Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
  • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
  • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D215/20—Oxygen atoms
  • C07D215/24—Oxygen atoms attached in position 8
  • C07D215/26—Alcohols; Ethers thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/08—Bronchodilators
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
  • C07D241/36—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings condensed with carbocyclic rings or ring systems
  • C07D241/38—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings condensed with carbocyclic rings or ring systems with only hydrogen or carbon atoms directly attached to the ring nitrogen atoms
  • C07D241/40—Benzopyrazines
  • C07D241/42—Benzopyrazines with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/04—Ortho-condensed systems
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

• 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.
• BK 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.
• 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.
• bradykinin receptors have been localized to nociceptive peripheral nerve pathways and bradykinin has been demonstrated to stimulate central fibers mediating pain sensation.
• bradykinin antagonists 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.
• 1 ' 3"6 Bradykinin has also been implicated in the production of symptoms in both allergic and viral rhinitis 7 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.
• 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.
• bradykinin antagonists that are increased potency and /or duration of action.
• 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.
• the non-peptidic multibinding compounds of the present invention will exhibit longer duration of activity vis-a-vis peptidic antagonists.
• 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.
• this invention provides a multibinding compound of Formula (I): (L) p (X) q
• 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):
• 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 1 is an integer of from 0 to 2);
• C is selected from the group consisting of a compound of formula (1) and (2):
• X 1 is -N- or -CR 4 where R 4 is alkyl
• X 2 is -N- or -CR 5 where R 5 is hydrogen or alkyl
• X 3 is -N- or -CR 6 where R 6 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;
• R 4 and R 5 are as defined above;
• R 1 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, and halo, or R 1 is a covalent bond linking the ligand to a linker;
• R 2 is selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, and halo, or R 2 is a covalent bond linking the ligand to a linker;
• R 3 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 -NR 7 R 8 [wherein R 7 is hydrogen, alkyl, or a covalent bond linking the ligand to a linker, and R 8 is hydrogen, -COOR 9 (where R 9 is aryl), -COR 10 (where R 10 is aryl, heteroaryl, or heterocyclyl)], or a group of formula:
• 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 -X 4 R a (where X 4 is -N-, -O-, or -S- and R is aryl, heteroaryl, or heterocyclyl each of which optionally links the ligand to a linker); and
• R 12 is selected from the group consisting of hydrogen and acylbiphenyl which optionally link the ligand to a linker; (ii) a compound of formula (b):
• a 1 is selected from the group consisting of alkylene and substituted alkylene
• R 13 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 14 is selected from the group consisting of hydrogen, halo, alkyl, and substituted alkyl, or R 14 is a covalent bond linking the ligand to a linker;
• R 15 is selected from the group consisting of halo and alkyl, or R" is a covalent bond linking the ligand to a linker; and
• R 16 is carboxy or a group of formula:
• Q 1 is alkylene or is a group of formula:
• R 19 is hydrogen or halo
• R 20 is selected from the group consisting of hydrog: and alkyl, or R 20 is a covalent bond linking the ligand to a linker, or R 20 and R 15 together form alkylene;
• R 21 is selected from the group consisting of hydrogen, alkyl, and aralkyl, or R 21 is a covalent bond linking the ligand to a linker, provided that A 2 is alkylene when R 20 is hydrogen;
• a 2 is selected from the group consisting of alkylene and a bond
• R !7 is selected from the group consisting of amino which optionally links the ligand to a linker, aminoacyl, cyano, hydroxy, and acyl;
• R 18 is selected from the group consisting of hydrogen and acyl; or (iii) a compound of formula (c):
• R 22 and R 23 are, independently of each other, halo or optionally link the ligand to a linker;
• a 3 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);
• R 24 and R 25 are, independently of each other, alkyl or optionally link the ligand to a linker;
• R 26 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 mo ⁇ holinocarbonyl; 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.
• 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
• m is an integer of from 0 to 20;
• X a 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.
