WO2010091876A2 - Small molecule bradykinin b1 receptor antagonists - Google Patents

Small molecule bradykinin b1 receptor antagonists Download PDF

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Publication number
WO2010091876A2
WO2010091876A2 PCT/EP2010/000883 EP2010000883W WO2010091876A2 WO 2010091876 A2 WO2010091876 A2 WO 2010091876A2 EP 2010000883 W EP2010000883 W EP 2010000883W WO 2010091876 A2 WO2010091876 A2 WO 2010091876A2
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optionally substituted
alkyl
methyl
mmol
fluoro
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PCT/EP2010/000883
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French (fr)
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WO2010091876A3 (en
Inventor
Karsten Schnatbaum
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Jerini Ag
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Priority to EP10705103A priority Critical patent/EP2396301A2/en
Priority to CA2752190A priority patent/CA2752190A1/en
Publication of WO2010091876A2 publication Critical patent/WO2010091876A2/en
Publication of WO2010091876A3 publication Critical patent/WO2010091876A3/en

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    • C07—ORGANIC CHEMISTRY
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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
    • C07D213/61—Halogen atoms or nitro radicals
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    • C07C281/00—Derivatives of carbonic acid containing functional groups covered by groups C07C269/00 - C07C279/00 in which at least one nitrogen atom of these functional groups is further bound to another nitrogen atom not being part of a nitro or nitroso group
    • C07C281/06—Compounds containing any of the groups, e.g. semicarbazides
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    • 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
    • C07D213/24—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 substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/36—Radicals substituted by singly-bound nitrogen atoms
    • C07D213/40—Acylated substituent nitrogen atom
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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/89—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members with hetero atoms directly attached to the ring nitrogen atom
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    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/24—Benzimidazoles; Hydrogenated benzimidazoles 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 in position 2
    • C07D235/30—Nitrogen atoms not forming part of a nitro radical
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    • 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
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    • C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
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    • C07D261/00—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings
    • C07D261/02—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings
    • C07D261/06—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having two or more double bonds between ring members or between ring members and non-ring members
    • C07D261/10—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having two 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, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D261/18—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen
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    • C07D271/00—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms
    • C07D271/02—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms not condensed with other rings
    • C07D271/06—1,2,4-Oxadiazoles; Hydrogenated 1,2,4-oxadiazoles
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    • C07D285/00—Heterocyclic compounds containing rings having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by groups C07D275/00 - C07D283/00
    • C07D285/01—Five-membered rings
    • C07D285/02—Thiadiazoles; Hydrogenated thiadiazoles
    • C07D285/04—Thiadiazoles; Hydrogenated thiadiazoles not condensed with other rings
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    • 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
    • C07D401/10—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 carbon chain containing aromatic rings
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    • 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
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/10—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing aromatic rings
    • C—CHEMISTRY; METALLURGY
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • 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
    • C07D413/10—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 carbon chain containing aromatic rings
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    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07D417/10—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 carbon chain containing aromatic rings
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    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07D417/14—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 three or more hetero rings

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 ah, 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.
  • 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.
  • X is CH or N
  • R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , and R 8 are each and independently of each other selected from hydrogen atom, halogen atom, hydroxy, cyano, amino, alkyl, or optionally substituted heteroalkyl;
  • R 5 is a halogen atom, hydroxy, cyano, amino, an alkyl, 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, or an optionally substituted heteroaralkyl;
  • R 9 is a hydrogen atom, an alkyl, or a heteroalkyl
  • R 10 is a hydrogen atom, an alkyl, an optionally substituted heteroalkyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aralkyl, or an optionally substituted heteroaralkyl;
  • R 11 is an alkyl, 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, or an optionally substituted heteroaralkyl;
  • R 12 is a hydrogen atom, an alkyl, an optionally substituted heteroalkyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, an optionally substituted aralkyl, or an optionally substituted heteroaralkyl;
  • B is O or N
  • Z 1 and Z 2 are each and independently of each other selected from C or N, and Z 3 and Z 4 are each and independently of each other selected from C, S, O or N;
  • R 13 is an alkyl, 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, or an optionally substituted heteroaralkyl;
  • R 14 and R 15 are
  • R 16 , R 17 , R 18 and R 19 are individually and independently selected from hydrogen atom, halogen atom, hydroxy, cyano, amino, alkyl, and optionally substituted heteroalkyl.
  • R 5 is a halogen atom, an alkyl, an optionally substituted heteroalkyl, or a moiety selected from
  • X 1 , X 2 , X 3 , and X 4 are each and independently of each other selected from C, N, O, S, NR", CR", or CR X R X , and X 5 is selected from carbon atom, N or CR X , wherein
  • R , R , R ,and R are each a hydrogen atom
  • R 2 , R 4 and R 6 are each and independently of each other selected from hydrogen atom, halogen atom, cyano, alkyl or heteroalkyl;
  • R 9 is a hydrogen atom or Me.
  • R 5 is Me, Et, cyano, CF 3 , Cl or a moiety selected from
  • R , R , R and R are each and independently of each other selected from hydrogen atom, halogen atom, alkyl or alkoxy, and R x5 , R x6 and R x7 are each and independently of each other selected from hydrogen atom or fluorine atom.
  • R xl is Me.
  • A is
  • R 1 ' is an alkyl, an optionally substituted heteroalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted aralkyl, or an optionally substituted heteroaralkyl.
  • A is
  • R 10 is an alkyl or an optionally substituted cycloalkyl.
  • R 12 is an alkyl, an optionally substituted heteroalkyl, or an optionally substituted cycloalkyl.
  • R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 are each and independently of each other selected from hydrogen atom, halogen atom, hydroxy, cyano, amino, alkyl, or optionally substituted heteroalkyl.
  • R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 are individually and independently selected from hydrogen, fluoro, chloro, methyl, ethyl, methoxy, CN or trifluoromethyl.
  • R 13 is an alkyl, an optionally substituted heteroalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted aralkyl, or an optionally substituted heteroaralkyl.
  • R 11 and R 13 are each and independently of each other selected from an alkyl, an optionally substituted heteroalkyl, or a moiety selected from
  • R yl , R y2 , and R y3 are each and independently of each other selected from hydrogen atom, halogen atom, alkyl or alkoxy.
  • R yl and R y2 are each and independently of each other selected from H, Me, Et, or OMe; and R y3 is Me.
  • R 11 is selected from Me, Et, iPr, tBu, CHF 2 , CH 2 F, CF 3 ,
  • R 10 and R 12 are selected from Me, Et, iPr or
  • R 13 is selected from Me, Et, iPr, tBu, CF 3 ,
  • R is H.
  • the problem underlying the present invention is solved by a compound, which compound is selected from compounds 1 to 293 of Table 1 , compounds 1001-1065 of Table 2 and compounds 2001-2115 of Table 3.
  • a pharmaceutical composition comprising one or more compounds according to the first or the second aspect of the present invention and, optionally, at least one carrier substance, excipient and/or adjuvant.
  • a fourth aspect which is also the first embodiment of the fourth aspect, the problem underlying the present invention is solved by the use of a compound according to the first or second aspect of the present invention or of a pharmaceutical composition according to the third aspect of the present invention for the manufacture of a medicament for the treatment and/or prevention of a disease or a condition.
  • condition or disease is selected from the group comprising inflammatory diseases, immunology disorders and pain.
  • a fifth aspect which is also the first embodiment of the fifth aspect, the problem underlying the present invention is solved by a method for the treatment of a subject which is in need of such treatment, comprising the administration of a compound according to the first aspect of the present invention or the second aspect of the present invention or of a pharmaceutical composition according to the third aspect of the present invention.
  • the compound is for use in a method for the treatment of a subject, preferably a subject in need of such treatment.
  • a 19 th embodiment of the first aspect which is also an embodiment of the first to the 18 th embodiment of the first aspect and also a third embodiment of the second aspect which is also an embodiment of the first and second embodiment of the second aspect the subject suffers from or is at risk of suffering from a disease selected from the group comprising inflammatory diseases, immunology disorders and pain.
  • the pharmaceutical composition is for use in a method for the treatment of a subject, preferably a subject in need of such treatment.
  • a third embodiment of the third aspect of the present invention which is also an embodiment of the first and second embodiment of the third aspect of the present invention the subject suffers from or is at risk of suffering from a disease selected from the group comprising inflammatory diseases, immunology disorders and pain.
  • the present invention more specifically relates to a compound of the formula (I):
  • X is CH or N
  • R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , and R 8 are each and independently of each other selected from hydrogen atom, halogen atom, hydroxy, cyano, amino, alkyl, or optionally substituted heteroalkyl;
  • R 5 is a halogen atom, hydroxy, cyano, amino, an alkyl, 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, or an optionally substituted heteroaralkyl;
  • R 9 is a hydrogen atom, an alkyl, or a heteroalkyl
  • R 10 is a hydrogen atom, an alkyl, an optionally substituted heteroalkyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aralkyl, or an optionally substituted heteroaralkyl;
  • R 11 is an alkyl, 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, or an optionally substituted heteroaralkyl;
  • R 12 is a hydrogen atom, an alkyl, an optionally substituted heteroalkyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, an optionally substituted aralkyl, or an optionally substituted heteroaralkyl;
  • Z 1 and Z 2 are each and independently of each other selected from C or N, and Z 3 and Z 4 are each and independently of each other selected from C, S, O or N;
  • R 13 is an alkyl, 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, or an optionally substituted heteroaralkyl;
  • R 14 and R 15 are
  • R 16 , R 17 , R 18 and R 19 are individually and independently selected from hydrogen atom, halogen atom, hydroxy, cyano, amino, alkyl, or optionally substituted heteroalkyl.
  • the pharmaceutical composition according to the present invention 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.
  • the present invention pertains to the use of a compound or of a pharmaceutical composition according to the present invention for the manufacture of a medicament for the treatment and/or prevention of a disease or a condition.
  • said condition or disease is responsive to bradykinin (BK BlR) modulation.
  • BK BlR bradykinin
  • a compound or a pharmaceutical composition according to the present invention is used for the manufacture of a medicament for the treatment and/or prevention of a disease or a condition 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.
  • pain is selected form the group comprising visceral pain, neuropathic pain, complex regional pain syndrome CRPS and inflammatory pain.
  • the present invention relates to 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 the present invention under conditions and in an amount sufficient to detectably inhibit binding of DAKD, KD and DABK to the BK Bl receptor.
  • the present invention concerns 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 present invention relates to 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 the present invention.
  • the compounds according to the present invention act or are used as BlR antagonists.
  • the BlR antagonists thus provided herein exhibit high activity on human BlR, i.e., an inhibition constant (IC 50 ) for competition with binding of labelled DAKD to human BlR of less than 5 micromolar.
  • Preferred compounds exhibit an IC 50 of 5000 nanomolar or less, preferably 1000 nanomolar or less, more preferably of 500 nanomolar or less, more preferably of 50 nanomolar or less and most preferably 5 nanomolar or less.
  • such BlR antagonists according to the present invention exhibit a high activity on BlR of species other than human.
  • the BlR antagonists exhibit an IC 50 for competition with binding of labelled DAKD to rabbit BlR and cynomolgus monkey of less than 5 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 38, 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 39.
  • Preferred compounds and thus BlR antagonists 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 IC 50 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 and preferably a BlR antagonist 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 herein.
  • 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 compound 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 provided 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 according to the present invention 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. It will be understood by a person skilled in the art that in a preferred embodiment, the term compounds according to the present invention comprises any prodrug of such 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 is oxo, i.e., —O
  • 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 (also referred to as Me herein), ethyl (also referred to as Et herein), propyl, iso-propyl, n-butyl, iso-butyl, sec- butyl, ter/-butyl, n-pentyl, n-hexyl, 2,2-dimethylbutyl or n-octyl group.
  • methyl also referred to as Me herein
  • Et ethyl
  • propyl iso-propyl
  • n-butyl iso-butyl
  • sec- butyl sec- butyl
  • ter/-butyl n-pentyl
  • n-hexyl 2,2-dimethylbutyl or n
  • 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.
  • alkenyl groups have one or two, more preferably one, double bond(s) and 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 comprise 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 -CO-R d , -Y a -NR c -CO-NR d -L, -Y a -NR c -CS-NR d -L, -Y a -O-CO-NR a R b , -Y a -CO-NR a R b , -O-Y a -CO-NR a R b , -Y a -NR c -CO-L, , -Y a -O-CO-O-R a R b , -Y a -CO-NR a R b
  • heteroalkyl groups are methoxy, trifluoromethoxy, ethoxy, n- propyloxy, isopropyloxy, t ⁇ rt-butyloxy, methoxymethyl, ethoxymethyl, methoxyethyl, methylamino, ethylamino, dimethylamino, diethylamino, isopropylethylamino, methylaminomethyl, ethylaminomethyl, diisopropylaminoethyl, enol ether, dimethyl- aminomethyl, dimethylaminoethyl, acetyl, propionyl, butyryloxy, acetyloxy, methoxycarbonyl, ethoxycarbonyl, isobutyrylamino-methyl, N-ethyl-N-methylcarbamoyl and N-methylcarbamoyl.
  • heteroalkyl groups comprise nitrile, isonitrile, cyanate, thiocyanate, isocyanate, isothiocyanate and alkylnitrile groups.
  • An example of a heteroalkylene group is a group of formulas -CH 2 CH(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 comprise a cyclopropyl, cyclobutyl, cyclopentyl, spiro[4,5]decanyl, norbornyl, cyclohexyl, cyclo- pentenyl, cyclohexadienyl, decalinyl, bicyclo[4.3.0]nonyl, tetralin, cyclopentylcyclohexyl, fluorocyclohexyl 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, nitrogen, 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 comprise 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 comprise alkylheterocycloalkyl, heterocycloalkylalkyl, alkylheterocycloalkenyl, alkenylheterocycloalkyl, alkynylhetero- cycloalkyl, heteroalkylcycloalkyl, heteroalkylheterocycloalkyl and hetero- alkylheterocycloalkenyl, 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 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, SH, NH 2 , CN or NO 2 groups.
  • Examples comprise 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 comprise 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 comprise 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 the 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 comprise arylheteroalkyl, arylheterocycloalkyl, aryl- heterocycloalkenyl, arylalkylheterocycloalkyl, arylalkenylheterocycloalkyl, arylalkynylheterocycloalkyl, arylalkylheterocycloalkenyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heteroarylheteroalkyl, heteroarylcycloalkyl, heteroarylcycloalkenyl, heteroarylheterocycloalkyl, heteroarylheterocycloalkenyl, heteroarylalkylcycloalkyl, heteroarylheterocycloalkenyl, heteroarylalkylcycloalkyl, heteroarylalkylheterocycloalkenyl, heteroarylalkylcycloalkyl, heteroarylalkyl
  • carbocyclic ring preferably refers to a ring system, where all ring atoms that form the ring system are carbon atoms.
  • heterocyclic ring preferably refers to a ring system, where one or more ring atom(s) that form the ring system are selected from N, O and S.
  • the nitrogen and sulfur heteroatoms may optionally be oxidized.
  • A is preferably selected from, but not limited to, the group comprising the optionally substituted 5-membered heterocyclic ring systems:
  • R 14 and R 15 can be joined to form a second carbocyclic or heterocyclic ring of a fused bicyclic systems.
  • R 14 and R 15 form a second ring A can be selected from, but is not limited to, the group comprising:
  • the resulting fused bicyclic system is an 8- or 9-membered bicyclic system, which is comprised of a first 5-membered ring and a second ring that has 5 or 6 members.
  • the carbocyclic or heterocyclic rings described herein may be substituted on one or more carbon or nitrogen atom(s) if the resulting compound is stable.
  • a nitrogen in a heterocyclic ring may optionally be quaternized.
  • examples of 5- or 6-membered carbocyclic or heterocyclic rings include, but are not limited to, rings represented by joining R 14 and R 15 to a ring system as shown below.
  • Such ring systems can optionally be substituted on one or more carbon or nitrogen atom(s) if the resulting compound is stable.
  • substituted means that such group, in one embodiment, is substituted, and, in another embodiment, it is not substituted.
  • a group which is substituted is, in a further embodiment, to a group in which one or more hydrogen atoms have been replaced each independently of the others by an unsubstituted Ci-C 6 alkyl, unsubstituted C 2 -C 6 alkenyl, unsubstituted C 2 -C 6 alkynyl, unsubstituted Ci-C ⁇ heteroalkyl, unsubstituted C 3 -Ci 0 cycloalkyl, unsubstituted C 2 -Cc)heterocycloalkyl, unsubstituted C 6 -Cioaryl, unsubstituted d-Cciheteroaryl, unsubstituted C 7 -Ci 2 aralkyl or unsubstituted C 2 -Ci 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.
  • Preferred compounds of formula (I) are those, wherein R 5 is a halogen atom, an alkyl, an optionally substituted heteroalkyl, or a moiety selected from
  • X 1 , X 2 , X 3 , and X 4 are each and independently of each other selected from carbon atom, N, O, S, NR X , CR X , or CR X R X> , and X 5 is selected from C, N or CR X , wherein
  • alkyl-amino refers to a group of formula -NR 8 R h , wherein R 8 and R h are each and independently of each other selected from hydrogen atom or alkyl.
  • alkyl-mercapto refers to a group of formula -SR 1 , wherein R 1 is alkyl. Further preferred are compounds of formula (I), wherein
  • R 1 , R 3 , R 7 , and R 8 are each a hydrogen atom
  • R 2 , R 4 and R 6 are each and independently of each other selected from hydrogen atom, halogen atom, cyano, alkyl or heteroalkyl;
  • R 9 is a hydrogen atom or Me.
  • R 2 , R 4 and R 6 are each and independently of each other selected from hydrogen atom, Cl or F.
  • R 5 is Me, Et, cyano, CF 3 , Cl or a moiety selected from
  • R xl , R" 2 , R" 3 and R x4 are each and independently of each other selected from hydrogen atom, halogen atom, alkyl or alkoxy
  • R x5 , R x6 and R x7 are each and independently of each other selected from hydrogen atom or fluorine atom.
  • R xl is Me.
  • the compound of formula (I) can also be a compound, wherein A is
  • R 11 is an alkyl, an optionally substituted heteroalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted aralkyl, or an optionally substituted heteroaralkyl.
  • R 10 is an alkyl or an optionally substituted cycloalkyl.
  • R 12 is an alkyl, an optionally substituted heteroalkyl, or an optionally substituted cycloalkyl.
  • R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 are each and independently of each other selected from hydrogen atom, halogen atom, hydroxy, cyano, amino, alkyl, or optionally substituted heteroalkyl.
  • the compound of formula (I) is a compound, wherein R 14 , R 15 , R 16 , R 17 , R 1 , and R 19 are each and independently selected from hydrogen atom, fluoro, chloro, methyl, ethyl, methoxy, CN or trifluoromethyl.
  • R 13 is an alkyl, an optionally substituted heteroalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted aralkyl, or an optionally substituted heteroaralkyl.
  • R 1 1 and R 13 are each and independently from each other selected from an alkyl, an optionally substituted heteroalkyl, or a moiety selected from
  • R yl , R y2 , and R y3 are each and independently of each other selected from hydrogen atom, halogen atom, alkyl or alkoxy.
  • the compound of formula (I) is a compound, wherein
  • R yl and R y2 are each and independently of each other selected from Me, Et, or OMe; and R y3 is Me.
  • the compound of formula (I) is a compound, wherein R 1 ' is selected from Me, Et, iPr, tBu, CHF 2 , CH 2 F, CF 3 ,
  • the compound of formula (I) is a compound, wherein R 10 and R 12 are selected
  • the compound of formula (I) is a compound, wherein R 13 is selected from Me, Et, iPr, tBu, CF 3 ,
  • the compound of formula (I) is a compound selected from compounds 1 to 293 of Table 1, compounds 1001-1065 of Table 2 and compounds 2001-2115 of Table 3.
  • the therapeutic use of the compounds according to the present invention, 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 the compounds according to the present invention as active ingredients in the preparation or manufacture of a medicament for the treatment and/or prevention of a disease or a condition.
  • compositions according to the present invention comprise at least one compound according to the present invention 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
  • 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 anti-rheumatic drug, a cytostatic drug, a drug with smooth muscle activity modulatory activity or mixtures of the aforementioned.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising one or more compounds according to the present invention and, optionally, at least one carrier substance, excipient and/or adjuvant.
  • 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 administration.
  • 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
  • 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 suh as, e.g., sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, 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
  • the 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 comprise 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.
  • compositions according to the present invention 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 appearance 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 topical 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.
  • the pharmaceutical composition according to the present invention 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 methods 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.
  • 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 500 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 according to the present invention 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 rectum to release the drug.
  • suitable excipients include, for example, cocoa butter and polyethylene glycols.
  • compositions according to the present invention 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 present invention provides a pharmaceutical composition, which 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.
  • a pharmaceutical composition which 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.
  • 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.
  • 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 therapeutically 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 antagonist(s) 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.
  • the compounds according to the present 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 prevention of 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
  • 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, vasculitis.
  • the Bl antagonists according to the present invention are preferably effective in the treatment/prevention of a condition or a disease responsive to BK B 1 R modulation as is for example apparent from the references cited herein. Accordingly, the compounds according to the present 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 ah, Am. J. Physiol. Gastrointest Liver Physiol. 2005, 289(2), G361-6; Devani et ah, Am. J.
  • the compounds may also be used in inflammatory skin diseases such as but not limited to psoriasis, eczema, atopic diseases, dermatitis and itching.
  • the compounds are effective in joint and bone diseases such as but not limited to 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.
  • 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 such as but not limited to 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 such as but not limited to multiple sclerosis, epilepsy, amyotrophic lateral sclerosis, Alzheimer's disease, stroke, Parkinson's disease. They may also be used in cardiovascular disease such as but not limited to 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 et al., Cardiovasc. Res. 2000, 48, 194-210).
  • CNS disorders such as but not limited to multiple sclerosis, epilepsy, amyotrophic lateral sclerosis, Alzheimer's disease, stroke, Parkinson's disease. They may also be used in cardiovascular disease such as but not limited to congestive heart failure, myocardial infarct, systemic
  • vascular disorders such as but not limited to 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 fibrotic 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; Ricupero et al, J. Biol. Chem. 2000, 275(17), 12475-80; Romero et al, J. Biol. Chem. 2005, 75, 14378-14384).
  • the compounds according to the present invention are also effective in the treatment of disorders of the respiratory pathways such as but not limited to asthma, atopic or non-atopic asthma, occupational asthma, exercise-induced bronchoconstriction, bronchitis, pneumoconiosis including aluminosis, anhracosis, 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.
  • disorders of the respiratory pathways such as but not limited to asthma, atopic or non-atopic asthma, occupational asthma, exercise-induced bronchoconstriction, bronchitis, pneumoconiosis including aluminosis, anhracosis, asbestosis, chalicosis, ptilosis, siderosis,
  • 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, neuralgic 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.
  • 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, visceral 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 the compounds according to the present invention are preferably effective against back pain, headache, cluster
  • the compounds according to the present invention are preferably used for the treatment and/or prevention of a disease or condition responsive to BK BlR modulation.
  • the disease or conditon 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.
  • pain preferably comprises visceral pain, neuropathic pain, complex regional pain syndrome CRPS and inflammatory pain.
  • the present invention also relates to 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 the invention.
  • the subject is preferably a human being and more preferably a subject such as a human being who suffers from or is at risk of developing a disease which is to be treated by the administration of a compound according to the present invention.
  • the disease may be any of the diseases as disclosed herein and which may be treated by the compounds according to the present invention.
  • the present invention also provides methods for localizing or detecting a BlR in a tissue, in vivo or in vitro, 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 38.
  • the present invention also provides methods of inhibiting the binding of DAKD or any other BlR ligand to a BlR, in vivo or in vitro, 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 39.
  • 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.
