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  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P9/00—Drugs for disorders of the cardiovascular system
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  • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
  • C07C237/28—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a non-condensed six-membered aromatic ring of the carbon skeleton
  • C07C237/42—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a non-condensed six-membered aromatic ring of the carbon skeleton having nitrogen atoms of amino groups bound to the carbon skeleton of the acid part, further acylated
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  • C07C281/00—Derivatives of carbonic acid containing functional groups covered by groups C07C269/00 - C07C279/00 in which at least one nitrogen atom of these functional groups is further bound to another nitrogen atom not being part of a nitro or nitroso group
  • C07C281/06—Compounds containing any of the groups, e.g. semicarbazides
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  • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
  • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
  • C07D207/06—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with radicals, containing only hydrogen and carbon atoms, attached to ring carbon atoms
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  • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D207/18—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member
  • C07D207/20—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
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  • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
  • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen 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
  • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
  • C07D213/81—Amides; Imides
  • C07D213/82—Amides; Imides in position 3
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
  • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
  • C07D213/89—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members with hetero atoms directly attached to the ring nitrogen atom
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  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
  • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
  • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
  • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
  • C07D239/32—One oxygen, sulfur or nitrogen atom
  • C07D239/42—One nitrogen atom
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
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  • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
  • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
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  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
  • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
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  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C2601/00—Systems containing only non-condensed rings
  • C07C2601/02—Systems containing only non-condensed rings with a three-membered ring

Definitions

• This invention relates to the field of biologically active pharmaceutical compounds and specifically to selective antagonists to the bradykinin Bl receptor (BlR) and their uses for treating conditions and diseases responsive to bradykinin Bl receptor such as inflammatory and pain-associated disorders.
• BlR bradykinin Bl receptor
• the biological actions of kinins are mediated by two major G-protein coupled receptors BlR and B2R.
• the B2R is constitutively expressed under physiological conditions in a variety of cells while the BlR is induced under pathophysiological conditions such as tissue damage or inflammation in several cell types including endothelial, smooth muscle cells, blood cells and neurons (Regoli and Barabe, Pharmacol. Rev. 1980, 32, 1-46; Marceau et al., Pharmacol. Rev. 1998, 50, 357-386).
• Activation of the BlR produces a range of pro-inflammatory effects including edema, pain and promotion of blood-borne leukocyte trafficking (Calixto et al., Br. J. Pharmacol. 2004, 143, 803-818).
• Bradykinin (BK) and Kallidin (KD) are peptidic kinins which act on the B2R and mediate acute physiological actions of kinins on the cardiovascular, renal, nervous and immune system.
• BK and kallidin are metabolized by carboxypeptidase N and M, which remove the carboxy-terminal arginine residue to generate des-Arg-9-BK (DABK) or des-Arg- 10-kallidin (DAKD).
• DAKD is the only known natural ligand for the human BlR whereas des-Arg-9-BK activates the BlR in rodents.
• DAKD acting specifically on the human BlR appears to be an important mediator of inflammation and pain in man (Leeb-Lundberg et al. , Pharmacol. Rev. 2005, 57, 27-77).
• WO03066577, WO03065789, WO05016886, WO04019868, US20060122236 disclose biphenyl compounds and WO9725315 discloses sulphonamide compounds that are BlR antagonists.
• WO9725315 discloses sulphonamide compounds that are BlR antagonists.
• the problem underlying the present invention is to provide highly selective BlR antagonists, preferably having improved properties over the BlR antagonists of the prior art.
• A is: i) an optionally substituted 5- or 6-membered cycloalkyl; ii) an optionally substituted S- or 6-membered heterocycloalkyl; iii) an optionally substituted 6-membered aryl; or iv) an optionally substituted 5- or 6-membered heteroaryl;
• T is a hydrogen atom, or joined to B to form i) an optionally substituted heterocycloalkyl; or ii) an optionally substituted heteroaryl;
• B is an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted heteroalkyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted alkylcycloalkyl, an optionally substituted heteroalkylcycloalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted aralkyl, an optionally substituted heteroaralkyl, or
• W is N, alkyl, heteroalkyl, alkenyl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
• R 4 is hydrogen atom, an optionally substituted alkyl, an optionally substituted heteroalkyl, an optionally substituted alkylcycloalkyl, an optionally substituted heteroalkylcycloalkyl, an optionally substituted aryl, an optionally substituted aralkyl, an optionally substituted heteroaralkyl or joined to R 5 to form, together with W, i) an optionally substituted cycloalkyl; ii) an optionally substituted heterocycloalkyl; Ui) an optionally substituted aryl; or iv) an optionally substituted heteroaryl;
• R 5 is hydrogen atom, an optionally substituted alkyl, an optionally substituted heteroalkyl, an optionally substituted heteroalkylcycloalkyl, an optionally substituted aryl, an optionally substituted aralkyl, an optionally substituted heteroaralkyl or joined to R 4 to form, together with W, i) an optionally substituted cycloalkyl; ii) an optionally substituted heterocycloalkyl;
• G if present, is an alkyl, a heteroalkyl, a cycloalkyl, a heterocycloalkyl, a alkylcycloalkyl, a heterocycloalkylalkyl, a heteroaryl, or a heteroaralkyl
• R 1 and R 2 are each independently selected from C2-C 6 alkyl, C2-C 6 alkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkyl, or form, together with N, an optionally substituted heterocycloalkyl, an optionally substituted heteroaryl, or an optionally substituted heteroaralkyl; and
• R 3 is a hydrogen atom, C 1 - or C2alkyl, cyano, or a halogen atom.
• X 1 , X 2 , and X 3 are each independently selected from N, O, S, NR X , CR X , or CR X R X' , wherein
• X is NH, O, or S.
• Y 1 , Y 2 , Y 3 and Y 4 are each independently selected from N, O, S, NR y , CR y , or CR y R y' , wherein
• a sixth embodiment of the first aspect which is also an embodiment of the first, fourth or fifth embodiment of the first aspect A is
• R yl is a hydrogen atom.
• R ⁇ is a hydrogen atom, halogen atom, or C 1 -C 6 alkyl
• R y4 is a hydrogen atom or halogen atom.
• R 5 * is a hydrogen atom.
• R y3 is a hydrogen atom or halogen atom.
• R y4 is a hydrogen atom or halogen atom.
• T is a hydrogen atom.
• a 17 th embodiment of the first aspect which is also an embodiment of the first to 16 th embodiment of the first aspect B is heteroalkyl, heteroaryl, or
• W is N, alkyl, alkenyl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl;
• R 4 is a hydrogen atom, alkyl, heteroalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, aralkyl, heteroaralkyl or joined to R 5 to form, together with N, i) an optionally substituted cycloalkyl; ii) an optionally substituted heterocycloalkyl; iii) an optionally substituted aryl; or iv) an optionally substituted heteroaryl; and
• R 5 is a hydrogen atom, an optionally substituted alkyl, an optionally substituted heteroalkyl, an optionally substituted heteroalkylcycloalkyl, an optionally substituted aryl, an optionally substituted aralkyl, an optionally substituted heteroaralkyl or joined to R 4 to form, together with N, i) an optionally substituted cycloalkyl; ii) an optionally substituted heterocycloalkyl; iii) an optionally substituted aryl; or iv) an optionally substituted heteroaryl.
• B is - Y ⁇ -CO-L 3 - or - Y ⁇ -CO-R 0 *-, wherein
• Y* 3 is a bond, a C 1 -C 6 alkylene, a C 2 -C 6 alkenylene or a C 2 -C 6 alkynylene;
• L B is a cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, aralkyl, or heteroaralkyl; and R cB is an optionally substituted C 1 -C 8 alkyl, an optionally substituted C 2 -C 8 alkenyl or an optionally substituted C 2 -C 8 alkynyl.
• R 4 is methyl, ethyl or isopropyl.
• a 22 nd embodiment of the first aspect which is also an embodiment of the first to 21 st embodiment of the first aspect E is -Y aE -CO-L E -, -Y aE -CO-R cE -, -Y aE -NR cE -CO-R dE -, -Y aE -NR cE -CO-L E -, -Y aE -NR cE -CO-NR dE -L E -, or -Y aE -NR cE -CS-NR dE -L E -, wherein Y ⁇ is a bond, a C 1 -C 6 alkylene, a C 2 -C 6 alkenylene or a C 2 -C 6 alkynylene;
• R cE is a hydrogen atom, an optionally substituted C 1 -C 8 alkyl, an optionally substituted C 2 -C 8 alkenyl or an optionally substituted C2-C 8 alkynyl, provided that R cE is not a hydrogen atom in -Y aE -CO-R cE -;
• R dE is a hydrogen atom, an optionally substituted C 1 -C 8 alkyl, an optionally substituted C 2 -C 8 alkenyl or an optionally substituted C2-C 8 alkynyl;
• L E is a cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, aralkyl, or heteroaralkyl.