• each Y a 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'
• this invention provides a pharmaceutical composition
• 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.
• 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.
• 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.
• 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:
• 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;
• this invention is directed to a method for identifying multimeric ligand compounds possessing multibinding properties for bradykinin receptor which method comprises:
• each linker comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand;
• 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).
• 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:
• 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;
• 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;
• 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.
• 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.
• 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 100A.
• 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.
• 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).
• 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.
• this method aspect is directed to a method for identifying multimeric ligand compounds possessing multibinding properties for bradykinin receptor which method comprises:
• 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.
• 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).
• 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.
• 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, w-propyl, wo-propyl, H-butyl, iso- butyl, ft-hexyl, rc-decyl, tetradecyl, and the like.
• 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.
• This term is exemplified by groups such as hydroxy methyl, hydroxyethyl, hydroxypropyl, 2- aminoethyl, 3-aminopropyl, 2-methylaminoethyl, 3-dimethylaminopropyl, 2- sulfonamidoethyl, 2-carboxyethyl, and the like.
• 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 2 -), ethylene
• 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
• 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.
• fused groups contain from 1 to 3 fused ring structures.
• 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.
• 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, «-propoxy, z ' s ⁇ -propoxy, «-butoxy, tert-butoxy, sec-butoxy, «-pentoxy, rz-hexoxy, 1,2-dimethylbutoxy, and the like.
• 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.
• 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.
• 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
• 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.
• 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, alkoxyamin
• 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.
• 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 2 C ⁇ CH) and the like.
• 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, alkoxya
• 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 (-CH 2 C ⁇ C-) and the like.
• 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, alkoxya
• 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.
• 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., mo ⁇ holino) wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.
• 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.
• 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.
• acyloxy refers to the groups alkyl-C(O)0-, 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)O- wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclic are as defined herein.
• 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.
• 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, thioaryl
• aryloxy refers to the group aryl-O- wherein the aryl group is as defined above including optionally substituted aryl groups as also defined above.
• 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.
• amino refers to the group -NH 2 .
• 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.
• carboxyalkyl or “alkoxycarbonyl” refers to the groups
• 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.
• 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, substituted thio
• 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.
• suitable cycloalkenyl groups include, for instance, cyclobut-2-enyl, cyclopent-3-enyl, cyclooct-3-enyl and the like.
• 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, heteroary
• halo or halogen refers to fluoro, chloro, bromo and iodo.
• 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).
• 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
• 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.
• heteroaryloxy refers to the group heteroaryl-O-.
• 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.
• heterocycle 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).
• 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
• heterocyclic groups can have a single ring or multiple condensed rings.
• Preferred heterocyclics include mo ⁇ holino, piperidinyl, and the like.
• 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, mo ⁇ holine, tetrahydrofuranyl, tetrahydrothiophen
• heterocyclooxy refers to the group heterocyclic-O-.
• heterocyclooxy refers to the group heterocyclic-S-.
• heterocyclene refers to the diradical group formed from a heterocycle, as defined herein, and is exemplified by the groups 2,6-mo ⁇ holino, 2,5-mo ⁇ holino and the like.
• 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.
• spiro-attached cycloalkyl group refers to a cycloalkyl group joined to another ring via one carbon atom common to both rings.
• thiol refers to the group -SH.
• thioalkoxy or “alkylthio” refers to the group -S-alkyl.
• substituted thioalkoxy refers to the group -S-substituted alkyl.
• thioaryloxy refers to the group aryl-S- wherein the aryl group is as defined above including optionally substituted aryl groups also defined above.
• heteroaryloxy refers to the group heteroaryl-S- wherein the heteroaryl group is as defined above including optionally substituted aryl groups as also defined above.
• amino acid residue refers to compounds having both carboxylic acid and amino functional groups and include both natural (Z-amino acids) and unnatural amino acids -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, -amino acids of naturally occurring L-amino acids, sarcosine, 1 -napthylalanine, and the like.