  • 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 present invention provides 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 the invention 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 compounds 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
  • AgNO 3 is silver nitrate
  • BlR is Bl receptor
  • BK is bradykinin
  • BSA bovine serum albumin cone, is concentrated
  • DAKD is des-Arg-10-kallidin
  • DCM is dichloromethane
  • DIBAL is diisobutylaluminium hydride
  • DIPEA is diisopropyethylamine
  • DMAP 4-iV,N-dimethylaminopyridine
  • DMEM Dulbecco's modified Eagle's medium
  • DMSO dimethylsulfoxide
  • EA is ethyl acetate
  • FBS is fetal bovine serum
  • FCS fetal calf serum h is hour
  • HATU is 2-(lH-7-azabenzotriazol-l-yl)-l,l,3,3-tetramethyl uranium hexafluorophosphate
  • HCl hydrochloric acid
  • ⁇ EK is human embryonic kidney
  • ⁇ EPES is 4-2-hydroxyethyl-l-piperazineethanesulfonic acid
  • ⁇ OBt is N-hydroxybenzotriazole
  • ⁇ PLC high performance liquid chromatography
  • IL is interleukine
  • LiOH is lithium hydroxide
  • MeOH is methyl alcohol
  • NaCl sodium chloride
  • Na 2 CO 3 is sodium carbonate
  • NaHCO 3 sodium hydrogencarbonate
  • NaOH sodium hydroxide
  • Na 2 SO 4 is sodium sulfate
  • PBS is phosphate-buffered saline
  • PIPES is piperazine-N,N'-bis(2-ethanesulfonic acid)
  • RT room temperature sat. is saturated tBu is tert. butyl
  • TFA is trifluoroacetate or trifluoroacetic acid
  • THF is tetrahydrofuran
  • WSC is (3-dimethylamino-propyl)-ethyl-carbodiimide hydrochloride
  • 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); N,iV-dimethylformamide (LAB, Merck Eurolab); dioxane (for synthesis, Aldrich); methanol (for synthesis, Merck Eurolab).
  • RP-HPLC-MS analyses For analytic chromatography a Hewlett Packard 1 100-system (degasser G1322A, quaternary pump G1311A, automatic sample changer G1313A, column heater G 1316A, variable UV detector Gl 314A) 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.
  • the mixture was heated to 90 °C under N 2 for 3 h, then cooled to RT and partitioned between EA and water.
  • the organic extract was washed with water and brine before being dried over NaSO 4 , filtered and concentrated.
  • the product was purified by silica gel chromatography to afford the corresponding Suzuki coupling product.
  • Methyl 4'-(aminomethyl)-3,3'-difluorobiphenyl-2-carboxylate (100 mg , 0.44 mmol) was dissolved in THF (4 mL), treated with triethylamine (183 ⁇ l, 0.132 mmol) and 4- nitrophenylchloroformiate (80 mg, 0.397 mmol) at 0°C and was stirred for additional 2 h at 0°C.
  • a solution of 9H-fluoren-9-yl)methyl 2-(chlorocarbonyl)-2-ethylhydrazinecarboxylate (101 mg, 0.357 mmol) in THF (6 mL) was added dropwise and the mixture was stirred overnight at RT.
  • N'-ethylidene-hydrazinecarboxylic acid tert-butyl ester was treated with DIBAL to afford the title compound after purification by flash chromatography on silica gel (n-hexane/EA).
  • N-(4-bromo-2-fluorobenzyl)-2-methylpropane-2- sulfinamide 500 mg, 1.6 mmol, example 7B
  • l-bromo-3,5-dichloro-2-(2,2- difluoroethoxy)benzene 587 mg, 1.92 mmol, example 10 G
  • 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-D AKD Perkin Elmer
  • the antagonist compound dilutions were prepared in a dilution plate which also included controls to assess total binding (1 nM 3H-D AKD) 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 min 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 NaCl, 0.2 M acetic acid) for 10 min on ice.
  • dissociation buffer 0.5 M NaCl, 0.2 M acetic acid
  • 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 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 40.000 cells/well. After an overnight incubation (appr. 12-16h) cells were treated with 1.36 ng/ mL human recombinant IL-l ⁇ in DMEM 15%FBS for 4h at 37°C to induce BlR upregulation.
  • 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°C.

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Abstract

The present invention is related to a compound of the formula (I): or a pharmacologically acceptable salt, solvate or hydrate thereof, wherein A is formula (II) (III) (IV), X is Ch or N; R1, R2, R3, R4, R6, R7, and R8 are each and independently of each other selected from hydrogen atom, halogen atom, hydroxy, cyano, amino, alkyl, or optionally substituted heteroalkyl; R5 is a halogen atom, hydroxy, cyano, amino, an alkyl, 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, or an optionally substituted heteroaralkyl; R9 is a hydrogen atom, an alkyl, or a heteroalkyl; R10 is a hydrogen atom, an alkyl, an optionally substituted heteroalkyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aralkyl, or an optionally substituted heteroaralkyl; R11 is an alkyl, 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, or an optionally substituted heteroaralkyl; R12 is a hydrogen atom, an alkyl, an optionally substituted heteroalkyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, an optionally substituted aralkyl, or an optionally substituted heteroaralkyl; B is O or N; Z1 and Z2 are each and independently of each other selected from C or N, and Z3 and Z4 are each and independently of each other selected from C, S, O or N; R13 is an alkyl, 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, or an optionally substituted heteroaralkyl; R14 and R15, if present, are (i) each and independently of each other selected from hydrogen atom, halogen atom, CN, hydroxy, =O, alkyl, C3-C6-cycloalkyl, heteroalkyl or alkoxy; or (ii) joined together to form a carbocyclic or heterocyclic 5- or 6-membered ring, which is substituted with 0 to 4 substituents selected from the group comprising R16, R17, R18 and R19, and is saturated, unsaturated, or aromatic, and, if heterocyclic, contains one or more heteroatom(s) each and independently selected from N, O and S; and R16, R17, R18 and R19 are individually and independently selected from hydrogen atom, halogen atom, hydroxy, cyano, amino, alkyl, and optionally substituted heteroalkyl.

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 ah, 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 and Johnson, Expert Opin. Ther. Targets 2007, 11, 21-35; Chen and Biswas, Progress in Medicinal Chemistry 2008, 46, 173-204). For instance, WO05016886, US20060122236, US20060111392, US20060173023, WO05063690, WO06132837 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.
The problem is solved by the subject matter of the attached independent claims. Specific embodiments may be taken from the dependent claims.
More specifically, in a first aspect which is also the first embodiment of the first aspect, the problem underlying the present invention is solved by a compound of the formula (I):
Figure imgf000005_0001
or a pharmacologically acceptable salt, solvate or hydrate thereof, wherein
A is
Figure imgf000005_0002
X is CH or N;
R1, R2, R3, R4, R6, R7, and R8 are each and independently of each other selected from hydrogen atom, halogen atom, hydroxy, cyano, amino, alkyl, or optionally substituted heteroalkyl;
R5 is a halogen atom, hydroxy, cyano, amino, an alkyl, 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, or an optionally substituted heteroaralkyl;
R9 is a hydrogen atom, an alkyl, or a heteroalkyl;
R10 is a hydrogen atom, an alkyl, an optionally substituted heteroalkyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aralkyl, or an optionally substituted heteroaralkyl;
R11 is an alkyl, 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, or an optionally substituted heteroaralkyl;
R12 is a hydrogen atom, an alkyl, an optionally substituted heteroalkyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, an optionally substituted aralkyl, or an optionally substituted heteroaralkyl;
B is O or N;
Z1 and Z2 are each and independently of each other selected from C or N, and Z3 and Z4 are each and independently of each other selected from C, S, O or N;
R13 is an alkyl, 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, or an optionally substituted heteroaralkyl;
R14 and R15, if present, are
(i) each and independently of each other selected from hydrogen atom, halogen atom, CN, hydroxy, =O, alkyl, C3-C6-cycloalkyl, heteroalkyl or alkoxy; or
(ii) joined together to form a carbocyclic or heterocyclic 5- or 6-membered ring, which is substituted with 0 to 4 substituents selected from the group comprising R16, R17, R18 and R19, and is saturated, unsaturated, or aromatic, and, if heterocyclic, contains one or more heteroatom(s) each and independently selected from N, O and S; and
R16, R17, R18 and R19 are individually and independently selected from hydrogen atom, halogen atom, hydroxy, cyano, amino, alkyl, and optionally substituted heteroalkyl.
In a second embodiment of the first aspect which is also an embodiment of the first embodiment of the first aspect, R5 is a halogen atom, an alkyl, an optionally substituted heteroalkyl, or a moiety selected from
Figure imgf000007_0001
wherein is a single or double bond;
X1, X2, X3, and X4 are each and independently of each other selected from C, N, O, S, NR", CR", or CRXRX , and X5 is selected from carbon atom, N or CRX, wherein
R" and Rx are each and independently of each other selected from hydrogen atom, halogen atom, =0, hydroxy, cyano, alkyl, alkyl-amino, alkyl-mercapto , or alkoxy.
In a third embodiment of the first aspect which is also an embodiment of the first and second embodiment of the first aspect,
R , R , R ,and R are each a hydrogen atom;
R2, R4 and R6 are each and independently of each other selected from hydrogen atom, halogen atom, cyano, alkyl or heteroalkyl; and
R9 is a hydrogen atom or Me.
In a fourth embodiment of the first aspect which is also an embodiment of the first, second and third embodiment of the first aspect,
R5 is Me, Et, cyano, CF3, Cl or a moiety selected from
Figure imgf000008_0001
and R , R , R and R are each and independently of each other selected from hydrogen atom, halogen atom, alkyl or alkoxy, and Rx5, Rx6 and Rx7 are each and independently of each other selected from hydrogen atom or fluorine atom.
In a fifth embodiment of the first aspect which is also an embodiment of the fourth embodiment of the first aspect, if present, Rxl is Me.
In a sixth embodiment of the first aspect which is also an embodiment of the first, second, third, fourth and fifth embodiment of the first aspect, A is
Figure imgf000009_0001
R1 ' is an alkyl, an optionally substituted heteroalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted aralkyl, or an optionally substituted heteroaralkyl.
In a seventh embodiment of the first aspect which is also an embodiment of the first, second, third, fourth, fifth and sixth embodiment of the first aspect, A is
Figure imgf000009_0002
R10 is an alkyl or an optionally substituted cycloalkyl.
In an eighth embodiment of the first aspect which is also an embodiment of the first, second, third, fourth and fifth embodiment of the first aspect, A is
Figure imgf000010_0001
R12 is an alkyl, an optionally substituted heteroalkyl, or an optionally substituted cycloalkyl.
In a ninth embodiment of the first aspect which is also an embodiment of the first, second, third, fourth and fifth embodiment of the first aspect A is selected from
Figure imgf000010_0002
R14, R15, R16, R17, R18, and R19 are each and independently of each other selected from hydrogen atom, halogen atom, hydroxy, cyano, amino, alkyl, or optionally substituted heteroalkyl.
In a tenth embodiment of the first aspect which is also an embodiment of the ninth embodiment of the first aspect, R14, R15, R16, R17, R18, and R19 are individually and independently selected from hydrogen, fluoro, chloro, methyl, ethyl, methoxy, CN or trifluoromethyl.
In an eleventh embodiment of the first aspect which is also an embodiment of the first to fifth and ninth to tenth embodiment of the first aspect, R13 is an alkyl, an optionally substituted heteroalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted aralkyl, or an optionally substituted heteroaralkyl.
In a twelfth embodiment of the first aspect which is also an embodiment of the first to seventh and ninth to eleventh embodiment of the first aspect, R11 and R13, are each and independently of each other selected from an alkyl, an optionally substituted heteroalkyl, or a moiety selected from
Figure imgf000011_0001
wherein Ryl, Ry2, and Ry3 are each and independently of each other selected from hydrogen atom, halogen atom, alkyl or alkoxy.
In a 13th embodiment of the first aspect which is also an embodiment of the twelfth embodiment of the first aspect, Ryl and Ry2 are each and independently of each other selected from H, Me, Et, or OMe; and Ry3 is Me.
In a 14th embodiment of the first aspect which is also an embodiment of the first to seventh , twelfth and 13th embodiment of the first aspect, R11 is selected from Me, Et, iPr, tBu, CHF2, CH2F, CF3,
Figure imgf000012_0001
Figure imgf000012_0002
In a 15th embodiment of the first aspect which is also an embodiment of the first to eighth and twelfth to 14th embodiment of the first aspect, R10 and R12 are selected from Me, Et, iPr or
Figure imgf000012_0003
In a 16th embodiment of the first aspect which is also an embodiment of the first to fifth and ninth to 14th embodiment of the first aspect, R13 is selected from Me, Et, iPr, tBu, CF3,
Figure imgf000013_0001
Figure imgf000013_0002
In a 17th embodiment of the first aspect which is also an embodiment of the ninth to 13th and 16th embodiment of the first aspect, R is H.
In a second aspect which is also the first embodiment of the second aspect and the 18th embodiment of the first aspect, the problem underlying the present invention is solved by a compound, which compound is selected from compounds 1 to 293 of Table 1 , compounds 1001-1065 of Table 2 and compounds 2001-2115 of Table 3.
In a third aspect which is also the first embodiment of the third aspect, the problem underlying the present invention is solved by a pharmaceutical composition comprising one or more compounds according to the first or the second aspect of the present invention and, optionally, at least one carrier substance, excipient and/or adjuvant.
In a fourth aspect which is also the first embodiment of the fourth aspect, the problem underlying the present invention is solved by the use of a compound according to the first or second aspect of the present invention or of a pharmaceutical composition according to the third aspect of the present invention for the manufacture of a medicament for the treatment 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 selected from the group comprising inflammatory diseases, immunology disorders and pain.
In a fifth aspect which is also the first embodiment of the fifth aspect, the problem underlying the present invention is solved by a method for the treatment of a subject which is in need of such treatment, comprising the administration of a compound according to the first aspect of the present invention or the second aspect of the present invention or of a pharmaceutical composition according to the third aspect of the present invention.
In an 18th embodiment of the first aspect which is also an embodiment of the first to the 17th embodiment of the first aspect and also a second embodiment of the second aspect the compound is for use in a method for the treatment of a subject, preferably a subject in need of such treatment.
In a 19th embodiment of the first aspect which is also an embodiment of the first to the 18th embodiment of the first aspect and also a third embodiment of the second aspect which is also an embodiment of the first and second embodiment of the second aspect the subject suffers from or is at risk of suffering from a disease selected from the group comprising inflammatory diseases, immunology disorders and pain.
In a second embodiment of the third aspect of the present invention the pharmaceutical composition is for use in a method for the treatment of a subject, preferably a subject in need of such treatment. In a third embodiment of the third aspect of the present invention which is also an embodiment of the first and second embodiment of the third aspect of the present invention the subject suffers from or is at risk of suffering from a disease selected from the group comprising inflammatory diseases, immunology disorders and pain.
The present invention more specifically relates to a compound of the formula (I):
Figure imgf000015_0001
or a pharmacologically acceptable salt, solvate or hydrate thereof, wherein
A is
Figure imgf000015_0002
X is CH or N;
R1, R2, R3, R4, R6, R7, and R8 are each and independently of each other selected from hydrogen atom, halogen atom, hydroxy, cyano, amino, alkyl, or optionally substituted heteroalkyl;
R5 is a halogen atom, hydroxy, cyano, amino, an alkyl, 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, or an optionally substituted heteroaralkyl;
R9 is a hydrogen atom, an alkyl, or a heteroalkyl;
R10 is a hydrogen atom, an alkyl, an optionally substituted heteroalkyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aralkyl, or an optionally substituted heteroaralkyl;
R11 is an alkyl, 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, or an optionally substituted heteroaralkyl;
R12 is a hydrogen atom, an alkyl, an optionally substituted heteroalkyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, an optionally substituted aralkyl, or an optionally substituted heteroaralkyl;
B is O or N; Z1 and Z2 are each and independently of each other selected from C or N, and Z3 and Z4 are each and independently of each other selected from C, S, O or N;
R13 is an alkyl, 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, or an optionally substituted heteroaralkyl;
R14 and R15, if present, are
(iii) each and independently of each other selected from hydrogen atom, halogen atom, CN, hydroxy, =0, alkyl, C3-C6-cycloalkyl, heteroaryl or alkoxy; or
(iv) joined together to form a carbocyclic or heterocyclic 5- or 6-membered ring, which is substituted with 0 to 4 substituents selected from the group comprising R16, R17, R18 and R19 and is saturated, unsaturated, or aromatic, and, if heterocyclic, contains one or more heteroatom(s) each and independently selected from N, O and S; and
R16, R17, R18 and R19 are individually and independently selected from hydrogen atom, halogen atom, hydroxy, cyano, amino, alkyl, or optionally substituted heteroalkyl.
Preferably, the pharmaceutical composition according to the present invention 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.
Moreover, the present invention pertains to the use of a compound or of a pharmaceutical composition according to the present invention for the manufacture of a medicament for the treatment and/or prevention of a disease or a condition. Preferably, said condition or disease is responsive to bradykinin (BK BlR) modulation. Preferably, a compound or a pharmaceutical composition according to the present invention is used for the manufacture of a medicament for the treatment and/or prevention of a disease or a condition selected from the group comprising inflammatory diseases, immunology disorders and pain.
More preferably, the inflammatory disease or immunology disorder is selected from the group comprising inflammatory bowel disease, rheumatoid arthritis, gouty arthritis, atherosclerosis and associated fibrotic conditions.
More preferably, pain is selected form the group comprising visceral pain, neuropathic pain, complex regional pain syndrome CRPS and inflammatory pain.
Furthermore, the present invention relates to 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 the present invention under conditions and in an amount sufficient to detectably inhibit binding of DAKD, KD and DABK to the BK Bl receptor.
Additionally, the present invention concerns 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 to the present invention under conditions that permit binding of the compound to the BK Bl 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 preferred embodiment of the method for localizing or detecting a BK Bl receptor in a tissue, the compound is radiolabeled, fluorescence labeled or luminescence labeled, or labeled with an antibody. Moreover, the present invention relates to 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 the present invention.
The compounds shown in the following Table 1 , Table 2 and Table 3 are embodiments of formula (I) and thus embodiments of the compound according to the present invention.
Figure imgf000020_0002
Figure imgf000020_0001
Figure imgf000021_0003
Figure imgf000021_0001
Figure imgf000021_0002
Figure imgf000022_0001
Figure imgf000022_0002
Figure imgf000023_0002
Figure imgf000023_0001
Figure imgf000023_0003
Figure imgf000024_0003
Figure imgf000024_0001
Figure imgf000024_0002
Figure imgf000025_0003
Figure imgf000025_0001
Figure imgf000025_0002
Figure imgf000026_0001
Figure imgf000026_0002
Figure imgf000027_0001
Figure imgf000027_0002
Figure imgf000028_0002
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000029_0002
Figure imgf000030_0003
Figure imgf000030_0001
Figure imgf000030_0002
Figure imgf000031_0002
Figure imgf000031_0001
Figure imgf000032_0002
Figure imgf000032_0001
Figure imgf000032_0003
Figure imgf000033_0001
Figure imgf000033_0002
Figure imgf000034_0001
Figure imgf000034_0002
Figure imgf000035_0002
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000036_0002
Figure imgf000037_0003
Figure imgf000037_0001
Figure imgf000037_0002
Figure imgf000038_0001
Figure imgf000038_0002
Figure imgf000039_0001
Figure imgf000039_0002
Figure imgf000040_0002
Figure imgf000040_0001
Figure imgf000040_0003
Figure imgf000041_0002
Figure imgf000041_0001
Figure imgf000042_0001
Table 2:
Figure imgf000043_0001
Figure imgf000043_0002
Figure imgf000044_0002
Figure imgf000044_0001
Figure imgf000045_0003
Figure imgf000045_0001
Figure imgf000045_0002
Figure imgf000046_0002
Figure imgf000046_0001
Figure imgf000047_0003
Figure imgf000047_0001
Figure imgf000047_0002
Figure imgf000048_0001
Figure imgf000049_0002
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000050_0002
Figure imgf000051_0002
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000052_0002
Figure imgf000053_0001
Figure imgf000054_0002
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000057_0002
Figure imgf000058_0002
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0002
Figure imgf000060_0001
It is within the present invention that the compounds according to the present invention act or are used as BlR antagonists. In general, the BlR antagonists thus provided herein exhibit high activity on human BlR, i.e., an inhibition constant (IC50) for competition with binding of labelled DAKD to human BlR of less than 5 micromolar. Preferred compounds exhibit an IC50 of 5000 nanomolar or less, preferably 1000 nanomolar or less, more preferably of 500 nanomolar or less, more preferably of 50 nanomolar or less and most preferably 5 nanomolar or less. In some embodiments, such BlR antagonists according to the present invention exhibit a high activity on BlR of species other than human. For example, in one embodiment the BlR antagonists exhibit an IC50 for competition with binding of labelled DAKD to rabbit BlR and cynomolgus monkey of less than 5 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 38, 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 39.
Preferred compounds and thus BlR antagonists 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 and preferably a BlR antagonist 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 herein.
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 encompassed. 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, RA, R1 -R19, X and 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 RA, the group may be unsubstituted or substituted with up to two RΛ groups and RA at each occurrence is selected independently from the definition of RΛ. 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 compound 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 provided 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 according to the present invention 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. It will be understood by a person skilled in the art that in a preferred embodiment, the term compounds according to the present invention comprises any prodrug of such 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., —O, 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(=O)- 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 (also referred to as Me herein), ethyl (also referred to as Et herein), propyl, iso-propyl, n-butyl, iso-butyl, sec- butyl, ter/-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) and 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 comprise groups of formulas -S-Ya-L, -S-Ya-CO-NRaRb, -Ya-NRc-CO-NRaRb, -Ya-NRc-CO-O-Rd, -Ya-NRc-CO-Rd, -Ya-NRc-CO-NRd-L, -Ya-NRc-CS-NRd-L, -Ya-O-CO-NRaRb, -Ya-CO-NRaRb, -O-Ya-CO-NRaRb, -Ya-NRc-CO-L, , -Ya-O-CO-O-Rc, -Ya-O-CO-Rc, -Ya-O-Rc, -Ya-CO-L, -Ya-NRaRb, Rc-S-Ya-, Ra-N(Rb)-Ya-, Rc-CO-Ya-, Rc-O-CO-Ya-, Rc-CO-O-Ya-, Rc-CO-N(Rb)-Ya-, Ra-N(Rb)-CO-Ya-, Rc-SO-Ya-, Rc-SO2-Ya-, -Ya-NRc-SO2-NRaRb, -Ya-SO2-NRaRb, -Ya-NRc-SO2-Rd, Ra-O-CO-N(Rb)-Ya-, Ra-N(Rb)-C(=NRd)-N(Rc)-Ya-, Rc-S-CO-Ya-, Rc-CO-S-Ya-, Rc-S-CO-N(Rb)-Ya-, Ra-N(Rb)-CO-S-Ya-, Rc-S-CO-O-Ya-, Rc-O-CO-S-Ya-, Rc-S-CO-S-Ya-; wherein Ra being a hydrogen atom, a Ci-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 Ci-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 Ci- Cgalkyl, an optionally substituted C2-Cgalkenyl or an optionally substituted C2-C8alkynyl ; Rd being a hydrogen atom, optionally substituted Ci-C8alkyl, optionally substituted C2-Cgalkenyl or optionally substituted C2-Cgalkynyl; L being a cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, aralkyl, or heteroaralkyl; and Ya being a bond, a Cj-Cόalkylene, 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. Specific examples of heteroalkyl groups are methoxy, trifluoromethoxy, ethoxy, n- propyloxy, isopropyloxy, tørt-butyloxy, methoxymethyl, ethoxymethyl, methoxyethyl, methylamino, ethylamino, dimethylamino, diethylamino, isopropylethylamino, methylaminomethyl, ethylaminomethyl, diisopropylaminoethyl, enol ether, dimethyl- aminomethyl, dimethylaminoethyl, acetyl, propionyl, butyryloxy, acetyloxy, methoxycarbonyl, ethoxycarbonyl, isobutyrylamino-methyl, N-ethyl-N-methylcarbamoyl and N-methylcarbamoyl. Further examples of heteroalkyl groups comprise nitrile, isonitrile, cyanate, thiocyanate, isocyanate, isothiocyanate and alkylnitrile groups. An example of a heteroalkylene group is a group of formulas -CH2CH(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 or NO2 groups, thus, for example, cyclic ketones such as, for example, cyclohexanone, 2- cyclohexenone or cyclopentanone. Further specific examples of a cycloalkyl group comprise a cyclopropyl, cyclobutyl, cyclopentyl, spiro[4,5]decanyl, norbornyl, cyclohexyl, cyclo- pentenyl, cyclohexadienyl, decalinyl, bicyclo[4.3.0]nonyl, tetralin, cyclopentylcyclohexyl, fluorocyclohexyl 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, nitrogen, 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 or NO2 groups. Examples comprise 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 comprise alkylheterocycloalkyl, heterocycloalkylalkyl, alkylheterocycloalkenyl, alkenylheterocycloalkyl, alkynylhetero- cycloalkyl, heteroalkylcycloalkyl, heteroalkylheterocycloalkyl and hetero- alkylheterocycloalkenyl, 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 replaced each independently of the others by fluorine, chlorine, bromine or iodine atoms or by OH, SH, NH2, CN or NO2 groups. Examples comprise 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 or NO2 groups. Examples comprise 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 comprise 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 the 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 comprise arylheteroalkyl, arylheterocycloalkyl, aryl- heterocycloalkenyl, arylalkylheterocycloalkyl, arylalkenylheterocycloalkyl, arylalkynylheterocycloalkyl, arylalkylheterocycloalkenyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heteroarylheteroalkyl, heteroarylcycloalkyl, heteroarylcycloalkenyl, heteroarylheterocycloalkyl, heteroarylheterocycloalkenyl, heteroarylalkylcycloalkyl, heteroarylalkylheterocycloalkenyl, heteroarylheteroalkylcycloalkyl, heteroarylheteroalkyl- cycloalkenyl, heteroalkylheteroarylalkyl and heteroarylheteroalkylheterocycloalkyl groups, the cyclic groups being saturated or mono-, di- or tri-unsaturated. Specific examples are a tetrahydroisoquinolinyl, 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 preferably 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, =O, SH, =S, NH2, =NH, CN or NO2 groups.