• G is alkyl, cycloalkyl, -Y ⁇ -O-R 00 , -Y aG -CO-NR aG R bG , -Y 8G -S-R 00 , -Y 1 ⁇ -SO-R 00 , -Y ⁇ -SO 2 -R' 0 , heteroaryl, alkylcycloalkyl, or heterocycloalkyl, wherein
• Y* 0 is a bond, a C 1 -C 6 alkylene, a C 2 -C 6 alkenylene or a C 2 -C 6 alkynylene;
• R"° is a hydrogen atom, a C 1 -C 6 alkyl, a C 2 -C 6 alkenyl, a C 2 -C 6 alkynyl, or is joined to R 60 to form a 4- to 10-membered cycloalkyl or heterocycloalkyl;
• R* 0 is a hydrogen atom, a C 1 -C 6 alkyl, a C ⁇ -C 6 alkenyl or a C 2 -C 6 alkynyl, or is joined to R"° to form a 4- to 10-membered cycloalkyl or heterocycloalkyl;
• R 00 is an optionally substituted C 1 -C 8 alkyl, an optionally substituted C 2 -C 8 alkenyl or an optionally substituted C 2 -C 8 alkynyl.
• Y 1 is N, CH or CF
• R 3 is a hydrogen atom or methyl
• R y2 is a hydrogen atom, halogen atom, or C 1 -C 6 alkyl
• R y3 is a hydrogen atom, halogen atom, C 1 -C 6 alkyl, or C 1 -C 6 heteroalkyl;
• R y4 is a hydrogen atom or halogen atom;
• B is -Y ⁇ -CO-lA, -Y ⁇ -CO-R 08 -, or
• Y* is a bond
• R cB is an optionally substituted C 1 -C 8 alkyl or an optionally substituted C2-C 8 alkenyl
• L B is a cycloalkyl, heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, or heteroaralkyl;
• W is alkyl or N
• R 4 if present, is a hydrogen atom, alkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aralkyl, or heteroaralkyl;
• R 5 if present, is a hydrogen atom or alkyl
• E is -Y bE -NR dE -C0-L E -, wherein
• Y bE is a bond, a C 1 -C 6 alkylene, or a C 2 -C 6 alkenylene
• R dE is a hydrogen atom, a Cj-C 6 alkyl, or C 2 -C 6 alkenyl
• L E is a cycloalkyl, heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, or heteroaralkyl.
• the compound is represented by formulas (III) or (IV):
• Y 1 is N, CH or CF
• R 3 is a hydrogen atom or methyl
• R y2 is a hydrogen atom, halogen atom, or C 1 -C 6 alkyl
• R y3 is a hydrogen atom, halogen atom, C 1 -C 6 alkyl, or C 1 -C 6 heteroalkyl
• R y4 is a hydrogen atom or halogen atom, C 1 -C 6 alkyl, or C 1 -C 6 heteroalkyl;
• E is -Y aE -NR cE -CO-R dE -, -Y aE -NR cE -CO-L E -, -Y aE -NR cE -CO-NR dE -L E -, or -Y aE -NR cE -CS-NR dE -L E -, wherein
• Y aE is a bond or C 1 -C 6 alkylene
• R cE is a hydrogen atom, C 1 -C 6 alkyl, or C 2 -C 6 alkenyl
• R dE is a hydrogen atom, an optionally substituted C 1 -C 8 alkyl, an optionally substituted C 2 -C 8 alkenyl or an optionally substituted C 2 -C 8 alkynyl group;
• L E is a cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, aralkyl, or heteroaralkyl;
• G if present, is cycloalkyl, -Y aG -CO-NR 80 R bG , -Y aG -O-R eG , -Y aG -S-R eG , -Y aG -SO-R eG , or -Y aG -SO 2 -R eG , wherein Y aG is a bond or C 1 -C 6 alkylene;
• R aG is a hydrogen atom or C 1 -C 6 alkyl
• R bG is a hydrogen atom or C 1 -C 6 alkyl; and R eG is C 1 -C 6 alkyl.
• Y 1 , K, R 3 , R y2 , R y3 , R y4 , and L E are as defined in 25 th embodiment of the first aspect.
• R y3 is a hydrogen atom.
• R* 2 is a hydrogen atom.
• Y 1 is N, CH or CF
• R* 4 is a hydrogen atom, halogen atom, C 1 -C 6 alkyl, or C 1 -C 6 heteroalkyl;
• E is -Y aE -CO-L E -, wherein
• Y aE is a bond or C 1 -C 6 alkylene
• L E is heterocycloalkyl
• G is alkylcycloalkyl, heterocycloalkylalkyl, aryl or heteroaryl.
• R y4 is a hydrogen atom or halogen atom.
• R 3 is methyl
• the compound exhibits an ICJO of 500 nM or less in a standard in vitro BK Bl receptor-mediated assay.
• the problem underlying the instant application is solved by a compound, which is preferably a compound according to any one of the first to 33 rd embodiment of the first aspect, which compound is selected from compounds 1 to 282 of Table 1.
• a pharmaceutical composition comprising one or more compounds according to any embodiment of the first and second aspect, optionally, at least one carrier substance, S excipient and/or adjuvant.
• the pharmaceutical composition is formulated as an aerosol, a cream, a gel, a pill, a capsule, a syrup, a solution, a transdermal therapeutic system, a 0 suppository, or a pharmaceutical device.
• the problem underlying the instant application is solved by the use of a compound or of a pharmaceutical composition according to any embodiment of the first to third aspect for the manufacture of a medicament for the treatment S and/or prevention of a disease or a condition.
• condition or disease is responsive to BK BlR modulation.
• condition or disease is selected from the group comprising inflammatory diseases, immunology disorders and pain.
• the inflammatory disease or immunology disorder is selected from the group comprising inflammatory bowel disease, rheumatoid arthritis, gouty arthritis, atherosclerosis and associated fibrotic conditions
• the pain is selected form the group comprising visceral pain, 2 neuropathic pain, complex regional pain syndrome CRPS and inflammatory pain.
• the problem underlying the instant application is solved by a method for inhibiting binding of DAKD, KD and DABK to a BK Bl receptor in S vitro, the method comprising contacting the BK Bl receptor with at least one compound or a pharmacologically acceptable salt, solvate, or hydrate thereof according to any embodiment of the first to third aspect under conditions and in an amount sufficient to detectably inhibit binding of DAKD, KD and DABK to the BK Bl receptor.
• a method for localizing or detecting a BK Bl receptor in a tissue, preferably a tissue section, in vitro comprising:
• the compound is radiolabeled, fluorescence-labeled or luminescence labeled, or labeled with an antibody.
• the problem underlying the instant application is solved by a method for the treatment of a subject which is in need of such treatment, comprising the administration of a compound or of a pharmaceutical composition according to any embodiment of the first to third aspect.
• BlR antagonists provided herein exhibit h activity on human BlR, i.e., an inhibition constant (IC50) for competition with binding of labelled DAKD to human BlR of less than S micromolar or very high activity on human BlR, i.e., an IC 50 for competition with the binding of labelled DAKD to human BlR of preferably less than 50 nanomolar.
• IC50 inhibition constant
• such antagonists exhibit a high activity on BlR of species other than human, i.e., an IC 50 for competition with binding of labelled DAKD to rabbit BlR and cynomolgus monkey of less than S micromolar.
• the activity and more specifically pharmacological activity of the BlR antagonists according to the present invention can be assessed using appropriate in vitro assays.
• the IC 50 values of the antagonists according to the present invention for BlR may be determined via a radioligand binding assay, such as the assay provided in Example 24, which is thus an embodiment of a standard in vitro BK BlR-mediated assay. Inhibitory effects of the
• BlR antagonists provided herein for BlR may be determined, for example, via calcium mobilization assay, such as the assay provided in Example 25.
• Preferred compounds of the invention have an IC 50 (half-maximal inhibitory concentration) of about 5 micromolar or less, still more preferably an IC 50 of about 500 nM or less, or even 50 nM or less, even more preferably an IC50 of about 10 nM or less, or even 1 nanomolar or less in the assays mentioned above.
• the present invention further provides, within other aspects, pharmaceutical compositions comprising at least one BlR modulator as described herein, in combination with a physiologically acceptable carrier or excipient. Processes for preparing such pharmaceutical compositions are also provided. Such compositions are particularly useful in the treatment of
• Recited compounds are further intended to encompass compounds in which one or more atoms are replaced with an isotope, i.e., an atom having the same atomic number but a different mass number.
• isotopes of hydrogen include tritium and deuterium and isotopes of carbon include 11 C, 13 C, and 14 C.
• a "pharmaceutically acceptable salt” of a pound disclosed herein is an acid or base salt that is generally considered in the art to be suitable for use in contact with the tissues of human beings or animals without excessive toxicity or carcinogenicity, and preferably without irritation, allergic response, or other problem or complication.
• Such salts include mineral and organic acid salts of basic residues such as amines, as well as alkali or organic salts of acidic residues such as carboxylic acids.