• acylbiphenyl refers to a biphenyl ring substituted with an acyl group as defined above.
• 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.
• the compounds of this invention include all stereochemical isomers arising from the substitution of these compounds.
• 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.
• 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, substituted cycloalkyl amines, substituted
• Suitable amines include, by way of example only, isopropylamine, trimethyl amine, diethyl amine, tri(w -propyl) amine, tri( «-propyl) amine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine, purines, piperazine, piperidine, mo ⁇ holine, N-ethylpiperidine, and the like.
• 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.
• 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, ?-toluene-sulfonic acid, salicylic acid, and the like.
• pharmaceutically-acceptable cation refers to the cation of a pharmaceutically-acceptable salt.
• library refers to at least 3, preferably from 10 2 to 10 9 and more preferably from 10 2 to 10 4 multimeric compounds. Preferably, these compounds are prepared as a multiplicity of compounds in a single solution or reaction mixture which permits facile synthesis thereof.
• the library of multimeric compounds can be directly assayed for multibinding properties.
• each member of the library of multimeric compounds is first isolated and, optionally, characterized. This member is then assayed for multibinding properties.
• selection 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 10 9 members and still more preferably from 10 to 10 4 members.
• 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).
• 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.
• protecting 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 nd Ed.).
• removable blocking group employed is not critical and preferred removable hydroxyl blocking groups include conventional substituents such as allyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidine, phenacyl, 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.
• t-BOC t-butyoxycarbonyl
• CBZ benzyloxycarbonyl
• FMOC fluorenylmethoxy-carbonyl
• ALOC allyloxycarbonyl
• 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.
• 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).
• 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.
• 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.
• 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.
• 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.
• 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)).
• 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.
• univalency 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.
• multivalency 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.
• 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.
• 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.
• selectivity 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 K d (i.e., the dissociation constants for each ligand-receptor complex) or, in cases where a biological effect is observed below the K d , the ratio of the respective EC 50 's (i.e., the concentrations that produce 50% of the maximum response for the ligand interacting with the two distinct ligand binding sites (receptors)).
• 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.
• 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.
• 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.
• antagonistagonism is well known in the art.
• 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.
• 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.
• the solvents used in the reactions described herein are inert solvents.
• treatment refers to any treatment of a pathologic condition in a mammal, particularly a human, and includes:
• 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.
• 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.
• 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.
• 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 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.
• 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.
• 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.
• 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.
• 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.
• preferred compounds are:
• R 13 is quinolyl, quinoxalinyl, benzimidazolyl, or imidazopyridyl each of which is substituted with one or more substituent(s) selected from halo, alkyl or alkoxy, preferably
• R 33 is chloro, bromo or iodo
• R 14 and R 15 are, independently of each other, hydrog m, alkyl, or halo, preferably hydrogen, methyl or chloro, most preferably methyl or chloro;
• R 21 is hydrogen or methyl, preferably methyl; and the terminal nitrogen atom attaches the ligand to a linker; or
• R 13 is quinolyl, quinoxalinyl, benzimidazolyl, or imidazopyridyl each of which is substituted with one or more substituent(s) selected from halo, alkyl or alkoxy, preferably
• R 33 is chloro, bromo or iodo
• R 14 and R 15 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;
• R 21 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
• R a is -COOH, -NH 2 , -CONR 28 R 29 (wherein R 28 is hydrogen or alkyl and R 29 is hydrogen, alkyl, or heteroaryl), -NR 30 COR 31 (where R 30 is hydrogen or alkyl and R 3 ' is alkyl), -NR 30 CONHR 32 (where R 30 is hydrogen or alkyl. and R 32 is alkyl), heteroaralkyl, heterocyclyl or a covalent bond linking the ligand to a linker, preferably -CONHCH 3 , -CON(CH 3 ) 2 , -NHCOCH 3 , -N(CH 3 )COCH 3 , -NHCONHCH 3 ,
• W is -CH- or -N-; or (iii) a compound of formula (IV):
• R 13 , R 14 and R 15 are as defined in preferred embodiment (i) above; and the terminal nitrogen atom attaches the ligand to a linker; or
• R 13 , R 14 , R 15 , W, and R a are as defined in the preferred embodiments (ii) above; and pharmaceutically acceptable salts thereof.