The expression carbocyclic ring preferably refers to a ring system, where all ring atoms that form the ring system are carbon atoms.
The expression heterocyclic ring preferably refers to a ring system, where one or more ring atom(s) that form the ring system are selected from N, O and S. The nitrogen and sulfur heteroatoms may optionally be oxidized.
If A in formula (I) is represented by the generic structure
Figure imgf000070_0001
then A is preferably selected from, but not limited to, the group comprising the optionally substituted 5-membered heterocyclic ring systems:
Figure imgf000070_0002
In the above generic structure of A R14 and R15, if both present, can be joined to form a second carbocyclic or heterocyclic ring of a fused bicyclic systems. For such bicyclic ring systems where R14 and R15 form a second ring A can be selected from, but is not limited to, the group comprising:
Figure imgf000071_0001
Preferably, the resulting fused bicyclic system is an 8- or 9-membered bicyclic system, which is comprised of a first 5-membered ring and a second ring that has 5 or 6 members. Additionally, the carbocyclic or heterocyclic rings described herein may be substituted on one or more carbon or nitrogen atom(s) if the resulting compound is stable. A nitrogen in a heterocyclic ring may optionally be quaternized. In addition to those exemplified elsewhere herein, examples of 5- or 6-membered carbocyclic or heterocyclic rings include, but are not limited to, rings represented by joining R14 and R15 to a ring system as shown below.
Figure imgf000072_0001
Figure imgf000072_0002
JWVW>
JO iI
Figure imgf000072_0003
N N
Figure imgf000072_0004
Figure imgf000072_0005
J (VWVV N> JwvxA,
Such ring systems can optionally be substituted on one or more carbon or nitrogen atom(s) if the resulting compound is stable.
The expression ,,optionally substituted" as preferably used herein in connection with any group means that such group, in one embodiment, is substituted, and, in another embodiment, it is not substituted. In case of the embodiment where such group is substituted, substituted means that one or more hydrogen atoms of such group have been replaced each independently of the others by fluorine, chlorine, bromine or iodine atoms or by OH, =0, SH, =S, NH2, =NH, CN or NO2 groups. Furthermore a group which is substituted, is, in a further embodiment, to a group in which one or more hydrogen atoms have been replaced each independently of the others by an unsubstituted Ci-C6alkyl, unsubstituted C2-C6alkenyl, unsubstituted C2-C6alkynyl, unsubstituted Ci-Cδheteroalkyl, unsubstituted C3-Ci0cycloalkyl, unsubstituted C2-Cc)heterocycloalkyl, unsubstituted C6-Cioaryl, unsubstituted d-Cciheteroaryl, unsubstituted C7-Ci2aralkyl or unsubstituted C2-Ci iheteroaralkyl 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.
Preferred compounds of formula (I) are those, wherein R5 is a halogen atom, an alkyl, an optionally substituted heteroalkyl, or a moiety selected from
Figure imgf000073_0001
wherein is a single or double bond, whereby, preferably, the number of double bonds is 0, 1 or 2 in said moiety;
X1, X2, X3, and X4 are each and independently of each other selected from carbon atom, N, O, S, NRX, CRX, or CRXRX>, and X5 is selected from C, N or CRX, wherein
Rx and Rx are each and independently of each other selected from hydrogen atom, halogen atom, =0, hydroxy, cyano, alkyl, alkyl-amino, alkyl-mercapto, or alkoxy.
The expression alkyl-amino refers to a group of formula -NR8Rh, wherein R8 and Rh are each and independently of each other selected from hydrogen atom or alkyl.
The expression alkyl-mercapto refers to a group of formula -SR1, wherein R1 is alkyl. Further preferred are compounds of formula (I), wherein
R1, R3, R7, and R8 are each a hydrogen atom;
R2, R4 and R6 are each and independently of each other selected from hydrogen atom, halogen atom, cyano, alkyl or heteroalkyl; and
R9 is a hydrogen atom or Me.
Preferred are compounds of formula (I), wherein
R2, R4 and R6 are each and independently of each other selected from hydrogen atom, Cl or F.
Also preferred are compounds of formula (I), wherein R5 is Me, Et, cyano, CF3, Cl or a moiety selected from
Figure imgf000074_0001
and wherein Rxl, R"2, R"3 and Rx4 are each and independently of each other selected from hydrogen atom, halogen atom, alkyl or alkoxy, and Rx5, Rx6 and Rx7 are each and independently of each other selected from hydrogen atom or fluorine atom. Further preferred are compounds of formula (I), wherein, if present, R xl is Me.
The compound of formula (I) can also be a compound, wherein A is
Figure imgf000075_0001
R11 is an alkyl, an optionally substituted heteroalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted aralkyl, or an optionally substituted heteroaralkyl.
Preferred are compounds of formula (I), wherein A is
Figure imgf000075_0002
R10 is an alkyl or an optionally substituted cycloalkyl.
Also preferred are compounds of formula (I), wherein A is
Figure imgf000075_0003
O ; and R12 is an alkyl, an optionally substituted heteroalkyl, or an optionally substituted cycloalkyl.
Further preferred compounds of formula (I) include compounds, wherein A is selected from
Figure imgf000076_0001
R14, R15, R16, R17, R18, and R19 are each and independently of each other selected from hydrogen atom, halogen atom, hydroxy, cyano, amino, alkyl, or optionally substituted heteroalkyl.
Preferably, the compound of formula (I) is a compound, wherein R14, R15, R16, R17, R1 , and R19 are each and independently selected from hydrogen atom, fluoro, chloro, methyl, ethyl, methoxy, CN or trifluoromethyl.
Further preferred are compounds of formula (I), wherein R13 is an alkyl, an optionally substituted heteroalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted aralkyl, or an optionally substituted heteroaralkyl.
Also preferred are compounds of formula (I), wherein R1 1 and R13, if present, are each and independently from each other selected from an alkyl, an optionally substituted heteroalkyl, or a moiety selected from
Figure imgf000076_0002
wherein Ryl, Ry2, and Ry3 are each and independently of each other selected from hydrogen atom, halogen atom, alkyl or alkoxy.
Preferably, the compound of formula (I) is a compound, wherein
Ryl and Ry2 are each and independently of each other selected from Me, Et, or OMe; and Ry3 is Me.
Preferably, the compound of formula (I) is a compound, wherein R1 ' is selected from Me, Et, iPr, tBu, CHF2, CH2F, CF3,
Figure imgf000077_0001
Preferably, the compound of formula (I) is a compound, wherein R10 and R12 are selected
Figure imgf000077_0002
Preferably, the compound of formula (I) is a compound, wherein R13 is selected from Me, Et, iPr, tBu, CF3,
Figure imgf000078_0001
Figure imgf000078_0002
Preferred are compounds of formula (I), wherein R9 is H.
Preferably, the compound of formula (I) is a compound selected from compounds 1 to 293 of Table 1, compounds 1001-1065 of Table 2 and compounds 2001-2115 of Table 3.
Also preferred are compounds, salts, solvates or hydrates of formula (I), wherein the compound exhibits an IC50 of 5000 nM or less, preferably 1000 nM or less, preferably 500 nM or less, more preferably 5OnM and most preferably 5 nM or less in a standard in vitro BK Bl receptor-mediated assay.
It is to be noted that the present invention also encompasses all possible combinations of all preferred embodiments.
The therapeutic use of the compounds according to the present invention, 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 the compounds according to the present invention as active ingredients in the preparation or manufacture of a medicament for the treatment and/or prevention of a disease or a condition.
The pharmaceutical compositions according to the present invention comprise at least one compound according to the present invention 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 anti-rheumatic drug, a cytostatic drug, a drug with smooth muscle activity modulatory activity or mixtures of the aforementioned.
Accordingly, the present invention provides a pharmaceutical composition comprising one or more compounds according to the present invention and, optionally, at least one carrier substance, excipient and/or adjuvant.
The 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 administration. 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.
The 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 suh as, e.g., sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, 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.
The 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 comprise 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.
The compounds according to the present invention 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 appearance 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 topical 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.
The pharmaceutical composition according to the present invention 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 methods 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 500 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.
The pharmaceutical compositions according to the present invention 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 rectum to release the drug. Suitable excipients include, for example, cocoa butter and polyethylene glycols.
The pharmaceutical compositions according to the present invention 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. Preferably, the present invention provides a pharmaceutical composition, which 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.
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 therapeutically 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 antagonist(s) 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.
The compounds according to the present 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 prevention of 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 present 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, 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 disease responsive to BK B 1 R modulation as is for example apparent from the references cited herein. Accordingly, the compounds according to the present 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 ah, Am. J. Physiol. Gastrointest Liver Physiol. 2005, 289(2), G361-6; Devani et ah, Am. J. Gastroenerol 2002, 97(S), 2026-32; Devani et ah, Dig. Liv. 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 such as but not limited to nephritis, bladder disease like cystitis, interstitial cystitis, eye diseases like uveitis, retinitis, glaucoma, and ear diseases auch as otitis media. They may also be used in inflammatory skin diseases such as but not limited to psoriasis, eczema, atopic diseases, dermatitis and itching. The compounds are effective in joint and bone diseases such as but not limited to 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 such as but not limited to 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 such as but not limited to multiple sclerosis, epilepsy, amyotrophic lateral sclerosis, Alzheimer's disease, stroke, Parkinson's disease. They may also be used in cardiovascular disease such as but not limited to 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 et al., Cardiovasc. Res. 2000, 48, 194-210). They may also be used in vascular disorders such as but not limited to 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 fibrotic 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; Ricupero et al, J. Biol. Chem. 2000, 275(17), 12475-80; Romero et al, J. Biol. Chem. 2005, 75, 14378-14384).
The compounds according to the present invention are also effective in the treatment of disorders of the respiratory pathways such as but not limited to asthma, atopic or non-atopic asthma, occupational asthma, exercise-induced bronchoconstriction, bronchitis, pneumoconiosis including aluminosis, anhracosis, 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 but not limited to 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, neuralgic 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(\), 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, visceral 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 the compounds according to the present invention 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.
Accordingly, the compounds according to the present invention are preferably used for the treatment and/or prevention of a disease or condition responsive to BK BlR modulation. Preferably, the disease or conditon is selected from the group comprising inflammatory diseases, immunology disorders and pain. Preferably, the inflammatory disease or immunology disorder is selected from the group comprising inflammatory bowel disease, rheumatoid arthritis, gouty arthritis, atherosclerosis and associated fibrotic conditions. As for pain, pain preferably comprises visceral pain, neuropathic pain, complex regional pain syndrome CRPS and inflammatory pain.
The present invention also relates to 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 the invention. It will be acknowledged by the persons skilled in the art that the subject is preferably a human being and more preferably a subject such as a human being who suffers from or is at risk of developing a disease which is to be treated by the administration of a compound according to the present invention. The disease may be any of the diseases as disclosed herein and which may be treated by the compounds according to the present invention.
Diagnostic and further applications
The present invention also provides methods for localizing or detecting a BlR in a tissue, in vivo or in vitro, 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 38.
The present invention also provides methods of inhibiting the binding of DAKD or any other BlR ligand to a BlR, in vivo or in vitro, 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 39.
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 disclosed 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.
Accordingly, the present invention provides 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 the invention under conditions and in an amount sufficient to detectably inhibit binding of DAKD, KD and DABK to the BK Bl receptor. Also provided is 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 B 1 receptor with a detectably labeled compound according to any one of claims 1 to 24 under conditions that permit binding of the compound to the BK Bl receptor; and
(b) detecting the compound bound to the BK Bl receptor or detecting the binding of the compound to the BK B 1 receptor.
Preferably, in the above mentioned applications and methods the compound is radiolabeled, fluorescence-labeled or luminescence labeled, or labeled with an antibody.
The compounds 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
AgNO3 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
DIBAL is diisobutylaluminium hydride
DIPEA is diisopropyethylamine
DMAP is 4-iV,N-dimethylaminopyridine
DMEM is Dulbecco's modified Eagle's medium
DMF is ΛyV-dimethylformamide
DMSO is dimethylsulfoxide
EA is ethyl acetate
FBS is fetal bovine serum
FCS is fetal calf serum h is hour
HATU is 2-(lH-7-azabenzotriazol-l-yl)-l,l,3,3-tetramethyl uranium hexafluorophosphate
HCl is hydrochloric acid
ΗEK is human embryonic kidney
ΗEPES is 4-2-hydroxyethyl-l-piperazineethanesulfonic acid
ΗOBt is N-hydroxybenzotriazole
ΗPLC is high performance liquid chromatography
IL is interleukine
LiOH is lithium hydroxide
MeOH is methyl alcohol
NaCl is sodium chloride
Na2CO3 is sodium carbonate
NaHCO3 is sodium hydrogencarbonate
NaOH is sodium hydroxide
Na2SO4 is sodium sulfate
NH4Cl is ammonium chloride
PBS is phosphate-buffered saline
PIPES is piperazine-N,N'-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-dimethylamino-propyl)-ethyl-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); N,iV-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 (Mϋhlheim/Main, Germany), Merck Eurolab (Darmstadt, Germany), Νeosystem (Strasbourg, France), Νovabiochem (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 1 100-system (degasser G1322A, quaternary pump G1311A, automatic sample changer G1313A, column heater G 1316A, variable UV detector Gl 314A) 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, C 18, 300 A with a pre column (Merck) was used at 30°C 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 (Rt) 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 Copyright® 1988-1998, Beilstein Institut fur 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.
General Procedure A: Synthesis of tert-butyl 2-alkylidene-hydrazinecarboxylates
To a stirred solution of tert-butyl hydrazinecarboxylate in toluene (0.5 molL"1) was added aldehyde (1.1 equiv.). The solution was heated to 50 °C for 1 h and then stirred at RT for 24 h. The mixture was concentrated to give the tert-butyl 2-alkylidene-hydrazinecarboxylates which were homogeneous by analytical HPLC (>95%).
General Procedure B: Synthesis of tert-butyl 2-alkylhydrazinecarboxylate
To tert-butyl 2-alkylidene-hydrazinecarboxylate in THF (0.3 molL"1) at -78 °C was added DIBAL (2.2 equiv.) as a 1.5 M solution in toluene. The reaction was maintained at -78 0C for 2 h and then at -40 °C for 2 h. The mixture was then warmed to RT before Rochelle's salt (aqueous potassium sodium tartrate) solution was added and the reaction mixture stirred at RT overnight. The organic phase was separated and the aqueous phase extracted twice with diethylether. The combined organic extracts were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. Purification by flash chromatography on silica gel gave tert-butyl 2-alkylhydrazinecarboxylate.
General Procedure C: Synthesis of ter/-butyl 2-ethyl-2-(alkyl- carbamoyl)hydrazinecarboxylates
To a stirred solution of phosgene in toluene (1.93 M, 4 equiv.) in dry THF (2 molL"1) at 0 °C was added a solution of ter t-butyl 2-alkylhydrazinecarboxylate and diisopropylethylamine (2.2 equiv.) in THF (0.5 molL"1) over 15 min. The mixture was stirred at 0 °C for 30 min before the excess phosgene and solvents were removed in vacuo. The residue was redissolved in THF (0.5 molL"1) with diisopropylethylamine (2.2 equiv.) and cooled to 0 °C before a solution of the desired amine (1.0 equiv.) and diisopropylethylamine (2.2 equiv.) was added all at once. The reaction was maintained at 0 °C (1 h) and then warmed to RT (16 h). The mixture was poured into EA and washed sequentially with HCl (1 M), sat. NaHCO3 and brine before being dried over Na2SO4, filtered and concentrated. The product was purified by flash chromatography on silica gel (hexane/EA) to afford the tert-buty\ 2-ethyl-2-(alkyl- carbamoyl)hydrazinecarboxylates.
General Procedure D: Cleavage of tert-butyl carbamate or tert-buty\ hydrazinecarboxylate to prepare the correspondent amine HCl salts
The TV-protected amine or semicarbazide derivative was dissolved in MeOH (0.5 molL'1) and a solution of HCl in MeOH (3 M, 20 mL/mmol) was added. The reaction mixture was stirred at RT for 2 h and then concentrated to dryness. No purification steps are required and the isolated product was utilized in the amide coupling reaction.
General Procedure E: Coupling of hydrazinyl HCl salts with carboxylic acids
The carboxylic acid (1.1 equiv.) was dissolved in DMF (0.3 molL"1), treated with DIPEA (4 equiv.) and HATU (1.1 equiv.) and stirred for 2 min at RT. The hydrazinyl HCl salt (1 equiv.) was added and the solution was stirred overnight at RT. Afterwards the reaction mixture was concentrated in vacuo and the residue was partitioned between EA (30 mL) and sat. NaHCO3 solution. After extraction of the aqueous layer with EA (2 x) the combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. Purification of the crude compound by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA yielded the desired compound as the TFA salt.
General Procedure F: Synthesis of rø-iV-benzylidene-2-methylpropane-2- sulfinamide from corresponding aldehydes
To a solution of fS/)-(-)-2-methyl-2-propanesulfinamide or ^-(+)-2-methyl-2- propanesulfinamide (1-1.5 equiv.) in CH2Cl2 (0.5 molL"1) was added the aldehyde (1.0 equiv.), pyridinium /?-toluenesulfonate (0.1 equiv.), magnesium sulphate (15 equiv.), and caesium carbonate (1 equiv.). The reaction was stirred at RT for 24 h. The mixture was filtered through celite, washed with CH2Cl2, and concentrated in vacuo. The resulting residue was subjected to silica gel chromatography to provide the corresponding imine
General Procedure G: Synthesis of 2-methyl-JV-((2?)-l-ary-ethyl)propane-2- sulfinamide from corresponding imines
To a solution of (/ζ)-7V-benzylidene-2-methylpropane-2-sulfinamide (1 equiv.) in CH2Cl2 (0.2 molL"1) at -50 °C was added methylmagnesium chloride (3.0 M solution in ether, 1.5 equiv.) dropwise. The reaction was stirred at -50 0C for 2 h, quenched with aqueous ammonium chloride, and the aqueous layer was extracted with CH2Cl2. The combined organics were dried over Na2SO4, filtered, and concentrated in vacuo. The resulting residue was subjected to silica gel chromatography to provide the corresponding 2-methy\-N-((R or S)- 1 -arylethyl)propane-2-sulfinamides.
General Procedure H: Synthesis of 7V-benzyl-2-methylpropane-2-sulfϊnamide from corresponding imines
To a solution of the (Z/)-Λr-benzylidene-2-methylpropane-2-sulfinamide in methanol (0.2 molL"1) at 0 °C was added sodium borohydride (1 equiv.) portionwise. The mixture was then warmed to RT during 1 h before the reaction was quenched with acetone. The reaction mixture was concentrated in vacuo, the residue was partitioned between EA and water and the aqueous layer was extracted with EA (2 x). The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The resulting residue was used in the following reaction without further purification.
General Procedure I: In situ synthesis of boronic esters und subsequent Suzuki coupling.
A mixture of the sulfinamide (1 equiv.), bis(pinacolato)diboron (1.5 equiv.), potassium acetate (3 equiv.), and (l,r-bis-(diphenylphosphino)ferrocene)palladium(II) dichloride (0.1 equiv.) in DMSO (0.3 molL*1) was heated to 90 0C under N2 for 3 h. The reaction mixture was cooled to RT, and the arylhalogenide (1 equiv.), and additional (1,1 '-bis- (diphenylphosphino)ferrocene)palladium(II) dichloride (0.1 equiv.) were added. The mixture was heated to 90 °C under N2 for 3 h, then cooled to RT and partitioned between EA and water. The organic extract was washed with water and brine before being dried over NaSO4, filtered and concentrated. The product was purified by silica gel chromatography to afford the corresponding Suzuki coupling product.
Example 1
Synthesis of methyl 4'-((2-acetyl-l-ethylhydrazinecarboxamido)methyl)-3,3'- difluorobiphenyl-2-carboxylate (compound 1)
Figure imgf000100_0001
A. Methyl 2-fluoro-6-iodobenzoate
To a mixture of 2-fluoro-6-iodo-benzoic acid (19.5 g, 73.3 mmol) in DCM (150 mL) was added thionyl chloride and the solution was stirred at RT for 48 h. The reaction mixture was carefully quenched with methanol at 0°C, warmed to RT and stirred for additional 30 min at RT. Afterwards the reaction mixture was concentrated in vacuo. Purification of the crude product by flash chromatography on silica gel (n-hexane/EA) obtained the title compound. GC/MS (m/z): 280.2
B. Methyl 4'-cyano-3,3'-difluorobiphenyl-2-carboxylate
To a solution of methyl 2-fluoro-6-iodobenzoate (2.0 g, 7.14 mmol), 4-cyano-3- flourobenzene boronic acid (1.33 g, 8.06 mmol) and bis(triphenylphosphine)palladium(II) dichloride (707 mg, 1.01 mmol) in dioxane (53 mL), was added a solution of sodium carbonate (3.53 g, 33.3 mmol) in water (26.5 mL). After stirring at 80°C overnight the reaction mixture was cooled to RT and diluted with EA. The organic layer was washed with brine (2x), dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (n-hexane/EA) to give the title compound.
C. Methyl 4'-(aminornethyl)-3,3'-difiuorobiphenyl-2-carboxylate To a mixture of methyl 4'-cyano-3,3'-difluorobiphenyl-2-carboxylate (750 mg, 2.75 mmol) and cobalt(ll)chloride hexahydrate (1.3 g, 5.46 mmol) in methanol (17 mL) was added sodium borohydride (1.0 g, 26.3 mmol) portionwise at 0°C. Afterwards the reaction mixture was warmed to RT and stirred overnight, the mixture was concentrated in vacuo and the residue was partitioned between EA and sat. NaHCO3. After extraction of the aqueous layer with EA (3x) 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): 277.8 [M+H+]
D. (9H-fluoren-9-yl)methyl hydrazinecarboxylate
Hydrazine monohydrate (28.7 mL, 0.59 mmol) was dissolved in ACN/water (230 mL, 1 :1) cooled to 0°C and a solution of 9-fluorenylmethyl chloroformate in ACN (915 mL) was added within 2 h. After stirring for 2 h at RT the solution was concentrated to about 200 mL. The precipitated solid was filtered off, washed with water (3x), n-hexane (3x) and dried in vacuo to give the title compound which was used for the next reaction without further purification. MS (m/z): 254.7 [M+H+]
E. (9//-fluoren-9-yl)methyl 2-ethylhydrazinecarboxylate
A mixture of (9H-fluoren-9-yl)methyl hydrazinecarboxylate (2.0 g, 7.87 mmol) and acetaldehyde (1.33 mL, 23.6 mmol) in ethanol (39 mL) was stirred at 85°C for 3 h and then concentrated to dryness. The resulting residue was dissolved in THF (39 mL) and after the addition of acetic acid (495 μl, 8.65 mmol) and sodium cyanoborohydride (136 mg, 8.65 mmol) the reaction mixture was stirred overnight. Afterwards the mixture was concentrated in vacuo and the residue was dissolved in EA, the organic layer was washed successively with potassium hydrogen sulfate (2 x 25 mL), brine (2 x 25 mL) and water (1 x 25 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo to give the title compound after flash chromatography on silica gel (n-hexane/EA). MS (m/z): 282.9 [M+H+]
F. (9H-fluoren-9-yl)methyl 2-(chlorocarbonyl)-2-ethylhydrazinecarboxylate According to general procedure C (9//-fluoren-9-yl)methyl 2-ethylhydrazinecarboxylate
(95 mg, 33.6 μmol) was treated with phosgene to provide the title compound which was used for the next reaction without further purification.