• Suitable pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic, methanesulfonic, benzene sulfonic, ethane disulfonic, 2- hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic, citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic, phenylacetic, alkanoic such as acetic, HOOC-(CH 2 ) n -COOH where n is any integer from 0 to 4, i.e., 0, 1, 2, 3, or 4, and the like.
• acids such as hydrochloric, phospho
• pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium and ammonium.
• a pharmaceutically acceptable acid or base salt can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.
• nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile, is preferred.
• each compound of formula (I) may, but need not, be present as a hydrate, solvate or non-covalent complex.
• the various crystal forms and polymorphs are within the scope of the present invention, as are prodrugs of the compounds of formula (I) provided herein.
• a “prodrug” is a compound that may not fully satisfy the structural requirements of the compounds provided herein, but is modified in vivo, following administration to a subject or patient, to produce a compound of formula (I) provided herein.
• a prodrug may be an acylated derivative of a compound as ded herein.
• Prodrugs include compounds wherein hydroxy, carboxy, amine or sulfhydryl groups are bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxy, carboxy, amino, or sulfhydryl group, respectively.
• prodrugs include, but are not limited to, acetate, formate, phosphate and benzoate derivatives of alcohol and amine functional groups within the compounds provided herein.
• Prodrugs of the compounds provided herein may be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved in vivo to generate the parent compounds.
• a “substituent,” as used herein, refers to a molecular moiety that is covalently bonded to an atom within a molecule of interest.
• a “ring substituent” may be a moiety such as a halogen, alkyl group, haloalkyl group or other substituent described herein that is covalently bonded to an atom, preferably a carbon or nitrogen atom, that is a ring member.
• substituted means that any one or more hydrogens on the designated atom is replaced with a selection from the indicated substituents, provided that the designated atom's normal valence is not exceeded, and that the substitution results in a stable compound, i.e., a compound that can be isolated, characterized and tested for biological activity.
• substituent oxo
• 2 hydrogens on the atom are replaced.
• a pyridyl group substituted by oxo is a pyridone.
• alkyl preferably refers to a saturated, straight-chain or branched hydrocarbon group that contains from 1 to 20 carbon atoms, preferably from 1 to 12 carbon atoms, more preferably from 1 to 6 carbon atoms, for example a methyl, ethyl, propyl, iso- propyl, n-butyl, iso-butyl, jec-butyl, terf-butyl, n-pentyl, n-hexyl, 2,2-dimethylbutyl or n-octyl group.
• alkenyl and alkynyl refer to at least partially unsaturated, straight-chain or branched hydrocarbon groups that contain from 2 to 20 carbon atoms, preferably from 2 to
• alkenyl groups have one or two, more preferably one, double bond(s) alkynyl groups have one or two, more preferably one, triple bond(s).
• alkyl, alkenyl and alkynyl refer to groups in which one or more hydrogen atoms have been replaced each independently of the others by a halogen atom, preferably F or Cl, such as, for example, a 2,2,2-trichloroethyl or a trifluoromethyl group.
• heteroalkyl preferably refers to an alkyl, alkenyl or alkynyl group, for example heteroalkenyl, heteroalkynyl, in which one or more, preferably 1, 2 or 3 carbon atoms have been replaced each independently of the others by an oxygen, nitrogen, phosphorus, boron, selenium, silicon or sulphur atom, preferably oxygen, sulphur or nitrogen.
• heteroalkyl furthermore preferably refers to a carboxylic acid or to a group derived from a carboxylic acid such as, for example, acyl, acylalkyl, alkoxycarbonyl, acyloxy, acyloxyalkyl, carboxyalkylamide, alkylcarbamoylalkyl, alkylcarbamoyloxyalkyl, alkylureidoalkyl, or alkoxycarbonyloxy.
• heteroalkyl groups are groups of formulas -S-Y a -L, -S-Y a -CO-NR a R b , -Y a -NR c -CO-NR a R b , -Y a -NR c -CO-O-R d , -Y a -NR c -C0-R d , -Y ⁇ -NR c -C0-NR d -L, -Y a -NR c -CS-NR d -L, -Y ⁇ O-CO-NR 8 R", -Y ⁇ CO-NR 8 R 5 , -O-Y a -CO-NR a R b , -Y a -NR c -CO-L, , -Y a -O-CO-O-R c , -Y a -O-CO-R c , -Y a -0-R c , -
• heteroalkyl groups are methoxy, trifluoromethoxy, ethoxy, n-propyloxy, isopropyloxy, tert- butyloxy, methoxymethyl, ethoxymethyl, methoxyethyl, methylamino, ethylamino, dimethyl- amino, diethylamino, isopropylethylamino, methylaminomethyl, ethylaminomethyl, diiso- propylaminoethyl, enol ether, dimethylaminomethyl, dimethylaminoethyl, acetyl, propionyl, butyryloxy, acetyloxy, methoxycarbonyl, ethoxycarbonyl, isobutyrylamino-methyl, N-ethyl- N-methylcarbamoyl and N-methylcarbamoyl.
• heteroalkyl groups are nitrile, isonitrile, cyanate, thiocyanate, isocyanate, isothiocyanate and alkylnitrile groups.
• An example of a heteroalkylene group is a group of formulas -CHiCH(OH)- or -CONH-.
• cycloalkyl preferably refers to a saturated or partially unsaturated cyclic group that contains one or more rings, preferably 1 or 2, containing from 3 to 14 ring carbon atoms, preferably from 3 to 10, more preferably 3, 4, 5, 6 or 7, ring carbon atoms.
• a partially unsaturated cyclic group has one, two or more double bonds, such as a cycloalkenyl group.
• a cycloalkyl group is a cyclo- propyl, cyclobutyl, cyclopentyl, spiro[4,5]decanyl, norbornyl, cyclohexyl, cyclopentenyl, cyclohexadienyl, decalinyl, bicyclo[4.3.0]nonyl, tetralin, cyclopentylcyclohexyl, fluorocyclo- hexyl or cyclohex-2-enyl group.
• heterocycloalkyl preferably refers to a cycloalkyl group as defined above in which one or more, preferably 1, 2 or 3, ring carbon atoms have been replaced each independently of the others by an oxygen, nitr silicon, selenium, phosphorus or sulphur atom, preferably oxygen, sulphur or nitrogen.
• a heterocycloalkyl group has preferably 1 or 2 ring(s) containing from 3 to 10, more preferably 3, 4, 5, 6 or 7, ring atoms.
• Examples are a piperidyl, piperazinyl, morpholinyl, urotropinyl, pyrrolidinyl, tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrofuryl or 2-pyrazolinyl group and also a lactam, a lactone, a cyclic imide and a cyclic anhydride.
• alkylcycloalkyl preferably refers to a group containing both cycloalkyl and also an alkyl, alkenyl or alkynyl group in accordance with the above definitions, for example alkylcycloalkyl, cycloalkylalkyl, alkylcycloalkenyl, alkenylcycloalkyl and alkynyl- cycloalkyl groups.
• An alkylcycloalkyl group preferably contains a cycloalkyl group that contains one or two ring systems having from 3 to 10, preferably 3, 4, 5, 6 or 7, carbon atoms, and one or two alkyl, alkenyl or alkynyl groups having 1 or 2 to 6 carbon atoms, the cyclic groups being optionally substituted.
• heteroalkylcycloalkyl preferably refers to alkylcycloalkyl groups as defined above in which one or more, preferably 1, 2 or 3, carbon atoms have been replaced each independently of the others by an oxygen, nitrogen, silicon, selenium, phosphorus or sulphur atom, preferably oxygen, sulphur or nitrogen.
• a heteroalkylcycloalkyl group preferably contains 1 or 2 ring systems having from 3 to 10, preferably 3, 4, 5, 6 or 7, ring atoms, and one or two alkyl, alkenyl, alkynyl or heteroalkyl groups having from 1 or 2 to 6 carbon atoms.
• Examples of such groups are alkylheterocycloalkyl, heterocycloalkylalkyl, alkylheterocycloalkenyl, alkenylheterocycloalkyl, alkynylheterocycloalkyl, heteroalkylcycloalkyl, heteroalkylheterocycloalkyl and heteroalkylheterocycloalkenyl, the cyclic groups being optionally substituted and saturated or mono-, di- or tri-unsaturated.
• aryl or Ar preferably refers to an aromatic group that contains one or more rings containing from 6 to 14 ring carbon atoms, preferably from 6 to 10, more preferably 6, ring carbon atoms.
• aryl (or Ar) preferably refers furthermore to groups in which one or more hydrogen atoms have been aced each independently of the others by fluorine, chlorine, bromine or iodine atoms or by OH, SH, NH2, CN, CF 3 or NO 2 groups. Examples are a phenyl, naphthyl, biphenyl, 2-fluorophenyl, anilinyl, 3-nitrophenyl or 4- hydroxyphenyl group.
