WO2009036996A2 - Small molecule bradykinin b1 receptor antagonists - Google Patents

Small molecule bradykinin b1 receptor antagonists Download PDF

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Publication number
WO2009036996A2
WO2009036996A2 PCT/EP2008/007919 EP2008007919W WO2009036996A2 WO 2009036996 A2 WO2009036996 A2 WO 2009036996A2 EP 2008007919 W EP2008007919 W EP 2008007919W WO 2009036996 A2 WO2009036996 A2 WO 2009036996A2
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Prior art keywords
optionally substituted
hydrogen atom
alkyl
mmol
compound
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PCT/EP2008/007919
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French (fr)
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WO2009036996A3 (en
Inventor
Elsa Locardi
Klaus Dinkel
Marco Schaudt
Uwe Richter
Dirk Scharn
Gerd Hummel
Ulrich Reineke
Ulf Reimer
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Jerini Ag
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Priority to JP2010525265A priority Critical patent/JP2010539204A/en
Priority to AU2008300822A priority patent/AU2008300822A1/en
Priority to EP08802425A priority patent/EP2195288A2/en
Priority to CA2700051A priority patent/CA2700051A1/en
Publication of WO2009036996A2 publication Critical patent/WO2009036996A2/en
Publication of WO2009036996A3 publication Critical patent/WO2009036996A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • 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
    • 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
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • 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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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • 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
    • C07D207/18—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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    • C07D213/00—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
    • C07D213/04—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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.

Abstract

Disclosed are compounds of formula (I) which are bradykinin B1 receptor (B1R) antagonists. These compounds are useful to treat diseases or relieve adverse symptoms associated with inflammation and pain. The invention encompasses novel compounds and acceptable derivatives thereof, pharmaceutical compositions and methods for prophylaxis and treatment of diseases involving inflammation and pain.

Description

Small molecule bradykinin Bl receptor antagonists
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.
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). This makes the BlR a particularly attractive drug target. 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).
Numerous peptide and non-peptide antagonists of BlR have been described in the prior art as novel therapeutics for the treatment of pain and inflammation (Chen et al., Expert Opin.
Ther. Targets 2007, 11, 21-35). For instance, WO03066577, WO03065789, WO05016886, WO04019868, US20060122236 disclose biphenyl compounds and WO9725315 discloses sulphonamide compounds that are BlR antagonists. In view of the severe conditions associated with overshooting and thus pathological inflammation and pain, both acute and chronic, there is a need for novel compounds that are effective in selectively blocking activation of Bl receptors.
Therefore, 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.
In a first embodiment of the first aspect the problem underlying the instant application is solved by a compound of the formula (I):
Figure imgf000003_0001
or a pharmacologically acceptable salt, solvate or hydrate thereof, wherein
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
Figure imgf000004_0001
wherein
W is N, alkyl, heteroalkyl, alkenyl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R4, if present, 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 R5 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;
R5, if present, 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 R4 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; E, if present, is a heteroalkyl or a heteroaralkyl;
G, if present, is an alkyl, a heteroalkyl, a cycloalkyl, a heterocycloalkyl, a alkylcycloalkyl, a heterocycloalkylalkyl, a heteroaryl, or a heteroaralkyl
K is
Figure imgf000005_0001
wherein
R1 and R2 are each independently selected from C2-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C2-C6heteroalkyl, or form, together with N, an optionally substituted heterocycloalkyl, an optionally substituted heteroaryl, or an optionally substituted heteroaralkyl; and
R3 is a hydrogen atom, C1- or C2alkyl, cyano, or a halogen atom.
In a second embodiment of the first aspect which is also an embodiment of the first embodiment of the first aspect A is
Figure imgf000005_0002
wherein X1, X2, and X3 are each independently selected from N, O, S, NRX, CRX, or CRXRX', wherein
Rx and Rx are each independently selected from hydrogen atom, halogen atom, =0, hydroxy, cyano, amino, nitro, mercapto, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl.
In a third embodiment of the first aspect which is also an embodiment of the first or the second embodiment of the first aspect A is selected from
Figure imgf000006_0001
wherein
Rx, Rxl, R"2, R*3, Rx>, Rxl>, R"2', and R"3' are each independently selected from hydrogen atom, halogen atom, =0, hydroxy, cyano, amino, nitro, mercapto, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and
X is NH, O, or S. In a fourth embodiment of the first aspect which is also an embodiment of the first embodiment of the first aspect A is
Figure imgf000007_0001
wherein Y1, Y2, Y3 and Y4 are each independently selected from N, O, S, NRy, CRy, or CRyRy', wherein
Ry and Ry are each independently selected from hydrogen atom, halogen atom, =0, hydroxy, cyano, amino, nitro, mercapto, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl.
In a fifth embodiment of the first aspect which is also an embodiment of the first embodiment or the fourth embodiment of the first aspect A is selected from
Figure imgf000008_0001
wherein
Ry, Ryl, Ry2, Ry3, Ry4, Ry>, Ryl >, Ry2', Ry3>, and Ry4' are each independently selected from hydrogen atom, halogen atom, =0, hydroxy, cyano, amino, nitro, mercapto, alkyl, alkeπyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl. In 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
Figure imgf000009_0001
wherein
Ryl, R5^, Ry3 and Ry4 are each independently selected from hydrogen atom, halogen atom, =0, hydroxy, cyano, amino, nitro, mercapto, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl.
In a seventh embodiment of the first aspect which is also an embodiment of the sixth embodiment of the first aspect
Ryl is a hydrogen atom.
In a eigth embodiment of the first aspect which is also an embodiment of the sixth or seventh embodiment of the first aspect
R^ is a hydrogen atom, halogen atom, or C1-C6alkyl; and
Ry4 is a hydrogen atom or halogen atom.
In a ninth embodiment of the first aspect which is also an embodiment of the sixth to eigth embodiment of the first aspect R5* is a hydrogen atom. In a tenth embodiment of the first aspect which is also an embodiment of the sixth to ninth embodiment of the first aspect Ry3 is a hydrogen atom or halogen atom.
In a eleventh embodiment of the first aspect which is also an embodiment of the sixth to tenth embodiment of the first aspect Ry4 is a hydrogen atom or halogen atom.
In a twelfth embodiment of the first aspect which is also an embodiment of the first to eleventh embodiment of the first aspect T is a hydrogen atom.
In a 13th embodiment of the first aspect which is also an embodiment of the first to twelfth embodiment of the first aspect K is selected from
Figure imgf000010_0001
In a 14th embodiment of the first aspect which is also an embodiment of the first to 13th embodiment of the first aspect K is selected from
Figure imgf000011_0001
In a 15th embodiment of the first aspect which is also an embodiment of the first to 14th embodiment of the first aspect K is
Figure imgf000011_0003
In a 16th embodiment of the first aspect which is also an embodiment of the first to 14th embodiment of the first aspect K is
Figure imgf000011_0004
In a 17th embodiment of the first aspect which is also an embodiment of the first to 16th embodiment of the first aspect B is heteroalkyl, heteroaryl, or
Figure imgf000011_0002
wherein
W is N, alkyl, alkenyl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl;
R4, if present, is a hydrogen atom, alkyl, heteroalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, aralkyl, heteroaralkyl or joined to R5 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
R5, if present, 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 R4 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.
In an 18th embodiment of the first aspect which is also an embodiment of the first to 17th embodiment of the first aspect
B is - Y^-CO-L3- or - Y^-CO-R0*-, wherein
Y*3 is a bond, a C1-C6alkylene, a C2-C6alkenylene or a C2-C6alkynylene;
LB is a cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, aralkyl, or heteroaralkyl; and RcB is an optionally substituted C1-C8alkyl, an optionally substituted C2-C8alkenyl or an optionally substituted C2-C8alkynyl.
In a 19th embodiment of the first aspect which is also an embodiment of the first to 18th embodiment of the first aspect B is
Figure imgf000013_0001
In a 20th embodiment of the first aspect which is also an embodiment of the first to 18th embodiment of the first aspect B is
wherein
Figure imgf000013_0002
R4 is methyl, ethyl or isopropyl.
In a 21st embodiment of the first aspect which is also an embodiment of the first to 17th embodiment of the first aspect B is
Figure imgf000013_0003
In a 22nd embodiment of the first aspect which is also an embodiment of the first to 21st embodiment of the first aspect E is -YaE-CO-LE-, -YaE-CO-RcE-, -YaE-NRcE-CO-RdE-, -YaE-NRcE-CO-LE-, -YaE-NRcE-CO-NRdE-LE-, or -YaE-NRcE-CS-NRdE-LE-, wherein Y^ is a bond, a C1-C6alkylene, a C2-C6alkenylene or a C2-C6alkynylene;
RcE is a hydrogen atom, an optionally substituted C1-C8alkyl, an optionally substituted C2-C8alkenyl or an optionally substituted C2-C8alkynyl, provided that RcE is not a hydrogen atom in -YaE-CO-RcE-;
RdE is a hydrogen atom, an optionally substituted C1-C8alkyl, an optionally substituted C2-C8alkenyl or an optionally substituted C2-C8alkynyl; and
LE is a cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, aralkyl, or heteroaralkyl.
In a 23rd embodiment of the first aspect which is also an embodiment of the first to 22nd embodiment of the first aspect G is alkyl, cycloalkyl, -Y^-O-R00, -YaG-CO-NRaGRbG, -Y8G-S-R00, -Y1^-SO-R00, -Y^-SO2-R'0, heteroaryl, alkylcycloalkyl, or heterocycloalkyl, wherein
Y*0 is a bond, a C1-C6alkylene, a C2-C6alkenylene or a C2-C6alkynylene;
R"° is a hydrogen atom, a C1-C6alkyl, a C2-C6alkenyl, a C2-C6alkynyl, or is joined to R60 to form a 4- to 10-membered cycloalkyl or heterocycloalkyl;
R*0 is a hydrogen atom, a C1-C6alkyl, a C∑-C6alkenyl or a C2-C6alkynyl, or is joined to R"° to form a 4- to 10-membered cycloalkyl or heterocycloalkyl; and
R00 is an optionally substituted C1-C8alkyl, an optionally substituted C2-C8alkenyl or an optionally substituted C2-C8alkynyl.
In a 24th embodiment of the first aspect which is also an embodiment of the first, fourth to six, twelfth, 17th or 22nd embodiment of the first aspect the compound is represented by formula (II):
Figure imgf000015_0001
wherein
Y1 is N, CH or CF;
K is selected from
Figure imgf000015_0002
R3 is a hydrogen atom or methyl;
Ry2 is a hydrogen atom, halogen atom, or C1-C6alkyl;
Ry3 is a hydrogen atom, halogen atom, C1-C6alkyl, or C1-C6heteroalkyl;
Ry4 is a hydrogen atom or halogen atom; B is -Y^-CO-lA, -Y^-CO-R08-, or
*\ S
K Z1X
wherein
Y* is a bond;
RcB is an optionally substituted C1-C8alkyl or an optionally substituted C2-C8alkenyl;
LB is a cycloalkyl, heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, or heteroaralkyl;
W is alkyl or N;
R4, if present, is a hydrogen atom, alkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aralkyl, or heteroaralkyl;
R5, if present, is a hydrogen atom or alkyl;
E is -YbE-NRdE-C0-LE-, wherein
YbE is a bond, a C1-C6alkylene, or a C2-C6alkenylene;
RdE is a hydrogen atom, a Cj-C6alkyl, or C2-C6alkenyl; and
LE is a cycloalkyl, heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, or heteroaralkyl. In a 25th embodiment of the first aspect which is also an embodiment of the first, fourth to sixth, twelfth, 13th, 17th to 20th, 22nd or 23rd embodiment of the first aspect the compound is represented by formulas (III) or (IV):
Figure imgf000017_0001
wherein
Y1 is N, CH or CF;
K is selected from
Figure imgf000017_0002
R3 is a hydrogen atom or methyl;
Ry2 is a hydrogen atom, halogen atom, or C1-C6alkyl; Ry3 is a hydrogen atom, halogen atom, C1-C6alkyl, or C1-C6heteroalkyl;
Ry4 is a hydrogen atom or halogen atom, C1-C6alkyl, or C1-C6heteroalkyl;
E is -YaE-NRcE-CO-RdE-, -YaE-NRcE-CO-LE-, -YaE-NRcE-CO-NRdE-LE-, or -YaE-NRcE-CS-NRdE-LE-, wherein
YaE is a bond or C1-C6alkylene;
RcE is a hydrogen atom, C1-C6alkyl, or C2-C6alkenyl;
RdE is a hydrogen atom, an optionally substituted C1-C8alkyl, an optionally substituted C2-C8alkenyl or an optionally substituted C2-C8alkynyl group;
LE is a cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, aralkyl, or heteroaralkyl;
G, if present, is cycloalkyl, -YaG-CO-NR80RbG, -YaG-O-ReG, -YaG-S-ReG, -YaG-SO-ReG, or -YaG-SO2-ReG, wherein YaG is a bond or C1-C6alkylene;
RaG is a hydrogen atom or C1-C6alkyl;
RbG is a hydrogen atom or C1-C6alkyl; and ReG is C1-C6alkyl.
In a 26th embodiment of the first aspect which is also an embodiment of the 25th embodiment of the first aspect the compound is represented by formula (V):
Figure imgf000019_0001
wherein Y1, K, R3, Ry2, Ry3, Ry4, and LE are as defined in 25th embodiment of the first aspect.
In a 27th embodiment of the first aspect which is also an embodiment of the 25th embodiment of the first aspect the compound is represented by formula (VI):
Figure imgf000019_0002
wherein Y1, K, R3, R*2, Ry3, Ry4, and LE are as defined in claim 25.
In a 28th embodiment of the first aspect which is also an embodiment of the 24th to 27th embodiment of the first aspect
Ry3 is a hydrogen atom. In a 29th embodiment of the first aspect which is also an embodiment of the 24th to 28th embodiment of the first aspect wherein
R*2 is a hydrogen atom.
In a 30th embodiment of the first aspect which is also an embodiment of the first, fourth to sixth, ninth, tenth, twelfth, 13th, 17th, 21st, 22nd or 23rd embodiment of the first aspect the compound is represented by formula (VII):
Figure imgf000020_0001
wherein
Y1 is N, CH or CF;
K is selected from
Figure imgf000020_0002
R*4 is a hydrogen atom, halogen atom, C1-C6alkyl, or C1-C6heteroalkyl;
E is -YaE-CO-LE-, wherein
YaE is a bond or C1-C6alkylene;
LE is heterocycloalkyl; and
G is alkylcycloalkyl, heterocycloalkylalkyl, aryl or heteroaryl.
In a 31st embodiment of the first aspect which is also an embodiment of the 30th embodiment of the first aspect
Ry4 is a hydrogen atom or halogen atom.
In a 32nd embodiment of the first aspect which is also an embodiment of the first to 31st embodiment of the first aspect wherein
the stereogenic center to which R3 is attached to is in the (JR) configuration; and
R3 is methyl.
In a 33rd embodiment of the first aspect which is also an embodiment of the first to 32nd embodiment of the first aspect the compound exhibits an ICJO of 500 nM or less in a standard in vitro BK Bl receptor-mediated assay.
In a first embodiment of the second aspect the problem underlying the instant application is solved by a compound, which is preferably a compound according to any one of the first to 33rd embodiment of the first aspect, which compound is selected from compounds 1 to 282 of Table 1. In a first embodiment of the third aspect the problem underlying the instant application is solved by 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.
In a second embodiment of the third aspect which is also an embodiment of the first embodiment of the third aspect,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.
In a first embodiment of the fourth aspect 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.
In a second embodiment of the fourth aspect which is also an embodiment of the first embodiment of the fourth aspect the condition or disease is responsive to BK BlR modulation. 0
In a third embodiment of the fourth aspect which is also an embodiment of the first or second embodiment of the fourth aspect the condition or disease is selected from the group comprising inflammatory diseases, immunology disorders and pain.
In a fourth embodiment of the fourth aspect which is also an embodiment of the third embodiment of the fourth aspect the inflammatory disease or immunology disorder is selected from the group comprising inflammatory bowel disease, rheumatoid arthritis, gouty arthritis, atherosclerosis and associated fibrotic conditions
In a fifth embodiment of the fourth aspect which is also an embodiment of the third embodiment of the fourth aspect the pain is selected form the group comprising visceral pain, 2 neuropathic pain, complex regional pain syndrome CRPS and inflammatory pain.
In a first embodiment of the fifth aspect 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. 0 In a first embodiment of the sixth aspect the problem underlying the instant application is solved by a method for localizing or detecting a BK Bl receptor in a tissue, preferably a tissue section, in vitro, comprising:
(a) contacting a sample of said tissue presumably containing the BK Bl receptor with a detectably labeled compound according to any embodiment of the first to third aspect under S conditions that permit binding of the compound to the BK B 1 receptor; and
(b) detecting the compound bound to the BK Bl receptor or detecting the binding of the compound to the BK Bl receptor.
In a second embodiment of the sixth aspect which is also an embodiment of the first embodiment of the sixth aspect the compound is radiolabeled, fluorescence-labeled or luminescence labeled, or labeled with an antibody.
In a first embodiment of the seventh aspect 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.
It will be acknowledged by the person skilled in the art that any compound which is generated by any combination of the individual generic groups of formula (I) as disclosed herein shall be encompassed by the instant invention. The compounds shown in the following Table 1 are embodiments of formula (I) according to the present invention.
Table 1:
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000040_0002
Figure imgf000041_0002
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000042_0002
Figure imgf000043_0002
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000044_0002
Figure imgf000045_0002
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0002
Figure imgf000047_0001
Figure imgf000048_0001
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 IC50 for competition with the binding of labelled DAKD to human BlR of preferably less than 50 nanomolar. In certain embodiments, such antagonists exhibit a high activity on BlR of species other than human, i.e., an IC50 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. For instance, the IC50 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 IC50 (half-maximal inhibitory concentration) of about 5 micromolar or less, still more preferably an IC50 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
BlR-mediated diseases as described below.
These and other aspects of the present invention will become apparent upon reference to the following detailed description.
Compounds are generally described herein using standard nomenclature. For compounds having asymmetric centers, it should be understood that, unless otherwise specified, all of the optical isomers and mixtures thereof are e mpassed. Compounds with two or more asymmetric elements can also be present as mixtures of diastereomers. In addition, compounds with carbon-carbon double bonds may occur in Z- and E- forms, with all isomeric forms of the compounds being included in the present invention unless otherwise specified. Where a compound exists in various tautomeric forms, a recited compound is not limited to any one specific tautomer, but rather is intended to encompass all tautomeric forms. 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. By way of general example, and without limitation, isotopes of hydrogen include tritium and deuterium and isotopes of carbon include 11C, 13C, and 14C.
Compounds according to the formulas provided herein, which have one or more stereogenic centers, have an enantiomeric excess of at least 50%. For example, such compounds may have an enantiomeric excess of at least 60%, 70%, 80%, 85%, 90%, 95%, or 98%. Some embodiments of the compounds have an enantiomeric excess of at least 99%. It will be apparent that single enantiomers (optically active forms) can be obtained by asymmetric synthesis, synthesis from optically pure precursors or by resolution of the racemates. Resolution of the racemates can be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example, a chiral HPLC column.
Certain compounds are described herein using a general formula that includes variables such as, e.g., A, R1, Ra, W, Y, Z. Unless otherwise specified, each variable within such a formula is defined independently of any other variable, and any variable that occurs more than one time in a formula is defined independently at each occurrence. Thus, for example, if a group is shown to be substituted with 0-2 R*, the group may be unsubstituted or substituted with up to two R* groups and R at each occurrence is selected independently from the definition of R*. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds, i.e., compounds that can be isolated, characterized and tested for biological activity. 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-(CH2)n-COOH where n is any integer from 0 to 4, i.e., 0, 1, 2, 3, or 4, and the like. Similarly, pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium and ammonium. Those of ordinary skill in the art will recognize further pharmaceutically acceptable salts for the compounds provided herein. In general, 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. Generally, the use of nonaqueous media, such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile, is preferred.
It will be apparent that each compound of formula (I) may, but need not, be present as a hydrate, solvate or non-covalent complex. In addition, 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. For example, 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. Examples of 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. For example, 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. The term "substituted," as used herein, 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. When a substituent is oxo, i.e., =0, then 2 hydrogens on the atom are replaced. An oxo group that is a substituent of an aromatic carbon atom results in a conversion of -CH- to -C(=0)- and a loss of aromaticity. For example a pyridyl group substituted by oxo is a pyridone.