G. (9H-fluoren-9-yl)methyl 2-((3 ,3 '-difluoro-2'-(methoxycarbonyl)biphenyl-4- yl)methylcarbamoyl)-2-ethylhydrazinecarboxylate
Methyl 4'-(aminomethyl)-3,3'-difluorobiphenyl-2-carboxylate (100 mg , 0.44 mmol) was dissolved in THF (4 mL), treated with triethylamine (183 μl, 0.132 mmol) and 4- nitrophenylchloroformiate (80 mg, 0.397 mmol) at 0°C and was stirred for additional 2 h at 0°C. A solution of 9H-fluoren-9-yl)methyl 2-(chlorocarbonyl)-2-ethylhydrazinecarboxylate (101 mg, 0.357 mmol) in THF (6 mL) was added dropwise and the mixture was stirred overnight at RT. Afterwards the reaction mixture was concentrated in vacuo purification of the crude product by flash chromatography on silica gel (n-hexane/EA) afforded the title compound. MS (m/z): 585.9 [M+H+]
H. Methyl 4'-((l-ethylhydrazinecarboxamido)methyl)-3,3'-difluorobiphenyl-2- carboxylate
(9H-fluoren-9-yl)methyl 2-((3,3'-difluoro-2'-(methoxycarbonyl)biphenyl-4- yl)methylcarbamoyl)-2-ethylhydrazinecarboxylate (80 mg, 0.137 mmol) was treated with 20% piperidine in DMF (1 mL) for 1 h at RT to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 363.9 [M+H+]
I.4'-((2-Acetyl-l-ethylhydrazinecarboxamido)methyl)-3,3'-difluorobiphenyl-2- carboxylate
4'-((l-Ethylhydrazinecarboxamido)methyl)-3,3'-difluorobiphenyl-2-carboxylate (4 mg, 1 μmol ) was dissolved in pyridine (300 μl) and acetic anhydride (150 μl) and stirred for 2 h at RT to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 405.9 [M+H+]
Example 2
Synthesis of methyl 4'-((l-ethyl-2-(3-fluoro-5-(trifluoromethyl)benzoyl) hydrazinecarboxamido)methyl)-3,3'-difluorobiphenyl-2-carboxylate (compound 3)
Figure imgf000103_0001
According to general procedure E 3-fluoro-5-(trifluoromethyl)benzoic acid (4.9 mg, 23 μmol) was activated with HATU and treated with methyl 4'-((I- ethylhydrazinecarboxamido)methyl)-3 ,3 '-difluorobiphenyl-2-carboxylate hydrochloride (8.5 mg, 21 μmol, example 1 H) to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 553.8 [M+H+]
Example 3
Synthesis of 1 -ethyl-N-((2'-ethyl-3-fluorobiphenyl-4-yl)methyl)-2-(5-
(trifluoromethyl)nicotinoyl)hydrazinecarboxamide (compound 7)
Figure imgf000103_0002
A. 2'-Ethyl-3 -fluorobiphenyl-4-carbonitrile
4-Cyano-3-flourobenzeneboronic acid (500 mg, 3.03 mmol) was reacted with l-ethyl-2- iodobenzene (623 mg, 2.68 mmol) according to the synthesis of methyl 4'-cyano-3,3'- difluorobiphenyl-2-carboxylate (example 1 B) to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA).
B . (2'-Ethyl-3 -fluorobiphenyl-4-yl)methanamine
2'-Ethyl-3-fluorobiphenyl-4-carbonitrile (509 mg, 2.26 mmol) was treated with cobalt(ll)chloride hexahydrate (1.1 g, 4.5 mmol) and sodium borohydride (823 mg, 22 mmol) according to the synthesis of methyl 4'-(aminomethyl)-3, 3 '-difluorobiphenyl-2-carboxylate (example 1 C). MS (m/z): 229.8 [M+H+]
C. (9H-fluoren-9-yl)methyl 2-ethyl-2-((2'-ethyl-3-fluorobiphenyl-4- yl)methylcarbamoyl)hydrazinecarboxylate
To a mixture of (2'-ethyl-3-fluorobiphenyl-4-yl)methanamine (150 mg, 0.654 mmol) and triethylamine (272 μl) in THF (6 mL) was added p-nitrophenylchloroformate (119 mg, 0.59 mmol) portionwise at 0°C. After stirring at 0°C for 2 h an ice cold solution of (9H-fiuoren-9- yl)methyl 2-(chlorocarbonyl)-2-ethylhydrazinecarboxylate (150 mg, 0.435 mmol, example 1 F) in THF (3 mL) was added dropwise and the reaction mixture was stirred at 0°C for 3 h. The mixture was concentrated to dryness and the resulting residue was dissolved in EA followed by washing with water (3x). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 537.9 [M+H+]
D. 1 -Ethyl-N-((2'-ethyl-3 -fluorobiphenyl-4-yl)methyl)hydrazinecarboxamide. According to the synthesis of methyl 4'-((l-ethylhydrazinecarboxamido)methyl)-3,3'- difluorobiphenyl-2-carboxylate (example 1 H) (9//-fiuoren-9-yl)methyl 2-ethyl-2-((2'-ethyl-3- fluorobiphenyl-4-yl)methylcarbamoyl)hydrazinecarboxylate (164 mg, 0.305 mmol) was treated with 20 % piperidine in DMF to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS
E. 5-(Trifiuoromethyl)nicotinic acid methyl ester
To a stirred solution of 3-bromo-5-(triflouromethyl)pyridine (4.35 g, 19.25 mmol) in DMSO (43.5 mL) was added l,r-bis(diphenylphosphino)ferrocene (783 mg, 1.35 mmol), palladium(II) acetate (303 mg, 1.35 mmol), methanol (17.4 mL) and trietylamine (16 mL). After the reaction mixture was stirred at 70°C for 24 h under carbon monoxide atmosphere, 1 , r-bis(diphenylphosphino)ferrocene (392 mg, 0.677 mmol) and palladium(II) acetate (151 mg, 0.677 mmol) were added again followed by additional stirring at 700C for 24 h under carbon monoxide atmosphere. The mixture was partitioned between EA and sat. NaHCO3 solution and after extraction of the aqueous layer with EA (2x) the combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (n-hexane/EA) to give the title compound. GC/MS (m/z): 205.0
F. 5-(Trifluoromethyl)nicotinic acid
A mixture of 5-(trifluoromethyl)nicotinic acid methyl ester (2.5 g, 12.19 mmol) in dioxane (10 mL) and LiOH (1 M, 10 mL) was stirred for 4 h at RT before again LiOH (4 mL, 1 M) was added. After additional stirring for 3 h at RT the organic solvent was removed in vacuo and HCl (1 M) was added to pH 4-5. Afterwards the aqueous layer was extracted with EA (4x), the combined organic layers were dried over Na2SO4, filtered and concentrated to dryness. No purification steps were required, and the isolated product was utilized in further reactions. GC/MS (m/z): 191.0
G. 1 -Ethyl-N-((2'-ethyl-3-fluorobiphenyl-4-yl)methyl)-2-(5- (trifluoromethyl)nicotinoyl)hydrazinecarboxamide
According to general procedure E 3-fluoro-5-(trifluoromethyl)benzoic acid (4.9 mg, 23 μmol) was activated with HATU and treated with methyl 4'-((I- ethylhydrazinecarboxamido)methyl)-3,3'-difluorobiphenyl-2-carboxylate hydrochloride (8.5 mg, 21 μmol) to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 488.8 [M+H+]
Example 4
Synthesis of (7?>3-(2-ethyl-2-(l-(2'-ethyl-3-fluorobiphenyl-4- yl)ethylcarbamoyl)hydrazinecarbonyl)-5-(trifluoromethyl)pyridine 1 -oxide (compound 9)
Figure imgf000105_0001
A. 2'-Ethyl-3 -fluorobiphenyl-4-carbaldehyde
3-Fluoro-4-formylphenylboronic acid (900 mg, 5.36 mmol) was reacted with l-ethyl-2- iodobenzene (1.1 g, 4.74 mmol) according to the synthesis of methyl 4'-cyano-3,3'- difluorobiphenyl-2-carboxylate (example 1 B) to yield the title compound after purification by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 228.9 [M+H+]
B. (S,E)-N-((2'-ethyl-3-fluorobiphenyl-4-yl)methylene)-2-methylpropane-2-sulfinamide According to the general procedure F 2'-ethyl-3-fluorobiphenyl-4-carbaldehyde (750 mg,
3.29 mmol) was reacted with (S|j-(-)-2-methyl-2-propanesulfinamide to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 331.8 [M+H+]
C. (S)-N-{(R)- 1 -(2l-Ethyl-3-fluorobiphenyl-4-yl)ethyl)-2-methylpropane-2-sulfinamide According to the general procedure G (S,E)-N-((2'-ethyl-3-fluorobiphenyl-4- yl)methylene)-2-methylpropane-2-sulfinamide (320 mg, 0.965 mmol) was reacted with methyl magnesium chloride to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 347.9 [M+H+]
D. (R)-I -(2'-Ethyl-3-fluorobiphenyl-4-yl)ethanamine
According to general procedure D ((5^-N-((7?j-l-(2'-ethyl-3-fluorobiphenyl-4-yl)ethyl)-2- methylpropane-2-sulfinamide (220 mg, 0.633 mmol) was treated with HCl in methanol to give the title compound as the HCl salt which was used for the next step without further purification. MS (m/z): 243.9 [M+H+]
E. (K;-(9H-Fluoren-9-yl)methyl 2-ethyl-2-(l -(2'-ethyl-3-fluorobiphenyl-4- yl)ethylcarbamoyl)hydrazinecarboxylate
(R)- l-(2'-Ethyl-3-fluorobiphenyl-4-yl)ethanamine (110 mg, 0.393 mmol) was reacted with (9H-fluoren-9-yl)methyl 2-(chlorocarbonyl)-2-ethylhydrazinecarboxylate (73 mg, 0.212 mmol, example 1 F) according to the synthesis of (9H-fluoren-9-yl)methyl 2-ethyl-2-((2'- ethyl-3-fluorobiphenyl-4-yl)methylcarbamoyl)hydrazinecarboxylate (example 3 C) to yield the title compound after purification by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 551.9 [M+Η+]
F. C^-I -Ethyl -N-(l-(2'-ethyl-3-fluorobiphenyl-4-yl)ethyl)hydrazinecarboxamide According to the synthesis of methyl 4'-((l-ethylhydrazinecarboxamido)methyl)-3,3'- difluorobiphenyl-2-carboxylate (example 1 H) fφ-(9H-fluoren-9-yl)methyl 2-ethyl-2-(l-(2'- ethyl-3-fluorobiphenyl-4-yl)ethylcarbamoyl)hydrazinecarboxylate (63 mg, 0.114 mmol) was treated with 20 % piperidine in DMF to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 329.8 [M+H+]
G. 3-Carboxy-5-(trifluoromethyl)pyridine 1 -oxide
A solution of 5-(trifluoromethyl)nicotinic acid (300 mg, 1.57 mmol, example 3 F) in acetic acid (1 mL) and hydrogen peroxide (1 mL) was stirred at 90 0C overnight. After the portionwise addition of an excess of manganese dioxide till pH 4, the purification of the crude product by flash chromatography on silica gel (n-hexane/EA) afforded the title compound. MS (m/z): 208.1 [M+H+]
H. f7?;-3-(2-Ethyl-2-(l-(2'-ethyl-3-fluorobiphenyl-4- yl)ethylcarbamoyl)hydrazinecarbonyl)-5-(trifluoromethyl)pyridine 1 -oxide
According to general procedure E 3-carboxy-5-(trifluoromethyl)pyridine 1 -oxide (5 mg, 24.1μmol) was activated with HATU and treated with (7?>l-ethyl-N-(l-(2'-ethyl-3- fluorobiphenyl-4-yl)ethyl)hydrazinecarboxamide hydrochloride (8.9 mg, 20.1 μmol) to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 518.8 [M+H+]
Example 5
Synthesis of f7?>N-(l-(3,5'-dichloro-3'-fluoro-2l-(5-methyl-l,2,4-oxadiazol-3-yl)biphenyl- 4-yl)ethyl)-l-ethyl-2-(3-methoxyisoxazole-5-carbonyl)hydrazinecarboxamide (compound 11)
Figure imgf000108_0001
A. 2-Bromo-4-chloro-6-fluorobenzonitrile
To a stirred solution of 2-bromo-4-chloro-6-fluoroaniline (5 g, 22.3 mmol) in DCM (40 mL) was added nitrosonium tetrafluoroborate (2.9 g, 24.5 mmol) and after stirring for 1 h at RT the reaction mixture was cooled to 0°C. Potassium cyanide (2.9 g, 44.6 mmol) was added and after stirring for 10 min, a solution of copper sulphate hexahydrate (11.2 g, 44.6 mmol) in water (40 mL) was added dropwise. After stirring at 0°C for additional 40 min, the reaction mixture was warmed to RT during 1 h, quenched with sat. Na2CO3 and then diluted with DCM. The mixture was filtered through celite, washed with DCM and the separated organic layer was washed with brine (2x). Then dried over Na2SO4, filtered and concentrated in vacuo to give the title compound after purification by flash chromatography on silica gel (n- hexane/EA). GC/MS (m/z): 233.2
B. 3-(2-Bromo-4-chloro-6-fluorophenyl)-5-methyl- 1 ,2,4-oxadiazole
A mixture of 2-bromo-4-chloro-6-fluorobenzonitrile (2.5 g, 10.6 mmol) in ethanol (10.6 mL) was reacted with hydroxylamine (10.6 mL, 50% in water) at 6O0C for 1.5 h. The reaction mixture was diluted with diethylether at RT and then washed with water (2x) and brine (2x). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo and the resulting residue was dissolved in N,N-dimethylacetamide dimethyl acetal (11 mL, 74.3 mmol). After 2 h at RT the reaction mixture was diluted with DCM and washed consecutively with water, 50 % brine/water and brine. The organic layer was dried over Na2SO4, filtered and concentrated in vacuo to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). GC/MS (m/z): 290.2 C. 2-Methyl-propane-2-sulfinic acid 4-bromo-2-chloro-benzylideneamide According to general procedure F 4-bromo-2-chlorobenzaldehyde (25 g, 0.11 mol) was reacted with (S^-(-)-2-methyl-2-propanesulfinamide (13.8 g, O.l lmmol) to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA) as a white solid. MS (m/z): 321.5 [M+H+]
D. 2-Methyl-propane-2-sulfinic acid (l-(4-bromo-2-chloro-phenyl)-ethyl)-amide According to the general procedure G 2-methyl-propane-2-sulfinic acid 4-bromo-2- chloro-benzylideneamide (5 g, 15.5 mmol) was reacted with methyl magnesium chloride to give the title compound after purification by flash chromatography on silica gel (n- hexane/EA). MS (m/z): 337.7 [M+H+]
E. 2-Methyl-propane-2-sulfinic acid (l-(2-chloro-4-(4,4,5,5-tetramethyl- ( 1 ,3 ,2)dioxaborolan-2-yl)-phenyl)-ethyl)-amide
A mixture of 2-methyl-propane-2-sulfinic acid (l-(4-bromo-2-chloro-phenyl)-ethyl)- amide (950 mg, 2.83 mmol), bis(pinacolato)diboron (1.4 g, 5.7 mmol), potassium acetate (823 mg, 8.5 mmol) and (l,r-bis-(diphenylphosphino)ferrocene)palladium(II) dichloride (346 mg, 0.42 mmol) in DMF (11 mL) was heated to 80 °C under N2 for 18 h. The reaction mixture was cooled to RT, diluted with EA and washed with HCl (1 M) and brine. The organic layer was dried over Na2SO4, filtered and concentrated in vacuo to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 385.9 [M+H+]
F. 2-Methyl-propane-2-sulfinic acid (l-(3,5'-dichloro-3'-fluoro-2'-(5-methyl-l,2,4- oxadiazol-3-yl)-biphenyl-4-yl)ethyl)-amide
A mixture of 2-methyl-propane-2-sulfinic acid (l-(2-chloro-4-(4,4,5,5-tetramethyl- (l,3,2)dioxaborolan-2-yl)-phenyl)-ethyl)-amide (785 mg, 2.03 mmol), 3-(2-bromo-4-chloro- 6-fluorophenyl)-5-methyl-l,2,4-oxadiazole (494 mg, 1.69 mmol), potassium carbonate (467 mg, 3.38 mmol) and (l,r-bis-(diphenylphosphino)ferrocene)palladium(II) dichloride (207 mg, 0.25 mmol) in DMSO (3.38 mL) and water (0.42 mL) was heated to 80 °C under N2 for 4 h. The reaction mixture was cooled to RT, diluted with EA and then washed with brine (2x). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 469.8 [M+H+]
G. l-(3,5'-Dichloro-3'-fluoro-2'-(5-methyl-l,2,4-oxadiazol-3-yl)-biphenyl-4-yl)- ethylamine
According to general procedure D 2-methyl-propane-2-sulfinic acid (l-(3,5'-dichloro-3'- fluoro-2'-(5 -methyl- l,2,4-oxadiazol-3-yl)-biphenyl-4-yl)ethyl)-amide (100 mg, 0.213 mmol) was treated with HCl in methanol to give the title compound as the HCl salt which was used for the next step without further purification. MS (m/z): 365.7 [M+H+]
H. N'-Ethylidene-hydrazinecarboxylic acid tert-butyl ester
According to general procedure A tert-butyl hydrazinecarboxylate (20 g, 0.151 mmol) was reacted with acetaldehyde (9.4 mL, 0.166 mmol) to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). GC/MS (m/z): 158.4
I. tert-Butyl 2-ethylhydrazinecarboxylate
According to general procedure B N'-ethylidene-hydrazinecarboxylic acid tert-butyl ester was treated with DIBAL to afford the title compound after purification by flash chromatography on silica gel (n-hexane/EA).
J. tert-Butyl 2-(chlorocarbonyl)-2-ethylhydrazinecarboxylate
According to general procedure C tert-butyl 2-ethylhydrazinecarboxylate (150 mg, 0.94 mmol) was treated with phosgene to provide the title compound which was used for the next reaction without further purification. MS (m/z): 222.9 [M+H+]
K. (R)-tert-Butyl 2-(l-(3,5'-dichloro-3'-fluoro-2'-(5-methyl-l,2,4-oxadiazol-3- yl)biphenyl-4-yl)ethylcarbamoyl)-2-ethylhydrazinecarboxylate
According to general procedure C l-(3,5'-dichloro-3'-fluoro-2'-(5-methyl-l,2,4-oxadiazol- 3-yl)-biphenyl-4-yl)-ethylamine (100 mg, 0.248 mmol) was reacted with tert-butyl 2- (chlorocarbonyl)-2-ethylhydrazinecarboxylate (109 mg, 0.489 mmol,) to give after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 551.7 [M+H+] L. fi?>N-(l-(3,5'-Dichloro-3l-fluoro-2'-(5-methyl-l,2,4-oxadiazol-3-yl)biphenyl-4- yl)ethyl)- 1 -ethylhydrazinecarboxamide
According to general procedure D (R)-tert-butyl 2-(l-(3,5'-dichloro-3'-fluoro-2'-(5- methyl-l,2,4-oxadiazol-3-yl)biphenyl-4-yl)ethylcarbamoyl)-2-ethylhydrazinecarboxylate (55 mg, 0.1 mmol) was treated with HCl in methanol to give the title compound as the HCl salt which was used for the next step without further purification. MS (m/z): 451.8 [M+H+]
M. (R)-N-(I -(3,5'-Dichloro-3'-fluoro-2'-(5-methyl-l ,2,4-oxadiazol-3-yl)biphenyl-4- yl)ethyl)-l-ethyl-2-(3-methoxyisoxazole-5-carbonyl)hydrazinecarboxamide
According to general procedure E f/?j-7V-(l-(3,5'-dichloro-3'-fluoro-2'-(5-methyl-l,2,4- oxadiazol-3-yl)biphenyl-4-yl)ethyl)-l -ethylhydrazinecarboxamide (10 mg, 22 μmol) was reacted with 3-methoxy-isoxazole-5-carboxylic (31 mg, 220 μmol) acid to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 576.7 [M+H+]
Example 6
Synthesis of methyl 4'-((l-ethyl-2-(5-fluoronicotinoyl)hydrazinecarboxamido)methyl)- 3,3'-difluorobiphenyl-2-carboxylate (compound 16)
Figure imgf000111_0001
A. ter/-Butyl 2-((3,3'-difluoro-2'-(methoxycarbonyl)biphenyl-4-yl)methylcarbamoyl)-2- ethylhydrazinecarboxylate
According to general procedure C methyl 4'-(aminomethyl)-3,3'-difluorobiphenyl-2- carboxylate (44 mg, 0.159 mmol, example 1 C) was reacted with tert-butyl 2- (chlorocarbonyl)-2-ethylhydrazinecarboxylate (70 mg, 0.314 mmol) to give the title compound after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA. MS (m/z): 463.9 [M+H+] B. Methyl 4'-((I -ethylhydrazinecarboxamido)methyl)-3,3'-difluorobiphenyl-2- carboxylate
According to general procedure D tert-butyl 2-((3,3'-difiuoro-2'- (methoxycarbonyl)biphenyl-4-yl)methylcarbamoyl)-2-ethylhydrazinecarboxylate (70 mg, 0.315 mmol) was treated with HCl in methanol to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 363.9 [M+H+]
C. Methyl 4'-((I -ethyl-2-(5-fluoronicotinoyl)hydrazinecarboxamido)methyl)-3,3'- difluorobiphenyl-2-carboxylate
According to general procedure E 3-fluoro-5-(trifluoromethyl)benzoic acid (5.4 mg, 39 μmol) was activated with HATU and treated with methyl 4'-((I- ethylhydrazinecarboxamido)methyl)-3,3'-difluorobiphenyl-2-carboxylate hydrochloride (7.0 mg, 19 μmol) to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 486.7 [M+H+]
Example 7
Synthesis of 3-chloro-5-(2-((5'-chloro-3,3'-difluoro-2'-(5-methyl-l ,2,4-oxadiazol-3- yl)biphenyl-4-yl)methylcarbamoyl)-2-ethylhydrazinecarbonyl)pyridine 1 -oxide (compound 19)
Figure imgf000112_0001
A. (£j-N-(4-Bromo-2-fluorobenzylidene)-2-methylpropane-2-sulfinamide
According to general procedure F 4-bromo-2-fluorobenzaldehyde (22 g, 0.108 mol) was reacted with (S/)-(-)-2-methyl-2-propanesulfinamide to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA) as a white solid. MS (m/z): 305.7 [M+H+]
B. N-(4-Bromo-2-fluorobenzyl)-2-methylpropane-2-sulfinamide
According to general procedure H (E)-./V-(4-bromo-2-fluorobenzylidene)-2- methylpropane-2-sulfinamide (250 mg, 0.816 mmol) was reduced with sodium borohydride to afford the title compound which was used for the next reaction without purification.
