60/088, 466, filed June 8,1998; U. S. Patent Application Serial No. 60/092,938, filed July 15,1998; and U. S. Patent Application Serial No. 60/125,751, filed March 23,1999; the disclosures of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION Field of the Invention This invention relates to novel multibinding compounds (agents) that are non-peptidic bradykinin antagonists, pharmaceutical compositions comprising such compounds, and methods of preparing these compounds. Accordingly, the multibinding compounds and pharmaceutical compositions of this invention are useful in the treatment and prevention of diseases mediated by bradykinin such as cancer, pain, shock, asthma, rhinitis, arthritis, inflammatory bowel disease, and the like.

References The following publications are cited in this application as superscript numbers: Burch et al.,"Bradykinin Receptor Antagonists"J. Med.

Chem., 30: 237-269 (1990).

2 Clark, W. G. Handbook of Expt. PharmacoL, Vol. XXV: Bradykinin, kallidin, and kallidrein. Erdo, E. G, 311-322.

3 Bames,"Inflammatory Mediator Receptors and Asthma'Am. Rev.

Respir. Dis., 135: S26-31 (1987).

4 Fuller et al,"Bradykinin-induced Bronchoconstriction in Humans", Am. Rev. Respir. Dis., 135: 176-180 (1987).

5 Jin et al,"Inhibition of Bradykinin-induced Bronchoconstriction in the Guinea-pig a Synthetic B2 Receptor Antagonist", Br. J.

Pharmacol., 9.7: 598-602 (1989).

6 Polosa et al,"Contribution of Histamine and Prostanoids to Bronchoconstriction Provoked by Inhaled Bradykinin in Atopic Asthma", Allergy, 45: 174-182 (1990).

7 Baumgarten et al,"Concentrations of Grandular Kallikrein in Human Nasal Secretions Increase During Experimentally Induce Allergic Rhinitis", J. Immunology, 137: 1323-1328 (1986).

All of the above publications are herein incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.

State of the Art Bradykinin (BK) is one of the most important kinins. It is derived by cleavage of precursor plasma proteins, through the kallikrein/kinin system. It is a potent inflammatory peptide whose generation in tissues and body fluids elicits many physiological responses including vasodilation, smooth muscle spasm, edema, as well as pain and hyperalgesia. There is increasing evidence that BK and related kinins contribute to the inflammatory response in acute and chronic diseases including allergic reactions, arthritis, asthma, sepsis, viral rhinitis, and inflammatory bowel disease. For example, bradykinin receptors have been localized to nociceptive peripheral nerve pathways and bradykinin has been demonstrated to stimulate central fibers mediating pain sensation.'-2 Numerous studies have also shown that bradykinin receptors are present in the lung and that bradykinin can cause bronchoconstriction in both animals and man and furthermore that

bronchoconstriction can be inhibited by treatment with bradykinin antagonists.'' Bradykinin has also been implicated in the production of symptoms in both allergic and viral rhinitis'and in the pathogenesis of human lung cancer. Therefore, the design and synthesis of specific, potent and stable bradykinin antagonists has long been considered a desirable goal in medicinal chemistry.

Stewart and Vavrek in"Chemistry of Peptide Bradykinin Antagonists", Bradykinin Antagonists : Basic and Chemical Research, R. M. Burch (Ed.), pages 51-96,1991 discuss peptide bradykinin antagonists and their possible use against effects of bradykinin. Regoli, D. et al Eur. J. of Pharmacology, 348,1-10,1998 disclose several peptidic and non-peptidic bradykinin antagonists and their potential use against effects of bradykinin. PCT Application No. 97/09347 discloses bradykinin antagonists dimers composed of peptidic bradykinin antagonists attached with a linking group for the treatment of cancer. PCT Application No.

94/11021 discloses heterodimers comprising a peptidic bradykinin antagonists covalently linked to a peptide or a non-peptide pharmacophore which is not a bradykinin antagonist via a linking group for the treatment of pain and inflammation. The major problems with presently available bradykinin antagonists are their low levels of potency and short duration of activity.

Accordingly, there is a need for bradykinin antagonists that are increased potency and/or duration of action.

SUMMARY OF THE INVENTION This invention is directed to novel multibinding compounds (agents) that are non-peptidic bradykinin antagonists and are useful in the treatment and prevention of diseases such as cancer, pain, shock, asthma, rhinitis, arthritis, inflammatory bowel disease, and the like. In addition, it is contemplated that the non-peptidic multibinding compounds of the present invention will exhibit longer duration of activity vis-a-vis peptidic antagonists.

Accordingly, in one aspect, this invention provides a multibinding compound comprising of from 2 to 10 ligands covalently attached to one or more

linkers, wherein each of said ligands comprises, independently of each other, a non- peptidic bradykinin antagonist, and pharmaceutically acceptable salts.

In a second aspect, this invention provides a multibinding compound of Formula (I) : (L)p(X)q (I) wherein: each L is, independently of each other, a non-peptidic bradykinin antagonist ; each X is independently a linker; p is an integer of from 2 to 10; and q is an integer of from 1 to 20, and pharmaceutically acceptable salts thereof.

Preferably, q is less than p in the multibinding compounds of this invention.

Preferably, each ligand, L, that is a non-peptidic bradykinin antagonist in the multibinding compound of Formula (I), is independently selected from the group consisting of : (i) a compound of formula (a): wherein: A is selected from the group consisting of alkylene and substituted alkylene; B is selected from the group consisting of-O-,-NH-, and-S (O) n' (where n' is an integer of from 0 to 2); C is selected from the group consisting of a compound of formula (1) and (2):

where: X'is-N-or-CR4 where R4 is alkyl ; X2 is-N-or-CR5 where R5 is hydrogen or alkyl ; X3 is-N-or-CR6 where R6 is selected from the group consisting of hydrogen, alkyl, alkoxy, halo, amino, aryl, carboxy, alkoxycarbonyl, substituted alkyl, substituted alkoxy, substituted amino,-CONHR (where R is hydrogen or alkyl), cycloalkyloxy, and N-containing heterocycl-N-yl group optionally substituted with alkyl ; R4 and R5 are as defined above; R'is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, and halo, or R'is a covalent bond linking the ligand to a linker; R is selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, and halo, or R2 is a covalent bond linking the ligand to a linker; RI is selected from the group consisting of hydroxy, nitro, alkoxy, substituted alkoxy, piperazinyl optionally substituted with one or two groups selected from acylalkyl, oxo, and-NRR' [wherein R'is hydrogen, alkyl, or a covalent bond linking the ligand to a linker, and R'is hydrogen,-COOR' (where R9 is aryl),-COR'° (where R'° is aryl, heteroaryl, or heterocyclyl)], or a group of formula: -(AA)-(CO-Q-R") n or-(AA)-R'2 where: n is 0 or 1 ;

AA is a amino acid residue wherein the terminal nitrogen atom of the amino acid residue optionally links the ligand to a linker when n is 0; Q is selected from the group consisting of alkylene, alkenylene, and a bond; R"selected from the group consisting of aryl, heteroaryl, heterocyclyl, and -X4Ra (where X'is-N-,-0-, or-S-and Ra is aryl, heteroaryl, or heterocyclyl each of which optionally links the ligand to a linker); and Rl2 is selected from the group consisting of hydrogen and acylbiphenyl which optionally link the ligand to a linker; (ii) a compound of formula (b): wherein: A'is selected from the group consisting of alkylene and substituted alkylene; R'3 is selected from the group consisting of quinolyl, quinazolinyl, quinoxalinyl, benzimidazolyl, benzofuryl, benzoxazolyl, and imidazopyridyl, each of which is optionally substituted with one or more substituent (s) selected from the group consisting of halo, alkyl, substituted alkyl, alkoxy, substituted alkoxy, amino, substituted amino, heteroaryl, and heterocyclyl; R'4 is selected from the group consisting of hydrogen, halo, alkyl, and substituted alkyl, or R'4 is a covalent bond linking the ligand to a linker; R'5 is selected from the group consisting of halo and alkyl, or R'5 is a covalent bond linking the ligand to a linker; and R"is carboxy or a group of formula: where: Q'is alkylene or is a group of formula:

where: R'9 is hydrogen or halo; R21 is selected from the group consisting ofhydrogn and alkyi, or R20 is a covalent bond linking the ligand to a linker, or R'° and R'5 together form alkylene; and R2'is selected from the group consisting of hydrogen, alkyl, and aralkyl, or R21 is a covalent bond linking the ligand to a linker, provided that A2 is alkylene when R21 is hydrogen; A2 is selected from the group consisting of alkylene and a bond; R"is selected from the group consisting of amino which optionally links the ligand to a linker, aminoacyl, cyano, hydroxy, and acyl ; and R'8 is selected from the group consisting of hydrogen and acyl ; or (iii) a compound of formula (c): wherein: R22 and R'-'are, independently of each other, halo or optionally link the ligand to a linker;

A3 is selected from the group consisting of a bond, alkylene,-CO-,-O-, and -S (O) n- (where n is an integer of 0 to 2); R24 and R25 are, independently of each other, alkyl or optionally link the ligand to a linker; R26 is selected from the group consisting of hydrogen, alkyl optionally substituted with one or two substituents selected from hydroxy, amino, substituted amino, pyridyl, carbamoyl, pyrrolidinocarbonyl, propylaminocarbonyl, piperidinocarbonyl or morpholinocarbonyl; piperidinyl optionally substituted on the nitrogen atom with alkyl or alkoxycarbonyl; cycloalkyl optionally substituted with one or two substituents selected from oxo, hydroxy, amino, alkylamino. dialkylamino, methoxybenzamido, or morpholino; C714 azacyclo, azabicyclo or azatricycloalkyl in which the nitrogen atom is optionally substituted with a substituent selected from alkyl, benzyl optionally substituted with one or two substituents selected from halo, trihaloalkyl, acyl or-COOR28 (where R'8 is alkyl optionally substituted with one or two halogen atoms); heteroaryl; heterocyclyl; cycloalkenyl; and phenyl fused to cycloalkyl ; or R26 optionally links the ligand to a linker; and pharmaceutically acceptable salts thereof.

Preferably, each linker, X, in the multibinding compound of Formula (I) is a non-peptidic linker.

More preferably, each linker, X, in the multibinding compound of Formula (I) independently has the formula: -xa z (ya-z) _xa wherein: m is an integer of from 0 to 20; Xa at each separate occurrence is selected from the group consisting of -O-,-S-,-NR-,-C (O)-,-C (O) O-,-C (O) NR-,-C (S),-C (S) O-,-C (S) NR- or a covalent bond where R is as defined below; Z at each separate occurrence is selected from the group consisting of alkylene, substituted alkylene, cycloalkylene, substituted cylcoalkylene, alkenylene,

substituted alkenylene, alkynylene, substituted alkynylene, cycloalkenylene, substituted cycloalkenylene, arylene, heteroarylene, heterocyclene, or a covalent bond; each ya at each separate occurrence is selected from the group consisting of -O-,-C (O)-,-OC (O)-,-C (O) O-,-NR-,-S (O) n-,-C (O) NR'-,-NR'C (O)-,-NR' C (O) NR'-,-NR'C (S) NR'-,-C (=NR')-NR'-,-NR'-C (=NR')-,-OC (O)-NR'-,-NR'- C (O)-O-,-N=C (Xa)-NR'-,-NR'-C (Xa) =N-,-P (O) (OR')-O-,-O-P (O) (OR')-,- S (O) CR' R"-,-S (O) ,-NR'-,-NR'-S (O) ,-,-S-S-, and a covalent bond; where n is 0, 1 or 2; and R, R'and R"at each separate occurrence are selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl and heterocyclic provided that at least one of Xa, Z, and ya is not a covalent bond.

In a third aspect, this invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a multibinding compound comprising from 2 to 10 ligands covalently attached to one or more linkers, wherein each of said ligands comprises, independently of each other, a non-peptidic bradykinin receptor antagonist and pharmaceutically acceptable salts thereof.

In a fourth aspect, this invention provides a method of treating diseases mediated by bradykinin in a mammal, said method comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a multibinding compound comprising from 2 to 10 ligands covalently attached to one or more linkers, wherein each of said ligands, comprises, independently of each other, a non-peptidic bradykinin antagonist, and pharmaceutically acceptable salts thereof.

In a fifth aspect, this invention is directed to general synthetic methods for generating large libraries of diverse multimeric compounds which multimeric compounds are candidates for possessing multibinding properties for bradykinin receptor. The diverse multimeric compound libraries provided by this invention are synthesized by combining a linker or linkers with a ligand or ligands to provide for a library of multimeric compounds wherein the linker and ligand each have complementary functional groups permitting covalent linkage. The library of linkers is preferably selected to have diverse properties such as valency, linker length, linker geometry and rigidity, hydrophilicity or hydrophobicity, amphiphilicity, acidity, basicity and polarization and/or polarizability. The library of ligands is preferably selected to have diverse attachment points on the same ligand, different functional groups at the same site of otherwise the same ligand, and the like.

In a sixth aspect, this invention is directed to libraries of diverse multimeric compounds which multimeric compounds are candidates for possessing multibinding properties for bradykinin receptor. These libraries are prepared via the methods described above and permit the rapid and efficient evaluation of what molecular constraints impart multibinding properties to a ligand or a class of ligands targeting bradykinin receptor.

Accordingly, in one of its method aspects, this invention is directed to a method for identifying multimeric ligand compounds possessing multibinding properties for bradykinin receptor which method comprises: (a) identifying a ligand or a mixture of ligands wherein each ligand contains at least one reactive functionality ; (b) identifying a library of linkers wherein each linker in said library comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand; (c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the ligand or mixture of ligands identified in (a) with the library of linkers identified in (b) under conditions wherein the

complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands; and (d) assaying the multimeric ligand compounds produced in (c) above to identify multimeric ligand compounds possessing multibinding properties for bradykinin receptor.

In another of its method aspects, this invention is directed to a method for identifying multimeric ligand compounds possessing multibinding properties for bradykinin receptor which method comprises: (a) identifying a library of ligands wherein each ligand contains at least one reactive functionality; (b) identifying a linker or mixture of linkers wherein each linker comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand; (c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the library of ligands identified in (a) with the linker or mixture of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands; and (d) assaying the multimeric ligand compounds produced in (c) above to identify multimeric ligand compounds possessing multibinding properties for bradykinin receptor.

The preparation of the multimeric ligand compound library is achieved by either the sequential or concurrent combination of the two or more stoichiometric equivalents of the ligands identified in (a) with the linkers identified in (b).

Sequential addition is preferred when a mixture of different ligands is employed to ensure heterodimeric or multimeric compounds are prepared. Concurrent addition of the ligands occurs when at least a portion of the multimer compounds prepared are homomultimeric compounds.

The assay protocols recited in (d) can be conducted on the multimeric ligand compound library produced in (c) above, or preferably, each member of the library is isolated by preparative liquid chromatography mass spectrometry (LCMS).

In one of its composition aspects, this invention is directed to a library of multimeric ligand compounds which may possess multivalent properties for bradykinin receptor which library is prepared by the method comprising: (a) identifying a ligand or a mixture of ligands wherein each ligand contains at least one reactive functionality ; (b) identifying a library of linkers wherein each linker in said library comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand; and (c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the ligand or mixture of ligands identified in (a) with the library of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands.

In another of its composition aspects, this invention is directed to a library of multimeric ligand compounds which may possess multivalent properties for bradykinin receptor which library is prepared by the method comprising: (a) identifying a library of ligands wherein each ligand contains at least one reactive functionality ; (b) identifying a linker or mixture of linkers wherein each linker comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand; and (c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the library of ligands identified in (a) with the linker or mixture of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands.

In a preferred embodiment, the library of linkers employed in either the methods or the library aspects of this invention is selected from the group comprising flexible linkers, rigid linkers, hydrophobic linkers, hydrophilic linkers, linkers of different geometry, acidic linkers, basic linkers, linkers of different polarization and/or polarizability, and amphiphilic linkers. For example, in one embodiment, each of the linkers in the linker library may comprise linkers of different chain length and/or having different complementary reactive groups. Such linker lengths can preferably range from about 2 to 1 OOA.

In another preferred embodiment, the ligand or mixture of ligands is selected to have reactive functionality at different sites on said ligands in order to provide for a range of orientations of said ligand on said multimeric ligand compounds. Such reactive functionality includes, by way of example, carboxylic acids, carboxylic acid halides, carboxyl esters, amines, halides, pseudohalides, isocyanates, vinyl unsaturation, ketones, aldehydes, thiols, alcohols, anhydrides, boronates, and precursors thereof. It is understood, of course, that the reactive functionality on the ligand is selected to be complementary to at least one of the reactive groups on the linker so that a covalent linkage can be formed between the linker and the ligand.

In other embodiments, the multimeric ligand compound is homomeric (i. e., each of the ligands is the same, although it may be attached at different points) or heteromeric (i. e., at least one of the ligands is different from the other ligands).

In addition to the combinatorial methods described herein, this invention provides for an iterative process for rationally evaluating what molecular constraints impart multibinding properties to a class of multimeric compounds or ligands targeting a receptor. Specifically, this method aspect is directed to a method for identifying multimeric ligand compounds possessing multibinding properties for bradykinin receptor which method comprises: (a) preparing a first collection or iteration of multimeric compounds which is prepared by contacting at least two stoichiometric equivalents of the ligand or mixture of ligands which target a receptor with a linker or mixture of linkers wherein said ligand or mixture of ligands comprises at least one reactive

functionality and said linker or mixture of linkers comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand wherein said contacting is conducted under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands; (b) assaying said first collection or iteration of multimeric compounds to assess which if any of said multimeric compounds possess multibinding properties; for bradykinin receptor (c) repeating the process of (a) and (b) above until at least one multimeric compound is found to possess multibinding properties for bradykinin receptor; (d) evaluating what molecular constraints imparted multibinding properties to the multimeric compound or compounds found in the first iteration recited in (a)- (c) above; (e) creating a second collection or iteration of multimeric compounds which elaborates upon the particular molecular constraints imparting multibinding properties to the multimeric compound or compounds found in said first iteration; (f) evaluating what molecular constraints imparted enhanced multibinding properties to the multimeric compound or compounds found in the second collection or iteration recited in (e) above; (g) optionally repeating steps (e) and (f) to further elaborate upon said molecular constraints.

Preferably, steps (e) and (f) are repeated at least two times, more preferably from 2-50 times, even more preferably from 3 to 50 times, and still more preferably at least 5-50 times.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 illustrates examples of multibinding compounds comprising 2 ligands attached in different formats to a linker.

FIG. 2 illustrates examples of multibinding compounds comprising 3 ligands attached in different formats to a linker.

FIG. 3 illustrates examples of multibinding compounds comprising 4 ligands attached in different formats to a linker.

FIG. 4 illustrates examples of multibinding compounds comprising >4 ligands attached in different formats to a linker.

FIG. 5 illustrates a synthesis of a compound of formula (b).

FIGS. 6-15 illustrate syntheses of bivalent multibinding compounds of Formula (I).

DETAILED DESCRIPTION OF THE INVENTION Definitions This invention is directed to multibinding compounds which are bradykinin receptor antagonists, pharmaceutical compositions containing such compounds and methods for treating diseases mediated by a bradykinin receptor in mammals.

When discussing such compounds, compositions or methods, the following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.

The term"alkyl"refers to a monoradical branched or unbranched saturated hydrocarbon chain preferably having from 1 to 40 carbon atoms, more preferably 1 to 10 carbon atoms, and even more preferably 1 to 6 carbon atoms. This term is exemplified by groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso- butyl, n-hexyl, n-decyl, tetradecyl, and the like.

The term"substituted alkyl"refers to an alkyl group as defined above, having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy,

heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,-SO-alkyl,-SO-substituted alkyl,-SO-aryl,-SO-heteroaryl, -SO2-alkyl,-SO2-substituted alkyl,-SO,-aryl and-SO,-heteroaryl. This term is exemplified by groups such as hydroxymethyl, hydroxyethyl, hydroxypropyl, 2- aminoethyl, 3-aminopropyl, 2-methylaminoethyl, 3-dimethylaminopropyl, 2- sulfonamidoethyl, 2-carboxyethyl, and the like.

The term"alkylene"refers to a diradical of a branched or unbranched saturated hydrocarbon chain, preferably having from 1 to 40 carbon atoms. more preferably 1 to 10 carbon atoms and even more preferably 1 to 6 carbon atoms.

This term is exemplified by groups such as methylene (-CH,-), ethylene (-CH, CH2-), the propylene isomers (e. g.,-CH2CH2CH2-and-CH (CH3) CH,-) and the like.

The term"substituted alkylene"refers to an alkylene group, as defined above, having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,-SO-alkyl,-SO-substituted alkyl,-SO-aryl,-SO-heteroaryl, -SO,-alkyl,-SO,-substituted alkyl,-SO,-aryl and-SO2-heteroaryl. Additionally, such substituted alkylene groups include those where 2 substituents on the alkylene group are fused to form one or more cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heterocyclic or heteroaryl groups fused to the alkylene group. Preferably such fused groups contain from 1 to 3 fused ring structures.

The term"alkaryl"or"aralkyl"refers to the groups-alkylene-aryl and -substituted alkylene-aryl where alkylene, substituted alkylene and aryl are defined herein. Such alkaryl groups are exemplified by benzyl, phenethyl and the like.