The expression 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.
The expressions 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
12 carbon atoms, more preferably from 2 to 6 carbon atoms, for example an ethenyl, allyl, acetylenyl, propargyl, isoprenyl or hex-2-enyl group. Preferably, alkenyl groups have one or two, more preferably one, double bond(s) alkynyl groups have one or two, more preferably one, triple bond(s).
Furthermore, the terms 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.
The expression 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. The expression 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.
Examples of heteroalkyl groups are groups of formulas -S-Ya-L, -S-Ya-CO-NRaRb, -Ya-NRc-CO-NRaRb, -Ya-NRc-CO-O-Rd, -Ya-NRc-C0-Rd, -Yβ-NRc-C0-NRd-L, -Ya-NRc-CS-NRd-L, -Y^O-CO-NR8R", -Y^CO-NR8R5, -O-Ya-CO-NRaRb, -Ya-NRc-CO-L, , -Ya-O-CO-O-Rc, -Ya-O-CO-Rc, -Ya-0-Rc, -Ya-CO-L, -Y1^NR8R6, Rc-S-Ya-, R^N(R1O-Y8-, Rc-C0-Ya-, R'-O-CO-Y8-, R^CO-O-Y8-, R'-CO-NtR^-Y8-, R^N(R1O-CO-Y11-, Rc-SO-Ya-, Rc-SO2-Ya-, -Ya-NRc-SO2-NRaRb, -Ya-Sθ2-NRaRb, -Ya-NRc-SO2-Rd, R8O-CO-N(R6)- Ya-, Ra-N(Rb)-C(=NRd)-N(Rc)-Y\ Rc-S-C0-Y8-, Rc-CO-S-Ya-, R^S-CO-N(R1O-Y8-, R^N(R1O-CO-S-Y8-, RC-S-CO-O-Y8-, Rc-O-CO-S-Ya-, RC-S-CO-S-Y8-; wherein Ra being a hydrogen atom, a C1-C6alkyl, a C2-C6alkenyl, a C2-C6alkynyl , or is joined to Rb to form a 4- to 10-membered cycloalkyl or heterocycloalkyl; Rb being a hydrogen atom, a C1-C6alkyl, a C2-C6alkenyl or a C2-C6alkynyl , or taken together with Ra to form a 4- to 10-membered cycloalkyl or heterocycloalkyl; Rc being a hydrogen atom, an optionally substituted C1- C8alkyl, an optionally substituted C2-C8alkenyl or an optionally substituted C2-C8alkynyl; Rd being a hydrogen atom, optionally substituted Q-Cealkyl, optionally substituted C2-C8alkenyl or optionally substituted C2-Csalkynyl; L being a cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, aralkyl, or heteroaralkyl; and Ya be bond, a C1-C6alkylene, a C2-C6alkenylene or a C2-C6alkynylene group; each heteroalkyl group containing at least one carbon atom and it being possible for one or more hydrogen atoms to have been replaced by fluorine or chlorine atoms. Certain compounds are described herein using formulas that include the variables captioned above which variables carry a superscripted B, E or G as an extension, for example, LB, Y"6, or R00. Unless otherwise specified, the definition of these variables corresponds to the above definition of the respective variable, i.e. LB and LE are defined as L; Y*8, YaE, and Y"° are defined as Ya; RcB, RcE, and R"0 are defined as Rc; etc. Specific examples of 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. Further examples of 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-.
The expression 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. In an embodiment a partially unsaturated cyclic group has one, two or more double bonds, such as a cycloalkenyl group. The expression cycloalkyl preferably refers furthermore to groups in which one or more hydrogen atoms have been replaced each independently of the others by fluorine, chlorine, bromine or iodine atoms or by OH, =0, SH, =S, NH2, =NH, CN, CF3 or NO2 groups, thus, for example, cyclic ketones such as, for example, cyclohexanone, 2- cyclohexenone or cyclopentanone. Further specific examples of 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.
The expression 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. The expression heterocycloalkyl preferably refers furthermore to groups in which one or more hydrogen atoms have been replaced each independently of the others by fluorine, chlorine, bromine or iodine atoms or by OH, =0, SH, =S, NH2, =NH, CN, CF3 or NO2 groups. 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.
The expression 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.
The expression 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.
The expression 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. The expression 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, CF3 or NO2 groups. Examples are a phenyl, naphthyl, biphenyl, 2-fluorophenyl, anilinyl, 3-nitrophenyl or 4- hydroxyphenyl group.
The expression 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. The expression heteroaryl preferably refers furthermore to groups in which one or more hydrogen atoms have been replaced each independently of the others by fluorine, chlorine, bromine or iodine atoms or by OH, =0, SH, NH2, =NH, CN, CF3 or NO2 groups. 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.
The expression 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. Specific examples of 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.
The expression 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.
Examples of heteroaralkyl groups are arylheteroalkyl, arylheterocycloalkyl, arylhetero- cycloalkenyl, arylalkylheterocycloalkyl, arylalkenylheterocycloalkyl, arylalkynylhetero- cycloalkyl, arylalkylheterocycloalkenyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heteroarylheteroalkyl, heteroarylcycloalkyl, heteroarylcycloalkenyl, heteroarylhetero- cycloalkyl, heteroarylheterocycloalkenyl, heteroarylalkylcycloalkyl, heteroarylalkyl- heterocycloalkenyl, heteroarylheteroalkylcycloalkyl, heteroarylheteroalkylcycloalkenyl, heteroalkylheteroarylalkyl and heteroarylheteroalkylheterocycloalkyl groups, the cyclic groups being saturated or mono-, di- or tri-unsaturated. Specific examples are a tetrahydroiso- quinolinyl, benzoyl, 2- or 3-ethylindolyl, 4-methylpyridino, 2-, 3- or 4-methoxyphenyl, 4-ethoxyphenyl, 2-, 3- or 4-carboxyphenylalkyl group.
The expressions cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl and heteroaralkyl refer to groups in which one or more hydrogen atoms of such groups have been replaced each independently of the others by fluorine, chlorine, bromine or iodine atoms or by OH, =0, SH, =S, NH2, =NH, CN, CF3 or NO2 groups.
The expression "optionally substituted" as used in connection with any group refers to a group in which one or more hydrogen atoms have been replaced each independently of the others by fluorine, chlorine, bromine or iodine atoms or by OH, =0, SH, =S, NH2, =NH, CN, CF3 or NO2 groups. 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- C6alkyl, unsubstituted C2-C6alkenyl, unsubstituted (VC6alkynyl, unsubstituted C1-C6hetero- alkyl, unsubstituted C3-C1ocycloalkyl, unsubstituted C2-C9heterocycloalkyl, unsubstituted Ce- C1oaryl, unsubstituted C1-C^heteroaryl, unsubstituted C7-C12aralkyl or unsubstituted C2- C1iheteroaralkyl group.
The expression "halogen" as preferably used herein means fluorine, chlorine, bromine, iodine.
As used herein 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. In other words, 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.
The pharmaceutical compositions according to the present invention 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. Furthermore, one or more other active ingredients may, but need not, be included in the pharmaceutical compositions provided herein. For instance, 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.
Pharmaceutical 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. The term 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. In certain embodiments, compositions in a form suitable for oral use are preferred. Such forms include, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs. Within yet other embodiments, 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. Such 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. For example, 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.
Aqueous suspensions contain the active ingredient(s) in admixture with excipients suitable for the manufacture of aqueous suspensions. Such 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 sorbitan monooleate. 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.
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. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, such as sweetening, flavoring and coloring agents, may also be present.
Pharmaceutical 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.
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. 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. Examples of such components are described in Martindale The Extra Pharmacopoeia (Pharmaceutical Press, London 1993) and Martin (ed.), Remington's Pharmaceutical Sciences. Formulations may comprise microcapsules, 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, like those of lotions and creams, 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. Suitable surfactants include, but are not limited to, nonionic, amphoteric, ionic and anionic surfactants. For example, one or more of dimethicone copolyol, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, lauramide DEA, cocamide DEA, and cocamide MEA, oleyl betaine, cocamidopropyl phosphatidyl PG-dimonium chloride, and 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. For inhalation formulations, the compounds provided herein may be delivered via any inhalation m ds known to those skilled in the art. Such 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. 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, wherein the carrier is a solid, 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.
Pharmaceutical 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.
Pharmaceutical 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. 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.
For the prevention and/or treatment of diseases mediated by BK or analogues thereof, 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
0.5 mg to about 7 g per patient per day. 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.
It will be understood, however, that 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. As used herein, the term "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.
Due to the involvement of the bradykinin Bl receptor in various diseases, 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. Apart from that, 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).
It is within the present invention that 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.
In the following the various diseases and conditions that are responsive to BlR modulation and the use of the compounds according to the present invention in specific methodology and diagnostics are further specified.
Inflammatory Diseases and Immunology Disorders
Within the present application the term "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.
Within the present application the term "immunology 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.
Due to the involvement of the bradykinin Bl receptor in various diseases, 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. Gastroenerol 2002, 97(8), 2026-32; Devani et al., Dig. Uv. Disease 2005, 37(9), 665-73), irritable bowel syndrome, enterocolitis, liver diseases, pancreatitis, gall bladder diseases, smooth muscle relaxants for the treatment of spasms of the gastrointestinal tract or uterus. They may also be used in kidney disease like nephritis, bladder disease like cystitis, interstitial cystitis, eye diseases like uveitis, retinitis, glaucoma, and ear diseases such as otitis media. They may also be used in inflammatory skin diseases such as psoriasis, eczema, atopic diseases, dermatitis and itching. 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. They 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. They may also be used in 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). 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. Furthermore the compounds may be used to treat fϊbrotic 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).
They are also effective in disorders of the respiratory pathways in 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.
These compounds according to the present invention may also be used to treat auto- inflammatory diseases such as familial Mediterranean fever (FMF), tumor-necrosis factor receptor associated periodic syndrome (TRAPS), neonatal onset multisystem inflammatory disease (NOMID), familial cold autoinflammatory syndrome (FCAS) including familial cold urticaria (FCU), pyogenic arthritis pyoderma gangrenosum acne (PAPA) syndrome and Muckle- Wells disease.
Pain
Within the present application the term "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. Ther. 2006, 318(1), 195-205; Ferreira et al, J. Neurosc. 2005, 25(9), 2405- 12; Conley et al, Eur. J. Pharm. 2005, 527(1-3), 44-51; Levy and Zochodne, Pain 2000, 86(3), 265-71; Yamaguchi-Sase et al, Inflamm. Res. 2003, 52(4), 164-9).
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 prophylactic and acute use, hyperalgesia, and fever. Furthermore, compounds of the invention are useful as analgesic agent for use during general and monitored anesthesia.
Diagnostic and further applications
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.
It is also within the present invention that 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.
For various applications, 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. 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.
The present invention is now further illustrated by the following examples from which further features, embodiments and advantages of the present invention may be taken.
EXAMPLES
Abbreviations used in the following examples are as follows: amu is atomic mass unit
ACN is acetonitrile
AgNC>3 is silver nitrate
BlR is Bl receptor BK is bradykinin
BSA is bovine serum albumin cone, is concentrated DAKD is des-Arg-10-kallidin DCM is dichloromethane DIP EA is diisopropyethylamine DMAP is 4-R^.V-dimethylaminopyridine S DMEM is Dulbecco's modified Eagle's medium
DMF is Λ^Λ/-dimethylformamide DMSO is dimethylsulfoxide EA is ethyl acetate FBS is fetal bovine serum 0 FCS is fetal calf serum h is hour
HATU is 2-(l//-7-azaben2otriazol-l-yl)-l,l,3,3-tetramethyl uranium hexafluorophosphate HCl is hydrochloric acid S HEK is human embryonic kidney
HEPES is 4-2-hydroxyethyl-l-piperazineethanesulfonic acid HOBt is N-hydroxybenzotriazole HPLC is high performance liquid chromatography K2CO3 is potassium carbonate IL is interleukine
LiOH is lithium hydroxide NaCl is sodium chloride Na2Cθ3 is sodium carbonate Na2S2Oa is sodium thiosulfate NaOH is sodium hydroxide
NaHCθ3 is sodium hydrogencarbonate NH4CI is ammonium chloride NH4OH is ammonia Pd(OAc)2 is 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
TPP is Techno Plastic Products AG
WSC is (3-dimeΛylamino-propyl)-eΛyl-carbodiimide hydrochloride
Materials and methods
The materials and methods as well as general methods are further illustrated by the following examples:
Solvents: Solvents were used in the specified quality without further purification. Acetonitrile
(Gradient grade, J.T. Baker); dichloromethane (for synthesis, Merck Eurolab); diethylether (for synthesis, Merck Eurolab); Λ^-dimethylformamide (LAB, Merck Eurolab); dioxane (for synthesis, Aldrich); methanol (for synthesis, Merck Eurolab).
Water:
Milli-Q Plus, Millipore, demineralized.
Chemicals:
Chemicals were synthesized according to or in analogy to literature procedures or purchased from Advanced ChemTech (Bamberg, Deutschland), Sigma-Aldrich-Fluka (Deisenhofen, Germany), Bachem (Heidelberg, Germany), J.T. Baker (Phillipsburg, USA), Lancaster (MUhlheim/Main, Germany), Merck Eurolab (Darmstadt, Germany), Neosystem (Strasbourg, France), Novabiochem (Bad Soden, Germany, from 2003 Merck Biosciences, Darmstadt, Germany) Acros (Geel, Belgium, distribution company Fisher Scientific GmbH, Schwerte, Germany), Peptech (Cambridge, MA, USA), Synthetech (Albany, OR, USA), Pharmacore (High Point, NC, USA) and Anaspec (San Jose, CA, USA) or other companies and used in the assigned quality without further purification. If not stated differently, concentrations are given as percent by volume.
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. For chromatographic separation a RP-18-column (Vydac 218 TP5215, 2.1 x 150 mm, 5 μm, C18, 300 A with a pre column (Merck) was used at 300C and a flow of 0.3 ml/min using a linear gradient for all chromatograms (5-95% B for 25 min, linear, A: 0.05% TFA in water and B: 0.05% TFA in CH3CN). UV detection was done at λ = 220 nm. The retention times (R4) are indicated in the decimal system (e.g. 1.9 min = 1 min 54 s) and are referring to detection in the mass spectrometer. The dead time between injection and UV detection (HPLC) was 1.65 min, and between UV detection and mass detection 0.21 min. The accuracy of the mass spectrometer was approx. ± 0.5 amu.
HPLC/MS analyses were performed by injection of 5 μl, using a linear gradient from 95:5 to 5:95 in 9.5 min (A: 0.05% TFA in water and B: 0.05% TFA in ACN). RP columns were from Phenomenex (Type Luna C-18, 3μm, 50 x 2.00 mm, flow 0.3 ml, HPLC at room temperature); Mass spectrometer: ThermoFinnigan Advantage and/or LCQ Classic (both ion trap), ESI ionization, helium served as impact gas in the ion trap. Excalibur version 1.3 and/or. 1.2 was used as software. Retention times (Rt) are indicated in the decimal system (e.g. 1.9 min = 1 min 54 s).
Preparative HPLC:
Preparative HPLC separations were done using Vydac R18-RP columns with the following gradient solvents: 0.05% TFA in H2O and B: 0.05% TFA in CH3CN
Compounds were named using AutoNom version 2.2 (Beilstein Informationssysteme Copyright0 1988-1998, Beilstein Institut fttr Literatur der Organischen Chemie licensed to Beilstein Chemiedaten and Software GmbH)
Preparation of compounds:
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.
Specific examples for the preparation of compounds of formula (I) are provided in the following examples. Unless otherwise specified all starting materials and reagents are of standard commercial grade, and are used without further purification, or are readily prepared from such materials by routine methods. Those skilled in the art of organic synthesis will recognize that starting materials and reaction conditions may be varied including additional steps employed to produce compounds encompassed by the present invention.