C. iV-((51-Chloro-3,3'-difluoro-2l-(5-methyl-l,2,4-oxadiazol-3-yl)biphenyl-4-yl)methyl)- 2-methylpropane-2-sulfϊnamide
According to general procedure I iV-(4-bromo-2-fluorobenzyl)-2-methylpropane-2- sulfinamide (100 mg, 0.324 mmol) was reacted with 3-(2-bromo-4-chloro-6-fluorophenyl)-5- methyl-l,2,4-oxadiazole (94.6 mg, 0.324 mmol, example 5 B) to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 439.8 [M+H+]
D. (5'-Chloro-3,3'-difluoro-2'-(5-methyl-l,2,4-oxadiazol-3-yl)biphenyl-4- yl)methanamine
According to general procedure D (5'-chloro-3,3'-difluoro-2'-(5-methyl-l,2,4-oxadiazol- 3-yl)biphenyl-4-yl)methanamine (50 mg, 0.114 mmol) was treated with HCl in methanol to give the title compound after purification by flash chromatography on silica gel (n- hexane/EA). MS (m/z): 335.8 [M+H+]
E. tert-Buty\ 2-((5'-chloro-3,3'-difluoro-2'-(5-methyl-l ,2,4-oxadiazol-3-yl)biphenyl-4- yl)rnethylcarbamoyl)-2-ethylhydrazinecarboxylate
According to general procedure C (5'-chloro-3,3'-difluoro-2'-(5-methyl-l,2,4-oxadiazol-3- yl)biphenyl-4-yl)methanamine (20.4 mg, 60.8 μmol) was reacted with tert-butyl 2- (chlorocarbonyl)-2-ethylhydrazinecarboxylate (26 mg, 121.5 μmol, example 5 J) to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 521.9 [M+H+]
F. N-((5'-Chloro-3,3t-difluoro-2'-(5-methyl-l,2,4-oxadiazol-3-yl)biphenyl-4- yl)methyl)- 1 -ethylhydrazinecarboxamide
According to general procedure D tert-butyl 2-((5'-chloro-3,3'-difluoro-2'-(5-methyl- 1 ,2,4-oxadiazol-3-yl)biphenyl-4-yl)methylcarbamoyl)-2-ethylhydrazinecarboxylate (26 mg, was treated with HCl in methanol to give the title compound as the HCl salt which was used for the next step without further purification. MS (m/z): 421.9 [M+H+]
G. 3 -Chloro-5 -(2-((5'-chloro-3 ,3 '-difluoro-2'-(5-methyl- 1 ,2,4-oxadiazol-3 -yl)biphenyl- 4-yl)methylcarbamoyl)-2-ethylhydrazinecarbonyl)pyridine 1 -oxide
According to general procedure E S-carboxy-S-chloropyridine 1 -oxide (3.4 mg, 20 μmol, example 4 H) was activated with HATU and treated with N-((5'-chloro-3,3'-difluoro-2'-(5- methyl-l,2,4-oxadiazol-3-yl)biphenyl-4-yl)methyl)-l-ethylhydrazinecarboxamide (3.0 mg, 7 μmol) to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 576.7 [M+H+]
Example 8
Synthesis of (^;-N-(l-(3,3'-difluoro-2'-(trifluoromethyl)biphenyl-4-yl)ethyl)-l-ethyl-2-(3- methoxyisoxazole-5-carbonyl)hydrazinecarboxamide (compund 20)
Figure imgf000114_0001
A. N-{(R)- 1 -(4-Bromo-2-fluorophenyl)ethyl)-2-methylpropane-2-sulfinamide According to the general procedure G fE>)-jV-(4-bromo-2-fluorobenzylidene)-2- methylpropane-2-sulfinamide (10 g, 32.7 mmol, example 7 A) was reacted with methyl magnesium chloride to give the title compound after purification by flash chromatography on silica gel (n-hexane/ΕA). MS (m/z): 321.8 [M+H+]
B. 2-Methyl-propane-2-sulfinic acid (l-(3,3l-difluoro-2'-trifluoromethyl-biphenyl-4-yl)- ethyl)-amide
According to general procedure I N-((R)-\ -(4-bromo-2-fluorophenyl)ethyl)-2- methylpropane-2-sulfinamide (500 mg, 1.55 mmol) was reacted with 2-bromo-6- fluorobenzotrifluoride (377 mg, 1.55 mmol) to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 405.8 [M+H+]
C. f7?^-l-(3,3'-difluoro-2'-(trifluoromethyl)biphenyl-4-yl)ethanamine
According to general procedure D 2-methyl-propane-2-sulfinic acid (l-(3,3'-difluoro-2'- trifluoromethyl-biphenyl-4-yl)-ethyl)-amide (100 mg, 0.247 mmol) was treated with HCl in methanol to give after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt.. MS (m/z): 301.8 [M+H+]
D. (R)-tert-Butyl 2-(l -(3,3'-difluoro-2'-(trifluoromethyl)biphenyl-4-yl)ethylcarbamoyl)- 2-ethylhydrazinecarboxylate
According to general procedure C (7?>)-l-(3,3'-difluoro-2'-(trifluoromethyl)biphenyl-4- yl)ethanamine (50 mg, 0.166 mmol) was reacted with tert-buty\ 2-(chlorocarbonyl)-2- ethylhydrazinecarboxylate ( 74 mg, 0.332 mmol, example 5 J) to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 487.7 [M+H+]
E. (R) -N-( 1 -(3 ,3 '-Difluoro-2'-(trifluoromethyl)biphenyl-4-yl)ethyl)- 1 - ethylhydrazinecarboxamide
According to general procedure D (R)-tert-buty\ 2-(l-(3,3'-difluoro-2'- (trifluoromethyl)biphenyl-4-yl)ethylcarbamoyl)-2-ethylhydrazinecarboxylate (74 mg, 0.333 mmol) was treated with HCl in methanol to give the title compound as the HCl salt which was used for the next step without further purification. MS (m/z): 387.8 [M+H+]
F. (7?;-N-(l-(3,3'-Difluoro-2'-(trifluoromethyl)biphenyl-4-yl)ethyl)-l-ethyl-2-(3- methoxyisoxazole-5-carbonyl)hydrazinecarboxamide
According to general procedure E 3-methoxy-isoxazole-5-carboxylic acid (19 mg, 13.3 μmol) was activated with HATU and treated with (R)-N-(I -(3,3 '-difluoro-2'- (trifluoromethyl)biphenyl-4-yl)ethyl)-l -ethylhydrazinecarboxamide hydrochloride (10 mg, 26 μmol) to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 512.8 [M+H+] Example 9
Synthesis of f7?>N-(l-(3,3'-difluoro-2'-(trifluoromethyl)biphenyl-4-yl)ethyl)-l-ethyl-2-(2- methylpyrimidine-S-carbony^hydrazinecarboxamide (compound 22)
Figure imgf000116_0001
According to general procedure E 2-methyl-pyrimidine-5-carboxylic acid (22 mg, 156 μmol) was activated with HATU and treated with f7?j-N-(l-(3,3'-difluoro-2'- (trifluoromethyl)biphenyl-4-yl)ethyl)-l-ethylhydrazinecarboxamide hydrochloride (10 mg, 26 μmol example 8 F) to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 507.8 [M+H+]
Example 10
Synthesis of (R)-N-(I -(5-(3,5-dichloro-2-(2,2-difluoroethoxy)phenyl)-3-fluoropyridin-2- yl)ethyl)-l -ethyl-2-(3,3,3-trifluoropropanoyl)hydrazinecarboxamide (compound 26)
Figure imgf000116_0002
A. 5-Bromo-3-fluoro-pyridine-2-carbonitrile
H2SO4 (ImL) was added to a stirred solution of tetrabutylammonium fluoride in THF (657 mL, 0.66 mol, IM). DMF (200 mL) was added at -40°C until the reaction mixture became a clear solution followed by the slowly addition of a solution of 5-bromo-3- nitropyridin-2-carbonitrile (50 g, 0.22 mol) in DMF (800 mL). After the solution was stirred at -40°C for 30 min. the reaction mixture was quenched with HCl (500 mL, 2 M) to afford pH 3 and the mixture was extracted with EA (2x). The combined organic layers were washed with water, dried over Na2SO4 and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel (n-hexane/EA); to obtain title compound as a light yellow solid.
B. 5-Bromo-3-fluoro-pyridine-2-carboxylic acid
Cone. HCl (300 mL) was added to 5-bromo-3-fluoro-pyridine-2-carbonitrile (21 g, 0.11 mol) and the mixture was stirred at 60°C overnight. After evaporation, diethylether was added and the suspension was stirred for additional 1 h. The solid was collected by filtration and dried in vacuo to obtain the title compound as a solid. This crude product was used without any further purification.
C. 5-Bromo-3-fluoro-pyridine-2-carboxylic acid methoxy-methyl-amide
To a stirred mixture of 5-bromo-3-fluoro-pyridine-2-carboxylic acid (55.5 g, 0.217 mol) in DMF (1000 mL) were added trietyhlamine (17.4 mL 0.13 mol), l-ethyl-3-(3'- dimethylaminopropyl) carbodiimide hydrochloride (83.2 g, 0.434 mol), HOBt (2.97 g, 0.022 mol) and N,O-dimethylhydroxylamine hydrochloride (25 g, 0.26 mol). After stirring overnight at RT the solvent was removed in vacuo. The resulting residue was partitioned between EA and brine and the aqueous layer was extracted with EA (3x). After the combined organic layers were washed with brine (2x), filtrated and dried over Na2SO4 the solvent was removed under reduced pressure. The crude product was purified by flash chromatography on silica gel yielded the title compound as a white solid. MS (m/z): 262.8 [M+H+]
D. 5-Bromo-3-fluoro-pyridine-2-carbaldehyde
To a stirred solution of 5-bromo-3-fluoro-pyridine-2-carboxylic acid methoxy-methyl- amide (29 g, 0.11 mol) in THF (350 mL) was slowly added lithiumaluminiumhydride (50 mL, 0.05 mol, 1 M in THF) at -78°C. After additional stirring at -78°C for 20 min the mixture was quenched with water (250 mL) and brine (200 mL) and allowed to warm to RT. Afterwards EA was added the suspension was filtered through celite, the aqueous layer was separated and extracted with EA. The combined organic layers were washed with brine (2x), dried over Na2SO4, filtrated and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel (n-hexane/EA) to obtain the title compound. MS (m/z): 205.8 [M+H+]
E. (]ζ)-7V-((5-Bromo-3-fluoropyridin-2-yl)methylene)-2-methylpropane-2-sulfinamide According to general procedure F 5-bromo-3-fluoro-pyridine-2-carbaldehyde (19.5 g, 0.1 mol) was treated with fi?>)-(+)-2-methyl-2-propansulfinamide (11.6 g, 0.096 mol) to afford the title compound after purification by chromatography on silica gel (n-hexane/EA) as a light brown oil. MS (m/z): 306.7 [M+H+]
F. N-{(R)- 1 -(5-Bromo-3-fluoropyridin-2-yl)ethyl)-2-methylpropane-2-sulfmamide According to general procedure G a solution of 2-methyl-propane-2-sulfinic acid 5- bromo-3-fluoro-pyridin-2-ylmethyleneamide (17.5 g, 0.06 mol) was treated with methyl magnesium chloride (29.9 mL, 0.086 mol, 22% in THF). The crude product was purified by flash chromatography on silica gel (n-hexane/EA) to obtain the title compound as a light yellow oil. The d.e. was determined by 1H-NMR. Integration of the aromatic signals indicated the presence of 2-3 % of the fS^-diastereoisomere. This corresponds with a d.e. of 94-96%. MS (m/z): 322.7 [M+H+]
G. 1 -Bromo-3,5-dichloro-2-(2,2-difluoroethoxy)benzene
To a stirred solution of 2-bromo-4,6-dichloro-phenol (882 mg, 3,65 mmol) in DMSO (12 mL) was added sodium hydride (220 mg, 5.5 mmol, 60 % suspension) portionwise at RT under argon. After additional stirring at RT for 15 min, l,l-difluoro-2-iodo-thane (840 mg, 4.4 mmol) was added and the reaction mixture was stirred at 70 0C overnight. l,l-diflouro-2- iodo-thane (840 mg, 4.38 mmol) was added followed by the portionwise addition of sodium hydride (220 mg, 5.5 mmol, 60% suspension) and the mixture was stirred again at 70°C overnight. Afterwards the reaction mixture was quenched with HCl (10 mL, 1 M), partitioned between EA and HCl (1 M) and the separated aqueous layer was extracted with EA (3x). The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo to give the title compound which was used for the next reaction without further purification. GC/MS (m/z): 304.0 H. N-(fK>l-(5-(3,5-Dichloro-2-(2,2-difluoroethoxy)phenyl)-3-fluoropyridin-2- yl)ethyl)-2-methylpropane-2-sulfinamide
According to general procedure I N-((7?^-l-(5-bromo-3-fluoropyridin-2-yl)ethyl)-2- methylpropane-2-sulfϊnamide (500 mg, 1.547 mmol was treated with l-bromo-3,5-dichloro- 2-(2,2-diflouro-ethoxy)-benzene (473 mg, 1.547 mmol) to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 468.8 [M+H+]
I. (R)- 1 -(5-(3,5-Dichloro-2-(2,2-difluoroethoxy)phenyl)-3-fluoropyridin-2-yl)ethanamine According to general procedure D N-((R)- 1-(5-(3, 5-dichloro-2-(2,2- difluoroethoxy)phenyl)-3-fluoropyridin-2-yl)ethyl)-2-methylpropane-2-sulfinamide (533 mg, 1.136 mmol) was treated with HCl in methanol to give the title compound as the HCl salt which was used for the next step without further purification. MS (m/z): 364.8 [M+H+]
J. (R)-tert-Butyl 2-(l -(5-(3,5-dichloro-2-(2,2-difluoroethoxy)phenyl)-3-fluoropyridin-2- yl)ethylcarbamoyl)-2-ethylhydrazinecarboxylate
According to general procedure C (7?j-l-(5-(3,5-dichloro-2-(2,2-difluoroethoxy)phenyl)- 3-fluoropyridin-2-yl)ethanamine (150 mg, 0.373 mmol) was treated with tert-butyl 2- (chlorocarbonyl)-2-ethylhydrazinecarboxylate (249 mg, 1.12 mmol, example 5 J) to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 552.8 [M+H+]
K. fK>N-(l-(5-(3,5-Dichloro-2-(2,2-difluoroethoxy)phenyl)-3-fluoropyridin-2- yl)ethyl)- 1 -ethylhydrazinecarboxamide
According to general procedure D (R)-tert-b\xty\ 2-(l-(5-(3,5-dichloro-2-(2,2- difluoroethoxy)phenyl)-3-fluoropyridin-2-yl)ethylcarbamoyl)-2-ethylhydrazinecarboxylate (100 mg, 0.20 mmol) was treated with HCl in methanol to give the title compound as the HCl salt which was used for the next step without further purification. MS (m/z): 450.9 [M+H+]
L. f7?>iV-(l-(5-(3,5-Dichloro-2-(2,2-difluoroethoxy)phenyl)-3-fluoropyridin-2- yl)ethyl)-l-ethyl-2-(3,3,3-trifluoropropanoyl)hydrazinecarboxamide
According to general procedure E cyanoacetic acid (17 mg, 132 μmol) was activated with HATU (18 mg, 132 μmol) and treated with rø-JV-(l-(5-(3,5-dichloro-2-(2,2- difluoroethoxy)phenyl)-3-fluoropyridin-2-yl)ethyl)-l-ethylhydrazinecarboxamide (10 mg, 22 μmol) to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 560.8 [M+H+]
Example 11
Synthesis of (R)-N-(I -(5-(3,5-dichloro-2-(2,2-difluoroethoxy)phenyl)-3-fluoropyridin-2- yl)ethyl)-l -ethyl-2-(2,2,2-trifluoroacetyl)hydrazinecarboxamide (compound 27)
Figure imgf000120_0001
A solution of f7?j-iV-(l-(5-(3,5-dichloro-2-(2,2-difluoroethoxy)phenyl)-3-fluoropyridin-2- yl)ethyl)-l-ethylhydrazinecarboxamide (example 10 L) in DCM (3 mL) was treated with trifluoroacetic anhydride (32 μL, 0.170 mmol) for 1 h at RT to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 546.7 [M+H+]
Example 12
Synthesis of (R)-N-(I -(5-(3,5-dichloro-2-(2,2-difluoroethoxy)phenyl)-3-fluoropyridin-2- yl)ethyl)-l -ethyl-2-(l ,2,3-thiadiazole-5-carbonyl)hydrazinecarboxamide (compound 28)
Figure imgf000120_0002
A. l,2,3-Thiadiazole-5-carboxylic acid
A solution of (l,2,3)-thiadiazole-5-carboxylic acid ethyl ester (1.0 g, 6.3 mmol) in ACN (5 mL) and LiOH (1 M, 10 mL) was stirred for 2 h at RT. After the ACN was evaporated in vacuo the resulting residue was partitioned between EA and HCl (1 M). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo to give the title compound which was used for the next reaction without further purification.
B. fi?;-N-(l-(5-(3,5-Dichloro-2-(2,2-difluoroethoxy)phenyl)-3-fluoropyridin-2- yl)ethyl)-l-ethyl-2-(l,2,3-thiadiazole-5-carbonyl)hydrazinecarboxamide
According to general procedure E l,2,3-thiadiazole-5-carboxylic acid (17 mg, 132 μmol) was activated with HATU and treated with (K>7V-(l-(5-(3,5-dichloro-2-(2,2- difluoroethoxy)phenyl)-3 -fluoropyridin-2-yl)ethyl)- 1 -ethylhydrazinecarboxamid hydrochloride (10 mg, 22 μmol, example 10 L) to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 563.4 [M+H+]
Example 13
Synthesis of 2-(2-cyanoacetyl)-N-((3',5'-dichloro-2'-(2,2-difluoroethoxy)-3- fluorobiphenyl-4-yl)methyl)- 1 -ethylhydrazinecarboxamide (compound 33)
Figure imgf000121_0001
A. N-((3',5'-Dichloro-2'-(2,2-difluoroethoxy)-3-fluorobiphenyl-4-yl)methyl)-2- methylpropane-2-sulfinamide
According to general procedure I N-(4-bromo-2-fluorobenzyl)-2-methylpropane-2- sulfinamide (500 mg, 1.6 mmol, example 7B) was reacted with l-bromo-3,5-dichloro-2-(2,2- difluoroethoxy)benzene (587 mg, 1.92 mmol, example 10 G) to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 453.8 [M+H+] B. (3',5'-Dichloro-2'-(2,2-difluoroethoxy)-3-fluorobiphenyl-4-yl)methanamine According to general procedure D N-((3',5'-dichloro-2'-(2,2-difluoroethoxy)-3- fluorobiphenyl-4-yl)methyl)-2-methylpropane-2-sulfinamide (250 mg, 0.55 mmol) was treated with HCl in methanol to give the title compound as the HCl salt which was used for the next reaction without further purification. MS (m/z): 349.8 [M+H+]
C. tert-Butyl 2-((3',5'-dichloro-2'-(2,2-difluoroethoxy)-3-fluorobiphenyl-4- yl)methylcarbamoyl)-2-ethylhydrazinecarboxylate
According to general procedure C (3',5'-dichloro-2'-(2,2-difiuoroethoxy)-3- fluorobiphenyl-4-yl)methanamine hydrochloride (80 mg, 29 μmol) was reacted with tert-butyl 2-(chlorocarbonyl)-2-ethylhydrazinecarboxylate (127 mg, 58 μmol, example 5 J) to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 536.0 [M+H+]
D. N-((3',5'-Dichloro-2'-(2,2-difluoroethoxy)-3-fluorobiphenyl-4-yl)methyl)-l- ethylhydrazinecarboxamide
According to general procedure D tert-buty\ 2-((3',5'-dichloro-2'-(2,2-difluoroethoxy)-3- fluorobiphenyl-4-yl)methylcarbamoyl)-2-ethylhydrazinecarboxylate (127 mg, 0.570 mmol) was treated with HCl in methanol to give the title compound as the HCl salt which was used for the next step without further purification. MS (m/z): 435.9 [M+H+]
E. 2-(2-Cyanoacetyl)-iV-((3',5'-dichloro-2'-(2,2-difluoroethoxy)-3-fluorobiphenyl-4- yl)methyl)- 1 -ethylhydrazinecarboxamide
According to general procedure E cyanoacetic acid (11 mg, 138 μmol) was activated with HATU and treated with N-((3',5'-dichloro-2'-(2,2-difluoroethoxy)-3-fluorobiphenyl-4- yl)methyl)-l -ethylhydrazinecarboxamide hydrochloride (10 mg, 23 μmol) to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 502.8 [M+H+]
Example 14
Synthesis of N-((5-(5-chloro-3-fluoro-2-(5-methyl-l ,2,4-oxadiazol-3-yl)phenyl)pyridin-2- yl)methyl)- 1 -ethyl-2-(l ,2,3-thiadiazole-5-carbonyl)hydrazinecarboxamide (compound 35)
Figure imgf000123_0001