The term"alkoxy"refers to the groups alkyl-O-, alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O-, where alkyl, alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein. Preferred alkoxy groups are alkyl-O-and include, by way of example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.

The term"substituted alkoxy"refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O-where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.

The term"alkenyl"refers to a monoradical of a branched or unbranched unsaturated hydrocarbon group preferably having from 2 to 40 carbon atoms, more preferably 2 to 10 carbon atoms and even more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-6 sites of vinyl unsaturation. Preferred alkenyl groups include ethenyl (-CH=CH), n-propenyl (-CH, CH=CH,), iso- propenyl (-C (CH3) =CH,), and the like.

The term"substituted alkenyl"refers to an alkenyl group as defined above having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,-SO-alkyl,-SO-substituted alkyl,-SO-aryl,-SO-heteroaryl, -SO,-alkyl,-SO,-substituted alkyl,-SO,-aryl and-SO,-heteroaryl.

The term"alkenylene"refers to a diradical of a branched or unbranched unsaturated hydrocarbon group preferably having from 2 to 40 carbon atoms, more preferably 2 to 10 carbon atoms and even more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-6 sites of vinyl unsaturation. This term is

exemplified by groups such as ethenylene (-CH=CH-), the propenylene isomers (e. g.,-CH, CH=CH-,-C (CH3) =CH-, and the like.

The term"substituted alkenylene"refers to an alkenylene group as defined above having from 1 to 5 substituents, and preferably from 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,-SO-alkyl,-SO-substituted alkyl,-SO-aryl, <BR> <BR> -SO-heteroaryl,-SO2-alkyl,-SO,-substituted alkyl,-SO,-aryl and-SO,-heteroaryl.

Additionally, such substituted alkenylene groups include those where 2 substituents on the alkenylene group are fused to form one or more cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heterocyclic or heteroaryl groups fused to the alkenylene group.

The term"alkynyl"refers to a monoradical of an unsaturated hydrocarbon preferably having from 2 to 40 carbon atoms, more preferably 2 to 20 carbon atoms and even more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-6 sites of acetylene (triple bond) unsaturation. Preferred alkynyl groups include ethynyl (-C--CH), propargyl (-CH, C=CH) and the like.

The term"substituted alkynyl"refers to an alkynyl group as defined above having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,-SO-alkyl,-SO-substituted alkyl,-SO-aryl,-SO-heteroaryl,

-SO,-alkyl,-SO,-substituted alkyl,-SO,-aryl, and-SO-heteroaryl.

The term"alkynylene"refers to a diradical of an unsaturated hydrocarbon preferably having from 2 to 40 carbon atoms, more preferably 2 to 10 carbon atoms and even more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-6 sites of acetylene (triple bond) unsaturation. Preferred alkynylene groups include ethynylene (-C=C-), propargylene (-CH2CEC-) and the like.

The term"substituted alkynylene"refers to an alkynylene group as defined above having from 1 to 5 substituents, and preferably 1 to 3 substituents. selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,-SO-alkyl,-SO-substituted alkyl,-SO-aryl,-SO-heteroaryl, -SO,-alkyl,-SO,-substituted alkyl,-SO2-aryl and-SO-heteroaryl.

The term"acyl"refers to the groups HC (O)-, alkyl-C (O)-, substituted alkyl- C (O)-, alkenyl-C (O)-, substituted alkenyl-C (O)-, cycloalkyl-C (O)-, substituted cycloalkyl-C (O)-, cycloalkenyl-C (O)-, substituted cycloalkenyl-C (O)-, aryl-C (O)-, heteroaryl-C (O)- and heterocyclic-C (O)- where alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term"acylamino"or"aminocarbonyl"refers to the group-C (O) NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, heterocyclic or where both R groups are joined to form a heterocyclic group (e. g., morpholino) wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term"aminoacyl"refers to the group-NRC (O) R where each R is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,

substituted amino, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term"aminoacyloxy"or"alkoxycarbonylamino"refers to the group -NRC (O) OR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term"acyloxy"refers to the groups alkyl-C (O) O-, substituted alkyl- C (O) O-, cycloalkyl-C (O) O-, substituted cycloalkyl-C (O) O-, aryl-C (O) O-* heteroaryl-C (O) O-, and heterocyclic-C (O) 0- wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclic are as defined herein.

The term"aryl"refers to an unsaturated aromatic carbocyclic group of from 6 to 20 carbon atoms having a single ring (e. g., phenyl) or multiple condensed (fused) rings (e. g., naphthyl or anthryl). Preferred aryls include phenyl, naphthyl and the like. Unless otherwise constrained by the definition for the aryl substituent, such aryl groups can optionally be substituted with from 1 to 5 substituents, preferably 1 to 3 substituents, selected from the group consisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl,-SO-substitutedalkyl,-SO-aryl,-SO-heteroaryl,-SO,- alkyl,-SO,- substituted alkyl,-SO2-aryl,-SO2-heteroaryl and trihalomethyl. Preferred aryl substituents include alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy.

The term"aryloxy"refers to the group aryl-O-wherein the aryl group is as defined above including optionally substituted aryl groups as also defined above.

The term"arylene"refers to the diradical derived from aryl (including substituted aryl) as defined above and is exemplified by 1,2-phenylene, 1. 3- phenylene, 1,4-phenylene, 1,2-naphthylene and the like.

The term"amino"refers to the group-NH,.

The term"substituted amino refers to the group-NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, acyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl. heteroaryl and heterocyclic provided that both R's are not hydrogen.

The term"carboxyalkyl"or"alkoxycarbonyl"refers to the groups "-C (O) O-alkyl","-C (O) 0-substituted alkyl","-C (O) O-cycloalkyl","-C (O) O- substituted cycloalkyl","-C (O) O-alkenyl","-C (O) 0-substituted alkenyl', "-C (O) O-alkynyl"and"-C (O) O-substituted alkynyl"where alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl and substituted alkynyl alkynyl are as defined herein.

The term"cycloalkyl"refers to cyclic alkyl groups of from 3 to 20 carbon atoms having a single cyclic ring or multiple condensed rings. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.

The term"substituted cycloalkyl"refers to cycloalkyl groups having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,-SO-alkyl,

-SO-substituted alkyl,-SO-aryl,-SO-heteroaryl,-SO,-alkyl,-SO,-substituted alkyl, -SO-aryl and-SO-heteroaryl.

The term"cycloalkenyl"refers to cyclic alkenyl groups of from 4 to 20 carbon atoms having a single cyclic ring and at least one point of internal unsaturation. Examples of suitable cycloalkenyl groups include, for instance, cyclobut-2-enyl, cyclopent-3-enyl, cyclooct-3-enyl and the like.

The term"substituted cycloalkenyl"refers to cycloalkenyl groups having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,-SO-alkyl,-SO- substituted alkyl,-SO-aryl,-SO-heteroaryl,-SO,-alkyl,-SO,-substituted alkyl,- SO-aryl and-SO-heteroaryl.

The term"halo"or"halogen"refers to fluoro, chloro, bromo and iodo.

The term"heteroaryl"refers to an aromatic group of from 1 to 15 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfur within at least one ring (if there is more than one ring). Unless otherwise constrained by the definition for the heteroaryl substituent, such heteroaryl groups can be optionally substituted with 1 to 5 substituents, preferably 1 to 3 substituents, selected from the group consisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy,-SO-alkyl,-SO-substituted alkyl,-SO-aryl,

-SO-heteroaryl,-SO,-alkyl,-SO,-substituted alkyl,-SO,-aryl,-SO,-heteroaryl and trihalomethyl. Preferred heteroaryl substituents include alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy. Such heteroaryl groups can have a single ring (e. g., pyridyl or furyl) or multiple condensed rings (e. g., indolizinyl or benzothienyl). Preferred heteroaryls include pyridyl, pyrrolyl and furyl.

The term"heteroaryloxy"refers to the group heteroaryl-O-.

The term"heteroarylene"refers to the diradical group derived from heteroaryl (including substituted heteroaryl), as defined above, and is exemplified by the groups 2,6-pyridylene, 2,4-pyridiylene, 1,2-quinolinylene, 1,8-quinolinylene, 1,4-benzofuranylene, 2,5-pyridnylene, 2,5-indolenyl, and the like.

The term"heterocycle"or"heterocyclyl"refers to a monoradical saturated unsaturated group having a single ring or multiple condensed rings, from 1 to 40 carbon atoms and from 1 to 10 hetero atoms, preferably 1 to 4 heteroatoms, selected from nitrogen, sulfur, phosphorus, and/or oxygen within the ring and further wherein one, two, or three of the ring carbon atoms may optionally be replaced with a carbonyl group (i. e., a keto group). Unless otherwise constrained by the definition for the heterocyclic substituent, such heterocyclic groups can be optionally substituted with 1 to 5, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,-SO-alkyl,-SO-substituted alkyl,-SO-aryl, -SO-heteroaryl,-SO,-alkyl,-SO,-substituted alkyl,-SO2-aryl and-SO-heteroaryl.

Such heterocyclic groups can have a single ring or multiple condensed rings.

Preferred heterocyclics include morpholino, piperidinyl, and the like.

Examples of heteroaryls and heterocycles include, but are not limited to, pyrrole, thiophene, furan, imidazole, pyrazole, pyridine, pyrazine, pyrimidine,

pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, pyrrolidine, piperidine, piperazine, indoline, morpholine, tetrahydrofuranyl, tetrahydrothiophene, and the like as well as N-alkoxy-nitrogen containing heterocycles.

The term"heterocyclooxy"refers to the group heterocyclic-O-.

The term"thioheterocyclooxy"refers to the group heterocyclic-S-.

The term"heterocyclene"refers to the diradical group formed from a heterocycle, as defined herein, and is exemplified by the groups 2,6-morpholino, 2,5-morpholino and the like.

The term"oxyacylamino"or"aminocarbonyloxy"refers to the group -OC (O) NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term"spiro-attached cycloalkyl group"refers to a cycloalkyl group joined to another ring via one carbon atom common to both rings.

The term"thiol"refers to the group-SH.

The term"thioalkoxy"or"alkylthio"refers to the group-S-alkyl.

The term"substituted thioalkoxy"refers to the group-S-substituted alkyl.

The term"thioaryloxy"refers to the group aryl-S-wherein the aryl group is as defined above including optionally substituted aryl groups also defined above.

The term"thioheteroaryloxy"refers to the group heteroaryl-S-wherein the heteroaryl group is as defined above including optionally substituted aryl groups as also defined above.

The term"amino acid residue"refers to compounds having both carboxylic acid and amino functional groups and include both natural (L-amino acids) and unnatural amino acids (D-amino acids). Natural amino acids, include by way of examples, glycine, alanine, valine, serine, glutamic acid, aspartic acid, lysine, and

the like. Unnatural amino acids, include by way of examples, D-amino acids of naturally occurring L-amino acids, sarcosine, 1-napthylalanine, and the like.

The term"acylbiphenyl"refers to a biphenyl ring substituted with an acyl group as defined above.

As to any of the above groups which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non- feasible. In addition, the compounds of this invention include all stereochemical isomers arising from the substitution of these compounds.

The term"pharmaceutically-acceptable salt"refers to salts which retain the biological effectiveness and properties of the multibinding compounds of this invention and which are not biologically or otherwise undesirable. In many cases, the multibinding compounds of this invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.

Pharmaceutically-acceptable base addition salts can be prepared from inorganic and organic bases. Salts derived from inorganic bases, include by way of example only, sodium, potassium, lithium, ammonium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, such as alkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines, di (substituted alkyl) amines, tri (substituted alkyl) amines, alkenyl amines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines, di (substituted alkenyl) amines, tri (substituted alkenyl) amines, cycloalkyl amines, di (cycloalkyl) amines, tri (cycloalkyl) amines, substituted cycloalkyl amines, disubstituted cycloalkyl amine, trisubstituted cycloalkyl amines, cycloalkenyl amines, di (cycloalkenyl) amines, tri (cycloalkenyl) amines, substituted cycloalkenyl amines, disubstituted cycloalkenyl amine, trisubstituted cycloalkenyl amines, aryl amines, diaryl amines, triaryl amines, heteroaryl amines, diheteroaryl amines, triheteroaryl amines, heterocyclic amines, diheterocyclic amines, triheterocyclic amines, mixed di-and tri-amines where at

least two of the substituents on the amine are different and are selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic, and the like. Also included are amines where the two or three substituents, together with the amino nitrogen, form a heterocyclic or heteroaryl group.

Examples of suitable amines include, by way of example only, isopropylamine, trimethyl amine, diethyl amine, tri (iso-propyl) amine, tri (n-propyl) amine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine, purines, piperazine, piperidine, morpholine, N-ethylpiperidine, and the like. It should also be understood that other carboxylic acid derivatives would be useful in the practice of this invention, for example, carboxylic acid amides, including carboxamides, lower alkyl carboxamides, dialkyl carboxamides, and the like.

Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids. Salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Salts derived from organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid, salicylic acid, and the like.

The term"pharmaceutically-acceptable cation"refers to the cation of a pharmaceutically-acceptable salt.

The term"library"refers to at least 3, preferably from 102 to 109 and more preferably from 102 to 104 multimeric compounds. Preferably, these compounds are prepared as a multiplicity of compounds in a single solution or reaction mixture which permits facile synthesis thereof. In one embodiment, the library of multimeric compounds can be directly assayed for multibinding properties. In

another embodiment, each member of the library of multimeric compounds is first isolated and, optionally, characterized. This member is then assayed for multibinding properties.

The term"collection"refers to a set of multimeric compounds which are prepared either sequentially or concurrently (e. g., combinatorially). The collection comprises at least 2 members; preferably from 2 to 109 members and still more preferably from 10 to 104 members.

The term"multimeric compound"refers to compounds comprising from 2 to 10 ligands covalently connected through at least one linker which compounds may or may not possess multibinding properties (as defined herein).

The term"pseudohalide"refers to functional groups which react in displacement reactions in a manner similar to a halogen. Such functional groups include, by way of example, mesyl, tosyl, azido and cyano groups.

The term"protecting group"or"blocking group"refers to any group which when bound to one or more hydroxyl, thiol, amino or carboxyl groups of the compounds (including intermediates thereof) prevents reactions from occurring at these groups and which protecting group can be removed by conventional chemical or enzymatic steps to reestablish the hydroxyl, thiol, amino or carboxyl group (See., T. W. Greene"Protective Groups in Organic Synthesis", 2"d Ed.).

The particular removable blocking group employed is not critical and preferred removable hydroxyl blocking groups include conventional substituents such as allyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidine, phenyl, t- butyl-diphenylsilyl and any other group that can be introduced chemically onto a hydroxyl functionality and later selectively removed either by chemical or enzymatic methods in mild conditions compatible with the nature of the product.

Preferred removable thiol blocking groups include disulfide groups, acyl groups, benzyl groups, and the like.

Preferred removable amino blocking groups include conventional substituents such as t-butyoxycarbonyl (t-BOC), benzyloxycarbonyl (CBZ), fluorenylmethoxy-carbonyl (FMOC), allyloxycarbonyl (ALOC), and the like which

can be removed by conventional conditions compatible with the nature of the product.

Preferred carboxyl protecting groups include esters such as methyl, ethyl, propyl, t-butyl etc. which can be removed by mild conditions compatible with the nature of the product.

The term"optional"or"optionally"means that the subsequently described event, circumstance or substituent may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

The term"ligand"or"ligands"as used herein denotes a compound that is a bradykinin receptor antagonist. The specific region or regions of the ligand that is (are) recognized by the receptor is designated as the"ligand domain". A ligand may be either capable of binding to the receptor by itself, or may require the presence of one or more non-ligand components for binding (e. g., Ca+2, Mg+2 or a water molecule is required for the binding of a ligand to various ligand binding sites).

Examples of ligands useful in this invention are described herein. Those skilled in the art will appreciate that portions of the ligand structure that are not essential for specific molecular recognition and binding activity may be varied substantially, replaced or substituted with unrelated structures (for example, with ancillary groups as defined below) and, in some cases, omitted entirely without affecting the binding interaction. The primary requirement for a ligand is that it has a ligand domain as defined above. It is understood that the term ligand is not intended to be limited to compounds known to be useful in binding to bradykinin receptor (e. g., known drugs). Those skilled in the art will understand that the term ligand can equally apply to a molecule that is not normally associated with bradykinin receptor binding properties. In addition, it should be noted that ligands that exhibit marginal activity or lack useful activity as monomers can be highly active as multivalent compounds because of the benefits conferred by multivalency.

The term"ligand"or"ligands"as used herein is intended to include the racemic forms of the ligands as well as individual enantiomers and diasteromers and non-racemic mixtures thereof.

The term"multibinding compound or agent"refers to a compound that is capable of multivalency, as defined below, and which has 2-10 ligands covalently bound to one or more linkers. In all cases, each ligand and linker in the multibinding compound is independently selected such that the multibinding compound includes both symmetric compounds (i. e., where each ligand as well as each linker is identical) and asymmetric compounds ( (i. e., where at least one of the ligands is different from the other ligand (s) and/or at least one linker is different from the other linker (s)). Additionally, the term is intended to include the racemic forms of the multibinding compound as well as individual enantiomers and diasteromers and non-racemic mixtures thereof. Multibinding compounds provide a biological and/or therapeutic effect greater than the aggregate of unlinked ligands equivalent thereto which are made available for binding. That is to say that the biological and/or therapeutic effect of the ligands attached to the multibinding compound is greater than that achieved by the same amount of unlinked ligands made available for binding to the ligand binding sites (receptors).

The phrase"increased biological or therapeutic effect"includes, for example: increased affinity, increased selectivity for target, increased specificity for target, increased potency, increased efficacy, decreased toxicity, improved duration of activity or action, increased ability to kill cells such as fungal pathogens, cancer cells, etc., decreased side effects, increased therapeutic index, improved bioavailibity, improved pharmacokinetics, improved activity spectrum, and the like.

The multibinding compounds of this invention will exhibit at least one and preferably more than one of the above-mentioned affects.

The term"univalency"as used herein refers to a single binding interaction between one ligand as defined herein with one ligand binding site as defined herein.

It should be noted that a compound having multiple copies of a ligand (or ligands) exhibit univalency when only one ligand is interacting with a ligand binding site. Examples of univalent interactions are depicted below.

The term"multivalency"as used herein refers to the concurrent binding of from 2 to 10 linked ligands (which may be the same or different) and two or more corresponding receptors (ligand binding sites) on one or more enzymes which may be the same or different.

For example, two ligands connected through a linker that bind concurrently to two ligand binding sites would be considered as bivalency; three ligands thus connected would be an example of trivalency. An example of trivalent binding, illustrating a multibinding compound bearing three ligands versus a monovalent binding interaction, is shown below: univalent interaction trivalent interaction

It should be understood that not all compounds that contain multiple copies of a ligand attached to a linker or to linkers necessarily exhibit the phenomena of multivalency, i. e., that the biological and/or therapeutic effect of the multibinding agent is greater than the sum of the aggregate of unlinked ligands made available for binding to the ligand binding site (receptor). For multivalency to occur, the ligands that are connected by a linker or linkers have to be presented to their ligand binding sites by the linker (s) in a specific manner in order to bring about the desired ligand-orienting result, and thus produce a multibinding event.

The term"potency"refers to the minimum concentration at which a ligand is able to achieve a desirable biological or therapeutic effect. The potency of a ligand is typically proportional to its affinity for its ligand binding site. In some cases, the potency may be non-linearly correlated with its affinity. In comparing the potency of two drugs, e. g., a multibinding agent and the aggregate of its unlinked ligand, the dose-response curve of each is determined under identical test conditions (e. g., in an in vitro or in vivo assay, in an appropriate animal model). The finding that the multibinding agent produces an equivalent biological or therapeutic effect at a lower concentration than the aggregate unlinked ligand is indicative of enhanced potency.

The term"selectivity"or"specificity"is a measure of the binding preferences of a ligand for different ligand binding sites (receptors). The selectivity of a ligand with respect to its target ligand binding site relative to another ligand binding site is given by the ratio of the respective values of Kd (i. e., the dissociation constants for each ligand-receptor complex) or, in cases where a biological effect is observed below the Kd, the ratio of the respective EC50's (i. e., the concentrations that produce 50% of the maximum response for the ligand interacting with the two distinct ligand binding sites (receptors)).

The term"ligand binding site"denotes the site on the bradykinin receptor that recognizes a ligand domain and provides a binding partner for the ligand. The ligand binding site may be defined by monomeric or multimeric structures. This interaction may be capable of producing a unique biological effect, for example,

agonism, antagonism, modulatory effects, may maintain an ongoing biological event, and the like.

It should be recognized that the ligand binding sites of the receptor that participate in biological multivalent binding interactions are constrained to varying degrees by their intra-and inter-molecular associations. For example, ligand binding sites may be covalently joined to a single structure, noncovalently associated in a multimeric structure, embedded in a membrane or polymeric matrix, and so on and therefore have less translational and rotational freedom than if the same structures were present as monomers in solution.

The term"antagonism"is well known in the art. The term"modulatory effect"refers to the ability of the ligand to change the activity of an agonist or antagonist through binding to a ligand binding site.