Example 1
Synthesis of (R)-pyrimidine-5-carboxylic acid (l-{l-[4-(2,5-ri$/frα/w-dimethyl- pyrrolidine-l-carbonyl)-phenyl]-ethylcarbamoyl}-cyclopropyl)-amide (Compound 1)
Figure imgf000075_0001
Method 1:
A. (R)-[l-(4-Bromo-phenyl)-ethyl]-carbamic acid tert-butyl ester (i.)-l-(4-Bromo-phenyl)-ethylamine hydrochloride (2.50 g, 10.57 mmol) was added portionwise to a stirred solution of di-/er/-butyl dicarbonate (2.77 g, 12.68 mmol) and triethylamine (3.24 mL, 23.25 mmol) in ACN (40 mL). After stirring for 18 h at RT the solvent was removed in vacuo and the residue was purified by flash chromatography on silica gel (elution with n-hexane/EA 4:1) to give the title compound. MS (m/z): 230.0 [M+H*- OtBu]
B. (R)-4-(l-/er/-Butoxycarbonylamino-ethyl)-benzoic acid methyl ester To a stirred suspension of (R)-[l-(4-brom henyl)-ethyl]-carbamic acid tert-butyl ester
(3.18 g, 10.58 mmol), l,4-bis-(diphenylphosphino)butane (0.32 g, 0.74 mmol) and Pd(OAc)2
(0.16 g, 0.74 mmol) in anhydrous DMSO (25 mL) under an atmosphere of nitrogen were added triethylamine (8.85 mL, 63.46 mmol) and anhydrous methanol (10 mL). After stirring at 700C under an atmosphere of carbon monoxide for 48 h the mixture was concentrated in vacuo and the residue was partitioned between EA (200 mL) and sat. NaHCθ3 solution (200 mL). After extraction of the aqueous layer with EA (2 x 200 mL) the combined organic layers were dried over Na2SO-I, filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (elution with n-hexane/EA 19:1) to give the title compound. MS (m/z): 208.2 [M+lf-OtBu]
C. (R)-4-(l-/βrr-Butoxycarbonylamino-ethyl)-benzoic acid
A mixture of (R)-4-(l-ter/-butoxycarbonylamino-ethyl)-benzoic acid methyl ester (1.16 g, 4.16 mmol) and LiOH (1 M solution in H20, 25 mL) in dioxane (25 mL) was stirred at RT for 3 h. The mixture was concentrated in vacuo and EA (150 mL) was added. The pH of the aqueous layer was adjusted to 4 by the addition of 1 N HCl solution and the aqueous layer was extracted with EA (2 x 150 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The resulting title compound was used for the next reaction without purification. MS (m/z): 209.9 [M+H^-tBu]
D. (R)- { 1 -[4-(2,5-cw/franj-Dimethyl-pyrrolidine-l -carbonyl)-phenyl]-ethyl} -carbamic acid tert-butyl ester
2,5-c/-5//rα/w-Dimethyl-pyrrolidine (0.76 mL, 6.27 mmol) was added to a stirred solution of (R)-4-(l-terf-butoxycarbonylamino-ethyl)-benzoic acid (0.83 g, 3.14 mmol), WSC (0.72 g, 3.76 mmol), HOBt (0.51 g, 3.76 mmol) and triethylamine (2.19 mL, 15.68 mmol) in DCM/DMF 9:1 (10 mL). After stirring overnight at RT the solvent was removed in vacuo and the residue was purified by flash chromatography on silica gel (elution with n-hexane/EA 4:1) to give the title compound. MS (m/z): 347.0 [M+H+]
E. (R)-[4-(l-Amino-ethyl)-phenyl]-(2,5-czj//rαΛ$i-dimethyl-pyrrolidin-l-yl)-methanone
A solution of (^^{l-^^.S-cw/rrans-dimethyl-pyrrolidine-l-carbony^-phenyll-ethyl}- carbamic acid ter/-butyl ester (0.56 g, 1.62 mmol) and TFA (5 mL) in DCM (45 mL) was stirred at RT for 4 h. The reaction mixture w oncentrated in vacuo and the residue was partitioned between DCM (50 mL) and 1 N NaOH solution (50 mL). After extraction of the aqueous layer with DCM (2 x 50 mL) the combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo to give the title compound. MS (m/z): 247.2 [M+H*]
F. (R)-(l-{l-[4-(2,5-cw//rαΛS-Dimethyl-pyrrolidine-l-carbonyl)-phenyl]- ethylcarbamoyl}-cyclopropyl)-carbamic acid ter/-butyl ester
(R)-[4-(l-Amino-ethyl)-phenyl]-(2,5-ci5/frα/ty-dimethyl-pyττolidin-l-yl)-methanone (10.0 mg, 0.041 mmol) was added to a stirred solution of 1-te/ϊ-butoxycarbonylamino- cyclopropanecarboxylic acid (9.8 mg, 0.049 mmol), WSC (9.3 mg, 0.049 mmol), HOBt (6.6 mg, 0.049 mmol) and #-methylmorpholine (22.3 μL, 0.203 mmol) in DMF (1 mL). After stirring overnight at RT the solvent was removed in vacuo and the residue was purified by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA to give the title compound as the TFA salt. MS (m/z): 429.9 [M+H4]
G. (R)-l-Amino-cyclopropanecarboxylic acid {l-[4-(2,5-cw//rajw-dimethyl-pyrrolidine- 1 -carbonyl)-phenyl]-ethyl} -amide
A solution of (R)-(l-{l-[4-(2,5-c/5/rrαns-dimethyl-pyrrolidine-l-carbonyl)-phenyI]- ethylcarbamoyl}-cyclopropyl)-carbamic acid tør/-butyl ester (8.0 mg, 0.019 mmol) and TFA (100 μL) in DCM (900 μL) was stirred at RT for 4 h. The reaction mixture was concentrated in vacuo to give the title compound as the TFA salt. MS (m/z): 330.0 [M+H*]
H. (R)-Pyrimidine-5-carboxylic acid (l-{l-[4-(2,5-c/j/f/"ΛΛr-dimethyl-pyrrolidine-l- carbonyl)-phenyl]-ethylcarbamoyl}-cyclopropyl)-amide (R)-l-Amino-cyclopropanecarboxylic acid {l-[4-(2,5-cw/frfl«j-dimethyl-pyrroIidine-l- carbonyl)-phenyl]-ethyl} -amide (10.0 mg, 0.030 mmol) was reacted with pyrimidine-5- carboxylic acid (4.5 mg, 0.036 mmol) according to the synthesis of (l-{l-[4-(2,5-c«//>wii:- dimethyl-pyrrolidine- 1 -carbonyl)-phenyl]-ethylcarbamoyl} -cyclopropyl)-carbamic acid tert- butyl ester to give the title compound as the TFA salt. MS (m/z): 436.1 [M+H+]
Method 2:
Reactions A-D were performed based upon literature (Dickins et al, Chem. Eur. J. 1999, 5, 1095-1105)
A. (R)-N-[I -(4-Bromo-phenyl)-ethyl]-acetamide
Acetyl chloride (2.13 mL, 30.0 mmol) was added dropwise at -15°C to a stirring solution of (R)-l-(4-bromo-phenyl)-ethylamine (5.00 g, 25.0 mmol) and triethylamine (4.53 mL, 32.5 mmol) in diethylether (750 mL). The reaction mixture was allowed to warm to RT over 2 h and then poured into 500 mL of water. The organic layer was washed with 0.1 N HCl solution (1 x 500 mL) and with water (1 x 500 mL), dried over K2CO3, filtered and concentrated in vacuo to give the title compound. MS (m/z): 243.8 [M+H*]
B. (R)-7/-[l-(4-Cyano-phenyl)-ethyl]-acetamide
To a stirred solution of (R)-JV-[l-(4-bromo-phenyl)-ethyl]-acetamide (3.47 g, 14.3 mmol) in anhydrous DMF (30 mL) under an atmosphere of nitrogen was added coppenT)cyanide (2.70 g, 30.1 mmol). After stirring at 1800C overnight the reaction mixture was concentrated in vacuo and the residue carefully poured into 6 N HCl solution (60 mL). The aqueous layer was extracted with DCM (3 x 100 mL). The organic layer was washed with water (1 x 100 mL), dried over Na2SO.), filtered and concentrated in vacuo to give the title compound. MS (m/z): 188.9 [M+H4]
C. (R)-4-(l-Amino-ethyl)-benzoic acid A solution of (R)-Λ^-[l-(4-cyano-phenyl)-ethyl]-acetamide (1.67 g, 8.87 mmol) in 6 N
HCl solution (27 mL) was stirred at 1200C overnight. The solvent was removed in vacuo and the solid was washed with diethylether, filtered and dried to give the title compound as the HCl salt. MS (m/z): 165.9 [M+H*]
D. (R)-4-(l-Amino-ethyl)-benzoic acid methyl ester
3 Drops of HCl cone, were added to a stirred solution of (R)-4-(l-amino-ethyl)-benzoic acid (1.79 g, 8.87 mmol) in methanol (25 mL). After stirring the mixture at 850C for 3 h the solvent was removed in vacuo to give the title compound as the HCl salt. MS (m/z): 179.8 [M+H*]
E. (R)-4-{l-[(l-Amino-cyclopropanecarbonyl)-amino]-ethyl}-benzoic acid methyl ester (R)-4-(l-Amino-ethyl)-benzoic acid methyl ester (420 mg, 2.34 mmol) was reacted with 1-tert-butoxycarbonylamino-cyclopropanecarb ic acid (560 mg, 2.81 mmol) according to the synthesis of (R)-{l-[4-(2,5-cis/trans-dimethyl-pyrrolidine-l-carbonyl)-phenyl]-ethyl}- carbamic acid tert-butyl ester to give (R)-4-{1-[(1-tert-butoxycarbonylamino- cyclopropanecarbonyl)-amino]-ethyl}-benzoic acid methyl ester. The tert-butyl ester (103 mg, 0.284 mmol) was treated with TFA according to the synthesis of (R)-l-amino- cyclopropanecarboxylic acid { 1 -[4-(2,5-cis/trans-dimethyl-pyrrolidine- 1 -carbonyl)-phenyl]- ethyl}-amide to give the title compound as the TFA salt. MS (m/z): 262.9 [M+H+]
F. (R)-4-[l-({l-[(Pyrimidine-5-carbonyl)-amino]-cyclopropanecarbonyl}-amino)-ethyl]- benzoic acid methyl ester
(R)-4-{l-[(l-Amino-cyclopropanecarbonyl)-amino]-ethyl}-benzoic acid methyl ester (74.5 mg, 0.284 mmol) was added to a stirred solution of pyrimidine-5-carboxylic acid (42.3 mg, 0.341 mmol), HATU (129.6 mg, 0.341 mmol) and DIPEA (247.3 μL, 1.420 mmol) in DMF (2 mL). After stirring overnight the reaction mixture was concentrated in vacuo and the residue was partitioned between EA (30 mL) and sat. NaHCO3 solution (50 mL). After extraction of the aqueous layer with EA (2 x 30 mL) the combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo to give the title compound which was used for the next reaction without further purification. MS (m/z): 368.9 [M+H*]
G. (R)-Pyrimidine-5-carboxylic acid (l-{l-[4-(2,5-cis/trans-dimethyl-pyrrolidine-l- carbonyl)-phenyl]-ethylcarbamoyl}-cyclopropyl)-amide
(R)-4-[l -({ 1 -[(Pyrimidine-5-carbonyl)-amino]-cyclopropanecarbonyl}-amino)-ethyl]- benzoic acid methyl ester (192 mg, 0.521 mmol) was reacted with LiOH according to the synthesis of (R)-4-(1-tert-butoxycarbonylamino-ethyl)-benzoic acid to give (R)-4-[1-({1- [(pyrimidine-5-carbonyl)-amino]-cyclopropanecarbonyl}-arnino)-ethyl]-benzoic acid.
Subsequent reaction with 2,5-cis/trans-dimethyl-pyrrolidine according to the synthesis of (R)- (1-{1-[4-(2,5-cis/trans-dimethyl-pyrrolidine-1-carbonyl)-phenyl]-ethylcarbamoyl}- cyclopropyl)-carbamic acid tert-butyl ester yielded the title compound as the TFA salt. MS (m/z): 436.1 [M+H*]
Example 2
Synthesis of (R)-N- { 1 -[ 1 -(4-diisopropylcarbamoyl-phenyl)-ethylcarbamoyl]- cyclopropyl}-5-trifluoromethyl-nicotinamide (C pound 43)
Figure imgf000080_0001
A. (R)-4- { 1 - [( 1 -tert-Butoxycarbonylamino-cyclopropanecarbonyl)-amino] -ethyl} - benzoic acid methyl ester (R)-4-(l-Amino-ethyl)-benzoic acid methyl ester (1.03 g, 4.77 mmol) was reacted with 1- tert-butoxycarbonylamino-cyclopropanecarboxylic acid (0.80 g, 3.97 mmol) according to the synthesis of (R)-4-[l -({ 1 -[(pyrimidine-5-carbonyl)-amino]-cyclopropaπecarboπyl} -amino)- ethyl] -benzoic acid methyl ester. The crude compound was purified by flash chromatography on silica gel (elution with n-hexane/EA 3:7) to give the title compound. MS (m/z): 362.7 [M+H+]
B. (R )-{ 1-[(1-Amino-cyclopropanecarbonyl)-amino]-ethyl}-N,N-diisopropyl- benzamide
(R)-4-{1-[(1-tert-Butoxycarbonylamino-cyclopropanecarbonyl)-amino]-ethyI}-benzoic acid methyl ester (423 mg, 1.17 mmol) was reacted with LiOH according to the synthesis of (R)-4-(1-tert-butoxycarbonylamino-ethyl)-benzoic acid to give (R)-4-{1-[1 -tert- butoxycarbonylamino-cyclopropanecarbonyl)-amino] -ethyl} -benzoic acid. The benzoic acid (59.6 mg, 0.171 mmol) was reacted with diisopropyl-amine (71.9 μL, 0.513 mmol) according to the synthesis of (R) -4-[1-({1-[(pyrimidine-5-carbonyl)-amino]- cyclopropanecarbonyl}-amino)-ethyl] -benzoic acid methyl ester. Purification of the crude compound by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA and subsequent reaction with TFA according to the synthesis of (R)-1-amino- cyclopropanecarboxylic acid { 1 -[4-(2,5-cis/trans-dimethyl-pyrrolidine- 1 -carbonyl)-phenyl]- ethyl}-amide yielded the title compound as the TFA salt. MS (m/z): 332.0 [M+H+]
C. (R)-N-{1-[1-(4-diisopropylcarbamoyl-phenyl)-ethylcarbamoyl]-cyclopropyl}-5- trifluoromethyl-nicotinamide
3-Bromo-5-trifluoromethyl-pyridine (1.00 g, 4.42 mmol) was reacted with carbon monoxide according to the synthesis of (R)-4-(l-tert-butoxycarbonylamino-ethyl)-benzoic acid methyl ester to give 5-trifluoromethyl-nicotinic acid methyl ester. Hydrolysis of the methyl ester according to the synthesis of (R)-4-(l-tert-butoxycarbonylamino-ethyl)-benzoic acid and subsequent reaction of the benzoic a (4.80 mg, 0.025 tnmol) with (R)-4-{l-[(l- amino-cyclopropanecarbonyl)-amino] -ethyl }-NJV-diisopropyl-benzamide (4.20 mg, 0.013 mmol) according to the synthesis of (R)-4-[l-({l-[(pyrimidine-5-carbonyl)-amino]- cyclopropanecarbonyl}-amino)-ethyl]-benzoic acid methyl ester yielded the title compound as the TFA salt (purification of the crude compound by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA). MS (m/z): 505.1 [M+H*]
Example 3
Synthesis of (R)-N- { l-[l-(4-diisopropylcarbamoyl-phenyl)-ethylcarbamoyl]- cyclopropyl} -S-fluoro-S-trifluoromethyl-benzamide (Compund 44)
Figure imgf000081_0001
(R)A- { 1 -[( 1 - Amino-cyclopropanecarbonyty-amino] -ethyl } -.V,-V-diisopropyl-benzamide (4.2 mg, 0.013 mmol) was reacted with 3-fluoro-5-trifluoromethyl-benzoic acid (5.3 mg, 0.025 mmol) according to the synthesis of (R)-4-[l-({l-[(pyrimidine-5-carbonyl)-amino]- cyclopropanecarbonyl}-amino)-ethyl]-benzoic acid methyl ester. Purification of the crude compound by reversed phase HPLC using a gradient of ACN in water with 0.1 % TFA yielded the title copound as the TFA salt. MS (m/z): 522.1 [M+H*]
Example 4 Synthesis of (J?)-pyrimidine-5-carboxylic acid (l-{l-[4-((2R,55)-2,5-dimethyl- pyrrolidine-l-carbonyl]-phenyl)-ethylcarbamoyl}-cyclopropyl)-amide (Compound 45)
Figure imgf000081_0002
Reactions A-C were performed based upon literature (Katritzky et al., J. Org. Chem. 1999, 64, 1979-1985) A. (35",5R,7aR)-5-(Benzotriazol-l-yl)-3-phenyl[2,l-b]oxazolopyrrolidine
A mixture of 2,5-dimethoxytetrahydrofuran (2.94 mL, 22.7 mmol) and 0.1 N HCl (90 mL) was refluxed for 1 h and then cooled to RT. A solution of benzotriazole (2.70 g, 22.7 mmol) and (£)-(+)-2-amino-2-phenyl-ethanol 1 g, 22.7 mmol) in DCM (220 mL) was added to the cooled mixture, which was then stiπed overnight. The reaction mixture was washed with 2 N NaOH (3 x 70 mL) and water (2 x 70 mL). The organic layer was dried over Na2SO4 and filtered. After removal of the solvent, the residue was recrystallized from EA to give the title compound. 1H NMR δ 2.20-2.26 (m, IH), 2.55-2.57 (m, 3H), 3.61-3.68 (m, IH), 4.44-4.45 (m, 2H), 5.16-5.17 (m, IH), 5.90-5.94 (m, IH), 7.03-7.08 (m, 5H), 7.21-7.26 (m, 2H), 7.44-7.46 (d, J= 7Hz, IH), 7.90-7.93 (d, J= 7Hz, IH)
B. (2S)-(2-(2/.,5S)-2,5-Dimethyltetrahydro- 1 H- 1 -pyrrolyl-2-phenylethan- 1 -ol A solution of methylmagnesiumbromide (3M in diethylether, 2.20 mL, 6.53 mmol) was added dropwise at O0C to a stirred solution of (3S,5R,7aR)-5-(benzotriazol-l-yl)-3-phenyl[2,l- b]oxazolopyrrolidine (500 mg, 1.63 mmol) in dry THF (33 mL) under nitrogen. After stirring at RT overnight the reaction mixture was washed with 2 N NaOH (3 x 40 mL) and water (2 x 40 mL). The organic layer was dried over Na2SO.;, filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (elution with n-hexane/EA 19:1) to give the title compound. MS (m/z): 220.0 [M+H*]
C. (2R,55)-2,5-Dimethyltetrahydro-l/-r-l-pyrrolidinium chloride (25)-(2-(2R,5S)-2,5-Dimethyltetrahydro-l/f-l-pyrrolyl-2-phenylethan-l-ol (100 mg, 0.46 mmol) was dissolved in methanol (9 mL). The solution was subjected to continuous flow hydrogenation utilizing a Thales H-Cube® flow hydrogenator (Thales Nanotechnology, H- 1031 Budapest, Zahony utca 7 (Graphisoft Park), Hungary. Website: http:// www.thalesnano.com), which contained a cartridge of 10% palladium on carbon as catalyst. The hydrogenation was done at RT under a pressure of 10 bar. The solution of the product was treated with 20 drops of HCl cone, while stirring at RT for 30 minutes. The solvent was evaporated in vacuo and the resulting product was washed with diethylether to give the title compound as the HCl salt. MS (m/z): 100.00 [M+H4]
D. (R)-pyrimidine-5-carboxylic acid (l-{l-[4-((2R,55)-2,5-dimethyl-pyrrolidine-l- carbonyl]-phenyl)-ethylcarbamoyl}-cyclopropyl)-amide
(R)-4-{l-[(l-rerr-butoxycarbonylamino-cyclopropanecarbonyl)-amino]-ethyl}-benzoic acid (30 mg, 0.086 mmol) was reacted with (2R,55)-2,5-dimethyltetrahydro-li¥-l- pyrrolidinium chloride (23 mg, 0.172 mmo cording to the synthesis of (R)-4-[l-({l- [(pyrimidine-5-carbonyl)-amino]-cyclopropanecarbonyl}-amino)-ethyl]-benzoic acid methyl ester to give (R)-(l-{l-[4-((2R,5S)-2,5-dimethyl-pyrrolidine-l-carbonyl)-phenyl]- ethylcarbamoyl}-cyclopropyl)-carbamic acid ter/-butyl ester. Subsequent reaction with TFA according to the synthesis of (R)-l-amino-cyclopropanecarboxylic acid {l-[4-(2,5-cis/trans- dimethyl-pyrrolidine-l-carbonyl)-phenyl]-ethyl}-amide yielded (R)-l-amino- cyclopropanecarboxylic acid { 1 -[4-((2R,5ιS)-2,5-dimethyl-pyrrolidine- 1 -carbonyl)-phenyl]- ethyl} -amide. Subsequent reaction with pyrimidine-5-carboxylic acid according to the synthesis of (R)-4-[l-({ l-[(pyrimidine-5-carbonyl)-amino]-cyclopropanecarbonyl}-amino)- ethylj-benzoic acid methyl ester and purification of the crude product by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA yielded the title compound as the TFA salt. MS (m/z): 436.1 [M-I-H+]
Example 5 Synthesis of (R)-M(l-{l-[4-(2,5-cw/fra»y-dimethyl-2,5-dihydro-pyiTole-l-carbonyl)- phenyl]-ethylcarbamoyl} -cycIopropyl)-3-fluoro-5-trifluoromethyl-benzamide (Compound 55)
Figure imgf000083_0001
(R)-A- { 1 -[(1 -/'er/-Butoxycarbonylamino-cycIopropanecarbonyl)-amino]-ethyl}-benzoic acid (60 mg, 0.17 mmol) was reacted with 2,5-cjj/fra/w-dimemyl-2,5-dihydro-l//-pyrrole (33 mg, 0.34 mmol) according to the synthesis of (R)-4-[l-({l-[(pyrimidine-5-carbonyl)-amino]- cyclopropanecarbonyl}-amino)-ethyl]-benzoic acid methyl ester to give (R)-(l-{l-[4-(2,5- cw//rαns-dimethyl-2,5-dihydro-pyrrole- 1 -carbonyl)-phenyl]-ethylcarbamoyl} -cyclopropyl)- carbamic acid ter/-butyl ester. Subsequent reaction with TFA according to the synthesis of (R)-l-amino-cyclopropanecarboxylic acid { l-[4-(2,5-cw/frαny-dimethyl-pyrrolidine-l- carbonyl)-phenyl]-ethyl} -amide yielded (R)- 1 -amino-cyclopropanecarboxylic acid { 1 -[4-(2,5- cw//rαΛS-dimethyl-2,5-dihydro-pyrrole-l-carbonyl)-phenyl]-ethyl}-amide. Subsequent reaction with 3-fluoro-5-trifluoromethyl-benzoic acid according to the synthesis of (R)-4-[l- ({l-[(pyrimidine-5-carbonyl)-amino]-cyclopropanecarbonyl}-amino)-ethyl]-benzoic acid methyl ester and purification of the crude product by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA yielded the ti ompound as the TFA salt. MS (m/z): 518.2 [M+H+]