A. (5 '-Chloro-3 '-fluoro-2'-(5 -methyl- 1 ,2,4-oxadiazol-3 -yl)-biphenyl-4-ylmethyl)- carbamic acid tert-butyl ester
According to the synthesis of 2-methyl-propane-2-sulfinic acid (l-(3,5'-dichloro-3'- fluoro-2'-(5-methyl-l,2,4-oxadiazol-3-yl)-biphenyl-4-yl)ethyl)-amide (example 5 F), 4-((tert- butoxycarbonylamino)methyl)phenylboronic acid (750 mg, 3.0 mmol) was reacted with 3-(2- bromo-4-chloro-6-fluorophenyl)-5 -methyl- 1,2,4-oxadiazole (436 mg, 1.5 mmol, example 5 B) to afford the title compound after purification by flash chromatography on silica gel (n- hexane/EA) as a colorless oil. MS (m/z): 360.8 [M+H+]
B. (5-(5-Chloro-3-fluoro-2-(5-methyl-l,2,4-oxadiazol-3-yl)phenyl)pyridin-2- yl)methanamine
According to general procedure D (5'-chloro-3'-fluoro-2'-(5-methyl-l,2,4-oxadiazol-3-yl)- biphenyl-4-ylmethyl)-carbamic acid tert-butyl ester (100 mg, 0.24 mmol) was treated with HCl in methanol to give the title compound as the HCl salt which was used for the next step without further purification. MS (m/z): 317.6 [M+H+]
C. tert-Butyl 2-((5-(5-chloro-3-fluoro-2-(5-methyl- 1 ,2,4-oxadiazol-3-yl)phenyl)pyridin- 2-yl)methylcarbamoyl)-2-ethylhydrazinecarboxylate
According to general procedure C (5-(5-chloro-3-fluoro-2-(5-methyl-l,2,4-oxadiazol-3- yl)phenyl)pyridin-2-yl)methanamine (40 mg, 16 μmol) was reacted with tert-butyl 2- (chlorocarbonyl)-2-ethylhydrazinecarboxylate (71 mg, 32 μmol, example 5 J) to give to after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 503.7 [M+H+]
D. N-((5-(5-Chloro-3-fluoro-2-(5-methyl-l,2,4-oxadiazol-3-yl)phenyl)pyridin-2- yl)methyl)- 1 -ethylhydrazinecarboxamide
According to general procedure D tert-butyl 2-((5-(5-chloro-3-fluoro-2-(5-methyl-l,2,4- oxadiazol-3-yl)phenyl)pyridin-2-yl)methylcarbamoyl)-2-ethylhydrazinecarboxylate (50 mg, 0.1 mmol) was treated with HCl in methanol to give the title compound as the HCl salt which was used for the next step without further purification. MS (m/z): 403.9 [M+H+]
E. N-((5-(5-Chloro-3-fluoro-2-(5-methyl-l,2,4-oxadiazol-3-yl)phenyl)pyridin-2- yl)methyl)-l-ethyl-2-(l,2,3-thiadiazole-5-carbonyl)hydrazinecarboxamide
According to general procedure E l,2,3-thiadiazole-5-carboxylic acid (17 mg, 138 μmol, example 11 A) was activated with HATU and treated with methyl 4'-((I- ethylhydrazinecarboxamido)methyl)-3 ,3 '-difluorobiphenyl-2-carboxylate hydrochloride (9 mg, 23 μmol) to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 515.8 [M+H+]
Example 15
Synthesis of N-((5'-chloro-3'-fluoro-2'-(5-methyl- 1 ,2,4-oxadiazol-3-yl)biphenyl-4- yl)methyl)- 1 -ethyl-2-(3-methoxyisoxazole-5-carbonyl)hydrazinecarboxamide (compound 37)
Figure imgf000124_0001
A. N-((5'-Chloro-3'-fluoro-2I-(5-methyl-l,2,4-oxadiazol-3-yl)biphenyl-4-yl)methyl)-l- ethyl-2-(3-methoxyisoxazole-5-carbonyl)hydrazinecarboxamide
According to general procedure E 3-methoxy-isoxazole-5-carboxylic acid (20 mg, 138 μmol) was activated with HATU and treated with methyl 4'-((I- ethylhydrazinecarboxamido)methyl)-3,3'-difluorobiphenyl-2-carboxylate hydrochloride (9 mg, 23 μmol) to afford the title compound after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 528.8 [M+H+] Example 16
Synthesis of (7?>N-(l-(5-(5-chloro-3-fluoro-2-(5-methyl-l,2,4-oxadiazol-3-yl)phenyl)-3- fluoropyridin-2-yl)ethyl)-l-ethyl-2-3-methoxyisoxazole-5-carbonyl)hydrazinecarboxamide (compound 39)
Figure imgf000125_0001
A. N-(fi?;-l-(5-(5-Chloro-3-fluoro-2-(5-methyl-l,2,4-oxadiazol-3-yl)phenyl)-3- fluoropyridin-2-yl)ethyl)-2-methylpropane-2-sulfinamide
According to general procedure I N-((7^-l-(5-bromo-3-fluoropyridin-2-yl)ethyl)-2- methylpropane-2-sulfϊnamide (170 mg, 0.526 mmol, example 10 F) was reacted with 3-(2- bromo-4-chloro-6-fluorophenyl)-5-methyl-l,2,4-oxadiazole (102 mg, 0.351 mmol, example 5 B) to give the title compound after purification by flash chromatography on silica gel (n- hexane/EA). MS (m/z): 454.8 [M+H+]
B. (R)A -(5-(5-Chloro-3-fluoro-2-(5-methyl-l ,2,4-oxadiazol-3-yl)phenyl)-3- fluoropyridin-2-yl)ethanamine
According to general procedure D N-((R)- l-(5-(5-chloro-3-fluoro-2-(5-methyl- 1,2,4- oxadiazol-3-yl)phenyl)-3-fluoropyridin-2-yl)ethyl)-2-methylpropane-2-sulfinamide (100 mg, 0.22 mmol) was treated with HCl in methanol to give the title compound as the HCl salt which was used for the next step without further purification. MS (m/z): 350.9 [M+H+]
C. (R)-tert-Butyl 2-(l -(5-(5-chloro-3-fluoro-2-(5-methyl-l ,2,4-oxadiazol-3-yl)phenyl)- 3-fluoropyridin-2-ylethylcarbamoyl)-2-ethylhydrazinecarboxylate
According to general procedure C ((R)- l-(5-(5-chloro-3-fluoro-2-(5-methyl- 1,2,4- oxadiazol-3-yl)phenyl)-3-fluoropyridin-2-yl)ethanamine (75 mg, 0.194 mmol) was reacted with tert-buXyl 2-(chlorocarbonyl)-2-ethylhydrazinecarboxylate (129 mg, 0.581 mmol, example 5 J) to give to after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 503.7 [M+H+]
D. (R)-N-(I -(5-(5-chloro-3-fluoro-2-(5-methyl-l ,2,4-oxadiazol-3-yl)phenyl)-3- fluoropyridin-2-yl)ethyl)- 1 -ethylhydrazinecarboxamide
According to general procedure D (R) -ter t-buty\ 2-(l-(5-(5-chloro-3-fluoro-2-(5-methyl- l,2,4-oxadiazol-3-yl)phenyl)-3-fluoropyridin-2-ylethylcarbamoyl)-2- ethylhydrazinecarboxylate (80 mg, 0.149 mmol) was treated with HCl in methanol to give the title compound as the HCl salt which was used for the next step without further purification. MS (m/z): 436.9 [M+H+]
E. (R)-N-(I -(5-(5-chloro-3-fluoro-2-(5-methyl-l ,2,4-oxadiazol-3-yl)phenyl)-3- fluoropyridin-2-yl)ethyl)-l-ethyl-2-3-methoxyisoxazole-5-carbonyl)hydrazinecarboxamide
According to general procedure E 3-methoxy-isoxazole-5-carboxylic acid (60 mg, 0.42 mmol) was activated with HATU and treated with (7?)-N-(l-(5-(5-chloro-3-fluoro-2-(5- methyl-l,2,4-oxadiazol-3-yl)phenyl)-3-fluoropyridin-2-yl)ethyl)-l- ethylhydrazinecarboxamide hydrochloride (40 mg, 0.09 mmol) to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 561.8 [M+H+]
Example 17
Synthesis of (7?>N-(l-(5-(5-chloro-3-fluoro-2-(5-methyl-l,2,4-oxadiazol-3-yl)phenyl)-3- fluoropyridin-2-yl)ethyl)ethyl-l-ethyl-2-(2,2,2-trifluoroacetyl)hydrazinecarboxamide (compound 57)
Figure imgf000126_0001
According to the synthesis of (7?^-N-(l-(5-(3,5-dichloro-2-(2,2-difluoroethoxy)phenyl)-3- fluoropyridin-2-yl)ethyl)- 1 -ethyl-2-(2,2,2-trifluoroacetyl)hydrazinecarboxamide (example 11), fT?>N-(l-(5-(5-chloro-3-fluoro-2-(5-methyl-l,2,4-oxadiazol-3-yl)phenyl)-3- fluoropyridin-2-yl)ethyl)-l -ethylhydrazinecarboxamide (5 mg, 11 μmol, example 16 D) was reacted with trifluoroacetic anhydride (1.6 μL, 12 μmol) to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 532.9 [M+H+]
Example 18
Synthesis of N-((5-(5-chloro-3-fluoro-2-(5-methyl-l ,2,4-oxadiazol-3-yl)phenyl)-3- fluoropyridin-2-yl)methyl)-l-ethyl-2-(3-methoxyisoxazole-5-carbonyl)hydrazinecarboxamide (compound 61)
Figure imgf000127_0001
A. (5-Bromo-3-fluoro-pyridin-2-yl)-rnethaneamine
To a stirred solution of 5-bromo-3-fluoro-pyridine-2-carbonitrile (1.0 g, 4.98 mmol, example 10 A) in THF (21 mL) was added DIBAL (16.6 mL, 24.9 mmol, 1.5 M in Toluol) at 0°C and the reaction mixture was stirred at RT overnight. After cooling to O0C the reaction was quenched with 5 % NaOH (20 mL) and stirred for additional 15 min. The reaction mixture was partitioned between EA and water and the separated aqueous layer was extracted with EA (4x). The combined organic layers were washed with 5 % NaOH, 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): 205.0 [M+H+]
B. (5-Bromo-3-fluoro-pyridin-2-ylmethyl)-carbamic acid tert-buty\ ester
To a stirred solution of (5-bromo-3-fluoro-pyridin-2-yl)-methaneamine (1.0 g, 4.98 mmol) in DCM (12.4 mL) were added di-tert-butyl dicarbonate (1.4 g, 6.62 mmol) and NaOH (13.2 mL, 1 M) and the reaction mixture was stirred overnight at RT. Afterwards the mixture was partitioned between DCM and water the separated aqueous layer was extracted with DCM (4x). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo to give the title compound after purification by flash chromatography on silica gel (n- hexane/EA). MS (nVz): 304.7 [M+H+] C . (3-Fluoro-5 -(4,4,5 ,5 -tetramethyl-( 1 ,3 ,2)dioxaborolan-2-yl)-pyridin-2-ylmethyl)- carbamic acid tert-butyl ester
According to the synthesis of 2-methyl-propane-2-sulfinic acid (l-(2-chloro-4-(4,4,5,5- tetramethyl-(l,3,2)dioxaborolan-2-yl)-phenyl)-ethyl)-amide (example 5 E) (5-bromo-3- fluoro-pyridin-2-ylmethyl)-carbamic acid tør/-butyl ester (163 mg, 0.534 mmol) was reacted with bis(pinacolato)diboron to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). GC/MS (m/z): 352.4
D. (5-(5-Chloro-3-fluoro-2-(5-methyl-l,2,4-oxadiazol-3yl)-phenyl)-3-fluoro-pyridin-2- ylmethyl)-carbamic acid ter/-butyl ester
According to the synthesis of 2-methyl-propane-2-sulfinic acid (l-(3,5'-dichloro-3'- fluoro-2'-(5-methyl-l,2,4-oxadiazol-3-yl)-biphenyl-4-yl)ethyl)-amide (example 5 F) (3- fluoro-5-(4,4,5,5-tetramethyl-(l,3,2)dioxaborolan-2-yl)-pyridin-2-ylmethyl)-carbamic acid ter/-butyl ester (140 mg, 0.397 mmol) was reacted 3-(2-bromo-4-chloro-6-fluorophenyl)-5- methyl-l,2,4-oxadiazole (1 16 mg, 0.397 mmol, example 5 B) to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 436.9 [M+H+]
E. (5-(5-Chloro-3-fluoro-2-(5-methyl-l,2,4-oxadiazol-3yl)-phenyl)-3-fluoro-pyridin-2- yl)-methaneamine
According to general procedure D (5-(5-chloro-3-fluoro-2-(5-methyl-l,2,4-oxadiazol- 3yl)-phenyl)-3-fluoro-pyridin-2-ylmethyl)-carbamic acid ter/-butyl ester (100 mg, 0.229 mmol) was treated with HCl in methanol to give the title compound as the HCl salt which was used for the next step without further purification. MS (m/z): 337.0 [M+H*]
F. tert-Butyl 2-((5-(5-chloro-3-fluoro-2-(5-methyl-l ,2,4-oxadiazol-3-yl)phenyl)-3- fluoropyridin-2-yl)methylcarbamoyl)-2-ethylhydrazinecarboxylate
According to general procedure C (5-(5-chloro-3-fluoro-2-(5-methyl-l,2,4-oxadiazol- 3yl)-phenyl)-3-fluoro-pyridin-2-yl)-methaneamine (50 mg, 0.154 mmol) was reacted with tert-buty\ 2-(chlorocarbonyl)-2-ethylhydrazinecarboxylate (102.9 mg, 0.462 mmol, example 5 J) to give after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 522.9 [M+H+] G. N-((5-(5-Chloro-3-fluoro-2-(5-methyl-l,2,4-oxadiazol-3-yl)phenyl)-3-fluoropyridin- 2-yl)methyl)- 1 -ethylhydrazinecarboxamide
According to general procedure D tert-butyl 2-((5-(5-chloro-3-fluoro-2-(5-methyl-l,2,4- oxadiazol-3-yl)phenyl)-3-fluoropyridin-2-yl)methylcarbamoyl)-2-ethylhydrazinecarboxylate (100 mg, 0.192 mmol) was treated with HCl in methanol to give the title compound as the HCl salt which was used for the next step without further purification. MS (m/z): 423.0 [M+H+]
H. N-((5-(5-Chloro-3-fluoro-2-(5-methyl- 1 ,2,4-oxadiazol-3-yl)phenyl)-3-fluoropyridin- 2-yl)methyl)- 1 -ethyl-2-(3 -methoxyisoxazole-5-carbonyl)hydrazinecarboxamide
According to general procedure E S-methoxy-isoxazole-S-carboxylic acid (7.5 mg, 54 μmol) was activated with HATU and treated with N-((5-(5-chloro-3-fluoro-2-(5-methyl- 1,2,4- oxadiazol-3 -yl)phenyl)-3 -fluoropyridin-2-yl)methyl)- 1 -ethylhydrazinecarboxamide hydrochloride (5 mg, 11 μmol) to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 547.9 [M+H+]
Example 19
Synthesis of N-((5 -(5-chloro-3 -fluoro-2-(5 -methyl- 1 ,2,4-oxadiazol-3 -yl)phenyl)-3 - fluoropyridin-2-yl)methyl)- 1 -ethyl-2-(3 -ethylisoxazole-5 -carbonyl)hydrazinecarboxamide (compound 69)
Figure imgf000129_0001
According to general procedure E S-ethyl-isoxazoleS-carboxylic acid (7.7 mg, 54 μmol) was activated with HATU and treated with N-((5-(5-chloro-3-fluoro-2-(5-methyl- 1,2,4- oxadiazol-3 -yl)phenyl)-3 -fluoropyridin-2-yl)methyl)- 1 -ethylhydrazinecarboxamide hydrochloride (5 mg, 11 μmol) to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 545.9 [M+H+] Example 20
Synthesis of fi?;-N-(l-(5-(3,5-dichloro-2-(2-methyl-2H-tetrazol-5-yl)phenyl)-3- fluoropyridin-2-yl)ethyl)- 1 -ethyl-2-(3 -methoxyisoxazole-5 -carbony^hydrazinecarboxamide (compound 71)
Figure imgf000130_0001
A. 2-Bromo-4,6-dichloro-benzonitrile
A solution of sodium nitrite (1.57 g, 22.8 mmol) in water (20 mL) was slowly added to a mixture of 2-bromo-4,6-dichloro-phenylarnine (5.0 g, 20.7 mmol) in water (15 mL), acetic acid (15 mL) and sulphuric acid (3 mL) at 0-50C and the mixture was stirred at 00C for 30 min (solution 1). A solution of potassium cyanide (6.7 g, 103.5 mmol) in water (25 mL) was added dropwise to a solution of coppersulfate (6.2 g, 39 mmol) in water (20 mL) (solution 2). After solution 1 was added dropwise to solution 2 at 0-5°C the reaction mixture was stirred at RT for additional 30 min, which was then diluted with methyl tert-butyl ether. The separated organic layer was washed with NaOH (1 M), sat. NaHCO3 and brine, dried over Na2SO4, filtered and concentrated in vacuo to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA).
B. 5-(2-Bromo-4,6-dichloro-phenyl)-2H-tetrazole
A mixture of 2-bromo-4,6-dichloro-benzonitrile (2.9 g, 1.6 mmol) and azidotrimethyltin (2.4 g, 11.6 mmol) in toluol (25 mL) was stirred at 1200C for 72 h. The reaction mixture was diluted with EA and HCl (0.5 M) was added until the mixture became a clear solution. The separated organic layer was washed with water and brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was redissolved in n-hexane to give the title compound after filtration as solid. MS (m/z): 292.7 [M+Η+]
C. 5-(2-Bromo-4,6-dichlorophenyl)-2-methyl-2H-tetrazole
To a stirred suspension of 5-(2-bromo-4,6-dichloro-phenyl)-2H-tetrazole (2.4 g, 8.16 mmol) and potassium carbonate (1.58 g, 11.43 mmol) in DMF (5 mL) was added methyl iodide (712 μL, 11.43 mmol) and stirred at RT overnight. The mixture was partitioned between EA and water and the separated organic layer was washed with water and brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (n-hexane/EA) to give the title compound. MS (m/z): 307.0 [M+H+]
D. 1 -(5-Bromo-3-fluoro-pyridin-2-yl)ethylamine
According to general procedure D N-((R)-\ -(5-bromo-3-fluoropyridin-2-yl)ethyl)-2- methylpropane-2-sulfInamide (930 mg, 2.88 mmol, example 10 F) was treated with HCl in methanol to give the title compound as the HCl salt which was used for the next step without further purification. MS (m/z): 218.9 [M+H+]
E. (l-(5-Bromo-3-fluoro-pyridin-2-yl)ethyl)carbamic acid tert-butyl ester According to the synthesis of (5-bromo-3-fluoro-pyridin-2-ylmethyl)-carbamic acid tert- butyl ester (example 18 B), l-(5-bromo-3-fluoro-pyridin-2-yl)ethylamine (738 mg, 2.88 mmol) was treated with di-tert-butyl dicarbonate (820 μL, 3.83 mmol ) to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 318.7 [M+H+]
F. (l-(5-(3,5-Dichloro-2-(2-methyl-2H-tetrazol-5-yl)-phenyl)-3-fluoro-pyridin-2-yl)- ethyl)-carbamic acid tert-butyl ester
According to general procedure I (l-(5-bromo-3-fluoro-pyridin-2-yl)ethyl)carbamic acid tert-buiy\ ester (97.5 mg, 0.305 mmol) was reacted with 5-(2-bromo-4,6-dichloro-phenyl)-2- methyl-2H-tetrazole (103.5 mg, 0.336 mmol) to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 466.9 [M+Η+]
G. l-(5-(3,5-Dichloro-2-(2-methyl-2H-tetrazol-5-yl)-phenyl)-3-fluoro-pyridin-2-yl)- etyhlamine
According to general procedure D (l-(5-(3,5-dichloro-2-(2-methyl-2//-tetrazol-5-yl)- phenyl)-3-fluoro-pyridin-2-yl)-ethyl)-carbamic acid ter/-butyl ester (34.6 mg, 74 μmol) was treated with HCl in methanol to give the title compound as the HCl salt which was used for the next step without further purification. MS (m/z): 366.9 [M+H+]
H. (R)-tert-Butyl 2-( 1 -(5-(3,5-dichloro-2-(2-methyl-2H-tetrazol-5-yl)phenyl)-3- fluoropyridin-2-yl)ethylcarbamoyl)-2-ethylhydrazinecarboxylate
According to general procedure C l-(5-(3,5-dichloro-2-(2-methyl-2H-tetrazol-5-yl)- phenyl)-3-fluoro-pyridin-2-yl)-etyhlamine (40.4 mg, 0.1 mmol) was reacted with tert-butyl 2- (chlorocarbonyl)-2-ethylhydrazinecarboxylate (62.3 mg, 0.28 mmol, example 5 J) to give after purification by reversed phase ΗPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 552.9 [M+Η+]
I. f2?>N-(l-(5-(3,5-dichloro-2-(2-methyl-2H-tetrazol-5-yl)phenyl)-3-fluoropyridin-2- yl)ethyl)- 1 -ethylhydrazinecarboxamide
According to general procedure D (R) -tert-butyl 2-(l-(5-(3,5-dichloro-2-(2-methyl-2H- tetrazol-5-yl)phenyl)-3-fluoropyridin-2-yl)ethylcarbamoyl)-2-ethylhydrazinecarboxylate (50.2 mg, 90 μmol) was treated with HCl in methanol to give the title compound as the HCl salt which was used for the next step without further purification. MS (m/z): 453.0 [M+FT1"]
J. 3-Methoxy-isoxazole-5-carbonyl chloride
To a stirred suspension of 3-methoxy-isoxazole-5-carboxylic acid (19.4 mg, 0.136 mmol) in DCM (270 μL) were added oxalyl chloride (32.7 mg, 0.258 mmol) and DMF (30 μL) and the mixture was stirred at RT for 30 min. After diluting with DCM (270 μL) the solution was used for the next step without further purification.
