Patent Publication Number: US-2004044075-A1

Title: Alpha acyloxyacetamides for kallikrein and urokinase inhibition

Description:
RELATED APPLICATION  
     [0001] This application claims the benefit of U.S. Provisional Application No. 60/385,264, filed May 31, 2002. The entire teachings of the above application is incorporated herein by reference. 
    
    
     
       BACKGROUND OF THE INVENTION  
       [0002] Human cancers generally require proteolytic enzymes to invade adjacent tissues and form metastases. The formation of new blood vessels is typically required for tumor growth. Urokinase (uPA), a member of the serine protease family, promotes tumor cell migration and invasion by converting the inactive zymogen plasminogen into the active serine protease, plasmin, which then cleaves extracellular matrix components including laminin, fibronectin and collagen. This process also promotes angiogenesis, the formation of new blood vessels, through the release of matrix-bound growth factors such as VEGF and bFGF. It has been shown that inhibition of uPA can decrease tumor size or even cause complete remission of cancers in mice. New and more potent inhibitors of urokinase are therefore needed for the treatment of human cancers.  
       [0003] Plasma kallikrein is responsible for cleaving kininigen to form bradykinin, a mediator of pain and inflammation. Bradykinin has been implicated in inflammatory diseases such as inflammatory bowel disease, rheumatoid arthritis, asthma and allergic rhinitis. Inhibitors of the kallikrein enzyme can provide clinical benefits by preventing production of bradykinin. In addition, kallikrein has been found to activate urokinase.  
       [0004] Thus, inhibitors of kallikrein should be effective against urokinase and therefore can also be used as treatments against cancers. Thus, new inhibitors of kallikrein are needed for the treatment of pain, inflammatory diseases and cancer.  
       SUMMARY OF THE INVENTION  
       [0005] It has now been found that certain alpha aryl acyloxyacetamides are potent inhibitors of kallikrein and urokinase. For example, many of the alpha aryl acyloxyacetamides shown in Tables 1 and 5 have an IC 50  less than 5.0 μM, and some less than 1.0 μM for the inhibition of kallikrein or urokinase (see Examples 5 and 9). Moreover, the alpha phenyl acyloxyacetamides shown in Tables 2 and 3 demonstrated analgesic activity when tested in mice with an acetic acid writhing assay (see Example 6) and/or anti-inflammatory activity when tested in rats with a carageenan assay (see Example 7); and Compound 26 had activity in a rat model of inflammatory bowel disease (Example 8). Based on these discoveries, novel alpha aryl acyloxyacetamides, methods of treating a subject with an inflammatory disorder and methods of treating a subject with cancer are disclosed herein.  
       [0006] One embodiment of the present invention is a compound represented by Structural Formula (I):  
                 