Example 6 Synthesis of fR.φ-pyrimidine-S-carboxylic acid (l-{l-[4-(2,5-c/V/rα/«-dimethyl- pyrrolidine- 1 -carbonyl)-2-fluoro-phenyl]-ethylcarbamoyl} -cyclopropyl)-amide (Compound 60)
Figure imgf000084_0001
A. 3-Fluoro-4-methyl-benzoic acid methyl ester Chloro-trimethyl-silane (3.64 mL, 28.8 mmol) was added to a stirring solution of 3- fluoro-4-methyl-benzoic acid (1.11 g, 7.20 mmol) in methanol (24 mL). After stirring at RT overnight the solvent was removed in vacuo and the residue was purified by flash chromatography on silica gel (elution with n-hexane) to give the title compound. GC/MS (m/z): 168.0
Reaction B was performed based upon literature (Gauuan et al, Bioorg. Med. Chem. 2002, 70, 3013-3021)
B. 3-Fluoro-4-formyl-benzoic acid methyl ester
After stirring a suspension of 3-fluoro-4-methyl-benzoic acid methyl ester (500 mg, 2.97 mmol), l-bromo-pyrrolidine-2,5-dione (1.73 g, 8.33 mmol) and benzoyl peroxide (53.1 mg, 0.47 mmol) in tetrachloromethane (36 mL) at 800C overnight the mixture was concentrated in vacuo and the residue was partitioned between EA (150 mL) and water (100 mL). The organic layer was washed with sat. NaHCC^ solution (1 x 100 mL), water (1 x 100 mL) and sat. NaCl solution (1 x 100 mL). The organic layer was dried over Na2SC>4, filtered and concentrated in vacuo. The residue, a mixture of 4-bromomethyl-3-fluoro-benzoic acid methyl ester and 4-dibromornethyl-3-fluoro-benzoic acid methyl ester, was used in the following step without further purification. The residue (50 mg, 0.30 mmol) was dissolved in acetone (2 mL) and water (0.4 mL) and AgNθ3 (171 mg, 0.87 mmol) were added. The flask was covered with aluminum foil to avoid decomposition of the AgNU3. The mixture was stirred at RT overnight. After filtration of th ixture and evaporation of the solvent, the residue was partitioned between EA (30 mL) and sat. NaHCO3 solution (15 mL). The organic layer was washed with water (1 x 15 mL) and sat. NaCl solution (1 x 15 mL), dried over Na2SO4, filtered and concentrated in vacuo to give the title compound. GC/MS (m/z): 182.2
C. fX,iξ)-3-Fluoro-4-(l-hydroxy-ethyl)-benzoic acid methyl ester Methylmagnesiumbromide (3M solution in diethylether, 0.99 mL, 2.96 mmol) was added dropwise at -8O0C to a stirring solution of 3-fluoro-4-formyl-benzoic acid methyl ester (450 mg, 2.47 mmol) in dry THF (10 mL) under nitrogen. After stirring for 80 minutes 5 mL 2 M HCl solution were added and the mixture was allowed to warm to RT. Sat. NaCl solution was added (100 mL) and the mixture was extracted with EA (4 x 100 mL). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (elution with n-hexane/EA 6:1) to give the title compound. GC/MS (m/z): 198.2
D. (2?,φ-4-(l-Azido-ethyl)-3-fluoro-benzoic acid methyl ester
Methanesulfonyl chloride (120 μL, 1.55 mmol) was dissolved in 500 μL DCM and then added dropwise at O0C to a stirred solution of (2?,φ-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 00C for 30 minutes, 10 mL water were added and the mixture was extracted with DCM (3 x 50 mL). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo to give c/s/frαΛy-3-fluoro-4-(l- rnethanesulfonyloxy-ethyl)-benzoic acid methyl ester, which was used in the following step without further purification. The residue was stirred with sodium azide (204 mg, 4.66 mmol) in ACN (4 mL) at 6O0C overnight. The solvent was removed in vacuo and the residue was partitioned between DCM (50 mL) and water (50 mL). The aqueous layer was extracted with DCM (2 x 20 mL). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (elution with n- hexane/EA 9:1) to give the title compound. GC/MS (m/z): 223.0
E. (R.S^-tl-Amino-ethytyO-fluoro-benzoic acid methyl ester (7?,5J-4-(l-Azido-ethyl)-3-fluoro>benzoic acid methyl ester (158 mg, 0.71 mmol) was dissolved in methanol (14 mL). The solution w ubjected to continuous flow hydrogenation utilizing a Thales H-Cube® flow hydrogenator, which contained a cartridge of 10% palladium on carbon as catalyst. The hydrogenation was done at RT under a pressure of 1 bar. The solvent was evaporated to give the title compound. MS (m/z): 197.8 [M+H*]
F. ^iS^-S-Fluoro^-fl-CIl-tCpyrimidine-S-carbonyO-aminolcyclopropanecarbonyl}- amino)-ethyl]-benzoic acid methyl ester f/J,5^-4-(l-Amino-ethyl)-3-fluoro-benzoic acid methyl ester (27.4 mg, 0.14 mmol) was reacted with l-/er/-butoxycarbonylamino-cyclopropanecarboxylic acid and then treated with TFA according to the synthesis of (R)-4- { 1 - [( 1 -amino-cyclopropanecarbonyty-amino] -ethyl } - benzoic acid methyl ester. Susequent reaction with pyrimidine-5-carboxylic acid according to the synthesis of (R)-4-[l-({l-[(pyrimidine-5-carbonyl)-amino]-cyclopropanecarbonyl}- amino)-ethyl] -benzoic acid methyl ester yielded the title coumpound. MS (m/z): 386.9 [M+H+]
G. ^R,S^-Pyrimidine-5-carboxylic acid (l-{l-[4-(2,5-ci,s/»>ians-dimethyl-pyrrolidine-l- carbonyl)-2-fluoro-phenyl] -ethylcarbamoyl } -cyclopropyl)-amide fR,5j-3-Fluoro-4-[l-({l-[φyrimidine-5-carbonyl)-amino]-cyclopropanecarbonyl}- amino)-ethyl]-benzoic acid methyl ester (73.0 mg, 0.19 mmol) was reacted with LiOH according to the synthesis of (R)-4-(l-ter/-butoxycarbonylamino-ethyl)-benzoic acid. Subsequent reaction of the benzoic acid (13.0 mg, 0.04 mmol) with 2,5-cw//rαns-dimethyl- pyrrolidine (4.30 μL, 0.04 mmol) according to the synthesis of (R)-4-[l-({l-[(pyrimidine-5- carbonyl)-amiπo]-cyclopropanecaτbonyl}-amino)-ethyl]-benzoic acid methyl ester and purification of the crude compound by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA yielded the title copound as the TFA salt . MS (m/z): 454.1 [M+H*]
Example 7
Synthesis of (X,iSj-.V-(l-{l-[5-(2,5-cw/fra/ty-dimethyl-pynolidine-l-carbonyl)-pyridin-2- yl]-ethylcarbamoyl}-cyclopropyl)-5-trifluoromethyl-nicotinamide (Compound 65)
Figure imgf000087_0001
Reaction A was performed based on literarture (Chong et al, J. Med. Chem. 2004, 47, 5230-5234)
A. 6-Hydroxymethyl-nicotinic acid methyl ester Sodiumborhydride (0.51 g, 13.5 mmol) was added portionwise at O0C to a stirring suspension of pyridine-2,5-dicarboxylic acid dimethyl ester (1.05 g, 5.38 mmol) and calcium choride (2.39 g, 21.5 mmol) in THF/methanol 1:2 (33 mL). After stirring overnight at O0C 20 mL water were added and the solvent was removed in vacuo. The aqueous residue was extraxted with DCM (3 x 30 mL), the organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (elution with n-hexane/EA 1 :1) to give the title compound. MS (m/z): 167.9 [M+H*]
B. 6-Formyl-nicotinic acid methyl ester
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.
GCMS (m/z): 165.2
C. (R,S;-6-(l-Hydroxy-ethyl)-nicotinic acid methyl ester
6-Formyl-nicotinic acid methyl ester (47 mg, 0.28 mmol) was reacted with methylmagnesium bromide according to the synthesis of (R,φ-3-fluoro-4-(l-hydroxy-ethyl)- benzoic acid methyl ester to give the title compound which was used in the following reation without further purification. MS (m/z): 182.0 [M+H4]
D. (X,5j-6-(l-Amino-ethyl)-nicotinic acid methyl ester
The reaction of (K,5>)-6-(l-hydroxy-ethyl)-nicotinic acid methyl ester (150 mg, 0.83 mmol) with methanesulfonyl chloride and subsequent reaction with sodium azide according to the synthesis of (R,S>4-(l-azido-ethyl)-3-fluoro-benzoic acid methyl ester yielded (R,S)-6- (l-azido-ethyl)-nicotinic acid methyl ester. H genation of the methyl ester according to the synthesis of (R,S)- 4-(1-amino-ethyl)-3-fluoro-benzoic acid methyl ester yielded the title compound. MS (m/z): 181.0 [M+H+]
E. (R,S)-(1 -{ 1 -[5-(2,5-cis/trans-Dimethyl-pyrrolidine-1 -carbonyl)-pyridin-2-yl]- ethylcarbamoyl}-cyclopropyl)-carbamic acid tert-butyl ester
(R,S)-6-(1- Amino-ethyl)-nicotinic acid methyl ester (116 mg, 0.64 mmol) was reacted with l-tert-butoxycarbonylamino-cyclopropanecarboxylic acid according to the synthesis of (R)-4-[1-({1-[(pyrimidine-5-carbonyl)-amino]-cyclopropanecarbonyl}-amino)-ethyl]-benzoic acid methyl ester. Purification of the crude compound by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA yielded (R,S)-6-{1-[(1-tert-butoxycarbonylamino- cyclopropanecarbonyl)-amino] -ethyl} -nicotinic acid methyl ester as the TFA salt. Reaction of the methyl ester with LiOH and subsequent reaction with 2,5-cis /trans- dimethyl-pyrrolidine according to the synthesis of (R)-pyrimidine-5-carboxylic acid (1-{1-[4-(2,5-cis/trans- dimethyl-pyrrolidine-1-carbonyl)-phenyl]-ethylcarbamoyl}-cyclopropyl)-amide yielded the title compound as the TFA salt. MS (m/z): 431.0 [M+H+]
F. (R,S)-N-(1-{1-[5-(2,5-cis/trans-DimethyI-pyrrolidine-l-carbonyl)-pyridin-2-yl]- ethylcarbamoyl}-cyclopropyl)-5-trifluoromethyl-nicotinamide (R,S)-(1-{1-[5-(2,5-cis/trans-Dimethyl-pyrrolidine-1-carbonyl)-pyridin-2-yl]- ethylcarbamoyl}-cyclopropyl)-carbamic acid tert-butyl ester (33.6 mg, 0.08 mmol) was treated with TFA according to the synthesis of (R)-l-amino-cyclopropanecarboxylic acid {1- [4-(2,5-cis/trans-dimethyl-pyrrolidine-l-carbonyl)-phenyl]-ethyl}-amide. Subsequent reaction with 5-trifluoromethyl-nicotinic acid according to the synthesis of (R)-4-[l-({l-[(pyrimidine- 5-carbonyl)-amino]-cyclopropanecarbonyl}-amino)-ethyl]-benzoic acid methyl ester and purification of the crude compound by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA yielded the title compound as the TFA salt. MS (m/z): 504.1 [M+H*]
Example 8 Synthesis of N-(1-(4-(2,5-dimethylpyrrolidine-1-carbonyl)phenyl)ethyl)-1-(l-hydroxy cyclopropanecarboxamido)cyclopropanecarboxamide (Compound 7)
Figure imgf000089_0001
(R)-l-Amino-cycIopropanecarboxylic acid { 1-[4-(2,5-cis/trans-dimethyl-pyrrolidine-1- carbonyl)-phenyl]-ethyl}-amide (10.0 mg, 0.03 mmol) was reacted with 1-hydroxy- cyclopropanecarboxylic acid (2.8 mg, 0.03 mmol) according to the synthesis of (1-{1-[4-(2,5- cis/trans-dimethyl-pyrrolidme-1-carbonyl)-phenyI]-emylcarbamoyl}-cyclopropyl)-carbamic acid tert-butyl ester to give the title copound as the TFA salt. MS (m/z): 414.1 [M+H+]
Example 9
Synthesis of N- {1- [4-(2,5-cis/trans-dimethyl-pyrrolidine- 1 -carbonyl)-2-fluoro- benzylcarbamoyl]-cyclopropyl}-5-trifluoromethyl-nicotinamide (Compound 78)
Figure imgf000089_0002
A. 3-Fluoro-4-formyl-benzoic acid
A solution of 3-fluoro-4-formyl-benzoic acid methyl ester (200 mg, 1.10 mmol) and
LiOH (1.5 mL) in ACN (1.5 mL) was stirred at RT for 1 h and then 1 h at 40°C. The solvent was removed in vacuo and the residue was purified by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA to give the title compound as the TFA salt. GC/MS (m/z):
168.0
B. 4-(2,5-cis/trans-Dimethyl-pyrrolidine- 1 -carbonyl)-2-fluoro-benzaldehyde 2,5-cis/trans-Dimethyl-pyrrolidine (264 mg, 2.67 mmol) was added to a stirring solution of 3-fluoro-4-formyl-benzoic acid (150 mg, 0.89 mmol), WSC (256 mg, 1.34 mmol) and HOBt (180 mg, 1.34 mmol) in DMF (3 mL). After stirring for 3 h at RT the solvent was removed in vacuo and the residue was purified by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA to give the title compound as the TFA salt. MS (m/z): 250.2 [M+H+]
C. (4-Aminomethyl-3-fluoro-phenyl)-(2,5-cis/trans-dimethyl-pyrrolidin-1-yl)-methanone A mixture of 4-(2,5-cis/trans-dimethyl-pyrrolidine-1-carbonyl)-2-fluoro-benzaldehyde (50.0 mg, 0.20 mmol), titanium-tetra-isopropylate (570 mg, 2.00 mmol) and ammonia (7N solution in ethanol, 1 mL) was stirred at RT overnight. Sodiumborhydride (23.0 mg, 0.60 mmol) was added. After stirring for 2 h the solvent was removed in vacuo and the residue was partitioned between EA (20 mL) and sat. NaHCO3 solution (20 mL). The aqueous layer was extracted with EA (3 x 20 mL), the organic layer was dried over Na2SO4, filtered and concentrated in vacuo to give the title compound. MS (m/z): 251.1 [M+H+]
D. {1-[4-(2,5-cis/trans-Dimethyl-pyrrolidine-l-carbonyl)-2-fluoro-benzylcarbamoyl]- cyclopropyl } -carbamic acid tert -butyl ester
(4-Aminomethyl-3-fluoro-phenyl)-(2,5-cis/trans-dimethyl-pyrrolidin-l-yl)-methanone (50.1 mg, 0.20 mmol) was added to a stirring solution of 1-tert-butoxycarbonylamino- cyclopropanecarboxylic acid (80.5 mg, 0.40 mmol), WSC (77.0 mg, 0.40 mmol), HOBt (54.0 mg, 0.40 mmol) and diisopropylamine (100 μL, 0.60 mmol) in DMF (1 mL). After stirring for 3 h at RT the solvent was removed in vacuo and the residue was purified by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA to give the title compound as the TFA salt. MS (m/z): 433.9 [M+H+]
E. 1-Amino-cyclopropanecarboxylic acid 4-(2,5-cis/trans-dimethyl-pyrrolidine-1- carbonyl)-2-fluoro-benzylamide
A solution of {1-[4-(2,5-cis/trans-dimethyl-pyrrolidine-l-carbonyl)-2-fluoro- benzylcarbamoyl] -cyclopropyl} -carbamic acid tert-butyl ester (33 mg, 0.08 mmol) and TFA
(300 μL) in DCM (2700 μL) was stirred at RT for 4 h. The reaction mixture was concentrated in vacuo. The residue was purified by reversed phase HPLC using a gradient of ACN hi water with 0.1% TFA to give the title compound as the TFA salt. MS (m/z): 334.1 [M+H+]
F. 1-Amino-cyclopropanecarboxylic acid 4-(2,5-cis/trans-dimethyl-pyrrolidine-l- carbonyl)-2-fluoro-benzylamide
1 -Amino-cyclopropanecarboxylic acid 4-(2,5-cis/trans-dimethyl-pyrrolidine-l-carbonyl)- 2-fluoro-benzylamide (13 mg, 0.04 mmol) was reacted with 5-trifluoromethyl-nicotinic acid
(33 mg, 0.08 mmol) according to the synthesis of {1-[4-(2,5-cis/trans-dimethyl-pyrrolidine-1- carbonyl)-2-fluoro-benzylcarbamoyl]-cyclopropyl}-carbamic acid tert-butyl ester to give the title compound as the TFA salt. MS (m/z): 507 M+H*]
Example 10
Synthesis of (R)-Λ/-(l-{l-[4-((2R,55)-2,5-dimethyl-pyrrolidine-l-carbonyl)-2-fluoro- phenylJ-ethylcarbamoyll-cyclopropy^-S-fluoro-S-trifluoromethyl-benzamide (Compound 90)
Figure imgf000091_0001
A. 3-Fluoro-4-[(2-methyl-propane-2-suli3nylimino)-methyl]-ben2oic acid methyl ester A mixture of 3-fluoro-4-formyl-benzoic acid methyl ester (529 mg, 2.91 mmol), (S)-(-)2- methyl-propane-2-sulfinic acid amide (352 mg, 2.91 mmol), pyridinium p-toluenesulfonate (36 mg, 0.15 mmol) and magnesium sulfate (5.20 g, 43.7 mmol) in DCM (8 mL) was stirred at RT for 2 days. The solvent was removed in vacuo and the residue was purified by flash chromatography on silica gel (elution with n-hexane/EA 9:1) to give the title compound. MS
(m/z): 285.8 [M+H+]
B. (R)-3-Fluoro-4-[l-(2-methyl-propane-2-sulfinylamino)-ethyl]-benzoic acid methyl ester
Methyl magnesium chloride (22% in THF, 520 μL, 1.49 mmol) was added dropwise at - 600C to a stirred solution of 3-fluoro-4-[(2-methyl-propane-2-sulfmylimino)-methyl]-benzoic acid methyl ester (386 mg, 1.35 mmol) in DCM (7 mL). After stirring for 4 h at -600C sat. ammonium chloride solution (25 mL) was added. The aqueous layer was extracted with DCM (4 x 25 mL), the organic layer was dried over Na2SO.), filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (elution with n-hexane/EA 1:1) to give the title compound. MS (m/z): 301.8 [M+H1]
C. (R)-4-(l-Amino-ethyl)-3-fluoro-benzoic acid methyl ester
A solution of (ϋ)-3-fluoro-4-[l-(2-methyl-propane-2-sulfinylamino)-ethyl]-benzoic acid methyl ester (61 mg, 0.21 mmol) and HCl (4N solution in dioxan, 320 μL, 1.28 mmol) was stirred at RT for 30 minutes. The solvent was removed in vacuo and the residue was recristallized from diethylether to give the title compound as the HCl salt. MS (m/z): 197.8 [M+H+]
D . (R)-3 -Fluoro-4-( 1 - { [ 1 -(3 -fluoro-5-trifluoromethyl-benzoylamino)- cyclopropanecarbonyl]-amino}-ethyl)-benzoic acid methyl ester (R)-4-(l-Amino-ethyl)-3-fluoro-benzoic acid methyl ester (35 mg, 0.15 mmol) was reacted with l-fer^butoxycarbonylamino-cyclopropanecarboxylic acid and then treated with TFA according to the synthesis of (R)-4-{l-[(l-amino-cyclopropanecarbonyl)-amino]-ethyl}- benzoic acid methyl ester. Susequent reaction with 3-fluoro-5-trifluoromethyl-benzoic acid according to the synthesis of (R)-4-[l-({l-[(pyrimidine-5-carbonyl)-amino]- cyclopropanecarbonyl}-amino)-ethyl]-benzoic acid methyl ester yielded the title compound. MS (m/z): 470.8 [M+H+]
E. (R)-N-( 1 - { 1 -[4-((2R,5S)-2,5-dimethyl-pyrrolidine- 1 -carbonyl)-2-fluoro-phenyl]- ethylcarbamoyl}-cyclopropyl)-3-fluoro-5-trifluoromethyl-benzamide (R)-3-Fluoro-4-(l-{[l-(3-fluoro-5-trifluoromethyl-benzoylamino)-cyclopropanecarbonyl]
-amino }-ethyl)-benzoic acid methyl ester (98 mg, 0.21 mmol) was reacted with LiOH according to the synthesis of (R)-4-(1-tert-butoxycarbonylamino-ethyl)-benzoic acid. Subsequent reaction of the benzoic acid (8.4 mg, 0.02 mmol) with (2R,5S)-2,5- dimethyltetrahydro-1H-1-pyrrolidinium chloride (3.2 mg, 0.024 mmol) according to the synthesis of (R)-4-[l-({l-[(pyrimidine-5-carbonyl)-amino]-cyclopropanecarbonyl}-amino)- ethyl]-benzoic acid methyl ester and purification of the crude compound by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA yielded the title compound as the TFA salt . MS (m/z): 538.3 [M+H+]
Example 11
Synthesis of {6-[4-(2,5-cis/trans-dimethyl-pyrrolidine-l-carbonyl)-2-iluoro- benzylamino] -pyridin-3 -yl } -(4-pyridin-4-y 1-piperazin- 1 -yl)-methanone (Compound 106)