The term"inert organic solvent"or"inert organic solvent"means a solvent which is inert under the conditions of the reaction being described in conjunction therewith including, by way of example only, benzene, toluene, acetonitrile, tetrahydrofuran, dimethylformamide, chloroform, methylene chloride, diethyl ether, ethyl acetate, acetone, methylethyl ketone, methanol, ethanol, propanol, isopropanol, t-butanol, dioxane, pyridine, and the like. Unless specified to the contrary, the solvents used in the reactions described herein are inert solvents.

The term"treatment"refers to any treatment of a pathologic condition in a mammal, particularly a human, and includes: (i) preventing the pathologic condition from occurring in a subject which may be predisposed to the condition but has not yet been diagnosed with the condition and, accordingly, the treatment constitutes prophylactic treatment for the disease condition; (ii) inhibiting the pathologic condition, i. e., arresting its development; (iii) relieving the pathologic condition, i. e., causing regression of the pathologic condition; or (iv) relieving the conditions mediated by the pathologic condition.

The term"pathologic condition which is modulated by treatment with a ligand"covers all disease states (i. e., pathologic conditions) which are generally acknowledged in the art to be usefully treated with a ligand for the bradykinin receptors in general, and those disease states which have been found to be usefully treated by a specific multibinding compound of our invention. Such disease states include, by way of example only, the treatment of a mammal afflicted with cancer, pain, shock, asthma, rhinitis, arthritis, inflammatory bowel disease, and the like.

The term"therapeutically effective amount"refers to that amount of multibinding compound which is sufficient to effect treatment, as defined above, when administered to a mammal in need of such treatment. The therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.

The term"linker", identified where appropriate by the symbol'X'refers to a group or groups that covalently attaches from 2 to 10 ligands (as identified above) in a manner that provides for a compound capable of multivalency. The term is intended to include the racemic forms of the linker as well as individual enantiomers and diasteromers and non-racemic mixtures thereof. Among other features, the linker is a ligand-orienting entity that permits attachment of multiple copies of a ligand (which may be the same or different) thereto. In some cases, the linker may itself be biologically active. The term"linker"does not, however, extend to cover solid inert supports such as beads, glass particles, fibers, and the like. But it is understood that the multibinding compounds of this invention can be attached to a solid support if desired. For example, such attachment to solid supports can be made for use in separation and purification processes and similar applications.

The extent to which multivalent binding is realized depends upon the efficiency with which the linker or linkers that joins the ligands presents these ligands to the array of available ligand binding sites. Beyond presenting these

ligands for multivalent interactions with ligand binding sites, the linker or linkers spatially constrains these interactions to occur within dimensions defined by the linker or linkers. Thus, the structural features of the linker (valency, geometry, orientation, size, flexibility, chemical composition, etc.) are features of multibinding agents that play an important role in determining their activities.

The linkers used in this invention are selected to allow multivalent binding of ligands to the ligand binding sites of a bradykinin receptor, whether such sites are located interiorly, both interiorly and on the periphery of the enzyme structure, or at any intermediate position thereof.

PREFERRED EMBODIMENTS While the broadest definition of this invention is set forth in the Summary of the Invention, certain compounds of Formula (I) are preferred.

(A) A preferred group is a multibinding compound of Formula (I) wherein: p is 2 or 3, preferably 2; and q is 1 or 2, preferably 1.

Within this group (A), a more preferred group of compounds is that wherein the ligands are a compound of formula (b) as defined in the Summary of the Invention.

Within compounds of formula (b), preferred compounds are: (i) a compound of formula (II): R13 iS quinolyl, quinoxalinyl, benzimidazolyl, or imidazopyridyl each of which is substituted with one or more substituent (s) selected from halo, alkyl or alkoxy, preferably wherein R 31 is chloro, bromo or iodo; R'4 and R'S are, independently of each other, hydrog m, alkyl, or halo, preferably hydrogen, methyl or chloro, most preferably methyl or chloro; R2'is hydrogen or methyl, preferably methyl; and the terminal nitrogen atom attaches the ligand to a linker; or (ii) a compound of formula (III) : wherein: R'3 is quinolyl, quinoxalinyl, benzimidazolyl, or imidazopyridyl each of which is substituted with one or more substituent (s) selected from halo, alkyl or alkoxy, preferably wherein R33 is chloro, bromo or iodo;

Rl4 and Rl5 are, independently of each other, hydrogen, alkyl, halo, or a covalent bond linking the ligand to a linker, preferably hydrogen, methyl or chloro, or a covalent bond linking the ligand to a linker, most preferably methyl or chloro; R2'is hydrogen, alkyl, or a covalent bond linking the ligand to a linker; preferably hydrogen, methyl or a covalent bond linking the ligand to a linker ; and Ra is-COOH,-NH2,-CoNR23R29 (wherein R23 is hydrogen or alkyl and R29 is hydrogen, alkyl, or heteroaryl),-NR30COR3' (where R30 is hydrogen or alkyl and R3'is alkyl),-NR30CONHR32 (where R"is hydrogen or alkyl, and R32 is alkyl), heteroaralkyl, heterocyclyl or a covalent bond linking the ligand to a linker, preferably-CONHCH3,-CON (CH3) 2,-NHCOCH3,-N (CH3) COCH3, -NHCONHCH3, or a covalent bond linking the ligand to a linker; W is-CH-or-N- ; or (iii) a compound of formula (IV): wherein: R'3, R'4 and R'5 are as defined in preferred embodiment (i) above; and the terminal nitrogen atom attaches the ligand to a linker; or (iv) a compound of formula (V):

wherein: R'3, R'4, R'5, W, and Ra are as defined in the preferred embodiments (ii) above; and pharmaceutically acceptable salts thereof.

In one embodiment, the multibinding compound comprises of identical ligands.

In one embodiment, the multibinding compound comprises of non-identical ligands.

Within these preferred and more preferred groups, even more preferred compounds are those shown below:

wherein R'3, R'4, R'5, Ra, X, and W are as defined in preferred embodiments above.

Within the above preferred groups, particularly preferred group of compounds is that wherein the linker, X, in the bivalent multibinding compound of Formula (I) independently has the formula: _Xa_Z_ (ya_Z) m_Xa_ wherein m is an integer of from 0 to 20, preferably 0 to 5; Xa at each separate occurrence is selected from the group consisting of -O-,-S-,-NR-,-C (O)-,-C (O) O-,-C (O) NR-,-C (S),-C (S) O-,-C (S) NR- or a covalent bond where R is as defined below; Z at each separate occurrence is selected from the group consisting of alkylene, substituted alkylene, cycloalkylene, substituted cylcoalkylene. alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, cycloalkenylene,

substituted cycloalkenylene, arylene, heteroarylene, heterocyclene, or a covalent bond, preferably alkylene; each ya at each separate occurrence is selected from the group consisting of -O-,-C (O)-,-OC (O)-,-C (O) O-,-NR-,-S (O) n-,-C (O) NR'-,-NR'C (O)-,-NR' C (O) NR'-,-NR'C (S) NR'-,-C (=NR')-NR'-,-NR'-C (=NR')-,-OC (O)-NR'-,-NR'- C (O)-O-,-N=C (Xa)-NR'-,-NR'-C (Xa) =N-,-P (O) (OR')-O-,-O-P (O) (OR')-,- S (O) nCR'R"-,-S (O) n-NR'-,-NR'-S (O) n-,-S-S-, and a covalent bond; where n is 0, 1 or 2; and R, R'and R"at each separate occurrence are selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl. alkynyl, substituted alkynyl, aryl, heteroaryl and heterocyclic provided that at least one of Xa, Z, and ya iS not a covalent bond, preferably-0-.

GENERAL SYNTHETIC SCHEME Compounds of this invention can be made by the methods depicted in the reaction schemes shown below.

The starting materials and reagents used in preparing these compounds are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wisconsin, USA), Bachem (Torrance, California, USA), Emka-Chemie, or Sigma (St. Louis, Missouri, USA) or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition), and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989).

The starting materials and the intermediates of the reaction may be isolated and purified if desired using conventional techniques, including but not limited to filtration, distillation, crystallization, chromatography, and the like. Such materials

may be characterized using conventional means, including physical constants and spectral data.

Furthermore, it will be appreciated that where typical or preferred process conditions (i. e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. The choice of a suitable protecting group for a particular functional group as well as suitable conditions for protection and deprotection are well known in the art. For example, numerous protecting groups, and their introduction and removal, are described in T. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York, 1991, and references cited therein.

These schemes are merely illustrative of some methods by which the compounds of this invention can be synthesized, and various modifications to these schemes can be made and will be suggested to one skilled in the art having referred to this disclosure.

Preparation of a multibinding compound of Formula CD In general, a bivalent multibinding compound of Formula (I) can be prepared by covalently attaching the ligands, L, wherein at least one of the ligand is selected from a compound of formula (a) as defined in the Summary of the Invention, to a linker, X, as shown in Scheme A below.

Scheme A Method (a) Method (b) FG L1 + FG2 X FG2PG b L1 X FG2PG [intermediate] (II) deprotectFG ----- L-X-FG2 + - L X L2 In method (a), a bivalent multibinding compound of Formula (I) is prepared in one step, by covalently attaching the ligands, L, to a linker, X, where FG'and FG2 represent a functional group such as halo, amino, hydroxy, thio, aldehyde, ketone, carboxy, carboxy derivatives such as acid halide, ester, amido, and the like.

This method is preferred for preparing compounds of Formula (I) where the ligands are the same.

In method (b), the compounds of Formula (I) are prepared in a stepwise manner by covalently attaching one equivalent of a ligand, L,, with a ligand X where where FG'and FG2 represent a functional group as defined above, and FG2PG is a protected functional group to give an intermediate of formula (II).

Deprotection of the second functional group on the ligand, followed by reaction with a ligand L,, which may be same or different than ligand L,, then provides a

compound of Formula (I). This method is suitable for preparing compounds of Formula (I) where the ligands are the non-identical.

The ligands are covalently attached to the linker using conventional chemical techniques providing for covalent linkage of the ligand to the linker.

Reaction chemistries resulting in such linkages are well known in the art and involve the use of complementary functional groups on the linker and ligand as shown in Table I below.

Table I Representative Complementarv Binding Ci. mistries First Reactive Group Second Reactive Group Linkage carboxyl amine amide sulfonyl halide amine sulfonamide hydroxyl alkyl/aryl halide ether hydroxyl isocyanate urethane amine epoxide p-hydroxyamine amine alkyl/aryl halide alkylamine hydroxyl carboxyl ester Reaction between a carboxylic acid of either the linker or the ligand and a primary or secondary amine of the ligand or the linker in the presence of suitable, well-known activating agents such as dicyclohexylcarbodiimide, results in formation of an amide bond covalently linking the ligand to the linker; reaction between an amine group of either the linker or the ligand and a sulfonyl halide of the ligand or the linker, in the presence of a base such as triethylamine, pyridine, an the like results in formation of a sulfonamide bond covalently linking the ligand to the linker; and reaction between an alcohol or phenol group of either the linker or the ligand and an alkyl or aryl halide of the ligand or the linker in the presence of a base such as triethylamine, pyridine, and the like, results in formation of an ether bond covalently linking the ligand to the linker.

Any compound which inhibits bradykinin receptor can be used as a ligand in this invention. As discussed in further detail below, numerous antagonists are known in the art and any of these known compounds or derivatives thereof may be employed as ligands in this invention. Typically, a compound selected for use as a ligand will have at least one functional group, such as an amino, hydroxyl, thiol or carboxyl group and the like, which allows the compound to be readily coupled to the linker. Compounds having such functionality are either known in the art or can be prepared by routine modification of known compounds using conventional reagents and procedures. The patents and publications set forth below provide numerous examples of suitably functionalized bradykinin receptor antagonist and intermediates thereof which may be used as ligands in this invention.

The compounds of formula (a) can be prepared as described in PCT Application NO. 96/13485. The compounds of formula (b) can be prepared by the methods described in PCT Application NO. 97/11069 and Y. Abe, et. al., J. Med.

Chem., 41, pages 564,4053,4062, and 4587, (1998). The compounds of formula (c) can be prepared by the methods described in PCT Application NO. 96/06082.

Some of the methods for preparing compounds of formulae (a) and (b) of the present invention are illustrated and described in Schemes B-F below.

Synthesis of compounds of formula fa) Ligands of formula (a) can be prepared as described in Scheme B below.

Scheme B Method (i) Method (ii) C BSA j C-B R1+ R-Q-COOH ! oracid derivative R7 AAH 4 'J R i i N-AA-CO-Q-R 3 R' (a)

In general, compounds of formula (a) can be prepared as shown in methods (i) or (ii) above.

In method (i), a compound of formula (a) is prepared by reacting a compound of formula 1 where X is a leaving group under nucleophilic displacement reaction conditions [such as tosylate, mesylate, or halo (such as chloro, bromo, or iodo)] with an amine or alcohol of formula 2 (B is-NH-or-O-). The reaction is typically carried out in the presence of a base such as triethylamine, and the like.

In method (ii), a compound of formula (a) is prepared by reacting a compound of formula 3 (where AAH is an amino acid residue) with an acid of formula 4 or its reactive derivative such as acid chloride, ester and the like. The reaction conditions used depend on the nature of compound 4. If 4 is an acid, the reaction is carried out in the presence of a coupling agent such as dicyclohexycarbodiimide. If 4 is an acid derivative such as acid chloride, then the reaction is carried out in the presence of a suitable base such as triethylamine, and

the like. Detailed descriptions of the synthesis of compounds of formula (a) via these methods are given in PCT Application NO. 96/13485.

Synthesis of compounds of formula (b ! Compounds of formula (b) can be prepared as illustrated and described in Schemes C-F below.

Compounds of formula (b) where Rl3, Rl4, R'5 are as described in the Summary of the Invention, A'is methylene, Ql is-NH-, A2 is a bond, and R"is acyl can be prepared as described below.

Scheme C XOR RJp, ROH RJ. pi5 reduction X oR13 56 - NOZ N02 5 6 OR13 OR13 14 RbCOCi R1 Ri5 7 (b) 7(b) Reaction of a 3-nitrophenyl compound of formula 5 where X is a leaving group under nucleophilic displacement reaction conditions such as tosylate, mesylate, or halo (such as chloro, bromo, or iodo), preferably mesylate, with an alcohol of formula R'30H where R'3 is as defined in the Summary of the Invention provides a compound of formula 6. The reaction is carried out in the presence of a base such as sodium hydride in an aprotic solvent such as dimethylformamide.

Reduction of the nitro group with a suitable reducing agent such as hydrazine monohydrate, iron (III) chloride hexahydrate and carbon provides an aniline of formula 7 which is then converted to a compound of formula (b) where Q'is-NH-, A2 is a bond, and R"is acyl by reaction 7 with an acylating agent such as as acid

chloride of formula RCOC1 where RCO-is an acyl group as defined in the Summary of the invention.

Compounds of formula 5 are either commercially available or they can be prepared by methods well known in the art. For example, compound 5 where R'4 and R'S are chloro and X is-OMs can be prepared by the method described in Abe, Y. et al, J. Med. Chem., 41,564,1998. Compounds of formula R'30H can be prepared by methods well known in the art. For example, 4-hydroxybenzimidazole derivatives, 8-hydroxy-2-methylquinazoline, 2-and 3-methylquinoxaline derivatives can be prepared by the methods described in Abe, Y. et al, J. Ated.

Chem., 41,4062,1998.

Compounds of formula (b) where A2, Rl3, Rl4, R'5 are as described in the Summary of the Invention, A'is methylene, Q'is-NR2'-, and R"is an aminoacyl (-NHCOR) group can be prepared as described below.

Scheme D oR13 oR13 1.alkylation R14 0 15 RCOCI R14 f 15 (optional) R R5 R R5 or acid derivatives 2.deprotection/ N-CaNH2 NH-CO.-NHCOR R 10 (b) 10 [R= alkyl, aryl, aralky, aralkenyl, heteroaralkenyl, etc] Treatment of a compound of formula 7 with an acid chloride of formula 8 where A2 is alkylene under the reaction conditions described in Abe, Y et al., J.

Med. Chem., 41,564,1998 provides N-phthaloyl derivatives of formula 9 where R

is hydrogen. Compound 9 where R2'is hydrogen can optionally be converted to a corresponding compound of formula 9 where R'-'is alkyl by treating it with an alkylating agent such as alkyl halide in the presence of a base such as sodium hydride. Treatment of 9 with hydrazine monohydrate provides a compound of formula 10 which can be converted to a compound of formula (b) where R"is aminoacyl by treating 10 with an acylating agent as described above. The reaction conditions employed depend on the nature of the acylationg agent. If the acylating agent agent is an acid chloride, the reaction reaction is carried out in the presence of a base such as triethylamine, pyridine or the like and in a suitable organic solvent such as dichloromethane, dimethylformamide, tetrahydrofuran, and the like. If it is an acid, then the reaction is carried out in the presence of a suitable coupling agent such as 1-ethoxy-3- [3- (dimethylamino) propyl] carbodiimide hydrochloride in the presence of 1-hydroxybenzotriazole as described in Abe, Y et al., J. Med. Chem., 41, 4053,1998.

Alternatively, compounds of formula (b) where A'-, R'3, R'a, R''are as described in the Summary of the invention, A'is methylene, Ql is-NR2l-, and R"is an aminoacyl (-NHCOR) group can be prepared as described below.

Scheme E OTBDPS OTBDPS 0 OTBDPS RR4 \ Rs +N-A N-A2-COCI omsOR 13 zu -g 12 omis 14 i.alkylation (opt.) \¢ kR15 o R130H R XRt5 ii.deprotection N-COA2NX ii. deprotection N-COA2NH2 iii. MsCI R21 t R21 13 0 ruz OR" oR13 R14 R15 N-CO-A-NHCOR '21 (b)

Treatment of a compound of formula 11 with an acid chloride of formula 8 where A2 is alkylene under the reaction conditions described in Abe, Y et al., J.

Med. Chem., 41,564,1998 provides N-phthaloyl derivatives of formula 12 where R2l is hydrogen. Removal of tert-butyldiphenylsilyl group with tetrabutylammonium flouride followed by treatment with mesyl chloride provides a compound of formula 13 which can then be converted to a ligand of formula (b) where R is arylalkenyl, heteroaralkenyl, alkyl, aralkyl, heteroaralkyl, etc as described in Scheme D above.

Compounds of formula (b) where R''', R'4, R'5 are as described in the Summary of the Invention, A'and A2 are methylene, R'7 is an aminoacyl (-NHCOR where R is aralkenyl or heteroaralkenyl) and Q'is a group of formula can be prepared as described in Scheme F below.

Scheme F OR" OR'3 R14R15 I, CH2NHz ORRA I "I R14 15 "W. \ R\, J\ R15 Da 15a COOH, \Y/ or acid derivatives 1 w OR 13 (b) H N ri4 ! 0 R 4 15 R , CHZNHZ i w Q1or i 15b

A compound of formula (b) where Q'is pyrrole or phenyl can be prepared by acylating a compound of formula 15a or 15b under the reaction conditions described above. Compounds 15a and 15b can be prepared by the methods described in Abe, Y et al, J. Med. Chem., 41,4587, (1998).

Synthesis of a compound of formula (b) where R'3 is as defined in the Summary of the Invention, R'4 is hydroxy, R'5 is chloro, Rl6 is-Q'-A2-R'7 where Q' is-N (CH3) CO, A 2is-CH,-, and R"is-NHCOR (where R is aralkenyl or heteroaralkenyl) is illustrated in Figure 5.

Syntheses of compounds of formula (b) where R'3, R'4, R'5, are as defined in the Summary of the Invention, R'6 is-Q'-A'--R"where Q'is-N (CH3) CO, A2 is -CH,-, and R'7 is-NHCOR (where R is aralkenyl or heteroaralkenyl) are illustrated in Figures 6 and 7.

Syntheses of some representative compounds of Formula (I) are illustrated in Figures 8-15 and described in detail in working examples 1-13 respectively.

It will be apparent to one skilled in the art that the above chemistries ARE not limited to preparing bivalent multibinding compounds of Formula (I) and can be used to prepare tri-, tetra-, etc., multibinding compounds of Formula (I).

The linker is attached to the ligand at a position such that it retains ligand domain-ligand binding site interaction and specifically which permits the ligand domain of the ligand to orient itself to bind to the ligand binding site. Such positions and synthetic protocols for linkage are well known in the art. The term linker embraces everything that is not considered to be part of the ligand.

The relative orientation in which the ligand domains are displayed derives from the particular point or points of attachment of the ligands to the linker, and on the framework geometry. The determination of where acceptable substitutions can be made on a ligand is typically based on prior knowledge of structure-activity relationships (SAR) of the ligand and/or congeners and/or structural information about ligand-receptor complexes (e. g., X-ray crystallography, NMR, and the like).

Such positions and the synthetic methods for covalent attachment are well known in the art. Following attachment to the selected linker (or attachment to a significant portion of the linker, for example 2-10 atoms of the linker), the univalent linker- ligand conjugate may be tested for retention of activity in the relevant assay.

The linker, when covalently attached to multiple copies of the ligands, provides a biocompatible, substantially non-immunogenic multibinding compound.

