Antithrombotic agents

Abstract: This invention relates to L-arginine aldehyde derivatives, having the formula I where X and Y have the values defined in the description, as well as pharmaceutical formulations containing those compounds and methods of their use as thrombin inhibitors, coagulation inhibitors, and thromboembolic disorder agents.

Description: This invention relates to thrombin inhibitors which are useful anticoagulants in mammals. In particular it relates to L-arginine aldehyde derivatives having high anticoagulant activity, antithrombotic activity, and oral bioavailability. The process of blood coagulation, thrombosis, is triggered by a complex proteolytic cascade leading to the formation of thrombin. Thrombin proteolytically removes activation peptides from the Aα-chains and the Bβ-chains of fibrinogen, which is soluble in blood plasma, initiating insoluble fibrin formation. Anticoagulation is currently achieved by the administration of heparins and coumarins. Parenteral pharmacological control of coagulation and thrombosis is based on inhibition of thrombin through the use of heparins. Heparins act indirectly on thrombin by accelerating the inhibitory effect of endogenous antithrombin III (the main physiological inhibitor of thrombin). Because antithrombin III levels vary in plasma and because surface-bound thrombin seems resistant to this indirect mechanism, heparins can be an ineffective treatment. Because coagulation assays are believed to be associated with efficacy and with safety, heparin levels must be monitored with coagulation assays (particularly the activated partial thromboplastin time (APTT) assay). Coumarins impede the generation of thrombin by blocking the posttranslational gamma-carboxylation in the synthesis of prothrombin and other proteins of this type. Because of their mechanism of action, the effect of coumarins can only develop slowly, 6-24 hours after administration. Further, they are not selective anticoagulants. Coumarins also require monitoring with coagulation assays (particularly the prothrombin time (PT) assay). Recently, interest in small synthetic peptides that are recognized by proteolytic enzymes in a manner similar to that of natural substrates has grown. Tripeptide aldehydes such as D-Phe-Pro-Arg-H, Boc-D-Phe-Pro-Arg-H, and D-MePhe-Pro-Arg-H, Bajusz et al., J. Med. Chem., 33 , 1729-1735 (1990) demonstrate potent direct inhibition of thrombin. Many investigators have synthesized analogs in an effort to develop pharmaceutical agents, for example Shuman et al., J. Med. Chem. , 36 , 314-319 (1993), as well as European Patent Applications, publication numbers 479489 and 542525. Early clinical studies which demonstrate that D-MePhe-Pro-Arg-H sulfate is an anticoagulant in man have been reported, see Simoons et al., Circulation, 80 , I-231, Abstr. 1241 (1994). EP-A-504064 relates to the use of novel polyfluorinated alkyl derivatives of certain tripeptides as thrombin inhibitors, while new isosteric peptides as inhibitors of thrombin are described in EP-A-530167. Inhibition of serine proteases by low molecular weight peptides and their derivatives is discussed in Annals N.Y. Academy of Sciences, 1981,756-784. Although the heparins and coumarins are effective anticoagulants, and no drug has yet emerged from the known tripeptide aldehydes, and despite the continuing promise for this class of compounds, there exists a need for anticoagulants that act selectively on thrombin, and independent of antithrombin III, exert inhibitory action shortly after administration, preferably by an oral route, and do not interfere with lysis of blood clots, as required to maintain hemostasis. The present invention is directed to the discovery that the compounds of the present invention, as defined below, are potent thrombin inhibitors that have high bioavailability following oral administration. Accordingly, it is a primary object of the present invention to provide novel L-arginine aldehyde derivatives that are potent thrombin inhibitors useful as anticoagulants. Other objects, features and advantages will be apparent to those skilled in the art from the following description and claims. The present invention provides a thrombin inhibiting compound having the formula I wherein X is an unsubstituted or substituted group selected from homoprolinyl, prolinyl, thiazolidinoyl, isothiazolidinoyl, thiomorpholinoyl, piperazinoyl, morpholinoyl, oxazolidinoyl and isoxazolidinoyl, and where in a sulfur containing group the sulfur may be oxidized with one or two oxygen atoms; Y is or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt thereof. In addition to the compounds of formula I, the present invention provides pharmaceutical formulations comprising a compound of formula I in association with a pharmaceutically acceptable carrier, diluent or excipient. The present invention also provides a method of inhibiting coagulation in mammals comprising administering to a mammal in need of treatment, a coagulation inhibiting dose of a compound of formula I. The present invention further provides a method of inhibiting thrombin comprising administering to a mammal in need of treatment, a thrombin inhibiting dose of a compound of formula I. Further, the present invention provides a method of treating a thromboembolic disorder comprising administering to a mammal in need of treatment, an effective dose of a compound of formula I. This invention relates to new inhibitors of thrombin, pharmaceutical compositions containing the compounds as active ingredients, and the use of the compounds as anticoagulants for prophylaxis and treatment of thromboembolic disorders such as venous thrombosis, pulmonary embolism, arterial thrombosis, in particular myocardial ischemia, myocardial infarction and cerebral thrombosis, general hypercoagulable states and local hypercoagulable states, such as following angioplasty and coronary bypass operations, and generalized tissue injury as it relates to the inflammatory process. The term "alkyl" by itself or as part of another substituent means a straight or branched chain alkyl radical having the stated number of carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl and sec-butyl. The term "alkoxy" means a straight or branched chain alkyl radical having the stated number of carbon atoms bonded to the parent moiety by an oxygen atom. The term "halo" means chloro, fluoro, bromo or iodo. The term "di(C 1 -C 4 alkyl)amino" means a group -N(C 1 -C 4 alkyl) 2 where each alkyl group, independently, has the stated number of carbon atoms. The group is referred to at times herein as homoprolinyl and abbreviated hPro. The term "azetidine" refers to an azetidine-2-carbonyl group and is abbreviated Azt. The terms "thiazolidinoyl, isothiazolidinoyl, thiomorpholinoyl, piperazinoyl, morpholinoyl, oxazolidinoyl, and isoxazolidinoyl" refer to the stated ring group having a carbonyl functionality bonded thereto so as to afford a stable structure. When X is a substituted group, including the fused bicyclic ring groups, there can be one to three of the same or different substituents that will afford a stable structure selected from halo, hydroxyl, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, amino (-NH 2 ), mono(C 1 -C 4 alkyl)amino, di(C 1 -C 4 alkyl)amino, mercapto, C 1 -C 4 alkylthio (-S(O) p (C 1 -C 4 alkyl)), -NHS(O) p (C 1 -C 4 alkyl), -NHC(O)C 1 -C 4 alkyl, -S(O) p NH2, -S(O) p NH(C 1 -C 4 alkyl), -S(O) p N(C 1 -C 4 alkyl) 2 , substituted or unsubstituted phenoxy, substituted or unsubstituted naphthyloxy, substituted or unsubstituted pyridyloxy, substituted or unsubstituted phenylthio; p is 0, 1 or 2; and the substituents on the phenoxy, naphthyloxy, pyridyloxy and phenyl thio groups are one or two of the same or different substituents selected from halo, hydroxyl, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, amino (-NH 2 ), mono(C 1 -C 4 alkyl)amino, di(C 1 -C 4 alkyl)amino, mercapto, C 1 -C 4 alkylthio (-S(O) p (C 1 -C 4 alkyl)), -NHS(O) p (C 1 -C 4 alkyl), -NHC(O)C 1 -C 4 alkyl, -S(O) p NH2, -S(O) p NH(C 1 -C 4 alkyl), -S(O) p N(C 1 -C 4 alkyl) 2 , and p is 0, 1 or 2. In the representation of formula I, the carbonyl functionality of group X is attached to the amine functionality of the Y group. The carbonyl functionality of Y is attached to the amino group drawn in Formula I. The asterisks in formula I and substituent Y denote a chiral center that is (L). In addition, diastereomers exist at the X substituent and, depending on substitutions on said X substituent, further diastereomers may exist. The compounds of the present invention include mixtures of two or more diastereomers as well as each individual isomer. The following compounds illustrate compounds contemplated within the scope of formula I: D-homoprolinyl-L-prolinyl-L-arginine aldehyde; D-prolinyl-L-prolinyl-L-arginine aldehyde; D-homoprolinyl-L-azetidin-2-carbonyl-L-arginine aldehyde; D-prolinyl-L-azetidin-2-carbonyl-L-arginine aldehyde; and D-(4-phenoxy)prolinyl-L-prolinyl-L-arginine aldehyde. Preferred compounds of the present invention are those compounds of formula I where X is unsubstituted or monosubstituted homoprolinyl, unsubstituted or monosubstituted prolinyl, unsubstituted or monosubstituted piperazinoyl, and pharmaceutically acceptable salts and solvates thereof. Particularly preferred compounds of the present invention are those compounds of formula I where X is homoprolinyl, prolinyl, 4-phenoxyprolinyl, piperazinoyl, and pharmaceutically acceptable salts or solvates thereof. As mentioned above, the invention includes pharmaceutically acceptable salts of the compounds defined by the above formula I. A particular compound of this invention can possess one or more sufficiently basic functional groups, and accordingly react with any of a number of nontoxic inorganic and organic acids, to form a pharmaceutically acceptable salt. Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p -toluene sulfonic, methanesulfonic acid, oxalic acid, p -bromo phenyl sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of such pharmaceutically acceptable salts thus are the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycollate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like. Preferred pharmaceutically acceptable acid addition salts are those formed with mineral acids such as hydrochloric acid, hydrobromic acid and sulfuric acid. As stated above, the present invention includes solvates of the compounds of formula I and the pharmaceutically acceptable salts thereof. A particular compound of the present invention or a pharmaceutically acceptable salt thereof may form solvates with water or common organic solvents. Such solvates are included within the scope of compounds of the present invention. A compound of formula I is prepared by removing simultaneously or sequentially the protecting group(s) P of a corresponding compound of Formula II wherein P on the guanidino group represents an amino protecting group and (P)X represent a radical X , which may bear an independently selected amino protecting group P for a compound of formula I in which X includes a basic NH moiety; whereafter, when a salt of the compound of formula I is required, forming the salt with a pharmaceutically acceptable acid. For example, a compound of Formula II in which the amino protecting group(s) is(are) benzyloxycarbonyl may be converted into the hydrochloride of the corresponding compound of formula I by hydrogenolysis at atmospheric pressure over palladium on carbon catalyst in dilute ethanolic hydrochloric acid. The compounds of formula I are prepared by known methods of peptide coupling. According to one such method the acid PX-COOH, where X -COOH is the acid equivalent of the X groups as defined for formula I, and P is an amino protecting group, is coupled with a carboxy protected proline (or azetidine-2-carboxy ester) to form the dipeptide. The carboxy protecting ester group of the proline moiety is then removed (deblocked or deesterified) and the free acid form of the dipeptide is coupled with the lactam form of arginine. The above reaction sequence is illustrated by the following Scheme 1: wherein P represents an amino protecting group. The coupled Arg(P) lactam product (c) is reacted with a hydride reducing agent, preferably lithium aluminum hydride or lithium tri-tert-butoxyaluminohydride, in an inert solvent or mixture of solvents to reduce the lactam ring and provide the tripeptide in the arginine aldehyde form represented by the formula wherein (P) represents amino protecting groups. The protecting groups are removed by procedures known to those skilled in the art such as hydrogenation over a metal catalyst. The lactam form of arginine is obtained by intramolecular coupling of amino protected arginine [Arg-OH]. For example, Boc-Arg(Cbz)OH, represented by the formula where Boc is t-butyloxycarbonyl and Cbz is benzyloxycarbonyl, is first converted to an active ester form, such as an active mixed anhydride, with a chloroformate ester, e.g. ethyl chloroformate to isobutyl chloroformate. The ester formation is carried out in the presence of a tertiary amine such as N-methylmorpholine. Addition of further or another tertiary amine base, such as triethylamine or diisopropylethylamine, effects the internal acylation to provide the lactam form of the di-amino protected arginine as shown below Prior to use in the coupling with the PX(C=O)-Pro-OH as shown in the above scheme, the Boc or other amine protecting group is selectively removed with trifluoroacetic acid or HCl to provide the requisite free amino group. The coupling of a PXCOOH compound with a proline ester, when X is as defined above for formula I, is carried out by first protecting the amino group of the amino acid. Conventional amino protecting groups commonly used for temporary protection or blocking of the amino group are employed. The amino-protecting group refers to substituents of the amino group commonly employed to block or protect the amino functionality while reacting other functional groups on the compound. Examples of such amino-protecting groups include the formyl group, the trityl group, the phthalimido group, the trichloroacetyl group, the chloroacetyl, bromoacetyl and iodoacetyl groups, urethane-type blocking groups such as benzyloxycarbonyl, t-butoxycarbonyl 4-phenylbenzyloxycarbonyl, 2-methylbenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 4-fluorobenzyloxycarbonyl, 4-chlorobenzyloxycarbonyl, 3-chlorobenzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 3-bromobenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-cyanobenzyloxycarbonyl, 2-(4-xenyl)isopropoxycarbonyl, 1,1-diphenyleth-1-yloxycarbonyl, 1,1-diphenylprop-1-yloxycarbonyl, 2-phenylprop-2-yloxycarbonyl, 2-(p-toluyl)prop-2-yloxycarbonyl, cyclopentanyloxycarbonyl, 1-methylcyclopentanyloxycarbonyl, cyclohexanyloxycarbonyl, 1-methylcyclohexanyloxycarbonyl, 2-methylcyclohexanyloxycarbonyl, 2-(4-toluylsulfonyl)ethoxycarbonyl, 2-(methylsulfonyl)ethoxycarbonyl, 2-(triphenylphosphino)ethoxycarbonyl, 9-fluoroenylmethoxycarbonyl ("FMOC"), 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, 1-(trimethylsilylmethyl)prop-1-enyloxycarbonyl, 5-benzisoxalylmethoxycarbonyl, 4-acetoxybenzyloxycarbonyl, 2,2,2-trichlorethoxycarbonyl, 2-ethynyl-2-propoxycarbonyl, cyclopropylmethoxycarbonyl, 4-(decyloxy)benzyloxycarbonyl, isobornyloxycarbonyl, 1-piperidyloxycarbonyl and the like; the benzoylmethylsulfonyl group, the 2-(nitro)phenylsulfenyl group, the diphenylphosphine oxide group, and the like amino-protecting groups. The species of amino-protecting group employed is not critical so long as the derivatized amino group is stable to the condition of subsequent reaction(s) on other positions of the molecule and can be removed at the appropriate point without disrupting the remainder of the molecule. Preferred amino- protecting groups are the benzyloxycarbonyl, allyloxycarbonyl, t-butoxycarbonyl, and trityl groups. Similar amino-protecting groups used in the cephalosporin, penicillin and peptide art are also embraced by the above terms. Further examples of groups referred to by the above terms are described by J. W. Barton, "Protective Groups in Organic Chemistry", J. G. W. McOmie, Ed., Plenum Press, New York, N.Y., 1973, Chapter 2, and T. W. Greene, "Protective Groups in Organic Synthesis", John Wiley and Sons, New York, N.Y., 1981, Chapter 7. The related term "protected amino" defines an amino group substituted with an amino-protecting group discussed above. In carrying out the coupling reaction, an ester protecting group for proline is employed which is removable by conditions under which the amino protecting group remains intact. The amino protecting group of the acylating acid PXCOOH thus remains in place for protection of the amino group during the subsequent coupling with the arginine lactam compound to form (c) in Scheme 1. The carboxy protecting ester group as used in the specification refers to one of the ester derivatives of the carboxylic acid group commonly employed to block or protect the carboxylic acid group while reactions are carried out on other functional groups on the compound. Examples of such carboxylic acid protecting groups include C 1 -C 4 alkyl, benzyl, 4-nitrobenzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl, 2,4-dimethoxybenzyl, 2,4,6-trimethoxybenzyl, 2,4,6-trimethylbenzyl, pentamethylbenzyl, 3,4-methylenedioxybenzyl, benzhydryl, 4,4'-dimethoxybenzhydryl, 2,2',4,4'-tetramethoxybenzhydryl, t-butyl, t-amyl, trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl, 4,4',4''-trimethoxytrityl, 2-phenylprop-2-yl, trimethylsilyl, t-butyldimethylsilyl, phenacyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(di(n-butyl)methylsilyl)ethyl, p-toluenesulfonylethyl, 4-nitrobenzylsulfonylethyl, allyl, cinnamyl, 1-(trimethylsilylmethyl)-prop-1-en-3-yl, and like moieties. The species of carboxy-protecting group employed is not critical so long as the derivatized carboxylic acid is stable to the conditions of subsequent reaction(s) on other positions of the molecule and can be removed at the appropriate point without disrupting the remainder of the molecule. In particular, it is important not to subject the carboxy-protected molecule to strong nucleophilic bases or reductive conditions employing highly activated metal catalysts such as Raney nickel. (Such harsh removal conditions are also to be avoided when removing amino-protecting groups discussed below.) Preferred carboxy protecting groups are C 1 -C 3 alkyl and benzyl. Further examples of these groups are found in E. Haslam, "Protective Groups in Organic Chemistry", J.G.W. McOmie, Ed., Plenum Press, New York, N.Y., 1973, Chapter 5, and T.W. Greene, "Protective Groups in Organic Synthesis", John Wiley and Sons, New York, N.Y., 1981, Chapter 5. The compounds of formula I where Y is azetidinyl (or prolinyl) are prepared in an analogous manner by known methods of peptide coupling. According to one such method, the cyclic lactam form of arginine (e) is prepared and coupled with an amino protected azetidine-2-carboxylic acid (d) as shown below to afford the dipeptide (f) where P represents an amino protecting group such as the benzyloxycarbonyl (Cbz) group, t-butoxycarbonyl (Boc), p-toluenesulfonyl, and the like. Preferably the amino protecting group used is removable by hydrogenation or treatment with mild acid (e.g. trifluoroacetic acid) or a strong acid (e.g. HCl). Examples of other suitable amino protecting groups are provided in "Protective Groups in Organic Synthesis", Second Edition, by T. W. Greene and Peter G. M. Wuts, Chapter 7, page 309-405 (1991), John Wiley & Sons, Inc., publishers. The Boc, or other suitable protecting group, is removed from the azetidine ring nitrogen which is then acylated with the desired amino acid acyl group to afford the tripeptide shown below. Although illustrated and described for those compound of the present invention where Y is azetidinyl-2-carbonyl, one skilled in the art will appreciate these procedures can also be used to afford those compounds of the present invention where Y is prolinyl. The coupled Arg(P) lactam product (g) is reduced with a hydride reducing agent, preferably lithium aluminum hydride or lithium tri-tert-butoxyaluminohydride, in an inert solvent or mixture of solvents to reduce the lactam and provide the tripeptide in the arginine aldehyde form represented by the formula PX(C=O)-Azt-Arg(P)-H wherein P represents an amino protecting group. The protecting groups are removed by procedures known to those skilled in the art such as hydrogenation over a metal catalyst. Alternatively, the compounds of the invention are prepared by coupling the PXCOOH acid with carboxy protected azetidine-2-carboxylic acid. The carboxy is deprotected as the dipeptide which is then coupled with the amino protected arginine in the lactam form prepared as described above. The tripeptide is then reduced to provide the amino protected arginal tripeptide as described above. The coupling of an PXCOOH compound is carried out by