• heteroaryl preferably refers to an aromatic group that contains one or more rings containing from 5 to 14 ring atoms, preferably from 5 to 10, more preferably 5 or 6, ring atoms, and contains one or more, preferably 1, 2, 3 or 4, oxygen, nitrogen, phosphorus or sulphur ring atoms, preferably O, S or N.
• Examples are 4-pyridyl, 2-imidazolyl, 3-phenylpyrrolyl, thiazolyl, oxazolyl, triazolyl, tetrazolyl, isoxazolyl, indazolyl, indolyl, benzimidazolyl, pyridazinyl, quinolinyl, purinyl, carbazolyl, acridinyl, pyrimidyl, 2,3 '-bifuryl, 3-pyrazolyl and isoquinolinyl.
• aralkyl preferably refers to a group containing both aryl and also alkyl, alkenyl, alkynyl and/or cycloalkyl groups in accordance with the above definitions, such as, for example, arylalkyl, arylalkenyl, arylalkynyl, arylcycloalkyl, arylcycloalkenyl, alkylaryl- cycloalkyl and alkylarylcycloalkenyl groups.
• aralkyls are toluene, xylene, mesitylene, styrene, benzyl chloride, o-fluorotoluene, lH-indene, tetralin, dihydro- naphthalene, indanone, phenylcyclopentyl, cumene, cyclohexylphenyl, fluorene and indan.
• An aralkyl group preferably contains one or two aromatic ring systems, 1 or 2 rings, containing from 6 to 10 carbon atoms and one or two alkyl, alkenyl and/or alkynyl groups containing from 1 or 2 to 6 carbon atoms and/or a cycloalkyl group containing 5 or 6 ring carbon atoms.
• heteroaralkyl preferably refers to an aralkyl group as defined above in which one or more, preferably 1, 2, 3 or 4, carbon atoms have been replaced each independently of the others by an oxygen, nitrogen, silicon, selenium, phosphorus, boron or sulphur atom, preferably oxygen, sulphur or nitrogen, that is to say to groups containing both aryl or heteroaryl and also alkyl, alkenyl, alkynyl and/or heteroalkyl and/or cycloalkyl and/or heterocycloalkyl groups in accordance with above definitions.
• a heteroaralkyl group preferably contains one or two aromatic ring systems, 1 or 2 rings, containing from 5 or 6 to 10 ring carbon atoms and one or two alkyl, alkenyl and/or alkynyl groups containing 1 or 2 to 6 carbon atoms and/or a cycloalkyl group containing 5 or 6 ring carbon atoms, 1, 2, 3 or 4 of those carbon atoms having been replaced each independently of the others by oxygen, sulphur or nitrogen atoms.
• heteroaralkyl groups are arylheteroalkyl, arylheterocycloalkyl, arylhetero- cycloalkenyl, arylalkylheterocycloalkyl, arylalkenylheterocycloalkyl, arylalkynylhetero- cycloalkyl, arylalkylheterocycloalkenyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heteroarylheteroalkyl, heteroarylcycloalkyl, heteroarylcycloalkenyl, heteroarylhetero- cycloalkyl, heteroarylheterocycloalkenyl, heteroarylalkylcycloalkyl, heteroarylheteroalkenyl, heteroarylalkylcycloalkyl, heteroarylalkyl- heterocycloalkenyl, heteroarylheteroalkylcycloalkyl
• This expression refers furthermore to a group in which one or more hydrogen atoms have been replaced each independently of the others by an unsubstituted Q- C 6 alkyl, unsubstituted C 2 -C6alkenyl, unsubstituted (VC 6 alkynyl, unsubstituted C 1 -C 6 hetero- alkyl, unsubstituted C3-C 1 ocycloalkyl, unsubstituted C 2 -C9heterocycloalkyl, unsubstituted Ce- C 1 oaryl, unsubstituted C 1 -C ⁇ heteroaryl, unsubstituted C 7 -C 12 aralkyl or unsubstituted C 2 - C 1 iheteroaralkyl group.
• halogen as preferably used herein means fluorine, chlorine, bromine, iodine.
• a wording defining the limits of a range of length such as, e. g., "from 1 to 5" means any integer from 1 to 5, i. e. 1, 2, 3, 4 and 5.
• any range defined by two integers explicitly mentioned is meant to comprise and disclose any integer defining said limits and any integer comprised in said range.
• the therapeutic use of compounds of formula (I), their pharmacologically acceptable salts, solvates and hydrates and also formulations and pharmaceutical compositions containing the same are within the scope of the present invention.
• the present invention also relates to the use of those compounds of formula (I) as active ingredients in the preparation or manufacture of a medicament.
• compositions comprise at least one compound of formula (I) and, optionally, one or more carrier substances, excipients and/or adjuvants.
• Pharmaceutical compositions may additionally comprise, for example, one or more of water, buffers such as, e.g., neutral buffered saline or phosphate buffered saline, ethanol, mineral oil, vegetable oil, dimethylsulfoxide, carbohydrates such as e.g., glucose, mannose, sucrose or dextrans, mannitol, proteins, adjuvants, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione and/or preservatives.
• buffers such as, e.g., neutral buffered saline or phosphate buffered saline
• ethanol mineral oil
• vegetable oil dimethylsulfoxide
• carbohydrates such as e.g., glucose, mannose, sucrose or dextrans, mannitol
• proteins e
• one or more other active ingredients may, but need not, be included in the pharmaceutical compositions provided herein.
• the compounds of the invention may advantageously be employed in combination with an antibiotic, anti-fungal, or anti-viral agent, an-anti histamine, a non-steroidal anti-inflammatory drug, a disease modifying antirheumatic drug, a cytostatic drug, a drug with smooth muscle activity modulatory activity or mixtures of the aforementioned.
• compositions may be formulated for any appropriate route of administration, including, for example, topical such as, e.g., transdermal or ocular, oral, buccal, nasal, vaginal, rectal or parenteral adm ration.
• parenteral as used herein includes subcutaneous, intradermal, intravascular such as, e.g., intravenous, intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial and intraperitoneal injection, as well as any similar injection or infusion technique.
• compositions in a form suitable for oral use are preferred.
• compositions provided herein may be formulated as a lyophilizate.
• Formulation for topical administration may be preferred for certain conditions such as, e.g., in the treatment of skin conditions such as burns or itch.
• compositions intended for oral use may further comprise one or more components such as sweetening agents, flavoring agents, coloring agents and/or preserving agents in order to provide appealing and palatable preparations.
• Tablets contain the active ingredient in admixture with physiologically acceptable excipients that are suitable for the manufacture of tablets.
• excipients include, for example, inert diluents such as, e.g., calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate, granulating and disintegrating agents such as, e.g., corn starch or alginic acid, binding agents such as, e.g., starch, gelatin or acacia, and lubricating agents such as, e.g., magnesium stearate, stearic acid or talc.
• the tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
• a time delay material such as glyceryl monosterate or glyceryl distearate may be employed.
• Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent such as, e.g., calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium such as, e.g., peanut oil, liquid paraffin or olive oil.
• an inert solid diluent such as, e.g., calcium carbonate, calcium phosphate or kaolin
• water or an oil medium such as, e.g., peanut oil, liquid paraffin or olive oil.
• Aqueous suspensions contain the active ingredient(s) in admixture with excipients suitable for the manufacture of aqueous suspensions.
• excipients include suspending agents such as, e.g., sodium carboxymethyl cellulose, methyl cellulose, hydropropylmethylcellulose, sodium alginate, p vinylpyrrolidone, gum tragacanth and gum acacia; and dispersing or wetting agents such as, e.g., naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with fatty acids such as polyoxyethylene stearate, condensation products of ethylene oxide with long chain aliphatic alcohols such as heptadecaethyleneoxycetanol, condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides such as polyethylene
• Aqueous suspensions may also comprise one or more preservatives, for example ethyl, or n-propyl p- hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
• preservatives for example ethyl, or n-propyl p- hydroxybenzoate
• coloring agents for example ethyl, or n-propyl p- hydroxybenzoate
• flavoring agents such as sucrose or saccharin.
• sweetening agents such as sucrose or saccharin.
• Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil such as, e.g., arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
• the oily suspensions may contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and/or flavoring agents may be added to provide palatable oral preparations.
• Such suspensions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
• Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
• a dispersing or wetting agent e.g., glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerin, glycerin, glycerin, glycerin, glycerin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol
• compositions may also be in the form of oil-in-water emulsions.
• the oily phase may be a vegetable oil such as, e.g., olive oil or arachis oil, a mineral oil such as, e.g., liquid paraffin, or a mixture thereof.
• Suitable emulsifying agents include naturally-occurring gums such as, e.g., gum acacia or gum tragacanth, naturally-occurring phosphatides such as, e.g., soy bean lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides such as, e.g., sorbitan monoleate, and condensation products of partial esters derived from fatty acids and hexitol with ethylene oxide such as, e.g., polyoxyethylene sorbitan monoleate.
• An emulsion may also com e one or more sweetening and/or flavoring agents.