The biological activity of the multibinding compound is highly sensitive to the valency, geometry, composition, size, flexibility or rigidity, etc. of the linker and, in turn, on the overall structure of the multibinding compound, as well as the presence or absence of anionic or cationic charge, the relative hydrophobicity/hydrophilicity of the linker, and the like on the linker. Accordingly, the linker is preferably chosen to maximize the biological activity of the multibinding compound. The linker may be chosen to enhance the biological activity of the molecule. In general, the linker may be chosen from any organic molecule construct that orients two or more ligands to their ligand binding sites to permit multivalency. In this regard, the linker can be considered as a"framework"on which the ligands are arranged in

order to bring about the desired ligand-orienting result, and thus produce a multibinding compound.

For example, different orientations can be achieved by including in the framework groups containing mono-or polycyclic groups, including aryl and/or heteroaryl groups, or structures incorporating one or more carbon-carbon multiple bonds (alkenyl, alkenylene, alkynyl or alkynylene groups). Other groups can also include oligomers and polymers which are branched-or straight-chain species. In preferred embodiments, rigidity is imparted by the presence of cyclic groups (e. g., aryl, heteroaryl, cycloalkyl, heterocyclic, etc.). In other preferred embodiments, the ring is a six or ten membered ring. In still further preferred embodiments, the ring is an aromatic ring such as, for example, phenyl or naphthyl.

Different hydrophobic/hydrophilic characteristics of the linker as well as the presence or absence of charged moieties can readily be controlled by the skilled artisan. For example, the hydrophobic nature of a linker derived from hexamethylene diamine (H, N (CH,) 6NH,) or related polyamines can be modified to be substantially more hydrophilic by replacing the alkylene group with a poly (oxyalkylene) group such as found in the commercially available"Jeffamines".

Different frameworks can be designed to provide preferred orientations of the ligands. Such frameworks may be represented by using an array of dots (as shown below) wherein each dot may potentially be an atom, such as C, O, N, S, P, H, F, Cl, Br, and F or the dot may alternatively indicate the absence of an atom at that position. To facilitate the understanding of the framework structure, the framework is illustrated as a two dimensional array in the following diagram, although clearly the framework is a three dimensional array in practice:

Each dot is either an atom, chosen from carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, or halogen, or the dot represents a point in space (i. e., an absence of an atom). As is apparent to the skilled artisan, only certain atoms on the grid have the ability to act as an attachment point for the ligands, namely, C, O, N, S and P.

Atoms can be connected to each other via bonds (single, double or triple bonds with acceptable resonance and tautomeric forms), with regard to the usual constraints of chemical bonding. Ligands may be attached to the framework via single, double or triple bonds (with chemically acceptable tautomeric and resonance forms). Multiple ligand groups (2 to 10) can be attached to the framework such that the minimal, shortest path distance between adjacent ligand groups does not exceed 100 atoms. Preferably, the linker connections to the ligand is selected such that the maximum spatial distance between two adjacent ligands is no more than 100A.

An example of a linker as presented by the grid is shown below for a biphenyl construct.

Nodes (1,2), (2,0), (4,4), (5,2), (4,0), (6,2), (7,4), (9,4), (10, 2), (9,0), (7,0) all represent carbon atoms. Node (10,0) represents a chlorine atom. All other nodes (or dots) are points in space (i. e., represent an absence of atoms).

Nodes (1,2) and (9,4) are attachment points. Hydrogen atoms are affixed to nodes (2,4), (4,4), (4,0), (2,0), (7,4), (10,2) and (7,0). Nodes (5,2) and (6,2) are connected by a single bond.

The carbon atoms present are connected by either a single or double bonds, taking into consideration the principle of resonance and/or tautomerism.

The intersection of the framework (linker) and the ligand group, and indeed, the framework (linker) itself can have many different bonding patterns. Examples of acceptable patterns of three contiguous atom arrangements are shown in the following diagram: CCC NCC OCC SCC PCC<BR> CCN NCN OCN SCN PCN<BR> CCO NCO OCO SCO PCO<BR> CCS NCS OCS SCS PCS<BR> CCP NCP OCP SCP PCP<BR> CNC NNC ONC SNC PNC<BR> CNO NNO ONO SNO PNO<BR> PO<BR> CNS NNS ONS SNS PNS<BR> PNP<BR> COC NOC OOC SOC POC<BR> COO NON T5N SON PON<BR> SOO COP <BR> <BR> CSC NSC<BR> CSN NSN OSC SSC PSC<BR> CSN NSN OSC SSC PSC<BR> CSO NSO OSN SSN PSN<BR> CSS NSS OSO SSO PSO<BR> OSP<BR> CPC NPC<BR> CPC NPC<BR> CPN NPN OPC SPC PPC<BR> CPO NPO OPN SPN CPS NPS OPO SPO PPO CPP NPP OPS SPS<BR> OPP SPP

One skilled in the art would be able to identify bonding patterns that would produce multivalent compounds. Methods for producing these bonding arrangements are described in March,"Advanced Organic Chemistry", 4th Edition, Wiley-Interscience, New York, New York (1992). These arrangements are described in the grid of dots shown in the scheme above. All of the possible arrangements for the five most preferred atoms are shown. Each atom has a variety of acceptable oxidation states. The bonding arrangements underlined are less acceptable and are not preferred.

Examples of molecular structures in which the above bonding patterns could be employed as components of the linker are shown below.

The identification of an appropriate framework geometry and size for ligand domain presentation are important steps in the construction of a multibinding compound with enhanced activity. Systematic spatial searching strategies can be used to aid in the identification of preferred frameworks through an iterative process. Figure 1 illustrates a useful strategy for determining an optimal framework display orientation for ligand domains. Various other strategies are known to those skilled in the art of molecular design and can be used for preparing compounds of this invention.

As shown in Figure 1, display vectors around similar central core structures such as a phenyl structure (Panel A) and a cyclohexane structure (Panel B) can be varied, as can the spacing of the ligand domain from the core structure (i. e., the length of the attaching moiety). It is to be noted that core structures other than

those shown here can be used for determining the optimal framework display orientation of the ligands. The process may require the use of multiple copies of the same central core structure or combinations of different types of display cores.

The above-described process can be extended to trimers (Figure 2) and compound of higher valency. (Figures 3 & 4) Assays of each of the individual compounds of a collection generated as described above will lead to a subset of compounds with the desired enhanced activities (e. g., potency, selectivity, etc.). The analysis of this subset using a technique such as Ensemble Molecular Dynamics will provide a framework orientation that favors the properties desired. A wide diversity of linkers is commercially available (see, e. g., Available Chemical Directory (ACD)). Many of the linkers that are suitable for use in this invention fall into this category. Other can be readily synthesized by methods well known in the art and/or are described below.

Having selected a preferred framework geometry, the physical properties of the linker can be optimized by varying the chemical composition thereof. The composition of the linker can be varied in numerous ways to achieve the desired physical properties for the multibinding compound.

It can therefore be seen that there is a plethora of possibilities for the composition of a linker. Examples of linkers include aliphatic moieties. aromatic moieties, steroidal moieties, peptides, and the like. Specific examples are peptides or polyamides, hydrocarbons, aromatic groups, ethers, lipids, cationic or anionic groups, or a combination thereof.

Examples are given below, but it should be understood that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. For example, properties of the linker can be modified by the addition or insertion of ancillary groups into or onto the linker, for example, to change the solubility of the multibinding compound (in water, fats, lipids, biological fluids, etc.), hydrophobicity, hydrophilicity, linker flexibility, antigenicity, stability, and the like. For example, the introduction of one or more

poly (ethylene glycol) (PEG) groups onto or into the linker enhances the hydrophilicity and water solubility of the multibinding compound, increases both molecular weight and molecular size and, depending on the nature of the unPEGylated linker, may increase the in vivo retention time. Further PEG may decrease antigenicity and potentially enhances the overall rigidity of the linker.

Ancillary groups which enhance the water solubility/hydrophilicity of the linker and, accordingly, the resulting multibinding compounds are useful in practicing this invention. Thus, it is within the scope of the present invention to use ancillary groups such as, for example, small repeating units of ethylene glycols, alcohols, polyols (e. g., glycerin, glycerol propoxylate, saccharides, including mono- , oligosaccharides, etc.), carboxylates (e. g., small repeating units of glutamic acid, acrylic acid, etc.), amines (e. g., tetraethylenepentamine), and the like) to enhance the water solubility and/or hydrophilicity of the multibinding compounds of this invention. In preferred embodiments, the ancillary group used to improve water solubility/hydrophilicity will be a polyether.

The incorporation of lipophilic ancillary groups within the structure of the linker to enhance the lipophilicity and/or hydrophobicity of the multibinding compounds described herein is also within the scope of this invention. Lipophilic groups useful with the linkers of this invention include, by way of example only, aryl and heteroaryl groups which, as above, may be either unsubstituted or substituted with other groups, but are at least substituted with a group which allows their covalent attachment to the linker. Other lipophilic groups useful with the linkers of this invention include fatty acid derivatives which do not form bilayers in aqueous medium until higher concentrations are reached.

Also within the scope of this invention is the use of ancillary groups which result in the multibinding compound being incorporated or anchored into a vesicle or other membranous structure such as a liposome or a micelle. The term"lipid" refers to any fatty acid derivative that is capable of forming a bilayer or a micelle such that a hydrophobic portion of the lipid material orients toward the bilayer while a hydrophilic portion orients toward the aqueous phase. Hydrophilic

characteristics derive from the presence of phosphato, carboxylic, sulfato. amino, sulfhydryl, nitro and other like groups well known in the art. Hydrophobicity could be conferred by the inclusion of groups that include, but are not limited to. long chain saturated and unsaturated aliphatic hydrocarbon groups of up to 20 carbon atoms and such groups substituted by one or more aryl, heteroaryl, cycloalkyl, and/or heterocyclic group (s). Preferred lipids are phosphglycerides and sphingolipids, representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidyl-ethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoyl-phosphatidylcholine or dilinoleoylphosphatidylcholine could be used. Other compounds lacking phosphorus, such as sphingolipid and glycosphingolipid families are also within the group designated as lipid. Additionally, the amphipathic lipids described above may be mixed with other lipids including triglycerides and sterols.

The flexibility of the linker can be manipulated by the inclusion of ancillary groups which are bulky and/or rigid. The presence of bulky or rigid groups can hinder free rotation about bonds in the linker or bonds between the linker and the ancillary group (s) or bonds between the linker and the functional groups. Rigid groups can include, for example, those groups whose conformational lability is restrained by the presence of rings and/or multiple bonds within the group, for example, aryl, heteroaryl, cycloalkyl, cycloalkenyl, and heterocyclic groups. Other groups which can impart rigidity include polypeptide groups such as oligo-or polyproline chains.

Rigidity can also be imparted electrostatically. Thus, if the ancillary groups are either positively or negatively charged, the similarly charged ancillary groups will force the presenter linker into a configuration affording the maximum distance between each of the like charges. The energetic cost of bringing the like-charged groups closer to each other will tend to hold the linker in a configuration that maintains the separation between the like-charged ancillary groups. Further

ancillary groups bearing opposite charges will tend to be attracted to their oppositely charged counterparts and potentially may enter into both inter-and intramolecular ionic bonds. This non-covalent mechanism will tend to hold the linker into a conformation which allows bonding between the oppositely charged groups. The addition of ancillary groups which are charged, or alternatively, bear a latent charge when deprotected, following addition to the linker, include deprotectation of a carboxyl, hydroxyl, thiol or amino group by a change in pH, oxidation, reduction or other mechanisms known to those skilled in the art which result in removal of the protecting group, is within the scope of this invention.

Rigidity may also be imparted by internal hydrogen bonding or by hydrophobic collapse.

Bulky groups can include, for example, large atoms, ions (e. g., iodine, sulfur, metal ions, etc.) or groups containing large atoms, polycyclic groups, including aromatic groups, non-aromatic groups and structures incorporating one or more carbon-carbon multiple bonds (i. e., alkenes and alkynes). Bulky groups can also include oligomers and polymers which are branched-or straight-chain species.

Species that are branched are expected to increase the rigidity of the structure more per unit molecular weight gain than are straight-chain species.

In preferred embodiments, rigidity is imparted by the presence of cyclic groups (e. g., aryl, heteroaryl, cycloalkyl, heterocyclic, etc.). In other preferred embodiments, the linker comprises one or more six-membered rings. In still further preferred embodiments, the ring is an aryl group such as, for example, phenyl or naphthyl.

In view of the above, it is apparent that the appropriate selection of a linker group providing suitable orientation, restricted/unrestricted rotation, the desired degree of hydrophobicity/hydrophilicity, etc. is well within the skill of the art.

Eliminating or reducing antigenicity of the multibinding compounds described herein is also within the scope of this invention. In certain cases, the antigenicity of a multibinding compound may be eliminated or reduced by use of groups such as, for example, poly (ethylene glycol).

As explained above, the multibinding compounds described herein comprise 2-10 ligands attached to a linker that attaches the ligands in such a manner that they are presented to the enzyme for multivalent interactions with ligand binding sites thereon/therein. The linker spatially constrains these interactions to occur within dimensions defined by the linker. This and other factors increases the biological activity of the multibinding compound as compared to the same number of ligands made available in monobinding form.

The compounds of this invention are preferably represented by the empirical Formula (L) p (X) q where L, X, p and q are as defined above. This is intended to include the several ways in which the ligands can be linked together in order to achieve the objective of multivalency, and a more detailed explanation is described below.

As noted previously, the linker may be considered as a framework to which ligands are attached. Thus, it should be recognized that the ligands can be attached at any suitable position on this framework, for example, at the termini of a linear chain or at any intermediate position.

The simplest and most preferred multibinding compound is a bivalent compound which can be represented as L-X-L, where each L is independently a ligand which may be the same or different and each X is independently the linker.

Examples of such bivalent compounds are provided in FIG. 2 where each shaded circle represents a ligand. A trivalent compound could also be represented in a linear fashion, i. e., as a sequence of repeated units L-X-L-X-L, in which L is a ligand and is the same or different at each occurrence, as can X. However, a trimer can also be a radial multibinding compound comprising three ligands attached to a central core, and thus represented as (L) 3X, where the linker X could include, for example, an aryl or cycloalkyl group. Illustrations of trivalent and tetravalent compounds of this invention are found in figures 2 and 3 respectively where, again, the shaded circles represent ligands. Tetravalent compounds can be represented in a linear array, e. g.,

L-X-L-X-L-X-L in a branched array, e. g., (a branched construct analogous to the isomers of butane--n-butyl, iso-butyl, sec- butyl, and t-butyl) or in a tetrahedral array, e. g.,

where X and L are as defined herein. Alternatively, it could be represented as an alkyl, aryl or cycloalkyl derivative as above with four (4) ligands attached to the core linker.

The same considerations apply to higher multibinding compounds of this invention containing 5-10 ligands as illustrated in FIG. 4 where, as before, the shaded circles represent ligands. However, for multibinding agents attached to a central linker such as aryl or cycloalkyl, there is a self-evident constraint that there must be sufficient attachment sites on the linker to accommodate the number of ligands present; for example, a benzene ring could not directly accommodate more than 6 ligands, whereas a multi-ring linker (e. g., biphenyl) could accommodate a larger number of ligands.

Certain of the above described compounds may alternatively be represented as cyclic chains of the form:

and variants thereof.

All of the above variations are intended to be within the scope of the invention defined by the Formula (L) p (X) q.

With the foregoing in mind, a preferred linker may be represented by the following: -xa-z (ya-z) _xa_ wherein m is an integer of from 0 to 20; Xa at each separate occurrence is selected from the group consisting of -O-,-S-,-NR-,-C (O)-,-C (O) O-,-C (O) NR-,-C (S),-C (S) O-,-C (S) NR- or a covalent bond where R is as defined below; Z at each separate occurrence is selected from the group consisting of alkylene, substituted alkylene, cycloalkylene, substituted cylcoalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, cycloalkenylene, substituted cycloalkenylene, arylene, heteroarylene, heterocyclene, or a covalent bond; each ya at each separate occurrence is selected from the group consisting of -O-,-C (O)-,-OC (O)-,-C (O) O-,-NR-,-S (O) n-,-C (O) NR'-,-NR'C (O)-,-NR' C (O) NR'-,-NR'C (S) NR'-,-C (=NR')-NR'-,-NR'-C (=NR')-,-OC (O)-NR'-,-NR'- C (O)-O-,-N=C (Xa)-NR'-,-NR'-C (Xa) =N-,-P (O) (OR')-O-,-O-P (O) (OR')-,- S (O) CR' R"-,-S (O) n-NR'-,-NR'-S (O) ,-,-S-S-, and a covalent bond; where n is 0, 1 or 2; and R, R'and R"at each separate occurrence are selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,

substituted alkynyl, aryl, heteroaryl and heterocyclic provided that at least one of Xa, Z, and ya is not a covalent bond.

Additionally, the linker moiety can be optionally substituted at any atom therein by one or more alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl and heterocyclic group.

In one embodiment of this invention, the linker (i. e., X, X'or X") is selected those shown in Table II : Table II Representative Linkers Linker -(CH2)n- where n is an integer of from 2-10 -(CH2)2-O-(CH2)2-O-(CH2)2- -CH,-Z-CH,-where Z is 1,4-phenylene - (CHCO, Et)- (CH,) 2- (CHCO, Et)- -CH,-Z-CH,-where Z is 2,3-quinoxaline (CH,) (CH=CH) (CH2) - (CH,)-C (O)-C (O)- (CH,)- - (CH,)- (C=C)- (CH2)- -CH,-Z-CH2-where Z is 1,2-phenylene - (CH,)-C (O)- (CH2)- - (CH,)-CH (COOH)- (CH2)- - (CH,)-CH (COOEt)- (CH2)- - (CH,)-CH (OH)- (CH2)- -CH(CH3)- (CH2)- -CH(CN)- (CH2)- -CH2-Z-CH,-where Z is 2,6-pyridyl - (CH,)-C (=CH,)- (CH,)-

Linker____ _ - (CH,)-CH (OH)-CH (OH)-(CH2)- -(CH2) 2-N (CH3)-(CH2) 2- - (CH,) 2-0- (CH,) 2- - (CH,)-Z- (CH,)- where Z is 4-biphenyl -CH,-Z-CH,-where Z is 1,2-phenylene -CH2-Z-CH2-where Z is 1,3-phenylene -CH,-O-C (O)-NH-Z-NH-C (O)-O-CH,- where Z is 1,3-phenylene -CH,-O-C(O)- (CH2) 8-C (O)-O-CH2- -CH,-O-C(O)- (CH,), o-C (O)-O-CH2- -CH,-O-C (O)-Z-C (O)-O-CH,- where Z is 1,3-phenylene -CH,-O-C (O)- (CH2) 6-C (O)-O-CH,- -CH,-O-C (O)-Z-C (O)-O-CH2-where Z is 1,4-phenylene -CH,-O-C (O)-Z-C (O)-O-CH,- where Z is 2,3- (5-norbomene) In view of the above description of the linker, it is understood that the term "linker"when used in combination with the term"multibinding compound" includes both a covalently contiguous single linker (e. g., L-X-L) and multiple covalently non-contiguous linkers (L-X-L-X-L) within the multibinding compound.

Combinatorial Libraries The methods described above lend themselves to combinatorial approaches for identifying multimeric compounds which possess multibinding properties.

Specifically, factors such as the proper juxtaposition of the individual ligands of a multibinding compound with respect to the relevant array of binding sites on a target or targets is important in optimizing the interaction of the multibinding compound with its target (s) and to maximize the biological advantage through multivalency. One approach is to identify a library of candidate

multibinding compounds with properties spanning the multibinding parameters that are relevant for a particular target. These parameters include: (1) the identity of ligand (s), (2) the orientation of ligands, (3) the valency of the construct, (4) linker length, (5) linker geometry, (6) linker physical properties, and (7) linker chemical functional groups.

Libraries of multimeric compounds potentially possessing multibinding properties (i. e., candidate multibinding compounds) and comprising a multiplicity of such variables are prepared and these libraries are then evaluated via conventional assays corresponding to the ligand selected and the multibinding parameters desired. Considerations relevant to each of these variables are set forth below: Selection of ligand (s) : A single ligand or set of ligands is (are) selected for incorporation into the libraries of candidate multibinding compounds which library is directed against a particular biological target or targets e. g., bradykinin receptor. The only requirement for the ligands chosen is that they are capable of interacting with the selected target (s). Thus, ligands may be known drugs, modified forms of known drugs, substructures of known drugs or substrates of modified forms of known drugs (which are competent to interact with the target), or other compounds.

Ligands are preferably chosen based on known favorable properties that may be projected to be carried over to or amplified in multibinding forms. Favorable properties include demonstrated safety and efficacy in human patients, appropriate PK/ADME profiles, synthetic accessibility, and desirable physical properties such as solubility, log P, etc. However, it is crucial to note that ligands which display an unfavorable property from among the previous list may obtain a more favorable property through the process of multibinding compound formation; i. e., ligands should not necessarily be excluded on such a basis. For example, a ligand that is not sufficiently potent at a particular target so as to be efficacious in a human patient may become highly potent and efficacious when presented in multibinding form. A ligand that is potent and efficacious but not of utility because of a non-

mechanism-related toxic side effect may have increased therapeutic index (increased potency relative to toxicity) as a multibinding compound. Compounds that exhibit short in vivo half-lives may have extended half-lives as multibinding compounds. Physical properties of ligands that limit their usefulness (e. g. poor bioavailability due to low solubility, hydrophobicity, hydrophilicity) may be rationally modulated in multibinding forms, providing compounds with physical properties consistent with the desired utility.