• the multibinding compound comprises of identical ligands.
• the multibinding compound comprises of non-identical ligands.
• R , R , R a , X, and W are as defined in preferred embodiments above.
• linker, X in the bivalent multibinding compound of Formula (I) independently has the formula:
• X a 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.
• 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.
• 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.
• reaction temperatures i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.
• 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.
• 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.
• 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.
• a bivalent multibinding compound of Formula (I) is prepared in one step, by covalently attaching the ligands, L, to a linker, X, where FG 1 and FG 2 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.
• 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 1 and FG 2 represent a functional group as defined above, and FG 2 PG is a protected functional group to give an intermediate of formula (II).
• FG 1 and FG 2 represent a functional group as defined above
• FG 2 PG 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 2 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.
• any compound which inhibits bradykinin receptor can be used as a ligand in this invention.
• numerous antagonists are known in the art and any of these known compounds or derivatives thereof may be employed as ligands in this invention.
• 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. 97/1 1069 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.
• a compound of formula (a) is prepared by reacting a compound of formula 1 where X is a leaving group under nucleophihc 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.
• 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.
• a coupling agent such as dicyclohexycarbodiimide
• an acid derivative such as acid chloride
• a suitable base such as triethylamine, and the like.
• a 2 is a bond
• R 17 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.
• compound 5 where R 14 and R 15 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 1 OH can be prepared by methods well known in the art.
• 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. Med. Chem., 41, 4062, 1998.
• 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 l-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.
• a suitable coupling agent such as l-ethoxy-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride in the presence of 1 -hydroxybenzotriazole as described in Abe, Y e
• a 1 and A 2 are methylene, R 17 is an aminoacyl (-NHCOR where R is aralkenyl or heteroaralkenyl) and Q 1 is a group of formula
• a compound of formula (b) where Q 1 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).
• R 16 is -Q'-A 2 -R 17 where Q 1 is -N(CH 3 )CO.
• a 2 is -CH 2 -
• R 17 is -NHCOR (where R is aralkenyl or heteroaralkenyl) are illustrated in Figures 6 and 7.
• 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.
• 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).
• SAR structure-activity relationships
• NMR nuclear magnetic resonance
• 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.
• 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.
• the linker may be chosen from any organic molecule construct that orients two or more ligands to their ligand binding sites to permit multivalency.
• 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.
• different orientations can be achieved by including in the framework groups containing mono- or polycyclic groups, including aryl and/or heteroaryl groups, or structures inco ⁇ orating 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.
• rigidity is imparted by the presence of cyclic groups (e.g., aryl, heteroaryl, cycloalkyl, heterocyclic, etc.).
• the ring is a six or ten membered ring.
• the ring is an aromatic ring such as, for example, phenyl or naphthyl.
• hydrophobic/hydrophilic characteristics of the linker as well as the presence or absence of charged moieties can readily be controlled by the skilled artisan.
• hydrophobic nature of a linker derived from hexamethylene diamine (H 2 N(CH 2 ) 6 NH 2 ) 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".
• 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.
• the framework is illustrated as a two dimensional array in the following diagram, although clearly the framework is a three dimensional array in practice: 8 7 6
• 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).
• 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.
• the linker connections to the ligand is selected such that the maximum spatial distance between two adjacent ligands is no more than 100 A.
• 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.
• FIG. 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.
• 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).
• 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)
• 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.
• 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.
• 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.
• PEG poly(ethylene glycol)
• 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.
