and cut into 3-4 cm segments. The longitudinal muscle was separated from the underlying circular muscle using a cotton applicator (Paton and Zar,  J. Physiol . (1968), 194:13). Muscle strips were connected to isometric force-displacement transducers (Grass or Gould) coupled to a physiograph and placed in tissue baths containing Tyrode&#39;s solution at 37° C. Each preparation was suspended under a resting tension of 2 g. 
     After equilibration of the tissues, appropriate volumes of bradykinin solutions were cumulatively added to the 10 Ml tissue baths to increase the concentration of bradykinin in the bath step-by-step without washing out after each single dose. Higher concentrations were added only after the preceding contraction had reached a steady value. When the next concentration step does not cause a further increase in contraction, it was assumed that the maximum effect had been obtained and the tissue was washed to remove bradykinin and allowed to recover for 15 minutes. Antagonism of bradykinin responses in the presence of antagonist were determined by repeating the cumulative addition procedure for bradykinin after the tissue has been exposed to the antagonist for 5 minutes. Three or four differentconcentrations of antagonist are studied sequentially in the same preparations. Responses were expressed as a percentage of the maximum contraction elicited by bradykinin in the absence of antagonist. pA2 values were calculated by Schild analysis. The following results were obtained. 
     
       
         
               
               
               
             
               
               
               
               
             
           
               
                   
               
               
                 Test 
                 Compound 
                 pA 2   
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1) 
                 D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic- 
                 6.85 
                   
               
               
                   
                 (Hydroxyproline  cis  methyl ether)-Arg 
               
               
                 2) 
                 D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic- 
                 6.65 
               
               
                   
                 (Hydroxyproline  cis  propyl ether)-Arg 
               
               
                 3) 
                 D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic- 
                 6.86 
               
               
                   
                 (Hydroxyproline  trans  propyl ether)-Arg 
               
               
                 4) 
                 D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic- 
                 6.85 
               
               
                   
                 (Hydroxyproline cis 2-nitrophenyl 
               
               
                   
                 ether)-Arg 
               
               
                 5) 
                 D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic- 
                 6.86 
               
               
                   
                 (Hydroxyproline cis 4-nitrophenyl 
                 +\−0.02 
               
               
                   
                 ether) -Arg 
               
               
                 6) 
                 D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic- 
                 7.29 
               
               
                   
                 (Hydroxyproline trans ethyl ether)- 
               
               
                   
                 Arg 
               
               
                 7) 
                 D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic- 
                 7.95 
               
               
                   
                 (Hydroxyproline-cis phenylthioether)- 
               
               
                   
                 Arg 
               
               
                 8) 
                 D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic- 
                 6.96 
               
               
                   
                 (Hydroxyproline-cis ethyl ether)- 
               
               
                   
                 Arg