Orientation: selection of ligand attachment points and linking chemistry : Several points are chosen on each ligand at which to attach the ligand to the linker. The selected points on the ligand/linker for attachment are functionalized to contain complementary reactive functional groups. This permits probing the effects of presenting the ligands to their receptor (s) in multiple relative orientations, an important multibinding design parameter. The only requirement for choosing attachment points is that attaching to at least one of these points does not abrogate activity of the ligand. Such points for attachment can be identified by structural information when available. For example, inspection of a co-crystal structure of a protease inhibitor bound to its target allows one to identify one or more sites where linker attachment will not preclude the enzyme: inhibitor interaction. Alternatively, evaluation of ligand/target binding by nuclear magnetic resonance will permit the identification of sites non-essential for ligand/target binding. See, for example, Fesik, et al., U. S. Patent No. 5,891,643. When such structural information is not available, utilization of structure-activity relationships (SAR) for ligands will suggest positions where substantial structural variations are and are not allowed. In the absence of both structural and SAR information, a library is merely selected with multiple points of attachment to allow presentation of the ligand in multiple distinct orientations. Subsequent evaluation of this library will indicate what positions are suitable for attachment.

It is important to emphasize that positions of attachment that do abrogate the activity of the monomeric ligand may also be advantageously included in candidate multibinding compounds in the library provided that such compounds bear at least

one ligand attached in a manner which does not abrogate intrinsic activity. This selection derives from, for example, heterobivalent interactions within the context of a single target molecule. For example, consider a receptor antagonist ligand bound to its target receptor, and then consider modifying this ligand by attaching to it a second copy of the same ligand with a linker which allows the second ligand to interact with the same receptor molecule at sites proximal to the antagonist binding site, which include elements of the receptor that are not part of the formal antagonist binding site and/or elements of the matrix surrounding the receptor such as the membrane. Here, the most favorable orientation for interaction of the second ligand molecule with the receptor/matrix may be achieved by attaching it to the linker at a position which abrogates activity of the ligand at the formal antagonist binding site. Another way to consider this is that the SAR of individual ligands within the context of a multibinding structure is often different from the SAR of those same ligands in momomeric form.

The foregoing discussion focused on bivalent interactions of dimeric compounds bearing two copies of the same ligand joined to a single linker through different attachment points, one of which may abrogate the binding/activity of the monomeric ligand. It should also be understood that bivalent advantage may also be attained with heterodimeric constructs bearing two different ligands that bind to common or different targets. For example, a 5HT4 receptor antagonist and a bladder-selective muscarinic M3 antagonist may be joined to a linker through attachment points which do not abrogate the binding affinity of the monomeric ligands for their respective receptor sites. The dimeric compound may achieve enhanced affinity for both receptors due to favorable interactions between the 5HT4 ligand and elements of the M3 receptor proximal to the formal M3 antagonist binding site and between the M3 ligand and elements of the 5HT4 receptor proximal to the formal 5HT4 antagonist binding site. Thus, the dimeric compound may be more potent and selective antagonist of overactive bladder and a superior therapy for urinary urge incontinence.

Once the ligand attachment points have been chosen, one identifies the types of chemical linkages that are possible at those points. The most preferred types of chemical linkages are those that are compatible with the overall structure of the ligand (or protected forms of the ligand) readily and generally formed, stable and intrinsically inocuous under typical chemical and physiological conditions, and compatible with a large number of available linkers. Amide bonds, ethers, amines, carbamates, ureas, and sulfonamides are but a few examples of preferred linkages.

Linkers: spanning relevant multibinding parameters through selection of valency, linker length, linker geometry, rigidity, physical properties, and chemical functional groups In the library of linkers employed to generate the library of candidate multibinding compounds, the selection of linkers employed in this library of linkers takes into consideration the following factors: Valency : In most instances the library of linkers is initiated with divalent linkers. The choice of ligands and proper juxtaposition of two ligands relative to their binding sites permits such molecules to exhibit target binding affinities and specificities more than sufficient to confer biological advantage. Furthermore, divalent linkers or constructs are also typically of modest size such that they retain the desirable biodistribution properties of small molecules.

Linker length : Linkers are chosen in a range of lengths to allow the spanning of a range of inter-ligand distances that encompass the distance preferable for a given divalent interaction. In some instances the preferred distance can be estimated rather precisely from high-resolution structural information of targets, typically enzymes and soluble receptor targets. In other instances where high-resolution structural information is not available (such as 7TM G-protein coupled receptors), one can make use of simple models to estimate the maximum distance between binding sites either on adjacent receptors or at different locations on the same receptor. In situations where two binding sites are present on the same target (or target subunit

for multisubunit targets), preferred linker distances are 2-20 A, with more preferred linker distances of 3-12 A. In situations where two binding sites reside on separate (e. g., protein) target sites, preferred linker distances are 20-100 A, with more preferred distances of 30-70 A.

Linker geometry and rigidity : The combination of ligand attachment site, linker length, linker geometry, and linker rigidity determine the possible ways in which the ligands of candidate multibinding compounds may be displayed in three dimensions and thereby presented to their binding sites. Linker geometry and rigidity are nominally determined by chemical composition and bonding pattern, which may be controlled and are systematically varied as another spanning function in a multibinding array.

For example, linker geometry is varied by attaching two ligands to the ortho, meta, and para positions of a benzene ring, or in cis-or trans-arrangements at the 1, 1- vs.

1,2- vs. 1,3- vs. 1,4- positions around a cyclohexane core or in cis-or trans- arrangements at a point of ethylene unsaturation. Linker rigidity is varied by controlling the number and relative energies of different conformational states possible for the linker. For example, a divalent compound bearing two ligands joined by 1, 8-octyl linker has many more degrees of freedom, and is therefore less rigid than a compound in which the two ligands are attached to the 4,4' positions of a biphenyl linker.

Linker physical properties : The physical properties of linkers are nominally determined by the chemical constitution and bonding patterns of the linker, and linker physical properties impact the overall physical properties of the candidate multibinding compounds in which they are included. A range of linker compositions is typically selected to provide a range of physical properties (hydrophobicity, hydrophilicity, amphiphilicity, polarization, acidity, and basicity) in the candidate multibinding compounds. The particular choice of linker physical properties is made within the context of the physical properties of the ligands they join and preferably the goal is to generate molecules with favorable PK/ADME properties. For example, linkers

can be selected to avoid those that are too hydrophilic or too hydrophobic to be readily absorbed and/or distributed in vivo.

Linker chemical functional groups : Linker chemical functional groups are selected to be compatible with the chemistry chosen to connect linkers to the ligands and to impart the range of physical properties sufficient to span initial examination of this parameter.

Combinatorial synthesis : Having chosen a set of n ligands (n being determined by the sum of the number of different attachment points for each ligand chosen) and m linkers by the process outlined above, a library of (n !) m candidate divalent multibinding compounds is prepared which spans the relevant multibinding design parameters for a particular target. For example, an array generated from two ligands, one which has two attachment points (Al, A2) and one which has three attachment points (B l, B2, B3) joined in all possible combinations provide for at least 15 possible combinations of multibinding compounds: A1-A1 A1-A2 A1-B1 A1-B2 A1-B3 A2-A2 A2-B1 A2-B2 A2-B3 B1-B1 B1-B2 B1-B3 B2-B2 B2-B3 B3-B3 When each of these combinations is joined by 10 different linkers, a library of 150 candidate multibinding compounds results.

Given the combinatorial nature of the library, common chemistries are preferably used to join the reactive functionalies on the ligands with complementary reactive functionalities on the linkers. The library therefore lends itself to efficient parallel synthetic methods. The combinatorial library can employ solid phase chemistries well known in the art wherein the ligand and/or linker is attached to a solid support. Alternatively and preferably, the combinatorial libary is prepared in the solution phase. After synthesis, candidate multibinding compounds are

optionally purified before assaying for activity by, for example, chromatographic methods (e. g., HPLC).

Analysis of array by biochemical, analytical, pharmacological, and computational methods: Various methods are used to characterize the properties and activities of the candidate multibinding compounds in the library to determine which compounds possess multibinding properties. Physical constants such as solubility under various solvent conditions and logD/clogD values can be determined. A combination of NMR spectroscopy and computational methods is used to determine low-energy conformations of the candidate multibinding compounds in fluid media. The ability of the members of the library to bind to the desired target and other targets is determined by various standard methods, which include radioligand displacement assays for receptor and ion channel targets, and kinetic inhibition analysis for many enzyme targets. In vitro efficacy, such as for receptor agonists and antagonists, ion channel blockers, and antimicrobial activity, can also be determined.

Pharmacological data, including oral absorption, everted gut penetration, other pharmacokinetic parameters and efficacy data can be determined in appropriate models. In this way, key structure-activity relationships are obtained for multibinding design parameters which are then used to direct future work.

The members of the library which exhibit multibinding properties, as defined herein, can be readily determined by conventional methods. First those members which exhibit multibinding properties are identified by conventional methods as described above including conventional assays (both in vitro and in vivo).

Second, ascertaining the structure of those compounds which exhibit multibinding properties can be accomplished via art recognized procedures. For example, each member of the library can be encrypted or tagged with appropriate information allowing determination of the structure of relevant members at a later time. See, for example, Dower, et al., International Patent Application Publication No. WO 93/06121; Brenner, et al., Proc. Natl. Acad. Sci., USA, 89: 5181 (1992);

Gallop, et al., U. S. Patent No. 5,846,839; each of which are incorporated herein by reference in its entirety. Alternatively, the structure of relevant multivalent compounds can also be determined from soluble and untagged libaries of candidate multivalent compounds by methods known in the art such as those described by Hindsgaul, et al., Canadian Patent Application No. 2,240,325 which was published on July 11,1998. Such methods couple frontal affinity chromatography with mass spectroscopy to determine both the structure and relative binding affinities of candidate multibinding compounds to receptors.

The process set forth above for dimeric candidate multibinding compounds can, of course, be extended to trimeric candidate compounds and higher analogs thereof.

Follow-up synthesis and analysis of additional array (s) : Based on the information obtained through analysis of the initial library, an optional component of the process is to ascertain one or more promising multibinding"lead"compounds as defined by particular relative ligand orientations, linker lengths, linker geometries, etc. Additional libraries can then be generated around these leads to provide for further information regarding structure to activity relationships. These arrays typically bear more focused variations in linker structure in an effort to further optimize target affinity and/or activity at the target (antagonism, partial agonism, etc.), and/or alter physical properties. By iterative redesign/analysis using the novel principles of multibinding design along with classical medicinal chemistry, biochemistry, and pharmacology approaches, one is able to prepare and identify optimal multibinding compounds that exhibit biological advantage towards their targets and as therapeutic agents.

To further elaborate upon this procedure, suitable divalent linkers include, by way of example only, those derived from dicarboxylic acids, disulfonylhalides, dialdehydes, diketones, dihalides, diisocyanates, diamines, diols, mixtures of carboxylic acids, sulfonylhalides, aldehydes, ketones, halides, isocyanates, amines and diols. In each case, the carboxylic acid, sulfonylhalide, aldehyde, ketone, halide, isocyanate, amine and diol functional group is reacted with a complementary

functionality on the ligand to form a covalent linkage. Such complementary functionality is well known in the art as illustrated in the following table: COMPLEMENTARY BINDING CHEMISTRIES First Reactive Group Second Reactive Group Linkage hydroxyl isocyanate urethane amine epoxide p-hydroxyamine hydroxyamine sulfonyl halide sulfonamide carboxyl acid amine amide hydroxyl alkyl/aryl halide ether aldehyde amine/NaCNBH3 amine ketone amine/NaCNBH3 amine amine isocyanate urea Exemplary linkers include the following linkers identified as X-l through X- 418 as set forth below: Diacids 0OH CH-z HO-11" ho 0 Q OH 0 x-I OH OH OH HO 0 X-2 X-3 H H3C O CH CH3 X-4 3 X-5 HOOH OU X-6X-7 X-8 OH OH OH HO 0 OH OH N 0 ho 0 o r-<9 c HO N H 0 CHj HO X-12 HO HO CH3 X-12 0 0 OH OH 0 HO OHX-14 X-13 O OH OH 00 0 HO p 0 HO HO OH H3C CH3 C 0 OHOH 0 0 , 0 HO OH , X 8 X-19 HO0 OH HO OH \H p 0 ,,,. HO w w S S. x-22 X-20 X-21 HO- H X-22 0 O O HOOH 0 0 OH OH X-240 X-23 0 0 HOOH L J -2J o OH on 0 0 0-N N HO S-, 5 0-0 OH SO X-26 0 ChiralN OH X-27 D HO Q Q HO 0 0 0 OH HO OH 0 CH ° X-29 OH p X-28X-30 HO 0 OH HO'SS OH 0 0 X-31 HO 0 N OH 0 Chiral X-32 \1 0w"T,. . -HO HO N HO C Oy N OH X34 X35 3 X-34 X-35 t N Chiro/ choral ho oeOx 0 0" oH 0 p HO 0 HO F F x_37 OH X-38 HO X-36 p OH o OH 0 0 OH CH OH 0 O--CN3 p S-N HO i 4 i i w w I Chiral X-39 0 X-40 C 3 H N X-41 H3 C CH3 H OH ° OH Ho cl cl i 0 H3 C CH3 0 OH X-44 HOOH O OH HO HO Ho MO V ii j 0 $ 0 OH HO x, < S HO ~ 5> < ° S O \< (OH X-45X-46 Chiroi H3 C OH X-47 X-48 HO 0 chu OHeO HA0 OH OH OHHO 0 N S-S N H p p 0 HO- F X_ 5 Ch ral 0ChiralF F X-52 X-50 X-50 H2NHN 0 HN0 N 0 HA - OH OH X55 0 c -55 HOChirol HO Chirol X-53X-54 0 oh oh 0 OH OH OH CHj OH 0 0 0 Ho X> f Chiral X-57 X-58 X-56 X-56 0 0 HO Ho ho / 0 Chiral X-60 X-590 OH nOH N HO S Chiral X-61X-62 C, H 0 Hj C CH3 H3 C O 0 0 NH o oH o-; o- 'i CH3 H3 C OH HO 5 0 0 NO 0 X-6i Chiral HO X-65 0 X-64 Oh zozo HO 0 HO HO X-67 HO 0 HO 0 0 0 -0 . JLJ o HOOH 0 0== ( N HOChoral X-69 X-70 0 OH 0 0 HO0 0 / HD'''OH Chiral S FFFFFFFF X-71 X-72 X-73 HO OH HO 0 HO ° CD NJ H S 'v <[ON ONZJ O HO ° +E X-75 X-76 X-74 0 oh oh on 'oh /C/VO 00 H3 C HO HO X-78 0 CN3 1-- 0X-79 c H c N CH3 0 ~OH CH-O f CH3 = p cl3 0 rot Oh X-80 0 OH X-81 0 OH i 0 0 OH OH 0 0 OH OH HO NEZ OH H N Hp 0 HO HO OH X-82 0 0 Ho X-85OH CH3 OH H 0N,,,. N OH o 0 zizi OH OU Chiral c X-88 HO 0 X-86 H X-87 HO0 OH 0 0 N p NND w C FT T - 0 OH, H 0 N f, r TI X-91 OH X-g 0 X-89 0 0 0 H f., 3 OH N,,. H H' S 0, OH O S X-93 X-94 CH3 OH Cl viral X-92 O o FF FF FF FF FF OU 0 0 HO HO--OHFF FF FF FF Fr- o OH X-97 X-95 X-96 0 OH HON,. w I OH OH ho _ CI H CH3 H3 0 CHj Ch iral 6r X-111 Ch iral X-llO X-112 OH OH 0 0 HOOH 0 OH 0 0, 0 0 HO 0 OH '_OH Q 0 X1 14 HO OH HO OH X-113 Chiral X-115 0r 0 -</v =\ 0 0 N Il 0 i ON N,,, OH\ I PU OH Chiral 0 OH OH X-116 0 OH HO OH Y - OH OH X-118X-120 X-ll9 0 0 oh 0 HO N-J HO S S'v'0 HO N f OH X-l21 v p X-122 0 0 - o/ YT pH ! 0 0 N--N-o OH OH H N OH0 N N OH OH X-724 =0 Chiral H2N X-125 X-125 Ch iral X-123 OH 0 OH HO 0X-128 WN OH OH N 0 0 N j0 0 Chircl 0 x-129 X-127 HO 0 H 0 00 0 0 f Hl"t, tOH nOH OH HHO 0 HO HO 0 X-131 OH X-130 X-132 Disulfonyl Halides °\\ P 0==S cul D Ip p X13311 X-lJ4 cl 0 p C% X-133 o X-135 <11 oS X-136 Fe,C/0 X-141 CH3 X-137 X-) 38 X-136 F. S N N S 0 S. CI 0 i w i w, In/ 0 0 s C/0//'F X-141 F 0=S =0 0 0 0, \\O N 0 SIF o9S r/ ' f-C W) X-142 H w I 0 S. F CH3 0 X-144 o x- 43 X-142 0 0 cl F S. :, /I 0 N N 0 . \\ 0 0 X-146 X-145 CH30 0 i 0 0 F i CI S 0 ir o chu H3C CH3 w 0 F i (CI CH3 X-148 HO 0 X-147 X-149 X-150 0 CI. I i 0" ° -2 c 0 X-152 0 X-151 u 0 X-1510 Dialdeh ydes i i l \ \ 0 I X-154 °x-156 CH '-/-70 X-156 o X-155 o o CH3 I o o. N I °l O X-157 CH3 X-158 0 0 o o r I oo w A-/9 c /v o X-160 0 ON 0 X-159 o ouzo X159 3 ° duN oo i X-161 i X-164 X-166 0 i i OH °X-165 X-167 -0H3Cuo 0 0=O 0HO X-168 s o ooh I X-169X-170 OH 0° X171 , l \ 0 S X-172 H X-174 X-1 3 Dihalidesi vCH3 X177 CIN, S Cls 75 Er Er Br X-175 Br~~Br Br Y Br OH OH X-178X-179 X-180 Diisocyanates p N N 0\ w I I/0 N N X-215 0 X-216 0 /~C < N 1 \ N 0 N N N I I I H3C-0 0-CH3 X-218 0 X-217 FF FF H3CsNX/'° >° 0=-N F F X-219 X-220 0 X-221 /NXNX \R o N N \ N N 0 Br CH 0 N<N CH3 X-222 H3C CH3 X-224 p X-223/ r nu 11 , N o0 w N 9 X-226 0 i X_ 26 p 0 2 CH3 CH3 N0/N/ I \ N\0 i X-225 X-229 X-228Cl CH, 3 N 0 CII) CHJ [t J cj cj CI CH3 N 0 C L J -22 --22 -22<9 c/ JL J. JJ c ()- X-231 X-232 X-230 0 o 0 N\\ 0 0 l NN,", 0 X-246 11 X-247 X-248 Diamines NO pN X-249 Nrr-//"""""""""""""2 NU N' N I X-251 H2N CH3 2 N H2N N zO X252 X 2 0 CH3 CH3 CH3 N H2NX NH2 H2N <NH2 X X X-253CH3 CH3 CH NH2 H2IV N X-254X256 H2N H2N NH2 H2N NH2 X-255 X-257 H2N nu w 2 H2NX-259 X-258X-262 C N N CH 2 X-261X-262 H2N X-265 -27 - . (I 2 s X-265 X-263H2N X-264 0 H2N NH2 0 NH X-J04 -JCJ X-303 CH3 ICH3 C C 2 0 0 C H2N X NH2 NH 2 X-305W NCH3 X307 Chiral X-306 NH2 NH2 CH3 CH3 nu i 3-- X308X-309 4 H2N NU2 0 H2N NH X-311 X-310 NH2 CH3 H2N CH3/w ''N H3C NH chu 2 CHU NU Chiral X-iii 2 X-312X-314 CH3 OH HO OH CH3 HO H3 C w HO -- OH X376 HO X-377 X-378 CH3 <CH3 0 HON XOH HO/0JOXOH c X-379X-380 HO SS OH HO 00 OH HO p OH X-381X-382 X-383 FFI HO F OH HOOH F F F X-384 F X-385 Dithiols HS HS HS \ SH SH SH CI X-386 X-387 X-388 SH HS CH vs X-389 X-391 X-390

Representative ligands for use in this invention include, by way of example. ligands of L-1 through L-3 wherein L-1 through L-3 are selected from the compounds of formulae (a)- (c) : L1 = (a). L2= (b). and L3= (c).