• Syrups and elixirs may be formulated with sweetening agents, such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also comprise one or more demulcents, preservatives, flavoring agents and/or coloring agents.
• sweetening agents such as glycerol, propylene glycol, sorbitol or sucrose.
• Such formulations may also comprise one or more demulcents, preservatives, flavoring agents and/or coloring agents.
• Compounds may be formulated for local or topical administration, such as for topical application to the skin or mucous membranes, such as in the eye.
• Formulations for topical administration typically comprise a topical vehicle combined with active agent(s), with or without additional optional components.
• Suitable topical vehicles and additional components are well known in the art, and it will be apparent that the choice of a vehicle will depend on the particular physical form and mode of delivery.
• Topical vehicles include water; organic solvents such as alcohols such as, e.g., ethanol or isopropyl alcohol or glycerin; glycols such as, e.g., butylene, isoprene or propylene glycol; aliphatic alcohols such as, e.g., lanolin; mixtures of water and organic solvents and mixtures of organic solvents such as alcohol and glycerin; lipid-based materials such as fatty acids, acylglycerols including oils, such as, e.g., mineral oil, and fats of natural or synthetic origin, phosphoglycerides, sphingolipids and waxes; protein-based materials such as collagen and gelatin; silicone-based materials, both non-volatile and volatile; and hydrocarbon-based materials such as microsponges and polymer matrices.
• organic solvents such as alcohols such as, e.g., ethanol or isopropyl alcohol or glycerin
• glycols
• a composition may further include one or more components adapted to improve the stability or effectiveness of the applied formulation, such as stabilizing agents, suspending agents, emulsifying agents, viscosity adjusters, gelling agents, preservatives, antioxidants, skin penetration enhancers, moisturizers and sustained release materials.
• stabilizing agents such as hydroxymethylcellulose or gelatin-microcapsules, liposomes, albumin microspheres, microemulsions, nanoparticles or nanocapsules.
• a topical formulation may be prepared in a variety of physical forms including, for example, solids, pastes, creams, foams, lotions, gels, powders, aqueous liquids, emulsions, sprays and skin patches.
• the physical appe ce and viscosity of such forms can be governed by the presence and amount of emulsifier(s) and viscosity adjuster(s) present in the formulation.
• Solids are generally firm and non-pourable and commonly are formulated as bars or sticks, or in particulate form; solids can be opaque or transparent, and optionally can contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product.
• Creams and lotions are often similar to one another, differing mainly in their viscosity; both lotions and creams may be opaque, translucent or clear and often contain emulsifiers, solvents, and viscosity adjusting agents, as well as moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product.
• Gels can be prepared with a range of viscosities, from thick or high viscosity to thin or low viscosity.
• These formulations may also contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product.
• Liquids are thinner than creams, lotions, or gels and often do not contain emulsifiers.
• Liquid topical products often contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product.
• Suitable emulsifiers for use in topical formulations include, but are not limited to, ionic emulsifiers, cetearyl alcohol, non-ionic emulsifiers like polyoxyethylene oleyl ether, PEG-40 stearate, ceteareth-12, ceteareth-20, ceteareth-30, ceteareth alcohol, PEG-100 stearate and glyceryl stearate.
• Suitable viscosity adjusting agents include, but are not limited to, protective colloids or non-ionic gums such as hydroxyethylcellulose, xanthan gum, magnesium aluminum silicate, silica, microcrystalline wax, beeswax, paraffin, and cetyl palmitate.
• a gel composition may be formed by the addition of a gelling agent such as chitosan, methyl cellulose, ethyl cellulose, polyvinyl alcohol, polyquaterniums, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carbomer or ammoniated glycyrrhizinate.
• a gelling agent such as chitosan, methyl cellulose, ethyl cellulose, polyvinyl alcohol, polyquaterniums, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carbomer or ammoniated glycyrrhizinate.
• Suitable surfactants include, but are not limited to, nonionic, amphoteric, ionic and anionic surfactants.
• dimethicone copolyol polysorbate 20
• polysorbate 40 polysorbate 60
• polysorbate 80 lauramide DEA, cocamide DEA, and cocamide MEA
• oleyl betaine cocamidopropyl phosphatidyl PG-dimonium chloride
• ammonium laureth sulfate may be used within al formulations.
• Suitable preservatives include, but are not limited to, antimicrobials such as methylparaben, propylparaben, sorbic acid, benzoic acid, and formaldehyde, as well as physical stabilizers and antioxidants such as vitamin E, sodium ascorbate/ascorbic acid and propyl gallate.
• Suitable moisturizers include, but are not limited to, lactic acid and other hydroxy acids and their salts, glycerin, propylene glycol, and butylene glycol.
• Suitable emollients include lanolin alcohol, lanolin, lanolin derivatives, cholesterol, petrolatum, isostearyl neopentanoate and mineral oils.
• Suitable fragrances and colors include, but are not limited to, FD&C Red No. 40 and FD&C Yellow No. 5.
• Other suitable additional ingredients that may be included in a topical formulation include, but are not limited to, abrasives, absorbents, anti-caking agents, anti-foaming agents, anti-static agents, astringents such as, e.g., witch hazel, alcohol and herbal extracts such as chamomile extract, binders/excipients, buffering agents, chelating agents, film forming agents, conditioning agents, propellants, opacifying agents, pH adjusters and protectants.
• An example of a suitable topical vehicle for formulation of a gel is: hydroxypropylcellulose (2.1%); 70/30 isopropyl alcohol/water (90.9%); propylene glycol (5.1%); and Polysorbate 80 (1.9%).
• An example of a suitable topical vehicle for formulation as a foam is: cetyl alcohol (1.1%); stearyl alcohol (0.5%); Quaternium 52 (1.0%); propylene glycol (2.0%); Ethanol 95 PGF3 (61.05%); deionized water (30.05%); P75 hydrocarbon propellant (4.30%). All percents are by weight.
• Typical modes of delivery for topical compositions include application using the fingers; application using a physical applicator such as a cloth, tissue, swab, stick or brush; spraying including mist, aerosol or foam spraying; dropper application; sprinkling; soaking; and rinsing.
• Controlled release vehicles can also be used, and compositions may be formulated for transdermal administration as a transdermal patch.
• a pharmaceutical composition may be formulated as inhaled formulations, including sprays, mists, or aerosols. Such formulations are particularly useful for the treatment of asthma or other respiratory conditions.
• the compounds provided herein may be delivered via any inhalation m ds known to those skilled in the art.
• inhalation methods and devices include, but are not limited to, metered dose inhalers with propellants such as CFC or HFA or propellants that are physiologically and environmentally acceptable.
• propellants such as CFC or HFA or propellants that are physiologically and environmentally acceptable.
• Other suitable devices are breath operated inhalers, multidose dry powder inhalers and aerosol nebulizers.
• Aerosol formulations for use in the subject method typically include propellants, surfactants and co-solvents and may be filled into conventional aerosol containers that are closed by a suitable metering valve.
• Inhalant compositions may comprise liquid or powdered compositions containing the active ingredient that are suitable for nebulization and intrabronchial use, or aerosol compositions administered via an aerosol unit dispensing metered doses.
• Suitable liquid compositions comprise the active ingredient in an aqueous, pharmaceutically acceptable inhalant solvent, e.g., isotonic saline or bacteriostatic water.
• the solutions are administered by means of a pump or squeeze-actuated nebulized spray dispenser, or by any other conventional means for causing or enabling the requisite dosage amount of the liquid composition to be inhaled into the patient's lungs.
• Suitable formulations, wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient.
• Formulations or compositions suitable for nasal administration include a coarse powder having a particle size, for example, in the range of 20 to SOO microns which is administered in the manner in which snuff is administered, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
• Suitable powder compositions include, by way of illustration, powdered preparations of the active ingredient thoroughly intermixed with lactose or other inert powders acceptable for intrabronchial administration.
• the powder compositions can be administered via an aerosol dispenser or encased in a breakable capsule which may be inserted by the patient into a device that punctures the capsule and blows the powder out in a steady stream suitable for inhalation.
• compositions may also be prepared in the form of suppositories such as e.g., for rectal administration.
• Such compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the re m to release the drug.
• suitable excipients include, for example, cocoa butter and polyethylene glycols.
• compositions may be formulated as sustained release formulations such as, i.e., a formulation such as a capsule that creates a slow release of modulator following administration.
• sustained release formulations such as, i.e., a formulation such as a capsule that creates a slow release of modulator following administration.
• Such formulations may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site.
• Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of modulator release.
• the amount of modulator contained within a sustained release formulation depends upon, for example, the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.
• the dose of the biologically active compound according to the invention may vary within wide limits and may be adjusted to individual requirements.
• Active compounds according to the present invention are generally administered in a therapeutically effective amount. Preferred doses range from about 0.1 mg to about 140 mg per kilogram of body weight per day, about
• the daily dose may be administered as a single dose or in a plurality of doses.