 
       [0007] R 1  is a substituted or unsubstituted aryl group or alkyl group.  
       [0008] R 2  is a substituted or unsubstituted aryl group or cycloalkyl group.  
       [0009] Ar is a substituted or unsubstituted aryl group.  
       [0010] X is a —CH 2 —, —O—, —S— or —CO.  
       [0011] m is an integer from zero to two.  
       [0012] n is an integer from 0-2 when X is —O—, —S— or 1-2 when X is —CH 2 — or —CO—.  
       [0013] In one aspect, when Ar is a substituted or unsubstituted phenyl group and X is —CH 2 — in Structural Formula (T), then R 1  is a cyclopropyl group or R 2  is a substituted or unsubstituted indolyl, pyrimidinyl, benzothienyl, cyclopentyl or cyclohexyl group.  
       [0014] Another embodiment of the present invention is a method of inhibiting kallikrein activity in a subject in need of a such inhibition. The method comprises administering to the subject an effective amount of a compound represented by Structural Formula (I).  
       [0015] Yet another embodiment of the present invention is a method of inhibiting urokinase activity in a subject in need of a such inhibition. The method comprises administering to the subject an effective amount of a compound represented by Structural Formula (I).  
       [0016] The compounds disclosed herein are effective inhibitors of kallikrein and/or urokinase and thus are useful as analgesics, in the treatment of cancer and/or the treatment of inflammatory diseases.  
       DETAILED DESCRIPTION OF THE INVENTION  
       [0017] The present invention is directed to compounds represented by Structural Formula (I) and the use of such compounds for inhibiting kallikrein activity and/or urokinase activity in a subject in need of such treatment. Definitions of the terms used to describe these inventions are provided below.  
       [0018] The term “aryl group”, e.g., the aryl groups represented by R 1 , R 2  and Ar, refers to carbocyclic aromatic groups such as phenyl, naphthyl, and anthracyl, and heteroaryl groups such as imidazolyl, isoimidazolyl, thienyl, furanyl, pyridyl, pyrimidyl, pyranyl, pyrazolyl, pyrrolyl, pyrazinyl, thiazolyl, isothiazolyl, oxazolyl, isooxazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, and tetrazolyl.  
       [0019] Aryl groups, such as the aryl groups represented by R 1 , R 2  and Ar, also include fused polycyclic aromatic ring systems in which a carbocyclic aromatic ring or heteroaryl ring is fused to one or more other heteroaryl rings. Examples include benzothienyl, benzofuranyl, indolyl, quinolinyl, benzothiazolyl, benzoisothiazolyl, benzooxazolyl, benzoisooxazolyl, benzimidazolyl, quinolinyl, isoquinolinyl and isoindolyl.  
       [0020] An alkyl group is a straight, branched or cyclic non-aromatic hydrocarbon which is completely saturated or which contains one or more units of unsaturation. Typically, a straight or branched alkyl group has from 1 to about 10 carbon atoms, preferably from 1 to about 4, and a cyclic aliphatic group has from 3 to about 10 carbon atoms, preferably from 3 to about 8. Examples of suitable straight or branched alkyl group include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl or octyl; and examples of suitable cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. A C1-C10 straight or branched alkyl group or a C3-C8 cyclic alkyl group are also referred to as a “lower alkyl” group.  
       [0021] Suitable substituents for an alkyl group, an aryl group (e.g., the aryl group represented by R 1 , R 2  and Ar) or a non-aromatic heterocyclic group are those which do not substantially interfere with the ability of the compound to inhibit kallikrein and/or urokinase activity. Examples of suitable substituents for any aryl (e.g., the aryl group represented by R 1 , R 2  or Ar), alkyl or non-aromatic heterocyclic group include —OH, halogen (—Br, —Cl, —I and —F), —OR a , —O—COR a , —COR a , —CN, —NO 2 , —COOH, —SO 3 H, —NH 2 , —NHR a , —N(R a R b ), —COOR a , —CHO, —CONH 2 , —CONHR a , —CON(R a R b ), —NHCOR a , —NR c COR a , —NHCONH 2 , NHCONR a H, —NHCON(R a R b ), —NR c CONH 2 , —NR c CONR a H, —NR c CON(R a R b ), —C(—NH)—NH 2 , —C(═NH)—NHR a , —C(═NH)—N(R a R b ), —C(═NR c )—NH 2 , —C(═NR c )—NHR a , —C(═NR c )—N(R a R b ), —NH—C(═NH)—NH 2 , —NH—C(═NH)—NHR a , —NH—C(═NH)—N(R a R b ), —NH—C(═NR c )—NH 2 , —NH—C(═NR c )—NHR a , —NH—C(═NR c )—N(R a R b ), _NR d H—C(═NH)—NH 2 , _NR d —C(═NH)—NHR a , _NR d —C(═NH)N(R a R b ), N(R a R b ) —NR d —C(═NR c )—NH 2 , —NR d —C(═NR c )-NHR a , —NR d —C(═NR c )-N(R a R b ), —SO 2 NH 2 , —SO 2 NHR a , —SO 2 NR a R b , —SH, —SO k R a  (k is 0, 1 or 2) and —NH—C(═NH)—NH 2 . R a -R d  are each independently an alkyl, substituted alkyl, benzyl, substituted benzyl, aryl or substituted aryl group, preferably an alkyl, benzylic or aryl group. In addition, —NR a R d , taken together, can also form a substituted or unsubstituted non-aromatic heterocyclic group, such as pyrollidinyl, piperidinyl, morpholinyl and thiomorpholinyl. A substituted aryl (including the aryl groups represented by R 1 , R 2  and Ar), alkyl and non-aromatic heterocyclic group can have more than one substitutent.  
       [0022] Preferred substituents for an aryl group (e.g., the aryl group represented by R 1 , R 2  and Ar) include methylenedioxy, —CO—NH 2 , —O(C1-C4 alkyl), —F, —Cl, —Br, —CN, C1-C4 alkyl, C1-C4 haloalkyl, —CF 3 , —SO 2 (C1-C4 alkyl), —COO(C1-C4 alkyl) and —S(C1-C4 alkyl). For example, zero, one or more of these substituents are typically present when R 1  is an optionally substituted phenyl group, Ar is an optionally substituted phenyl or naphthyl group and R 2  is an optionally substituted phenyl or pyrimidyl group.  
       [0023] Particularly preferred substituents for a phenyl group represented by R 1  and R 2  are methoxy or methylenedioxy, for example, 3,4-dimethoxyphenyl or 3,4,5-trimethoxyphenyl, 3,4-methylenedioxy for R 1  and monosubstituted phenyl (e.g., 4-methoxyphenyl) and disubstituted phenyl (e.g., 2,5-dimethoxyphenyl) for R 2 . Ar is typically substituted at the 3, 4 and/or 5 position; cyano and bromo are particularly preferred as substitutents for a phenyl group represented by Ar.  
       [0024] Preferred substituents for an alkyl group include —O(C1-C4 alkyl), —S(C1-C4 alkyl), —COO(C1-C4 alkyl).  
       [0025] Also included in the present invention are pharmaceutically acceptable salts of the compounds described herein. Compounds disclosed herein which possess a sufficiently acidic, a sufficiently basic, or both functional groups, and accordingly can react with any of a number of organic or inorganic bases, and inorganic and organic acids, to form a salt. Acids commonly employed to form acid addition salts from compounds with basic groups are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of such salts include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, 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, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like.  
       [0026] Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Such bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the like.  
       [0027] The disclosed compounds can be used to inhibit kallikrein activity in a subject in need of such treatment. Subjects “in need of kallikrein inhibition” are those with a disease, condition or abnormality resulting from excessive kallikrein activity or insufficient kallikrein inhibition. Subjects “in need of kallikrein inhibition” also include those for whom a beneficial therapeutic or prophylactic effect can be achieved by kallikrein inhibition. For example, kallikrein cleaves kinnigen to form bradykinnin, a mediator of pain and inflammation. Therefore, the disclosed compounds can be used as analgesics and to treat, inter alia, subjects with inflammatory diseases such as inflammatory bowel disease (e.g., colititis and Crohn&#39;s disease) and rheumatoid arthritis. Because kallikrein activates urokinase activity, the kallikrein inhibitors disclosed herein can also be used to inhibit urokinase activity and thus be used in the treatment of cancer.  
       [0028] The disclosed compounds can also be used to inhibit urokinase activity in a subject in need such treatment. Subjects “in need of urokinase inhibition” are those with a disease, condition or abnormality resulting from excessive urokinase activity or insufficient urokinase inhibition. Subjects “in need of urokinase inhibition” also include those for whom a beneficial therapeutic or prophylactic effect can be achieved by urokinase inhibition. For example, urokinase inhibition has been shown to be useful in the treatment of cancer and in the inhibition of angiogenesis. Because urokinase inhibition modulates growth factor release from extra-cellular matrix, the disclosed urokinase inhibitors are also expected to act as immunomodulators.  
       [0029] An “effective amount” is the quantity of compound in which a beneficial clinical outcome (prophylactic or therapeutic) is achieved when the compound is administered to a subject in need of treatment. For the treatment of inflammatory disorders, a “beneficial clinical outcome” includes a reduction in the severity of the symptoms associated with the disease (e.g., pain and inflammation), an increase in the longevity of the subject and/or a delay in the onset of the symptoms associated with the disease compared with the absence of the treatment. For the treatment of cancer, a beneficial clinical outcome includes a reduction in tumor mass, a reduction in the rate of tumor growth, a reduction in metastasis, a reduction in the severity of the symptoms associated with the cancer and/or an increase in the longevity of the subject compared with the absence of the treatment. The precise amount of compound administered to a subject will depend on the type and severity of the disease or condition and on the characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease or condition. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. Effective amounts of the disclosed compounds typically range between about 0.1 mg/kg body weight per day and about 1000 mg/kg body weight per day, and preferably between 1 mg/kg body weight per day and 100 mg/kg body weight per day.  
       [0030] The disclosed compounds are administered by any suitable route, including, for example, orally in capsules, suspensions or tablets or by parenteral administration. Parenteral administration can include, for example, systemic administration, such as by intramuscular, intravenous, subcutaneous, or intraperitoneal injection. The compounds can also be administered orally (e.g., dietary), topically, by inhalation (e.g., intrabronchial, intranasal, oral inhalation or intranasal drops), or rectally, depending on the type of cancer to be treated. Oral or parenteral administration are preferred modes of administration.  
       [0031] The disclosed compounds can be administered to the subject in conjunction with an acceptable pharmaceutical carrier as part of a pharmaceutical composition for treatment of cancer. Formulation of the compound to be administered will vary according to the route of administration selected (e.g., solution, emulsion, capsule). Suitable pharmaceutical carriers may contain inert ingredients which do not interact with the compound. Standard pharmaceutical formulation techniques can be employed, such as those described in Remington&#39;s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% mg/ml benzyl alcohol), phosphate-buffered saline, Hank&#39;s solution, Ringer&#39;s-lactate and the like. Methods for encapsulating compositions (such as in a coating of hard gelatin or cyclodextran) are known in the art (Baker, et al, “Controlled Release of Biological Active Agents”, John Wiley and Sons, 1986).  
       [0032] When used to treat inflammatory diseases, the disclosed compounds can be coadministered with other analgesics and/or anti-inflammatory agents, such as aspirin, acetaminophen, REMICADE or a COX2 inhibitor. When used to treat cancer, the disclosed compounds can be advantageously used in combination with other anti-cancer therapy, including drugs (e.g., taxol, flurouracil, methatrexate, cisplatin, and the like), radiation and/or surgery.  
       [0033] A “subject” is a mammal, preferably a human, but can also be an animal in need of veterinary treatment, e.g., companion animals (e.g., dogs, cats, rabbits and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, and the like).  
       [0034] Compounds represented by Structural Formula (I), wherein m is 0, can be prepared by reacting an aryl aldehyde (ArCHO), an aryl isocyanide (R 1 N + ═C − ) and an aryl alkanoic acid, as shown below in Scheme I:  
                 