Combinations of ligands CD and linkers (X) per this invention include. by way example only. homo-and hetero-dimers wherein the first ligand is selected from L-1 through L-3 above and the second ligand and linker is selected from the following: <BR> <BR> L-1/X-I-L-l/X-2-L-1/X-3-L-l/X-4-L-1/X-5-L-l/X-6- L-1/X-7- L-1/X-8- L-1/X-9- L-1/X-10- L-1/X-11- L-1/X-12- L-1/X-13- L-1/X-14- L-1/X-15- L-1/X-16- L-1/X-17- L-1/X-18- L-1/X-19- L-1/X-20- L-1/X-21- L-1/X-22- L-1/X-23- L-1/X-24- <BR> <BR> L-1/X-25-L-I/X-26-L-1/X-27-L-I/X-28-L-1/X-29-L-I/X-30-<BR > L-1/X-31-L-I/X-32-L-I/X-33-L-I/X-34-L-I/X-35-L-I/X-36-<BR > L-I/X-37-L-I/X-38-L-1/X-39-L-1/X-40-L-I/X-41-L-I/X-42-<BR > L-I/X-43-L-I/X-44-L-l/X-45-L-1/X-46-L-1/X-47-L-I/X-48-<BR > L-l/X-49-L-1/X-50-L-1/X 51-L-l/X-52-L-1/X-53-L-1/X-54-<BR> L-IIX-55-L-l/X-56-L-l/X-57-L-1/X-58-L-IIX-59-L-IIX-60- L-1/X-61- L-1/X-62- L-1/X-63- L-1/X-64- L-1/X-65- L-1/X-66- L-1/X-67- L-1/X-68- L-1/X-69- L-1/X-70- L-1/X-71- L-1/X-72- <BR> <BR> L-I/X-73-L-I/X-74-L-I/X-75-L-I/X-76-L-I/X-77-L-I/X-78-<BR > L-I/X-79-L-I/X-80-L-I/X-81-L-I/X-82-L-I/X-83-L-I/X-84-<BR > L-I/X-85-L-1/X-86-L-I/X-87-L-1/X-88-L-I/X-89-L-I/X-90-<BR > L-IIX-91-L-1/X-92-L-1/X-93-L-1/X-94-L-l/X-95-L-IIX-96- L-1/X-97- L-1/X-98- L-1/X-99- L-1/X-100- L-1/X-101- L-1/X-102- <BR> <BR> L-l/X-103-L-IIX-104-L-IIX-105-L-1/X-106-L-l/X-107-L-IIX-108- L-1/X-109- L-1/X-110- L-1/X-111- L-1/X-112- L-1/X-113- L-1/X-114- <BR> <BR> L-1/X-115-L-1/X-116-L-1/X-117-L-1/X-118-L-I/X-119-L-1/X-120- <BR> L-I/X-121-L-I/X-122-L-I/X-123-L-1/X-124-L-I/X-125-L-I/'X-126 -<BR> L-liX-127-L-l/X-128-L-l/X-129-L-l/X-130-L-l/X-131-L-l/X-132- L-1/X-133- L-1/X-134- L-1/X-135- L-1/X-136- L-1/X-137 L-1/X-138- L-1X-139- L-1/X-140- L-1/X-141- L-1/X-142- L-1/X-143- L-1/X-144- L-1/X-145- L-1/X-146- L-1/X-147- L-1/X-148- L-1/X-149- L-1/X-150- <BR> <BR> L-I/X-151-L-I/X-152-L-I/X-153-L-I/X-154-L-I/X-155-L-I/X-156-

L-1/X-157- L-1/X-158- L-1/X-159- L-1/X-160- L-1/X-161- L-1/X-162- L-1/X-163- L-1/X-164- L-1/X-165- L-1/X-166- L-1/X-167- L-1/X-168- L-l/X-169-L-l/X-170-L-l/X-171-L-l/X-172- L-1/X-173- L-1/X-174- L-1/X-175- L-1/X-176- L-1/X-177- L-1/X-178- L-1/X-179-L-I/X-180-L-I/X-181-L-I/X-182-L-I/X-183-L-I/X-184- <BR> L-I/X-185-L-I/X-186-L-1/X-187-L-I/X-188-L-I/X-189-L-1/X-190- L-l1X-191- L-1/X-192- L-1/X-193- L-1/X-194- L-1/X-195- L-1/X-196- L-l/X-197-L-l/X-198-L-I/X-199-L-I/X-200-L-I/X-201-L-1/X-202- L-1/X-203- L-1/X-204- L-1/X-205- L-1/X-206- L-1/X-207- L-1/X-208- L-t/X-209-L-I/X-210-L-l/X-211-L-t/X-212-L-I/X-213-L-t/X-214- L-1/X-215- L-1/X-216- L-1/X-217- L-1/X-218- L-1/X-219- L-1/X-220- L-t/X-22 !-L-I/X-222-L-t/X-223-L-I/X-224-L-I/X-225-L-i/X-226-<BR&g t; L-I/X-227-L-I/X-228-L-I/X-229-L-I/X-230-L-I/X-231-L-l/X-232- <BR> L-1/X-233-L-1/X-234-L-l/X-235-L-I/X-236-L-I/X-237-L-l/X-238- <BR> L-l/X-239-L-l/X-240-L-I/X-241-L-1/X-242-L-I/X-243-L-1/X-244- L-1/X-245- L-1/X-246- L-1/X-247- L-1/X-248- L-1/X-249- L-1/X-250- L-t/X-251-L-i/X-252-L-t/X-253-L-)/X-254-L-I/X-255-L-I/X-256- <BR> L-I/X-257-L-l/X-258-L-I/X-259-L-l/X-260-L-1/X-261-L-1/X-262- <BR> L-l/X-263-L-I/X-264-L-I/X-265-L-I/X-266-L-I/X-267-L-I/X-268- <BR> L-I/X-269-L-I/X-270-L-I/X-271-L-I/X-272-L-l/X-273-L-I/X-274- <BR> L-I/X-275-L-I/X-276-L-I/X-277-L-I/X-278-L-I/X-279-L-I/X-280- <BR> L-l/X-281-L-I/X-282-L-1/X-283-L-I/X-284-L-I/X-285-L-1/X-286- <BR> L-I/X-287-L-I/X-288-L-l/X-289-L-l/X-290-L-l/X-291-L-l/X-292- <BR> L-I/X-293-L-1/X-294-L-I/X-295-L-I/X-296-L-I/X-297-L-I/X-298- <BR> L-I/X-299-L-1/X-300-L-l/X-301-L-I/X-302-L-l/X-303-L-I/X-304- <BR> L-t/X-305-L-l/X-306-L-I/X-307-L-t/X-308-L-I/X-309-L-I/X-310- <BR> L-1/X-311-L-I/X-312-L-l/X-313-L-l/X-314-L-I/X-315-L-I/X-316- <BR> L-l/X-317-L-1/X-318-L-l/X-319-L-l/X-320-L-I/X-321-L-l/X-322- <BR> L-l/X-323-L-l/X-324-L-l/X-325-L-l X-326-L-l X-327-L-l/X-328-<BR> L-l/X-329-L-l/X-330-L-l/X-331-L-l X-332-L-l/X-333-L-l/X-334-<BR> L-l/X-335-L-l/X-336-L-l/X-337-L-l/X-338-L-l/X-339-L-l/X-340- <BR> L-I/X-341-L-l/X-342-L-I/X-343-L-I/X-344-L-1/X-345-L-l/X-346- L-1/X-347- L-1/X-348- L-1/X-349- L-1/X-350- L-1/X-351- L-1/X-352- L-l/X-353-L-I/X-354-L-l/X-355-L-I/X-356-L-I/X-357-L-I/X-358- L-I/X-359-L-I/X-360-L-l/X-361-L-I/X-362-L-1/X-363-L-1/X-364- L-1/X-365- L-1/X-366- L-1/X-367- L-1/X-368- L-1/X-369- L-1/X-370-

L-l/X-371-L-I/X-372-L-l/X-373-L-l/X-374-L-I/X-375-L-I/X-376- <BR> L-l/X-377-L-I/X-378-L-I/X-379-L-1/X-380-L-1/X-381-L-l/X-382- <BR> L-t/X-383-L-l/X-384-L-I/X-385-L-I/X-386-L-t/X-387-L-I/X-388- <BR> L-1/X-389-L-1/X-390-L-1/X-391-L-1/X-392-L-1/X-393-L-1/X-394- <BR> L-1/X-395-L-I/X-396-L-I/X-397-L-1/X-398-L-1/X-399-L-l/X-400- <BR> L-l/X-401-L-l/X-402-L-l X-403-L-l/X-404-L-l/X-405-L-l X-406-<BR> L-l/X-407-L-l/X-408-L-l/X-409-L-l/X-410-L-l/X-411-L-l/X-412- L-1/X-413- L-1/X-414- L-1/X-415- L-1/X-416- L-1/X-417- L-1/X-418- L-2/X-1-L-2/X-2-L-2/X-3-L-2/X-4-L-2/X-5-L-2/X-6- <BR> <BR> L-2/X-7-L-2/X-8-L-2/X-9-L-2/X-10-L-2 X-11-L-2/X-12-<BR> L-2/X-13-L-2/X-14-L-2/X-15-L-2/X-16-L-2/X-17-L-2/X-18-<BR > L-2/X-19-L-2/X-20-L-2/X-21-L-2/X-22-L-2/X-23-L-2/X-24-<BR > L-2/X-25-L-2/X-26-L-2/X-27-L-2/X-28-L-2/X-29-L-2/X-30-<BR > L-2/X-31-L-2/X-32-L-2/X-33-L-2/X-34-L-2/X-35-L-2 X-36-<BR> L-2/X-37-L-2/X-38-L-2/X-39-L-2/X-40-L-2/X-41-L-2/X-42-<BR > L-2/X-43-L-2/X-44-L-2/X-45-L-2/X-46-L-2/X-47-L-2/X-48-<BR > L-2/X-49-L-2/X-50-L-2/X-51-L-2/X-52-L-2/X-53-L-2/X-54-<BR > L-2/X-55-L-2/X-56-L-2/X-57-L-2/X-58-L-2/X-59-L-2/X-60- L-2/X-6I-L-2/X-62-L-2/X-63-L-2/X-64-L-2/X-65-L-2/X-66- L-2/X-67-L-2/X-68-L-2/X-69-L-2/X-70-L-2/X-71-L-2/X-72- <BR> <BR> L-2/X-73-L-2/X-74-L-2/X-75-L-2/X-76-L-2/X-77-L-2/X-78-<BR > L-2/X-79-L-2/X-80-L-2/X-81-L-2/X-82-L-2/X-83-L-2/X-84-<BR > L-2/X-85-L-2/X-86-L-2/X-87-L-2/X-88-L-2/X-89-L-2/X-90- L-2/X-91-L-2/X-92-L-2/X-93-L-2/X-94-L-2/X-95-L-2/X-96- <BR> <BR> L-2/X-97-L-2/X-98-L-2/X-99-L-2/X-100-L-2/X-101-L-2/X-102- L-2/X-103-L-2/X-104-L-2/X-105-L-2/X-106-L-2/X-107-L-2/X-108- <BR> <BR> L-2/X-109-L-2/X-I10-L-2/X-I I I-L-2/X-112-L-2/X-113-L-2/X-114-<BR> L-2/X-115-L-2/X-116-L-2/X-117-L-2/X-118-L-2/X-119-L-2/X-120- <BR> L-2/X-121-L-2/X-122-L-2/X-123-L-2/X-124-L-2/X-125-L-2/X-126- <BR> L-2/X-127-L-2/X-128-L-2/X-129-L-2/X-130-L-2/X-131-L-2/X-132- <BR> L-2/X-133-L-2/X-134-L-2/X-135-L-2/X-136-L-2/X-137-L-2/X-138- <BR> L-2/X-139-L-2/X-140-L-2/X-141-L-2/X-142-L-2/X-143-L-2/X-144- <BR> L-2/X-145-L-2/X-146-L-2/X-147-L-2/X-148-L-2/X-149-L-2/X-150- L-2/X-151-L-2/X-152-L-2/X-153-L-2/X-154-L-2/X-155-L-2/X-156- <BR> <BR> L-2/X-157-L-2/X-158-L-2/X-159-L-2/X-160-L-2/X-161-L-2/X-162-

L-2/X-163- L-2/X-164- L-2/X-165- L-2/X-166- L-2/X-167- L-2X-168- L-2/X-169-L-2/X-170-L-2/X-171-L-2 : X-172- L-2/X-173- L-2/X-174- L-2/X-175- L-2/X-176- L-2/X-177- L-2/X-178- L-2/X-179- L-2/X-180- L-2/X-181- L-2/X-182- L-2/X-183- L-2/X-184- L-2/X-185- L-2/X-186- L-2/X-187- L-2/X-188- L-2/X-189- L-2/X-190- L-2/X-191-L-2/X-192-L-2/X-193-L-2/X-194-L-2/X-195-L- ?/X-196-<BR> L-2/X-197-L-2/X-198-L-2/X-199-L-2'X-200-L-2/X-20I-L-2 :'X-202-<BR> L-2/X-203-L-2/X-204-L-2/X-205-L-21X-206-L-2/X-207-L-/X-208-& lt;BR> L-2/X-209-L-2/X-210-L-2/X-211-L-2/X-212-L-2/X-213-L-2/X-214- <BR> L-2/X-215-L-2/X-216-L-2/X-217-L-2/X-218-L-2/X-219-L-2/X-220- <BR> L-2/X-22 I-L-2/X-222-L-2/X-223-L-2/X-224-L-2/X-225-L-2/X-226- L-2/X-227- L-2/X-228- L-2/X-229-L-2/X-230- L-2/X-231- L-2/X-232- L-2/X-233- L-2/X-234- L-2/X-235- L-2/X-236- L-2/X-237- L-2/X-238-<BR> L-2/X-233- L-2/X-234- L-2/X-235- L-2/X-236- L-2/X-237- L-2/X-238-<BR> L-2/X-239-L-2/X-240-L-2/X-241-L-2/X-242-L-2/X-243-L-2/X-244- <BR> L-2/X-245-L-2/X-246-L-2/X-247-L-2/X-248-L-2/X-249-L-2/X-250- L-2/X-25I-L-2/X-252-L-2/X-253-L-2/X-254-L-2/X-255-L-2/X-256- <BR> <BR> L-2/X-257-L-2/X-258-L-2/X-259-L-2/X-260-L-2/X-261-L-2/X-262- <BR> L-2/X-263-L-2/X-264-L-2/X-265-L-2/X-266-L-2/X-267-L-2/X-268- <BR> L-2/X-269-L-2/X-270-L-2/X-271-L-2/X-272-L-2/X-273-L-2/X-274- <BR> L-2/X-275-L-2/X-276-L-2/X-277-L-2/X-278-L-2/X-279-L-2/X-280- <BR> L-2/X-281-L-2/X-282-L-2/X-283-L-2/X-284-L-2/X-285-L-2/X-286- <BR> L-2'X-287-L-2/X-288-L-2/X-289-L-2/X-290-L-2/X-291-L-2/X-292- <BR> L-2/X-293-L-2/X-294-L-2/X-295-L-2/X-296-L-2/X-297-L- ? ;'X-298-<BR> L-2iX-299-L-21X-300-L-2/X-301 ~ L-2/X-302-L-2/X-303-L-2/X-304-<BR> L-2/X-305-L-2/X-306-L-2/X-307-L-2/X-308-L-2/X-309-L-2/X-310- <BR> L-2/X-311-L-2/X-312-L-2/X-313-L-2/X-314-L-2/X-315-L-2/X-316- <BR> L-2/X-317-L-2/X-318-L-2/X-319-L-2/X-320-L-2/X-321-L-2/X-322- <BR> L-2/X-323-L-2/X-324-L-2/X-325-L-2/X-326-L-2/X-327-L-2/X-328- <BR> L-2/X-329-L-2/X-330-L-2/X-331 ~ L-2/X-332-L-2/X-333-L-21X-334-<BR> L-2/X-335-L-2/X-336-L-2/X-337-L-2/X-338-L-2/X-339-L-2/X-340- <BR> L-2 X-341-L-21X-342-L-2/X-343-L-2/X-344-L-2/X-345-L-21X-346-< BR> L-2iX-347-L-21X-348-L-21X-349-L-2/X-350-L-2/X-351-L-2/X-352- <BR> L-2/X-353-L-2/X-354-L-2/X-355-L-2/X-356-L-2/X-357-L-2/X-358- <BR> L-2X-359-L-2/X-360-L-2xX-361-L-2 X-36-L-2, X-363-L-X-364- L-2/X-365-L-2/X-366-L-2/X-367-L-2/X-368-L-2/X-369-L-2/X-370- L-2/X-371- L-2/X-372- L-2/X-373- L-2/X-374- L-2/X-375- L-2/X-376-

L-2/X-377- L-2/X-378- L-2/X-379- L-2/X-380- L-2/X-381- L-2/X-382- L-2/X-383- L-2/X-384- L-2/X-385- L-2/X-386- L-2/X-387- L-2/X-388-<BR> L-2/X-383-L-2/X-384-L-2/X-385-L-2/X-386-L-2/X-387-L-2/X-388- <BR> L-2/X-389-L-2 ! X-390-L-2'X-391-L-2, X-392-L-2/X-393-L-2,'X-394-<BR> L-2,'X-395-L-2/X-396-L-2/X-397-L-2/X-398-L-2/X-399-L-2/X-400 -<BR> L-2, X-401-L-2/X-402-L-2/X-403-L-2/X-404-L-2/X-405-L-2/X-406-< BR> L-2/X-407-L-2/X-408-L-2 X-409-L-2/X-4iO-L-2/X-411-L-2/X-412-<BR> L-2/X-413-L-2/X-414-L-2/X-415-L-2/X-416-L-2/X-417-L-2/X-418- <BR> L-3/X-I-L-3/X-2-L-3/X-3-L-3/X-4-L-3/X-5-L-3/X-6-<BR> L-3/X-7-L-31X-8-L-3/X-9-L-3/X-10-L-3/X-1 I-L-3/X-12-<BR> L-3/X-13-L-3/X-14-L-3/X-15-L-3/X-16-L-3/X-17-L-3/X-18-<BR > L-3/X-19-L-3/X-20-L-3/X-21-L-3/X-22-L-3/X-23-L-3/X-24-<BR > L-3/X-25-L-3/X-26-L-3/X-27-L-3/X-28-L-3/X-29-L-3/X-30-<BR > L-3/X-31-L-3/X-32-L-3/X-33-L-3/X-34-L-3/X-35-L-3/X-36-<BR > L-3/X-37-L-3/X-38-L-3/X-39-L-3/X-40-L-3/X-41-L-3/X-42- L-3/X-43-L-3/X-44-L-3/X-45-L-3/X-46-L-3/X-47-L-3/X-48- L-3/X-49-L-3/X-50-L-3/X-51-L-3/X-52-L-3/X-53-L-3/X-54-<BR > L-3/X-55-L-3/X-56-L-3/X-57-L-3/X-58-L-3/X-59-L-3/X-60- L-3/X-61-L-3/X-62-L-3/X-63-L-3/X-64-L-3/X-65-L-3/X-66- L-3/X-67-L-3/X-68-L-3/X-69-L-3/X-70-L-3/X-71-L-3/X-72- L-3/X-73-L-3/X-74-L-3/X-75-L-3/X-76-L-3/X-77-L-3/X-78- L-3/X-79-L-3/X-80-L-3/X-81-L-3/X-82-L-3/X-83-L-3/X-84-<BR > L-3/X-85-L-3/X-86-L-3/X-87-L-3/X-88-L-3/X-89-L-3/X-90-<BR > L-3/X-91-L-3/X-92-L-3/X-93-L-3/X-94-L-3/X-95-L-3/X-96-<BR > L-3/X-97-L-3/X-98-L-3/X-99-L-3/X-100-L-3/X-101-L-3/X-102- L-3/X-103-L-3/X-104-L-3/X-105-L-3/X-106-L-3/X-107-L-3/X-108- L-3/X-109-L-3/X-I 10-L-3/X-I I I-L-3/X-112-L-3/X-113-L-3/X-114-<BR> L-3/X-115-L-3/X-116-L-3/X-117-L-3/X-118-L-3/X-119-L-3/X-120- <BR> L-3/X-121-L-3/X-122-L-3/X-123-L-3/X-124-L-3/X-125-L-3/X-126- <BR> L-3/X-127-L-3/X-128-L-3/X-129-L-3/X-130-L-3/X-131-L-3/X-132- <BR> L-3/X-133-L-3/X-134-L-3/X-135-L-3/X-136-L-3/X-137-L-3/X-138- <BR> L-3/X-139-L-3/X-140-L-3/X-141-L-3/X-142-L-3/X-143-L-3/X-144- <BR> L-3/X-145-L-3/X-146-L-3/X-147-L-3/X-148-L-3/X-149-L-3/X-150- <BR> L-3/X-151-L-3eX-152-L-3,'X-153-L-3/X-154-L-3vX-155-L-3/X-156 -<BR> L-3/X-157-L-3X-158-L-3/X-159-L-3xX-160-L-3/X-161-L-3/X-162-