• the ancillary group used to improve water solubility/hydrophilicity will be a polyether .
• 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.
• 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.
• 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.
• lipid 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.
• 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.
• 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.
• rigidity is imparted by the presence of cyclic groups (e.g., aryl, heteroaryl, cycloalkyl, heterocyclic, etc.).
• the linker comprises one or more six-membered rings.
• the ring is an aryl group such as, for example, phenyl or naphthyl.
• 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).
• 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.
• the linker may be considered as a framework to which ligands are attached.
• 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.
• a trimer can also be a radial multibinding compound comprising three ligands attached to a central core, and thus represented as (L) 3 X, 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
• X and L are as defined herein.
• X and L could be represented as an alkyl, aryl or cycloalkyl derivative as above with four (4) ligands attached to the core linker.
• a preferred linker may be represented by the following:
• 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.
• the linker i.e., X, X' or X
• the linker is selected those shown in Table II:
• n is an integer of from 2-10
• 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.
• 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.
• a single ligand or set of ligands is (are) selected for inco ⁇ oration 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).
• 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.
• 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.
• 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
• 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.
• 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.
• 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.
• 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.
• a 5HT 4 receptor antagonist and a bladder-selective muscarinic M 3 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 5HT 4 ligand and elements of the M 3 receptor proximal to the formal M 3 antagonist binding site and between the M 3 ligand and elements of the 5HT 4 receptor proximal to the formal 5HT 4 antagonist binding site.
• the dimeric compound may be more potent and selective antagonist of overactive bladder and a superior therapy for urinary urge incontinence.
• 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
• 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.
• the preferred distance can be estimated rather precisely from high-resolution structural information of targets, typically enzymes and soluble receptor targets.
• high-resolution structural information is not available (such as 7TM G-protein coupled receptors)
• preferred linker distances are 2-20 A, with more preferred linker distances of 3-12 A.
• preferred linker distances are 20-100 A, with more preferred distances of 30-70 A.
• 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 tr ⁇ s-arrangements at the 1,1- vs. 1,2- vs. 1,3- vs.
• Linker rigidity is varied by controlling the number and relative energies of different conformational states possible for the linker.
• 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.
• 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.
• 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 (BI, B2, B3) joined in all possible combinations provide for at least 15 possible combinations of multibinding compounds:
• combinatorial libraries Given the combinatorial nature of the library, common chemistries are preferably used to join the reactive fiinctionalies 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.
• the combinatorial libary is prepared in the solution phase.
• candidate multibinding compounds are optionally purified before assaying for activity by, for example, chromatographic methods (e.g., HPLC).
• Pharmacological data including oral abso ⁇ tion, 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 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• complementary functionality is well known in the art as illustrated in the following table:
• First Reactive Group Second Reactive Group Linkage hydroxyl isocyanate urethane amine epoxide ⁇ -hydroxyamine hydroxyamine sulfonyl halide sulfonamide carboxyl acid amine amide hydroxyl alkyl/aryl halide ether aldehyde amine/NaCNBH 3 amine ketone amine/NaCNBH 3 amine amine isocyanate urea
• Exemplary linkers include the following linkers identified as X-1 through X- 418 as set forth below:
• Combinations of ligands (L) 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:
• L-l/X-109- L-l X-1 10- L-l/X-111- L-l/X-112- L-l/X- 1 13- L-l/X-114- -l/X-1 15- L-l/X-1 16- L-l/X-1 17- L-l/X-1 18- L- l/X-1 19- L-l/X-120-
• L-3/X-209 L-3/X-210- L-3/X-21 1- 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-
• 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.
• 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.
• compositions 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.
• 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.
• the excipient when it 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.
• 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.
• the active compound 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.
• excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, 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 by employing procedures known in the art.
• 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.
• 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.
• 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.
• 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.
• preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may 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.
• 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.
• enteric layers 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.
• 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 corn 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.
• 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.