• the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Dosage unit forms will generally contain between from about 1 mg to about 500 mg of an active ingredient.
• the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination, i.e. other drugs being used to treat the patient, and the severity of the particular disease undergoing therapy.
• Preferred compounds of the invention will have certain pharmacological properties. Such properties include, but are not limited to oral bioavailability, such that the preferred oral dosage forms discussed above can provide ther tically effective levels of the compound in vivo.
• BlR antagonists of formula (I) according to the present invention may be used preferably as antagonists of BlR in a variety of applications, both in vitro and in vivo.
• BlR antagonists according to the present invention may be used to inhibit the binding of BK BlR ligands such as, e.g., DAKD, to BlR in vitro or in vivo.
• BK BlR antagonists) provided herein are preferably administered to a patient such as, e.g., a human, orally or topically, and are present within at least one body fluid or tissue of the patient while modulating BK BlR activity. Accordingly, the present invention further provides methods for treating patients suffering from conditions responsive to BlR modulation as mentioned above.
• treatment encompasses both disease-modifying treatment and symptomatic treatment, either of which may be prophylactic, i.e., before the onset of symptoms, in order to prevent, delay or reduce the severity of symptoms, or therapeutic, i.e., after the onset of symptoms, in order to reduce the severity and/or duration of symptoms.
• a condition is "responsive to BlR modulation” if modulation of BlR activity results in alleviation or curing of the condition or a symptom thereof.
• Patients may include but are not limited to primates, especially humans, domesticated companion animals such as dogs, cats, horses, and livestock such as cattle, pigs, sheep, with dosages as described herein.
• the Bl antagonists according to the present invention are effective in the treatment and/or prevention of a condition or a disease responsive to BK BlR modulation as is apparent from the prior art and in particular from references recited herein.
• Animal models of these diseases are generally well known in the art and may be used to evaluating compounds of the present invention for their potential utilities.
• the compounds according to the present invention are also useful in diagnosis of such diseases and other applications including, but not limited to, their use as research tools in vivo and in vitro.
• Compounds of this invention are antagonists of the BK BlR and as such are useful in the treatment and prevention of diseases and conditions mediated through the BK receptor pathway such as inflammation, immunology disorders and pain.
• the compounds are preferably effective in the treatment or prevent f inflammation such as, but not limited to, persistent or chronic inflammatory diseases, immunology disorders, autoimmune diseases, neurogenic inflammation, inflammation associated edema and fibrosis. This includes but is not limited to gastrointestinal inflammation, septic shock, diseases of the skin, diseases of the respiratory pathway and vasculopathies.
• the compounds are preferably also effective in the treatment or prevention of pain such as chronic pain, inflammatory pain, visceral pain and neuropathic pain. This includes but is not limited to complex regional pain syndrome (CRPS).
• CRPS complex regional pain syndrome
• the compounds according to the invention are used as or for the manufacture of a diagnostic agent, whereby such diagnostic agent is for the diagnosis of the diseases and conditions which can be addressed by the compounds of the present invention for therapeutic purposes as disclosed herein.
• inflammatory diseases preferably encompasses, but is not limited to, disorders such as acute-phase reaction, local and systemic inflammation and inflammation caused by other diseases whatever type, etiology or pathogenesis and caused by those inflammatory diseases specified within this application.
• immunosuppression disorders preferably encompasses, but is not limited to, disorders such as hyperesthesia, autoimmune disorders, graft rejection in transplantation, transplant toxicity, granulomatous inflammation / tissue remodelling, myasthenia gravis, immunosuppression, immune-complex diseases, over- and underproduction of antibodies and vasculitis.
• the Bl antagonists according to the present invention are preferably effective in the treatment/prevention of a condition or a disea sponsive to BK BlR modulation as is i.a. apparent from the references cited herein. Accordingly, the compounds according to the invention are effective in the treatment/prevention of inflammatory diseases such as but not limited to inflammatory bowel disease including Crohn's disease and ulcerative colitis (Stadnicki et al, Am. J. Physiol. Gastrointest Liver Physiol. 2005, 289(2), G361-6; Devani et al, Am. J.
• the compounds are effective in joint and bone diseases such as juvenile or adult onset rheumatoid arthritis and gouty arthritis (Cassim et al, Pharmacol. Ther. 2002, 94, 1-34; Sharma et al, Exp. Toxic Pathol. 1994, 46, 421-433; Brechter et al, Arthr. Rheum. 2007, 56(3), 910-923), ankylosing spondylitis, adult onset or pediatric onset like systemic onset juvenile idiopathic arthritis, Still's disease, psoriatic arthritis and osteoarthritis.
• the compounds are also effective in edema associated with burns, sprains or fracture, cerebral edema, closed head injury and angioedema.
• diabetic vasculopathy may be used to treat immunology disorders like diabetic vasculopathy, type I diabetes, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, diabetic vasculopathy, post capillary resistance or diabetic syndromes associated with insulits such as, e.g. , hyperglycemia, diuresis, proteinuria and increased nitrite and kallikrein urinary excretion. Additionally they may be used in CNS disorders like multiple sclerosis, epilepsy, amyotrophic lateral sclerosis, Alzheimer's disease, stroke, Parkinson's disease.
• immunology disorders like diabetic vasculopathy, type I diabetes, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, diabetic vasculopathy, post capillary resistance or diabetic syndromes associated with insulits such as, e.g. , hyperglycemia, diuresis, proteinuria and increased nitrite and kallikrein urinary excretion.
• cardiovascular disease such as congestive heart failure, myocardial infarct, systemic inflammatory response syndrome (SIRS), ischemia-reperfusion injury and atherosclerosis (Raidoo et al, Immunopharmacol 1997, 36(2-3), 153-60; McLean etal, Cardiovasc. Res. 2000, 48, 194-210).
• SIRS systemic inflammatory response syndrome
• ischemia-reperfusion injury and atherosclerosis Rosclerosis
• They may also be used in vascular disorders like vasculitis, septic shock, antihypovolemic and/or anti- hypotensive agents, closed head trauma, cancer, sepsis, gingivitis, osteoporosis, benign prostatic hyperplasia and hyperactive bladder.
• the compounds may be used to treat f ⁇ brotic diseases such as but not limited to pulmonary fibrosis, renal fibrosis, liver fibrosis, progressive sclerosis and recurrent stricture formation in Crohn's disease (Goldstein et al, J. Biol. Chem. 1984, 259(14), 9263-8; pero et al, J. Biol. Chem. 2000, 275(17), 12475-80; Romero et al., J. Biol. Chem. 2005, 15, 14378-14384).
• f ⁇ brotic diseases such as but not limited to pulmonary fibrosis, renal fibrosis, liver fibrosis, progressive sclerosis and recurrent stricture formation in Crohn's disease (Goldstein et al, J. Biol. Chem. 1984, 259(14), 9263-8; pero et al, J. Biol. Chem. 2000, 275(17), 12475-80; Romero et al., J
• asthma atopic or non- atopic asthma
• occupational asthma exercise-induced bronchoconstriction
• bronchitis pneumoconiosis including aluminosis, anthracosis, asbestosis, chalicosis, ptilosis, siderosis, silicosis, tabaccosis and byssinosis
• chronic obstructive pulmonary disease including emphysema, adult respiratory distress syndrome, pneumonia, allergic rhinitis, vasomotor rhinitis and pleurisy.
• FMF familial Mediterranean fever
• TRAPS tumor-necrosis factor receptor associated periodic syndrome
• NOMID neonatal onset multisystem inflammatory disease
• FCAS familial cold autoinflammatory syndrome
• FCU familial cold urticaria
• PAPA pyogenic arthritis pyoderma gangrenosum acne
• pain preferably encompasses, but is not limited to, centrally and peripherally mediated pain, vascular pain, visceral pain, inflammatory mediated pain, neural eic pain, referred pain, nociceptive pain, reflectory pain, psychosomatic pain, acute pain such as caused by acute injury, trauma or surgery of bones, muscle, tissue, soft tissue, organs, pain after insectbites, post-stroke pain syndrome, post-surgery pain, progressive disease related pain and chronic pain (Calixto et al., Br. J. Pharmacol. 2004, 143, 803-818; Chen et al., Expert Opin. Ther. Targets 2007, 77(1), 21-35; Porreca et al., J. Pharmacol. Exp.
• the compounds according to the present invention are preferably also effective in the treatment and/or prevention of pain including but not limited to inflammatory pain of various origins such as rheumatoid arthritis or gout, v al pain as pancreatitis, interstitial cystitis, renal or gall bladder colic, neuropathic pain as postherpetic neuralgia, complex regional pain syndrome, phantom limb pain, root avulsions, trigeminal neuralgia, painful traumatic mononeuropathy, painful polyneuropathy, vulvodynia, central pain syndromes potentially caused by any lesion at any level of the peripheral and/or central nervous system, postsurgical pain syndromes as postmastectomy syndrome, bone and joint pain, repetitive motion pain, dental pain, cancer pain, myofascial pain as muscular injury and fibromyalgia, perioperative pain as from general surgery, chronic pain, dysmennorhea as well as pain associated with angina. Additionally these compounds are preferably effective against back pain, headache, cluster headache, migraine including pro
• the present invention also provides methods for localizing or detecting a BlR in a tissue, preferably a tissue section, which methods involve contacting the tissue sample presumably containing BlR with a detectably labelled compound according to the present invention under conditions that permit binding of the compound to the BlR and detecting the bound compound.