L-3/X-163-L-3/X-164-L-3/X-165-L-3/X-166-L-3/X-167-L-3/X-168- <BR> L-3/X-169-L-3/X-170-L-3/X-171-L-3/X-172-<BR> L-3/X-173-L-3/X-174-L-3/X-175-L-3/X-176-L-3/X-177-L-3/X-178- <BR> L-3/X-179-L-3/X-180-L-3/X-181-L-3/X-182-L-3/X-183-L-3/X-184- <BR> L-3/X-185-L-3/X-186-L-3/X-187-L-3/X-188-L-3/X-189-L-3/X-190- <BR> L-3/X-191-L-3/X-192-L-3/X-193-L-3/X-194-L-3/X-195-L-3/X-196- <BR> L-3/X-197-L-3/X-198-L-3/X-199-L-3/X-200-L-3/X-201-L-3/X-202- <BR> L-3/X-203-L-3/X-204-L-3/X-205-L-3/X-206-L-3/X-207-L-3/X-208- <BR> L-3/X-209-L-3/X-210-L-3/X-211-L-3/X-212-L-3/X-213-L-3/X-214- L-3/X-215-L-3/X-216-L-3/X-217-L-3/X-218-L-3/X-219-L-3/X-220- <BR> <BR> L-3/X-221-L-3/X-222-L-3/X-223-L-3/X-224-L-3/X-225-L-3/X-226- <BR> L-3/X-227-L-3/X-228-L-3/X-229-L-3/X-230-L-3/X-231-L-3/X-232- <BR> L-3/X-233-L-3/X-234-L-3/X-235-L-3/X-236-L-3/X-237-L-3/X-238- <BR> L-3/X-239-L-3/X-240-L-3/X-241-L-3/X-242-L-3/X-243-L-3/X-244- <BR> L-3/X-245-L-3/X-246-L-3/X-247-L-3/X-248-L-3/X-249-L-3/X-250- <BR> L-3/X-251-L-3/X-252-L-3/X-253-L-3/X-254-L-3/X-255-L-3/X-256- <BR> L-3/X-257-L-3/X-258-L-3/X-259-L-3/X-260-L-3/X-261-L-3/X-262- <BR> L-3/X-263-L-3/X-264-L-3/X-265-L-3/X-266-L-3/X-267-L-3/X-268- <BR> L-3/X-269-L-3/X-270-L-3/X-271-L-3/X-272-L-3/X-273-L-3/X-274- L-3/X-275-L-3/X-276-L-3/X-277-L-3/X-278-L-3/X-279-L-3/X-280- <BR> <BR> L-3/X-281-L-3/X-282-L-3/X-283-L-3/X-284-L-3/X-285-L-3/X-286- <BR> L-3/X-287-L-3/X-288-L-3/X-289-L-3/X-290-L-3/X-291-L-3/X-292- <BR> L-3/X-293-L-3/X-294-L-3/X-295-L-3/X-296-L-3/X-297-L-3/X-298- <BR> L-3/X-299-L-3/X-300-L-3/X-301-L-3/X-302-L-3/X-303-L-3/X-304- <BR> L-3/X-305-L-3/X-306-L-3/X-307-L-3/X-308-L-3/X-309-L-3/X-310- <BR> L-3/X-311-L-3/X-312-L-3/X-313-L-3/X-314-L-3/X-315-L-3/X-316- <BR> L-3/X-317-L-3/X-318-L-3/X-319-L-3/X-320-L-3/X-321-L-3/X-322- <BR> L-3/X-323-L-3/X-324-L-3/X-325-L-3/X-326-L-3/X-327-L-3/X-328- <BR> L-3/X-329-L-3/X-330-L-3/X-331-L-3/X-332-L-3/X-333-L-3/X-334- <BR> L-3/X-335-L-3/X-336-L-3/X-337-L-3/X-338-L-3/X-339-L-3/X-340- <BR> L-3/X-341-L-3/X-342-L-3/X-343-L-3/X-344-L-3/X-345-L-3/X-346- <BR> L-3/X-347-L-3/X-348-L-3/X-349-L-3/X-350-L-3/X-351-L-3/X-352- <BR> L-3/X-353-L-3/X-354-L-3/X-355-L-3/X-356-L-3/X-357-L-3/X-358- <BR> L-3/X-359-L-3/X-360-L-3/X-361-L-3/X-362-L-3/X-363-L-3/X-364- <BR> L-3/X-365-L-3/X-366-L-3/X-367-L-3/X-368-L-3/X-369-L-3/X-370- <BR> L-3/X-371-L-3/X-372-L-3/X-373-L-3/X-374-L-3/X-375-L-3/X-376-

L-3/X-377-L-3/X-378-L-3/X-379-L-3/X-380-L-3/X-381-L-3/X-382- <BR> L-3/X-383-L-3/X-384-L-3/X-385-L-3/X-386-L-3/X-387-L-3/X-388- <BR> L-3/X-389-L-3/X-390-L-3/X-391-L-3/X-392-L-3/X-393-L-3/X-394- <BR> L-3/X-395-L-3/X-396-L-3/X-397-L-3/X-398-L-3/X-399-L-3/X-400- <BR> L-3/X-401-L-3/X-402-L-3/X-403-L-3/X-404-L-3/X-405-L-3/X-406- <BR> L-3/X-407-L-3/X-408-L-3/X-409-L-3/X-410-L-3/X-411-L-3/X-412- <BR> L-3/X-413-L-3/X-414-L-3/X-415-L-3/X-416-L-3/X-417-L-3/X-418- Utility, Testing, and Administration Utility The multibinding compounds of this invention are bradykinin antagonists.

Accordingly, the multibinding compounds and pharmaceutical compositions of this invention are useful in the treatment and prevention of diseases mediated by bradydinin such as cancer, pain, shock, asthma, rhinitis, arthritis, inflammatory bowel disease, and the like.

Testing The in vitro bradykinin antagonist activity of the compounds of Formula (I) may be tested by the assay described in Example 24. The effectiveness of the compounds of Formula (I) in inhibiting bradykinin-induced brochoconstriction can be tested using an asthma model as described in Example 25. The effectiveness of the compounds of Formula (I) in inhibiting bradykinin-induced inflammation can be tested using the carrageenin-induced paw edema model as described in Example 26 and bradykinin-induced pancreatitis can be tested using Caerulein-induced pancreatitis model as described in Example 27.

Pharmaceutical Formulations When employed as pharmaceuticals, the compounds of this invention are usually administered in the form of pharmaceutical compositions. These compounds can be administered by a variety of routes including oral, rectal,

transdermal. subcutaneous. intravenous intramuscular. and intranasal. These compounds are effective as both injectable and oral compositions. Such compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.

This invention also includes pharmaceutical compositions which contain. as the active ingredient. one or more of the compounds described herein associated with pharmaceutically acceptable carriers. In making the compositions of this invention. the active ingredient is usually mixed with an excipient. diluted by an excipient or enclosed within such a carrier which can be in the form of a capsule. sachet. paper or other container. When the excipient serves as a diluent. it can be a solid. semi-solid, or liquid material. which acts as a vehicle. carrier 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), ointments containing, for example, up to 10% by weight of the active compound. soft and hard gelatin capsules, suppositories, sterile injectable solutions. and sterile packaged powders.

In preparing a formulation. it may be necessary to mill the active compound to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble. it ordinarily is milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble. the particle size is normally adjusted by milling to provide a substantially uniform distribution in the formulation, e. g. about 40 mesh.

Some examples of suitable excipients include lactose. dextrose. sucrose. sorbitol. mannitol, starch, gum acacia. calcium phosphate. alginates. tragacanth. gelatin, calcium silicate. microcrystalline cellulose, polyvinylpyrrolidone. cellulose. sterile water. syrup. and methyl cellulose. The formulations can additionally include: lubricating agents such as talc. magnesium stearate. and mineral oil : wetting agents : emulsifying and suspending agents: preserving agents such as methyl-and propylhydroxy-benzoates : sweetening agents : and flavoring agents.

The compositions of the invention can be formulated so as to provide quick.

sustained or delayed release of the active ingredient after administration to the patient bv employing procedures known in the art.

The compositions are preferably formulated in a unit dosage form. each dosage containing from about 0.001 to about 1 g. more usually about 1 to about 30 mg. of the active ingredient. The term"unit dosage forms"refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect. in association with a suitable pharmaceutical excipient. Preferably, the compound of Formula (I) above is employed at no more than about 20 weight percent of the pharmaceutical composition. more preferably no more than about 15 weight percent, with the balance being pharmaceutically inert carrier (s).

The active compound is effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. It, will be understood, however. that the amount of the compound actually administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated. the chosen route of administration, the actual compound administered and its relative activity, the age, weight. and response of the individual patient. the severity of the patient's symptoms. and the like.

For preparing solid compositions such as tablets. the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition mav be readily subdivided into equally effective unit dosage forms such as tablets. pills and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above containing from. for example. 0.1 to about 500 mg of the active ingredient of the present invention.

The tablets or pills of the present invention may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.

For example. the tablet or pill can comprise an inner dosage and an outer dosage component. the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric lavers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac. cetyl alcohol. and cellulose acetate.

The liquid forms in which the novel compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions. suitably flavored syrups. aqueous or oil suspensions. and flavored emulsions with edible oils such as com oil. cottonseed oil. sesame oil. coconut oil. or peanut oil. as well as elixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include'solutions and suspensions in pharmaceutically acceptable. aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra. Preferably the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions in preferably pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device or the nebulizing device may be attached to a face mask tent, or intermittent positive pressure breathing machine. Solution. suspension. or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.

EXAMPLES The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. They should not be considered as limiting the scope of the invention. but merely as being illustrative and representative thereof.

In the examples below. the following abbreviations have the following meanings. Unless otherwise stated. all temperatures are in degrees Celsius. If an abbreviation is not defined. it has its generally accepted meaning.

DMF = N dimethylformamide g=gram HPLC=high performance liquid chromatography mg-milligram ml milliliter<BR> mmol millimol N normal THF tetrahvdrofuran µg = micrograms Na, CO3 = sodium carbonate Na2SO4 = sodium sulfate NaOH = sodium hydroxide K, C03 sodium carbonate NaH sodium hydride TLC = thin layer chromatography EtOH-ethanol NaHCO, sodium bicarbonate Pd/C palladium on carbon MeCN=acetonitrile HCl = hydrochloric acid

Synthetic Examples Example 1 (Following Fig. 8) Preparation of a compound of Formula I wherein p is 2 and q is 1 and the ligands L, are a compound of formula (II) (where R''is 2-methylquinolin-8-yl and R'a and R'5 are chloro) which are linked via the terminal amine nitrogen

A solution of compound (II) (where Rl3 is 2-methylquinolin-8-yl and R' and R'5 are chloro) (2 mmols), triethylamine (2 mmols), and a dibromo linker molecule (1 mmol) in DMF (5 ml) is warmed. The course of the reaction is followed by thin layer chromatography. After reaction occurs, the reaction solution is quenched in water and the aqueous mixture is extracted with methylene chloride.

The organic layer is dried (Na2SOa), filtered, and concentrated under reduced pressure to give the crude product. The desired compound is obtained by purification of the crude product by use of HPLC.

Example 2 (Following Fig. 8) Preparation of a compound of Formula I wherein p is 2 and q is 1 and the ligands L. are a compound of formula (II) fn here R'3 is 2-bromo--methvlimidazof 1 2- a) pvridin-8-vl, and R'4 and R'5 are chloro} which are linked via the terminai amine nitrogen A solution of compound (II) (where R"is 2-bromo-3-methylimidazo (1, 2- a) pyridin-8-yl and R'4 and R'5 are Cl) (2 mmols) and benzene-1.4-bisacetic acid (1 mmol) in methylene chloride (5 ml) is prepared under argon in a flask equipped with magnetic stirrer and a drying tube. To this solution is added dicyclohexylcarbodiimide (solid. 2.1 mmols). The course of the reaction is followed by thin layer chromatography. After reaction occurs. the reaction solution is quenched in water and the aqueous mixture is extracted with methylene chloride.

The organic layer is washed with aqueous Na, C03, with water. and is dried (Na2SO,), filtered and concentrated under reduced pressure to give the crude product. The desired compound is obtained by purification of the crude product by use of HPLC.

Example 3 (Following Fig 8) Preparation of a compound of Formula I wherein p is 2 and q is 1 and the ligands.

L. are a compound of formula (II ! (where R'3 is 2-bromo-3-methvlimidazo- (1. ? pvridin-8-vl and R'4 and Rl5 are chloro ? which are linked via the terminal amine nitrogen

A solution of the benzene-1, 4-bisacetyl chloride (1 mmol) in methylene chloride is added slowly to a solution of compound (II) where (R¹³ is 2-bromo-3- methylimidazo (1, 2-a) pyridin-8-yl and R'4 and R'5 are Cl) (2 mmols) in methylene chloride (5 ml) and pyridine (0.5 ml) in a flask equipped with a magnetic stirrer and a drying tube and which is cooled in an ice-water bath. The course of the reaction is followed by thin layer chromatography. After reaction occurs, the reaction solution is quenched in water and the aqueous mixture is extracted with ethyl acetate. The organic layer is washed with aqueous Na-, C03, with water, and is dried (Na2SO4), filtered and concentrated under reduced pressure to give the crude product. The desired compound is obtained by purification of the crude product by use of HPLC.

Example 4 (Following Fig. 8) Preparation of a compound of Formula I wherein p is 2 and q is 1 and the ligands.

L, are a compound of formula (11) (where R'3 is 2-methvlquinoxalin-8-vl. and R' and R'5 are chloro ? which are linked via the terminal amine nitrogen A solution of compound (II) (where R'3 is 2-methylquinoxal-8-yl and R' and R'5 are Cl) (2 mmols) in methanol (4 ml) is acidified with acetic acid to pH 6.6 (pH meter) under a nitrogen atmosphere. 1, 6-Hexanedial (1 mmol) is added neat followed by sodium cyanoborohydride (1. 1 mmol). The course of the reaction is followed by thin layer chromatography. After reaction occurs, the reaction solution is quenched in water and the pH of the aqueous mixture is adjusted to greater than 10 with aqueous NaOH. The mixture is extracted with ether, the organic extracts are washed with half-saturated saline, dried (Na2SO4), filtered and concentrated under reduced pressure to give the crude product. The desired compound is obtained by purification of the crude product by use of HPLC.

Example 5 (Following Fig. 9) Preparation of a Compound of Formula I wherein p is 2. q is 1. and the ligands. L. are a of formula (III ? where R'3 is 2-methvlquinolin-8-vl. R'4 and R'5 are chloro R2' is methyl. and W is-CH- A solution of compound (III) (where R'3 is 2-methylquinolin-8-yl, R'4 and R'5 are chloro, R2'is methyl, and W is-CH-) (2 mmols) and 1, 5-bisamino-3- oxapentane (1 mmol) in methylene chloride (20 ml) is prepared under argon in a flask equipped with magnetic stirrer and drying tube. To this solution is added dicyclohexylcarbodiimide (solid, 2.1 mmols). The course of the reaction is followed by thin layer chromatography while stirring at room temperature. After reaction occurs, the reaction solution is diluted with ethyl acetate and washed with water and with aqueous Na, C03. The organic layer is dried (Na2SO4), filtered and concentrated under reduced pressure to give the crude product. The desired compound is obtained by purification of the crude product by use of HPLC.

Example 6 (Following Fig. 9) Preparation of a Compound of Formula I wherein p is 2. q is 1. and the ligands. L. are a compound of formula fill) where R'3 is 2-methvlquinolin-8-vl R'4 and R'5 are chloro.R'-'is methyl, and W is-CH-

A solution of compound (III) (where R'3 is 2-methylquinolin-8-yl, R'4 and R 15 are chloro, R2'is methyl, and W is-CH-) (2 mmols) and benzene-1. 4-bisacetic acid (1 mmol) in methylene chloride (20 ml) is prepared under argon in a flask equipped with magnetic stirrer and drying tube. To this solution is added dicyclohexylcarbodiimide (solid, 2.1 mmols) while stirring at room temperature.

The course of the reaction is followed by thin layer chromatography. After reaction occurs, the reaction solution is diluted with ethyl acetate and washed with water and with aqueous Na, C03. The organic layer is dried (Na2SO4), filtered and concentrated under reduced pressure to give the crude product. The desired compound is obtained by purification of the crude product by use of HPLC.

Example 7 (Following Fig. 9) Preparation of a Compound of Formula I wherein p is 2. q is l, and the ligands. L, are a compound of formula (III) where Rl3 is 2-methvlimidazo (l. 2-a !- pvridin-8-vl, R'S is chloro. R'-'is methyl, and W is-CH-

A solution of compound (III) (where R'3 is 2-methylimidazo (1, 2-a) pyridin- 8-yl, R'4 is hydroxy, R'5 is chloro, R2'is methyl, and W is-CH-) (2 mmols) and 1,4-bisiodomethylbenzene (1 mmol) in acetone (5 ml) containing K, C03 is stirred and heated at reflux temperature under an inert atmosphere. The course of the reaction is followed by thin layer chromatography. After reaction occurs, the reaction solution is diluted with ethyl acetate and washed with water and with aqueous Na, C03. The organic layer is dried (Na2SO4), filtered and concentrated under reduced pressure to give the crude product. The desired compound is obtained by purification of the crude product by use of HPLC.

Example 8 (Following Fig. 10) Preparation of a Compound of Formula I wherein p is 2. q is 1. and the ligands. L. are a compound of formula (III) where R'3 is 2-methvlquinolin-8-vl.

R' and R"are chloro. R'is methyl, and W is-CH- Step l A solution of compound (III) (where R'3 is 2-methylquinolin-8-yL R'4 and R'5 are chloro, R2'is methyl, and W is-CH-) (1 mmol) and an aminoacid, methyl ester linker molecule (1 mmol) in methylene chloride (20 ml) is stirred under argon in a flask equipped with magnetic stirrer and drying tube. To this solution is added dicyclohexylcarbodiimide (solid, 1.1 mmol). The course of the reaction is followed by thin layer chromatography while stirring at room temperature. After reaction occurs, the reaction solution is diluted with ethyl acetate and washed with water and with aqueous Na, C03. The organic layer is dried (Na, SO,). filtered and concentrated under reduced pressure to give the crude product. The desired compound is obtained by purification of the crude product by use of HPLC.

Step 2 A solution of the product from Step 1 above, and lithium hydroxide (10 mmols) in methanol (6 ml) and water (2 ml) is stirred at room temperature. The reaction is followed by thin layer chromatography. After reaction occurs, the pH of the solution is adjusted to 7 by the addition of dilute aq. hydrochloric acid. The

solvent is removed by lyophilization and the dry, crude product is used directly in the next reaction.

Step 3 A solution containing the crude product from Step 2 above, and a compound of formula (III) (where R'3 is 2-methylquinolin-8-yl, R'4 and R'5 are chloro, and W is-CH-) (1 mmol) in methylene chloride (20 ml) is prepared under argon in a flask equipped with magnetic stirrer and drying tube. To this solution is added dicyclohexylcarbodiimide (solid, 1.1 mmols). The course of the reaction is followed by thin layer chromatography while stirring at room temperature. After reaction occurs, the reaction solution is diluted with ethyl acetate and washed with water and with aqueous Na,, C03. The organic layer is dried (Na2SO,), filtered and concentrated under reduced pressure to give the crude product. The desired compound is obtained by purification of the crude product by use of HPLC.

Example 9 (Following Fig. 11) Preparation of a Compound of Formula I wherein p is 2, q is 1. and one of the ligands L, s is a compound of formula (III) wherein R'3 is 2-methylquinolin-8-yl.

R'4 and R'5 are chloro. and Ra is-NHCOCH3 and the other ligand.

L2, is a compound of formula din wherein R''is 2-methyl- quinolin-8-vl. R'4 and R'5 are chloro. and R2'is methyl

Step l A mixture of NaH (1.1 mmol) and DMF (1 ml) is prepared under an inert atmosphere in a flask equipped with a stirring bar and a drying tube. To this is added a solution of phthaloyl derivative 9 (where R'3 is 2-methylquinolin-8-yl and R'4 and Rl5 are Cl) (1 mmol) and an N-Cbz-bromomethyl linker molecule in dry DMF (5 ml). The resulting mixture is stirred and the course of the reaction is followed by thin layer chromatography. After reaction occurs, the reaction is quenched with cold dilute aq. Na, C03 and extracted with methylene chloride. The organic layer is dried (Na2SO4), filtered and concentrated under reduced pressure to give the crude product. The desired compound 16 is obtained by purification of the crude product by use of HPLC.

Step' Product 16 obtained in Step 1 above. is suspended in EtOH (5 mi) and hydrazine hydrate (2 mmols) is added. The reaction mixture is warmed and the course of the reaction followed by TLC. After reaction occurs. the reaction solution is filtered to remove solids and the filtrate is quenched with cold dilute aq. Na. CO. and extracted with methylene chloride. The organic layer is dried (Na., SO,), filtered and concentrated under reduced pressure to give the crude product. The desired compound is obtained by purification of the crude product by use of HPLC.

Step 3 The product obtained from Step 2 above. is carefully dried and placed in a solution in dry DMF (5 ml) with the carboxylic acid 18 (where W is-CH-and Ra is -NHAc) (1 mmol) and 1-hydroxybenzotriazole (1.4 mmols) under an inert atmosphere. The solution is stirred, cooled in an ice-water bath and protected from the atmosphere with a drying tube. To the stirred solution is added I-ethoxy-3- [3- (dimethylamino) propyl] carbodiimide hydrochloride (1.1 mmol). The course of the reaction is followed by tlc. The cooling bath is removed and after reaction occurs, the reaction mixture is partitioned between methylene chloride and saturated aqueous NaHC03. The organic layer is washed with water and brine. dried and concentrated under reduced pressure. The desired product 17 is obtained by purification of the crude product by use of HPLC.