K. ^;-N-(l-(5-(3,5-dichloro-2-(2-methyl-2H-tetrazol-5-yl)phenyl)-3-fluoropyridin-2- yl)ethyl)-l-ethyl-2-(3-methoxyisoxazole-5-carbonyl)hydrazinecarboxamide
To a stirred solution of (7?>iV-(l-(5-(3,5-dichloro-2-(2-methyl-2H-tetrazol-5-yl)phenyl)-3- fluoropyridin-2-yl)ethyl)-l -ethylhydrazinecarboxamide (12 mg, 25 μmol) in DCM (1 mL) was added 3-methoxy-isoxazole-5-carbonyl chloride (0.5 mmol/mL in DCM, 27 μmol) and then DIPEA until pΗ 9 and the mixture was stirred at RT overnight. After the mixture was concentrated in vacuo, purification by reversed phase ΗPLC using a gradient of ACN in water with 0.1% TFA afforded the title compound as the TFA salt. MS (m/z): 577.9 [M+Η+] Example 21
Synthesis of (R)-N-(I -(5-(5-chloro-3-fluoro-2-(2-methyl-2H-tetrazol-5-yl)phenyl)-3- fluoropyridin-2-yl)ethyl)- 1 -ethyl-2-(2,2,2-trifluoroacetyl)hydrazinecarboxamide (compound 73)
Figure imgf000133_0001
According to the synthesis of (T^-N-(I -(5-(3,5-dichloro-2-(2,2-difluoroethoxy)phenyl)-3- fluoropyridin-2-yl)ethyl)-l-ethyl-2-(2,2,2-trifluoroacetyl)hydrazinecarboxamide (example 11) fϊ?;-N-(l-(5-(3,5-dichloro-2-(2-methyl-2H-tetrazol-5-yl)phenyl)-3-fluoropyridin-2-yl)ethyl)-l- ethylhydrazinecarboxamide hydrochloride (10.7 mg, 23 μmol, example 20 I) was reacted with trifluoroacetic anhydride (10 μL, 20 μmol) to afford after purification by reversed phase ΗPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 546.7 [M+Η+]
Example 22
Synthesis of N-((5-(3,5-dichloro-2-(2,2-difluoroethoxy)phenyl)-3-fluoropyridin-2- yl)methyl)-l -ethyl-2-(3-methoxyisoxazole-5-carbonyl)hydrazinecarboxamide (compound 75)
Figure imgf000133_0002
A. (5-(3,5-Dichloro-2-(2,2-diflouro-ethoxy)-phenyl)-3-flouro-pyridin-2-ylmethyl)- carbamiδc acid ter/-butyl ester
According to the synthesis of 2-methyl-propane-2-sulfinic acid (l-(3,5'-dichloro-3'- fluoro-2'-(5-methyl-l,2,4-oxadiazol-3-yl)-biphenyl-4-yl)ethyl)-amide (example 5 F) (3- fluoro-5-(4,4,5,5-tetramethyl-(l,3,2)dioxaborolan-2-yl)-pyridin-2-ylmethyl)-carbamic acid tert-butyl ester (300 mg, 0.852 mmol, example 18 C) was reacted with l-bromo-3,5-dichloro- 2-(2,2-difluoroethoxy)benzene (312 mg, 1.02 mmol, example 10 G) to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 450.8 [M+H+]
B . (5 -(3 ,5 -Dichloro-2-(2,2-difluoroethoxy)phenyl)-3 -fluoropyridin-2-yl)methanamine According to the general procedure D (5-(3,5-dichloro-2-(2,2-diflouro-ethoxy)-phenyl)-3- flouro-pyridin-2-ylmethyl)-carbamic acid tert-buty\ ester (320 mg, 0.711 mmol) was treated with HCl in methanol to give the title compound as the HCl salt. MS (m/z): 350.9 [M+H+]
C. tert-Butyl 2-((5-(3,5-dichloro-2-(2,2-difluoroethoxy)phenyl)-3-fluoropyridin-2- yl)methylcarbamoyl)-2-ethylhydrazinecarboxylate
According to general procedure C (5-(3,5-dichloro-2-(2,2-difluoroethoxy)phenyl)-3- fluoropyridin-2-yl)methanamine (49.4 mg, 0.141 mmol) was reacted with tert-buty\ 2- (chlorocarbonyl)-2-ethylhydrazinecarboxylate (94.0 mg, 0.422 mmol, example 5 J) to give after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 536.9 [M+H+]
D. iV-((5-(3,5-Dichloro-2-(2,2-difluoroethoxy)phenyl)-3-fluoropyridin-2-yl)methyl)- 1 - ethylhydrazinecarboxamide
According to general procedure D tert-buty\ 2-((5-(3,5-dichloro-2-(2,2- difluoroethoxy)phenyl)-3-fluoropyridin-2-yl)methylcarbamoyl)-2-ethylhydrazinecarboxylate (10.0 mg, 20 μmol) was treated with HCl in methanol to give the title compound as the HCl salt which was used for the next step without further purification. MS (m/z): 437.0 [M+H+]
E. N-((5-(3,5-Dichloro-2-(2,2-difluoroethoxy)phenyl)-3-fluoropyridin-2-yl)methyl)-l- ethyl-2-(3-methoxyisoxazole-5-carbonyl)hydrazinecarboxamide
According to general procedure E S-methoxy-isoxazole-S-carboxylic acid (14.6 mg, 106 μmol) was activated with HATU and treated with N-((5-(3,5-dichloro-2-(2,2- difluoroethoxy)phenyl)-3 -fluoropyridin-2-yl)methyl)- 1 -ethylhydrazinecarboxamide hydrochloride (10 mg, 21 μmol) to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z):
561.9 [M+H+] Example 23
Synthesis of (R)-N-(I -(5-(5-chloro-3-fluoro-2-(thiophen-2-yl)phenyl)-3-fluoropyridin-2- yl)ethyl)- 1 -ethyl-2-(2,2,2-trifluoroacetyl)hydrazinecarboxamide (compound 121)
Figure imgf000135_0001
A. 1 -Bromo-5 -chloro-3 -fluoro-2-iodobenzene
To a stirred solution of 2-bromo-4-chloro-6-fluoro-phenylamine (5.0 g, 22.3 mmol) in DCM (50 ml) was added nitronium tetrafluoroborat (3.7 g, 31.2 mmol) at RT and the mixture was stirred for 90 min before sodium iodide (6.7 g, 44.6 mmol) was added at 0°C. The reaction mixture was allowed to warm to RT overnight and diluted with EA. Then washed with brine (2x), dried over Na2SO4, filtered and concentrated in vacuo to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). GC/MS (m/z): 334.2
B. 2-(2-Bromo-4-chloro-6-fluorophenyl)thiophene
According to the synthesis of methyl-propane-2-sulfinic acid (l-(3,5'-dichloro-3'-fluoro- 2'-(5-methyl-l,2,4-oxadiazol-3-yl)-biphenyl-4-yl)ethyl)-amide (example 5 F) l-bromo-5- chloro-3 -fluoro-2-iodobenzene (300 mg, 0.895 mmol) in dioxane (7.5 mL) was reacted with thiophene-2-boronic acid (137.4 mg, 1.07 mmol) to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). GC/MS (m/z): 290.2
C. (R) -ter t-Butyl l-(5-bromo-3-fluoropyridin-2-yl)ethylcarbamate
According to the synthesis of 2-methyl-propane-2-sulfinic acid (l-(2-chloro-4-(4,4,5,5- tetramethyl-(l,3,2)dioxaborolan-2-yl)-phenyl)-ethyl)-amide (example 5 E) (R) -ter t-butyl l-(5- bromo-3-fluoropyridin-2-yl)ethylcarbamate (2.58 g, 8.08 mmol, example 20 E) was reacted with bis(pinacolato)diboron (2.46 g, 9.7 mmol) to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). GC/MS (m/z): 310.6 [M-tert-Bu]+ D. (R)-tert-Buty\ 1 -(5-(5-chloro-3-fluoro-2-(thiophen-2-yl)phenyl)-3-fluoropyridin-2- yl)ethylcarbamate
According to the synthesis of 2-methyl-propane-2-sulfinic acid (l-(3,5'-dichloro-3'- fluoro-2'-(5-methyl-l,2,4-oxadiazol-3-yl)-biphenyl-4-yl)ethyl)-amide (example 5 F), (R)-tert- butyl 1 -(3-fluoro-5-(4,4,5,5-tetramethyl- 1 ,2,3-dioxaborolan-2-yl)pyridin-2-yl)ethylcarbamate (49.9 mg, 0.136 mmol,) was reacted 2-(2-bromo-4-chloro-6-fluorophenyl)thiophene (39.8 mg, 0.136 mmol) to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 450.8 [M+H+]
E. fi?;-l-(5-(5-Chloro-3-fluoro-2-(thiophen-2-yl)phenyl)-3-fluoropyridin-2- yl)ethanamine
According to the general procedure D (R) -ter t-butyl l-(5-(5-chloro-3-fluoro-2-(thiophen- 2-yl)phenyl)-3-fluoropyridin-2-yl)ethylcarbamate (15.6 mg, 36 μmol) was treated with HCl in methanol to give the title compound as the HCl salt. MS (m/z): 350.9 [M+H+]
F. (R)-tert-Buty\ 2-(l -(5-(5-chloro-3-fluoro-2-(thiophen-2-yl)phenyl)-3-fluoropyridin- 2-yl)ethylcarbarnoyl)-2-ethylhydrazinecarboxylate
According to general procedure C (7?^-l-(5-(5-chloro-3-fluoro-2-(thiophen-2-yl)phenyl)- 3-fluoropyridin-2-yl)ethanamine (49.7 mg, 0.142 mmol) was reacted with tert-butyl 2- (chlorocarbonyl)-2-ethylhydrazinecarboxylate (88.3 mg, 0.397 mmol, example 5 J) to give after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 536.9 [M+H+]
G. (7?;-iV-(l-(5-(5-Chloro-3-fluoro-2-(thiophen-2-yl)phenyl)-3-fluoropyridin-2- yl)ethyl)- 1 -ethylhydrazinecarboxamide
According to general procedure D
Figure imgf000136_0001
2-(l-(5-(5-chloro-3-fluoro-2-(thiophen- 2-yl)phenyl)-3-fluoropyridin-2-yl)ethylcarbamoyl)-2-ethylhydrazinecarboxylate (52.9 mg, 98.7 μmol) was treated with HCl in methanol to give the title compound as the HCl salt which was used for the next step without further purification. MS (m/z): 436.9 [M+H+1 H. (7?;-N-(l-(5-(5-chloro-3-fluoro-2-(thiophen-2-yl)phenyl)-3-fluoropyridin-2-yl)ethyl)- l-ethyl-2-(2,2,2-trifluoroacetyl)hydrazinecarboxamide
According to the synthesis of (T^-N-(I -(5-(3,5-dichloro-2-(2,2-difluoroethoxy)phenyl)-3- fluoropyridin-2-yl)ethyl)- 1 -ethyl -2-(2,2,2-trifluoroacetyl)hydrazinecarboxamide (example 11), fi?;-N-(l-(5-(5-chloro-3-fluoro-2-(thiophen-2-yl)phenyl)-3-fluoropyridin-2-yl)ethyl)-l- ethylhydrazinecarboxamide (10 mg, 21.3 μmol) was reacted with trifluoroacetic anhydride (3.5 μL, 25 μmol) to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 532.9 [M+H+]
Example 24
Synthesis of f7?>iV-(l-(5-(5-chloro-3-fluoro-2-(4-fluoro-lH-pyrazol-l-yl)phenyl)-3- fluoropyridin-2-yl)ethyl)- 1 -ethyl-2-(2,2,2-trifluoroacetyl)hydrazinecarboxamide (compound 123)
Figure imgf000137_0001
A. (2-Bromo-4-chloro-6-fluoro-phenyl)-hydrazine
To a mixture of 2-bromo-4-chloro-6-fluoro-phenylamine (1.5 g, 6.68 mmol) in HCl (62 ml, 6 M) was slowly added a solution of sodium nitrite (553 mg, 8.02 mmol) in water (2 ml) at -7°C. After stirring at 0-5°C for 30 min, tindichloride dihydrate (2.19 g, 8.69 mmol) in HCl (3 ml, 6 M) was added over 30 min at -15-10 °C and the mixture was stirred for additional 30 min. The reaction mixture was concentrated in vacuo and the resulting residue was diluted with EA and sat. NaHCO3 solution was added until pH 8. The separated aqueous layer was extracted with EA (4x) and the combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). GC/MS (m/z): 238.2
B. Toluene-4-sulfonic acid 2,3,3-trifluoro-propyl ester
To a stirred solution of 2,3,3-trifluoro-propan-l-ol (3.96 g, 30.0 mmol) and 4-methyl- benzenesulfonyl chloride (6.9 g, 36.0 mmol) in DCM (20 mL) was added triethylamine (5.0 ml, 36.0 mmol) dropwise at RT. After stirring at RT for 3 h the reaction mixture was quenched with brine (50 mL) and the separated aqueous layer was extracted with diethylether (3x). The combined organic layers were washed with brine (3x), dried over Na2SO4, filtered and concentrated in vacuo to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). GC/MS (m/z): 268.2
C. ^ζ)-2,3,3-Trifluoroprop-l-enyl 4-methylbenzenesulfonate
To a stirred solution of toluene-4-sulfonic acid 2,3,3-trifluoro-propyl ester (1.72 g, 6 mmol) in THF (24 mL) was added buthyllithium (11.3 mL, 18 mmol) at -780C. After stirring at -78°C for 45 min the reaction mixture was quenched with an icecold 10% HCl (14 mL) and extracted with diethylether (3x). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). GC/MS (m/z): 266.2
D. l-(2-Bromo-4-chloro-6-fluorophenyl)-4-fluoro-lH-pyrazole
To a solution of (Z^^^-trifluoroprop- 1-enyl 4-methylbenzenesulfonate (209 mg, 0.786 mmol) in ACN (2.1 mL) was added diethylamine (408 μL, 3.929 mmol) and the mixture was stirred at 70°C for 1 h and then cooled to RT. Then a solution of (2-bromo-4-chloro-6-fluoro- phenyl)-hydrazine (93.5 mg, 0.393 mmol) in ethanol (5.3 mL) and HCl (50%ig, 0.786 mmol) were added. After stirring at 80 °C for 45 min the reaction mixture was concentrated in vacuo and the resulting residue was partioned between DCM and sat. NaHCO3 solution. The aqueous layer was extracted with DCM (3x), dried over Na2SO4, filtered and concentrated in vacuo to give the title compound after purification by flash chromatography on silica gel (n- hexane/EA). GC/MS (m/z): 292.2
E. (R)-tert-Buty\ l-(5-(5-chloro-3-fluoro-2-(4-fluoro-lH-pyrazol-l-yl)phenyl)-3- fluoropyridin-2-yl)ethylcarbamate
According to the synthesis of 2-methyl-propane-2-sulfinic acid (l-(3,5'-dichloro-3'- fluoro-2'-(5-methyl-l,2,4-oxadiazol-3-yl)-biphenyl-4-yl)ethyl)-amide (example 5 F), (R)-tert- butyl 1 -(3-fluoro-5-(4,4,5,5-tetramethyl-l ,2,3-dioxaborolan-2-yl)pyridin-2-yl)ethylcarbamate (100.4 mg, 0.274 mmol, example 23 C) was reacted l-(2-brorno-4-chloro-6-fluorophenyl)-4- fiuoro-lH-pyrazole (80 mg, 0.274 mmol) to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 452.8 [M+H+]
F. (R)- 1 -(5-(5-Chloro-3-fluoro-2-(4-fluoro- lH-pyrazol- 1 -yl)phenyl)-3-fluoropyridin-2- yl)ethanamine
According to the general procedure D (R)-tert-buiy\ l-(5-(5-chloro-3-fluoro-2-(4-fluoro- lH-pyrazol-l-yl)phenyl)-3-fluoropyridin-2-yl)ethylcarbamate (124 mg, 0.274 mmol) was treated with HCl in methanol to give the title compound as the HCl salt. MS (m/z): 352.9 [M+Η+]
G. (R)-tert-Butyl 2-(l-(5-(5-chloro-3-fluoro-2-(4-fluoro-lH-pyrazol-l-yl)phenyl)-3- fluoropyridin-2-yl)ethylcarbamoyl)-2-ethylhydrazinecarboxylate
According to general procedure C ^-l-(5-(5-chloro-3-fluoro-2-(4-fluoro-lH-pyrazol-l- yl)phenyl)-3-fluoropyridin-2-yl)ethanamine (80 mg, 0.138 mmol) was reacted with tert-butyl 2-(chlorocarbonyl)-2-ethylhydrazinecarboxylate (61.3 mg, 0.276 mmol, example 5 J) to give after purification by reversed phase ΗPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 538.9 [M+Η+]
H. fi?>N-(l-(5-(5-Chloro-3-fluoro-2-(4-fluoro-lH-pyrazol-l-yl)phenyl)-3- fluoropyridin-2-yl)ethyl)- 1 -ethylhydrazinecarboxamide
According to general procedure D
Figure imgf000139_0001
2-(l-(5-(5-chloro-3-fluoro-2-(4-fluoro- 1 H-pyrazol- 1 -yl)phenyl)-3 -fluoropyridin-2-yl)ethylcarbamoyl)-2-ethylhydrazinecarboxylate (47 mg, 87.3 μmol) was treated with HCl in methanol to give the title compound as the HCl salt which was used for the next step without further purification. MS (m/z): 439.0 [M+Η+]
I. f7?>N-(l-(5-(5-Chloro-3-fluoro-2-(4-fiuoro-lH-pyrazol-l-yl)phenyl)-3-fluoropyridin-2- yl)ethyl)-l-ethyl-2-(2,2,2-trifluoroacetyl)hydrazinecarboxamide
According to the synthesis of (T^-N-(I -(5-(3,5-dichloro-2-(2,2-difluoroethoxy)phenyl)-3- fluoropyridin-2-yl)ethyl)- 1 -ethyl-2-(2,2,2-trifluoroacetyl)hydrazinecarboxamide (example 11 ), (R)-N-(I -(5-(5-chloro-3-fluoro-2-(4-fluoro- 1 H-pyrazol- 1 -yl)phenyl)-3 -fluoropyridin-2- yl)ethyl)-l -ethylhydrazinecarboxamide (10 mg, 22.8 μmol) was reacted with trifluoroacetic anhydride (7 μL, 50.2 μmol) to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 534.9 [M+H+]
Example 25
Synthesis of fK;-N-(l-(5-(5-chloro-3-fluoro-2-(l-methyl-lH-pyrrol-3-yl)phenyl)-3- fluoropyridin-2-yl)ethyl)-l-ethyl-2-(3-methoxyisoxazole-5-carbonyl)hydrazinecarboxamide (compound 136)
Figure imgf000140_0001
A. 4-(2-Bromo-4-chloro-6-fluorophenyl)- 1 -methyl- 1 H-pyrazole
To a stirred solution of l-bromo-5-chloro-3-fluoro-2-iodobenzene (100 mg, 0.298 mmol, example 23 A) in TΗF (5 mL) was added butyllithium (204 μl, 0.328 mmol) at -90°C and afterwards zinc(II) chloride (954 μl, 0.954 mmol. The mixture was stirred for additional 1 h and allowed to warm to -300C. This cold solution was added dropwise to a solution of 4-iodo- 1 -methyl- 1 H-pyrazole (93.1 mg, 0.447 mmol) and tetrakis(triphenylphosphin)palladium(0) (24.1 mg, 0.021 mmol) in dioxane (2 mL) at RT. The reaction mixture was stirred at 55°C for 1.5 h and then quenched with methanol (1 mL) at RT. After the mixture was evaporated to dryness the resulting residue was portioned between DCM and water. Cone, ammonium hydroxide was added to the separated aqueous layer until pΗ 11-12 and then extracted with DCM (2x). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo to give the title compound after purification by flash chromatography on silica gel (n- hexane/EA). MS (m/z): 289.1 [M+Η+]
B. (R)-tert-Butyl 1 -(5-(5-chloro-3-fluoro-2-(l -methyl- 1 H-pyrazol-4-yl)phenyl)-3 - fluoropyridin-2-yl)ethylcarbamate
According to the synthesis of 2-methyl-propane-2-sulfinic acid (l-(3,5'-dichloro-3'- fluoro-2'-(5-methyl-l,2,4-oxadiazol-3-yl)-biphenyl-4-yl)ethyl)-amide (example 5 F), (R)-tert- butyl 1 -(3-fiuoro-5-(4,4,5,5-tetramethyl- 1 ,2,3-dioxaborolan-2-yl)pyridin-2-yl)ethylcarbamate (94.4 mg, 0.258 mmol, example 23 C) was reacted with 4-(2-bromo-4-chloro-6- fluorophenyl)-! -methyl- 1 H-pyrazole (74.6 mg, 0.258 mmol) to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 448.8 [M+H+]
C. (R)- 1 -(5 -(5 -Chloro-3 -fluoro-2-( 1 -methyl- 1 H-pyrazol-4-yl)phenyl)-3 -fluoropyridin-2- yl)ethanamine
According to the general procedure D (R)-tert-butyl l-(5-(5-chloro-3-fluoro-2-(l-methyl- lH-pyrazol-4-yl)phenyl)-3-fluoropyridin-2-yl)ethylcarbamate (82.9 mg, 0.185 mmol) was treated with HCl in methanol to give the title compound as the HCl salt. MS (m/z): 348.8 [M+Η+]
D. (R)-tert-Butyl 2-(l -(5-(5-chloro-3-fluoro-2-(l -methyl- 1 H-pyrazol-4-yl)phenyl)-3- fluoropyridin-2-yl)ethylcarbamoyl)-2-ethylhydrazinecarboxylate
According to general procedure C (7?j-l-(5-(5-chloro-3-fluoro-2-(l-methyl-lH-pyrazol-4- yl)phenyl)-3-fluoropyridin-2-yl)ethanamine (64.5 mg, 0.185 mmol) was reacted with tert- butyl 2-(chlorocarbonyl)-2-ethylhydrazinecarboxylate (115.3 mg, 0.518 mmol, example 5 J) to give the title compound after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA as the TFA salt. MS (m/z): 534.9 [M+H+]
E. ^>N-(l-(5-(5-Chloro-3-fluoro-2-(l-methyl-lH-pyrazol-4-yl)phenyl)-3- fluoropyridin-2-yl)ethyl)- 1 -ethylhydrazinecarboxamide
According to general procedure D
Figure imgf000141_0001
2-(l-(5-(5-chloro-3-fluoro-2-(l-methyl- l//-pyrazol-4-yl)phenyl)-3-fluoropyridin-2-yl)ethylcarbamoyl)-2-ethylhydrazinecarboxylate (65.4 mg, 0.122 mmol) was treated with HCl in methanol to give the title compound as the HCl salt which was used for the next step without further purification. MS (m/z): 434.9 [M+Η+]
F. f/?>iV-(l-(5-(5-chloro-3-fluoro-2-(l-methyl-lH-pyrrol-3-yl)phenyl)-3-fluoropyridin- 2-yl)ethyl)-l-ethyl-2-(3-methoxyisoxazole-5-carbonyl)hydrazinecarboxamide
To fK>N-(l-(5-(5-chloro-3-fluoro-2-(l-methyl-lH-pyrazol-4-yl)phenyl)-3-fluoropyridin-2- yl)ethyl)-l -ethylhydrazinecarboxamide hydrochloride (10 mg, 0.022 mmol) in DCM (2 mL) was added a solution of 3-methoxy-isoxazole-5-carboxylic acid chloride (242 μL, 0.024 mmol) in DCM (224 μL) and the reaction mixture was stirred at RT for 2 h. Then evaporation to dryness and purification by reversed phase ΗPLC using a gradient of ACΝ in water with 0.1% TFA afforded the title compound as the TFA salt. MS (m/z): 559.8 [M+H+]
Example 26
Synthesis of (7?;-3-(l-(5-(5-chloro-3-fluoro-2-(5-methyl-l ,2,4-oxadiazol-3-yl)phenyl)-3- fluoropyridin-2-yl)ethyl)-l -hydroxy- 1-methylurea (compound 1004)
Figure imgf000142_0001
(R)- 1 -(5-(5-chloro-3-fluoro-2-(5-methyl- 1 ,2,4-oxadiazol-3-yl)phenyl)-3-fluoropyridin-2- yl)ethanamine was reacted with N-methylhydroxylamine hydrochloride (2.2 mg, 26 μmol, example 16 B) to afford after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 423.8 [M+H*]
Example 27
Synthesis of 3-((5-chloro-3-fluoro-2-(2-methyl-2H-tetrazol-5-yl)phenyl)-3-fluotopyridin- 2-yl)methyl)-l -hydroxy- 1-isopropylurea (compound 1023)
Figure imgf000142_0002
A. 2-Bromo-4-chloro-6-fluoro-benzonitrile
To a mixture of 2-bromo-4-chloro-6-fluoro-phenylamine (13.8 g, 61.5 mmol) in tetrafluoroboronic acid solution (200 mL, 48 wt. % in water) was added sodium nitrite (5 g, 73.8 mmol) in water (40 mL) dropwise at 0-5°C. After additional 10 min at 0°C potassium cyanide (20.0 g, 307.5 mmol) was added followed by an ice-cooled solution of copper sulfate (19.6 g, 123 mmol) in water (40 mL). After the reaction mixture was stirred at 0°C for 30 min and at RT for 1 h the mixture was quenched with NaOH (10 M) at 0°C until pΗ 8-9, diluted with EA and filtered through Celite. The separated aqueous was extracted with EA and the combined organic layers were washed with sat. NaHCO3 solution and brine, dried over Na2SO4, filtered and concentrated in vacuo to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). GC/MS (m/z): 234.5
B. 5-(2-Bromo-4-chloro-6-fluoro-phenyl)-2-methyl-2H-tetrazole
A mixture of 2-bromo-4-chloro-6-fluoro-benzonitrile (3.5 g, 14.9 mmol), dibutyltin oxide (3.7 g, 14.9 mmol) and trimethylsilyl azide (9.9 ml, 74.5 mmol) in toluol (70 mL) was stirred at 1100C overnight. Afterwards the mixture was evaporated to dryness, the resulting residue was dissolved in EA, extracted with HCl (0.5 M) and brine, dried over Na2SO4, filtered and concentrated in vacuo. To a stirred mixture of the crude product in DMF (25 mL) were added potassium carbonate (2.48 g) and then methyl iodide (1.1 mL, 18.0 mmol) dropwise at 0°C. After stirring at RT for 30 min the mixture was diluted with EA, washed with water and brine, dried over Na2SO4, filtered and concentrated in vacuo to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA).