first protecting the amino group of the amino acid. Conventional amino protecting groups commonly used for temporary protection or blocking of the amino group are employed. Examples of such protecting groups are described above. The coupling reactions described above are carried out in the cold preferably at a temperature between about -20 °C and about 15 °C. The coupling reactions are carried out in an inert organic solvent such as dimethylformamide, dimethylacetamide, tetrahydrofuran, methylene chloride, chloroform, and like common solvents or a mixture of such solvents. Generally anhydrous conditions are used when, in the coupling reaction, an active ester of the acylating acid is used. The compounds of the invention are isolated best in the form of acid addition salts. Salts of the compounds of formula I formed with acids such as those mentioned above are useful as pharmaceutically acceptable salts for administration of the antithrombotic agents and for preparation of formulations of these agents. Other acid addition salts may be prepared and used in the isolation and purification of the peptides. For example, the salts formed with the sulfonic acids such as methanesulfonic acid, n-butanesulfonic acid, p-toluenesulfonic acid and naphthalenesulfonic acid may be so used. The preferred method for purifying the compounds of formula I, while at the same time preparing a desired stable salt form, is that described in U.S. Patent 5,250,660. According to the method, stable sulfates or hydrochlorides are provided by preparative purification over C 18 reversed-phase chromatography in which the aqueous component comprises sulfuric acid or hydrochloric acid at pH 2.5 and acetonitrile is the organic component. The pH of the acidic eluant is adjusted to between about pH 4 and about 6 with an anion exchange resin in the hydroxyl form, e.g. Bio-Rad AG-1X8. After adjustment of the pH, the solution of tripeptide sulfate or hydrochloride salt is lyophilized to provide the pure salt in dry powder form. In an example of the process, crude D-hPro-L-Azt-L-Arg-H sulfate is dissolved in water and the solution is loaded on Vydac C 18 RPHPLC 5 cm X 50 cm column. A gradient of 2-20 percent B (A = 0.01 percent H 2 SO 4 ; B = acetonitrile) over 10 hours is used. Multiple fractions are collected and those containing product as determined by analytical RPHPLC are pooled. The pH of the pooled fractions is adjusted to pH 4.0 - 4.5 with AG-1X8 resin in hydroxide form (Bio-Rad, 3300 Ragatta Blvd., Richmond, CA 94804). The solution is filtered and the filtrate is lyophilized to provide the pure D-,L-,L- tripeptide in the form of the sulfate salt. The optically active isomers of the diastereomers of the X substituent are also considered part of this invention. Such optically active isomers may be prepared from their respective optically active precursors by the procedures described above, or by resolving the racemic mixtures. This resolution can be carried out by derivatization with a chiral reagent followed by chromatography or by repeated crystallization. Removal of the chiral auxiliary by standard methods affords substantially optically pure isomers of the compounds of the present invention or their precursors. Further details regarding resolutions can be obtained in Jacques, et al., Enantiomers, Racemates, and Resolutions , John Wiley & Sons, 1981. The compounds employed as initial starting materials in the synthesis of the compounds of this invention are well known and, to the extent not commercially available, are readily synthesized by standard procedures commonly employed by those of ordinary skill in the art. The following Examples are provided to further describe the invention and are not to be construed as limitations thereof. The R f values in the following examples unless otherwise stated, were determined by silica gel thin layer chromatography using Kieselgel 60F-254 (Merck, Darmstadt) in the following solvent systems: (A) chloroform-methanol-acetic acid, 135:15:1, v:v:v (B) ethyl acetate-acetic acid-absolute ethanol, 90:10:10, v:v:v (C) chloroform-methanol-acetic acid, 90:30:5, v:v:v The analytical HPLC methods used in the examples were as follows: Method 1 . Waters 600E using a Vydac C 18 reversed-phase column of 0.46 cm x 10 cm. The chromatogram was monitored on an LDC at 214 nM using a gradient of A = water containing 0.1 percent (v:v)TFA and B = acetonitrile containing 0.1 percent (v:v) TFA. Method 2 . Pharmacia FPLC using a Vydac C 18 reversed-phase column measuring 0.46 cm x 10.0 cm. Monitoring was done on a Pharmacia UV-M at 214 nM using a gradient of either A = water containing 0.1 percent (v:v) TFA or B = acetonitrile containing 0.1 percent (v:v) TFA. Method 3 . Hitachi L-6200 using a Vydac C 18 reversed-phase column of 0.46 cm x 10 cm. Samples were eluted using a gradient composed of A (0.1% (v:v) aqueous TFA) and B (0.1% TFA in acetonitrile). The chromatogram was monitored at 214 nm using a L-4000 UV detector. The abbreviations used in the examples have the following meanings. Amino acids: Arg = arginine, Pro = proline, hPro = homoproline, Azt = azetidine-2-carboxylic acid, Phe = phenylalamine, hPhe = homophenylalanine Boc = t-butyloxycarbonyl (t-butoxycarbonyl) Bzl = benzyl Cbz = benzyloxycarbonyl DCC = dicyclohexylcarbodiimide DMF = dimethylformamide DMSO = dimethylsulfoxide EtOAc = ethyl acetate Et 2 O = diethyl ether EtOH = ethanol FAB-MS = fast atom bombardment mass spectrum FD-MS = field desorption mass spectrum HOBT = 1-hydroxybenzotriazole hydrate HPLC = High Performance Liquid Chromatography IR = Infrared Spectrum LAH = Lithium Aluminum Hydride NMR = Nuclear Magnetic Resonance MOC = methoxycarbonyl RPHPLC = Reversed Phase High Performance Liquid Chromatography TFA = trifluoroacetic acid THF = tetrahydrofuran TLC = thin layer chromatography Unless otherwise stated, pH adjustments and work up are with aqueous acid or base solutions. Example 1 Preparation of D-Homoprolinyl-L-Prolinyl-L-Arginine Aldehyde Dihydrochloride (D-hPro-Pro-Arg-H·2HCl) A) Cbz-D-homoproline D-Pipecolic acid (5.0 g, 38.7 mmol) was dissolved in tetrahydrofuran (100 mL) and water (30mL). The pH of the solution was adjusted to 9.5 with 2 N NaOH, and benzyl chloroformate (5.5 mL, 38.7 mmol) was added dropwise and the pH maintained at 9.5 with 2 N NaOH. The reaction was stirred for an additional 1 hour at room temperature. The organic solvent was evaporated in vacuo , diethyl ether (100mL) and water (50 mL) were added to the residue. The aqueous layer was separated, the pH of the solution was adjusted to 2.8 with 3 N HCl and ethyl acetate (150 mL) was added. The organic layer was separated and dried (MgSO 4 ); the filtrate was concentrated in vacuo to give a clear oil of the title compound (9.6 g; 95 percent yield): FD-MS 264 (MH + ); TLC R f (A) 0.37; 1 HNMR (CDCl 3 ) δ1.22-1.58 (m, 2H), 1.60-1.80 (m, 2H), 2.20-2.35 (m, 1H), 2.98-3.18 (m, 1H), 4.00-4.20 (m, 1H), 4.85-5.05 (m, 1H) 5.20 (s, 2H), 7.30-7.40 (d, 5H); [α] D +39.0° (C= 0.5 / MeOH). B) Cbz-D-homoprolinyl-Proline Cbz-D-homoproline (A) (9.5 g, 36 mmol) was dissolved in EtOAc (100mL) and the solution cooled to 0 °C. Added to the solution was 2,4,5 trichlorophenol (7.1g, 36 mmol) and dicyclohexylcarbodiimide (7.4 g, 36 mmol). The reaction was stirred for 1 hour at 0 °C and 1 hour at room temperature. The precipitate was filtered and the filtrate concentrated in vacuo to an oil. The oil was dissolved in pyridine (100mL), L-proline (4.2 g, 36mmol), and triethylamine (5.0 mL, 36 mmol) were added. The reaction was stirred at room temperature (24 hours). The reaction solvent was removed in vacuo to an oil. The residue was dissolved in water (100mL), diethyl ether (50mL) was added and the pH adjusted to 9.5 with 2 N NaOH. The aqueous layer was extracted twice with diethyl ether. The aqueous layer was separated, the pH adjusted to 2.8 with 3 N HCl and EtOAc (150mL) was added. The organic layer was separated, dried (M g SO 4 ), and the filtrate evaporated in vacuo to an amorphous solid (11.4 g; 88 percent yield); FD-MS 361 (MH + ); TLC R f (A) 0.78; [α] D = -2.7° (C= 0.5 / Trifluoroethanol); Elemental Analysis calculated for C 19 H 24 N 2 O 5 : C 63.32, H 6.71, N 7.77; Found C 63.42, H 6.84, N 7.96. C) Boc-Arg(Cbz)-OH Boc-Arg(HCL)-OH (82.1 g, 250 mmol) was dissolved in 5 N NaOH (240 mL) in a 3 necked flask. The reaction mixture was chilled to -5 °C and the pH was maintained at 13.2-13.5 using 5 N NaOH (250 mL) while adding benzyl chloroformate (143 mL, 1.0 mol) dropwise (55 min). The reaction mixture was stirred for an additional 1 hour at -5 °C and diluted with H 2 O (100 mL) and Et 2 O (500 mL). The aqueous layer was separated and extracted with Et2O (2 X 500 mL). The aqueous layer was acidified to pH 3.0 with 3 N H 2 SO 4 (560 mL) and extracted with EtOAc (550 mL). The aqueous layer was separated and extracted once with EtOAc. The combined EtOAc layers were washed with water and dried (MgSO 4 ). The organic layers were concentrated to dryness in vacuo to give the title compound (66.1 g; 65 percent yield): TLC R f (C) 0.43; FD-MS 408 (M + ); 1 HNMR (CDCl 3 ) δ 1.42 (s,9H), 1.61-1.91 (m,4H), 3.23-3.41 (m,2H), 4.17 (d,lH), 5.21 (s,2H), 5.62 (d,lH), 7.30-7.42 (m,6H), 8.37 (m,1H). D) Boc-Arg(Cbz)-Lactam Boc-Arg(Cbz)-OH (C) (66.0 g, 0.162 mol) was dissolved in THF (230 mL) and cooled to -10 °C. To the reaction mixture was added N-methylmorpholine (18.7 mL, 0.17 mol) followed by isobutyl chloroformate (22.5 mL, 0.17mol). The reaction mixture was stirred 5 minutes at -10 °C and triethylamine (23.5 mL, 0.17 mol) was added. The reaction mixture was stirred for 1 hour at -10 °C and 1 hour at room temperature. The reaction mixture was poured into 1 L of ice-water and the resulting precipitate filtered, washed with cold water, and dried in vacuo . The product was crystallized from EtOAc to give the title compound (38.05 g; 60 percent yield): TLC R f (A) 0.77; FD-MS 391 (MH + ); 1 HNMR (CDCl 3 ) δ 1.48 (s, 9H), 1.78-1.98 (m, 2H), 2.50 (m, 1H), 3.41 (m, 1H), 4.43 (m, 1H), 4.90 (m, 1H), 5.16 (s, 2H), 5.27 (m, 1H), 7.28-7.45 (m, 6H), 9.41 (m, 1H), 9.68 (m, 1H). E) HCl·Arg(Cbz)-Lactam A solution of HCl(g) saturated in EtOAc (7.2 L) was added dropwise over 30 minutes to a solution of Boc-Arg(Cbz)-Lactam (D) (641 g, 1.64 mol) dissolved in CH 2 Cl 2 (3 L) at -10°C. The reaction was allowed to stir 1 hour at -10 °C and slowly warmed to room temperature over 3 hours. Diethyl ether (12 L) was added and the precipitate was filtered, washed with diethyl ether, dried (MgSO 4 ) and