 
       [0035] R 1 , R 2 , Ar, X and n in Scheme 1 defined as in Structural Formula (I). The reaction is carried out by mixing the reagents together in a suitable solvent, for example, an alcoholic solvent. Optionally, the reaction can be heated to bring about more rapid product formation, for example, from about 40° C. to about 70° C. Illustrative conditions for carrying out the reaction are provided in Examples 1 and 3.  
       [0036] Another method for preparing the compounds of the present invention is to amidate an O-protected aryl mandelic acid with a suitable amine and then remove the O-protecting group. Amidation can be carried out by a variety of methods known to one of ordinary skill in the art, including first converting the carboxylic acid group to an acid chloride with thionyl chloride or oxalyl chloride, for example, and then reacting the acid chloride thus formed with the amine. The free alcohol group is then used to esterify a suitable alkanoic acid using a carboxylic acid activating agent such as dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride (EDCI) or other coupling reagents commonly known or typically used in peptide synthesis. Esterification reactions of this type are also known to one of ordinary skill in the art. This reaction sequence is illustrated in Scheme (2) below:  
                 
 
       [0037] R 1 , R 2 , Ar, X, n and m in Scheme 2 are as defined in Structural Formula (I); and R is a hydroxyl protecting group such as acetyl. Illustrative conditions for carrying out these reactions are provided in Examples 2 and 4.  
       [0038] In one preferred embodiment, the compound of the present invention is represented by Structural Formula (II):  
                 
 
       [0039] The variables in Structural Formula (II) are as defined above for Structural Formula (I). Preferably, Ar is a substituted or unsubstituted phenyl or naphthyl group.  
       [0040] When the compound represented by Structural Formula (II) is being used to inhibit kallikrein activity in a subject, then R 2  in Structural Formula (II) is preferably a substituted or unsubstituted phenyl, cyclohexyl or indolyl group. More preferably, n is 0, X is —CH 2 —, and R 2  is an optionally substituted phenyl or indolyl group. Alternatively, n is 2, X is —CO— and R 2  is a substituted or unsubstituted phenyl group. Specific examples of kallikrein inhibitors represented by Structural Formula (II) are shown below as Compounds (1)-(3):  
                 