Figure imgf000092_0001
A. 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).
After stirring for 4 days at RT the solvent was removed in vacuo and the residue was purified by flash chromatography on silica gel (elution with n-hexane/EA 9:1) to give the title compound. MS (m/z): 252.8 [M+H4]
B. 6-ferr-Butoxycarbonylamino-nicotinic acid
A mixture of ό-ferr-butoxycarbonylamino-nicotinic acid methyl ester (1.62 g, 6.40 mmol) and LiOH (IM solution in H2O, 40 mL) in dioxane (40 mL) was stirred at RT for 2 h. The mixture was concentrated in vacuo and EA (150 mL) was added. The pH of the aqueous layer was adjusted to 4 by the addition of 1 N HCl solution and the aqueous layer was extracted with EA (2 x 150 mL). The combined organic layers were dried over Na2SO^ filtered and concentrated in vacuo. The resulting title compound was used for the next reaction without purification. MS (m/z): 238.8 [M+H*]
C. [5-(4-Pyridin-4-yl-piperazine-l-carbonyl)-pyridin-2-yl]-carbamic acid ter/-butyl ester l-Pyridin-4-yl-piperazine (123 mg, 0.75 mmol) was added to a stirring solution of 6-terf- butoxycarbonylamino-nicotinic acid (90 mg, 0.38 mmol), WSC (86 mg, 0.45 mmol) and HOBt (61 mg, 0.45 mmol) in DMF (3 mL). After stirring for 3 h at RT the solvent was removed in vacuo and the residue was purified by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA to give the title compound as the TFA salt. MS (m/z): 384.0 [M+H*]
D. (6-Amino-pyridin-3-yl)-(4-pyridin-4-yl-piperazin-l-yl)-methanone A solution of [5-(4-pyridin-4-yl-piperazine-l-carbonyl)-pyridin-2-yl]-carbamic acid tert- butyl ester (24 mg, 0.06 mmol) and TFA (5 mL) in DCM (5 mL) was stirred at RT for 4 h. The reaction mixture was concentrated in vacuo to give the title compound as the TFA salt. MS (m/z): 284.2 [M+H+]
E. 4-(2,5-cw//rflns-Dimethyl-pyrrolidine- 1 -carbonyl)-2-fluoro-benzaldehyde
3-Fluoro-4-formyl-benzoic acid (46 mg, 0.28 mmol) was reacted with 2,5-cis/trans- dimethyl-pyrrolidine (34 μL, 0.28 mmol) according to the synthesis of [5-(4-pyridin-4-yl- piperazine-l-carbonyO-pyridin^-yll-carbamic d tert-butyl ester to give the title compound as the TFA salt. MS (m/z): 250.1 [NH-H+]
F. {6-[4-(2,5-cw/frαn.?-DimeΛyl-pyπolidine- 1 -carbonyl)-2-fluoro-benzylamino]-pyridin- 3-yl}-(4-pyridin-4-yl-piperazin-l -yl)-methanone
A mixture of (6-amino-pyridin-3-yl)-(4-pyridin-4-yl-piperazin-l-yl)-methanone (17.5 mg,
0.062 mmol), 4-(2,5-cw//rαΛS-dimethyl-pyrτolidine-l-carbonyl)-2-fluoro-benzaldehyde (11.0 mg, 0.044 mmol) and titanium-tetra-isopropylate (62.5 mg, 0.220 mmol) in dichloroethane (2 mL) was stirred at RT overnight. Sodiumtriacetoxyborhydride (18.6 mg, 0.088 mmol) was added and the mixture was stirred at RT for additional 3 h. The solvent was removed in vacuo and the residue was partitioned between EA (20 mL) and sat. NaHCθ3 solution (20 mL). The aqueous layer was extracted with EA (2 x 20 mL), the organic layer was dried over Na2SO-J, filtered and concentrated in vacuo. The residue was purified by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA to give the title compound as the TFA salt. MS (m/z): 517.3 [M-I-H+]
Example 12
Synthesis of {6-[4-(2,5-c/V/rαns-dimethyl-2,5-dihydro-pyrrole- 1 -carbonyl)-2-fluoro- benzylamino]-pyridin-3-yl}-(4-pyridin-4-yl-piperazin-l-yl)-methanone (Compound 111)
Figure imgf000094_0001
A. 3-Fluoro-4-{[5-(4-pyridin-4-yl-piperazine-l-carbonyl)-pyridin-2-ylamino]-methyl}- benzoic acid methyl ester
(6-Amino-pyridin-3-yl)-(4-pyridin-4-yl-piperazin-l-yl)-methanone (92 mg, 0.32 mmol) was reacted with 3-fluoro-4-formyl-benzoic acid methyl ester (65 mg, 0.35 mmol) according to the synthesis of {6-[4-(2,5-cw/frany-dimethyl-pyrrolidine-l-carbonyl)-2-fluoro- benzylamino]-pyridin-3-yl}-(4-pyridin-4-yl-piperazin-l-yl)-methanone to give the title compound, which was used in the following step without further purification. MS (m/z):450.3 [M-J-H+] B . { 6- [4-(2,5 -c/5/frαΛS-Dimethyl-2,5 -dihy pyrrole- 1 -carbonyl)-2-fluoro- benzylaminol-pyridin-S-ylJ^-pyridin^-yl-piperazin-l-y^-methanone
3-Fluoro-4- { [5 -(4-pyridin-4-yl-piperazine- 1 -carbonyl)-pyridin-2-ylamino] -methyl } - benzoic acid methyl ester (110 mg, 0.23 mmol) was reacted with LiOH according to the synthesis of 3-fluoro-4-formyl-benzoic acid to give 3-fluoro-4- { [5-(4-pyridin-4-yl-piperazine- l-carbonyl)-pyridin-2-ylamino]-methyl}-benzoic acid. The benzoic acid (18 mg, 0.04 mmol) was reacted with 2,5-c/5/rrαΛS-dimethyl-2,5-dihydro-lH-pyrrolidin (25 μL, 0.20 mmol) according to the synthesis of [5-(4-pyridin-4-yl-piperazine-l-carbonyl)-pyridin-2-yl]- carbamic acid terf-butyl ester to give the title compound as the TFA salt. MS (m/z): 515.2 PvRH+]
Example 13
Synthesis of (R)-pyrimidine-5-carboxylic acid (l-{l-[4-(ethyl-isopropyl-carbamoyl)- phenyl]-ethylcarbamoyl}-l-methyl-ethyl)-amide (Compound 115)
Figure imgf000095_0001
A. (R)-4-[l-(9i¥-Fluoren-9-ylmethoxycarbonylamino)-ethyl] -benzoic acid
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 chlorofoπnate (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 resulting aqueous solution was extracted with diethylether (2 x 150 mL) and the organic layer was dried over Na2SO4, filtered and concentrated in vacuo to give the title compound. MS (m/z): 387.7 [M+H4]
B. Resin-bound (R)-4-(l-amino-ethyl)-benzoic acid 4-[l-(9/-r-Fluoren-9-ylmethoxycarbonylamino)-ethyl]-benzoic acid (296 mg, 0.8 mmol) and DIPEA (332 μL, 1.9 mmol) were dissolved in DCM (8 mL). The solution was added to 2- chlorotrityl chloride resin (loading: 1.4 mmol/g, 1.09 g, 1.5 mmol) swollen in DCM (8 mL) and the mixture was shaken at RT for 30 minu The mixture was quenched with methanol (800 μL). 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).
C. Resin-bound (R)-4-[l-(2-amino-2-methyl-propionylamino)-ethyl]-benzoic acid
A solution of 2-(9/-r-fluoren-9-ylmethoxycarbonylamino)-2-methyl-propionic acid (63 mg, 0.19 mmol), HATU (74 mg, 0.19 mmol) and DIPEA (68 μL, 0.39 mmol) in DMF (1 mL) was stirred at RT for 5 minutes and then added to resin-bound (R)-4-(l-amino-ethyl)-benzoic acid (100 mg, 0.07 mmol) swollen in DCM (1 mL). The mixture was shaken at RT for 1.5 h and then quenched with methanol. The resin was filtered and washed with DMF (4 x 2 mL) to give resin-bound 4-{l-[2-(9//-fluoren-9-ylmethoxycarbonylamino)-2-methyl- propionylamino]-ethyl} -benzoic acid. A test cleavage with TFA showed the right product. MS (m/z): 472.9 [M+H*]. Piperidin (25% solution in DMF, 4 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.
D. Resin-bound (R)-4-(l-{2-methyl-2-[(pyrimidine-5-carbonyl)-amino]-propionyl amino }-ethyl)-benzoic acid
Resin-bound (R)-4-[l-(2-amino-2-methyl-propionylamino)-ethyl]-benzoic acid (100 mg,
0.065 mmol) was reacted with 5-pyrimidinecarboxylic acid according to the synthesis of resin-bound 4-{l-[2-(9H-fluoren-9-ylmethoxycarbonylamino)-2-methyl-propionylamino]- ethyl} -benzoic acid. A test cleavage with TFA showed the right product. MS (m/z): 356.9 [M+rT]
E. (R)-A-(I - {2-Methyl-2-[(pyrimidine-5-carbonyl)-amino]-propionylamino } -ethyl)- benzoic acid
A suspension of resin-bound (R)-4-(l-{2-methyl-2-[(pyrimidine-5-carbonyl)-amino]- propionylamino}-ethyl)-benzoic acid (100 mg, 0.065 mmol) in l,l,l,3,3,3,-hexafluoro-2- propanol/DCM (4:1, 2.5 mL) was shaken at RT for 3 h. The resin was filtered and washed with DCM (2 x 3 mL). The filtrate was concentrated in vacuo to give the title compound. MS (m/z): 356.9 [M+H*]
F. (^-Pyrimidine-S-carboxylic acid (l-{l-[4-(ethyl-isopropyl-carbamoyl)-phenyl]- ethylcarbamoyl } - 1 -methyl-ethyl)-amide Λf-Ethylisopropylamine (41 μL, 0.337 mmol) was added to a stirred solution of (R)-4-(l-
{2-methyl-2-[(pyrimidine-5-carbonyl)-amino]-propionylamino}-ethyl)-benzoic acid (12.0 mg, 0.034 mmol) and HATU (12.8 mg, 0.034 mmol) in DMF (0.4 mL). After stirring overnight the reaction mixture was concentrated in vacuo and the residue was purified by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA to give the title compound as the TFA salt. MS (m/z): 426.1 [M+H*]
Example 14
Synthesis of (R)-.V-(cyclopropyl-{ 1 -[4-(2,5-cis//rα/w-dimethyl-pyrrolidine-l -carbonyl)- phenyl]-ethylcarbamoyl}-methyl)-3-fluoro-5-trifluoromethyl-benzamide (Compound 116)
Figure imgf000097_0001
A. Resin-bound (R)-4-[l-(2-amino-2-cyclopropyl-acetylamino)-ethyl]-benzoic acid Resin-bound (R)-4-(l-amino-ethyl)-benzoic acid (58.7 mg, 0.038 mmol) was reacted with
(ϋ)-cyclopropyl-[(9i/-fluoren-9-ymiethoxycarbonylamino)]-acetic acid (38.6 mg, 0.114 mmol) according to the synthesis of 4-{l-[2-(9//-fluoren-9-ylmethoxycarbonylamino)-2- methyl-propionylamino]-ethyl}-benzoic acid. A test cleavage with TFA showed the right product 4-{l-[2-cyclopropyl-2-(9/-'-fluoren-9-ylmethoxycarbonylamino)-acetylamino]- ethyl} -benzoic acid. MS (m/z): 485.0 [M+H*]. Deprotection with piperidin according to the synthesis of resin-bound (i?)-4-[l-(2-amino-2-methyl-propionylamino)-ethyl]-benzoic acid yielded the title compound.
B. (R)-JV-(Cyclopropyl- { 1 -[4-(2,5-c/-f//ra/is-dimethyl-pyrrolidine- 1 -carbonyl)-phenyl]- ethylcarbamoyl}-methyl)-3-fluoro-5-trifluoromethyl-benzamide
Resin-bound (R)-4-[l-(2-anτino-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. Cleavage with l,l,l,3,3,3,-hexafluoro-2- propanol/DCM according to the synthesis of (R)-4-(l-{2-methyl-2-[(pyrimidine-5-carbonyl)- amino]-propionylamino}-ethyl)-benzoic acid (12 mg, 0.034 mmol) yielded 4-{l-[2- cyclopropyl-2-(3-fluoro-5-trifluoromethyl-benzoylamino)-acetylamino]-ethyl}-benzoic acid. Reaction of the acid (12.5 mg, 0.028 mmol) with 2,5-cw/rrαfw-dimethylpyrrolidin (6.8 μL, 0.055 mmol) according to the synthesis of (i?)-pyrimidine-5-carboxylic acid (l-{l-[4-(ethyl- isopropyl-carbamoyl)-phenyl]-ethylcarbamoyl}-l-methyl-ethyl)-amide yielded the title compound as the TFA salt. MS (m/z): 534.1 [M+H*]
Example 15
Synthesis of (R1S)-N-(I -{ 1 -[2-chloro-4-((2R,5R)-2,5-dimethyl-pyrrolidine-l-carbonyl)- phenylJ-ethylcarbamoy^-cyclopropyO-S-fluoro-S-trifluoromethyl-benzamide (Compound 100)
Figure imgf000098_0001
A. 3-Chloro-4-methyl-benzoic acid methyl ester
Chloro-trimethyl-silane (6.0 mL, 46.9 mmol) was added to a stirring solution of 3-chloro- 4-methyl-benzoic acid (2.0 g, 11.7 mmol) in methanol (40 mL). After stirring at RT overnight the solvent was removed in vacuo to give the title compound, which was used in the following step without further purification. GC/MS (m/z): 184
B. 3-Chloro-4-formyl-benzoic acid methyl ester
After stirring a suspension of 3-chloro-4-methyl-benzoic acid methyl ester (1.04 g, 5.61 mmol), l-bromo-pyrτolidine-2,5-dione (3.26 g, 15.7 mmol) and benzoyl peroxide (100.2 mg, 0.88 mmol) in tetrachloromethane (68 mL) at 800C overnight the mixture was concentrated in vacuo and the residue was partitioned between EA (300 mL) and water (200 mL). The organic layer was washed with sat. NaHCθ3 solution (1 x 200 mL), water (1 x 200 mL) and sat. NaCl solution (1 x 200 mL). The organic layer was dried over Na2SO4, 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 AgNO3 (3.22 g, 16.3 mmol) were added. The flask was covered with aluminum foil to avoid decomposition of the AgNU3. The mixture was stirred at RT overnight. After filtration of the mixture and evaporation of the solvent, the residue was partitioned between EA (700 mL) and sat. NaHCC>3 solution (350 mL). The organic layer was washed with water (1 x 350 mL) and sat. NaCl solution (1 x 350 mL), dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (elution with n-hexane/EA 9:1) to give the title compound. GC/MS (m/z): 198
C. fR,ι9-4-(l-Azido-ethyl)-3-chloro-benzoic acid methyl ester
3-Chloro-4-formyl-benzoic acid methyl ester (398 mg, 2.01 mmol) was reacted with methylmagnesiumbromide according to the synthesis of ^R,iS^-3-fluoro-4-(l-hydroxy-ethyl)- benzoic acid methyl ester. Reaction of the ester with methanesulfonyl chloride and subsequent reaction with sodium azide according to the synthesis of (ftiSJM-O-azido-ethyl)-
3-fluoro-benzoic acid methyl ester yielded the title compound. GC/MS (m/z): 239
D. fK,iS^-4-(l-Amino-ethyl)-3-chloro-benzoic acid methyl ester
(R,φ-4-(l-Azido-ethyl)-3-chloro-benzoic acid methyl ester (181 mg, 0.76 mmol) was subjected to continuous flow hydrogenation utilizing a Thales H-Cube® flow hydrogenator according to the synthesis of ^R,-S^-4-(l-amino-ethyl)-3-fluoro-benzoic acid methyl ester to give the title compound. MS (m/z): 213.8 [M+H*]
E. fK,5>3-Chloro-4-(l-{[l-(3-fluoro-5-trifluoromethyl-benzoylamino)- cyclopropanecarbonyl]-amino}-ethyl)-benzoic acid methyl ester
(/Z,iS)-4-(l-Amino-ethyl)-3-chloro-benzoic acid methyl ester (17.7 mg, 0.083 mmol) was reacted with l-/er/-butoxycarbonylamino-cyclopropanecarboxylic acid and then treated with TFA according to the synthesis of (R)-4-{l-[(l-amino-cyclopropanecarbonyl)-amino]-ethyl}- benzoic acid methyl ester. Susequent reaction with 3-fluoro-5-trifluoromethyl-benzoic acid according to the synthesis of (R)-4-[l-({l-[(pyrimidine-5-carbonyl)-amino]- cyclopropanecarbonyl}-amino)-ethyl] -benzoic methyl ester yielded the title compound. MS (m/z): 472.9 [M+H+]
F. (R,S) -N-(l-{l-[2-Chloro-4-((2R,5R)-2,5-dimethyl-pyrrolidine-1-carbonyl)-phenyl]- ethylcarbamoyl} -cyclopropyl)-3-fluoro-5-trifluoromethyl-benzamide
(R,S)-3-Chloro-4-(1-{[1-(3-fluoro-5-trifluoromethyl-benzoylamino)-cyclopropane carbonyl]-amino}-ethyl)-benzoic acid methyl ester (40.3 mg, 0.083 mmol) was treated with LiOH (1.4 ml) to give (R,S)-3-chloro-4-(1-{[1-(3-fluoro-5-trifluoromethyl-benzoylamino)- cyclopropanecarbonyl]-amino}-ethyl)-benzoic acid. Subsequent reaction of the benzoic acid with (2R,5R)-2,5-dimethyl-pyrrolidine according to the synthesis of (R,S)-pyrimidine-5- carboxylic acid (1-{ 1-[4-(2,5-cis/trans-dimethyl-pyrrolidine-1-carbonyl)-2-fluoro-phenyl]- ethylcarbamoyl}-cyclopropyl)-amide yielded the title compound as the TFA salt. MS (m/z): 554.0 [M+H+]
Example 16
Synthesis of (R,S)-N-(1-{1-[2-Chloro-4-((2S,5S)-2,5-dimethyl-pvrrolidine-1-carbonyl)- phenyl]-ethylcarbamoyl} -cyclopropyl)-3-fluoro-5-trifluoromethyl-benzamide (Compound 99)
Figure imgf000100_0001
Reactions A-D were performed based on literarture (Schlessinger et al, Tetrahedron Lett. 1987, 28, 2083-2086)
A. (2-Hydroxy-l(-S)-methyl-ethyl)-carbamic acid benzyl ester
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. To the combined THF solutions was added 2 N NaOH solution (170 mL) and benzyl chloroformate (25.3 g, 0.15 mol). After stirring 1 h, the biphasic system was separated and the aqueous layer was extracted with EA (1 x 50 mL). The org layer was dried over Na2SO4, filtered and concentrated in vacuo. Crystallisation of the crude compound from THF/cyclohexane yielded the title compound.
B. (ιS)-Toluene-4-sulfonic acid 2-benzyloxycarbonylamino-propyl ester
To a solution of (2-hydroxy-l(5)-methyl-ethyl)-carbamic acid benzyl ester (12.0 g, 0.06 mol) in pyridine (190 mL) was added p-toluenesulfonyl chloride (4.40 g, 0.11 mol) at 00C. After stirring at RT overnight, diethyl ether (50 mL) was added. The mixture was filtered and the solids were washed with diethyl ether. The organic extract was washed with 0.5N H2SO4 solution (3 x 20 mL), 5% NaHCO3 solution (1 x 20 mL) and sat. NaCl solution (1 x 20 mL), dried over Na2SO4, filtered and concentrated in vacuo. Crystallisation of the crude compound from THF/n-hexane yielded the title compound.