concentrated to dryness in vacuo to give the title compound (580 g): TLC R f (C) 0.29; FD-MS 291 (MH + ). F) Cbz-D-hPro-Pro-Arg(Cbz)-Lactam In flask 1 Cbz-hPro-Pro-OH (B) (11.1 g, 30.8 mmol) was dissolved in DMF (75 mL), cooled to -15 °C and N-methylmorpholine (3.4 mL, 30.8 mmol) was added followed by isobutyl chloroformate (4.0 mL, 30.8 mmol). The reaction mixture was stirred at -15 °C for 2 minutes. In flask 2 HCl·Arg(Cbz)-Lactam (E) (10.1 g, 30.8 mmol) was dissolved in DMF (75 mL), cooled to 0 °C, and diisopropylethylamine (10.7 mL, 61.6 mmol) was added. The reaction mixture was stirred at 0 °C for 2 minutes. The contents of flask 2 were added to flask 1 in one portion and the reaction mixture was stirred for 4 hours at -15 °C. The reaction mixture was slowly warmed to room temperature (24 hours). To the reaction mixture was added 1 N NaHCO 3 (5 mL) and the reaction solvent was removed in vacuo . To the oil was added EtOAc (200 mL) and water (100 mL), the organic layer was separated, washed with 1 N NaHCO 3 , water, 1.5 N citric acid, and water. The organic layer was dried (MgS0 4 ), and the filtrate evaporated to an amorphous solid of the title compound (17.4 g, 89 percent yield): TLC R f (A) 0.66; FAB-MS 633 (MH + ). G) Cbz-D-hPro-Pro-Arg(Cbz)-H Cbz-D-hPro-Pro-Arg(Cbz)-Lactam (F) (17.2 g, 27.1 mmol) was dissolved in anhydrous THF (200 mL) and placed in a flask under a N 2 atmosphere. The reaction mixture was cooled to -65 °C and lithium aluminum hydride 1 M in THF (27.1 mL, 27.1 mmol) was added dropwise over 5 minutes. The reaction mixture was stirred at -65 °C for 30 minutes. A solution of 5 mL of THF and 5 mL of 0.5 N H 2 SO 4 was added dropwise to the reaction mixture over 5 minutes. The reaction mixture was diluted with EtOAc (150 mL), and water (50 mL) and the organic layer separated. The organic layer was washed with water (2 X 100 mL) and dried (MgSO 4 ). The filtrate was concentrated to dryness in vacuo to an amorphous solid to give the title compound (14.1 g; 82 percent yield): TLC R f (A) 0.33; FAB-MS 635 (MH + ). H) D-hPro-Pro-Arg-H·2HCl•1.5 H 2 O Cbz-D-hPro-Pro-Arg(Cbz)-H (G) (14.0 g, 22.0 mmol) was dissolved in ethanol (150 mL), water (50 mL), and 1 N HCl (55 mL). To the solution was added 5 percent Pd/C (5.0 g) and the reaction was hydrogenated at ambient temperature and pressure for 3 hours and the reaction purged with nitrogen for 5 minutes. The catalyst was removed by filtration through a diatomaceous earth pad and the filtrate concentrated in vacuo down to 100 mL. An additional 50 mL of H 2 O was added to the reaction and pH of solution adjusted to 4.0 with BioRad AG1-X8 resin (hydroxide form). The resin was removed by filtration and the solution lyophilized to give 8.29g (86 percent) of crude title compound. The crude material in two portions was dissolved in 20 mL 0.05 percent HCl (pH 2.5) and applied to two 5 X 25 cm columns (Vydac C 18 resin) connected in series. A gradient system consisting of (A) 0.05 percent HCl and (B) CH 3 CN was used to elute the pure peptide. The gradient used was an increasing concentration of CH 3 CN from 2 percent to 10 percent. Fractions were collected and pooled on the basis of analytical RPHPLC profile. The combined fractions were adjusted to pH 4.0 using AG1-X8 resin (Bio-Rad analytical anion exchange resin 50-100 mesh) in hydroxide form. The solution was filtered, and the filtrate was lyophilized to dryness resulting in pure title compound (3.1 g; 61 percent yield): FAB-MS 367 (MH + ); Amino acid analysis: hPro, 1.00; Pro, 0.98; [α] D = -88.4° (C= 0.5 / 0.1 N HCl); Elemental Analysis calculated for C 17 H 30 N 6 O 3 ·2 HCl·1.5 H 2 O: C 43.78, H 7.56, N 18.02; Found C 43.48, H 7.25, N 18.00. The following compounds were synthesized using methods substantially equivalent to those described in Example 1 above or as described elsewhere herein. Example 2 Preparation of D-Prolinyl-L-Prolinyl-L-Arginine Aldehyde Dihydrochloride (D-Pro-Pro-Arg-H·2HCl) Elemental Analysis calculated for C 16 H 30 N 6 O 3 Cl 2 : C 45.18, H 7.11, N 19.76; Found C 44.96, H 6.90, N 19.56. Example 3 Preparation of D-Homoprolinyl-L-Azetidinyl-L-Arginine Aldehyde Dihydrochloride (D-hPro-Azt-Arg-H·2HCl) Elemental Analysis calculated for C 16 H 34 N 6 O 5 Cl 2 : C 41.65, H 7.43, N 18.22; Found C 42.05, H 7.35, N 18.37. Example 4 Preparation of D-Thiazolidinyl-4-Carbonyl-L-Prolinyl-L-Arginine Aldehyde Dihydrochloride FAB-MS 371 (MH + ); [α] D = -36.2° (C-0.5/0.1 N HCl). Example 5 Preparation of D-2-Isopropyl-5,5-Dimethylthiazolidinyl-4-Carbonyl-L-Prolinyl-L-Arginine Aldehyde Dihydrochloride A) D-2,2,5,5-Tetramethylthiazolidine A solution of D-penicillamine (29.8 g, 0.2 mol) in acetone (1800 mL) was reacted with 12 N HCl (18.3 mL) at 50 °C for 4 hours. The reaction mixture was filtered, the filtrate was concentrated down in vacuo to 1500 mL and was allowed to stand at 4 °C for 24 hours. The solid was filtered and dried to give pure title compound (39.1 g, 86 percent yield): mp 188-191 °C. B) D-5,5-Dimethyl-2-isopropylthiazolidine A solution of D-2,2,5,5 tetramethylthiazolidine (A) (11.25 g, 0.050 mol) was dissolved in dioxane (150 mL), isobutyraldehyde (14 mL, 0.153 mol) was added and the reaction mixture heated 2 hours at reflux. The reaction mixture was cooled to room temperature and allowed to stand for 24 hours. The precipitate was filtered, and re-crystallized from ethanol (EtOH) (45 mL)/diethyl ether (125 mL) to afford pure title compound (9.0 g, 77 percent yield): mp 214-216 °C. C) D-2-Isopropyl-5,5-dimethylthiazolidinyl-4-carbonyl-L-prolinyl-L-arginine Aldehyde Dihydrochloride By substantially following the procedures of Steps B through H of Example 1, the title compound was prepared: FAB-MS 441 (MH + ); [α] D -88.4° (C=0.5/0.01 N HCl); Elemental Analysis calculated for C 20 H 40 N 6 O 4 Cl 2 S: C 45.20, H 7.57, N 15.81, S 6.03; Found C 45.44, H 7.39, N 15.86, S 5.87. Example 6 Preparation of trans-4-(2-Naphthyloxy)-D-Prolinyl-L-Prolinyl-L-Arginine Aldehyde Trihydrochloride Monohydrate A) N-Cbz- cis -4-hydroxy-D-proline methyl ester A 5 °C solution of (30 g; 229 mmol) of cis-4-hydroxy-D-proline in 115 mL of 2 N aq NaOH was treated simultaneously with 36 mL (252 mmol) of benzyl chloroformate and 115 mL of 2 N aq NaOH. After the pH of the reaction had stabilized, the mixture was washed with Et 2 O (2 x 150 mL) and was acidified to pH 2 with 5 N aq HCl. The reaction was extracted EtOAc (4 x 200 mL) and the combined EtOAc extracts were dried over Na 2 SO 4 and evaporated in vacuo to give 64.1 g of the crude N-Cbz-protected acid as a gum. A mixture of the crude acid and 33.0 g (239 mmol) of K 2 CO 3 in 300 mL of DMF was treated with 14.5 mL (233 mmol) of MeI in a dropwise manner. After stirring for 54 hours at room temperature, the reaction was poured into 300 mL of H 2 O and the mixture extracted with EtOAc (5 x 200 mL). The combined organic extracts were washed with H 2 O (3 x 200 mL), were dried over Na 2 SO 4 and were evaporated in vacuo to give 65.3 g of an oil. Purification by flash chromatography (SiO 2 ; 25 percent EtOAc in hexanes) afforded 47.3 g (169 mmol; 74 percent from cis-4-hydroxy-D-proline) of the title compound as a viscous oil. FD-MS m/e 279 (M + ); Elemental Analysis calculated for C 14 H 17 NO 5 : C 60.21, H 6.13, N 5.01; Found C 59.95, H 6.11, N 4.92. B) N-Cbz-trans-4-(2-naphthyloxy)-D-proline methyl ester A solution of 15.0 g (53.7 mmol) of N-Cbz- cis -4-hydroxy-D-proline methyl ester, 11.3 g (78.4 mmol) of β-naphthol, and 20.5 g (78.2 mmol) of triphenylphosphine in 300 mL of THF was treated with 12.3 mL (78.1 mmol) of diethyl azidodicarboxylate over 0.5 hour. The reaction was stirred at room temperature for 18 hours and was quenched by the addition of 100 mL sat'd aq NaCl. The two layers were separated and the organic solution dried (Na 2 SO 4 ). Evaporation of the solvent gave 46.2 g of an oil which was purified by flash chromatography (SiO 2 ; gradient of 25 percent to 50 percent EtOAc in hexanes) to afford 15.2 g (37.5 mmol; 70 percent) of the title compound. FD-MS m/e 405 (M + ); IR (film) 3014, 1749, 1705, 1630, 1422, 1357, 1179, 1121 cm -1 . Elemental Analysis calculated for C 24 H 23 NO 5 : C 71.10, H 5.72, N 3.46; Found C 71.04, H 5.73, N 3.59. C) trans -4-(2-naphthyloxy)-D-Proline-L-Proline-L-Arginine Aldehyde Trihydrochloride Monohydrate By substantially following the procedures of Example 1 except using lithium tri-t-butoxyalumino hydride, rather than lithium aluminum hydride, to reduce the coupled amino-protected Arg lactam, N-Cbz-trans-4-(2-naphthyloxy)-D-proline methyl ester was converted to the title compound which was isolated as the trihydrochloride monohydrate. FAB-MS 495 (MH + ); [α] D = -5.11° (C=0.01, MeOH). Elemental Analysis calculated for C 26 H 39 Cl 3 N 6 O 5 : C 50.21, H 6.32, N 13.51; Found C 50.11, H 6.07, N 13.72. Example 7 Preparation of D,L-Piperazin-2-oyl-L-Prolinyl-L-Arginine Aldehyde Dihydrochloride The title compound was prepared from D,L-piperazine-2-carboxylic acid dihydrochloride by substantially following the procedures of Example 1 except using lithium tri-t-butoxyaluminohydride, rather than lithium aluminum hydride, to reduce the coupled amino-protected Arg lactam: FAB-MS m/e 368 (MH + ); Elemental Analysis calculated for C 16 H 31 Cl 2 N 7 O 3 : C 43.64, H 7.10, N 22.26; Found C 43.17, H 7.78, N 15.21. Example 8 Preparation of D,L-Thiazolidinyl-2-carbonyl-L-prolinyl-L-arginine aldehyde dihydrochloride A) Cbz-D,L-thiazolidinyl-2-carbonyl-L-Prolinyl-L-Arginyl Lactam The title compound was prepared from D,L-thiazolidinyl-2-carboxylic acid by substantially following the procedures described in Example 7. FD-MS: m/e 636 (M + ) [α] D = -74.26° (C = 0.01, CH 2 Cl 2 ) Elemental Analysis calculated for C 31 H 36 N 6 O 7 S: C 58.48, H 5.70, N 13.20; Found C 58.49, H 5.57, N 12.95. B) Cbz-D,L-thiazolidinyl-2-carbonyl-L-Proline-Cbz-L-Arginine Aldehyde A -25 °C solution of 12.3 g (19.0 mmol) of Cbz-D,L-thiazolidinyl-2-carbonyl-L-prolyinyl-L-arginyl lactam in 200 mL THF was treated with 29 mL (1 M in THF; 29 mmol) of Li(t-BuO) 3 AlH solution at a rate that did not warm the reaction temperature to above -20 °C. The reaction was stirred at -25 °C for 3 hours and was poured into 100 mL HCl. The mixture was extracted with 1:1 THF-hexane (2 x 100 mL) and EtOAc (2 x 100 mL). The EtOAc layer was dried over Na 2 SO 4 and evaporated in vacuo to afford 6.96 g (10.9 mmol; 58 percent yield) of the crude product as a white foam. The presence of the desired product was confirmed by mass spec. [FD-MS; m/e 638 (M + )] and the mixture taken on to the next reaction without further purification. C) D,L-thiazolidinyl-2-carbonyl-L-prolinyl-L-arginine aldehyde dihydrochloride To a mixture of the protected aldehyde (B) (6.72 g; 10.5 mmol) and p-cresol (7.0 mL) was added 35 mL of liquid HF in a Teflon/Kel-F apparatus. The mixture was stirred at 0 °C for 20 min, and then the HF was removed in vacuo . The residue was triturated with Et 2 O to give a white solid which was purified by reverse phase chromatography using a 5 x 25 cm Vydac C 18 RPHPLC column using a gradient of 2 percent CH 3 CN in 0.5 percent aq HCl to 40 percent CH 3 CN in 0.5 percent aq HCl. The pure fractions were combined and lyopholized to afford 2.65 g (6.0 mmol; 60 percent) of the title compound as the dihydrochloride. FAB-MS m/e 370 (M + ) Elemental Analysis calculated for C 15 H 28 Cl 2 N 6 O 3 S: C 40.63, H 6.37, N 18.95; Found C 40.84, H 6.19, N 18.80. Example 9 Preparation of D,L-thiomorpholinyl-2-carbonyl-L-Proline-L-Arginine Aldehyde Dihydrochloride The title compound was prepared by substantially following the procedure used in the synthesis of D,L-thiazolidinyl-2-carbonyl-L-proline-arginine aldehyde dihydrochloride (Example 8): FAB-MS m/e 385 (M + ); [α] D = -58.43° (C = 0.01, MeOH); Elemental Analysis calculated for C 16 H 30 Cl 2 N 6 O 3 S: C 42.01, H 6.61, N 18.37; Found C 40.73, H 6.73, N 15.09. Example 10 Preparation of D-Cis-(4-phenoxy)Prolinyl-L-Prolinyl-L-Arginine Aldehyde Trihydrochloride Monohydrate By substantially following the procedures of Example 6, the title compound was prepared: FAB-MS 445 (MH + ); Elemental Analysis calculated for C 22 H 32 N 6 O 4 •3 HCl•H 2 O C 46.20, H 6.52, N 14.69; Found C 46.04, H 6.73, N 14.44. Example 11 Preparation of 4-(3-pyridyloxy)-D-prolinyl-L-prolinyl-L-arginine aldehyde hydrochloride hydrate A) N-CBz-trans-4-(3-pyridyloxy)-D-Proline Methyl Ester The title compound was prepared from 3-hydroxy pyridine and N-Cbz- cis -4-hydroxy-D-proline methyl ester by substantially following the procedure used in the preparation of N-Cbz- trans -4-(2-naphthyloxy)-D-proline methyl ester, Example 6, Steps A and B. FD-MS 356 (M + ); Elemental Analysis calculated for C 19 H 20 N 2 O 5 : C 64.04, H 5.66, N 7.86; Found C 64.22, H 5.81, N 7.76. B) 4-(3-pyridyloxy)-D-Proline-L-Proline-Arginine Aldehyde Hydrochloride Hydrate The title compound was prepared from N-Cbz-trans-4-(3-pyridyloxy)-D-proline methyl ester by substantially following the procedures of Example 7. FAB-MS 368 (M + ); Elemental Analysis calculated for C 21 H 34 ClN 7 O 5 : C 50.45, H 6.38, N 19.61; Found C 50.62, H 6.61, N 19.60. Example 12 Preparation of trans-4-phenylthio-D-prolinyl-L-prolinyl-L-arginine aldehyde trihydrochloride trihydrate A) N-Cbz-cis-4-tosyl-D-proline methyl ester A solution of 20 g (71.6 mmol) of N-Cbz- cis -4-hydroxy-D-proline methyl ester, 15 mL (107 mmol) of triethylamine and 0.4 g (3.3 mmol) of 4-dimethylaminopyridine in 200 mL of CHCl 3 was treated with 15.1 g (79.2 mmol) of p-toluenesulfonyl chloride in portions. The reaction was stirred at room temperature for 18 hours and was washed successively with 100 mL of H 2 O, 100 mL of 1 N aqueous citric acid, and 100 mL of H 2 O. The organic fraction was dried over Na 2 SO 4 and evaporated in vacuo to give 31.4 g of an oil which was purified by flash chromatography (SiO 2 ; 50 percent EtOAc in hexanes) to afford 18.2 g (42 mmol; 59 percent) of the title compound as a white solid. FD-MS m/e 433 (M + ); Elemental Analysis calculated for C 21 H 23 NO 7 S: C 58.19, H 5.35, N 3.23; Found C 58.43, H 5.33, N 3.16. B) N-Cbz- trans -4-Phenylthio-D-Proline ethyl ester Thiophenol (3.3 mL; 32.2 mmol) was added to a solution of 35.6 mmol of sodium ethoxide in 40 mL EtOH (generated from adding 820 mg of Na to 40 mL EtOH). The mixture was stirred for 15 min and was treated with 6.0 g (15 mmol) of solid N-Cbz-cis-4-tosyl-D-proline methyl ester. The reaction was stirred at 40 °C for 19 hours at which time it was cooled and diluted with 100 mL of H 2 O. The EtOH was evaporated in vacuo and the aqueous layer extracted with EtOAc (3 x 100 mL). The combined organic extracts were dried over Na 2 SO 4 and evaporated in vacuo to give 7.40 g of an oil which was purified by flash chromatography (SiO 2 ; 5 percent EtOAc in hexanes) to afford 4.60 g (12 mmol; 79 percent) of the title compound as a clear oil. FD-MS m/e 385 (M + ); Elemental Analysis calculated for C 14 H 17 NO 5 : C 65.43, H 6.01, N 3.63; Found C 65.39, H 6.01, N 3.85. C) trans-4-Phenylthio-D-Proline-L-Proline-Arginine Aldehyde Trihydrochloride Trihydrate The title compound was prepared from N-Cbz- trans -4-phenylthio-D-proline ethyl ester by substantially following the procedures used in the synthesis of D,L-thiazolidinyl-2-carbonyl-L-Proline-L-Arginine Aldehyde dihydrochloride, Example 8. FAB-MS m/e 461 (M + ). High Resolution Mass Spec. (HRMS) (MH + ), C 22 H 33 N 6 O 3 S. Theory 461.2341, Found: 461.2318. Elemental Analysis calculated for C 22 H 35 Cl 3 N 6 O 3 S•3H 2 O: C 42.35, H 6.62, N 13.47; Found C 42.46, H 5.73, N 13.53. The compounds of the invention are believed to selectively inhibit thrombin over other proteinases and nonenzyme proteins involved in blood coagulation without appreciable interference with the body's natural clot lysing ability (the compounds have a low inhibitory effect on fibrinolysis). Further, such selectivity is believed to permit use with thrombolytic agents' without substantial interference with thrombolysis and fibrinolysis. The invention in one of its aspects provides compounds useful in a method of inhibiting thrombin in mammals comprising administering to a mammal in need of treatment an effective (thrombin inhibiting) dose of a compound of formula I. In another of its aspects, the invention provides compounds useful in a method of treating a thromboembolic disorder comprising administering to a mammal in need of treatment an effective (thromboembolic disorder therapeutic and/or prophylactic amount) dose of a compound of formula I. The invention in another of its aspects provides compounds useful in a method of inhibiting coagulation in mammals comprising administering to a mammal in need of treatment an effective (coagulation inhibiting) dose of a compound of formula I. The thrombin inhibition, coagulation inhibition and thromboembolic disorder treatment contemplated by the present method includes both medical therapeutic and/or prophylactic treatment as appropriate. In a further embodiment the invention relates to treatment, in a human or animal, of conditions where inhibition of thrombin is required. The compounds of the invention are expected to be useful in animals, including man, in treatment or prophylaxis of thrombosis and hypercoagulability in blood and tissues. Disorders in which the compounds have a potential utility are in treatment or prophylaxis of thrombosis and hypercoagulability in blood and tissues. Disorders in which the compounds have a potential utility, in treatment and/or prophylaxis, include venous thrombosis and pulmonary embolism, arterial thrombosis, such as in myocardial ischemia, myocardial infarction, unstable angina, thrombosis-based stroke and peripheral arterial thrombosis. Further, the compounds have expected utility in the treatment or prophylaxis of atherosclerotic disorders (diseases) such as coronary arterial disease, cerebral arterial disease and peripheral arterial disease. Further, the compounds are expected to be useful together with thrombolytics in myocardial infarction. Further, the compounds have expected utility in prophylaxis for reocclusion after thrombolysis, percutaneous transluminal angioplasty (PTCA) and coronary bypass operations. Further, the compounds have expected utility in prevention of rethrombosis after microsurgery. Further, the compounds are expected to be useful in anticoagulant treatment in connection with artificial organs and cardiac valves. Further, the compounds have expected utility in anticoagulant treatment in hemodialysis and disseminated intravascular coagulation. A further expected utility is in rinsing of catheters and mechanical devices used in patients in vivo, and as an anticoagulant for preservation of blood, plasma and other blood products in vitro. Still further, the compounds have expected utility in other diseases where blood coagulation could be a fundamental contributing process or a source of secondary pathology, such as cancer, including metastasis, inflammatory diseases, including arthritis, and diabetes. The anti-coagulant compound is administered orally, parenterally e.g. by intravenous infusion (iv), intramuscular injection (im) or subcutaneously (sc). The specific dose of a compound administered according to this invention to obtain therapeutic and/or prophylactic effects will, of course, be determined by the particular circumstances surrounding the case, including, for example, the compound administered, the rate of administration, the route of administration, and the condition being treated. A typical daily dose for each of the above utilities is between about 0.01 mg/kg and about 1000 mg/kg. The dose regimen may vary e.g. for prophylactic use a single daily dose may be administered or multiple doses such as 3 or 5 times daily may be appropriate. In critical care situations a compound of the invention is administered by iv infusion at a rate between about 0.01 mg/kg/h and about 20 mg/kg/h and preferably between about 0.1 mg/kg/h and about 5 mg/kg/h. The method of this invention also is practiced in conjunction with a clot lysing agent e.g. tissue plasminogen activator (t-PA), modified t-PA, streptokinase or urokinase. In cases when clot formation has occurred and an artery or vein is blocked, either partially or totally, a clot lysing agent is usually employed. A compound of the invention can be administered prior to or along with the lysing agent or subsequent to its use, and preferably further is administered along with aspirin to prevent the reoccurrence of clot formation. The method of this invention is also practiced in conjunction with a platelet glycoprotein receptor (IIb/IIIa) antagonist, that inhibits platelet aggregation. A compound of the invention can be administered prior to or along with the IIb/IIIa antagonist or subsequent to its use to prevent the occurrence or reoccurrence of clot formation. The method of this invention is also practiced in conjunction with aspirin. A compound of the invention can be administered prior to or along with aspirin or subsequent to