 
       [0041] Other specific examples of kallikrein inhibitors represented by Structural Formula (II) are shown in Table 1 of Example 5.  
       [0042] When the compound represented by Structural Formula (II) is being used to inhibit urokinase activity in a subject, R 2  in Structural Formula (II) is preferably a substituted or unsubstituted phenyl, C5-C6 cycloalkyl, pyrimidyl or indolyl group. More preferably, n is 0, X is —CH 2 —, and R 2  is an optionally substituted phenyl or indolyl group (e.g., 4-methoxyphenyl, 3-indolyl or 5-bromo-3-indolyl). Alternatively, n is 1, X is —S—, and R 2  is an optionally substituted pyrimidyl group (e.g., 2-pyrimidyl). In another alternative, n is 1, X is —CH 2 — and R 2  is a cyclopentyl or cyclohexyl group. Specific examples of urokinase inhibitors represented by Structural Formula (II) are shown below as Compounds (4)-(7):  
                 
 
       [0043] Other specific examples of urokinase inhibitors represented by Structural Formula (II) are shown in Table 5 of Example 9.  
       [0044] In another preferred embodiment, the compound of the present invention is represented by Structural Formula (III) or (IV):  
                 
 
       [0045] The variables in Structural Formulas (III) and (TV) are as described in Structural Formula (I) above. Phenyl Rings A-C are substituted or unsubstituted.  
       [0046] When the compound represented by Structural Formula (III) or (IV) is being used to inhibit kallikrein activity, R 1  is preferably a substituted or unstubstituted phenyl group. More preferably, R 1  is a substituted or unstubstituted phenyl group and R 2  is a substituted or unsubstitutted phenyl, indolyl, pyrimidyl or benzothienyl group. Even more preferably, R 1  and R 2  are as defined above in Structural Formula (I) (e.g., a phenyl group optionally substituted with one or more methoxy groups), m is 0 or 1; n is 2 and X is —CO—. Kallikrein inhibitors that are particularly preferred are represented by Structural Formula (V):  
                 
 
       [0047] In Structural Formula (V), m is 0 or 1; R 3  is —H or —OCH 3 ; and Ar is naphthyl or phenyl optionally monosubstituted with —CN or —Br (e.g., 2-napthyl, 4-bromophenyl or 3-cyanophenyl).  
       [0048] Specific examples of kallikrein inhibitors represented by Structural Formulas (III)-(V) are shown in Table 1 of Example 5.  
       [0049] When the compound represented by Structural Formulas (III) and (IV) is being used to inhibit urokinase, R 1  in one preferred embodiment is a substituted or unsubstituted alkyl group (e.g., C1-C10 alkyl group, preferably C1-C4 alkyl group). More preferably m is 0; n is 0 or 1; X is —CH 2 — or —S—; the alkyl represented by R 1  has from one to four carbon atoms and is optionally substituted with —O(C1-C4 alkyl), —S(C1-C4 alkyl) or —COO(C1-C4 alkyl); Ar is an optionally substituted phenyl group; and R 2  is an optionally substituted phenyl or pyrimidyl group.  
       [0050] When the compounds represented by Structural Formulas (III) and (IV) are used to inhibit urokinase, R 1  in another preferred embodiment is a substituted or unsubstituted phenyl group. When R 1  is a substituted or unstubstituted phenyl group, R 2  is preferably a substituted or unsubstituted pyrimidyl or phenyl group. More preferably, R 1  and R 2  are as described above and n is 0 or 1; X is —CH 2 — or —S—; and m is 1 or 2. For preferred urokinase inhibitors represented by Structural Formulas (III) or (IV), R 1  is a phenyl group substituted with one or more methoxy groups (e.g., 4-methoxyphenyl, 3,4-dimethoxyphenyl or 3,4,5-trimethoxyphenyl group) and/or methylenedioxy groups (e.g., 3,4-methlyenedioxyphenyl); Ar is a phenyl group optionally substituted with a halogen or cyano group (e.g., phenyl, 3-bromo, 4-bromo, 3-cyano or 4-cyanophenyl); and R 2  is a pyrimidyl group (e.g., 2-pyrimidyl group) or phenyl group monosubstituted with a methoxy group. Variables n, m and X are as defined in the prior paragraph. Specific examples of urokinase inhibitors represented by Structural Formula (III) are shown below as Compounds (8)-(10):  
                 
 
       [0051] Other specific examples of urokinase inhibitors represented by Structural Formulas (IV) and (V) are shown in Table 5 of Example 9.  
       [0052] Another embodiment of the present invention is a compound represented by Structural Formula (I), (II), (III) or (IV), wherein R 1  corresponds to the group at the corresponding position in any one of the compounds shown in Tables 1 and 5. Also included is a method utilizing such compounds for kallikrein and/or urokinase inhibition in a subject, as described herein.  
       [0053] Another embodiment of the present invention is a compound represented by Structural Formula (I), (II), (III) or (IV), wherein R 2  corresponds to the group at the corresponding position in any one of the compounds shown in Tables 1 and 5. Also included a method utilizing such compounds for kallikrein and/or urokinase inhibition in a subject, as described herein.  
       [0054] Another embodiment of the present invention is a compound represented by Structural Formula (I), (II), (III) or (IV), wherein R 1  and R 2  are each selected to correspond to the group at the corresponding position of any one of the compounds shown in Tables 1 and 5. Also included is a method utilizing such compounds for kallikrein and/or urokinase inhibition in a subject, as described herein.  
       [0055] Another embodiment of the present invention is a compound represented by Structural Formula (I), (II), (III) or (IV), wherein R 1  and Ar are each selected to correspond to the group at the corresponding position of any one of the compounds shown in Tables 1 and 5. Also included is a method utilizing such compounds for kallikrein and/or urokinase inhibition in a subject, as described herein.  
       [0056] Another embodiment of the present invention is a compound represented by Structural Formula (I)-(IV), wherein Ar and R 2  are each selected to correspond to the group at the corresponding position of any one of the compounds shown in Tables 1 and 5. Also included is a method utilizing such compounds for kallikrein and/or urokinase inhibition in a subject, as described herein.  
       [0057] The invention is illustrated by the following examples which are not intended to be limiting in any way. 
     