C. (iS)-Toluene-(2-iodo-l-methyl-ethyl)-carbamic acid benzyl ester To a solution of (S)-toluene-4-sulfonic acid 2-benzyloxycarbonylamino-propyl ester (12.3 g, 0.03 mol) in acetone (113 mL) was added solid sodium iodide (50.7 g, 0.34 mol) at 00C. After stirring at 00C for 30 minutes, the reaction mixture was stirred at RT for 48 h. The solvent was removed in vacuo at RT. The residue was suspended in EA. The mixture was filtered and the solids were washed with EA. The organic extract was washed with H2O (2 x 20 mL), 5% Na2S2O3 solution (1 x 20 mL) and sat. NaCl solution (1 x 20 mL), dried over Na2SO4, filtered and concentrated in vacuo. Crystallisation of the crude compound from THF/n-hexane yielded the title compound.
D. (S)- (l-Methyl-pent-4-enyl)-carbamic acid benzyl ester AUyI magnesium chloride (19.0 mL, 0.038 mol, 2 M solution in THF) was added dropwise to a stirring suspension of copper iodide (3.60 g, 0.019 mol) in anhydrous THF (64 mL) at -780C. Upon completion of the addition, the mixture was warmed to -36°C for 5 minutes, then cooled to -78°C, and (5)-toluene-(2-iodo-l-methyl-ethyl)-carbamic acid benzyl ester (4.00 g, 0.013 mol) in THF (19 mL) was added dropwise. After stirring at -36°C for 6 h, sat. NH4Cl solution (10 mL) was added and the mixture was allowed to warm to RT. The solvent was removed in vacuo. The residue was suspended in diethyl ether. The mixture was filtered and the solids were washed with diethyl ether. The organic extract was washed with sat. NaCl solution (1 x 20 mL), NH4OH solu (3 x 20 mL) and sat. NaCl solution (1 x 20 mL), dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (elution with n-hexane/diethyl ether 1:1) to give the title compound. MS (m/z): 233.8 [M+H4]
Reaction E was performed based on literature (Harding et al, J.Org.Chem. 1981, 46, 3920-3922)
E. (2-?,5S)-2,5-Dimethyl-pyrrolidine-l-carboxylic acid benzyl ester
Mercuric acetate (2.05 g, 6.43 mmol) was added to a stirring solution of (S)-(I -methyl- pent-4-enyl)-carbamic acid benzyl ester (1.00 g, 4.29 mmol) in THF (43 mL). The mixture was purged with nitrogen, covered with aluminum foil and stirred at RT overnight. A solution of sodium borhydride (160 mg, 4.24 mmol) in 2 N NaOH (0.32 mL) was added dropwise and the mixture was stirred at RT overnight. Sat. Na2CO3 solution (3 mL) was added. After stirring at RT for 4 h, the solvent was removed in vacuo. The residue was diluted with diethyl ether and extracted with sat. Na2CO3 solution (3 x 15 mL). The aqueous layer was extracted with diethyl ether (3 x 20 mL). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel
(elution with DCM/ methanol 30:1) to give the title compound. MS (m/z): 233.8 [M+H*]
F. (2S',55)-2,5-Dimethyl-pyrrolidine
(25,5S)-2,5-Dimethyl-pyrrolidine-l-carboxylic acid benzyl ester (100 mg, 0.43 mmol) was dissolved in methanol (9 mL). The solution was subjected to continuous flow hydrogenation utilizing a Thales H-Cube® flow hydrogenator, which contained a cartridge of 10% palladium on carbon as catalyst. The hydrogenation was done at RT under a pressure of 10 bar. 20 drops of concentrated HCl were added. The solvent was evaporated to give the title compound as the HCl salt. MS (m/z): 100.0 [M+H*]
G. (K,5;-N-(l-{l-[2-Chloro-4-((25,55)-2,5-dimethyl-pyrrolidine-l-carbonyl)-phenyl]- emylcarbamoylJ-cyclopropyl^-fluoro-S-trifluoromemyl-benzamide f'i?,5/)-3-Chloro-4-(l-{[l-(3-fluoro-5-trifluoromethyl-benzoylamino)-cyclopropane carbonyl]-amino}-ethyl)-benzoic acid was reacted with (25r,5S)-2,5-dimethyl-pyrrolidine according to the synthesis of (K,5>pyrimidine-5-carboxylic acid (l-{\-[4-(2,5-cis/trans- dimethyl-pyrrolidine-l-carbonyl)-2-fluoro-phe ethylcarbamoyl}-cyclopropyl)-amide to give the title compound as the TFA salt. MS (m/z): 554.0 [M+H*]
Example 17 Synthesis of N-(( 1 R)- 1 -(4-(2,5-cw//ft7n$-dimethylpyrrolidine- 1 -carbonyl)phenyl)ethyl)-2-
(3-fluoro-5-(trifluoromethyl)benzoyl)hydrazinecarboxamide (Compound 150)
Figure imgf000103_0001
First step of reaction A was performed based on literarture (Gibson et al, J. Org. Chem. 1999, 64, 7388-7394) A. Resin-bound (ϋ)-4-(l-(hydrazinecarboxamido)ethyl) benzoic acid
Hydrazinecarboxylic acid 9/-r-fluoren-9-ylmethyl ester (150 mg, 0.59 mmol) was suspended in DCM (5 mL) and sat. NaHCU3 solution (5 mL). The biphasic mixture was stirred at 00C 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 00C for 10 minutes, the layers were separated and the aqueous layer was extracted with DCM (3 x 10 mL). The combined organic layers were dried over Na2SO.j, filtered and concentrated in vacuo to give 5-(9H-fluoren-9-ylmethoxy)-3/f-[l,3,4]oxadiazol- 2-one. The crude product (350 mg, 0.483 mmol) was dissolved in DCM (2 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 μL, 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). A test cleavage with TFA showed the right product (R)-4-(\-(2-(((?H- fluoren-9-yl)methoxy)carbonyl)hydrazinecarboxamido)ethyl) benzoic acid. MS (m/z): 445.8 [M-I-H+]. Piperidin (20% solution in DMF, 4 mL) was added and the mixture was shaken for 15 minutes. The resin was filtered, washed with DMF (4 x 2 mL), methanol (4 x 2 mL) and DCM (4 x 2 mL) and dried in vacuo. A test cleavage with TFA showed the right product. MS (m/z): 223.8 [M+H*]
B. (R)-4-(l-(2-(3-Fluoro-5-(trifluoromethyl)benzoyl)hydrazinecarboxamido)ethyl) benzoic acid
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 μL, 0.345 mmol) in DCM (1 mL) was added to resin- bound (R)-4-(l-(hydrazinecarboxamido)ethyl)benzoic acid (106 mg, 0.069 mmol). The mixture was shaken 2 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). The suspension was shaken at RT for 30 minutes. The resin was filtered and washed with DCM (2 x 2 mL). The filtrate was concentrated in vacuo. The residue was purified by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA to give the title compound. MS (m/z): 413.8 [M+H*]
C. iV-((lR)-l-(4-(2,5-ciV/rαΛS-Dimethylpyrrolidine-l-carbonyl)phenyl)ethyl)-2-(3- fluoro-5-(trifluoromethyl)benzoyl)hydrazinecarboxamide
2,5-c/j//rΛΛy-Dimethyl-pyrrolidine (4.7 mg, 0.047 mmol) was added to a solution of (R)- 4-(l-(2-(3-fluoro-5-(trifluoromethyl)benzoyl)hydrazinecarboxamido)ethyl)benzoic acid (16.2 mg, 0.039 mmol), WSC (9.0 mg, 0.047 mmol) and collidine (25.8 μL, 0.196 mmol) in DCM/DMF 9:1 (1 mL). After stirring overnight at RT, the mixture was concentrated in vacuo. The residue was purified by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA to give the title compound. MS (m/z): 495.1 [M+H*]
Example 18
Synthesis of N-((li?)-l-(4-(2,5-cw//rfln-f-dimethylpyrrolidine-l-carbonyl)phenyl)ethyl)-2- (3-fluoro-5-(trifluoromethyl)benzoyl)-l-methylhydrazinecarboxamide (Compound 151)
Figure imgf000104_0001
Reaction A and the first step of reaction B were performed based on literarture (Gibson et al, J. Org.Chem. 1999, 64, 7388-7394)
A. JV-Methyl-hydrazinecarboxylic acid 9iJ-fiuoren-9-ylmethyl ester
Methylhydrazine (173 μl, 3.26 mmol) was added at -780C 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 μl, 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 mixture was concentrated in vacuo and the residue was purified by flash chromatography on silica gel (elution with n-hexane/EA 7:3) to give the title compound MS (m/z): 268.8 [M+H*]
B . (R)-4-( 1 -(2-(3 -Fluoro-5-(trifluoromethyl)benzoyl)- 1 -methylhydrazinecarboxamido) ethyl) benzoic acid
Phosgene (20% solution in toluene, 3.43 ml, 6.51 mmol) was added dropwise at 100C to a stirred solution of iV-methyl-hydrazinecarboxylic acid 9i/-fluoren-9-ylmethyl ester (1.81 g, 3.26 mmol) in dioxane (15 ml). The mixture was stirred at 1O0C for 5 minutes and then stirred at RT for 1.5 h. The mixture was concentrated in vacuo to give (9itf-fluoren-9-yl)methyl 2- (chlorocarbonyl)-2-methylhydrazinecarboxylate. 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 μL, 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). A test cleavage with TFA showed the right product (R)-4-(l-(2-(((9/-r-fluoren-9-yl)methoxy)carbonyl)-l- methylhydrazinecarboxamido)ethyl)benzoic acid. MS (m/z): 459.8 [M+H*]. Piperidin (20% solution in DMF, 4 mL) was added and the mixture was shaken for 30 minutes to give resin- bound (R)-4-(l-(l-methylhydrazinecarboxamido)ethyl)benzoic acid. 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 μL, 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). The suspension was shaken at RT for 20 minutes. The resin was filtered and washed with DCM (2 x 2 mL). The filtrate was concentrated in vacuo. The residue was purified by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA to give the title compound. MS (m/z): 427.7 [M+H+]
C. -v'-((l^)-l-(4-(2,5-cw/rrawj-DimethylpyrroIidine-l-carbonyl)phenyl)ethyl)-2-(3- fluoro-5-(trifluoromethyl)benzoyl)-l-methylhy inecarboxamide
2,5-c/s//rα/w-Dimethyl-pym>lidine (2.8 mg, 0.028 mmol) was added to a solution of (R)- 4-(l-(2-(3-fluoro-5-(trifluoromethyl)benzoyl)-l-methylhydrazinecarboxamido)ethyl)benzoic acid (10 mg, 0.023 mmol), WSC (5.4 mg, 0.028 mmol) and collidine (15.4 μL, 0.117 mmol) in DCM/DMF 9:1 (1 mL). After stirring overnight at RT, the mixture was concentrated in vacuo. The residue was purified by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA to give the title compound. MS (m/z): 509.0 [M+H*]
Example 19 Synthesis of ΛT-((R)-l-(5-((2R,5S)-2,5-Dimethylpyrrolidine-l-carbonyl)-3-fluoropyridin-
2-yl)ethyl)-l-ethyl-2-(3-fluoro-5-(trifluoromethyl)benzoyl)hydra2inecarboxamide (Compound 268)
Figure imgf000106_0001
A. 5 -Bromo-3 -fluoro-pyridine-2-carbonitrile H2SO4 (1 ml, 5 μmol) was added to a TBAF solution in THF (657 ml, 0.66 mol, 1 M).
DMF (200 mL) were added at -400C until the suspension became a clear solution. 5-bromo-3- nitropyridin-2-carbonitrile (50.0 g, 0.22 mol) in DMF (800 mL) were added slowly. The solution was stirred at -400C for 30 min, till starting material could not be detected by TLC (n-hex/EA 5:1). The reaction was quenched with 2 M HCl (500 ml) at -4O0C to afford pH 3. The mixture was extracted twice with EA. The combined organic layers were washed with water, dried over sodium sulfate and concentrated in vacuo. The crude product was purifieded by chromatography on silica gel.
B. 5-Bromo-3-fluoro-pyridine-2-carboxylic acid Cone. HCl (300 mL) were added to 5-Bromo-3-fluoro-pyridine-2-carbonitrile (21.0 g,
0.11 mol) and stirred over night at 600C. After evaporation ether was added and the suspension was stirred for additional time. The solid was collected and dried. The crude product was used without any further purification. MS (m/z): 219.8 [M+H*] C. 5-Bromo-3-fluoro-pyridine-2-carboxylic acid methoxy-methyl-amide
TEA (17.4 ml, 0.13 mol), EDCl (83.2 g, 0.43 mol), HOBt (2.90 g, 0.02 mol) and N,O- Dimethylhydroxylamine x HCl (25.0 g, 0.26 mol) were added to a solution of 5-Bromo-3- fluoro-pyridine-2-carboxylic acid (55.5 g, 0.22 mol) in DMF (1000 mL). After stirring overnight the solvent was reduced in vacuo. EA and brine were added and the aq. layer was extracted with EA. The combined organic layers were washed with brine, dried over sodium sulfate and the solvent was removed under reduced pressure. The crude product was purified by chromatography on silica gel. MS (m/z): 262.8 [M+H*]
D. 5-Bromo-3-fluoro-pyridine-2-carbaldehyde
IM LiAlH4 in THF (50 ml, 0.05 mol) was added slowly at -78°C to a solution of 5- bromo-3-fluoro-pyridine-2-carboxylic acid methoxy-methyl-amide (29.0 g, 0.11 mol) in THF (350 mL) After 20 min stirring at -78°C the mixture was quenched with water and brine and allowed to come to room temperature. EA was added and the suspension was filtered over celite. The aq. layer was extracted with EA and the combined organic layers were washed with brine and dried over sodium sulfate. After evaporation the crude product was purified by chromatography on silica gel (n-hexan/EA). MS (m/z): 203.9 [M+H*]
E. 2-Methyl-propane-2-sulfinic acid 5-bromo-3-fluoro-pyridin-2-ylmethyleneamide
(R)-(+)-2-Methyl-2-propansulfinamide (11.6 g, 0.10 mol), magnesium sulfate (115 g,
0.10 mol), PPTS (2.40 g, 0.01 mol) and cesium carbonate (33.7 g, 0.10 mol) were added to a solution of 5-bromo-3-fluoro-pyridine-2-carbaldehyd (19.5 g, 0.10 mol) in DCM (600 ml).
After stirring over night the reaction was filtered over celite and the solvent was removed in vacuo. The crude product was purified by chromatography on silica gel (n-hexan/EA).
MS (m/z): 306.6 [M+H+]
F. 2-Methyl-propane-2-sulfinic acid [l-(5-bromo-3-fluoro-pyridin-2-yl)-ethyl]-amide
Methylmagnesium chloride solution (29.9 ml, 0.09 mol, 22% in THF) was added slowly to a solution of 2-methyl-propane-2-sulflnic acid 5-bromo-3-fluoro-pyridin-2- ylmethyleneamide (17.5 g, 0.06 mol) in DCM (570 mL) at -550C. The reaction mixture was quenched with brine after stirring for 20 minutes at -550C. Celite was added and the suspension was filtered. The layers were separ and the aq. layer was extracted with DCM. The combined organic layers were dried over sodium sulfate and the solvent was removed in vacuo. The crude product was purified by chromatography on silica gel (n-hexan/EA). MS (m/z): 322.6 [M+H+]
G. 5-Fluoro-6-[l-(2-methyl-propane-2-sulfinylamino)-ethyl]-nicotinic acid methyl ester To a stirred suspension of 2-Methyl-propane-2-sulfinic acid [l-(5-bromo-3-fluoro- pyridin-2-yl)-ethyl] -amide (1.40 g, 4.33 mmol), l,4-bis-(diphenylphosphino)ferrocene (0.53 g, 0.96 mmol) and Pd(OAc)2 (0.21 g, 0.91 mmol) in anhydrous DMSO (10 mL) under an atmosphere of nitrogen were added triethylamine (3.6 mL, 26.0 mmol) and anhydrous methanol (4 mL). After stirring at 700C under an atmosphere of carbon monoxide for 14 h the mixture was concentrated in vacuo and the residue was partitioned between EA (200 mL) and sat. NaHCO3 solution (200 mL). After extraction of the aqueous layer with EA (2 x 200 mL) the combined organic layers were dried over Na2SO^ filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (elution with n-hexane/EA 19: 1) to give the title compound. MS (m/z): 302.7 [M+H*]
H. ό-^l^-l^l.l-DimethylethylsulfinamidoJethylJ-S-fluoronicotinic acid To a solution of 5-fluoro-6-[l-(2-methyl-propane-2-sulfϊnylamino)-ethyl]-nicotinic acid methyl ester (910 mg, 3.01 mmol) in MeOH (1.5 mL) and H2O (0.5 mL) was added LiOH (215 mg, 9.03 mmol). The solution was stirred overnight then concentrated. The residue was dissolved in 15% MeOH/CH2Cl2 and filtered through a short silica column to remove inorganic salts. Crude ό-^l^-l^l.l-dimethylethylsulfinamido^thyO-S-fluoronicotinic acid was determined to be of sufficient purity for the next reactions. MS (m/z): 288.7 [M+H+]
I. N-((R)-l-(5-((2R,5S)-2,5-Dimethylpyrrolidine-l-carbonyl)-3-fluoropyridin-2- yl)ethyl)-2-methyIpropane-2-sulfinamide
6-((1/J)-I-(I, l-Dimethylethylsulfinamido)ethyl)-5-fluoronicotinic acid (50.0 mg, 0.17 mmol) was added to a stirred solution of (2R,5iS)-2,5-dimethyltetrahydro-l/-r-l-pyrrolidinium chloride (Example 4C) (35.3 mg, 0.26 mmol), HATU (65.9 mg, 0.17 mmol) and DIPEA (151 μL, 0.87 mmol) in DMF (1.7 mL). After stirring overnight the reaction mixture was concentrated in vacuo and the residue was partitioned between EA (30 mL) and sat. NaHCO3 solution (50 mL). After extraction of the aqu layer with EA (2 x 30 mL) the combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo to give the crude title compound which was purified by flash chromatography on silica gel (elution with n- hexane/EA 5:1). MS (m/z): 369.8 [IvH-H+]
J. (6-((R)-l-Aminoethyl)-5-fluoropyridin-3-yl)((2R,5S)-2,5-dimethylpyrrolidin-l- yl)methanone hydrochloride
To //-((R)-l-(5-((2R,5S)-2,5-dimethylpyrrolidine-l-carbonyl)-3-fluoropyridin-2-yl)ethyl)-
2-methylpropane-2-sulfinamide (50 mg, 0.14 mmol) is added a solution of HCl in MeOH (3 N, 1 mL). The reaction mixture is stirred at RT for 2 h and then concentrated to dryness. No purification step is required, and the isolated title compound is utilized in the next reaction.
MS (m/z): 265.9 [M+H+]
K. Λ?r/-Butyl 2-((R)-l -(5-((2R,5.S)-2,5-dimeΛylpyrrolidine-l -carbonyl)-3-fluoropyridin- 2-yl)ethylcarbamoyl)-2-ethylhydrazinecarboxylate
Terf-butyl 2-(chlorocarbonyl)-2-ethylhydrazinecarboxylate were synthesised according to literature (Bailey et al. J. Med Chem. 2004, 47, 3788-3799). tert-Butyl 2-(chlorocarbonyl)-2-ethylhydrazinecarboxylate (46 mg, 0.21 mmol) was dissolved in THF (2 mL) treated with DIPEA (361 μL, 2.07 mmol) and cooled to 00C before a solution of (6-((R)-l-aminoethyl)-5-fluoropyridin-3-yl)((2i-,55)-2,5-dimethylpyrrolidin-l- yl)methanone hydrochloride (25 mg, 0.08 mmol) with DIPEA (32 μL, 0.18 mmol) in THF (2 mL) was added all at once. The reaction was maintained at 00C (1 h) and then stirred at RT for 14 h. The mixture was poured into EA and washed sequentially with HCl (1 N), sat.