its use to prevent the occurrence or reoccurrence of clot formation. As stated above, preferably a compound of the present invention is administered in conjunction with a clot lysing agent and aspirin. This invention also provides pharmaceutical formulations for use in the above described therapeutic method. Pharmaceutical formulations of the invention comprise an effective thrombin inhibiting amount of a compound of formula I in association with a pharmaceutically acceptable carrier, excipient or diluent. For oral administration the antithrombotic compound is formulated in gelatin capsules or tablets which may contain excipients such as binders, lubricants, disintegration agents and the like. For parenteral administration the antithrombotic is formulated in a pharmaceutically acceptable diluent e.g. physiological saline (0.9 percent), 5 percent dextrose, Ringer's solution and the like. The compound of the present invention can be formulated in unit dosage formulations comprising a dose between about 0.1 mg and about 1000 mg. Preferably the compound is in the form of a pharmaceutically acceptable salt such as for example the sulfate salt, acetate salt or a phosphate salt. An example of a unit dosage formulation comprises 5 mg of a compound of the present invention as a pharmaceutically acceptable salt in a 10 ml sterile glass ampoule. Another example of a unit dosage formulation comprises about 10 mg of a compound of the present invention as a pharmaceutically acceptable salt in 20 ml of isotonic saline contained in a sterile ampoule. The compounds can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal. The compounds of the present invention are preferably formulated prior to administration. Another embodiment of the present invention is a pharmaceutical formulation comprising an effective amount of a compound of Formula I or a pharmaceutically acceptable salt or solvate thereof in association with a pharmaceutically acceptable carrier, diluent or excipient therefor. The active ingredient in such formulations comprises from 0.1 percent to 99.9 percent by weight of the formulation. By "pharmaceutically acceptable" it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The present pharmaceutical formulations are prepared by known procedures using well known and readily available ingredients. The compositions of this invention may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art. In making the compositions of the present invention, the active ingredient will usually be admixed with a carrier, or diluted by a carrier, or enclosed within a carrier which may be in the form of a capsule, sachet, paper or other container. When the carrier serves as a diluent, it may be a solid, semi-solid or liquid material which acts as a vehicle, excipient or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, (as a solid or in a liquid medium), soft and hard gelatin capsules, suppositories, sterile injectable solutions, sterile packaged powders, and the like. The following formulation examples are illustrative only. "Active ingredient," of course, means a compound according to Formula I or a pharmaceutically acceptable salt or solvate thereof. Formulation 1 Hard gelatin capsules are prepared using the following ingredients: Quantity (mg/capsule) Active ingredient 250 Starch, dried 200 Magnesium stearate 10 Total 460 mg Formulation 2 A tablet is prepared using the ingredients below: Quantity (mg/capsule) Active ingredient 250 Cellulose, microcrystalline 400 Silicon dioxide, fumed 10 Stearic acid 5 Total 665 mg The components are blended and compressed to form tablets each weighing 665 mg. Formulation 3 An aerosol solution is prepared containing the following components: Weight Active ingredient 0.25 Ethanol 25.75 Propellant 22 (Chlorodifluoromethane) 70.00 Total 100.00 The active compound is mixed with ethanol and the mixture added to a portion of the propellant 22, cooled to -30 °C and transferred to a filling device. The required amount is then fed to a stainless steel container and diluted with the remainder of the propellant. The valve units are then fitted to the container. Formulation 4 Tablets, each containing 60 mg of active ingredient, are made as follows: Active ingredient 60 mg Starch 45 mg Microcrystalline cellulose 35 mg Polyvinylpyrrolidone (as 10 % solution in water) 4 mg Sodium carboxymethyl starch 4.5 mg Magnesium stearate 0.5 mg Talc 1 mg Total 150 mg The active ingredient, starch and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly. The aqueous solution containing polyvinylpyrrolidone is mixed with the resultant powder, and the mixture then is passed through a No. 14 mesh U.S. sieve. The granules so produced are dried at 50 °C and passed through a No. 18 mesh U.S. Sieve. The sodium carboxymethyl starch, magnesium stearate and talc, previously passed through a No. 60 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 150 mg. Formulation 5 Capsules, each containing 80 mg of active ingredient, are made as follows: Active ingredient 80 mg Starch 59 mg Microcrystalline cellulose 59 mg Magnesium stearate 2 mg Total 200 mg The active ingredient, cellulose, starch, and magnesium stearate are blended, passed through a No. 45 mesh U.S. sieve, and filled into hard gelatin capsules in 200 mg quantities. Formulation 6 Suppositories, each containing 225 mg of active ingredient, are made as follows: Active ingredient 225 mg Saturated fatty acid glycerides 2,000 mg Total 2,225 mg The active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2 g capacity and allowed to cool. Formulation 7 Suspensions, each containing 50 mg of active ingredient per 5 ml dose, are made as follows: Active ingredient 50 mg Sodium carboxymethyl cellulose 50 mg Syrup 1.25 ml Benzoic acid solution 0.10 ml Flavor q.v. Color q.v. Purified water to total 5 ml The active ingredient is passed through a No. 45 mesh U.S. sieve and mixed with the sodium carboxymethyl cellulose and syrup to form a smooth paste. The benzoic acid solution, flavor and color are diluted with a portion of the water and added, with stirring. Sufficient water is then added to produce the required volume. Formulation 8 An intravenous formulation may be prepared as follows: Active ingredient 100 mg Isotonic saline 1,000 ml The solution of the above ingredients generally is administered intravenously to a subject at a rate of 1 ml per minute. The ability of the compounds of the present invention to be an effective and orally active thrombin inhibitor are evaluated in one or more of the following assays. The compounds provided by the invention (formula 1) selectively inhibit the action of thrombin in mammals. The inhibition of thrombin is demonstrated by in vitro inhibition of the amidase activity of thrombin as measured in an assay in which thrombin hydrolyzes the chromogenic substrate, N-benzoyl-L-phenylalanyl-L-valyl-L-arginyl-p-nitroanilide, N-benzoyl-L-Phe-L-Val-L-Arg-p-nitroanilide. The assay is carried out by mixing 50 µl buffer (0.03M Tris, 0.15M NaCl, pH 7.4) with 25 µl of human thrombin solution (purified human thrombin, Enzyme Research Laboratories, South Bend, Indiana, at 8 NIH units/ml) and 25 µl of test compound in a solvent (50% aqueous methanol (v:v)). Then 150 µl of an aqueous solution of the chromogenic substate (at 0.25 mg/ml) are added and the rates of hydrolysis of the substrate are measured by monitoring the reactions at 405 nm for the release of p-nitroaniline. Standard curves are constructed by plotting free thrombin concentration against hydrolysis rate. The hydrolysis rates observed with test compounds are then converted to "free thrombin" values in the respective assays by use of the standard curves. The bound thrombin (bound to test compound) is calculated by subtracting the amount of free thrombin observed in each assay from the known initial amount of thrombin used in the assay. The amount of free inhibitor in each assay is calculated by subtracting the number of moles of bound thrombin from the number of moles of added inhibitor (test compound). The Kass value is the hypothetical equilibrium constant for the reaction between thrombin and the test compound (I). Kass = [Thrombin-I] [(Thrombin) x (I)] Kass is calculated for a range of concentrations of test compounds and the mean value reported in units of liter per mole. By substantially following the procedures described above for human thrombin, and using other human blood coagulation system serine proteases and using fibrinolytic system serine proteases, with the appropriate chromogenic substrates, identified below, the selectivity of the compounds of the present invention with respect to the coagulation factor serine proteases and to the fibronolytic serine proteases are evaluated as well as their substantial lack of interference with human plasma clot fibrinolysis. Human factors X, Xa, IXa, XIa, and XIIa are purchased from Enzyme Research Laboratories, South Bend, Indiana; human urokinase from Leo Pharmaceuticals, Denmark; and recombinant activated Protein C (aPC) is prepared at Eli Lilly and Co. substantially according to U.S. Patent 4,981,952. Chromogenic substrates: N-Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (for factor Xa); N-Cbz-D-Arg-Gly-Arg-p-nitroanilide (for factor IXa assay as the factor Xa substrate); Pyroglutamyl-Pro-Arg-p-nitroanilide (for Factor XIa and for aPC); H-D-Pro-Phe-Arg-p-nitroanilide (for factor XIIa); and Pyroglutamyl-Gly-Arg-p-nitroanilide (for urokinase); are purchased from KabiVitrum, Stockholm, Sweden, or from Midwest Biotech, Fishers, Indiana. Bovine trypsin is purchased from Worthington Biochemicals, Freehold, New Jersey, and human plasma kallikrein from Kabi Vitrum, Stockholm, Sweden. Chromogenic substrate H-D-Pro-Phe-Arg-p-nitroanilide for plasma kallikrein is purchased from Kabi Vitrum, Stockholm, Sweden. N-Benzoyl-Phe-Val-Arg-p-nitroanilide, the substrate for human thrombin and for trypsin, is synthesized according to procedures described above for the compounds of the present invention, using known methods of peptide coupling from commercially available reactants, or purchaed from Midwest Biotech, Fishers, Indiana. Human plasmin is purchased from Boehringer Mannheim, Indianapolis, Indiana; nt-PA is purchased as single chain activity reference from American Diagnostica, Greenwich, Connecticut; modified-t-PA6 (mt-PA6) is prepared at Eli Lilly and Company by procedure known in the art (See, Burck, et al., J. Biol. Chem. , 265 , 5120-5177 (1990). Plasmin chromogenic substrate H-D-Val-Leu-Lys-p-nitroanilide