    
    
     EXEMPLIFICATION  
     Example 1  
     Preparation of (4-Methoxy-Phenyl)-Acetic Acid (4-Cyano-Phenyl)-(3,4-Dimethoxy-Phenylcarbamoyl)-Methyl Ester  
     [0058]                   
     [0059] 4-Cyanobenzaldehyde (52 mg, 0.40 mmole), 4-methoxyphenylacetic acid (70 mg, 0.44 mmole) and 3,4-dimethoxyphenyl isocyanide (64 mg, 0.40 mmole) were dissolved in 5 ml methanol and warmed to 50° C. The mixture was allowed to stir at this temperature overnight. The reaction was worked up by concentrating in-vacuo and dissolving the residue in 10 ml ethyl acetate. This was then washed with 1N HCl, saturated sodium bicarbonate, and saturated sodium chloride, dried over Na 2 SO 4  and concentrated in-vacuo. The compound was then purified by flash chromatography on silica gel (1:1 ethyl acetate/hexane).  1 H NMR (300 MHz, CDCl 3 ) δ 7.64 (d, 2H), 7.54 (d, 2H), 7.42 (bs, 1H), 7.30 (d, 2H), 7.11 (d, 1H), 6.95 (d, 2H), 6.75 (d, 1H), 6.58 (dd, 1H), 6.21 (s, 1H), 3.85 (s, 3H), 3.83 (s, 3H), 3.81 (s, 3H), 3.77 (s, 2H).  
     Example 2  
     Preparation of 4-(2,5-Dimethoxyphenyl)-4-Oxo-Butyric Acid (4-Bromophenyl)-(3,4,5-Trimethoxy Benzylcarbamoyl)-Methyl Ester  
     [0060]                   
     [0061] Step 1—Preparation of Acetoxy-(4-bromophenyl)-acetic Acid  
     [0062] Bromomandelic acid (10.30 g, 0.045 mole) was dissolved in 25 ml pyridine. Acetic anhydride (5.01 g, 0.050 mole) was added dropwise to this mixture over 15 minutes. The reaction was allowed to stir at room temperature overnight. The reaction was concentrated in-vacuo and dissolved in 75 ml ethyl acetate. This was then washed with 1 N HCl and saturated sodium chloride, dried over MgSO 4  and concentrated in-vacuo to give a colorless oil.  1 H NMR (300 MHz, CDCl 3 ) δ 9.28 (bs, 1H), 7.56 (d, 2H), 7.38 (d, 2H), 5.88 (s, 1H), 2.19 (s, 3H).  
     [0063] Step 2 Preparation of Acetic Acid (4-bromophenyl)-(3,4,5-trimethoxybenzylcarbamoyl)-methyl Ester  
     [0064] To the product of step 1 (6.10 g, 0.022 mole), dissolved in 30 ml dichloromethane, was added dimethylformamide (0.1 ml, catalytic). The mixture was cooled in an ice water bath to 0° C. and oxalyl chloride (16.5 ml, 0.033 mole) was added drop-wise over 15 minutes. The reaction mixture was allowed to warm to room temperature and stirred for 1 hour. The reaction was then concentrated in-vacuo and dried under vacuum. The acid chloride was dissolved in 20 ml dichloromethane and then added over fifteen minutes to a solution of 3,4,5-trimethoxybenzylamine (4.84 g, 0.025 mole) and triethylamine (2.48 g, 0.025 mole) at 0° C. The reaction was then allowed to warm to room temperature and stirred for 4 hours. The reaction was diluted with 30 ml dichloromethane and this solution was then washed twice with 1M HCl, twice with saturated sodium bicarbonate and twice with saturated sodium chloride. The organic layer was then dried over MgSO 4  and concentrated in-vacuo to give a golden foamy solid.  1 H NMR (300 MHz, CDCl 3 ) δ 7.41 (d, 2H), 7.30 (d, 2H), 6.90 (bt, 1H), 6.28 (s, 2H), 5.92 (s, 1H), 4.26 (m, 2H), 3.77 (s, 3H), 3.63 (s, 6H), 2.10 (s, 3H).  
     [0065] Step 3-Preparation of 2-(4-bromophenyl)-2-hydroxy-N-(3,4,5-trimethoxybenzyl)-acetamide  
     [0066] To the amide-acetate from step 2 (7.90 g, 0.0175 mole), dissolved in 40 ml tetrahydrofuran, was added lithium hydroxide monohydrate (1.10 g, 0.0262 mole) dissolved in 15 ml water. The resulting mixture was stirred at room temperature. After 2 hours, 10 ml water and 30 ml diethyl ether were added, resulting in the precipitation of a white solid. The solid was filtered and washed with diethyl ether. The filtrate was then collected and the organic layer was separated and concentrated in-vacuo to one-third volume. Additional solid precipitated, which was collected, as described above. The combined solid was dried in a vacuum oven at 40° C. for 4 hours.  1 H NMR (300 MHz, CDCl 3 ) δ 7.48 (d, 2H), 7.31 (d, 2H), 6.50 (bt, 1H), 6.32 (s, 2H), 5.06 (s, 1H), 4.38 (m, 2H), 3.81 (s, 3H), 3.76 (s, 6H).  
     [0067] Step 4—Preparation of 4-(2,5-Dimethoxyphenyl)-4-oxo-butyric acid (4-bromophenyl)(3,4,5-trimethoxy-benzylcarbamoyl)-methyl Ester  
     [0068] The hydroxy amide from step 3 (4.10 g, 0.010 mole) and 2,5-dimethoxybenzoyl propionic acid (2.50 g, 0.011 mole) were mostly dissolved 125 ml dichloromethane. To this reaction was added dimethylaminopyridine (0.122 g, 0.001 mole) and then EDCI (3.30 g, 0.011 mole) dissolved in 25 ml dichloromethane. The reaction was allowed to stir at room temperature for 16 hours. The reaction mixture was then transferred to a separatory funnel and washed twice with 1M HCl, twice with saturated sodium bicarbonate and twice with saturated sodium chloride. The organic layer was then dried over MgSO 4  and concentrated in-vacuo. The compound was then purified by flash chromatography on silica gel eluted with 2:1 ethyl acetate/hexane.  1 H NMR (300 MHz, CDCl 3 ) δ 7.55 (bt, 1H), 7.49 (d, 2H), 7.38 (d, 2H), 7.18 (d, 1H), 7.05 (dd, 1H), 6.92 (d, 1H), 6.42 (s, 2H), 6.09 (s, 1H), 4.46 (m, 2H), 3.87 (s, 3H), 3.76 (s, 3H), 3.73 (s, 6H), 3.69 (s, 3H), 3.45 (m, 2H), 2.84 (m, 1H), 2.66 (m, 1H).  
     Example 3  
     Preparation of 4-Methoxyphenyl Acetic Acid [(Benzo[1,3] Dioxol-5-ylmethyl)-Carbamoyl](4-Cyanophenyl) Methyl Ester  
     [0069]                   
     [0070] 4-Cyanobenzaldehyde (131 mg, 1.00 mmole), 4-methoxyphenylacetic acid (166 mg, 1.00 mmole) and 3,4-methylenedioxybenzyl isocyanide (161 mg, 1.00 mmole) were dissolved in 25 ml methanol and warmed to 600 C. The reaction was allowed to stir at this temperature overnight. The reaction was then worked up by concentrating in-vacuo and dissolving the residue in 50 ml ethyl acetate. This was then washed with 1N HCl, saturated sodium bicarbonate, and saturated sodium chloride, dried over Na 2 SO 4  and concentrated in-vacuo. The compound was then purified by flash chromatography on silica gel (1:1 ethyl acetate/hexane).  1 H NMR (300 MHz, CDCl 3 ) δ 7.61 (d, 2H), 7.48 (d, 2H), 7.15 (d, 2H), 6.80 (d, 2H), 6.74 (d, 1H), 6.58 (d, 2H), 6.14 (bs, 1H), 6.12 (s, 1H), 5.96 (s, 2H), 4.10 (d, 2H), 3.77 (s, 3H), 3.69 (s, 2H).  
     Example 4  
     Preparation of 3-(Cyclohexyl)-Propionic Acid (4-Cyanophenyl)(Cyclopropylcarbamoyl)-Methyl Ester  
     [0071]                   
     [0072] Step 1—Preparation of Acetoxy-(4-cyanophenyl)-acetic Acid  