Step 4 Ammonium formate (96 mg, 1. 5 mmol) and 10% Pd/C (50 mg) are added to a solution of compound 17 in methanol (2 ml) and THF (1 ml). The mixture is stirred at room temperature. The reaction is monitored by tic and after reaction occurs. the mixture is filtered through Celite and rinsed with ethyl acetate. The filtrate is washed successively with aq. NaHCO3 and with half-saturated brine. then filtered and concentrated under reduced pressure to give the crude product. The desired compound is obtained by purification of the crude product 19 by use of HPLC.

Step5

Product 19 is placed in a dry DMF solution (3 ml) with formula (III) compound (where R'3 is 2-methylquinolin-8-yl, R'''and R'"are CI, W is-CH-, and Q'is-N (CH) 3C (O)-) (0.8 mmols) under argon in a flask equipped with magnetic stirrer and drying tube. To this solution is added dicyclohexylcarbodiimide (solid, 1.1 mmols). The course of the reaction is followed by thin layer chromatography while stirring at room temperature. After reaction occurs, the reaction solution is diluted with ethyl acetate and washed with water and with aqueous Na. C03. The organic layer is dried (Na2SO4), filtered and concentrated under reduced pressure to give the crude product. The desired compound (I) is obtained by purification of the crude product by use of HPLC.

Example 10 (Following Fig. l') Preparation of a compound of Formula I wherein p is 2 and q is 1 and the ligands.

L. are a compound of formula (IIIlwhere R''is 2-methylquinolin-8-vl. R''and R'' are chloro, and R"is-NHCOCH) which is linked via the anilide nitrogen

Step I To a stirred mixture of NaH (2.1 mmol) in dry DMF (3 ml) under argon is added phthaloyl derivative 9 (where R'3 is 2-methylquinolin-8-yl and R'4 and R'5 are chloro) (2 mmol). After stirring for 20 min, 1, 5-dibromopentane (neat. 1 mmol) is added and stirring is continued. The course of the reaction is followed by thin layer chromatography and the reaction mixture is warmed to complete the reaction.

After reaction occurs. the reaction solution is poured into water and the aqueous mixture extracted with methylene chloride. The organic extracts are washed with water and with brine. dried (Na. SO,), filtered and concentrated under reduced pressure to give the crude product. The desired compound 20 is obtained by purification of the crude product by use of HPLC. The pure product is used in the following reaction.

steep 2 To a solution of compound 20 in 95% EtOH (5 ml) is added hydrazine hydrate (2 mmols). The stirred mixture is warmed and the course of the reaction followed by tlc. After reaction occurs, the reaction solution is filtered to remove solids and the filtrate is quenched with cold dilute aq. Na, C03 and extracted with methylene chloride. The organic layer is dried (Na2SO4), filtered and concentrated under reduced pressure to give the crude product. The desired compound 21 is obtained by purification of the crude product by use of HPLC.

Step 3 The product 21 from Step 2 above is carefully dried and placed in a solution in dry DMF (5 ml) with the carboxylic acid 18 (where W is-CH-and Ra is-NHAc) (1 mmol) and 1-hydroxybenzotriazole (1.4 mmols) under an inert atmosphere. The solution is stirred, cooled in an ice-water bath and protected from the atmosphere with a drying tube. To the stirred solution is added 1-ethoxy-3- [3- (dimethylamino)- propyl] carbodiimide hydrochloride (1. 1 mmol). The course of the reaction is followed by tlc. The cooling bath is removed and after reaction occurs, the reaction mixture is partitioned between methylene chloride and saturated aqueous NaHCO3.

The organic layer is washed with water and brine, dried and concentrated under reduced pressure. The desired compound of Formula I, is obtained by purification of the crude product by use of HPLC.

Example 11 (Following Fig. 13) Preparation of a compound of Formula I wherein p is 2, q is 1, and one of the ligands. L,, is a compound of formula (111) wherein R'3 is (3-bromo-2- methylimidazo [1. 2-a] pyridin-8-yl and R''* and R' are methyl, and Ra is -NHCOCH3 and the other ligand L2* is a compound of formula (III) wherein R¹³ is (3-bromo-2-methyl)imidazo [1.2-a] pyridin-8-yl, and R14 is chloro

Step 1 A mixture of NaH (1.1 mmol) and DMF (1 ml) is prepared under an inert atmosphere in a flask equipped with a stirring bar and a drying tube. To this is added a solution of phthaloyl derivative 9 [where Rl3 is 8-(3-bromo-2-methyl)-8- imidazo [1, 2-a] pyridyl and R14 and R15 are Me] (1 mmol) and a tert- butyldimethylsilyl-protected hydroxymethyl-bromomethyl linker molecule (1 mmol) in dry DMF (5 ml). The resulting mixture is stirred and the course of the reaction is followed by thin layer chromatography. After reaction occurs, the reaction is quenched with cold dilute aq. Na, C03 and extracted with methylene chloride. The organic layer is dried (Na2SO4), filtered and concentrated under

reduced pressure to give the crude product. The desired compound 22 is obtained by purification of the crude product by use of HPLC.

Step 2 To a solution of the product 22 in 95% EtOH (5 ml) is added hydrazine hydrate (2 mmols). The stirred mixture is warmed and the course of the reaction followed by TLC. After reaction occurs, the reaction solution is filtered to remove solids and the filtrate is quenched with cold dilute aq. Na, C03 and extracted with methylene chloride. The organic layer is dried (Na2SO4), filtered and concentrated under reduced pressure to give the crude product. The desired compound is obtained by purification of the crude product by use of HPLC.

Step 3 The product obtained in Step 2 is carefully dried and placed in a solution in dry DMF (5 ml) with the carboxylic acid 18 (where W is-CH= and Ra is-NHAc) (1 mmol) and 1-hydroxybenzotriazole (1.4 mmols) under an inert atmosphere. The solution is stirred, cooled in an ice-water bath and protected from the atmosphere with a drying tube. To the stirred solution is added 1-ethoxy-3- [3- (dimethylamino)- propyl] carbodiimide hydrochloride (1. 1 mmol). The course of the reaction is followed by tlc. The cooling bath is removed and after reaction occurs, the reaction mixture is partitioned between methylene chloride and saturated aqueous NaHCO3.

The organic layer is washed with water and brine, dried and concentrated under reduced pressure. The desired product 23 is obtained by purification of the crude product by use of HPLC.

Step 4 A solution compound 23 obtained from Step 3 above and Et3N- (HF) 3 in MeCN (5 ml) is stirred at room temperature. After reaction occurs as detected by tlc, the solution is diluted with EtOAc and then washed with water-brine. The organic layer is dried (Na2SO4), filtered and concentrated under reduced pressure to give the crude product. The desired hydroxy compound is obtained by purification of the crude product with the use of HPLC.

Step 5

A solution of the hydroxy compound obtained in Step 4 above in methylene chloride (5 ml) and triethyl amine (5 drops) is stirred under argon and cooled in an ice-water bath. Methanesulfonyl chloride (1.2 mmol) in methylene chloride (0.5 ml) is added and the solution is stirred and allowed to warm to room temperature.

The progress of the reaction is followed by TLC and after reaction occurs, the solution is poured into aqueous Na2CO3. The mixture is extracted with methylene chloride and the organic layer is washed with water and with brine, dried (Na. SO,), filtered and concentrated under reduced pressure to give the crude product. The desired mesylated compound is obtained by purification of the crude product by use of HPLC.

Step 6 A mixture of the mesylated compound obtained in Step 5 above, KI (10 mmol), and 18-crown-6 (1 mmol) in DMF (2 ml) is stirred and warmed. The progress of the reaction is followed by tlc and after reaction occurs, the mixture is poured into water and extracted with ethyl acetate. The combined organic extract solution is washed with water, with brine, dried (Na2SO4), filtered and concentrated under reduced pressure to give the crude product. The desired compound 24 is obtained by purification of the crude product by use of HPLC.

Step 7 A solution of the compound 24 and compound (III) (where R'3 is 8-(2- methyl) imidazo (1, 2-a) pyridinyl and W is-CH-) in acetone (5 ml) containing K, C03 is stirred and heated at reflux temperature under an inert atmosphere. The course of the reaction is followed by thin layer chromatography. After reaction occurs, the reaction solution is diluted with ethyl acetate and washed with water and with aqueous Na, C03. The organic layer is dried (Na2SO,), filtered and concentrated under reduced pressure to give the crude product. The desired compound (I) is obtained by purification of the crude product by use of HPLC.

Example 12 (Following Fig 14) Preparation of a compound of Formula I wherein p is 2, q is 1. and one of the ligands L,. is a compound of formula (III) wherein Rl3 is 2-methvlquinolin8-vh R' and R15 are chloro. and R²¹ is methyl. and the other ligand L, is a compound of formula (III) wherein R'3 is 2-methylimidazo [1. 2-a]- pyridin-8-yl. R' is chloro and R''is methyl Step 1 A solution of compound (III) (where R'3 is 2-methylquinolin-8-yl, R'4 and Rl5 are Cl, W is-CH-, and R2'is methyl) (1 mmol) and a tert-butyldimethylsilyl- protected hydroxymethyl amine linker molecule (lmmol) in methylene chloride (20 ml) is prepared under argon in a flask equipped with magnetic stirrer and drying tube. To this solution is added dicyclohexylcarbodiimide (solid, 1. 1 mmol). The course of the reaction is followed by thin layer chromatography while stirring at room temperature. After reaction occurs, the reaction solution is diluted with ethyl acetate and washed with water and with aqueous Na, C03. The organic layer is dried (Na2SO4), filtered and concentrated under reduced pressure to give the crude product. The desired compound 25 is obtained by purification of the crude product by use of HPLC and is then converted to a compound of formula 26 as described in Example 12, Steps 2-4 above.

Step 2

A solution of the compound 26 and of formula (III) compound (where R'3 is 8- (2-methyl) imidazo (l. 2-a) pyridinyi and W is-CH-) in acetone (5 ml a containing K, CO, is stirred and heated at reflux temperature under an inert atmosphere. The course of the reaction is followed by thin laver chromaiography. After reaction occurs. the reaction solution is diluted with ethvl acetate and washed with water and with aqueous Na. CO3. The organic layer is dried (Na. SO,), filtered and concentrated under reduced pressure to give the crude product. The desired compound (I) is obtained by purification of the crude product by use of HPLC.

Example 13 (Following Fig. 15) Preparation of a compound of Formula I wherein p is 2. q is I * and one of the ligands. L, is a compound of formula (TIP wherein R'3 is 2-methvlquinolin-8-vl, R'4 and R'5 are chloro. and Rz'is methyl, and the other ligand. L2. is a compound of formula (wherein R'3 is (2-methvl) imidazo [1. 2-a] pvridin-8-, vl. R'4 is chloro. R2' is diethyl. and Ra is-CONHCH3 Step l A solution of compound (III) (where R'3 is 9-methylquinolin-8-yl, R'4 and R's are Cl W is-CH-and R''is methvl) (l mmol) and a t-butyldimethylsilyl- protected hvdroxymethyl carboxylic acid linker molecule (Immol) in methylene chloride (20 mi) is prepared under argon in a flask equipped with magnetic stirrer and drying tube. To this solution is added dicyclohexylcarbodiimide (solid. 1.1

mmol). The course of the reaction is followed by thin layer chromatography while stirring at room temperature. After reaction occurs, the reaction solution is diluted with ethyl acetate and washed with water and with aqueous Na03. The organic layer is dried (Na., SO,), filtered and concentrated under reduced pressure to give the crude product. The desired compound 27 is obtained by purification of the crude product by use of HPLC which is then converted to a compound of formula 28 as described in Example 12, Steps 2-4 above.

Step 2 A solution of the compound 28 and a compound (III) (where A is 8- (2- methyl) imidazo (1, 2-a) pyridinyl and W is-CH-) in acetone (5 ml) containing K2CO3 is stirred and heated at reflux temperature under an inert atmosphere. The course of the reaction is followed by thin layer chromatography. After reaction occurs, the reaction solution is diluted with ethyl acetate and washed with water and with aqueous Na, C03. The organic layer is dried (Na2SO4), filtered and concentrated under reduced pressure to give the crude product. The desired compound (I) is obtained by purification of the crude product by use of HPLC.

Formulation Examples Example 14 Hard gelatin capsules containing the following ingredients are prepared: Quantity Ingredient (mg/capsule Active Ingredient 30.0 Starch 305.0 Magnesium stearate 5.0 The above ingredients are mixed and filled into hard gelatin capsules in 340 mg quantities.

Example 15 A tablet Formula is prepared using the ingredients below: Quantity Ingredient tablet Active Ingredient 25.0 Cellulose, microcrystalline 200.0 Colloidal silicon dioxide 10.0 Stearic acid 5. 0 The components are blended and compressed to form tablets, each weighing 240 mg.

Example 16 A dry powder inhaler formulation is prepared containing the following components: Ingredient Weight % Active Ingredient 5 Lactose 95 The active ingredient is mixed with the lactose and the mixture is added to a dry powder inhaling appliance.

Example 17 Tablets, each containing 30 mg of active ingredient, are prepared as follows: Quantity Ingredient tablet Active Ingredient 30.0 mg Starch 45.0 mg Microcrystalline cellulose 35. 0 mg Polyvinylpyrrolidone (as 10% solution in sterile water) 4.0 mg Sodium carboxymethyl starch 4.5 mg Magnesium stearate 0.5 mg Talc 1.0 mg Total 120 mg

The active ingredient, starch and cellulose are passed through a No. 20 mesh U. S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with the resultant powders, which are then passed through a 16 mesh U. S. sieve.

The granules so produced are dried at 50° to 60°C and passed through a 16 mesh U. S. sieve. The sodium carboxymethyl starch, magnesium stearate, and talc, previously passed through a No. 30 mesh U. S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 120 mg.

Example 18 Capsules, each containing 40 mg of medicament are made as follows: Quantity Ingredient (mg/capsule Active Ingredient 40.0 mg Starch 109.0 mg Magnesium stearate 1.0 mg Total 150.0 mg The active ingredient, starch, and magnesium stearate are blended, passed through a No. 20 mesh U. S. sieve, and filled into hard gelatin capsules in 150 mg quantities.

Example 19 Suppositories, each containing 25 mg of active ingredient are made as follows: Ingredient Amount Active Ingredient 25 mg Saturated fatty acid glycerides to 2,000 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 2.0 g capacity and allowed to cool.

Example 20 Suspensions, each containing 50 mg of medicament per 5.0 mL dose are made as follows: Ingredient Amount Active Ingredient 50.0 mg Xanthan gum 4.0 mg Sodium carboxymethyl cellulose (11%) Microcrystalline cellulose (89%) 50.0 mg Sucrose 1.75 g Sodium benzoate 10.0 mg Flavor and Color q. v.

Purified water to 5.0 mL The active ingredient, sucrose and xanthan gum are blended, passed through a No. 10 mesh U. S. sieve, and then mixed with a previously made solution of the microcrystalline cellulose and sodium carboxymethyl cellulose in water. The sodium benzoate, flavor, and color are diluted with some of the water and added with stirring. Sufficient water is then added to produce the required volume.

Example 21 A formulation may be prepared as follows: Quantity Ingredient (mg/capsule Active Ingredient 15. 0 mg Starch 407.0 mg Magnesium stearate 3.0 mg Total 425.0 mg The active ingredient, starch, and magnesium stearate are blended, passed through a No. 20 mesh U. S. sieve, and filled into hard gelatin capsules in 425.0 mg quantities.

Example 22 A formulation may be prepared as follows: Ingredient Quantity Active Ingredient 5.0 mg Corn Oil 1.0 mL Example 23 A topical formulation may be prepared as follows: Ingredient Quantity Active Ingredient 1-10 g Emulsifying Wax 30 g Liquid Paraffin 20 g White Soft Paraffin to 100 g The white soft paraffin is heated until molten. The liquid paraffin and emulsifying wax are incorporated and stirred until dissolved. The active ingredient is added and stirring is continued until dispersed. The mixture is then cooled until solid.

Another preferred formulation employed in the methods of the present invention employs transdermal delivery devices ("patches"). Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, e. g., U. S. Patent 5,023,252, issued June 11,1991, herein incorporated by reference in its entirety. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.

Other suitable formulations for use in the present invention can be found in Remington's Pharmaceutical Sciences, edited by E. W. Martin (Mack Publishing Company, 18th ed., 1990).

Biological Examples Example 24 Bradykinin Antagonist Activity.... In assay The bradykinin antagonistic activity of the compounds of the invention can be tested as described below.

Tissue Preparation : The specific binding of [3H] BK (a high affinity B, ligand) is assayed as follows. Male Hartley guinea pigs are killed by exsanguination under anesthesia.

The ilea are removed and homogenized in ice-cold buffer (5JmM sodium (trimethylamino) ethanesulfonate (TES) and 1mM l, 10-phenanthroline, pH6.8) with a Polytron homogenizer (PT-10, Brinkmann Instruments, Inc., Westbury, NY).

Cellular debris is removed by centrifuging at 1000g at 4°C for 20 minutes. The supernatant is centrifuged at 100000g at 4°C for 60 minutes. The pellet is then resuspended in ice-cold assay buffer containing 50mM TES, 1mM 1,10- phenanthroline, 140ug/ml bacitracin, 1mM dithiothreiol, luM captopril, and 0.1% bovine serum albumin (BSA), pH 6.8. The amount of protein is determined by the method of Lowry et al. using a kit (Catalog # P5656, Sigma Chemical Co., St.

Louis, MO). The pellet is stored at-80 °C until use.

Receptor binding : The bradykinin antagonist activity of the compounds of the invention is tested as follows. 0.2mg/ml of the receptor is incubated with 0.06 nM [3H] bradykinin and varying concentrations of either a test compound or unlabeled BK at room temperature for 60 minutes. Receptor bound [3H] bradykinin is harvested by filtration through Whatman glass fiber filters (Catalog # GF/B, Whatman, Inc., Clifton, NJ) under reduced pressure, and the filter is washed five times with 300 ml of ice-cold buffer (50mM Tris HCI). The radioactivity retained on the filter is measured with a scintillation counter. Specific binding is calculated by subtracting the nonspecific binding from total binding.

Example 25 Inhibition of bradykinin-induced bronchoconstriction... in vivo asthma model The ability of the compounds of the invention to inhibit bradykinin induced bronchoconstriction can be tested using an asthma model as described below. Male Hartley guinea pigs, obtained from Charles River, weighing 470-750 g are used.

The guinea pigs are fasted overnight and anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg/kg). The trachea, jugular vein, and esophagus are cannulated. The animals are ventilated at a tidal volume of 10 mg/kg with a frequency of 60 breathes/min through the tracheal cannula. To suppress spontaneous respiration, alcuronium chloride (0.5 mg/kg) is administered intravenously through the jugular vein cannula. Then, propanolol (10 mg/kg is administered subcutaneously. After 10 min., 5, ug/kg bradykinin is dissolved in saline with 0.1% BSA and admininstered intravenously via the jugular vein cannula. Bronchoconstriction is measured as the peak increase of pulmonary insufflation pressure. Each dose of the test compound or control compound is suspended in 0.5% methylcellulose solution and administered through the esophageal cannula after the first bradykinin-induced bronchoconstriction. The bradykinin is administered again at 30 min. and bronchoconstriction is measured. A 0% response is determined as peak increase of pulmonary insufflation pressure before administration of bradykinin and the 100% response is determined as the first bradykinin-induced bronchoconstriction before drug administration. The percent response was calculated from the following formula: % response = change in peak increase of pulmonary insufflation after drug/peak increase in pulmonary insufflation before drug) X 100.

Example 26 Inhibition of inflammation... in vivo Carrageenan-Induced paw edema model Carrageenin induced paw edema in rats is a standard model of inflammation.

In this model, male Sprague Dawley rats are deprived of food overnight and then various concentrations of test compounds, dissolved in 0.05 N HC1, are administered orally. After 15 minutes, 0. 1 ml of carrageenan (1 % in saline) is injected into the right hind paw intraplantar. Paw volume is measured using a water plethysmometer (Catalog # Ugo Basile 57140, Stoelting Co., Wood Dale, IL) before, and 1, 2,3, and 4 hours after injection of carrageenin. The ability of the test compounds to inhibit bradykinin induced inflammation is measured as duration of action.

Example 27 Inhibition of bradvkinin-induced Pancreatitis... In vivo Caerulein-Induced Pancreatitis model Female Sprague Dawley rats (9-l Oweeks old) are deprived of food for 18hours and caerulein (20g/ml) is injected intraperitoneally 4 times at hourly intervals. Test compounds, dissolved in 0. 05N HCl or vehicle, are administered orally 30 minutes before the first caerulein injection. Three hours after the last caerulein injection, a blood sample is taken from the abdominal artery with heparin under anesthesia (diethyl ether inhalation), and the animal is killed by exsanguination. Serum amylase and lipase levels are determined using a Vet Test 8000 chemistry analyzer model (see, Gukovskaya et al.,"Pancreatic Acinar Cells Produce, Release, and Respond to Tumor Necrosis Factor-a: Role in Regulating Cell Death and Pancreatitis"J. Clin. Invest., 100 (7), 1853-1862. (1997)).

The foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity and understanding. It will be obvious to one of skill in the art that changes and modifications may be practiced within the scope of the appended claims. Therefore, it is to be understood that the above description is

intended to be illustrative and not restrictive. The scope of the invention should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the following appended claims, along with the full scope of equivalents to which such claims are entitled.

All patents, patent applications and publications cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual patent, patent application or publication were so individually denoted.