• Such methods and their respective conditions are known to those skilled in the art and include, for example, the radioligand binding assay disclosed in Example 24.
• the present invention also provides methods of inhibiting the binding of DAKD or any other BlR ligand to a BlR which methods involve contacting a solution containing a BlR antagonist compound disclosed herein with cells expressing BlR under conditions and in an amount sufficient to detectably inhibit binding of DAKD or any other substance to BlR.
• Such methods and their respective conditions are known to those skilled in the art and include, for example, the calcium mobilization assay disclosed in Example 25.
• the compounds according to the invention are used as or for the manufacture of a diagnostic agent, whereby such diagnostic agent is for the diagnosis of the diseases and conditions which can be addressed by the compounds of the present invention for therapeutic purposes as d sed herein.
• the compounds of the invention can be labelled by isotopes, fluorescence or luminescence markers, antibodies or antibody fragments, any other affinity label like nanobodies, aptamers, peptides etc., enzymes or enzyme substrates.
• These labelled compounds of this invention are useful for mapping the location of BK receptors in vivo, ex vivo, in vitro and in situ such as, e.g. in tissue sections via autoradiography and as radiotracers for positron emission tomography (PET) imaging, single photon emission computerized tomography (SPECT) and the like to characterize those receptors in living subjects or other materials.
• PET positron emission tomography
• SPECT single photon emission computerized tomography
• the labelled compounds according to the present invention may be used in therapy, diagnosis and other applications such as research tools in vivo and in vitro, in particular the applications disclosed herein.
• the compounds of formula (I) according to the present invention have improved properties when compared to BlR antagonists known in the state of the art, especially, improved selectivity, low toxicity, low drug drug interaction, improved bioavailability especially with regard to oral administration, improved metabolic stability, improved stability in microsomal degradation assay, and improved solubility.
• ACN is acetonitrile
• AgNC> 3 is silver nitrate
• BlR is Bl receptor
• BK is bradykinin
• BSA bovine serum albumin cone, is concentrated
• DAKD des-Arg-10-kallidin
• DCM dichloromethane
• EA diisopropyethylamine
• DMAP 4-R ⁇ .V-dimethylaminopyridine
• S DMEM Dulbecco's modified Eagle's medium
• DMF is ⁇ /-dimethylformamide
• DMSO dimethylsulfoxide
• EA is ethyl acetate
• FBS is fetal bovine serum 0
• FCS is fetal calf serum h is hour
• HATU 2-(l//-7-azaben2otriazol-l-yl)-l,l,3,3-tetramethyl uranium hexafluorophosphate
• HCl hydrochloric acid
• S HEK is human embryonic kidney
• HEPES is 4-2-hydroxyethyl-l-piperazineethanesulfonic acid
• HOBt is N-hydroxybenzotriazole
• HPLC high performance liquid chromatography
• K 2 CO 3 is potassium carbonate IL is interleukine
• LiOH is lithium hydroxide
• NaCl sodium chloride
• Na 2 C ⁇ 3 is sodium carbonate
• Na 2 S 2 Oa is sodium thiosulfate
• NaOH is sodium hydroxide
• NaHC ⁇ 3 sodium hydrogencarbonate
• NH 4 CI ammonium chloride
• NH 4 OH ammonia
• Pd(OAc) 2 palladium (II) acetate
• PBS is phosphate-buffered saline
• PIPES is piperazine-M_V'-bis(2-ethanesulfonic acid)
• RP is reverse phase
• RT is room temperature sat. is saturated
• tBu is tert. butyl
• TFA is trifluoroacetate or trifluoroacetic acid
• THF is tetrahydrofuran
• WSC is (3-dime ⁇ ylamino-propyl)-e ⁇ yl-carbodiimide hydrochloride
• Solvents Solvents were used in the specified quality without further purification. Acetonitrile
• RP-HPLC-MS analyses For analytic chromatography a Hewlett Packard 1100-system (degasser G 1322 A, quaternary pump G1311A, automatic sample changer G1313A, column heater G 1316A, variable UV detector G1314A) together with an ESI-MS (Finnigan LCQ ion trap mass spectrometer) was used. The system was controlled by "navigator version 1.1 spl" software (Finnigan). As impact gas in the ion trap helium was used.
• the compounds of the present invention can be prepared in a number of ways well known to one skilled in the art of organic synthesis.
• the compounds of the present invention can be synthesized using the methods described below, together with synthetic methods known in the art of synthetic organic chemistry, or variations thereon as appreciated by those skilled in the art. Preferred methods include but are not limited to those methods described below.
• Each of the references cited below are hereby incorporated herein by reference.
• Reaction B was performed based upon literature (Gauuan et al, Bioorg. Med. Chem. 2002, 70, 3013-3021)
• Methanesulfonyl chloride 120 ⁇ L, 1.55 mmol was dissolved in 500 ⁇ L DCM and then added dropwise at O 0 C to a stirred solution of (2?, ⁇ -3-fluoro-4-(l-hydroxy-ethyl)-benzoic acid methyl ester (154 mg, 0.78 mmol), DMAP (4.7 mg, 0.04 mmol) and triethylamine (1.08 mL, 7.77 mmol) in DCM (4 mL). After stirring at 0 0 C for 30 minutes, 10 mL water were added and the mixture was extracted with DCM (3 x 50 mL).
• Dess-Martin reagent (207 mg, 0.49 mmol) was added to a stirring solution of 6- hydroxymethyl-nicotinic acid methyl ester (55 mg, 0.33 mmol) in DCM (17 mL). After stirring for 3 h the solvent was removed in vacuo. The residue was purified by flash chromatography on silica gel (elution with n-hexane/EA 4:1) to give the title compound.
• 6-tert-Butoxycarbonylamino-nicotinic acid methyl ester 6-Amino-nicotinic acid methyl ester (1.0 6.S7 mmol) was added portionwise to a stirring solution of di-fer/-butyl dicarbonate (1.72 g, 7.89 mmol) in terf-butanol (20 mL).
• Trimethylsilyl chloride (3.14 mL, 24.8 mmol) was added to a solution of (R)-4-(l-amino- ethyl)-benzoic acid (2.00 g, 9.92 mmol) in DCM (50 mL). The mixture was heated to reflux for 2 h then cooled to RT and triethylamine (5.53 mL, 39.7 mmol) was added dropwise followed by 9-fluorenylmethyl chlorofo ⁇ nate (2.82 g, 10.9 mmol). The reaction mixture was stirred at RT overnight and then concentrated in vacuo. The residue was stirred in sat. sodium carbonate solution and acidified with 2 N hydrochloric acid.
• the resin was filtered and washed with DCM/methanol/DIPEA (16:3:3, 1 x 10 mL) and DMF (3 x 10 mL). Piperidin (25% solution in DMF, 10 mL) was added and the mixture was shaken for 20 minutes. The resin was filtered, washed with DMF (6 x 10 mL) and dried in vacuo to give the title compound with an expected loading of 45% (0.65 mmol/g).
• Resin-bound (R)-4-[l-(2-an ⁇ ino-2-cyclopropyl-acetylamino)-ethyl]-benzoic acid (58.7 mg, 0.038 mmol) was reacted with 3-fluoro- fluoromethylbenzoic acid (15.8 mg, 0.076 mmol) according to the synthesis of 4- ⁇ l-[2-(9//-fluoren-9-ylmethoxycarbonyIaniino)-2- methyl-propionylamino]-ethyl ⁇ -benzoic acid.
• the organic layer was dried over Na 2 SO 4 , filtered and concentrated in vacuo.
• the residue a mix of 4-bromomethyl-3-chloro-benzoic acid methyl ester and 4-dibromomethyl-3-chloro-benzoic acid methyl ester, was used in the following step without further purification.
• the residue (1.48 g, 5.61 mmol) was dissolved in acetone (45 mL) and water (9 mL) and AgNO 3 (3.22 g, 16.3 mmol) were added.
• the flask was covered with aluminum foil to avoid decomposition of the AgNU 3 .
• the mixture was stirred at RT overnight.
• Lithium aluminium hydride (10.2 g, 0.27 mol) was suspended in anhydrous THF (450 mL). To this mixture L-Alanine (12.0 g, 0.13 mol) was added portionwise at 0°C. The mixture was refluxed overnight and then cooled to 0°C. 2 N NaOH solution (70 mL) was added. After stirring at RT for 3 h, the mixture was filtered and the solids were washed with THF. The solids were suspended in THF (250 mL) and the mixture was refluxed for 1 h. The solution was filtered and the solids were washed with THF. This procedure was repeated twice.