     [0073] 4-Cyanomandelic acid (8.90 g, 0.050 mole) was dissolved in 50 ml pyridine. Acetic anhydride (6.15 g, 0.060 mole) was then added dropwise to this solution over 15 minutes. The reaction was then allowed to stir at room temperature overnight. The reaction was then concentrated in-vacuo and dissolved in 100 ml dichloromethane. This solution was then washed with 1N HCl (2×) and saturated sodium chloride, dried over MgSO 4  and concentrated in-vacuo to give a colorless oil.  1 H NMR (300 MHz, CDCl 3 ) δ 8.94 (bs, 1H), 7.69 (d, 2H), 7.61 (d, 2H), 5.98 (s, 1H), 2.21 (s, 3H).  
     [0074] Step 2—Preparation of Acetic Acid (4-cyanophenyl)-(cyclopropylcarbamoyl)-methyl Ester  
     [0075] The acetate prepared in step 1 (9.10 g, 0.041 mole) was converted to the acid chloride by dissolving in 50 ml dichloromethane. Dimethylformamide (0.1 ml, catalytic) was added and the reaction mixture cooled in an ice water bath to  0 ° C. Oxalyl chloride (25 ml, 0.050 mole) was added drop-wise over 15 minutes. The reaction was then allowed to warm to room temperature and stirred for 1 hour. The reaction was then concentrated in-vacuo and dried under vacuum. The resulting acid chloride was dissolved in 40 ml dichloromethane and added dropwise over 45 minutes to a solution of cyclopropylamine (2.81 g, 0.045 mole) and triethylamine (4.58 g, 0.045 mole) at 0° C. The reaction was then allowed to warm to room temperature and stirred overnight. The sample was diluted with 25 ml dichloromethane and this solution was then washed with 1M HCl (2×), saturated sodium bicarbonate (2×) and saturated sodium chloride. The organic layer was then dried over MgSO 4  and concentrated in-vacuo to give a solid. The product was triturated in 100 ml diethyl ether for 30 minutes, filtered and dried to give a solid.  1 H NMR (300 MHz, CDCl 3 ) δ 7.64 (d, 2H), 7.54 (d, 2H), 6.49 (bs, 1H), 6.03 (s, 1H), 2.70 (m, 1H), 2.19 (s, 3H), 0.78 (m, 2H), 0.49 (m, 2H).  
     [0076] Step 3—2-(4-Cyanophenyl)-2-hydroxy-N-(cyclopropyl)-acetamide  
     [0077] The amide-acetate prepared in step 2 (6.76 g, 0.0262 mole) was dissolved in 40 ml tetrahydrofuran. To this was added lithium hydroxide monohydrate (1.65 g, 0.0393 mole) dissolved in 15 ml water. The resulting mixture was allowed to stir at room temperature. After 2 hours, 20 ml water and 25 ml ethyl acetate were added. The aqueous layer was separated and extracted with ethyl acetate. The organic layers were combined and washed with saturated sodium chloride. The organic layer was then dried over MgSO4 and concentrated in-vacuo to give the product as a solid. This solid was triturated with diethyl ether filtered and dried to give the product.  1 H NMR (300 MHz, CDCl 3 ) δ 7.63 (d, 2H), 7.58 (d, 2H), 6.49 (bs, 1H), 5.05 (s, 1H), 2.68 (m, 1H), 0.78 (m, 2H), 0.49 (m, 2H).  
     [0078] Step 4—Preparation of 3-(Cyclohexyl)-Propionic Acid (4-Cyanophenyl)-(Cyclopropylcarbamoyl)-Methyl Ester  
     [0079] The hydroxy amide prepared in step 3 (500 mg, 2.31 mmole) and cyclohexanepropionic acid (397 mg, 2.54 mmole) were mostly dissolved in 20 ml chloroform and 1 ml dimethylformamide. To this reaction was added dimethylaminopyridine (5.0 mg, 0.04 mmole) and then EDCI (756 mg, 2.54 mmole) dissolved in 1 ml chloroform. The reaction was allowed to stir at room temperature for 16 hours. The reaction mixture was then diluted with 20 ml chloroform and transferred to a separatory funnel and washed with 1M HCl (2×), saturated sodium bicarbonate (2×) and saturated sodium chloride. The organic layer was then dried over MgSO4 and concentrated in-vacuo. The compound was then purified by flash chromatography on silica gel (2:1 ethyl acetate/hexane).  1 H NMR (300 MHz, CDCl 3 ) δ 7.67 (d, 2H), 7.54 (d, 2H), 6.31 (bs, 1H), 6.08 (s, 1H), 2.74 (m, 1H), 2.45 (m, 2H), 1.68 (m, 5H), 1.54 (m, 2H), 1.17 (m, 2H), 0.90 (m, 2H), 0.82 (m, 2H), 0.51 (m, 2H).  
     Example 5  
     Kallikrein Inhibition Assay  
     [0080] Kallikrein activity was assayed fluorometrically with an aminomethyl coumarin-containing (AMC-containing) substrate according to procedures described in Zimmerman et al.,  Proc. Natl. Acad. Sci. USA  75:750 (1978), the entire teachings of which are incorporated herein by reference. Specifically, enzyme assays with varying concentrations of the fluorogenic substrate H-Pro-Phe-Arg-AMC were conducted at 24° C in 0.05 M 4-(2-hydroxyethyl)-piperazineethanesulfonic acid (HEPES) buffer, pH 7.5/5% (vol/vol) dimethyl sulfoxide. The final volume was 0.1 ml in each assay. Fluorescence was read after ten minutes at 360 nm (activation wavelength) and 460 nm (emission wavelength).  
     [0081] The results for representative compounds of the present invention are provided below in Table 1.  
               TABLE 1                          Kallikrein Inhibition Data For Compounds of the Present Invention                         Compound Number   Structure   IC50 (μM)                                                 Compound (11)                         0.442               Compound (12)                         0.737               Compound (13)                         0.874               Compound (14)                         1.18               Compound (15)                         0.577               Compound (16)                         0.253               Compound (17)                         0.384               Compound (18)                         6.47               Compound (19)                         1.69               Compound (20)                         1.48               Compound (21)                         1.18               Compound (22)                         0.428               Compound (23)                         0.576               Compound (24)                         0.247               Compound (25)                         0.275               Compound (26)                         0.191               Compound (27)                         0.364               Compound (28)                         0.680               Compound (29)                         0.310               Compound (30)                         0.714               Compound (31)                         0.543               Compound (32)                         1.345               Compound (33)                         0.459               Compound (34)                         0.731               Compound (35)                         0.391               Compound (36)                         3.67               Compound (37)                         1.67               Compound (3)                         1.06               Compound (38)                         3.77               Compound (2)                         0.167               Compound (39)                         2.68               Compound (1)                         0.174                  
 