NaHCθ3, and brine before being dried (MgSO4), filtered, and concentrated. The product was purified by flash chromatography on silica gel (elution with n-hexane/EA 3:1). MS (m/z):
451.9 [M+H+]
L. N-HR)- 1 -(5-((2R,5S)-2,5-Dimethylpyrrolidine- 1 -carbonyl)-3 -fluoropyridin-2- yl)ethyl)-l-ethyl-2-(3-fiuoro-5-(trifluoromethyl)benzoyl)hydrazinecarboxamide To tert-Znrtyl 2-((R)-l-(5-((2R,5S)-2,5-dimethylpyrrolidine-l-carbonyl)-3-fluoropyridin-2- yl)ethylcarbamoyl)-2-ethylhydrazinecarboxylate (25 mg, 0.06 mmol) is added a solution of HCl in MeOH (3N, 1 mL). The reaction mixture is stirred at RT for 2 h and then concentrated to dryness. No purification step is required, a he isolated hydrochloride is utilized in the next reaction.
The hydrochloride (20 mg, 0.05 mmol) is dissolved in DMF (0.5 mL), treated with
DIPEA (90 μL, 0.52 mmol) and 3-fluoro-5-(trifluoromethyl)benzoyl chloride (10 mg, 0.08 mmol) and stirred 2 h at RT. Then the mixture was concentrated in vacuo and the residue was purified by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA to give the title compound. MS (m/z): 541.8 [M+H+]
Example 20 Synthesis of (R)-7V-(l-(2-chloro-4-(diethylcarbamoyl)phenyl)ethyl)-l-ethyl-2-(3-fluoro-5-
(trifluoromethyl)benzoyl)hydrazinecarboxamide (Compound 256)
Figure imgf000110_0001
A. Methyl 4-((ter/-butylsulfinylimino)methyl)-3-chlorobenzoate
The synthesis of 3-chloro-4-formyl-benzoic acid methyl ester is described in Example 15B.
(5)-(-)-2-Methyl-2-propansulfinamide (1.26 g, 10.4 mol), magnesium sulfate (12.5 g, 104 mmol), PPTS (0.52 g, 2.08 mol) were added to a solution of 3-chloro-4-formyl-benzoic acid methyl ester (2.07 g, 10.4 mmol) in DCM (52 ml). After stirring over night the reaction was filtered over celite and the solvent was removed in vacuo. The crude product was purified by chromatography on silica gel (n-hexan/EA). MS (m/z): 301.7 [M+H*]
B. 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. The combined organic layers were dried over sodium sulfate and the solvent was removed in vacuo. The crude product was purified by chromatography on silica gel (n-hexan/EA). M m/z): 317.8 [M+H*]
C. (R)-Methyl 4-(l-aminoethyl)-3-chlorobenzoate hydrochloride
To methyl 3-chloro-4-((lR)-l-(l,l-dimeΛyleΛylsulfinamido)ethyl)benzoate (199 mg, 0.63 mmol) is added a solution of HCl in MeOH (3N, 10 mL). The reaction mixture is stirred at RT for 1 h and then concentrated to dryness. No purification step is required, and the isolated title compound is utilized in the next reaction. MS (m/z): 213.8 [M+H*]
D. (/.)-tert-Butyl 2-(l -(Z-chloro^^methoxycarbonyOphenyl^thylcarbamoy^-Z-ethyl hydrazinecarboxylate
Terr-butyl 2-(chlorocarbonyl)-2-ethylhydrazinecarboxylate were synthesised according to literature (Bailey etal. J. Med. Chem. 2004, 47, 3788-3799).
Tert-butyl 2-(chlorocarbonyl)-2-ethylhydrazinecarboxylate (417 mg, 1.87 mmol) was dissolved in THF (2 mL) treated with DIPEA (718 μL, 4.11 mmol) and cooled to 00C before a solution of (R)-methyl 4-(l-aminoethyl)-3-chlorobenzoate hydrochloride (79 mg, 0.32 mmol) with DIPEA (121 μL, 0.69 mmol) in THF (2 mL) was added all at once. The reaction was maintained at O0C (1 h) and then stirred at RT for 14 h. The mixture was poured into EA and washed sequentially with HCl (1 N), sat. NaHCC>3, and brine before being dried (MgSO-O, filtered, and concentrated. The product was purified by flash chromatography on silica gel (elution with n-hexane/EA). MS (m/z): 399.7 [M+H+]
E. (R)-Methyl 3-chloro-4-(l -(I -ethyl-2-(3-fluoro-5-(trifluoromethyl)benzoyl) hydrazinecarboxamido)ethyl)benzoate
To (R)-tør/-butyl 2-(l -(2-chloro-4-(methoxycarbonyl)phenyl)ethylcarbamoyl)-2- ethylhydrazinecarboxylate (75 mg, 0.19 mmol) is added a solution of HCl in MeOH (3 N, 2 mL). The reaction mixture is stirred at RT for 14 h and then concentrated to dryness. No purification step is required, and the isolated title compound is utilized in the next reaction.
The hydrochloride (63 mg, 0.19 mmol) is dissolved in DMF (1.0 mL), treated with DIPEA (163 μL, 0.94 mmol) and 3-fluoro-5-(trifluoromethyl)benzoyl chloride (47 mg, 0.21 mmol) and stirred 2 h at RT. Then the mixture was concentrated in vacuo and the residue was purified by chromatography on silica gel (n-hexan/EA). MS (m/z): 489.6 [M+H*] F. (R)--V-(l -(2-Chloro-4-(diethylcarbamo enyl)ethyl)-l -ethyl-2-(3-fluoro-5- (trifluoromethyl)benzoyl)hydrazinecarboxamide
To a solution of (R)-methyl 3-chloro-4-(l-(l-ethyl-2-(3-fluoro-5- (trifluoromethyl)benzoyl)hydrazinecarboxamido)ethyl)benzoate (92 mg, 0.19 mmol) in Dioxan (2 mL) was added a 2 N solution of LiOH in water (1 mL) and the reaction was stirred for 3 h. Then the organic solvent is removed in vacuo, the water layer was extracted twice with EA and the combined organic layers were dried over sodium sulfate and the solvent was removed in vacuo. The crude product was used in the next step without further purification. (R)-3-Chloro-4-(l -(I -ethyl-2-(3-fluoro-5-(trifluoromethyl)benzoyl)hydrazine carboxamido)ethyl)benzoic acid (27.0 mg, 0.06 mmol) was added to a stirred solution of diethylamine (44.0 mg, 0.60 mmol) and HATU (22.9 mg, 0.06 mmol) in DMF (1.5 mL). Alter stirring for 2 h the reaction mixture was concentrated in vacuo and the residue was purified by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA to give the title compound. MS (m/z): 530.9 [M+H*]
Example 21
Synthesis of N-((R)-\ -(3-chloro-5-((2R,55)-2,5-dimethylpyrrolidine- 1 -carbonyl)pyridin- 2-yl)ethyl)-l-ethyl-2-(3-fluoro-5-(trifluoromethyl)benzoyl)hydrazinecarboxamide (Compound 263)
Figure imgf000112_0001
A. Dimethyl 3-chloropyridine-2,5-dicarboxylate
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. Na2WO4 (0.6 g). To this solution was added H2O2 in water (30% w/w, 34 mL, 315 mmol). The resulting mixture was stirred and heated at 80-850C 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 Λf-oxide, which was used in the next step without further purification.
To a solution of thionyl chloride (17.4 mL, 240 mmol) in DCM (500 mL) was added
DMF (5 mL) at O0C. Pyridine-2,5-dicarboxylic acid N-oxide from the first step was added into this mixture portionwise. The resulting mixture was heated at 650C for 2 hours. The reaction was cooled to RT then placed in an ice-bath. The reaction was quenched with methanol (100 mL) slowly at 0 0C. The solvent was removed in vacuo and the crude product was partitioned between DCM (200 mL) and aq. NaHCθ3 (sat. 100 mL). The organic layer was separated and the aqueous layer was extracted with additional DCM (2 x 200 mL). The combined organic layer was dried (Na2SO4), filtered and the solvent was removed in vacuo.
Purification of the crude oil through a short plug of silica gel using hexane/ethyl acetate (4:1) afforded dimethyl 3-chloropyridine-2,5-dicarboxylate. GC/MS (m/z): 229.0
B. Methyl 5-chloro-6-(hydroxymethyl)nicotinate To a solution of dimethyl 3-chloropyridine-2,5-dicarboxylate (1.0 g, 4.4 mmol) in THF
(10 mL) and MeOH (20 mL) was added powdered calcium chloride (3.9 g, 35 mmol). The suspension was cooled to 00C and sodium borohydride (416 mg, 11.0 mmol) was added portionwise. The resulting mixture was stirred for 3 h at 00C and poured into ice-water (200 mL), and extracted with DCM (2 x 100 mL). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo to yield methyl 5-chloro-6- (hydroxymethyl)nicotinate which was used in the next step without further purification. GC/MS (m/z): 201.0
C. Methyl 5-chloro-6-formylnicotinate To a solution of methyl 5-chloro-6-(hydroxymethyl)nicotinate (3.2 g, 15.8 mmol) in
DCM (20 mL) was added Dess-Martin periodinane (10.1 g, 23.7 mmol). The solution was stirred for 1 h, diluted with DCM and filtered over a pad of Celite. After evaporation of the solvent in vacuo the crude material was purified by flash chromatography (hexane/ethyl acetate 3:1) to give the title compound. GC/MS (m/z): 201.0
D. (R)-Methyl 6-((ferr-butylsulfinylimino)methyl)-5-chloronicotinate
To a solution of (R)-(-)-2-methyl-2-propanesulfinamide (0.73 g, 6.0 mmol) in DCM (25 mL) was added methyl S-chloro-ό-formyln nate (1.2 g, 6.0 mmol), pyridinium p- toluenesulfonate (0.15 mg, 0.6 mmol), magnesium sulphate (7.2 g, 60 mmol) and freshly powdered caesium carbonate (1.9 g, 6.0 mmol). The reaction was stirred at RT for 6 h. The mixture was filtered through celite, washed with DCM, and concentrated in vacuo. The resulting residue was subjected to silica gel chromatography (0-10% ethyl acetate in hexanes) to provide the title compound. MS (m/z): 302.6 [M+H*]
E. 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 - 600C 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 0C, sat. ammonium chloride solution (40 mL) was added. The aqueous layer was extracted with DCM (4 x 25 ml), the organic layer was dried over Na2SO_j, filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (elution with hexane/EA 1:1) to give the tiltle compound. MS (m/z): 318.8 [M+H*]
F. (R)-Methyl 6-(l-aminoethyl)-5-chloronicotinate hydrochloride
A solution of methyl 5-chloro-6-((R)-l -((R)- l,l-dimethylethylsulfinamido)ethyl) nicotinate (230 mg, 0.72 mmol) and HCl (3N solution in MeOH, 2 mL) was stirred at RT for 1.5 h. The solvent was removed in vacuo and the residue was recristallized from diethylether to give the title compound as the HCl salt. MS (m/z): 214.8 [M+H*]
G. (R)-Methyl 6-(l-(2-(/er/-butoxycarbonyl)-l-ethylhydrazinecarboxamido)ethyl)-5- chloronicotinate 7erf-butyl 2-(chlorocarbonyl)-2-ethylhydrazinecarboxylate were synthesised according to literature (Bailey etal. J. Med. Chem. 2004, 47, 3788-3799).
Terr-butyl 2-(chlorocarbonyl)-2-ethylhydrazinecarboxylate (443 mg, 1.99 mmol) was dissolved in THF (2.5 mL) treated with DIPEA (705 μL, 3.98 mmol) and cooled to 00C before a solution of (R)-methyl 6-(l-aminoethyl)-5-chloronicotinate hydrochloride (100 mg, 0.40 mmol) with DIPEA (155 μL, 0.88 mmol) in THF (2.5 mL) was added all at once. The reaction was maintained at O0C (1 h) and then stirred at RT for 14 h. The mixture was poured into EA and washed sequentially with HCl (1 N), sat. NaHCC^, and brine before being dried (MgSO4), filtered, and. concentrated. The prod was purified by flash chromatography on silica gel (elution with hexane/EA). MS (m/z): 400.8 [M+H+]
H. (R)-6-(l -(2-(tert-Butoxycarbonyl)- 1 -ethylhydrazinecarboxamido)ethyl)-5- chloronicotinic acid
(R)-Methyl 6-(l-(2-(rerr-butoxycarbonyl)-l-ethylhydrazinecarboxamido)ethyl)-5- chloronicotinate (220 mg, 0.55 mmol) was dissolved in ACN (5 mL), treated with LiOH (IM solution in water, 3.5 mL) and stirred 13 h at RT. The mixture was concentrated in vacuo and EA (150 mL) was added. The pH of the aqueous layer was adjusted to 4 by the addition of 1 N HCl solution and the aqueous layer was extracted with EA (3 x) and DCM/iPrOH (4:1, 3 x). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The resulting title compound was used for the next reaction without purification. MS (m/z): 386.8 [M-I-H+]
I. ter/-Butyl 2-((R)-l-(3-chloro-5-((2R,55)-2,5-dimethylpyrrolidine-l- carbonyl)pyridin-2-yl)ethylcarbamoyl)-2-ethylhydrazinecarboxylate
(2R,5S)-2,5-Dimethylpyrrolidine hydrochloride (105 mg, 0.78 mmol) was added to a stirred solution of (/-)-6-(l-(2-(rer/-butoxycarbonyl)-l-ethylhydrazinecarboxamido)ethyl)-5- chloronicotinic acid (150 mg, 0.39 mmol), WSC (149 mg, 0.78 mmol), HOBt (105 mg, 0.78 mmol) and DIPEA (330 μL, 1.94 mmol) in DCM (2.5 mL). After stirring overnight at RT the solvent was removed in vacuo and the crude product was partitioned between EE and aq. Na2Cθ3. The organic layer was separated and the aqueous layer was extracted with additional EA (2 x). The combined organic layer was dried (Na2SO4), filtered and the solvent was removed in vacuo. The resulting title compound was used for the next reaction without purification. MS (m/z): 467.9 [M-I-H+]
J. N-((R)- 1 -(3-chloro-5-((2R,55)-2,5-dimethylpyrrolidine- 1 -carbonyl)pyridin-2- yl)ethyl)-l-ethyl-2-(3-fluoro-5-(trifluoromethyl)benzoyl)hydrazinecarboxamide
To ter/-Butyl 2-((R)-l-(3-chloro-5-((2R,5S)-2,5-dimethylpyrrolidine-l-carbonyl)pyridin- 2-yl)ethylcarbamoyl)-2-ethylhydrazinecarboxylate (180 mg, 0.39 mmol) is added a solution of HCl in MeOH (3N, 20 mL). The reaction mixture is stirred at RT for 13 h and then concentrated to dryness. No purification step is required, and the isolated hydrochloride is utilized in the next reaction.
N-(I[Ry 1 -(3-Chloro-5-((2R,5-S)-2,5-dimethylpyrrolidine-l -carbonyl)pyridin-2-yl)ethyl)- 1 - ethylhydrazinecarboxamide hydrochloride (8.0 mg, 0.02 mmol) was added to a stirred solution of 3-fluoro-5-(trifluoromethyl)benzoic acid (9.1 mg, 0.04 mmol), WSC (8.3 mg, 0.04 mmol), HOBt (5.9 mg, 0.04 mmol) and DIPEA (19 μL, 0.11 mmol) in DMF (0.2 mL). After stirring overnight at RT the residue was purified by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA to give the title compound. MS (m/z): 557.9 [M+H*]
Example 22 Synthesis of (R)-5-chloro-N^-diethyl-6-(l-(l-ethyl-2-(3-fluoro-5-(trifluoromethyl) benzoyl)hydrazinecarboxamido)ethyl)nicotinamide (Compound 265)
Figure imgf000116_0001
A. (R)-tert-Buty\ 2-(l-(3-chloro-5-(diethylcarbamoyl)pyridin-2-yl)ethylcarbamoyl)-2- ethylhydrazinecarboxylate Diethylamine (31 mg, 0.42 mmol) was added to a solution of (R)-6-(l-(2-(tert- butoxycarbonyl)-l-ethylhydrazinecarboxamido)ethyl)-5-chloronicotinic acid (82 mg, 0.21 mmol), WSC (82 mg, 0.42 mmol), HOBt (57 mg, 0.42 mmol) and DIPEA (180 μL, 1.06 mmol) in DCM (1.5 mL). After stirring overnight at RT the solvent was removed in vacuo and the crude product was partitioned between EE and aq. Na2CO3. The organic layer was separated and the aqueous layer was extracted with additional EA (2 x). The combined organic layer was dried (Na2SO4), filtered and the solvent was removed in vacuo. The resulting title compound was used for the next reaction without purification. MS (m/z): 441.9 [M+H+]
B. ^-((R)-l-(3-Chloro-5-((2R,5.S)-2,5-dimethylpyrrolidine-l-carbonyl)pyridin-2- yl)ethyl)-l-ethyl-2-(3-fluoro-5-(trifluoromethyl)benzoyl)hydrazinecarboxamide
To (R)-tert-buty\ 2-(l-(3-chloro-5-(diethylcarbamoyl)pyridin-2-yl)ethylcarbamoyl)-2- ethylhydrazinecarboxylate (93 mg, 0.21 mmol) is added a solution of HCl in MeOH (3N, 15 mL). The reaction mixture is stirred at RT f 3 h and then concentrated to dryness. No purification step is required, and the isolated hydrochloride is utilized in the next reaction.
(R)-5-Chloro-iV^V-diethyl-6-(l -( 1 -ethylhydrazinecarboxamido)ethyl)nicotinamide hydrochloride (8.3 mg, 0.02 mmol) was added to a stirred solution of 3-fluoro-5- (trifluoromethyl)benzoic acid (10 mg, 0.05 mmol), WSC (9.3 mg, 0.05 mmol), HOBt (6.6 mg, 0.05 mmol) and DIPEA (22 μL, 0.12 mmol) in DMF (0.2 mL). After stirring overnight at RT the residue was purified by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA to give the title compound. MS (m/z): 531.8 [M+H*]
Example 23
Synthesis of (R)-N-(I -(4-(diethylcarbamoyl)-2-fiuorophenyl)ethyl)- 1 -ethyl-2-(3-fluoro-5- (trifluoromethyl)benzoyl)hydrazinecarboxamide (Compound 221)
Figure imgf000117_0001
A. (R)-4-(\ -(((9/-r-Fluoren-9-yl)methoxy)carbonylamino)ethyl)-3-fluorobenzoic acid (R)-4-(l-Amino-ethyl)-3-fluoro-benzoic acid methyl ester (Example 10C) (100 mg, 0.43 mmol) was stirred in 6 N HCl solution (10 mL) at reflux overnight. Solvent was reduced in vacuo to give (R)-4-(l-aminoethyl)-3-fluorobenzoic acid hydrochloride. To the crude product (94 mg, 0.43 mmol) dissolved in DCM (2 mL), chlorotrimethylsilane (135 μL, 1.07 mmol) was added. After stirring at reflux for few minutes, (9/-r-fluoren-9-yl)methyl carbonochloridate and triethylamine (230 mL, 1.7 mmol) were added at RT and the solution was stirred overnight. The residue was concentrated in vacuo and sat. Na∑COa solution (50 mL) was added. After acidification of the aqueous solution with 2 N HCL (pH=3), the aqueous layer was extracted with EA (2 x 30 mL) and the combined organic layers were dried over Na2SO1J, filtered and concentrated in vacuo to give the crude title compound which was purified by flash chromatography on silica gel (elution with DCM/methanol 2:1). MS (m/z): 405.8 [M-I-H+]
B. Resin-bound (R)-4-(l-aminoethyl)-3-fluorobenzoic acid (R)-4-(l -(((9H-fluoren-9-yl)methoxy)carb amino)ethyl)-3-fluorobenzoic acid (324 mg, 0.8 mmol) and DIPEA (332 μL, 1.9 mmol) were dissolved in DCM (8 mL). The solution was added to 2-chlorotrityl chloride resin (loading: 1.4 mmol/g, 1.09 g, 1.5 mmol) swollen in
DCM (8 mL) and the mixture was shaken at RT for 30 minutes. The mixture was quenched with methanol (800 μL). 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).