and tissue plasminogen activator (t-PA) substrate H-D-Ile-Pro-Arg-p-nitroanilide are purchased from Kabi Vitrum, Stockholm, Sweden. In the chromogenic substrates described above the three-letter symbols Ile, Glu, Gly, Pro, Arg, Phe, Val, Leu and Lys are used to indicate the corresponding amino acid group isoleucine, glutamic acid, glycine, proline, arginine, phenylalanine, valine, leucine and lysine, respectively. Table 1 which follows lists the Kass values obtained with the indicated compound represented by formula I. Human Thrombin Inhibition Levels Example Kass X 10 6 (1/mole) 1 46 2 12 3 45 4 5 5 4 6 21 7 121 8 27 9 10 25 11 12 37 Thrombin inhibitors preferably should spare fibrinolysis induced by urokinase, tissue plasminogen activator (t-PA) and steptokinase. This would be important to the therapeutic use of such agents as an adjunct to streptokinase, t-PA or urokinase thrombolytic therapy and to the use of such agents as an endogenous fibrinolysis-sparing (with respect to t-PA and urokinase) antithrombotic agents. In addition to the lack of interference with the amidase activity of the fibrinolytic proteases, such fibrinolytic system sparing can be studied by the use of human plasma clots and their lysis by the respective fibrinolytic plasminogen activators. Materials Dog plasma is obtained from conscious mixed-breed hounds (either sex Hazelton-LRE, Kalamazoo, Michigan, U.S.A.) by venipuncture into 3.8 percent citrate. Fibrinogen is prepared from fresh dog plasma and human fibrinogen is prepared from in-date ACD human blood at the fraction I-2 according to previous procedures and specifications. Smith, Biochem. J. , 185 , 1-11 (1980); and Smith, et al., Biochemistry , 11 , 2958-2967, (1972). Human fibrinogen (98 percent pure/plasmin free) is from American Diagnostica, Greenwich, Connecticut. Radiolabeling of fibrinogen I-2 preparations is performed as previously reported. Smith, et al., Biochemistry , 11 , 2958-2967, (1972). Urokinase is purchased form Leo Pharmaceuticals, Denmark, as 2200 Ploug units/vial. Streptokinase is purchased from Hoechst-Roussel Pharmaceuticals, Somerville, New Jersey. Methods - Effects on Lysis of Human Plasma Clots by t-PA Human plasma clots are formed in micro test tubes by adding 50 ul thrombin (73 NIH unit/ml) to 100 ul human plasma which contains 0.0229 uCi 125-iodine labeled fibrinogen. Clot lysis is studied by overlaying the clots with 50 ul of urokinase or streptokinase (50, 100, or 1000 unit/ml) and incubating for 20 hours at room temperature. After incubation the tubes are centrifuged in a Beckman Microfuge. 25 ul of supernate is added into 1.0 ml volume of 0.03 M tris/0.15 M NaCl buffer for gamma counting. Counting controls 100 percent lysis are obtained by omitting thrombin (and substituting buffer). The thrombin inhibitors are evaluated for possible interference with fibrinolysis by including the compounds in the overlay solutions at 1, 5, and 10 ug/ml concentrations. Rough approximations of IC 50 values are estimated by linear extrapolations from data points to a value which would represent 50 percent of lysis for that particular concentration of fibrinolytic agent. Anticoagulant Activity Materials Dog plasma and rat plasma are obtained from conscious mixed-breed hounds (either sex, hazelton-LRE, Kalamazoo, Michigan, U.S.A.) or from anesthetized male Sprague-Dawley rats (Harlan Sprague-Dawley, Inc., Indianapolis, Indiana, U.S.A.) by venipuncture into 3.8 percent citrate. Fibrinogen is prepared from in-date ACD human blood as the fraction I-2 according to previous procedures and specifications. Smith, Biochem. J. , 185 , 1-11 (1980); and Smith, et al., Biochemistry, 11 , 2958-2967 (1972). Human fibrinogen is also purchased as 98 percent pure/plasmin free from American Diagnostica, Greenwich, Connecticut. Coagulation reagents ACTIN, Thromboplastin, and Human plasma are from Baxter Healthcare Corp., Dade Division, Miami, Florida. Bovine thrombin from Parke-Davis (Detroit, Michigan) is used for coagulation assays in plasma. Methods Anticoagulation Determinations Coagulation assay procedures are as previously described. Smith, et al., Thrombosis Research , 50 , 163-174 (1988). A CoAScreener coagulation instrument (American LABor, Inc.) is used for all coagulation assay measurements. The prothrombin time (PT) is measured by adding 0.05 ml saline and 0.05 ml Thromboplastin-C reagent to 0.05 ml test plasma. The activated partial thromboplastin time (APTT) is measured by incubation of 0.05 ml test plasma with 0.05 ml Actin reagent for 120 seconds followed by 0.05 ml CaCl 2 (0.02 M). The thrombin time (TT) is measured by adding 0.05 ml saline and 0.05 ml thrombin (10 NIH units/ml) to 0.05 ml test plasma. The compounds of formula I are added to human or animal plasma over a wide range of concentrations to determine prolongation effects on the APTT, PT, and TT assays. Linear extrapolations are performed to estimate the concentrations required to double the clotting time for each assay. Animals Male Sprague Dawley rats (350-425 gm, Harlan Sprague Dawley Inc., Indianapolis, IN) are anesthetized with xylazine (20 mg/kg, s.c.) and ketamine (120 mg/kg, s.c.) and maintained on a heated water blanket (37 °C). The jugular vein(s) is cannulated to allow for infusions. Arterio-Venous shunt model The left jugular vein and right carotid artery are cannulated with 20 cm lengths of polyethylene PE 60 tubing. A 6 cm center section of larger tubing (PE 190) with a cotton thread (5 cm) in the lumen, is friction fitted between the longer sections to complete the arterio-venous shunt circuit. Blood is circulated through the shunt for 15 min before the thread is carefully removed and weighed. The weight of a wet thread is subtracted from the total weight of the thread and thrombus (see J.R. Smith, Br J Pharmacol, 77 :29,1982). FeCl 3 model of arterial injury The carotid arteries are isolated via a midline ventral cervical incision. A thermocouple is placed under each artery and vessel temperature is recorded continuously on a strip chart recorder. A cuff of tubing (0.058 ID x 0.077 OD x 4 mm, Baxter Med. Grade Silicone), cut longitudinally, is placed around each carotid directly above the thermocouple. FeCl3 hexahydrate is dissolved in water and the concentration (20 percent) is expressed in terms of the actual weight of FeCl 3 only. To injure the artery and induce thrombosis, 2.85 ul is pipetted into the cuff to bathe the artery above the thermocouple probe. Arterial occlusion is indicated by a rapid drop in temperature. The time to occlusion is reported in minutes and represents the elapsed time between application of FeCl 3 and the rapid drop in vessel temperature (see K.D. Kurz, Thromb. Res ., 60 :269,1990). Spontaneous thrombolysis model In vitro data suggests that peptide thrombin inhibitors inhibit thrombin and at higher concentration may inhibit, other serine proteases, such as plasmin and tissue plasminogen activator. To assess if the compounds inhibit fibrinolysis in vivo, the rate of spontaneous thrombolysis is determined by implanting a labeled whole blood clot into the pulmonary circulation. Rat blood (1 ml) is mixed rapidly with bovine thrombin (4 IU, Parke Davis) and 125 I human fibrogen (5 µCi, ICN), immediately drawn into silastic tubing and incubated at 37 °C for 1 hour. The aged thrombus is expelled from the tubing, cut into 1 cm segments, washed 3X in normal saline and each segment is counted in a gamma counter. A segment with known counts is aspirated into a catheter that is subsequently implanted into the jugular vein. The catheter tip is advanced to the vicinity of the right atrium and the clot is expelled to float into the pulmonary circulation. One hour after implant, the heart and lungs are harvested and counted separately. Thrombolysis is expressed as a percentage where: % Thrombolysis = (injected cpm - lung cpm) injected cpm x 100 The fibrinolytic dissolution of the implanted clot occurs time-dependently (see J.P. Clozel, Cardiovas. Pharmacol ., 12 :520, 1988). Coagulation parameters Plasma thrombin time (TT) and activated partial thromboplastin time (APTT) are measured with a fibrometer. Blood is sampled from a jugular catheter and collected in syringe containing sodium citrate (3.8 percent, 1 part to 9 parts blood). To measure TT, rat plasma (0.1 ml) is mixed with saline (0.1 ml) and bovine thrombin (0.1 ml, 30 U/ml in TRIS buffer; Parke Davis) at 37 °C. For APTT, plasma (0.1 ml) and APTT solution (0.1 ml, Organon Teknika) are incubated for 5 minutes (37 °C) and CaCl 2 (0.1 ml, 0.025M) is added to start coagulation. Assays are done in duplicate and averaged. Index of Bioavailability A measure of bioactivity, plasma thrombin time (TT), serves as a substitute for the assay of parent compound on the assumption that increments in TT resulted from thrombin inhibition by parent only. The time course of the effect of the thrombin inhibitor upon TT is determined after i.v bolus administration to anesthetized rats and after oral treatment of fasted conscious rats. Due to limitations of blood volume and the number of points required to determine the time course from time of treatment to the time when the response returns to pretreatment values, two populations of rats are used. Each sample population represents alternating sequential time points. The average TT over the time course is used to calculate area under the curve (AUC). The index of bioavailability is calculated by the formula shown below and is expressed as percent relative activity. The area under the curve (AUC) of the plasma TT time course is determined and adjusted for the dose. This index of bioavailability is termed "% Relative Activity" and is calculated as %Relative Activity = AUC po AUC iv X Dose iv Dose po X100 Compounds Compound solutions are prepared fresh daily in normal saline and are injected as a bolus or are infused starting 15 minutes before and continuing throughout the experimental perturbation which is 15 minutes in the arteriovenous shunt model and 60 minutes in the FeCl 3 model of arterial injury and in the spontaneous thrombolysis model. Bolus injection volume is 1 ml/kg for i.v., and 5 ml/kg for p.o. and infusion volume is 3 ml/hr. Statistics Results are expressed as means +/- SEM. One-way analysis of variance is used to detect statistically significant differences and then Dunnett's test is applied to determine which means are different. Significance level for rejection of the null hypothesis of equal means is P<0.05. Index of Bioavailability Example