• L. are a compound of formula (II) (where R' 3 is 2-methylquinolin-8-yl and R' 4 and
• R' 5 are chloro) which are linked via the terminal amine nitrogen
• 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 fll) (where R' 3 is 2-bromo-3-methylimidazo(1.2- a)pyridin-8-yl. and R' 4 and R' 5 are chloro) which are linked via the terminal amine nitrogen
• L. are a compound of formula (II) (where R' 3 is 2-methylquinoxalin-8-yl. and R' 4 and R 1S are chloro) which are linked via the terminal amine nitrogen
• the mixture is extracted with ether, the organic extracts are washed with half-saturated saline, 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.
• 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 (HI) where R' 3 is 2-methylquinolin-8-yl. R 14 and R 15 are chloro. R 2 ' is methyl, and W is -CH-
• R' 4 and R 15 are chloro.
• R 21 is methyl.
• W is -CH-
• a solution containing the crude product from Step 2 above, and a compound of formula (III) (where R 13 is 2-methylquinolin-8-yl, R 14 and R 15 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.
• 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 2 CO 3 .
• 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.
• R 14 and R 15 are chloro. and R a is -NHCOCH 3 and the other ligand.
• L 2 . is a compound of formula (HI) wherein R' 3 is 2-methyl- quinolin-8-yl. R 14 and R 15 are chloro, and R 2 ' is methyl
• Stepl 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 13 is 2-methylquinolin-8-yl and R 14 and R 15 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 2 CO 3 and extracted with methylene chloride. The organic layer is dried (Na,SO 4 ), 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 2
• 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 R a 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 l-ethoxy-3-[3- (dimethylamino)propyl]carbodiimide hydrochloride (1.1 mmol). The course of the reaction is followed by tic.
• Step 5 Product 19 is placed in a dry DMF solution (3 ml) with formula (III) compound (where R 13 is 2-methylquinolin-8-yl, R 14 and R 15 are Cl, W is -CH-, and Q 1 is -N(CH) 3 C(O)-) (0.8 mmols) under argon in a flask equipped with magnetic stirrer and drying tube.
• 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 2 CO 3 .
• the organic layer is dried (Na,SO 4 ), 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.
• L. are a compound of formula (HI) (where R' 3 is 2-methylquinolin-8-yl. R' 4 and R' 5 are chloro. and R a is -NHCOCH 3 ) which is linked via the anilide nitrogen
• 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 R a 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 l-ethoxy-3-[3-(dimethylamino)- propyljcarbodiimide hydrochloride (1.1 mmol). The course of the reaction is followed by tic.
• the cooling bath is removed and after reaction occurs, the reaction mixture is partitioned between methylene chloride and saturated aqueous NaHCO 3 .
• 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.
• L 2 . is a compound of formula (HI) wherein
• R' 3 is (3-bromo-2-methyl)imidazo[1.2-a]pyridin-8-yl, and R 14 is chloro
• 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.
• a solution of phthaloyl derivative 9 [where R' 3 is 8-(3-bromo-2-methyl)-8- imidazo[l,2- ⁇ ] pyridyl and R 14 and R 15 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.
• 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 l-ethoxy-3-[3-(dimethylamino)- propyljcarbodiimide hydrochloride (1.1 mmol). The course of the reaction is followed by tic.
• Step 5 A solution compound 22 obtained from Step 3 above and Et 3 N-(HF) 3 in MeCN (5 ml) is stirred at room temperature. After reaction occurs as detected by tic, the solution is diluted with EtOAc and then washed with water-brine. The organic layer is dried (Na,SO ), 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.
• Step 7 A solution of the compound 24 and compound (III) (where R 13 is 8-(2- methyl)imidazo(l,2- ⁇ )pyridinyl and W is -CH-) in acetone (5 ml) containing K 2 CO 3 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 2 CO 3 . The organic layer is dried (Na,SO 4 ), 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 A solution of the compound 24 and compound (III) (where R 13 is 8-(2- methyl)imidazo(l,2- ⁇ )pyridinyl and W is -CH-) in acetone (5 ml) containing K 2 CO 3 is stirred and heated at reflux temperature under an inert atmosphere.