C. (5-(5-Chloro-3-fluoro-2-(2-methyl-2H-tetrazol-5-yl)-phenyl)-3-fluoro-pyridin-2- ylmethyl)-carbamic acid tert-butyl ester
According to the synthesis of 2-methyl-propane-2-sulfinic acid (l-(3,5'-dichloro-3'- fluoro-2'-(5-methyl-l,2,4-oxadiazol-3-yl)-biphenyl-4-yl)ethyl)-amide (example 5 F), (3- fluoro-5-(4,4,5,5-tetramethyl-(l,3,2)dioxaborolan-2-yl)-pyridin-2-ylmethyl)-carbamic acid tert-buty\ ester (1.0 g, 2.90 mmol, example 18 C) was reacted with 5-(2-bromo-4-chloro-6- fluoro-phenyl)-2-methyl-2H-tetrazole (844 mg, 2.90 mmol) to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 436.8 [M+Η+]
D. (5-(5-Chloro-3-fluoro-2-(2-methyl-2H-tetrazol-5-yl)-phenyl)-3-fluoro-pyridin-2- yl)methanamine
According to general procedure D (5-(5-chloro-3-fluoro-2-(2-methyl-2//-tetrazol-5-yl)- phenyl)-3-fluoropyridin-2-ylmethyl)-carbamic acid tert-butyl ester (641 mg, 1.47 mmol) was treated with HCl in methanol to give the title compound as the HCl salt. MS (m/z): 336.9 [M+Η+]
E. 3-((5-Chloro-3-fluoro-2-(2-methyl-2H-tetrazol-5-yl)ρhenyl)-3-fluoropyridin-2- yl)methyl)- 1 -hydroxy- 1 -isopropylurea
A mixture of di-ter/-butyl dicarbonate (5.5 μL, 26 μmol), 4-dimethylaminopyridine (1 1.1 mg, 2 μmol) and (5-(5-chloro-3-fluoro-2-(2-methyl-2H-tetrazol-5-yl)-phenyl)-3-fluoro- pyridin-2-yl)methanamine (9.1 mg, 24 μmol) in DCM (900 μL) was stirred at RT for 30 min. After the addition of DIPEA (9.6 μL, 55 μmol) and N-isopropylhydroxylamine hydrochloride (4.1 mg, 37 μmol) the reaction mixture was stirred at 40°C overnight and then concentrated to dryness. Purification by reversed phase ΗPLC using a gradient of ACN in water with 0.1% TFA afforded the title compound as the TFA salt. MS (m/z): 437.9 [M+Η+]
Example 28
Synthesis of 3-((5-(5-chloro-3-fluoro-2-(2-methyl-2H-tetrazol-5-yl)phenyl)-3- fluoropyridin-2-yl)methyl)- 1 -hydroxy- 1-methylurea (compound 1025)
Figure imgf000144_0001
According to the synthesis of 3-((5-chloro-3-fluoro-2-(2-methyl-2H-tetrazol-5-yl)phenyl)- 3-fluotopyridin-2-yl)methyl)-l -hydroxy- 1 -isopropylurea (example 27 E), (5-(5-chloro-3- fluoro-2-(2-methyl-2Η-tetrazol-5-yl)-phenyl)-3-fluoro-pyridin-2-yl)methanamine (11.5 mg, 31 μmol) was reacted with jV-methylhydroxylamine hydrochloride to give the title compound after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA as the TFA salt. MS (m/z): 409.9 [M+H+]
Example 29
Synthesis of (R)-3-(\ -(3,5'-dichloro-3'-fluoro-2'-(5-methyl-l ,2,4-oxadiazol-3-yl)biphenyl- 4-yl)ethyl)-l -hydroxy- 1-methylurea (compound 1026)
OH
Figure imgf000144_0002
To a stirred mixture of l-(3,5'-dichloro-3'-fluoro-2'-(5-methyl-l,2,4-oxadiazol-3-yl)- biphenyl-4-yl)-ethylamine hydrochloride (50 mg, 0.122 mmol, example 5 F) and triethylamine (51 μl, 0.366 mmol) in THF (2 mL) was added a solution of 4- nitrophenylchloroformiate (27 mg, 0.134 mmol) in THF (0.5 mL) dropwise at -78°C. The mixture was stirred at RT for 15 min and concentrated to dryness. The resulting residue was dissolved in DMF (1.25 mL) and triethylamine (102 mL, 0.61 mmol) as well as N- methylhydroxylamine hydrochloride (20 mg, 0.244 mmol) were added. After the reaction mixture was stirred at RT for 24 h, purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA afforded the title compound as the TFA salt. M S (m/z): 438.8 [M+H+]
Example 30
Synthesis of (R)-I-(I -(5-(3,5-dichloro-2-(2,2-difluoroethoxy)phenyl)-3-fluoropyridin-2- yl)ethyl)-l -hydroxy- 1-methylurea (compound 1027)
Figure imgf000145_0001
According to the synthesis of f7?;-3-(l-(3,5'-dichloro-3'-fluoro-2'-(5-methyl-l,2,4- oxadiazol-3-yl)biphenyl-4-yl)ethyl)- 1 -hydroxy- 1 -methylurea, (R)- 1 -(5-(3,5-dichloro-2-(2,2- difluoroethoxy)phenyl)-3-fluoropyridin-2-yl)ethanamine hydrochloride (30 mg, 0.069 mmolmol, example 10 1) was reacted with iV-methylhydroxylamine (11.6 mg, 0.138 mmol) to give the title compound after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% as the TFA salt. MS (m/z): 437.8 [M+H+]
Example 31
Synthesis of (7?>3-(l-(5-(5-chloro-3-fluoro-2-(5-methyl-l,2,4-oxadiazol-3-yl)phenyl)-3- methylpyridin-2-yl)ethyl)- 1 -hydroxy- 1 -methylurea (compound 1015)
Figure imgf000145_0002
A. (R,E)-N-(l-(5-Bromo-3-methylpyridin-2-yl)ethylidene)-2-methylpropane-2- sulfmamide According to general procedure F 3-bromo-2-formyl-3-picoline (2.5 g, 12.5 mmol) was treated with (7?χ+)-2-methyl-2-propansulfinamide (1.5 g, 12.5 mmol) to afford the title compound after purification by chromatography on silica gel (n-hexane/EA) as light brown oil. MS (m/z): 317.8 [M+H+]
B. (R)-N-{(R)-\ -(5-Bromo-3-methylpyridin-2-yl)ethyl)-2-methylpropane-2-sulfinamide According to the general procedure G (R,E)-iV-(l-(5-bromo-3-methylpyridin-2- yl)ethylidene)-2-methylpropane-2-sulfinamide (1.0 g, 3.36 mmol) was reacted with methyl magnesium chloride to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 320.8 [M+H+]
C. fT?>N-(^>l-(5-(5-Chloro-3-fluoro-2-(5-methyl-l,2,4-oxadiazol-3-yl)phenyl)-3- methylpyridin-2-yl)ethyl)-2-methylpropane-2-sulfinamide
According to general procedure I (7^-N-((7^-l-(5-bromo-3-methylpyridin-2-yl)ethyl)-2- methylpropane-2-sulfinamide (206 mg, 0.645 mmol) was reacted with 3-(2-bromo-4-chloro- 6-fluorophenyl)-5-methyl-l,2,4-oxadiazole (395 mg, 1.36 mmol, example 5 B) to give the title compound after purification by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 450.9 [M+H+]
D. ^;-l-(5-(5-Chloro-3-fluoro-2-(5-methyl-l,2,4-oxadiazol-3-yl)phenyl)-3- methylpyridin-2-yl)ethanamine
According to general procedure D (7?>)-iV-(^)-l-(5-(5-chloro-3-fluoro-2-(5-methyl-l,2,4- oxadiazol-3-yl)phenyl)-3-methylpyridin-2-yl)ethyl)-2-methylpropane-2-sulfinamide ( 107 mg, 0.24 mmol) was treated with HCl in methanol to give the title compound as the HCl salt which was used for the next step without further purification. MS (m/z): 346.9 [M+H+]
E. (7?>3-(l-(5-(5-Chloro-3-fluoro-2-(5-methyl-l,2,4-oxadiazol-3-yl)phenyl)-3- methylpyridin-2-yl)ethyl)- 1 -hydroxy- 1 -methylurea
According to the synthesis of (9H-fluoren-9-yl)methyl 2-((3,3'-difluoro-2'- (methoxycarbonyl)biphenyl-4-yl)methylcarbamoyl)-2-ethylhydrazinecarboxylate (example 1 J), (7?;-l-(5-(5-chloro-3-fluoro-2-(5-methyl-l,2,4-oxadiazol-3-yl)phenyl)-3-methylpyridin-2- yl)ethanamine hydrochloride (10 mg, 26 μmol) was reacted with N-methylhydroxylamine hydrochloride (4.4 mg, 52 μmol) to give the title compound after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% as the TFA salt. MS (m/z): 346.9 [M+H+]
Example 32
Synthesis of 3-((5-(5-chloro-3-fluoro-2-(2-methyl-2H-tetrazol-5-yl)phenyl)-3- fluoropyridin-2-yl)methyl)- 1 -hydroxy- 1 -(tetrahydrosulfon-3-yl)urea (compound 1028)
Figure imgf000147_0001
According to the synthesis of 3-((5-chloro-3-fluoro-2-(2-methyl-2H-tetrazol-5-yl)phenyl)- 3 -fluoropyridin-2-yl)methyl)-l -hydroxy- 1 -isopropylurea (example 24 E), (5-(5-chloro-3- fluoro-2-(2-methyl-2H-tetrazol-5-yl)-phenyl)-3-fluoro-pyridin-2-yl)methanamine (12.3 mg, 0.033 mmol) was reacted with (hydroxyamino)trtrahydrothophene- 1,1 -dioxide (30.9 mg, 0.165 mmol) to give the title compound after purification by reversed phase ΗPLC using a gradient of ACN in water with 0.1% as the TFA salt. MS (m/z): 515.9 [M+Η+]
Example 33
Synthesis of N-(2-((5'-chloro-3,3'-difluoro-2'-(5-methyl-l ,2,4-oxadiazol-3-yl)-biphenyl-4- ylmethyl)-amino)-benzoimidazol- 1 -yl)-2-cyano-acetamide (compound 2008)
Figure imgf000147_0002
A. Nitrilo-acetic acid N'-(2-nitro-phenyl)-hydrazide
A suspension of 1 -fluoro-2-nitrobenzene (25.4 g, 0.18 mmol) and 2-cyanoacetic hydrazide (44.6 g, 0.45 mmol) in DMF (200 mL) was stirred at 90 0C for 18 h. The reaction mixture was diluted with water (1000 mL) and then NaOH (1 M) was added until pH 6-7 before the precipitated solid was collected by filtration. Recrystalization of the resulting residue in EA afforded the title compound as yellow solid. GC/MS (m/z): 206.2
B. Nitrilo-acetic acid N'-(2-amino-phenyl)-hydrazide
A mixture of nitrilo-acetic acid N'-(2-nitro-phenyl)-hydrazide (4.8 g, 21.8 mmol) in methanol (750 mL) was treated with palladium on cabon (500 mg) and hydrogen at RT for 3 h. The reaction mixture was filtrated through celite and concentrated in vacuo to give the title compound after purification by crystalisation with EA.
C. N-(2-((5t-Chloro-3,3'-difluoro-2'(5-methyl-l,2,4-oxadiazol-3-yl)-biphenyl-4- ylmethyl)-amino)-benzoimidazol- 1 -yl)-2-cyano-acetamide
To stirred suspension of (5'-chloro-3,3'-difluoro-2'-(5-methyl-l,2,4-oxadiazol-3- yl)biphenyl-4-yl)methanamine (34 mg, 0.1 mmol) in DCM (2 mL) and 50 % NaHCO3 (2 mL) was added thiophosgene (11.5 μl, 0.15 mmol) at 0°C, which was then stirred for 30 min at 0°C. The mixture was diluted with EA and the separated organic phase was washed with brine, filtrated and concentrated in vacuo. The residue was redissolved in DMF (1 mL) and nitrilo-acetic acid N'-(2-amino-phenyl)-hydrazide (29 mg, 0.15 mmol) was added and the mixture was stirred at 60 °C for 1.5 h. EDC (16.5 ml, 0.1 μmol) was added to the reaction mixture and after additional stirring at 60 0C for 1.5 h, purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA afforded the title compound as the TFA salt. MS (m/z): 534.0 [M+H+]
Example 34
Synthesis of S-methoxy-isoxazole-S-carboxylic acid (2- ((5-(5-chloro-3-fluoro-2-(5- methyl- 1 ,2,4-oxadiazol-3 -yl)-phenyl)-3 -fluoro-pyridin-2-ylmethyl)-amino)-benzoimidazol- 1 - yl)-amide (compound 2058)
Figure imgf000148_0001
A. l-((3-Methoxy-isoxazole-5-carbonyl)-amino)-lH-benzoimidazole-2-sulfonic acid To a stirred solution of S-methoxy-isoxazole-S-carboxylic acid (840 mg, 5.9 mmol) in DCM (5 mL) was added l-chlor-2-methyl-l-dimethylamino-l-propen (783 μl, 5.88 mmol) and after 5 min at RT l-aminobenzimidazole-2-sulfonic acid (500 mg, 2.35 mmol), followed by DIPEA at 0°C. Afterwards the mixture was evaporated to dryness, the resulting residue was dissolved in water (30 mL), NaOH (5 mL, 1 M) and EA (1 mL) and HCl (1 M) was slowly added until pH 1. The precipitating solid was collected by filtration and dried in vacuo to afford the title compound as a white solid which was used fir the next step without further purification. MS (m/z): 338.8 [M+H+]
B. S-Methoxy-isoxazole-S-carboxylic acid (2- ((5-(5-chloro-3-fluoro-2-(5-methyl-l,2,4- oxadiazol-3 -yl)-phenyl)-3 -fluoro-pyridin-2-ylmethyl)-amino)-benzoimidazol- 1 -yl)-amide
A mixture of l-((3-methoxy-isoxazole-5-carbonyl)-amino)-lH-benzoimidazole-2-sulfonic acid (20 mg, 5.95 μmol), (5-(5-chloro-3-fluoro-2-(5-methyl-l,2,4-oxadiazol-3yl)-phenyl)-3- fluoro-pyridin-2-yl)-methaneamine (20 mg, 5.95 mmol, example 18 E) in DMF (300 μL) was stirred at 90°C for 18 h. Purification by reversed phase ΗPLC using a gradient of ACN in water with 0.1% TFA afforded the title compound as the TFA salt. MS (m/z): 593.0 [M+Η+]
Example 35
Synthesis of S-methoxy-isoxazole-S-carboxylic acid ((2-((5-(3,5-dichloro-2-(2,2-difluoro- ethoxy)-phenyl)-3-fluoro-pyridin-2-ylmethyl)-amino)-benzoimidazol- 1 -yl)-amide (compound 2059)
Figure imgf000149_0001
A mixture of (5-(3,5-dichloro-2-(2,2-difluoro-ethoxy)-phenyl)-3-fluoro-pyridin-2-yl)- methanamine (21 mg, 5.92 μmol, example 22 B) and l-((3-methoxy-isoxazole-5-carbonyl)- amino)-lH-benzoimidazole-2-sulfonic acid (20 mg, 5.92 μmol, example 31 A) in isopropanole (300 μL) was stirred at 80°C over the weekend to give after purification by reversed phase HPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 607.0 [M+H+]
Example 36
Synthesis of S-methoxy-isoxazole-S-carboxylic acid (2-((5-(3,5-dichloro-2-(5-methyl- 1 ,2,4-oxadiazol-3-yl)-phenyl)-3-fluoro-pyridin-2-ylmethyl)-amino)-benzimidazol- 1 -yl)-amide (compound 2060)
Figure imgf000150_0001
A. 2-Bromo-4,6-dichloro-N-hydroxy-benzamidine
A mixture of 2-bromo-4,6-dichloro-benzonitrile (2.6 g, 10.36 mmol, example 20A), hydroxylamine hydrochloride (938 mg, 13.5 mmol) and sodium tert-butanolat (1.49 g, 15.54 mmol) in ethanol (15 mL) was stirred at 100°C overnight. The reaction mixture was concentrated to dryness and the resulting to residue was dissolved in DCM and the organic layer was washed with HCl (IM). After the separated aqueous layer was extracted twice with DCM, followed by extraction with DCM (3x) at pH 9. The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo to give the title compound which was used for the next step without further purification. MS (m/z): 365.0 [M-OH]+
B. 3-(2-Bromo-4,6-dichloro-phenyl)-5-methyl-l,2,4-oxadiazole 2-Bromo-4,6-dichloro-N-hydroxy-benzamidine (1.74 g, 6.1 mmol) was reacted with N ,7V- dimethylacetamide dimethylacetal (896 μL, 6.1 mmol) at RT for 1 h to afford the title compound after the resulting solid residue was purified by flash chromatography on silica gel (n-hexane/EA). GC/MS (m/z): 308.0
C. (5-(3,5-Dichloro-2-(5-methyl-l,2,4-oxadiazol-3-yl)-phenyl)-3-fluoro-pyridin-2- ylmethyl)-carbamic acid tert-butyl ester According to the synthesis of 2-methyl-propane-2-sulfinic acid (l-(3,5'-dichloro-3'- fluoro-2'-(5-methyl-l,2,4-oxadiazol-3-yl)-biphenyl-4-yl)ethyl)-amide (example 5 F) (3- fluoro-5-(4,4,5,5-tetramethyl-(l,3,2)dioxaborolan-2-yl)-pyridin-2-ylmethyl)-carbamic acid tert-buty\ ester (300 mg, 0.852 mmol, example 18 C) was reacted with 3-(2-bromo-4,6- dichloro-phenyl)-5-methyl-l,2,4-oxadiazole (314 mg, 0.695 mmol) to afford the title comound after the resulting solid residue was purified by flash chromatography on silica gel (n-hexane/EA). MS (m/z): 457.7 [M+H+]
D. (5-(3,5-Dichloro-2-(5-methyl-l,2,4-oxadiazol-3-yl)-phenyl-3-fluoro-pyridin-2-yl)- methanamine
According to the general procedure D (5-(3,5-dichloro-2-(5-methyl-l,2,4-oxadiazol-3-yl)- phenyl)-3-fluoro-pyridin-2-ylmethyl)-carbamic acid tert-butyl ester (190 mg, 0.42 mmol) was treated with HCl in methanol to give the title compound as the HCl salt. MS (m/z): 352.9 [M^-H+]
E. S-Methoxy-isoxazole-S-carboxylic acid (2-((5-(3,5-dichloro-2-(5-methyl- 1,2,4- oxadiazol-3 -yl)-phenyl)-3 -fluoro-pyridin-2-ylmethyl)-amino)-benzimidazol- 1 -yl)-amide
According to the synthesis of 3-methoxy-isoxazole-5-carboxylic acid ((2-((5-(3,5- dichloro-2-(2,2-difluoro-ethoxy)-phenyl)-3-fluoro-pyridin-2-ylmethyl)-amino)- benzoimidazol-l-yl)-amide (example 32) (5-(3,5-dichloro-2-(5-methyl-l,2,4-oxadiazol-3-yl)- phenyl-3-fluoro-pyridin-2-yl)-methanamine (21 mg, 5.92 mmol) was reacted with l-((3- methoxy-isoxazole-5-carbonyl)-amino)-lH-benzoimidazole-2-sulfonic acid (20 mg, 5.92 μmol, example 31 A) to give after purification by reversed phase ΗPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 609.0 [M+Η+]
Example 37
Synthesis of S-methoxy-isoxazole-S-carboxylic acid (2-((5-(5-chloro-3-fluoro-2-(2- methyl-2H-tetrazol-5-yl)phenyl)-3-fluoro-pyridin-2-ylmethyl)-amino)-benzoimidazol-l-yl)- amide (compound 2063)
Figure imgf000152_0001
According to the synthesis of S-methoxy-isoxazole-S-carboxylic acid ((2-((5-(3,5- dichloro-2-(2,2-difluoro-ethoxy)-phenyl)-3-fluoro-pyridin-2-ylmethyl)-amino)- benzoimidazol-l-yl)-amide (example 32) (5-(5-chloro-3-fluoro-2-(2-methyl-2H-tetrazol-5- yl)-phenyl)-3-fluoro-pyridin-2-yl)methanamine (20 mg, 5.92 μmol, example 24 D) was treated with l-((3-methoxy-isoxazole-5-carbonyl)-amino)-lH-benzoimidazole-2-sulfonic acid (20 mg, 5.92 μmol, example 31 B ) to give after purification by reversed phase ΗPLC using a gradient of ACN in water with 0.1% TFA the title compound as the TFA salt. MS (m/z): 593.0 [M+Η+]
Biological evaluation:
Example 38
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-D AKD (Perkin Elmer) and the antagonist compound dilutions were prepared in a dilution plate which also included controls to assess total binding (1 nM 3H-D AKD) 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 min 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 NaCl, 0.2 M acetic acid) for 10 min 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 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). IC50 values were calculated by fitting a 4- parameter logistic function to the concentration-response data using non-linear regression (Xlfit, 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.
Example 39
Functional assay for BK Bl 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 40.000 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 37°C 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 37°C. 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 (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.
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 imgf000156_0001
(I) or a pharmacologically acceptable salt, solvate or hydrate thereof, wherein
A is
Figure imgf000156_0002
X is CH or N;
R ι l , D R^ , D R3J, I RJ44, Γ R>66, Γ R> 77, and R are each and independently of each other selected from hydrogen atom, halogen atom, hydroxy, cyano, amino, alkyl, or optionally substituted heteroalkyl;
R5 is a halogen atom, hydroxy, cyano, amino, an alkyl, 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, or an optionally substituted heteroaralkyl;
R9 is a hydrogen atom, an alkyl, or a heteroalkyl;
R10 is a hydrogen atom, an alkyl, an optionally substituted heteroalkyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aralkyl, or an optionally substituted heteroaralkyl;
R11 is an alkyl, 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, or an optionally substituted heteroaralkyl;
R12 is a hydrogen atom, an alkyl, an optionally substituted heteroalkyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, an optionally substituted aralkyl, or an optionally substituted heteroaralkyl;
B is O or N;
Z1 and Z2 are each and independently of each other selected from C or N, and Z3 and Z4 are each and independently of each other selected from C, S, O or N;
R13 is an alkyl, 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, or an optionally substituted heteroaralkyl;
R14 and R15, if present, are
(i) each and independently of each other selected from hydrogen atom, halogen atom, CN, hydroxy, =O, alkyl, C3-C6-cycloalkyl, heteroalkyl or alkoxy; or
(ii) joined together to form a carbocyclic or heterocyclic 5- or 6-membered ring, which is substituted with 0 to 4 substituents selected from the group comprising R16, R17, R18 and R19, and is saturated, unsaturated, or aromatic, and, if heterocyclic, contains one or more heteroatom(s) each and independently selected from N, O and S; and
R16, R17, R18 and R19 are individually and independently selected from hydrogen atom, halogen atom, hydroxy, cyano, amino, alkyl, and optionally substituted heteroalkyl.
2. The compound according to Claim 1, wherein R5 is a halogen atom, an alkyl, an optionally substituted heteroalkyl, or a moiety selected from
Figure imgf000158_0001
wherein is a single or double bond;
X1, X2, X3, and X4 are each and independently of each other selected from C, N, O, S, NRX, CR", or CRXRX', and X5 is selected from carbon atom, N or CRX, wherein
Rx and Rx are each and independently of each other selected from hydrogen atom, halogen atom, =0, hydroxy, cyano, alkyl, alkyl-amino, alkyl-mercapto, or alkoxy.
3. The compound according to Claim 1 or Claim 2, wherein
R1, R3, R7,and R8 are each a hydrogen atom;
R2, R4 and R6 are each and independently of each other selected from hydrogen atom, halogen atom, cyano, alkyl or heteroalkyl; and
R9 is a hydrogen atom or Me.
4. The compound according to any one of Claims 1 to 3, wherein R5 is Me, Et, cyano, CF3, Cl or a moiety selected from
Figure imgf000159_0001
and Rxl, R1^2, R^ and Rx4 are each and independently of each other selected from hydrogen atom, halogen atom, alkyl or alkoxy, and Rx5, Rx6 and Rx7 are each and independently of each other selected from hydrogen atom or fluorine atom.
5. The compound according to Claim 4, wherein, if present, Rxl is Me.
6. The compound according to anyone of Claims 1 to 5, wherein A is
Figure imgf000160_0001
O ; and
R11 is an alkyl, an optionally substituted heteroalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted aralkyl, or an optionally substituted heteroaralkyl.
7. The compound according to anyone of Claims 1 to 6, wherein A is
Figure imgf000160_0002
R1 is an alkyl or an optionally substituted cycloalkyl.
8. The compound according to anyone of Claims 1 to 5, wherein A is
Figure imgf000160_0003
R12 is an alkyl, an optionally substituted heteroalkyl, or an optionally substituted cycloalkyl.
9. The compound according to anyone of Claims 1 to 5, wherein A is selected from
Figure imgf000161_0001
R14, R15, R16, R17, R18, and R19 are each and independently of each other selected from hydrogen atom, halogen atom, hydroxy, cyano, amino, alkyl, or optionally substituted heteroalkyl.
10. The compound according to Claim 9, wherein R14, R15, R16, R17, R18, and R19 are individually and independently selected from hydrogen, fluoro, chloro, methyl, ethyl, methoxy, CN or trifluoromethyl.
11. The compound according to anyone of Claims 1 to 5, 9 and 10, wherein R13 is an alkyl, an optionally substituted heteroalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted aralkyl, or an optionally substituted heteroaralkyl.
12. The compound according to anyone of Claims 1 to 7, and 9 to 11, wherein R and R13, are each and independently of each other selected from an alkyl, an optionally substituted heteroalkyl, or a moiety selected from
Figure imgf000161_0002
wherein Ryl, Ry2, and Ry3 are each and independently of each other selected from hydrogen atom, halogen atom, alkyl or alkoxy.
13. The compound according to Claim 12, wherein
Ryl and Ry2 are each and independently of each other selected from H, Me, Et, or OMe; and Ry3 is Me.
14. The compound according to anyone of Claims 1 to 7, 12 and 13, wherein R11 is selected from Me, Et, iPr, tBu, CHF2, CH2F, CF3,
Figure imgf000162_0001
Figure imgf000162_0002
15. The compound according to anyone of Claims 1 to 8 and 12 to 14, wherein R 10 and R12 are selected from Me, Et, iPr or
Figure imgf000162_0003
16. The compound according to anyone of Claims 1 to 5 and 9 to 14, wherein R13 is selected from Me, Et, iPr, tBu, CF3,
Figure imgf000163_0001
Figure imgf000163_0002
17. The compound according to anyone of Claims 9 to 13 and 16, wherein R is H.
18. A compound, preferably a compound according to any one of claims 1 to 17, which compound is selected from compounds 1 to 293 of Table 1, compounds 1001-1065 of Table 2 and compounds 2001-2115 of Table 3.
19. A pharmaceutical composition comprising one or more compounds according to any one of claims 1 to 18 and, optionally, at least one carrier substance, excipient and/or adjuvant.
20. Use of a compound or of a pharmaceutical composition according to any one of claims 1 to 19 for the manufacture of a medicament for the treatment and/or prevention of a disease or a condition.
21. The use according to claim 20, wherein the condition or disease is selected from the group comprising inflammatory diseases, immunology disorders and pain.
22. 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 19.
23. The compound according to any of claims 1 to 18, for use in a method for the treatment of a subject, preferably a subject in need of such treatment.
24. The compound according to claim 23, wherein the subject suffers from or is at risk of suffering from a disease selected from the group comprising inflammatory diseases, immunology disorders and pain.
25. The pharmaceutical composition according to claim 19, for use in a method for the treatment of a subject, preferably a subject in need of such treatment.
26. The pharmaceutical composition according to claim 25, wherein the subject suffers from or is at risk of suffering from a disease selected from the group comprising inflammatory diseases, immunology disorders and pain.
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WO2015039172A1 (en) 2013-09-17 2015-03-26 Vectus Biosystems Pty Ltd Compositions for the treatment of hypertension and/or fibrosis
EP3046902A4 (en) * 2013-09-17 2017-01-25 Vectus Biosystems Limited Compositions for the treatment of hypertension and/or fibrosis
RU2661878C2 (en) * 2013-09-17 2018-07-20 Вектус Байосистемс Лимитед Compositions for the treatment of hypertension and/or fibrosis
US11498903B2 (en) 2017-08-17 2022-11-15 Bristol-Myers Squibb Company 2-(1,1′-biphenyl)-1H-benzodimidazole derivatives and related compounds as apelin and APJ agonists for treating cardiovascular diseases

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