Percent Relative Activity 1 38% 2 36% 3 32% 4 16% 5 6 7 19% 8 5% 9 10 16% 11 12 Animals Male dogs (Beagles; 18 months - 2 years; 12-13 kg, Marshall Farms, North Rose, New York 14516) are fasted overnight and fed Purina certified Prescription Diet (Purina Mills, St. Louis, Missouri) 240 minutes after dosing. Water is available ad libitum. The room temperature is maintained between 66-74°F; 45-50 percent relative humidity; and lighted from 0600-1800 hours. Pharmacokinetic model. Test compound is formulated immediately prior to dosing by dissolving in sterile 0.9 percent saline to a 5 mg/ml preparation. Dogs are given a single 2 mg/kg dose of test compound by oral gavage. Blood samples (4.5 ml) are taken from the cephalic vein at 0.25, 0.5, 0.75, 1,2,3,4 and 6 hours after dosing. Samples are collected in citrated Vacutainer tubes and kept on ice prior to reduction to plasma by centrifugation. Plasma samples are derivatized with dinitrophenylhydrazine and analyzed by HPLC (Zorbax SB-C8 column) eluting with methanol/500 mM sodium acetate adjusted to pH7 with phosphoric acid (60:40, v/v). Plasma concentration of test compound is recorded and used to calculate the pharmacokinetic parameters: elimination rate constant, Ke; total clearance, Clt; volume of distribution, V D ; time of maximum plasma test compound concentration, Tmax; maximum concentration of test compound of Tmax, Cmax; plasma half-life, t0.5; and area under the curve, A.U.C.; fraction of test compound absorbed, F. Pharmacokinetic Parameters Example Ke (min-1) Clt/F (L/hr.kg) VD/F (L/kg) Tmax (hr) Cmax (ng/ml) t0.5 (min) A.U.C. (ng.hr/ml) o-infinity 1 0.0104 0.437 0.729 1-2 1676 67 4651 ±0.0009 ±0.032 ±0.120 ±202 range=57-86 ±319 Canine Model of Coronary Artery Thrombosis Surgical preparation and instrumentation of the dogs are as described in Jackson, et al., Circulation , 82 , 930-940 (1990). Mixed-breed hounds (aged 6-7 months, either sex, Hazelton-LRE, Kalamazoo, MI, U.S.A.) are anesthetized with sodium pentobarbital (30 mg/kg intravenously, i.v.), intubated, and ventilated with room air. Tidal volume and respiratory rates are adjusted to maintain blood PO 2 , PCO 2 , and pH within normal limits. Subdermal needle electrodes are inserted for the recording of a lead II ECG. The left jugular vein and common carotid artery are isolated through a left mediolateral neck incision. Arterial blood pressure (ABP) is measured continuously with a precalibrated Millar transducer (model (MPC-500, Millar Instruments, Houston, TX, U.S.A.) inserted into the carotid artery. The jugular vein is cannulated for blood sampling during the experiment. In addition, the femoral veins of both hindlegs are cannulated for administration of test compound. A left thoracotomy is performed at the fifth intercostal space, and the heart is suspended in a pericardial cradle. A 1- to 2-cm segment of the left circumflex coronary artery (LCX) is isolated proximal to the first major diagonal ventricular branch. A 26-gauge needle-tipped wire anodal electrode (Teflon-coated, 30-gauge silverplated copper wire) 3-4 mm long is inserted into the LCX and placed in contact with the intimal surface of the artery (confirmed at the end of the experiment). The stimulating circuit is completed by placing the cathode in a subcutaneous (s.c.) site. An adjustable plastic occluder is placed around the LCX, over the region of the electrode. A precalibrated electromagnetic flow probe (Carolina Medical Electronics, King, NC, U.S.A.) is placed around the LCX proximal to the anode for measurement of coronary blood flow (CBF). The occluder is adjusted to produce a 40-50 percent inhibition of the hyperemic blood flow response observed after 10-s mechanical occlusion of the LCX. All hemodynamic and ECG measurements are recorded and analyzed with a data acquisition system (model M3000, Modular Instruments, Malvern, PA. U.S.A.). Thrombus Formation and Compound Administration Regimens Electrolytic injury of the intima of the LCX is produced by applying 100-µA direct current (DC) to the anode. The current is maintained for 60 min and then discontinued whether the vessel has occluded or not. Thrombus formation proceeds spontaneously until the LCX is totally occluded (determined as zero CBF and an increase in the S-T segment). Compound administration is started after the occluding thrombus is allowed to age for 1 hour. A 2-hour infusion of the compounds of the present invention at doses of 0.5 and 1 mg/kg/hour is begun simultaneously with an infusion of thrombolytic agent (e.g. tissue plasminogen activator, streptokinase, APSAC). Reperfusion is followed for 3 hour after administration of test compound. Reocclusion of coronary arteries after successful thrombolysis is defined as zero CBF which persisted for ≥ 30 minutes. Hematology and template bleeding time determinations Whole blood cell counts, hemoglobin, and hematocrit values are determined on a 40-µl sample of citrated (3.8 percent ) blood (1 part citrate:9 parts blood) with a hematology analyzer (Cell-Dyn 900, Sequoia-Turner. Mount View, CA, U.S.A.). Gingival template bleeding times are determined with a Simplate II bleeding time device (Organon Teknika Durham, N.C., U.S.A.). The device is used to make 2 horizontal incisions in the gingiva of either the upper or lower left jaw of the dog. Each incision is 3 mm wide x 2 mm deep. The incisions are made, and a stopwatch is used to determine how long bleeding occurs. A cotton swab is used to soak up the blood as it oozes from the incision. Template bleeding time is the time from incision to stoppage of bleeding. Bleeding times are taken just before administration of test compound (0 min), 60 min into infusion, at conclusion of administration of the test compound (120 min), and at the end of the experiment. All data are analyzed by one-way analysis of variance (ANOVA) followed by Student-Neuman-Kuels post hoc t test to determine the level of significance. Repeated-measures ANOVA are used to determine significant differences between time points during the experiments. Values are determined to be statistically different at least at the level of p<0.05. All values are mean ± SEM. All studies are conducted in accordance with the guiding principles of the American Physiological Society. Further details regarding the procedures are described in Jackson, et al., J. Cardiovasc . Pharmacol., 21 , 587-599 (1993). Canine Model of Coronary Artery Thromboses Example Dose Time to Occlusion mg/kg.hr (min) 1 0.25 60 0.50 150 1.00 >225 The compound of Example 1 was also evaluated in the Template Bleeding Time assay at 0.25, 0.50 and 1.0 mg/kg.hr. Over a 240 minute time, the compound of Example 1 showed no significant effect on template bleeding time.

Claims: A compound having the formula I wherein X is an unsubstituted or substituted group selected from homoprolinyl, prolinyl, thiazolidinoyl, isothiazolidinoyl, thiomorpholinoyl, piperazinoyl, morpholinoyl, oxazolidinoyl and isoxazolidinoyl, where n is 1-3 and m is 2 or 3 and where in a sulfur containing group the sulfur may be oxidized with one or two oxygen atoms; Y is or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt thereof; and further wherein when X is a substituted group, including the fused bicyclic ring groups, there can be one to three of the same or different substituents that will afford a stable structure selected from halo, hydroxyl, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, amino (-NH 2 ), mono(C 1 -C 4 alkyl)amino, di(C 1 -C 4 alkyl)amino, mercapto, C 1 -C 4 alkylthio (-S(O) p (C 1 -C 4 alkyl)), -NHS(O) p (C 1 -C 4 alkyl), -NHC(O)C 1 -C 4 alkyl, -S(O) p NH2, -S(O) p NH(C 1 -C 4 alkyl), -S(O) p N(C 1 -C 4 alkyl) 2 , substituted or unsubstituted phenoxy, substituted or unsubstituted naphthyloxy, substituted or unsubstituted pyridyloxy, substituted or unsubstituted phenylthio; p is 0, 1 or 2; and the substituents on the phenoxy, naphthyloxy, pyridyloxy and phenyl thio groups are one or two of the same or different substituents selected from halo, hydroxyl, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, amino (-NH 2 ), mono(C 1 -C 4 alkyl)amino, di(C 1 -C 4 alkyl)amino, mercapto, C 1 -C 4 alkylthio (-S(O) p (C 1 -C 4 alkyl)), -NHS(O) p (C 1 -C 4 alkyl), -NHC(O)C 1 -C 4 alkyl, -S(O) p NH2, -S(O) p NH(C 1 -C 4 alkyl), -S(O) p N(C 1 -C 4 alkyl) 2 , and p is 0, 1 or 2. A compound or salt or solvate thereof as claimed in Claim 1 wherein X is unsubstituted or monosubstituted homoprolinyl, unsubstituted or monosubstituted prolinyl, unsubstituted or monosubstituted piperazinoyl. A compound or salt or solvate thereof as claimed in Claim 2 wherein X is homoprolinyl, prolinyl, 4-phenoxyprolinyl or piperazinoyl. A compound or salt or solvate thereof as claimed in Claim 1 or 2 wherein, alkyl by itself or as part of another substituent is methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl or sec-butyl; and halo is chloro, fluoro, bromo or iodo. A compound or salt or solvate thereof as claimed in Claim 1 which compound is selected from: D-homoprolinyl-L-prolinyl-L-arginine aldehyde; D-prolinyl-L-prolinyl-L-arginine aldehyde; D-homoprolinyl-L-azetidin-2-carbonyl-L-arginine aldehyde; D-prolinyl-L-azetidin-2-carbonyl-L-arginine aldehyde; and D-(4-phenoxy)prolinyl-L-prolinyl-L-arginine aldehyde. A pharmaceutical formulation comprising in association with a pharmaceutically acceptable carrier, diluent, or excipient, a compound of the formula I, or a pharmaceutically acceptable salt or solvate thereof, as claimed in any one of the Claims 1-5. A process for preparing a compound having the formula I as claimed in any one of Claims 1-5 which comprises removing simultaneously or sequentially the protecting group(s) P of a corresponding compound of Formula II wherein P on the guanidino group represents an amino protecting group and (P)X represent a radical X , which may bear an independently selected amino protecting group P for a compound of formula I in which X includes a basic NH moiety; whereafter, when a salt of the compound of formula I is required, forming the salt with a pharmaceutically acceptable acid.

Inventors: KURZ KENNETH DEAN, ROTHENBERGER ROBERT BERKEY, SALL DANIEL JON, SHUMAN ROBERT THEODORE, SMITH GERALD FLOYD, WILEY MICHAEL ROBERT, KURZ, KENNETH DEAN, ROTHENBERGER, ROBERT BERKEY, SALL, DANIEL JON, SHUMAN, ROBERT THEODORE, SMITH, GERALD FLOYD, WILEY, MICHAEL ROBERT
Applicants: LILLY CO ELI, ELI LILLY AND COMPANY
Application year: 1995
Main classification: C07D401
IPC codes: C07C279, A61P7, C07D403, A61K38, C07K5