C. (9/f-Fluoren-9-yl)methyl 2-ethylhydrazinecarboxylate
To a solution of (9/f-fluoren-9-yl)methyl hydrazinecarboxylate (500 mg, 1.97 mmol) in ethanol (9.8 mL), acetaldehyde (86.7 mg, 1.97 mmol) was added and the mixture was refluxed overnight. To this solution acetic acid (124 μL, 2.16 mmol) and sodium cyanotrihydroborate (136 mg, 2.16 mmol) were added. After stirring at RT overnight the solvent was reduced in vacuo and the residue was partitioned between EA (30 mL) and sat. NaCl solution (50 mL). After extraction of the aqueous layer with EA (2 x 30 mL) the combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo to give the crude title compound which was purified by flash chromatography on silica gel (elution with n-hexane/EA 1:1). MS (m/z): 282.7 [M+H*]
D. (R)-4-(l-(l-E%l-2-(3-fluoro-5<trifluoromethyl)benzoyl)hydrazinecarboxamido) ethyl)-3-fluorobenzoic acid
Phosgene (20% solution in toluene, 0.463 ml, 0.88 mmol) was added dropwise at 00C to a stirred solution of (9H-fluoren-9-yl)methyI 2-ethylhydrazinecarboxylate (125 mg, 0.44 mmol) in DCM (4.4 ml). The mixture was stirred at 00C for 30 minutes and then concentrated in vacuo to give (9H-fIuoren-9-yl)methyl 2-(chlorocarbonyl)-2-ethylhydrazinecarboxylate. 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 μL, 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). A test cleavage with TFA showed the right product (R)-4-(l-(2-(((9//- fluoren-9-yl)methoxy)carbonyl)- 1 -ethy lhydraz arboxamido)ethyl)-3 -fluorobenzoic acid. MS (m/z): 491.7 [M-I-H+]. Piperidin (20% solution in DMF, 4 mL) was added and the mixture was shaken for 30 min to give resin-bound (R)-4-(l-(l-ethylhydrazinecarboxamido)ethyl)-3- fluorobenzoic acid. 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 μL, 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). The suspension was shaken at RT for 20 minutes. The resin was filtered and washed with DCM (2 x 2 mL). The filtrate was concentrated in vacuo. The residue was purified by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA to give the title compound. MS (m/z): 459.6 [M+H*]
E. (R)-N-(I -(4-(Diethylcarbamoyl)-2-fluorophenyl)ethyl)-l -ethyl-2-(3-fluoro-5- (trifluoromethyl)benzoyl)hydrazinecarboxamide
(R)A-(I -(I -EthyI-2-(3-fluoro-5-(trifluoromethyl)benzoyl)hydrazinecarboxamido) ethyl)-
3-fluorobenzoic acid (12 mg, 0.028 mmol) was added to a stirred solution of diethylamine
(5.7 μL, 0.055 mmol), HATU (10.6 mg, 0.028 mmol) and DIPEA (23.4 μL, 0.14 mmol) in
DMF (1.5 mL). After stirring overnight the reaction mixture was concentrated in vacuo and the residue was partitioned between EA (30 mL) and sat. NaHCOj solution (50 mL). After extraction of the aqueous layer with EA (2 x 30 mL) the combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo to give the crude title compound which was purified by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA to give the title compound. MS (m/z): 514.8 [M+H*]
Biological evaluation:
Example 24
Radioligand binding assay
Assessing the affinity of selected compounds to bind to the BK Bl receptor of different species.
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.
For all receptor types 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 5μg/ 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.
The cell plates were washed 2x with icecold PBS and kept on ice during the whole assay. 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 CaCl2, 1 mM MgCl2, 60 mM NaOH; pH 7.4) containing the protease inhibitors Captopril (lOOμM), 1,10-Phenanthroline (20 μM) and Bacitracin (500 μM). InM 3H-DAKD (Perkin Elmer) and 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 μL 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 μL dissociation buffer (0.5 M Cl, 0.2 M acetic acid) for 10 minutes on ice.
Supernatants were transferred into 6 mL scintillation vials (Sarstedt) containing 2 mL of scintillation solution (Ultima Gold, Perkin-Elmer), mixed well and subsequently measured in S a Packard Topcount Scintillation counter. Specific binding to BlR was defined as the difference between total binding and non-specific binding wells. Total specific binding was set to 100% binding and 0% inhibition. Specific counts in the presence of compounds were plotted against compound concentration (log M). ICso values were calculated by fitting a 4- parameter logistic function to the concentration-response data using non-linear regression0 (Xlfit, IDBS ID Business Solutions Ltd.). The compounds of this invention have affinity for the BlR in the above assay as demonstrated by results of less than 5 μM.
Example 25
S Functional assay for BK B 1 antagonists
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.
Calcium-indicator-loaded cells were preincubated in the absence or presence of different concentrations of test compounds followed by stimulation of with selective BlR agonist peptide. 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
DMEM high (4.5g/l) glucose supplemented with 15% FCS and 4mM Glutamine. Confluent cells were harvested by trypsinization after 5d of culture and seeded into black wall/clear bottom 96-well plates (Costar# 3603) at 40 cells/well. After an overnight incubation (appr. 12-16h) cells were treated with 1.36 ng/ mL human recombinant IL- lβ in DMEM 15%FBS for 4h at 370C to induce BlR upregulation. Thereafter cells were washed 2x with prewarmed Hank's balanced salt solution/20 mM HEPES buffer and subsequently loaded with fluorescent calcium indicator dye no-wash Calcium 3 assay kit (Molecular Devices) at 37°C for Ih in the presence of anion transport inhibitor probenecid at 2.5 mM. 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 μL 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 370C. The addition of 50 μL of the BlR agonist desArgKallidin (DAKD, Bachem) was carried out in the FlexStation II while continuously monitoring Ca-dependent fluorescence increase. Fluorescence peak heights were calculated by subtracting mean of baseline from maximum peak height. Peak heights were plotted as a function of test compound concentration while peak height without antagonist addition was defined as 100% response and 0% inhibition. Relative fluorescence peak heights were used to calculate the degree of of inhibition of the BlR agonist response by test compound. IC50 values were calculated by fitting a 4-parameter logistic function to the concentration-response data using non-linear regression (Xlfϊt, IDBS ID Business Solutions Ltd.). The compounds of this invention have affinity for the B 1 R in the above assay as demonstrated by results of less than 5 μM.
Most preferred compounds of formula (I) exhibited ICJO values of less than 50 nanomolar in this assay.
The features of the present invention disclosed in the specification, the claims and/or the drawings may both separately and in any combination thereof be material for realizing the invention in various forms thereof.

Claims

Claims
1. A compound of the formula (I):
Figure imgf000123_0001
or a pharmacologically acceptable salt, solvate or hydrate thereof, wherein
A is: i) an optionally substituted 5- or 6-membered cycloalkyl; ii) an optionally substituted 5- 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
Figure imgf000124_0001
wherein
W is N, alkyl, heteroalkyl, alkenyl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R4, if present, 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 R5 to form, together with W, v) an optionally substituted cycloalkyl; vi) an optionally substituted heterocycloalkyl; vii) an optionally substituted aryl; or viii) an optionally substituted heteroaryl;
R5, if present, 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 R4 to form, together with W, v) an optionally substituted cycloalkyl; vi) an optionally substituted heterocycloalkyl; vii) an optionally substituted aryl; or viii) an optionally substituted heteroaryl;
E, if present, is a heteroalkyl or a heteroaralkyl;
G, if present, is an alkyl, a heteroalkyl, a cycloalkyl, a heterocycloalkyl, a alkylcycloalkyl, a heterocycloalkylalkyl, a heteroaryl, or a heteroaralkyl K iS
Figure imgf000125_0001
wherein
R1 and R2 are each independently selected from C2-C6alkyl, C2-C6alkenyl, Cj-C6alkynyl,
C2-C6heteroalkyl, or form, together with N, an optionally substituted heterocycloalkyl, an optionally substituted heteroaryl, or an optionally substituted heteroaralkyl; and
R3 is a hydrogen atom, C1- or C2alkyl, cyano, or a halogen atom.
2. The compound according to claim 1, wherein A is
Figure imgf000125_0002
wherein X1, X2, and X3 are each independently selected from N, O, S, NRX, CRX, or
CRXRX>, wherein
Rx and Rx are each independently selected from hydrogen atom, halogen atom, =0, hydroxy, cyano, amino, nitro, mercapto, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl.
3. The compound according to claim 1 or c 2, wherein A is selected from
Figure imgf000126_0001
wherein
Rx, Rxl, Rx2, Rx3, Rx', Rx1', Rx2', and Rx3' are each independently selected from hydrogen atom, halogen atom, =0, hydroxy, cyano, amino, nitro, mercapto, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and
X is NH, O, or S.
4. The compound according to claim 1, wherein A is
Figure imgf000126_0002
wherein Y1, Y2, Y3 and Y4 are each independently selected from N, O, S, NRy, CRy, or CRyRy', wherein
Ry and Ry are each independently selected from hydrogen atom, halogen atom, =O, hydroxy, cyano, amino, nitro, mercapto, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl.
5. The compound according to claim 1 or claim 4, wherein A is selected from
Figure imgf000127_0001
wherein
Ry, RyI, R5*, Ry3, R^, Ry>, Ryl>, R"2', Ry3', and Ry4> are each independently selected from hydrogen atom, halogen atom, =0, hydroxy, cyano, amino, nitro, mercapto, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl.
6. The compound according to any one of claims 1, 4 or 5, wherein A is
Figure imgf000128_0001
wherein
Ryl, Ry2, Ry3 and R5* are each independently selected from hydrogen atom, halogen atom,
=0, hydroxy, cyano, amino, nitro, mercapto, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl.
7. The compound according to claim 6, wherein
Ryl is a hydrogen atom.
8. The compound according to claim 6 or claim 7, wherein
Ry2 is a hydrogen atom, halogen atom, or C1-C6alkyl; and
Ry4 is a hydrogen atom or halogen atom.
9. The compound according to any one of claims 6 to 8, wherein Ry2 is a hydrogen atom.
10. The compound according to any one of claims 6 to 9, wherein Ry3 is a hydrogen atom or halogen atom.
11. The compound according to any one of claims 6 to 10, wherein Ry4 is a hydrogen atom or halogen atom.
12. The compound according to any one of claims 1 to 11, wherein T is a hydrogen atom.
13. The compound according to any one of claims 1 to 12, wherein K is selected from
Figure imgf000129_0001
14. The compound according to any one o ims 1 to 13, wherein K is selected from
Figure imgf000130_0001
15. The compound according to any one of claims 1 to 14, wherein K is
Figure imgf000130_0003
16. The compound according to any one of claims 1 to 14, wherein K is
Figure imgf000130_0004
17. The compound according to any one of claims 1 to 16, wherein B is heteroalkyl, heteroaryl, or
Figure imgf000130_0002
wherein W is N, alkyl, alkenyl, cycloalkyl, alkylcy kyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl;
R4, if present, is a hydrogen atom, alkyl, heteroalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aiyl, aralkyl, heteroaralkyl or joined to R5 to form, together with N, v) an optionally substituted cycloalkyl; vi) an optionally substituted heterocycloalkyl; vii) an optionally substituted aryl; or viii) an optionally substituted heteroaryl; and
R5, if present, 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 R4 to form, together with N, v) an optionally substituted cycloalkyl; vi) an optionally substituted heterocycloalkyl; vii) an optionally substituted aryl; or viii) an optionally substituted heteroaryl.
18. The compound according to any one of claims 1 to 17, wherein
B is -Y^-CO-lΛ or -Y^-CO-R0*-, wherein
Y*8 is a bond, a C1-C6alkylene, a C2-C6alkenylene or a C2-C6alkynylene;
LB is a cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, aralkyl, or heteroaralkyl; and
RcB is an optionally substituted C1-C8alkyl, an optionally substituted C2-C8alkenyl or an optionally substituted C2-C8alkynyl.
19. The compound according to any one o ims 1 to 18, wherein B is
Figure imgf000132_0001
20. The compound according to any one of claims 1 to 18, wherein B is
wherein
Figure imgf000132_0002
R4 is methyl, ethyl or isopropyl.
21. The compound according to any one of claims 1 to 17, wherein B is
Figure imgf000132_0003
22. The compound according to any one of claims 1 to 21, wherein E is -YaE-CO-LE-, -YaE-CO-RcE-, -YaE-NRcE-CO-RdE-, -YaE-NRcE-CO-LE-, -YaE-NRcE-CO-NRdE-LE-, or -YaE-NRcE-CS-NRdE-LE-, wherein
YaE is a bond, a C1-C6alkylene, a C2-C6alkenylene or a C2-C6alkynylene;
RcE is a hydrogen atom, an optionally substituted C1-C8alkyl, an optionally substituted C2- C8alkenyl or an optionally substituted C2-C8alkynyl, provided that RcE is not a hydrogen atom in -YaE-CO- RcE-; RdE is a hydrogen atom, an optionally subs ed C1-C8alkyl, an optionally substituted C2- C8alkenyl or an optionally substituted C2-C8alkynyl; and
LE is a cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, aralkyl, or heteroaralkyl.
23. The compound according to any one of claims 1 to 22, wherein G is alkyl, cycloalkyl, -Y8G-O-R00, -YaG-C0-NRaGRb0, -Y^-S-R60, -Y^-SO-R00, -Y^-SO2-R* heteroaryl, alkylcycloalkyl, or heterocycloalkyl, wherein
Y*° is a bond, a C1-C6alkylene, a C2-C6alkenylene or a C2-C6alkynylene;
R"0 is a hydrogen atom, a C1-C6alkyl, a C2-C6alkenyl, a C2-C6alkynyl, or is joined to R60 to form a 4- to 10-membered cycloalkyl or heterocycloalkyl;
R60 is a hydrogen atom, a C1-C6alkyl, a C2-C6alkenyl or a C2-C6alkynyl, or is joined to R*0 to form a 4- to 10-membered cycloalkyl or heterocycloalkyl; and
R00 is an optionally substituted C1-C8alkyl, an optionally substituted C2-C8alkenyl or an optionally substituted Ci-C8alkynyl.
24. The compound according to any one of claims 1, 4 to 6, 12, 17, or 22, wherein the compound is represented by formula (II):
Figure imgf000133_0001
wherein
Y1 is N, CH or CF;
K is selected from
Figure imgf000134_0001
R3 is a hydrogen atom or methyl; Ry2 is a hydrogen atom, halogen atom, or C1-C6alkyl;
Ry3 is a hydrogen atom, halogen atom, C1-C6alkyl, or C1-C6heteroalkyl;
Ry4 is a hydrogen atom or halogen atom;
B is -Y^-CO-LB, -YaB-CO-RcB-, or
Figure imgf000134_0002
wherein
YaB is a bond; R ,cB is an optionally substituted C1-C8alkyl n optionally substituted C2-C8alkenyl;
LB is a cycloalkyl, heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, or heteroaralkyl;
W is alkyl orN;
R4, if present, is a hydrogen atom, alkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aralkyl, or heteroaralkyl;
R5, if present, is a hydrogen atom or alkyl;
E is -YbE-NRdE-C0-LE-, wherein
YbE is a bond, a C1-C6alkylene, or a C^-C6alkenylene;
RdE is a hydrogen atom, a C1-C6alkyl, or C2-C6alkenyl; and
LE is a cycloalkyl, heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, or heteroaralkyl.
25. The compound according to any one of claims 1, 4 to 6, 12, 13, 17 to 20, 22 or 23, wherein the compound is represented by formulas (III) or (IV):
Figure imgf000135_0001
wherein
Y1 is N, CH or CF;
K is selected from
Figure imgf000136_0001
R3 is a hydrogen atom or methyl;
Ry2 is a hydrogen atom, halogen atom, or C1-C6alkyl;
Ry3 is a hydrogen atom, halogen atom, C1-C6alkyl, or C1-C6heteroalkyl;
Ry4 is a hydrogen atom or halogen atom, C1-C6alkyl, or C1-C6heteroalkyl;
E is -YaE-NRcE-CO-RdE-, -YaE-NRcE-CO-LE-, -YaE-NRcE-CO-NRdE-LE-, or -YaE-N RcE-CS-NRdE-LE-, wherein
YaE is a bond or C1-C6alkylene;
RcE is a hydrogen atom, C1-C6alkyl, or C2-C6alkenyl;
RdE is a hydrogen atom, an optionally substituted C1-C8alkyl, an optionally substituted C2- C8alkenyl or an optionally substituted C2-C8alkynyl group; LE is a cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, optionally substituted aiyl, optionally substituted heteroaryl, aralkyl, or heteroaralkyl;
G, if present, is cycloalkyl, -YaG-CO-NRaGRbG, -YaG-0-ReG, -YaG-S-ReG -YaG-SO-ReG, or
-YaG-SO2-ReG, wherein
YaG is a bond or C1-C6alkylene;
RaG is a hydrogen atom or C1-C6alkyl;
RbG is a hydrogen atom or C1-C6alkyl; and
ReG is C1-C6alkyl.
26. The compound according to claim 25, wherein the compound is represented by formula (V):
Figure imgf000137_0001
wherein Y1, K, R3, Ry2, Ry3 Ry4, and LE are as defined in claim 25.
27. The compound according to claim wherein the compound is represented by formula (VI):
Figure imgf000138_0001
wherein Y1, K, R3, Ry2, Ry3, Ry4, and LE are as defined in claim 25.
28. The compound according to any one of claims 24 to 27, wherein
Ry3 is a hydrogen atom.
29. The compound according to any one of claims 24 to 28, wherein
Ry2 is a hydrogen atom.
30. The compound according to any one of claims 1, 4 to 6, 9, 10, 12, 13, 17, 21, 22, or3, wherein the compound is represented by formula (VII):
Figure imgf000138_0002
wherein
Y1 is N, CH or CF;
K is selected from
Figure imgf000139_0001
Ry4 is a hydrogen atom, halogen atom, C1-C6alkyl, or C1-C6heteroalkyl;
E is -YaE-CO-LE-, wherein YaE is a bond or C1-C6alkylene;
LE is heterocycloalkyl; and
G is alkylcycloalkyl, heterocycloalkylalkyl, aryl or heteroaryl.
31. The compound according to claim 30, wherein
Ry4 is a hydrogen atom or halogen atom.
32. The compound according to any one of claims 1 to 31, wherein the stereogenic center to which R3 is attached to is in the (R) configuration; and R3 is methyl.
33. The compound, salt, solvate or hydrate thereof, according to any one of claims 1 to 32, wherein the compound exhibits an IC50 of 500 nM or less in a standard in vitro BK Bl receptor-mediated assay.
34. A compound, preferably a compound according to any one of claims 1 to 33, which compound is selected from compounds 1 to 282 of Table 1.
35. A pharmaceutical composition comprising one or more compounds according to any one of claims 1 to 34 and, optionally, at least one carrier substance, excipient and/or adjuvant.
36. The pharmaceutical composition according to claim 35, wherein 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 suppository, or a pharmaceutical device.
37. Use of a compound or of a pharmaceutical composition according to any one of claims 1 to 36 for the manufacture of a medicament for the treatment and/or prevention of a disease or a condition.
38. The use according to claim 37, wherein the condition or disease is responsive to BK BlR modulation.
39. The use according to claim 37 or claim 38, wherein the condition or disease is selected from the group comprising inflammatory diseases, immunology disorders and pain.
40. The use according to claim 39, wherein the inflammatory disease or immunology disorder is selected from the group comprising inflammatory bowel disease, rheumatoid arthritis, gouty arthritis, atherosclerosis and associated fibrotic conditions
41. The use according to claim 39, wherein pain is selected form the group comprising visceral pain, neuropathic pain, complex regional pain syndrome CRPS and inflammatory pain.
42. A method for inhibiting binding of DAKD, KD and DABK to a BK Bl receptor in 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 one of claims 1 to 36 under conditions and in an amount sufficient to detectably inhibit binding of DAKD, KD and DABK to the BK Bl receptor.
43. A method for localizing or detecting a BK Bl receptor in a tissue, preferably a tissue section, in vitro, comprising:
(a) contacting a sample of said tissue presumably containing the BK Bl receptor with a detectably labeled compound according to any one of claims 1 to 36o under conditions that permit binding of the compound to the BK B 1 receptor; and
(b) detecting the compound bound to the BK Bl receptor or detecting the binding of the compound to the BK Bl receptor.
44. The method according to claim 43, wherein the compound is radiolabeled, fluorescence-labeled or luminescence labeled, or labeled with an antibody.
45. 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 one of claims 1 to 36.
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