     Example 6  
     Acetic Acid Writhing Assay  
     [0082] The analgesic activity of a number of compounds of the present invention were tested in an acetic acid writhing assay disclosed in Inoue et al.,  Arzneimittel Forschung Drug Research  41:228 (1991), the entire teachings of which are incorporated herein by reference. The test substance was administered intraperitoneally (500, 100 and 20 mg/kg) to groups of 3 ICR derived male or female mice weighing 22±2 g one hour before the injection of acetic acid (3%, 10 ml/kg, IP). Reduction in the number of writhes by 50% or more per group of animals observed during the 5-10 minutes period after acetic acid administration, relative to a vehicle-treated control group (challenge with acetic acid), indicates analgesic activity.  
     [0083] The results for the compounds tested are shown below in Table 2.  
               TABLE 2                          Results for Test Compounds in the Acetic Acid Writhing Test                         Treatment   Dose (IP)   % Reduction of Writhes                                     Vehicle (1% Cremophor EL)   20   ml/kg    0*       (Challenge with acetic acid)       Vehicle (1% Cremophor EL)   20   ml/kg   ND       (No challenge with acetic       acid)       Compound (24)   500   mg/kg   83           100   mg/kg   25           20   mg/kg    0       Compound (26)   500   mg/kg   75           100   mg/kg   58           20   mg/kg   25       Compound (27)   500   mg/kg   92           100   mg/kg   25           20   mg/kg    8       Compound (29)   500   mg/kg   83           100   mg/kg   33           20   mg/kg   17                                  
 