• Reaction E was performed based on literature (Harding et al, J.Org.Chem. 1981, 46, 3920-3922)
• Hydrazinecarboxylic acid 9/- r -fluoren-9-ylmethyl ester 150 mg, 0.59 mmol was suspended in DCM (5 mL) and sat. NaHCU 3 solution (5 mL). The biphasic mixture was stirred at 0 0 C for 5 minutes. The two layers were allowed to separate and phosgene (20% solution in toluene, 1.3 mL, 2.95 mmol) was added via syringe to the organic phase. The mixture was stirred at 0 0 C for 10 minutes, the layers were separated and the aqueous layer was extracted with DCM (3 x 10 mL).
• Reaction A and the first step of reaction B were performed based on mechanical and mechanical properties (Gibson et al, J. Org.Chem. 1999, 64, 7388-7394)
• Methylhydrazine (173 ⁇ l, 3.26 mmol) was added at -78 0 C to a stirring solution ofdi-tert- butyl dicarbonate (924 mg, 4.23 mmol) in DCM (5 mL). The solution was allowed to warm to RT and stirred at RT for 30 minutes. 9/- r -Fluore methyl chloroformate (842 mg, 3.26 mmol) and ethyl-diisopropyl-amine (664 ⁇ l, 3.91 mmol) were added dropwise and the mixture was stirred at RT for 8 h. TFA (S ml) was added and the mixture stirred for additional 2 h.
• the crude product (68.5 mg, 0.207 mmol) was dissolved in DCM (1 mL) and added to a suspension of resin-bound (R)-4-(l-amino- ethyl)-benzoic acid (106 mg, 0.069 mmol), swollen in DCM (5 mL) and collidine (136 ⁇ L, 1.035 mmol). The mixture was shaken 90 minutes at RT. The resin was filtered and washed with DMF (4 x 2 mL), methanol (4 x 2 mL) and DCM (4 x 2 mL).
• the resin was filtered, washed with DMF (4 x 2 mL), methanol (4 x 2 mL) and DCM (4 x 2 mL) and a solution of 3- fluoro-5-trifluoromethyl-benzoic acid (17.2 mg, 0.083 mmol), HATU (31.5 mg, 0.083 mmol) and collidine (45.4 ⁇ L, 0.345 mmol) in DCM/DMF 2:1 (1.5 ml) was added. The mixture was shaken 4 h at RT. The resin was filtered, washed with DMF (4 x 2 mL), methanol (4 x 2 mL) and DCM (4 x 2 mL) and suspended in DCM (1 mL) and TFA (1 mL).
• Methyl 3-chloro-4-((lR)-l-(l,l-dimethylethylsulfinamido)ethyl)benzoate Methylmagnesium chloride solution (3.4 ml, 9.75 mmol, 22% in THF) was added slowly to a solution of methyl 4-((terNbutylsulfinyIimino)methyl)-3-chlorobenzoate (1.73 g, 5.73 mmol) in DCM (30 mL) at -55°C. The reaction mixture was quenched with brine after stirring for 1 h at -55°C. Celite was added and the suspension was filtered. The layers were separated and the aq. layer was extracted with DCM.
• Dimethyl 3-chloropyridine-2,5-dicarboxylate was synthesized according to literature (Hendrickson et al. Org. Lett. 2004, 3-5). Pyridine-2,5-dicarboxylic acid (10.0 g, 60.0 mmol) was suspended in 300 mL 0.2% (w/w) aq. Na 2 WO 4 (0.6 g). To this solution was added H 2 O 2 in water (30% w/w, 34 mL, 315 mmol). The resulting mixture was stirred and heated at 80-85 0 C for 10 hours. The resulting solid was collected by filtration and washed with cold water. Drying the material under high vacuum overnight yielded pyridine-2,5-dicarboxylic a ⁇ f-oxide, which was used in the next step without further purification.
• Methyl 5-chloro-6-((R)- 1 -((R)- 1 , 1 -dimethylethylsulfinamido)ethyl)nicotinate Methyl magnesium chloride (22% in THF, 2.1 mL, 6.0 mmol) was added dropwise at - 60 0 C to a stirred solution of (R)-methyl 6-((/er/-butylsulfinylimino)methyl)-5- chloronicotinate (1.2 g, 4.0 mmol) in DCM (30 mL). After stirring for 30 min at -60 0 C, sat. ammonium chloride solution (40 mL) was added.
• the crude product (68.5 mg, 0.207 mmol) was dissolved in DCM (1 mL) and added to a suspension of resin-bound (R)-4-(l-aminoethyl)-3-fluorobenzoic acid (106 mg, 0.069 mmol), swollen in DCM (5 mL) and collidine (136 ⁇ L, 1.035 mmol). The mixture was shaken 90 minutes at RT. The resin was filtered and washed with DMF (4 x 2 mL), methanol (4 x 2 mL) and DCM (4 x 2 mL).
• the resin was filtered, washed with DMF (4 x 2 mL), methanol (4 x 2 mL) and DCM (4 x 2 mL) and a solution of 3-fluoro-5-trifluoromethyl-benzoic acid (17.2 mg, 0.083mol), HATU (31.5 mg, 0.083 mmol) and collidine (45.4 ⁇ L, 0.345 mmol) in DCM/DMF 2:1 (1.5 ml) was added. The mixture was shaken 4 h at RT. The resin was filtered, washed with DMF (4 x 2 mL), methanol (4 x 2 mL) and DCM (4 x 2 mL) and suspended in DCM (1 mL) and TFA (1 mL).
• Radioligand binding assays were performed using HEK923 cells that can be induced by tetracycline to express the human, rabbit, mouse, dog, pig or rat Bl receptor.
• the genes for the BK BlR of human, rat, mouse, rabbit, dog, and pig were generated synthetically (GENEART, Regensburg) using a gene design and a codon usage optimized for stable expression in human cells. All receptor genes were stably and isogenically expressed by insertion into an identical position in the genome by using the FIp-In system from Invitrogen with the host cell line FIp-In T-REx HEK293 (human embryonic kidney) together with either the pcDNA5/FRT/TO-vector (for stable but tetracyclin-inducible expression). The latter was used in particular for all BlR subtypes, to avoid problems with a potential constitutive activity of these receptors that may prohibit the growth and selection of stably expressing clones.
• the respective HEK293 cells bearing the respective transgene were cultured in DMEM high glucose medium supplemented with 10% FCS. At 80% confluence cells were harvested from culture flasks by trypsinization and resuspended in DMEM 10% FCS containing 5 ⁇ g/ mL tetracycline. Cells were seeded in poly-lysine coated flat bottom 96 well plates (TPP) at appr. 80.000 cells/well and used in the binding assay after an overnight incubation at 37°C.
• TPP poly-lysine coated flat bottom 96 well plates
• test compounds were assayed at 10 different concentrations diluted in half-log steps in duplicate wells.
• Compounds were diluted in assay binding buffer (4OmM PIPES, 109 mM NaCl, 5mM KCl, 0.1% Glucose, 0.05% BSA, 2mM CaCl 2 , 1 mM MgCl 2 , 60 mM NaOH; pH 7.4) containing the protease inhibitors Captopril (lOO ⁇ M), 1,10-Phenanthroline (20 ⁇ M) and Bacitracin (500 ⁇ M).
• InM 3H-DAKD Perkin Elmer
• the antagonist compound dilutions were prepared in a dilution plate which also included controls to assess total binding (1 nM 3H-DAKD) and non-specific binding (1 nM 3H DAKD + 10 mM DAKD).
• 100 ⁇ L from the dilution plate was added to the cell plate and incubated for 90 minutes on ice. After that supernatants were aspirated and the plates were washed 4x with icecold PBS followed by the addition of 200 ⁇ L dissociation buffer (0.5 M Cl, 0.2 M acetic acid) for 10 minutes on ice.
• the potency and efficacy of the compounds in this invention to antagonize the BK BlR was determined in a cell-based fluorescent calcium-mobilization assay.
• the assay measures the ability of test compounds to inhibit BK BlR agonist-induced increase of intracellular free Calcium in different cell lines. Endogenous BlR expression was induced by IL-I beta pretreatment of human embryonal lung fibroblasts IMR-90. BlR of several non-human species (rat, mouse, rabbit, dog and pig) were expressed recombinantly after Tetracycline induction in HEK 293 cells bearing the respective transgene.
• BlR agonist-induced calcium mobilization was monitored using the FlexStation fluorescence imaging plate reader platform.
• IMR-90 human embryonal lung fibroblast cells (ATCC# CCL 186) were cultured in
• test compounds were assayed at 7 concentrations in duplicate wells.
• Compound addition plates contain 5x final concentrations of test compounds or controls in 5% DMSO. The test compounds were added in 5 ⁇ L followed by a 10 minute equilibration phase at 37°C in an incubator. Plates were then placed in the FlexStation II unit (Molecular Devices) which was set to 37 0 C.

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