• R 14 and R' 5 are chloro. and R 2 ' is methyl, and the other ligand.
• L 2 . is a compound of formula (HI) wherein R' 3 is 2-methylimidazo[1.2-a]- pyridin-8-yl. R 14 is chloro and R 2 ' is methyl
• R 15 are Cl, W is -CH-, and R 21 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 2 CO 3 .
• Step 2 A solution of the compound 26 and of formula (III) compound (where R 13 is 8-(2-methyl)imidazo(l,2- ⁇ )pyridinyl and W is -CH-) in acetone (5 ml) containing K 2 CO 3 is stirred and heated at reflux temperature under an inert atmosphere. The course of the reaction is followed by thin layer chromatography.
• reaction solution is diluted with ethyl acetate and washed with water and with aqueous Na 2 CO 3 .
• the organic layer is dried (NajSO , 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.
• R' 4 and R' 5 are chloro. and R 21 is methyl, and the other ligand.
• L 2 . is a compound of formula (III) wherein R' 3 is (2-methyl)imidazo[1.2-a]pyridin-8-yl. R' 4 is chloro. R 2 ' is methyl, and R a is -CONHCH,
• reaction solution is diluted with ethyl acetate and washed with water and with aqueous Na 2 CO 3 .
• the organic layer is dried (Na,SO 4 ), 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 which is then converted to a compound of formula 28 as described in Example 12, Steps 2-4 above.
• Example 14 Hard gelatin capsules containing the following ingredients are prepared:
• Quantity Ingredient (mg/capsule)
• Example 15 The above ingredients are mixed and filled into hard gelatin capsules in 340 mg quantities.
• Example 15 The above ingredients are mixed and filled into hard gelatin capsules in 340 mg quantities.
• a tablet Formula is prepared using the ingredients below:
• Quantity Ingredient (mg/tablet)
• 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:
• the active ingredient is mixed with the lactose and the mixture is added to a dry powder inhaling appliance.
• Tablets each containing 30 mg of active ingredient, are prepared as follows:
• Quantity Ingredient (mg/tablet)
• 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.
• Capsules each containing 40 mg of medicament are made as follows:
• 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.
• Suppositories each containing 25 mg of active ingredient are made as follows:
• 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:
• 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.
• a formulation may be prepared as follows:
• Example 22 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 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.
• a formulation may be prepared as follows:
• Example 23 A topical formulation may be prepared as follows:
• transdermal delivery devices 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 inco ⁇ orated by reference in its entirety.
• patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.
• [3H]BK a high affinity B 2 ligand
• Male Hartley guinea pigs are killed by exsanguination under anesthesia.
• the ilea are removed and homogenized in ice-cold buffer (50mM sodium (trimethylamino)ethanesulfonate (TES) and ImM 1, 10-phenanthroline, pH6.8) with a Polytron homogenizer (PT-10, Brinkmann Instruments, Inc., Westbury, NY).
• TES trimethylamino)ethanesulfonate
• PT-10 Polytron homogenizer
• the pellet is then resuspended in ice-cold assay buffer containing 50mM TES, ImM 1,10- phenanthroline, 140 ⁇ g/ml bacitracin, ImM dithiothreiol, l ⁇ M captopril, and 0.1% bovine serum albumin (BSA), pH 6.8.
• BSA bovine serum albumin
• 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 [ 3 H] bradykinin and varying concentrations of either a test compound or unlabeled BK at room temperature for 60 minutes.
• Receptor bound [ 3 H] 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...
• 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.
• 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 ⁇ g/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.
• % response change in peak increase of pulmonary insufflation after drug/peak increase in pulmonary insufflation before drug
• 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)).

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