     [0084] The four compounds tested showed dose responsive activity and statistically significant responses at the highest dose (500 mg/kg). Compound (26) had significant response at both the 500 and 100 mg/kg doses.  
     Example 7  
     Carageenan Assay  
     [0085] The assay was performed according to procedures disclosed in Winter et al,  Proc. Exper. Biol. Med  111: 544 (1962), the entire teachings of which are incorporated herein by reference. Specifically, the test substance was administered intraperitoneally (300, 100 and 30 mg/kg) to groups of 3 Long Evans derived male or female overnight fasted rats weighing 150±20 grams two hours before right hind paw injection of carageenan (0.1 ml of 1% suspension intraplantar). Hind paw edema, as a measure of inflammation, was measured 3 hours after carageenan administration using a plethysmometer (Ugo Basile Catalog No. 7150) with water cell (25 mm diameter, Catalog No. 7157). Reduction of hind paw edema by 30% or more indicates significant acute anti-inflammatory activity. The results are shown below in Table 3.  
               TABLE 3                          Results For Test Compounds in The Carageenan Assay                                     Edema           Treatment   Dose (IP)   (ml)   Inhibition                                         Vehicle (1% Cremophor in   5   ml/kg   0   —       saline)       (No carageenan administration)       Vehicle (1% Cremophor EL)   5   ml/kg   0.94   —       (With carageenan administration)       Compound (26)   300   mg/kg   0.62   34           100   mg/kg   0.76   19           30   mg/kg   0.97    0       Indomethacin (control)   10   mg/kg   0.44   53                  
 
     [0086] Compound (26) showed dose responsive activity and statistically significant response at the highest dose (300 mg/kg).  
     Example 8  
     Compound 26 Inhibits Inflammation in a Rat Model of Inflammatory Bowel Disease  
     [0087] Compounds were tested according to procedures described in C M Hogaboam et al.,  Eur. J. Pharmacol.  309:261 (1996), the entire teachings of which are incorporated herein by reference. Groups of 3 overnight fasted male rats weighing 150±10 grams were used. Distal colitis was induced by intra-colonic instillation of 0.5 ml/rat of DNBS (2,4-dinitrobenzene sulfonic acid, 60 mg/ml in ethanol 30%) after which air (2 ml) was gently injected through the cannula to ensure that the solution remained in the colon. Test compound was administered orally 24 and 2 hours before DNBS instillation and then daily for 5 days in total of 7 doses. The animals were sacrificed 24 hours after the final dose of test compound administration and each colon was removed and weighed. A 30 percent or more (≧30%) inhibition in net increase of colon to 100 grams body weight ratio, relative to vehicle-control-2 group is considered significant.  
               TABLE 4                          Results for test compounds in a rat model of       inflammatory bowel disease.                                                 Inhi-                   Net   bi-       Treatment   Dose (PO) 1     Ave. 2     Inc. 3     tion 4                                               Group 1: Vehicle (1% Cremophor   10 ml/kg   × 7   0.487   —   —       in Dist. Water) (No DNBS       administration)       Group 2: Vehicle (1% Cremophor   10 ml/kg   × 7   1.040   0.533   —       in Dist. Water) (With DNBS       administration)       Group 3: Compound 26   75 mg/kg   × 7   0.857   0.370   33       (With DNBS administration)   20 mg/kg   × 7   0.947   0.460   17           4 mg/kg   × 7   1.061   0.574    0       Group 4: Sulfasalazine (With   500 mg/kg   × 7   0.865   0.378   32       DNBS administration)                                                  
 
     [0088] As can be seen from the results in Table 4, Compound 26 showed dose responsive activity and statistically significant response at the highest dose (75 mg/kg).  
     Example 9  
     Urokinase Assay  
     [0089] Urokinase activity was assayed fluorometrically with an aminomethyl coumarin-containing (AMC-containing) substrate according to procedures described in Zimmerman et al,  Proc. Natl. Acad. Sci. USA  75:750 (1978), the entire teachings of which are incorporated herein by reference. Specifically, enzyme assays with varying concentrations of the fluorogenic substrate Cbz-Gly-Gly-Arg-AMC were conducted at 24° C. in 0.05 M 4-(2-hydroxyethyl)-piperazineethanesulfonic acid (HEPES) buffer, pH 7.5/5% (vol/vol) dimethyl sulfoxide; the final volume was 0.1 ml in each assay.  
     [0090] Fluorescence was read after ten minutes at 360 nm (activation wavelength) and 460 nm (emission wavelength).  
     [0091] The results for representative compounds of the present invention are provided below in Table 5.  
               TABLE 5                          Inhibition of urokinase by compound of the present invention       Urokinase Inhibition Data                         Compound Number   Structure   IC 50  (μM)                                                 Compound (40)                         6.09               Compound (9)                         0.923               Compound (41)                         2.32               Compound (42)                         1.97               Compound (43)                         3.39               Compound (44)                         1.78               Compound (8)                         0.112               Compound (45)                         1.41               Compound (10)                         0.783               Compound (46)                         2.93               Compound (47)                         2.38               Compound (48)                         5.38               Compound (49)                         1.37               Compound (50)                         3.10               Compound (51)                         1.22               Compound (52)                         0.628               Compound (7)                         0.110               Compound (53)                         1.06               Compound (6)                         0.032               Compound (54)                         0.814               Compound (55)                         0.151               Compound (56)                         0.055               Compound (4)                         0.086               Compound (5)                         0.029                  
 
     [0092] As can be seen from this data, compounds of the present invention are effective inhibitors of urokinase.  
     [0093] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.